MASARYKOVA UNIVERZITA

MASARYKOVA UNIVERZITA Přírodovědecká fakulta Výzkumné centrum pro chemii životního prostředí a ekotoxikologii

Soňa MARVANOVÁ

Genotoxicita polyaromatických sloučenin v jaterních progenitorových buňkách

Disertační práce

Školitel: RNDr. Miroslav Machala, CSc.

Brno, 2009

Bibliografické informace

Jméno a příjmení autora:

Soňa Marvanová

Název disertační práce: Genotoxicita polyaromatických sloučenin v jaterních progenitorových buňkách Název disertační práce anglicky: Genotoxicity of polyaromatic compounds in liver progenitor cells Studijní program:

Chemie

Studijní obor (směr), kombinace oborů:

Chemie životního prostředí

Školitel:

RNDr. Miroslav Machala, CSc.

Rok obhajoby:

2009

Klíčová slova v češtině: Polyaromatické sloučeniny; genotoxicita; apoptóza; protein p53; proliferace; Ah receptor; jaterní progenitorové buňky. Klíčová slova v angličtině: Polyaromatic compounds; genotoxicity; apoptosis; protein p53; proliferation; Ah receptor; liver progenitor cells.

© Soňa Marvanová, Masarykova univerzita, 2009

Poděkování

Můj největší dík patří mému školiteli RNDr. Miroslavu Machalovi, CSc. za jeho vedení, cenné rady a velkou podporu. Za spolupráci děkuji také Honzovi Vondráčkovi, Zdeňkovi Andrysíkovi, Katce Pěnčíkové a všem kolegyním a kolegům z Výzkumného ústavu veterinárního lékařství, kteří zde vytvářeli příjemné pracovní prostředí. Můj dík za spolupráci také náleží Zuzce Valovičové a RNDr. Aleně Gábelové, CSc. z Ústavu experimentální onkologie SAV a Ing. Janu Topinkovi, CSc. z Ústavu experimentální medicíny AV ČR. Za pomoc s péčí o naši dcerušku velmi děkuji svému muži Hynkovi, který mi tím umožnil tuto práci dokončit.

Abstrakt Játra představují centrální orgán metabolismu xenobiotik. Obsahují několik typů buněk, včetně buněk progenitorových, které mohou v případě rozsáhlého poškození jater proliferovat a nahradit poškozené buňky. Vedle této role při regeneraci jater se mohou progenitorové buňky také stát cílem chemických karcinogenů a hrát roli v hepatokarcinogenezi. Přesto je většina studií zabývajících se účinky polyaromatických uhlovodíků (PAU), významných environmentálních kontaminantů, dosud zaměřena na diferencované buňky nebo jejich deriváty. V prezentované práci byly jako buněčný model pro studium genotoxických efektů PAU použity buňky WB-F344, potkaní jaterní epiteliální buněčná linie, sdílející řadu vlastností s progenitorovými buňkami. Z vybraných karcinogenních PAU byly dibenzo[a,l]pyren (DBalP), benzo[a]pyren, benzo[g]chrysen a dibenzo[a,e]pyren schopny vytvářet v buňkách WB-F344 stabilní DNA adukty s následnou aktivací proteinu p53 a apoptózou. Naproti tomu další karcinogenní PAU chrysen, benz[a]anthracen, benzo[b]fluoranthen a dibenzo[a,h]anthracen tyto genotoxické účinky nevyvolávaly a na základě předchozích studií lze předpokládat, že v jejich působení na jaterní progenitorové buňky hrají významnější roli tumor-promoční efekty. Na příkladu DBalP bylo za použití inhibitorů cytochromu P450 (CYP) 1B1 potvrzeno, že CYP1B1 hraje jednu z klíčových rolí v genotoxických a cytotoxických procesech indukovaných PAU. Některé monomethylované deriváty benzo[a]anthracenu (MeBaA) ve starších studiích projevovaly tumor-iniciační, karcinogenní a mutagenní účinky v hlodavčích kožních a bakteriálních modelech. V modelu jaterních progenitorových buněk WB-F344 pouze 10-MeBaA vyvolával mírně genotoxické účinky, a to tvorbu stabilních aduktů DNA, fosforylaci proteinu p53, apoptózu, zvýšené procento buněk v S-fázi buněčného cyklu a zvýšenou produkci reaktivních kyslíkových radikálů. Ostatní MeBaA v buňkách WB-F344 tyto genotoxické účinky nevyvolávaly, ale byly schopny aktivovat receptor AhR silněji než jejich parentální sloučenina BaA. Tímto mechanismem, aktivitou zprostředkovanou receptorem AhR, mohou významně přispívat k toxicitě environmentálních směsí. Výsledky také poukazují na rozdílnou tkáňovou specifitu účinků PAU. Environmentální polutant 7H-dibenzo[c,g]karbazol (DBC) a jeho syntetický derivát 5,9-dimethyldibenzo[c,g]karbazol (diMeDBC), vykazující striktní hepatokarcinogenitu, v buňkách WB-F344 vyvolávaly efekty související s jejich silnou genotoxicitou (fosforylace p53, akumulace buněk v S-fázi a apoptóza). Další syntetický derivát, sarkomagenní N-methyldibenzo[c,g]karbazol (N-MeDBC) tyto účinky nevyvolával. DiMeDBC ale překvapivě nevytvářel adukty s DNA a na druhé straně N-MeDBC vyvolával vznik zlomů na DNA i mikrojader v buňkách WB-F344. Podrobnější studium genotoxických efektů ukázalo, že zatímco příčinou genotoxicity DBC jsou stabilní DNA adukty, v případě diMeDBC by mohlo být za genotoxicitu zodpovědné oxidativní poškození nebo modifikace bází. N-MeDBC vyvolával sice nízkou hladinu aduktů na DNA, ale pravděpodobně dostačující pro

vznik zlomů na DNA a mikrojader. Tato poškození byla nicméně účinně opravena a neprojevila se na buněčné úrovni. Buňky WB-F344 byly dále použity jako model pro studium genotoxických a tumorpromočních efektů vzorku ovzduší - městského prachu, standardního referenčního materiálu SRM 1649a a jeho chromatografických frakcí. Extrakt SRM 1649a a polární frakce vyvolávaly genotoxické účinky (apoptóza, fosforylace p53 a snížení počtu buněk) pouze v nejvyšších koncentracích. Mezi DNA adukty, vytvořenými po expozici extraktem SRM, byly identifikovány pouze adukty BaP-dihydrodiolepoxidu a ve velmi malé míře adukty DBalP-dihydrodiolepoxidu. Efekty asociované s nádorovou promocí (aktivace receptoru AhR, indukce mRNA CYP1A1 a CYP1B1 a proliferace) se projevovaly už od nízkých koncentrací celkového extraktu SRM, polární frakce i neutrální frakce obsahující PAU. Tyto výsledky potvrzují, že genotoxicita PAU ve směsích není aditivní, ale její výsledný efekt je slabší, pravděpodobně díky inhibici enzymů metabolické aktivace CYP1 některými PAU přítomnými ve směsi, a dále naznačují významnost tumor-promočních efektů, projevujících se na rozdíl od genotoxicity v nižších koncentracích.

Abstract (in English) The liver represents a central organ of xenobiotic metabolism. It consists of several types of cells, including progenitor cells, which are able to proliferate in case of extensive liver damage and replace the damaged cells. Besides their role in liver regeneration, these progenitor cells might be a target of chemical carcinogens and thus play a role in hepatocarcinogenesis. Nevertheless, most of the studies dealing with the effects of polyaromatic hydrocarbons (PAHs), important environmental contaminants, are focused on the differentiated cells or their derivatives. In this thesis, rat liver epithelial cell line WBF344, sharing many characteristics with progenitor cells, was used as a cellular model for the research of genotoxic effects of PAHs. From the group of selected carcinogenic PAHs, dibenzo[a,l]pyrene (DBalP), benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene were able to induce the formation of stable DNA adducts with subsequent protein p53 activation and apoptosis in the cell line WB-F344. In contrast, the other selected carcinogenic PAHs chrysene, benz[a]anthracene, benzo[b]fluoranthene and dibenzo[a,h]anthracene did not induce these genotoxic effects and on the basis of previous studies it could be assumed that their tumor-promoting effects might play a more important role in their activity in liver progenitor cells. Next, using DBalP and the inhibitors of cytochrome P450 (CYP) 1B1 activity, we confirmed that CYP1B1 played a significant role in genotoxic and cytotoxic processes induced by PAHs. In former studies, several monomethylated derivatives of benz[a]anthracene (MeBaAs) have exhibited tumor-initiating, carcinogenic and mutagenic effects in rodent skin and bacterial models. In the WB-F344 cell line, only 10-MeBaA induced slight genotoxic effects, such as formation of stable DNA adducts, phosphorylation of protein p53, apoptosis, accumulation of cells in S-phase of the cell cycle and increased formation of reactive oxygen species. Other MeBaAs did not induce these genotoxic effects in the WB-F344 cells, but were able to activate the Ah receptor stronger than their parental compound BaA. Therefore they may contribute significantly to the toxicity of environmental mixtures through AhR-mediated activity. The results of the study also point to different tissue specificity of methylated PAHs. Environmental contaminant 7H-dibenzo[c,g]carbazole (DBC) and its synthetic derivative 5,9-dimethyldibenzo[c,g]carbazole (diMeDBC), strict hepatocarcinogen, induced effects connected with their strong genotoxicity in cells WB-F344 (phosphorylation of p53, cell accumulation in S-phase and apoptosis). Another synthetic derivative of DBC, sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC) did not induce these effects. Surprisingly, diMeDBC did not induced formation of DNA adducts, while N-MeDBC caused formation of DNA strand breaks and micronuclei in WB-F344 cells. More detailed study of genotoxic effects showed that while the cause of DBC genotoxicity is the formation of DNA adducts, in case of diMeDBC, the oxidative damage or base modifications might be responsible for its genotoxicity. N-MeDBC induced low level of DNA adducts, but these were

probably sufficient to form DNA strand breaks and micronuclei. Nevertheless, this damage was efficiently repaired and did not exert on the cell level. Next, the WB-F344 cells were used as a model for study of genotoxic and tumorpromoting effects of air particulate sample (urban dust), Standard Reference Material SRM 1649a and its chromatographic fractions. The extract of SRM 1649a and polar fraction induced genotoxic effects (phosphorylation of p53, apoptosis and decreased number of cells) only at the highest doses used. Of DNA adducts formed by SRM extract, only BaPdihydrodiolepoxide adducts were identified, as well as a very small amount of DBalPdihydrodiolepoxide adducts. Crude extract of SRM, neutral fraction containing PAHs and polar fraction exerted effects associated with tumor promotion (AhR activation, induction of mRNA CYP1A1 and CYP1B1 and proliferation), already in low doses. These results confirmed that genotoxicity of PAHs in mixtures is not additive, but instead, their final effect is lower than expected, most probably due to inhibition of xenobiotic metabolizing enzymes CYP1 by some PAHs present in the mixture. The results also indicate the importance of tumor-promoting effects, observed already in lower doses, contrary to genotoxicity.

Obsah 1.

Úvod ................................................................................................................................. 11 1.1. Polyaromatické uhlovodíky, jejich zdroje a expozice .............................................. 11 1.2. Metabolismus a účinky PAU.................................................................................... 12 1.2.1. Metabolická aktivace a genotoxické účinky PAU............................................ 12 1.2.1.1. Aktivace receptoru AhR ............................................................................... 12 1.2.1.2. Tvorba dihydrodiolepoxidů .......................................................................... 13 1.2.1.3. Tvorba o-chinonů a ROS.............................................................................. 13 1.2.1.4. Tvorba kationtových radikálů....................................................................... 14 1.2.1.5. Typy genotoxických poškození a odpovědi na poškození DNA.................. 15 1.2.2. Negenotoxické účinky PAU ............................................................................. 16 1.3. Metody detekce genotoxických a negenotoxických účinků PAU ............................ 18 1.3.1. Testy in vivo ..................................................................................................... 18 1.3.2. Testy in vitro..................................................................................................... 19 1.3.2.1. Testy genotoxických efektů.......................................................................... 19 1.3.2.2. Testy negenotoxických efektů ...................................................................... 20 1.4. Charakteristika vybraných skupin PAU ................................................................... 22 1.4.1. Abundantní nížemolekulární PAU ................................................................... 22 1.4.2. Skupina PAU s molekulovou hmotností 228 ................................................... 23 1.4.3. Skupina PAU s molekulovou hmotností 252 ................................................... 23 1.4.4. Skupina PAU s molekulovou hmotností 278 ................................................... 24 1.4.5. Skupina PAU s molekulovou hmotností 302 ................................................... 25 1.4.6. Methylované deriváty chrysenu a benz[a]anthracenu ...................................... 26 1.5. Vybrané N-heterocyklické aromatické uhlovodíky....................................................... 27 2. Cíle práce.......................................................................................................................... 28 3. Materiál a metody............................................................................................................. 29 3.1. Buněčná linie ............................................................................................................ 29 3.2. Metody...................................................................................................................... 30 3.2.1. Analýza DNA aduktů ....................................................................................... 30 3.2.2. Detekce mRNA metabolizačních enzymů pomocí real time-PCR................... 30 3.2.3. Detekce proteinu p53 metodou Western blotting ............................................. 31 3.2.4. Detekce apoptózy ............................................................................................. 31 3.2.5. Hodnocení buněčné proliferace a buněčného cyklu ......................................... 32 3.2.6. Detekce zlomů na DNA pomocí gelové elektroforézy jednotlivých buněk..... 32 4. Výsledky........................................................................................................................... 34 4.1. Tvorba DNA aduktů a indukce apoptózy v potkaních jaterních buňkách WB-F344 exponovaných karcinogenním PAU..................................................................................... 35 4.1.1. Tvorba stabilních DNA aduktů ........................................................................ 35 4.1.2. Akumulace a fosforylace proteinu p53............................................................. 36 4.1.3. Indukce apoptózy.............................................................................................. 37 4.1.4. Aktivace metabolizačních enzymů................................................................... 39 4.1.5. Role CYP1B1 v metabolické aktivaci genotoxinu DBalP ............................... 39 4.2. Toxické efekty methylovaných benz[a]anthracenů v jaterních buňkách ................. 41 4.2.1. Aktivace metabolizačních enzymů................................................................... 41 4.2.2. Narušení buněčného cyklu a indukce buněčné proliferace .............................. 42 4.2.3. Fosforylace proteinu p53 a apoptóza................................................................ 43 4.2.4. Tvorba DNA aduktů a oxidativního stresu....................................................... 45 4.3. Genotoxické a tumor-promoční efekty 7H-dibenzo[c,g]karbazolu a jeho syntetických derivátů v jaterních epiteliálních buňkách WB-F344 ..................................... 46

4.3.1. Aktivace metabolizačních enzymů................................................................... 47 4.3.2. Narušení buněčného cyklu a indukce proliferace............................................. 47 4.3.3. Indukce apoptózy a fosforylace proteinu p53 .................................................. 49 4.3.4. Poškození DNA - tvorba stabilních DNA aduktů, zlomů a mikrojader ........... 49 4.3.5. Kinetika oprav zlomů na DNA......................................................................... 51 4.3.6. Oxidativní poškození DNA a oxidativní stres.................................................. 51 4.4. Účinky komplexní směsi PAU vázaných na částice ovzduší SRM 1649a v jaterních progenitorových buňkách ..................................................................................................... 53 4.4.1. Aktivace metabolizačních enzymů................................................................... 54 4.4.2. Tvorba DNA aduktů ......................................................................................... 54 4.4.3. Fosforylace proteinu p53 a apoptóza................................................................ 55 4.4.4. Vliv SRM na buněčnou proliferaci a cyklus .................................................... 57 5. Diskuze a závěry............................................................................................................... 59 6. Použitá literatura............................................................................................................... 63 7. Souhrnný přehled publikací a odborných příspěvků ........................................................ 73 Přílohy

Úvod

1.

Úvod

1.1. Polyaromatické uhlovodíky, jejich zdroje a expozice Polycyklické aromatické uhlovodíky (PAU) jsou velkou a rozmanitou skupinou organických sloučenin, obsahujících dvě a více kondenzovaných benzenových jader. Mohou existovat také ve formě alkyl-, nitro-, amino-, sulfo-, halogen- a dalších derivátů. Strukturou, účinky a výskytem jsou jim velmi blízké heterocyklické aromatické uhlovodíky, které obsahují jeden nebo více atomů dusíku, síry nebo kyslíku v aromatických kruzích. PAU představují hojně rozšířené kontaminanty životního prostředí s negativními, především karcinogenními, účinky na živé organismy. PAU vznikají nedokonalým spalováním organických materiálů během antropogenních i přírodních procesů. Největší podíl na jejich produkci má lokální vytápění, silniční doprava, průmyslová výroba (např. výroba hliníku, koksu, asfaltu, plastů), výroba energie spalováním fosilních paliv a spalování odpadu (Boström, 2002; IARC, 1983; WHO, 1998). Nejvýznamnější cestou expozice člověka PAU je potrava (v případě nekuřáků). Hlavním zdrojem kontaminace zeleniny a obilovin je atmosférická depozice malých částic obsahujících PAU, zatímco maso je kontaminováno během úpravy, především při uzení a pečení na otevřeném ohni (rev. viz Phillips, 1999). K další expozici člověka PAU dochází dýcháním ovzduší kontaminovaného cigaretovým kouřem, výfukovými plyny, emisemi z domácích topenišť, průmyslovými emisemi, příp. expozicí na některých pracovištích. Expozice je také možná prostřednictvím dermálního kontaktu s ropnými produkty (např. dehtem) nebo vodou jimi kontaminovanou (Boström, 2002). PAU se vyskytují v prostředí ve směsích. Řada směsí a materiálů obsahujících PAU a expozic v zaměstnání byla Mezinárodní agenturou pro výzkum rakoviny (IARC) klasifikována jako prokazatelně nebo potenciálně karcinogenní. Do skupiny 1, jako prokazatelně karcinogenní pro člověka, byly zařazeny např. uhelný dehet, asfalt, saze, cigaretový kouř, výroba hliníku, zplyňování uhlí a výroba koksu. Do skupiny 2A, jako pravděpodobně karcinogenní pro člověka, byly zařazeny např. výfukové plyny nebo rafinace ropy (IARC, 1984a,b; IARC, 1985; IARC, 1987; IARC, 1989a,b).

11

Úvod

1.2. Metabolismus a účinky PAU PAU vyvolávají řadu genotoxických a negenotoxických účinků, které se na úrovni organismu mohou projevit vznikem různých chorob, včetně vzniku nádoru. Proces karcinogeneze je třístupňový a zahrnuje fázi iniciace, promoce a progrese. Genotoxické účinky se uplatňují během iniciace, kdy dochází k nevratným změnám na DNA (Trosko, 1997). Ve druhé fázi, nádorové promoci, se projevují především negenotoxické efekty, vyvolané dlouhodobým působením nádorového promotoru. Negenotoxické efekty vedou ke vzniku nezávislosti iniciovaných buněk na homeostatické kontrole tkáně, jejímž výsledkem je potom nekontrolovaná proliferace iniciovaných buněk (Trosko, 1998), a dalším procesům jako inhibice apoptózy, rezistence vůči protirůstovým signálům, angiogeneze atd. (Hanahan and Weinberg, 2000).

1.2.1. Metabolická aktivace a genotoxické účinky PAU Pro rozvoj svých mutagenních a karcinogenních vlastností vyžadují PAU metabolickou aktivaci, tzn. fungují jako promutageny, resp. prokarcinogeny (Miller, 1966; Miller, 1978). Metabolická aktivace vede k produkci reaktivních metabolitů PAU a následně ke vzniku DNA aduktů, které jsou jednou z hlavních příčin genotoxických efektů PAU. Existuje několik cest metabolické aktivace PAU, přičemž všechny mohou přispívat ke genotoxickým účinkům PAU. Během metabolické aktivace PAU může docházet také k oxidativnímu stresu a následně k oxidativnímu poškození DNA. Význam příspěvku každého mechanismu závisí na chemických a biologických vlastnostech jednotlivých sloučenin, druhu organismu, úrovni exprese aktivačních enzymů apod. (Xue, 2005).

1.2.1.1.

Aktivace receptoru AhR

Významnou úlohu v metabolické aktivaci PAU hraje cytosolový receptor AhR (Aryl Hydrocarbon Receptor). Po vazbě PAU na AhR dojde k uvolnění receptoru z komplexu s dalšími proteiny a k dimerizaci AhR s jeho translokátorem ARNT. Vzniklý dimer interaguje v jádře s promotery obsahujícími specifickou sekvenci - xenobiotický responzivní element (XRE). Tato interakce vyústí v indukci genové exprese řady proteinů první a druhé fáze biotransformace xenobiotik, při níž jsou PAU transformovány na reaktivní metabolity, které mohou být buď konjugovány a vyloučeny z buňky, nebo se mohou vázat na DNA a proteiny (Whitlock, 1999). Aktivace AhR vede ale také ke změně exprese řady genů regulujících 12

Úvod

buněčný cyklus a další buněčné procesy; tyto negenotoxické efekty jsou spojeny s nádorovou promocí, vývojovými a obecně endokrinními poruchami (Puga, 2002).

1.2.1.2.

Tvorba dihydrodiolepoxidů

Jednou z cest metabolické aktivace je biotransformace PAU cytochromy P450 (CYP), zejména CYP1A1, 1A2 a 1B1, a epoxidhydrolázou za vzniku dihydrodiolepoxidů, které se mohou kovalentně vázat na purinové báze a vytvářet stabilní DNA adukty (obr. 2) (Baird, 2005). V druhé fázi biotransformace se uplatňují enzymy glutation-S-transferázy, UDPglukuronyltransferázy a sulfotransferázy, které vytvářejí konjugáty s metabolity PAU z první fáze nebo přímo s některými substituovanými deriváty PAU (Cooper, 1983; Sims, 1981; Yuzuru, 1978). Schopnost dihydrodiolepoxidů vázat se na DNA souvisí s přítomností tzv. „bay“ a „fjord“ oblasti ve struktuře molekuly (obr. 1). Reaktivní metabolity PAU s epoxidovou skupinou navázanou v pozici sousedící s touto oblastí mohou mít silnější genotoxické účinky, především pokud PAU obsahuje strukturu označovanou jako „fjord“ (Baird, 2005). Tyto oblasti, zejména pokud jsou v sousedství s epoxidovou skupinou, představují natolik významné sférické zábrany pro enzymy druhé fáze biotransformace, že vzniklé metabolity s touto strukturou jsou nedostatečně konjugovány a reagují s DNA (Baird, 2005).

„bay“ oblast

„fjord“ oblast

Obr. 1: „Bay“ a „fjord“ oblast v molekule benzo[a]pyrenu a dibenzo[a,l]pyrenu.

1.2.1.3.

Tvorba o-chinonů a ROS

V alternativní dráze metabolické aktivace slouží dihydrodiol, vzniklý hydrolýzou epoxidu, jako substrát pro dihydrodioldehydrogenázy, které patří mezi aldo-ketoreduktázy (AKR). Dihydrodiol je oxidován AKR na ketol, který spontánně přechází na katechol (obr. 2). Katechol je nestabilní a podstupuje další neenzymovou oxidaci. První jednoelektronovou oxidací vzniká o-semichinonový aniontový radikál a peroxid vodíku. Druhou jednoelektronovou oxidací vzniká o-chinon a superoxidový anion (Penning, 1996). Vzniklé ochinony jsou vysoce reaktivní a mohou vytvářet stabilní i nestabilní DNA adukty (McCoull, 1999; Shou, 1993). Mohou také dvouelektronovou redukcí znovuvytvořit katechol za účasti 13

Úvod

NADPH-chinonoxidoreduktázy (NQO1) nebo jednoelektronovou redukcí dalšími cytosolovými reduktázami znovuvytvořit o-semichinonový aniontový radikál (Flowers-Geary, 1992; Flowers-Geary, 1996). Takto může docházet k několikanásobnému opakování redoxního cyklu a opakované tvorbě reaktivních forem kyslíku (ROS), které mohou vyvolat oxidativní poškození DNA bází vedoucí k mutacím (obr. 2).

PAU CYP1/Epoxidhydroláza H O

Dihydrodioldehydrogenáza

konjugace H O

H O H O

2 O

katechol

.-

AKR

2 O 2 H

e 1

dihydrodiol CYP1

O

NADPH-chinonoxidoreduktáza

....

-O

O

NAD(P)+

oxidativní poškození DNA

2 O

H O

o-semichinonový radikál

2 O

dihydrodiolepoxid

O

konjugace

oxidativní poškození DNA

tvorba aduktu O

DNA adukty

.-

e 1

-

H O

NAD(P)H

o-chinon PAU

Obr. 2: Role cytochromů a aldo-ketoreduktáz v metabolické aktivaci trans-dihydrodiolů PAU (upraveno podle Burczynski, 2000).

1.2.1.4.

Tvorba kationtových radikálů

Ke vzniku DNA aduktů může vést i tvorba kationtových radikálů PAU katalyzovaná P450 peroxidázou (Cavalieri, 1995) nebo v případě methylsubstituovaných PAU tvorba benzylkarboniových kationtů, vzniklých hydroxylací methylové skupiny s následnou tvorbou reaktivních benzylesterů nesoucích dobře odstupující skupinu, např. sulfátovou (Surh, 1994).

14

Úvod

1.2.1.5.

Typy genotoxických poškození a odpovědi na poškození DNA

Změny v genotypu organismu mohou být způsobeny různými typy genotoxických poškození. Genotoxické efekty na řetězci DNA se označují jako genové mutace a mohou vzniknout inzercí, tj. zařazením jednoho nebo více nukleotidů, delecí, tj. ztrátou jednoho nebo více nukleotidů, nebo substitucí, tj. náhradou báze. Chromozomální aberace představují změny struktury nebo počtu chromozomů. Strukturní změny chromozomů vznikají jako následek chromozomální nestability (zlomů). Jako euploidie se označuje znásobení celé chromozomové sady, jako aneuploidie znásobení jednoho z chromozomů (Rosypal, 2002). Zachování integrity a přesnosti genomu je nezbytné pro správné fungování a přežití všech organismů. V eukaryotických buňkách se vyvinul komplexní systém odpovědí na poškození DNA, který má zabránit projevu jeho škodlivých následků. Tento systém zahrnuje modulaci tzv. kontrolních bodů buněčného cyklu, aktivaci mechanismů opravujících poškozenou DNA a iniciaci apoptózy v případě vážného poškození. Aktivace kontrolních bodů po rozpoznání poškození DNA nebo přerušení replikace zastaví postup buněčným cyklem a tím poskytne více času k opravě DNA (Zhou, 2000). Klíčovými regulátory mechanismů fungování kontrolních bodů v G1, S a G2 fázi buněčného cyklu jsou v savčích buňkách proteinkinázy ATM a ATR, podílející se na detekci poškození DNA a na přenosu signálu na transduktorové a efektorové proteiny (Bakkenist, 2004) (obr. 3). Kinázy ATM a ATR kontrolují mimo jiné jeden z nejvíce studovaných procesů odpovědi na poškození DNA, kterým je akumulace a aktivace tumor-supresorového proteinu p53. Protein p53 funguje jako transkripční faktor a má důležitou úlohu v regulaci buněčného cyklu a prevenci karcinogeneze. Bylo zjištěno, že v řadě lidských nádorů se vyskytuje v mutované, nefunkční formě (Levine, 1997). Za normálních podmínek je v nestresovaných buňkách přítomen inaktivní a v relativně malém množství. K jeho aktivaci a akumulaci dochází působením buněčného stresu, jako je právě poškození DNA. Protein p53 může regulovat zastavení buněčného cyklu v G1 fázi prostřednictvím svého transkripčního produktu, proteinu p21 (El Deiry, 1994), v S fázi (Binková, 2000; Khan, 2002), zastavení v G2 fázi prostřednictvím p21 nebo GADD45 (Smith, 1994; Luch, 1999) nebo indukovat v poškozených buňkách apoptózu. Pro apoptózu je nezbytná aktivace proapoptotických genů a represe antiapoptotických genů. Apoptózu zprostředkovanou p53 může podporovat několik cílových genů proteinu p53, aktivujících paralelní apoptotické cesty. Jedná se o geny aktivující tzv. receptory smrti (např. CD95/Fas, TNF) a geny indukující změny v mitochondriích vedoucí k apoptóze (např. Bcl-2, Bax, Noxa, PUMA) (rev. viz Bourdon 2003). Pro všechny alternativní apoptotické signály je obdobná následná kaskádová aktivace kaspáz, vedoucí až k samotné apoptóze.

15

Úvod

p21

Obr. 3: Schematické znázornění složek přenosu signálu po poškození DNA v lidských buňkách. Poškození DNA je detekováno senzory (proteinové komplexy obsahující např. proteinkinázy ATM a ATR), které za pomoci mediátorů přenášejí signál na checkpoint-kinázy Chk1 a Chk2. Ty aktivují nebo inaktivují další proteiny (efektory), např. p53 nebo Cdc25, které se účastní inhibice přechodu z G1 do S-fáze, postupu S-fází nebo přechodu z G2 do M-fáze. (Převzato a upraveno podle Sancar, 2004).

1.2.2. Negenotoxické účinky PAU Vedle genotoxicity mohou PAU vyvolávat také řadu negenotoxických účinků, zejména účinky spojené s aktivací receptoru AhR, inhibicí mezibuněčné komunikace a další efekty zmíněné níže; jejich výskyt po působení PAU navzájem nekoreluje. Modulace genové exprese a mezibuněčné komunikace se může projevit na buněčné úrovni ve změnách v buněčném cyklu, diferenciaci, proliferaci, apoptóze nebo adaptivní odpovědi. Tyto změny na tkáňové úrovni mohou vyústit v narušení rovnováhy, expanzi transformovaných buněk, vznik dysplasií a neoplasií, vedoucích ke karcinogenezi. Negenotoxické mechanismy se uplatňují především v procesech nádorové promoce a endokrinní disrupce. Aktivace receptoru AhR po expozici PAU vede k indukci biotransformačních enzymů, jejímž následkem je zvýšená metabolická aktivace prokarcinogenů (Guengerich, 1998). 16

Úvod

Aktivace AhR však také patří mezi negenotoxické mechanismy; dalším možným mechanismem PAU přispívajícím k nádorové promoci je AhR-dependentní modulace proliferace, v případě jaterních progenitorových buněk uvolnění buněk z kontaktní inhibice růstu (Chramostová, 2004; Andrysík, 2007). Dalšími typy škodlivých účinků PAU jsou aktivace nebo inhibice aktivity jaderných receptorů pro steroidní hormony vedoucí k endokrinní disrupci (Arcaro, 1999; Vinggaard, 2000; Vondráček, 2002). Rovnovážný stav organismu a chování buněk v tkáních jsou regulovány prostřednictvím komunikace na úrovni mimo-, mezi- a vnitrobuněčné a PAU mohou tuto komunikaci narušovat. Endogenní extracelulární signální molekuly (hormony, růstové faktory, cytokiny a neurotransmitery), stejně jako různé exogenní chemické látky (složky potravy, léčiva a kontaminanty prostředí), mohou spouštět různé vnitrobuněčné signální dráhy, které ovlivňují expresi genů, a mohou ovlivňovat také mezibuněčnou komunikaci (Trosko, 1998). Důležitou roli v nádorové promoci představuje inhibice mezibuněčné komunikace zprostředkované spojením „gap junction“ (Gap Junctional Intercellular Communication, GJIC), která uvolňuje buňky z regulace růstu (Trosko and Ruch, 1998; Upham, 1998; Yamasaki, 1995). Schopnost PAU inhibovat mezibuněčnou komunikaci (Bláha, 2002) nekoreluje s jejich genotoxickou potencí, nýbrž s karcinogenitou (Rosenkranz, 2000). Mezi další významné efekty PAU patří modulace dalších vnitrobuněčných signálních drah a aktivit transkripčních faktorů, které mohou být spojeny s indukcí buněčné proliferace nebo modulací buněčné diferenciace a apoptózy (Li, 2004). Jedná se např. o narušení hladiny vápenatých iontů (Tannheimer, 1997), modulaci aktivity proteinkináz aktivovaných mitogenem (MAPK) (Patten Hitt, 2002; Rummel, 1999; Tan, 2002; Andrysík, 2006), modulaci metabolismu kyseliny arachidonové (Sumida, 1993). Modulací cytokinů, regulujících hematopoézu, mohou PAU způsobovat poškození imunitního systému (Page, 2004). Imunotoxicita PAU je způsobena také indukcí apoptózy buněk imunitního systému metabolity PAU (Page, 2002).

17

Úvod

1.3. Metody detekce genotoxických a negenotoxických účinků PAU Od 19. století s rozvojem industrializace docházelo k narůstajícímu počtu pozorování lékařů, že pracovní expozice některým chemikáliím nebo směsím mají za následek karcinogenní efekty. Na začátku 20. století proto vzrostla potřeba vyvinout metody experimentálního vyvolání nemocí v testovacích systémech pro potřeby systematického výzkumu. Prvními, komu se podařilo experimentálně vyvolat maligní epiteliální nádory po aplikaci černouhelného dehtu na uši králíků, byli japonští patologové Yamagiwa a Ichikawa v roce 1915. Tento experiment je považován za přechod do moderní éry experimentálního výzkumu rakoviny. V roce 1930 Kenneway a Hieger poprvé prokázali, že jednotlivý PAU, dibenzo[a,h]anthracen, vyvolává nádory v myší kůži. Roku 1933 byl izolován benzo[a]pyren a byla dokázána jeho karcinogenita v myší kůži. Roku 1935 Boyland a Levi navrhli hypotézu, že toxické PAU mohou být v organismu přeměněny na aktivnější patogenní látky nebo mohou být metabolizovány (rev. viz Luch, 2005). S rozvojem testovacích metod se objevily snahy o harmonizaci testovacích strategií a standardů. Jednou z prvních snah bylo hodnocení mezilaboratorní shody výsledků různých testovacích systémů a vývoj testovacích doporučení Světové zdravotnické organizace (WHO) v rámci programu IPCS (International Programme on Chemical Safety) (Ashby, 1988). Hlavní roli ve vývoji doporučení pro mezinárodně harmonizované testovací protokoly hraje v současné době Organizace pro ekonomickou spolupráci a rozvoj (OECD). Směrnice Evropské unie pro testování chemikálií se shodují se směrnicemi OECD, pouze z testů genotoxicity zahrnují navíc test na morfologické transformace buněk.

1.3.1. Testy in vivo Základní informaci o karcinogenních schopnostech PAU poskytují in vivo testy na zvířatech, zejména hlodavcích. Jedná se o schopnost iniciovat vznik nádoru, působit nádorovou promoci nebo kompletní karcinogenezi. V testu na tumorovou iniciaci je zvířeti jednorázově podána testovaná látka a opakovaně je podáván modelový tumorový promoter, např. forbolester. Pokud dojde ke vzniku nádoru, má testovaná látka tumor-iniciační schopnosti (Iyer, 1980; Wislocki, 1982). Ve dvoustupňovém testu karcinogenity je testovaná látka podávána opakovaně a pokud dojde ke vzniku nádoru, jedná se o kompletní karcinogen (Stevenson and von Haam, 1965; Lacassagne, 1968; NIH, 2006). Vedle dospělých hlodavců se také k testování používá model novorozených myší (Grover, 1975; Rice, 1987). 18

Úvod

Nevýhodou dat získaných z těchto testů je, že nepodávají informaci o mechanismech karcinogenity a nádorové promoce a že se tato data obtížně extrapolují mezi živočišnými druhy, včetně člověka.

1.3.2. Testy in vitro 1.3.2.1.

Testy genotoxických efektů

Vzhledem k náročnosti testování na zvířatech byla jako alternativa vyvinuta řada testů genotoxicity a metod stanovujících parametry tumorové promoce. První test mutagenity založený na sledování reverzních mutací v bakterii Salmonella typhimurium (Amesův test) (Ames, 1973; Ames, 1975) detekuje genové mutace a je zahrnut ve směrnicích OECD a EU pro testování chemikálií. Je používán ve variantě bez a s metabolickou aktivací mikrosomální a cytosolovou jaterní frakcí z hlodavců, obsahující biotransformační enzymy, nebo přímo intaktními jaterními buňkami. Dalšími metodami využívajícími bakteriální modely jsou umuC test (Hansen, 1998) a SOS Chromotest (Quillardet, 1993), které však nejsou zahrnuty ve směrnicích OECD a EU. Navzdory používání metabolické aktivace v bakteriálních testech genotoxicity zůstává tento systém poměrně vzdálený savčímu, resp. lidskému organismu. Nevýhodami tohoto testovacího systému je potřeba experimentálních zvířat pro získání frakce jaterních enzymů, rozdíly v profilech metabolitů v závislosti na použitém typu aktivačních systémů, rozdíly mezi metabolity produkovanými testovaným systémem a organismy in vivo a skutečnost, že externě vytvořené metabolity v testovacím systému nemusí proniknout až k DNA použité cílové buňky (Rueff, 1996). Vhodnější jsou proto testy na savčích primárních buňkách nebo modelových buněčných liniích. Pro testování účinků sloučenin vyžadujících metabolickou aktivaci jsou nejvhodnějšími modely metabolicky kompetentní buňky, zejména jaterní, např. často používaná hepatoma linie HepG2, ale také plicní, např. lidské buňky A549 nebo fibroblasty z čínského křečka V79, které se používají transfekované jednotlivými biotransformačními enzymy CYP. Další hojně používanou buněčnou linií jsou buňky odvozené z prsního karcinomu MCF-7. Následující testy využívající savčí buňky jsou zahrnuty ve směrnicích OECD a EU pro testování chemikálií. Ze savčích systémů se v testech na genové mutace používají buněčné linie lymfocytů myší, křečků nebo lidské lymfoblastické linie, v nichž se detekují mutace v určitých specifických genech (TK, HPRT a XPRT) (Liber, 1982; Moore, 1989; Aaron, 1989). Z hlediska PAU jsou významné práce Duranta et al. s výsledky mutagenity řady PAU stanovované v lokuse TK na lidských B-lymfoblastoidních buňkách transfekovaných lidskou cDNA kódující CYP1A1 (Durant, 1996; Durant, 1999). Zlomy DNA jsou běžně detekovány testem na chromozomové aberace (Preston, 1987; 19

Úvod

Tice, 1994), v němž je možné používat různé buněčné linie, primární buněčné kultury nebo buňky získané po expozici zvířete in vivo, zejména buňky kostní dřeně. Test na chromozomové aberace se také používá k analýze periferních lymfocytů lidí jako expoziční test in vivo, sloužící ke sledování reálného genetického rizika prostředí (Georgiadis, 2005; Topinka, 2007). Poškození chromozomů nebo mitotického aparátu může vyvolat indukci mikrojader, obsahujících fragmenty chromozomů nebo celé chromozomy. V testu na mikrojádra se nejčastěji používají erytrocyty in vivo, možné je ale i použití jiných typů buněk in vivo nebo in vitro (Heddle, 1973; Schmid, 1975; Heddle, 1983). Testy na neplánovanou syntézu DNA, související s poruchami oprav poškozené DNA, a na výměny sesterských chromatid, zahrnující zlomy a znovuspojení chromatid, využívají inkorporaci radioaktivně značeného thymidinu a bromdeoxyuridinu (Warshawsky, 1995). Vedle metod zahrnutých do směrnic OECD pro testování chemikáliií existuje a je vyvíjena řada dalších. Zlomy DNA detekuje gelová elektroforéza jednotlivých buněk (SCGE, tzv. comet assay), která může využít prakticky jakékoli buňky in vivo nebo in vitro (Singh, 1988; Tice, 2000). Předchůdcem metody SCGE byla metoda alkalického vymývání DNA (alkaline elution) (Ahnström, 1974) a metoda alkalického rozvinutí DNA (alkaline DNA unwinding) (Kohn, 1973). Další metoda využívá skutečnost, že přítomnost zlomů DNA zvyšuje citlivost k denaturaci jaderné DNA in situ (Gorczyca, 1993). Barvivo akridinová oranž se váže na denaturované jednořetězcové úseky DNA a nedenaturované dvouřetězcové úseky DNA a emituje záření o rozdílné vlnové délce, které je detekováno průtokovou cytometrií. Tato metoda je využívána zejména k detekci poškození spermií jako tzv. metoda SCSA (Sperm Chromatin Structure Assay) (Evenson, 1980). Často je také používána imunochemická detekce proteinů indukovaných následkem poškození DNA, např. p53, p21 a dalších nebo detekce jejich mRNA pomocí PCR (Chramostová, 2004; Helt, 2005). Je možné také měřit přímo adukty reaktivních metabolitů s DNA za použití radioaktivního značení a rozdělení vysokoúčinnou kapalinovou chromatografií nebo tenkovrstvou chromatografií (Gupta, 1985; Mahadevan, 2001). Jako marker genotoxicity je také možné využít zvýšenou apoptózu. Ranné fáze apoptózy jsou detekovatelné průtokovou cytometrií po vazbě annexinu-V (Koopman, 1994) a pozdní fáze mohou být určeny mikroskopicky po barvení DAPI (Chramostová, 2004).

1.3.2.2.

Testy negenotoxických efektů

K určení aktivace receptoru AhR se často používá stanovení aktivity CYP1 enzymů pomocí modelových substrátů, především O-deethylace 7-ethoxyresorufinu (EROD) (Prough, 1978) nebo hydroxylace aromatických uhlovodíků (AHH) (Piskorska-Pliszczynska, 1986). Hojně užívané jsou také metody detekující indukci genové exprese specifických reporterových genů pod kontrolou dioxin-responzivního elementu. Jako reporterový gen se 20

Úvod

využívá zejména luciferáza (metoda DR-CALUX) (Murk, 1996), β-galaktosidáza nebo zelený fluorescenční protein (Nagy, 2002). Metodou PCR může být měřena indukce mRNA cytochromů P450, transkripčních produktů aktivovaného AhR (Umannová, 2008). Z in vitro metod se k určení aktivace receptoru ER obdobně jako u měření aktivace AhR využívají metody založené na aktivaci stejných reporterových genů pod kontrolou estrogen-responzivního elementu (např. luciferáza v testu ER-CALUX) (Legler, 1999). Obdobné systémy jsou v současné době vyvinuty také pro stanovení (anti)androgenní aktivity, modulaci aktivity glukokortikoidního receptoru apod. (Sonneveld, 2005). Alternativním testem je také E-screen, test indukce proliferace závislé na ER v buněčných linií karcinomu prsu (Gupta, 1998; Combes, 2000; Plíšková, 2005). V posledních deseti letech byla vyvinuta nová metoda – tzv. DNA microarray, umožňující sledovat expresi tisíců genů na jednom čipu. Změny v genové expresi mohou sloužit jako citlivý parametr toxicity a indikovat mechanismy působení (Nuwaysir, 1999). Změny v buněčném cyklu bývají detekovány průtokovou cytometrií, proliferace může být stanovována různými metodami jako počet buněk nebo měřením inkorporace bromdeoxyuridinu průtokovou cytometrií (Chramostová, 2004). Fáze buněčného cyklu i počet pozdně apoptotických buněk obsahujících zlomy na DNA mohou být určeny na průtokovém cytometru také pracnější metodou TUNEL (Terminal Deoxynucleotidyl Transferase Assay) (Tuschl, 2005). Inhibice mezibuněčné komunikace (GJIC) může být určena mikroskopicky metodou scrape-loading, která spočívá v měření vzdálenosti proniknutí fluorescenční barvy buňkami od místa řezu (El-Fouly, 1987). Také je možná detekce změn hladin a post-translačních modifikací (fosforylací) konexinů, proteinů tvořících kanálky „gap junction“, imunochemickými metodami (Bager, 1994; Tateno, 1994).

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Úvod

1.4. Charakteristika vybraných skupin PAU Obecně lze říci, že PAU působí celou řadou mechanismů toxicity a tyto efekty jsou silně závislé na molekulové hmotnosti (MW), struktuře, poloze substituce atd. Z velké řady PAU a jejich derivátů jsou v této kapitole charakterizovány ty skupiny, kterými se zabývá experimentální část této práce nebo skupiny obsahující tzv. prioritní PAU vybrané Americkou agenturou pro ochranu životního prostředí (US EPA) pro rutinní chemické monitorování prostředí.

1.4.1. Abundantní nížemolekulární PAU Anthracen, fenanthren, fluoranthen, pyren a fluoren (obr. 4) se většinou vyskytují v životním prostředí ve vyšších koncentracích než ostatní PAU. Patří mezi šestnáct tzv. prioritních PAU (US EPA). Podle IARC nejsou klasifikovány jako lidské karcinogeny (IARC, 1983; IARC, 1987).

anthracen

fenanthren

fluoren

fluoranthen

pyren

Obr. 4: Abundantní PAU

Anthracen a fluoren nemají genotoxické ani karcinogenní účinky (WHO, 1998) a neaktivují AhR (Machala, 2001). Důkazy o schopnosti fenanthrenu a pyrenu vyvolávat genotoxické a karcinogenní účinky nejsou jednoznačné, nicméně fluoranthen vykazoval v některých testech slabou genotoxicitu (WHO, 1998). Pyren a fluoranthen také patří mezi slabé induktory aktivity AhR (Machala, 2001). Pouze u fluoranthenu (v nejvyšší testované koncentraci 10 µM) byla zjištěna schopnost vyvolávat proliferaci v modelu jaterních progenitorových buněk (Chramostová, 2004) a aktivaci estrogenního receptoru (Vondráček, 2002). Kromě anthracenu jsou tyto PAU silnými inhibitory mezibuněčné komunikace, zejména fluoranthen, který je mezi PAU dosud nejsilnějším identifikovaným inhibitorem GJIC (Bláha, 2002). U fluoranthenu byla navíc zjištěna schopnost vyvolávat imunotoxické účinky (Hinoshita, 1992). 22

Úvod

1.4.2. Skupina PAU s molekulovou hmotností 228 Nejvýznamnějšími zástupci jsou benzo[a]anthracen (BaA), chrysen (Chry), benzo[c]fenanthren (BcPhe) a trifenylen (obr. 5). Z nich patří BaA a Chry mezi prioritní PAU podle US EPA. BaA, Chry a BcPhe jsou zařazeny do kategorie 2B jako možné lidské karcinogeny, trifenylen není klasifikován jako lidský karcinogen (IARC, 1983; IARC, 1987; IARC, 2008). Mají genotoxické a (kromě trifenylenu) karcinogenní účinky v myších a krysích modelech (WHO, 1998). Vyvolávají aktivaci AhR a slabou inhibici GJIC, pouze BcPhe je silnějším inhibitorem GJIC (Machala, 2001; Bláha, 2002). BaA vyvolává estrogenní efekty (Plíšková, 2005).

benz[a]anthracen

chrysen

benzo[c]fenanthren

trifenylen

Obr. 5: PAU s molekulovou hmotností 228.

1.4.3. Skupina PAU s molekulovou hmotností 252 Do této skupiny patří např. benzo[a]pyren (BaP), benzo[e]pyren (BeP), benzo[k]fluoranthen (BkF), benzo[b]fluoranthen (BbF), benzo[j]fluoranthen (BjF) (obr. 6) a některé další. BaP je klasifikován jako karcinogenní pro člověka (kategorie 1), BkF, BbF a BjF jako potenciální karcinogeny (kategorie 2B) a BeP není klasifikován jako karcinogen (IARC 1983; IARC, 1987; IARC, 2008). Tyto PAU mají genotoxické a karcinogenní účinky (WHO, 1998) a aktivují AhR. Zejména benzofluorantheny jsou silnými induktory aktivity AhR (Machala, 2001), vyvolávají také proliferaci a zvyšují procento buněk v S-fázi v modelu jaterních progenitorových buněk (Chramostová, 2004). Neinhibují GJIC vůbec nebo jen slabě (BaP, BbF) (Bláha, 2002). Z této skupiny patří mezi prioritní PAU BaP, BkF a BbF. BaP je silný genotoxin in vivo a v řadě in vitro modelů a je široce používán jako referenční genotoxický a karcinogenní PAU (WHO, 1998). BaP v genotoxických koncentracích aktivuje protein p53, vyvolává fragmentaci buněčného jádra a další apoptotické procesy, zároveň aktivuje AhR a indukuje kompenzující proliferaci buněk (Chramostová, 2004).

23

Úvod

benzo[a]pyren

benzo[e]pyren

benzo[b]fluoranthen

benzo[j]fluoranthen

benzo[k]fluoranthen Obr. 6: PAU s molekulovou hmotností 252.

1.4.4. Skupina PAU s molekulovou hmotností 278 Z této skupiny PAU jsou významné především dibenzo[a,h]anthracen (DBahA), dibenzo[a,c]anthracen (DBacA), dibenzo[a,j]anthracen (DBajA), benzo[g]chrysen (BgChry), benzo[c]chrysen (BcChry) a benzo[b]chrysen (BbChry) (obr. 7). DBahA je klasifikován jako pravděpodobný lidský karcinogen (kategorie 2A), DBacA a DBajA nejsou (kategorie 3) (IARC, 1983; IARC, 1987; IARC, 2008). Z této skupiny je na seznam prioritních PAU zařazen pouze DBahA. Dibenzoanthraceny jsou silnými induktory aktivity AhR (Machala, 2001), DBahA má genotoxické a karcinogenní účinky (WHO, 1998); z dibenzoanthracenů pouze DBacA vyvolává inhibici GJIC (Bláha, 2002). BgChry a BbChry nejsou klasifikovány jako karcinogeny (kategorie 3) (IARC, 2008). Molekuly BcChry a BgChry obsahují ve své struktuře zároveň tzv. „bay“ a „fjord“ oblast, přičemž v obou částech molekuly mohou vznikat dihydrodiolepoxidy. Zejména BgChry má velmi silné genotoxické účinky. BcChry i BgChry vyvolávaly tvorbu DNA aduktů v kůži myší (Giles, 1996; Giles, 1997). BcChry dihydrodiolepoxid lokalizovaný v sousedství „fjord“ regionu indukoval, na rozdíl od dihydrodiolepoxidu umístěném v „bay“ oblasti, vznik nádoru v prsních žlázách krys (Amin, 2003). BbChry a zejména BcChry indukovaly EROD aktivitu jaterních CYP1A1/2 v krysách in vivo, BgChry nebyl v experimentu zahrnut (Cheung, 1993).

24

Úvod

dibenzo[a,c]anthracen

benzo[b]chrysen

dibenzo[a,j]anthracen

benzo[c]chrysen

dibenzo[a,h]anthracen

benzo[g]chrysen


1.4.5. Skupina PAU s molekulovou hmotností 302 Nejvýznamnějšími

zástupci této skupiny jsou dibenzo[a,l]pyren (DBalP) a dibenzo[a,e]pyren (DBaeP) (obr. 8). DBalP je klasifikován jako pravděpodobný lidský karcinogen (kategorie 2A), ale není zařazen na seznam prioritních PAU. DBaeP není klasifikován jako karcinogen (IARC, 1983; IARC, 1987; IARC, 2008). Mají genotoxické a karcinogenní účinky (WHO, 1998), jsou slabšími aktivátory AhR (Machala, 2001) a jen velmi slabými inhibitory GJIC (Bláha, 2002). DBalP je používán jako modelový PAU a vykazuje nejsilnější genotoxický a karcinogenní účinek z doposud testovaných PAU (Cavalieri, 1991).

dibenzo[a,l]pyren

dibenzo[a,e]pyren


25

Úvod

1.4.6. Methylované deriváty chrysenu a benz[a]anthracenu V životním prostředí se vyskytuje také velké množství methylovaných derivátů PAU (naftalenů, anthracenů, fenanthrenů, pyrenů, chrysenů, benzanthracenů a benzo[a]pyrenů). Mezi monomethylovanými deriváty chrysenu má unikátní karcinogenní vlastnosti 5-methylchrysen (5-MeChry) (obr. 9), pravděpodobně díky přítomnosti tzv. „methyl bay“ oblasti (Hecht, 1987). 5-MeChry je zařazen do kategorie 2B (IARC, 1973; IARC, 1987; IARC, 2008). 5-MeChry je také silným aktivátorem AhR (Machala, 2001), silným inhibitorem GJIC (Bláha, 2002) a vyvolává proliferaci a nárůst procenta jaterních progenitorových buněk v S-fázi (Chramostová, 2004). 2-, 3-, 4- a 6-MeChry mají výrazně slabší karcinogenní účinky, zatímco 1-MeChry je neaktivní (Hecht, 1974), podle IARC nejsou klasifikovány jako lidské karcinogeny (IARC, 1973; IARC, 1987; IARC, 2008). Všechny deriváty silně indukovaly aktivitu jaterních CYP1A1 v krysách in vivo, měřenou jako O-deethylace ethoxyresorufinu (Cheung, 1993). 7,12-dimethylbenz[a]anthracen (DMBA) (obr. 9) je jedním z nejsilnějších karcinogenních PAU. Může indukovat vznik kožních (Boyland, 1965), plicních (Walters, 1966), jaterních a prsních nádorů (Huggins, 1961). Tvoří vysoké hladiny aduktů s DNA v řadě experimentálních modelů. Aktivuje AhR (Machala, 2001) a inhibuje GJIC (Bláha, 2002). Z monomethylovaných derivátů benz[a]anthracenu (MeBaA) mají na kožních hlodavčích modelech karcinogenní účinky 6-, 7-, 8- a 12-MeBaA, zatímco ostatní deriváty mají velmi slabé nebo žádné schopnosti vyvolat nádor v těchto modelech (Dunning, 1960; Stevenson, 1965). Nejsilnější tumor-iniciační účinky vykazoval 7-MeBaA, který byl ve srovnání s DMBA středně silný. Dalšími aktivními deriváty byly 8- a 12-MeBaA, následované 6- a 9-MeBaA (Wislocki, 1982). S karcinogenitou MeBaA v kožních modelech přibližně korelovala jejich mutagenita v Amesově testu za použití intaktních potkaních hepatocytů jako metabolizujícího systému (Utesch, 1987). 2 3

1

3 H C

1

2 1

4

2 1

1 1 0 1

2

1 1

5

9

4

9

3

0 1

6

3 H

7C

8

6

7

3 H C

5

8

5-methylchrysen

7,12-dimethylbenz[a]anthracen

Obr. 9: Příklady methylovaných derivátů PAU s vysokou karcinogenní potencí.

26

Úvod

1.5. Vybrané N-heterocyklické aromatické uhlovodíky Ze skupiny N-heterocyklických aromatických uhlovodíků byl v této práci vybrán ke studiu 7H-dibenzo[c,g]karbazol (DBC) a jeho syntetické deriváty 5,9-dimethyldibenzo[c,g]karbazol (diMeDBC) a N-methyldibenzo[c,g]karbazol (N-MeDBC) (obr. 10). DBC je environmentální polutant, přítomný v řadě směsí vzniklých nedokonalým spalováním organického materiálu, jako např. v cigaretovém kouři, asfaltu, sazích a automobilových exhalátech (rev. viz Warshawsky, 1996). Je to silný karcinogen, vyvolávající tvorbu nádorů na různých místech organismu a v různých druzích zvířat (Szafarz, 1988; Warshawsky, 1996). Je klasifikován jako možný lidský karcinogen (kategorie 2B) (IARC, 1983). Pro studium vztahu mezi chemickou strukturou a biologickou aktivitou byly syntetizovány deriváty DBC substitucí methylovou skupinou na různých pozicích jeho molekuly. Z těchto derivátů vykazují N-MeDBC a diMeDBC specifické účinky. N-MeDBC tvoří DNA adukty, nádory a mutace především na kůži (Périn, 1984; Schurdak, 1987), zatímco diMeDBC je striktní hepatokarcinogen, který tvoří nádory, adukty a mutace v játrech, ale nemá žádnou aktivitu na kůži (Valéro, 1984; Renault, 1998). Experimenty na geneticky upravených buňkách čínského křečka V79, stabilně produkujících buď CYP1A1 nebo CYP1A2, ukázaly, že po metabolické aktivaci CYP1A1, resp. CYP1A2 vyvolává DBC a NMeDBC tvorbu DNA aduktů, zatímco diMeDBC jen velmi málo, resp. vůbec (Gábelová, 2004). 3

2 1

3 1

2 1 1 1

1

3

2

3 1

2 1 1 1

0 1

4

0 1

4

0 1

4

1

3

2

3 1

2 1 1 1

6

3

H C

N

7

8

5

9

6

3

5,9-dimethyldibenzo[c,g]karbazol

H C

H

H

C 3 H

N

7

8

5

9

6

N

7

8

5

9

7H-dibenzo[c,g]karbazol

N-methyldibenzo[c,g]karbazol

Obr. 10: N-heterocyklické aromatické uhlovodíky.

27

Cíle práce

2.

Cíle práce

Játra představují centrální orgán metabolismu xenobiotik. Obsahují několik typů buněk, včetně buněk progenitorových, které mohou v případě poškození jater proliferovat a nahradit poškozené buňky. Vedle této role při regeneraci jater se mohou progenitorové buňky také stát cílem chemických karcinogenů a hrát roli v hepatokarcinogenezi. Přesto je většina studií zabývajících se účinky polyaromatických uhlovodíků (PAU), významných environmentálních kontaminantů, dosud zaměřena na diferencované buňky (hepatocyty) nebo buněčné hepatoma linie odvozené z hepatocytů. Cílem této práce byl výzkum genotoxických účinků PAU a referenčního abiotického vzorku prostředí v potkaních buňkách WB-F344, jaterní progenitorové epiteliální buněčné linii. Práce byla zaměřena na: •

studium genotoxických efektů karcinogenních PAU;

•

studium genotoxických a tumor-promočních efektů methylovaných benz[a]anthracenů;

•

studium genotoxických a tumor-promočních efektů 7H-dibenzo[c,g]karbazolu a jeho derivátů a mechanismu jejich působení;

•

studium genotoxických a tumor-promočních efektů vzorku pevných částic z ovzduší SRM1649a.

28

Materiál a metody

3.

Materiál a metody

3.1. Buněčná linie

Experimenty byly provedeny v linii jaterních epiteliálních buněk WB-F344, původně izolovaných z jater dospělého samce potkana Fischer 344 (Tsao, 1984). Tuto linii nám poskytnul prof. James E. Trosko, Michigan State University, East Lansing, USA. Buňky WBF344 představují model jaterní progenitorové linie (Tsao, 1984). Jaterní progenitorové buňky, označované u zvířat také jako oválné buňky (Libbrecht, 2002), se mohou jak u lidí, tak u zvířat diferencovat na hepatocyty a buňky žlučových kanálků (Haque, 1996; Yasui, 1997; Roskams, 1998; Yang, 2004). Progenitorové buňky bývají v játrech aktivovány v případě, že hepatocyty a/nebo cholangiocyty jsou poškozeny nebo je inhibována schopnost jejich replikace (Roskams, 2003). Tato kompenzační expanze jaterních progenitorových buněk hraje roli v zachování tkáňové integrity jater a omezuje poškození jater (Yang, 2004). Zároveň tyto buňky představují možný cíl hepatokarcinogenů, jelikož hepatocelulární karcinomy mohou vznikat z jaterních buněk v různých stadiích diferenciace, včetně oválných buněk (Roskams, 2006; Wu, 2006). Linie WB-F344 obsahuje receptor AhR, dále významné hladiny biotransformačních enzymů, zejména CYP1A1, CYP1B1, AKR1C9 a nemutovaný protein p53. V těchto buňkách jsou také funkční mezibuněčná spojení typu gap junctions. Z hlediska in vitro studií je důležité, že se jedná o nenádorovou a netransformovanou buněčnou linii, což zvyšuje výpovědní hodnotu získaných výsledků.

29

Materiál a metody

3.2. Metody

3.2.1. Analýza DNA aduktů Buňky byly pěstovány na miskách o ploše 60 cm2 a exponovány 24 hod testovaným sloučeninám. Po expozici byly omyty PBS, seškrábnuty do mikrozkumavek, zcentrifugovány a buněčný pelet byl zamražen na -80°C. Buněčné pelety byly poté homogenizovány v roztoku 10 mM Tris-HCl, 100 mM EDTA a 0,5% SDS, pH 8,0. DNA byla izolována za použití RNAáz A a T1 a proteinázy K a fenol/chloroform/isoamylalkoholové precipitace (Binková, 2000). Vzorky byly analyzovány metodou 32P-postlabeling (Phillips and Castegnaro, 1999; Reddy and Randerath, 1986). Značené DNA adukty byly detekovány pomocí dvousměrné tenkovrstvé chromatografie. Autoradiografie byla provedena při -80°C po dobu 6, 24 a 96 hod. Radioaktivita jednotlivých skvrn byla měřena kapalinovým scintilačním počítačem. Ve vzorcích bylo také určeno přesné množství DNA pomocí HPLC s UV detekcí. Hladina DNA aduktů byla vyjádřena jako počet aduktů na 108 nukleotidů. Stanovení stabilních DNA aduktů bylo rutinně provedeno v laboratoři ÚEM AV ČR v Praze.

3.2.2. Detekce mRNA metabolizačních enzymů pomocí real time-PCR Buňky byly po expozici 24 hod sklizeny do lyzačního roztoku a celková RNA byla vyizolována pomocí kitu NucleoSpin RNA II (Macherey-Nagel). Amplifikace vzorků byly provedeny za použití kitu QuantiTect Probe RT-PCR (Qiagen GmbH, Hilden, Německo) podle návodu výrobce. Všechny proby byly značeny 6-karboxyfluoresceinem na 5’-konci a barvivem Black Hole 1 na 3’-konci. Amplifikace byly provedeny na přístroji LightCycler (Roche Diagnostics). Genová exprese pro každý vzorek byla vyjádřena jako tzv. „threshold cycle“ (Ct), normalizovaný na housekeeping gen porphobilinogen deaminázu (∆Ct). Hodnoty ∆Ct vzorků exponovaných PAU byly srovnány s negativní kontrolou (0,1% DMSO) a byla spočítána hodnota ∆∆Ct (∆Ct[kontrola] – ∆Ct[PAU]). Konečný výsledek je vyjádřen jako 2-∆∆Ct (Livak, 2001).

30

Materiál a metody

3.2.3. Detekce proteinu p53 metodou Western blotting Metoda byla užita pro sledování aktivace (fosforylace) a akumulace proteinu p53, tj. markeru genotoxických procesů. Exponované buňky byly sklizeny do lyzačního pufru (1% SDS, 10% glycerol, 100 mM Tris, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4) a zamraženy na -75°C přes noc. Poté byly lyzáty sonikovány. Koncentrace proteinů byla určena za použití kyseliny bicinchoninové a síranu měďnatého (Sigma-Aldrich). Vzorky byly upraveny na stejnou koncentraci bílkovin a byl k nim přidán redukující roztok (65 mM Tris pH 6.8; 10% glycerol; 2% SDS; 5% merkaptoethanol; bromfenolová modř 50 µg/ml) v poměru 1:2 (1 díl redukujícího roztoku : 2 díly vzorku). Proteiny byly rozděleny na standardní SDS-PAGE (Bio-Rad, Hercules, CA, USA) a pomocí polosuchého blottingu přeneseny na PVDF membránu Hybond-F (GE Healthcare). Paralelně byly použity standardy molekulových hmotností (Bio-Rad, Hercules, CA, USA). Membrány byly po zablokování v 2,5% roztoku odtučněného mléka nebo bovinního sérového albuminu v promývacím pufru (20 mM TrisHCl pH 7,4; 100 mM NaCl; 0,1% Tween-20) inkubovány s primární protilátkou vázající se na cílový protein. Inkubace s primární protilátkou probíhala ve stejném roztoku, v jakém byly membrány blokovány. Na navázanou primární protilátku se poté navázala sekundární protilátka, konjugovaná s křenovou peroxidázou. Pro ověření stejného množství proteinů v jednotlivých jamkách byla použita detekce β-aktinu. Byly použity tyto protilátky: a) protilátka na protein p53 fosforylovaný na Ser15, #9284 (Cell Signaling Technology, Beverly, MA, USA), konjugát SWAR (Sevapharma, Praha, Česká republika); b) protilátka na celkový p53 v potkaních buňkách - p53(R-19), sc-1313 (Santa Cruz Biotechnology, CA, USA), konjugát proti kozímu imunoglobulinu, A 5420 (Sigma-Aldrich); c) protilátka proti β-aktinu, klon AC-15, A 1978 (Sigma-Aldrich), konjugát proti myšímu imunoglobulinu, A 9044 (Sigma-Aldrich). Proteiny byly vizualizovány použitím chemiluminiscenčního systému ECL Plus (GE Healthcare) podle návodu výrobce.

3.2.4. Detekce apoptózy Buněčná smrt byla detekována s využitím průtokové cytometrie a mikroskopického hodnocení. V rané fázi apoptózy dochází k přemístění fosfatidylserinu z vnitřní strany cytoplazmatické membrány na vnější stranu. Fosfatidylserin představuje signál pro makrofágy, které odstraňují apoptotické buňky (Fadok, 1992). Po expozici byly buňky sklizeny včetně média obsahujícího buňky uvolněné od podkladu. Externalizovaný fosfatidylserin byl značen Annexinem-V-Fluos (Roche Diagnostics, Mannheim, Německo) dle návodu výrobce. Pro odlišení nekrotických buněk nebo buněk v pozdní fázi apoptózy, jejichž membrána byla permeabilizována, od buněk raně apoptotických s intaktní membránou, 31

Materiál a metody bylo použito značení propidium jodidem (PI) (Vermes, 1995). Vzorky byly analyzovány na průtokovém cytometru FACSCalibur a hodnoceny programem CellQuest (Becton Dickinson, San Jose, CA, USA). Pozdní fáze apoptózy byly kvantifikovány mikroskopicky na základě morfologických parametrů kondenzace a fragmentace jádra. Buňky fixované v 70% ethanolu byly barveny DAPI v konečné koncentraci 1 µg/ml 5 min při pokojové teplotě. Poté byly zcentrifugovány (200 g, 5 min), smíchány s potřebným množstvím roztoku Mowiolu (10% roztok Mowiolu připravený v 25% glycerolu, 100 mM Tris-HCl, pH 8,5 a DABCO 0,6%) a přeneseny na mikroskopické sklo. Za použití fluorescenčního mikroskopu bylo hodnoceno minimálně tři sta jader.

3.2.5. Hodnocení buněčné proliferace a buněčného cyklu Proliferativní účinky testovaných látek byly hodnoceny v konfluentních buňkách WBF344 po expozici 72 hod s každodenní výměnou média a testovaných látek. Po skončení expozice byly buňky uvolněny z kultivační desky roztokem trypsinu a spočítány na přístroji Coulter Counter (model ZM, Coulter Electronics, Luton, Velká Británie). Poté byly buňky promyty PBS a zafixovány v 70% ethanolu při 4°C minimálně přes noc. Pro změření distribuce buněk v jednotlivých fázích buněčného cyklu byly fixované buňky promyty PBS a barveny PI ve Vindelovově roztoku: 1M Tris-HCl, pH 8,0; 0,1% Triton X-100; 10 mM NaCl; propidium jodid 50 µg/ml; RNAza A 50 Kunitzových jednotek/ml (Vindelov, 1977). Buňky byly inkubovány 30 min při 37°C v temnu a změřeny na průtokovém cytometru FACSCalibur (Becton Dickinson). Data byla analyzována programem ModFit LT verze 3.0 (Verity Software House, Topsham, ME, USA).

3.2.6. Detekce zlomů na DNA pomocí gelové elektroforézy jednotlivých buněk Gelová elektroforéza jednotlivých buněk (Single Cell Gel Electrophoresis, SCGE, comet assay) je rychlá a citlivá fluorescenční mikroskopická metoda pro detekci primárního poškození DNA na úrovni jednotlivých buněk (Singh, 1988; Tice, 2000). Tato metoda je založena na schopnosti zlomů na DNA rozvolnit řetězec DNA. Ve slabém elektrickém poli rozvolněná DNA migruje ven z jádra směrem k anodě a za jádrem vytváří útvar podobný kometě vizualizovaný pomocí fluorescenční mikroskopie. Buňky s více poškozenou DNA vykazují větší migraci DNA z jádra. Za použití alkalických podmínek (pH > 12,6) během elektroforézy metoda detekuje poškození způsobené dvouřetězcovými i jednořetězcovými 32

Materiál a metody zlomy, křížové vazby, místa nekompletní excizní opravy a alkali-labilní místa na DNA. Při použití pH v rozmezí 12,1 až 12,4 se neprojeví alkali-labilní místa, zatímco v neutrálních podmínkách se detekují pouze dvouřetězcové zlomy a křížové vazby. Byl použit postup Singha et al. (Singh, 1988), modifikovaný Collinsem et al. a Gábelovou et al. (Collins, 1995; Gábelová, 1997). Buňky rozsuspendované v 0,75% LMP agaróze byly v tenké vrstvě umístěny na mikroskopické sklíčko potažené 1% NMP agarózou v PBS pufru. Sklíčka byla na 1 hod umístěna do lyzačního roztoku (2,5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, pH 10 a 1% Triton X-100) při 4°C. V experimentech zaměřených na detekci oxidačního poškození DNA byla sklíčka třikrát promyta po dobu 5 min v endonukleázovém pufru (40 mM HEPES-KOH, 0,1 M KCl, 0,5 mM EDTA, pH 8) a inkubována s formamidopyrimidin-DNA glykozylázou/AP nukleázou (Fpg) 30 min při 37°C. Sklíčka byla umístěna do elektroforetické vany a zalita alkalickým roztokem (300 mM NaOH, 1 mM Na2EDTA, pH>13) na 40 min při 4°C, kdy probíhalo rozvíjení DNA. Poté byla sklíčka neutralizována promytím v Tris-HCl (0,4 M, pH 7,4) třikrát na 5 min a barvena 20 µl ethidium bromidu (EtBr 10 µg/ml). Takto nabarvené komety byly hodnoceny pomocí fluorescenčního mikroskopu Olympus BX51 (Olympus Europa, Německo) a analýzou obrazu za použití programu Komet 5.5 (Kinetic Imaging Ltd., Velká Británie). Jako parametr poškození DNA bylo použito procento DNA v ohonu komety (% tail DNA). Na každém sklíčku bylo počítáno sto komet.

33

Výsledky

4.

Výsledky

Autorčin příspěvek k prezentovaným studiím Kap. 4.1. - Topinka, 2008 Soňa Marvanová se podílela na designu a organizaci experimentu, exponovala a sklízela buňky pro detekci DNA aduktů a mRNA cytochromů P450, prováděla western bloty a detekci apoptózy na průtokovém cytometru, zpracovávala a interpretovala výsledky. Významně se podílela na napsání rukopisu. Kap. 4.2. - Marvanová, 2008 Soňa Marvanová se podílela na designu a organizaci experimentu, exponovala a sklízela buňky pro detekci DNA aduktů a mRNA cytochromů P450, prováděla western bloty a detekci apoptózy na průtokovém cytometru i mikroskopicky, podílela se na detekci proliferace a buněčného cyklu, zpracovávala a interpretovala výsledky. Významně se podílela na napsání rukopisu. Kap. 4.3. - Vondráček, 2006 Soňa Marvanová se podílela na western blotech a detekci apoptózy. Kap. 4.3. - Valovičová, v tisku. Soňa Marvanová se podílela na designu a organizaci experimentu, exponovala a sklízela buňky pro detekci DNA aduktů, podílela se na detekci zlomů na DNA pomocí gelové elektroforézy jednotlivých buněk. Podílela se na revizi a korekci rukopisu. Kap. 4.4. - Andrysík, rukopis v přípravě. Soňa Marvanová exponovala a sklízela buňky pro detekci DNA aduktů, podílela se na provádění western blotů, detekci apoptózy, proliferace a buněčného cyklu. Podílí se významně i na napsání rukopisu.

34

Výsledky

4.1. Tvorba DNA aduktů a indukce apoptózy v potkaních jaterních buňkách WB-F344 exponovaných karcinogenním PAU Uveřejněno v: Topinka, J., Marvanová, S., Vondráček, J., Sevastyanova, O., Nováková, Z., Krčmář, P., Pěnčíková, K. and Machala, M. (2008). DNA adducts formation and induction of apoptosis in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aromatic hydrocarbons. Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis 638, p.122–132.

4.1.1. Tvorba stabilních DNA aduktů Cílem této práce bylo prokázat genotoxické a apoptotické efekty PAU v zavedené buněčné linii WB-F344. Jako modelové karcinogenní PAU bylo na základě předchozích studií (Durant, 1996; Sjogren, 1996; Durant, 1999; Machala, 2001; Bláha, 2002; Chramostová, 2004) vybráno několik sloučenin s rozdílnou molekulovou hmotností, chemickou strukturou a odlišnými genotoxickými a tumor-promočními schopnostmi. Tvorba stabilních DNA aduktů byla studována v buňkách WB-F344 po expozici 24 hod vybraným PAU metodou 32P-postlabeling. Přestože tento buněčný model je široce využíván pro studium tumor-promočních efektů xenobiotik (Weis, 1998; Bláha, 2002; Dietrich, 2002; Chramostová, 2004), je málo známo o indukci genotoxických efektů v těchto buňkách. Největší schopnost vytvářet DNA adukty měl DBalP, působil už od koncentrace 1 nM. BaP a BgChry vytvářely DNA adukty od koncentrace 100 nM. Koncentrace 10 µM byly méně účinné než 1 µM, pravděpodobně kvůli cytotoxicitě (tab. 1). DBaeP vytvářel adukty až v koncentraci 10 µM. Naproti tomu DBahA, BbF, BaA a Chry vytvářely pouze nízké množství stabilních DNA aduktů (tab. 1).

35

Výsledky Tab. 1: Hladiny DNA aduktů indukované po 24 hod expozice vybraným PAU v buňkách WB-F344

Sloučenina Koncentrace DBalP

a

8

Množství DNA aduktů / 10 nukleotidů 0.1 mM

1 mM

297 ± 90

n.a.

b

10 mM n.a.

BaP

6.7 ± 0.2

68.0 ± 6.5

43.8 ± 7.9

BgChry

8.1 ± 0.3

26.0 ± 2.1

11.4 ± 3.4

DBaeP

0.5 ± 0.1

1.2 ± 0.3

6.9 ± 2.3

DBahA

0.8 ± 0.1

1.2 ± 0.1

0.8 ± 0.1

BbF

0.9 ± 0.2

1.1 ± 0

0.7 ± 0.2

BaA

n.a.

n.a.

1.2 ± 0.4

Chry

n.a.

n.a.

0.7 ± 0.1

Data byla získána nejméně ze tří nezávislých inkubací a jsou vyjádřena jako průměr ± SD. a

DBalP indukoval DNA adukty v koncentraci 1 nM – 10.2 aduktů / 108 nukleotidů a 10 nM – 54.5 aduktů / 108

nukleotidů b n.a.: nebyly analyzovány

4.1.2. Akumulace a fosforylace proteinu p53 Tumor supresorový protein p53 je důležitý transkripční faktor, který se v buňkách akumuluje v odpovědi na signály následující po genotoxickém poškození PAU (Luch, 1999; Binková, 2004). Protein p53 může být posttranslačně modifikován a v řadě studií bylo ukázáno, že fosforylace na Ser15 může hrát významnou roli ve stabilizaci a funkční aktivaci p53 během genotoxického stresu (She, 2004). Akumulace proteinu p53 a jeho fosforylace na Ser15 byla proto sledována po expozici 24 hod PAU v koncentraci 10 µM, pouze DBalP byl použit v koncentraci 100 nM. Pouze DBalP a BgChry byly silnými induktory fosforylace p53 na Ser15 a tyto sloučeniny také významně zvyšovaly stabilizaci a akumulaci celkového p53 (obr. 11). Ostatní testované PAU indukovaly nízké hladiny fosforylace na Ser15 (BaP, DBaeP) nebo na ně neměly žádný vliv (obr. 11).

36

Výsledky

Obr. 11: Účinek PAU na hladiny celkového proteinu p53 a p53 fosforylovaného na Ser15. Buňky byly exponovány 24 hod 10 µM koncentracím PAU s vyjímkou DBalP (100 nM). Detekce β-aktinu byla použita pro kontrolu rovnoměrnosti množství bílkovin. Uveden je reprezentativní obrázek ze tří nezávislých experimentů.

4.1.3. Indukce apoptózy Apoptóza byla detekována pomocí průtokové cytometrie za použití barvení fosfatidylserinu Annexinem-V v kombinaci s barvením PI. Jak ukazuje obr. 12, tři nejsilnější genotoxiny (DBalP, BgChry a BaP) vyvolávaly vysokou hladinu apoptózy (až přes 40 %) v závislosti na koncentraci. Úroveň indukce apoptózy korespondovala se zvýšenou akumulací DNA aduktů a indukcí p53. Nižší, ale také významný nárust apoptózy byl zjištěn po expozici DBaeP. Na druhou stranu byl pozorován pouze nízký nárust procenta apoptotických buněk exponovaných DBahA, BbF a Chry a expozice BaA neměla na apoptózu žádný signifikantní efekt. Vysoké dávky testovaných PAU také indukovaly nekrózu, která může souviset s jejich cytotoxicitou anebo postupem apoptózy do pozdních stádií.

37

Výsledky

Obr. 12: Reprezentativní diagramy barvení buněk Annexinem-V a propidium jodidem (PI) po 48 hod expozici karcinogenním PAU a detekci průtokovou cytometrií. Region R1 představuje raně apoptotické buňky (AnnexinV pozitivní, PI negativní), region R2 živé buňky (Annexin-V a PI negativní) a region R3 pozdně apoptotické/nekrotické buňky (Annexin-V a PI pozitivní). Data představují průměry z minimálně tří nezávislých experimentů ± SD. Data byla statisticky hodnocena Studentovým t-testem. Průměry jsou významně odlišné od kontroly při * p < 0.05 a ** p < 0.01.

38

Výsledky

4.1.4. Aktivace metabolizačních enzymů Indukce mRNA CYP1A1 a CYP1B1 byla určena metodou RT-PCR (obr. 13). Testované PAU kromě DBalP, BgChry a BaA indukovaly maximální hladinu mRNA CYP1A1 srovnatelně s modelovým ligandem receptoru AhR TCDD. DBalP také velmi slabě, narozdíl od všech ostatních PAU, indukoval mRNA CYP1B1. Výsledky ukazují, že indukce exprese hlavních enzymů CYP1, které jsou zahrnuty v metabolické aktivaci PAU, nepředstavuje limitující faktor genotoxicity PAU. Dokonce částečná indukce po expozici DBalP byla dostatečná pro tvorbu vysokých hladin DNA aduktů.

mRNA CYP1B1 6 násobky indukce

160 140 120 100 80 60 40 20 0

4 3 2 1

hr y C

Ba A

Bb F

hr y Ba eP D Ba hA D

Ba P

Bg C

D

Ba lP D

SO

TC D

M D

hr y C

Ba A

Bb F

Ba P Bg C hr y D Ba eP D Ba hA

D

Ba lP D

SO M D

5

0

TC D

násobky indukce

mRNA CYP1A1

Obr. 13: Indukce mRNA CYP1A1 a CYP1B1 po 24 hod expozici studovaným 1 µM PAU ve srovnání s modelovým AhR ligandem TCDD. Výsledky byly získány metodou RT-PCR a jsou vyjádřeny jako průměry ze tří nezávislých experimentů ± SD. Inducibilita byla srovnána s maximální indukcí získanou po expozici buněk 1 nM TCDD.

4.1.5. Role CYP1B1 v metabolické aktivaci genotoxinu DBalP Některé nížemolekulární PAU, přítomné ve vysokých koncentracích v environmentálních směsích, jako např. fluoranthen, jsou schopny inhibovat enzymy CYP1A1 a CYP1B1 (Shimada, 2006). DBalP je metabolizován na aktivní metabolity převážně CYP1B1 (Buters, 2002). Proto jsme vyzkoušeli, zda inhibice CYP1B1 jeho specifickým inhibitorem 2,4,3',5'-tetramethoxystilbenem (TMS) (Chun, 2005) nebo fluoranthenem může zabránit apoptóze indukované silným genotoxinem DBalP. Na obr. 14 je ukázáno, že jak TMS, tak fluoranthen inhibovaly v závislosti na své koncentraci apoptózu vyvolanou DBalP. Naproti tomu benzo[c]fenanthren, inhibující CYP1A1 (Shimada, 2006), byl schopen zabránit apoptóze pouze částečně. Tento výsledek potvrzuje klíčovou roli CYP1B1 v metabolické aktivaci DBalP v buňkách WB-F344.

39

Výsledky

60 pozdní apoptóza / nekróza ranná apoptóza

50 **

% buněk

40

** ***

***

**

30

***

20

*

10

µM Fl a

10

µM 1 Fl a

Fl a

0. 5

µM

µM cP he B

TM S

1

1

µM

µM S

0. 5

µM TM

0. 25 S TM

al P B D

D

M SO

10 0

0. 2

nM

%

0

+ DBalP 100 nM

Obr. 14: Inhibice apoptózy vyvolané DBalP po působení 2,4,3',5'-tetramethoxystilbenu (TMS), benzo[c]phenanthrenu (BcPhe) a fluoranthenu (Fla). Buňky byly předinkubovány s testovanými sloučeninami 30 min a poté byl na dalších 24 hod přidán DBalP. Buňky byly barveny Annexinem-V a PI a apoptóza byla hodnocena průtokovou cytometrií. Výsledky z minimálně tří nezávislých experimentů jsou vyjádřeny jako průměry ± SD. Data byla analyzována Studentovým t-testem. Průměry se odlišují od pozitivní kontroly (DBalP) na hladině významnosti * p < 0.05 a ** p < 0.01.

40

Výsledky

4.2. Toxické efekty methylovaných benz[a]anthracenů v jaterních buňkách Uveřejněno v: Marvanová, S., Vondráček, J., Pěnčíková, K., Trilecová, L., Krčmář, P., Topinka, J., Nováková, Z., Milcová, A. and Machala, M. (2008). Toxic effects of methylated benz[a]anthracenes in liver cells. Chemical Research in Toxicology 21, p. 503 - 512.

4.2.1. Aktivace metabolizačních enzymů Monomethylované deriváty benz[a]anthracenu jsou důležitou skupinou methylovaných PAU vyskytujících se v životním prostředí. Dosud byly testovány pouze jejich karcinogenní a tumor-iniciační účinky převážně na kožních myších nebo potkaních modelech (viz kap. 1.4.6). Jejich účinky v jaterních buňkách nebyly dosud testovány. Exprese mRNA metabolizačních enzymů v jaterních epiteliálních buňkách WB-F344 byla stanovena po expozici dvanácti MeBaA, BaA nebo DMBA (obr. 15) metodou RT-PCR. Všechny MeBaA byly schopny maximálně indukovat CYP1A1, CYP1B1 a také zvyšovaly indukci mRNA AKR1C9. mRNA CYP1B1 7

35

6

násobky indukce

40 30 25 20 15 10 5

4 3 2 1 0

D B 1- aA M eB 2- a M A e 3- Ba M A eB 4- a A M e 5- Ba A M e 6- Ba A M e 7- Ba A M e 8- Ba A M e 9- Ba A M 10 e Ba -M A 11 eBa -M A 12 eB -M aA eB a DM A BA

TC D

SO

0 DM

5

DM SO TC D D 1- BaA M eB 2- a A M e 3- Ba A M e 4- Ba A M e 5- Ba A M e 6- Ba M A eB 7- a A M e 8- Ba A M e 9- Ba M A 10 e Ba -M A 11 eBa -M A 12 eBa -M A eB a DM A BA

násobky indukce

mRNA CYP1A1

9 8 7 6 5 4 3 2 1 0 DM SO TC D D 1- BaA M eB 2- a A M e 3- Ba A M e 4- Ba A M e 5- Ba A M e 6- Ba A M e 7- Ba A M e 8- Ba A M e 9- Ba A M 10 e Ba -M A 11 eBa -M A 12 eBa -M A eB a DM A BA

násobky indukce

mRNA AKR1C9

Obr. 15: Indukce mRNA CYP1A1, CYP1B1 a AKR1C9 stanovená RT-PCR po 24 hod expozici MeBaA nebo BaA (1 µM), DMBA (100 nM) a modelovému ligandu AhR, TCDD (1 nM). Data jsou vyjádřena jako průměr ze tří nezávislých experimentů ± SD.

41

Výsledky

4.2.2. Narušení buněčného cyklu a indukce buněčné proliferace Naše předchozí studie naznačují, že mírně zvýšené procento buněk v S-fázi (5 - 10 % buněk) v kontaktně inhibovaných buňkách WB-F344 souvisí s proliferativními účinky negenotoxických ligandů AhR, zatímco silné genotoxiny indukují výraznou akumulaci buněk v S-fázi (nad 20 % buněk) (Chramostová, 2004; Andrysík, 2007; Švihálková-Šindlerová, 2007). U všech MeBaA byla pozorována mírná akumulace buněk v S-fázi s vyjímkou 10MeBaA, který vyvolával vyšší akumulaci (nad 20 %) už v koncentraci 1 µM (obr. 16). Silný genotoxin DMBA indukoval ještě výraznější akumulaci buněk v S-fázi než 10-MeBaA. Tyto výsledky naznačily, že ze skupiny MeBaA pouze 10-MeBaA má genotoxické účinky. To bylo potvrzeno i hodnocením buněčné proliferace. Zatímco všechny MeBaA významně zvýšily proliferaci, po expozici 10-MeBaA počet buněk nebyl ovlivněn a po expozici DMBA došlo vlivem silné genotoxicity k poklesu počtu buněk (obr. 17). 7-MeBaA

D

5

0.

1%

**

M SO

5 nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

M SO

D D

D

TC

* *

nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

** *

D

% S-fáze

45 40 35 30 25 20 15 10 5 0

TC D

45 40 35 30 25 20 15 10 5 0

10-MeBaA

12-MeBaA

** *

5 D

M SO

0.

1%

*

D

5 nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

D

0.

1%

**

*

*

nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

**

TC D

M SO D

45 40 35 30 25 20 15 10 5 0

TC D

45 40 35 30 25 20 15 10 5 0

DMBA

Koncentrace (M)

Obr. 16: Procenta buněk v S-fázi buněčného cyklu po 72 hod expozici buněk WB-F344 7-, 10-, 12-MeBaA a DMBA, stanovená průtokovou cytometrií za použití barvení propidium jodidem. Data představují průměry ± SD ze tří nezávislých experimentů provedených v duplikátech. Průměry jsou významně odlišné od kontroly při * p < 0,05 a ** p < 0,01.

42

Výsledky 1-MeBaA

2-MeBaA 700

700 600

*

**

**

500

400

200

200

100

100

0

0

DM SO TC DD 5 nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

300

DM SO TC D D 5 nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E -0 6 1. 00 E05

5-MeBaA

6-MeBaA

600

**

800

700

700

200

200

200

100

100

100

0

0

0

**

**

600

1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

BaA

-0 6

-0 5

00 E

00 E08

1.

00 E09

1.

1.

-0 5 00 E

1.

-0 7 00 E

-0 8

00 E09

** **

**

DM SO TC DD 5 nM 1. 00 E10 1. 00 E09 1. 00 E08 1. 00 E07 1. 00 E06 1. 00 E05

nM

-1 0 1. 00 E

5 DD

DM SO

500

TC

1.

1.

1.

1.

1.

-1 0 1.

00 E

5

00 E05

300

00 E06

300

00 E07

300

00 E08

400

00 E09

400

nM

500

400

DM SO

600

500

**

*

12-MeBaA

800

600

TC DD

nM

TC

11-MeBaA

800

DMBA

800

800

700

5

DM SO

00 E05

1.

00 E06

1.

00 E07

1.

00 E08

00 E09

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Obr. 17: Modulace buněčné proliferace v buňkách WB-F344 hodnocená jako počty buněk po 72 hod expozici testovaným sloučeninám. Data představují průměry ± SD ze tří nezávislých experimentů provedených v duplikátech. Průměry jsou významně odlišné od kontroly při * p < 0,05 a ** p < 0,01.

4.2.3. Fosforylace proteinu p53 a apoptóza Fosforylace proteinu p53 na Ser15 byla sledována po 24 hod expozici MeBaA v koncentraci 10 µM, zároveň byly testovány i účinky BaA a jako pozitivní kontrola byl použit silný genotoxin DMBA. Obr. 18 ukazuje, že z MeBaA pouze 10-MeBaA vyvolával fosforylaci p53. 43

Výsledky Apoptóza byla stanovena pomocí průtokové cytometrie a mikroskopického hodnocení fragmentace jader. Všechny MeBaA indukovaly mírný nárust počtu buněk s fragmentovaným jádrem (obr. 19), stejně jako mírný nárust raně apoptotických buněk, barvených AnnexinemV (data nejsou ukázána). Nejsilnější apoptotickou odpověď vyvolávala expozice 10-MeBaA (obr. 19, 20). Tento účinek byl ale výrazně nižší než po expozici silnému genotoxinu DMBA. aA BaA BaA BaA BaA eB e e e e BA SO M M M M M 134DM 25DM

P-p53 (Ser15) β-aktin aA aA BaA BaA BaA eB eB e e e BA SO M M M M M M M 0 9 6 D 7 D 8 1

P-p53 (Ser15) β-aktin DMBA aA BaA eB e A -M 2 -M 1 10 µM 11 Ba 0.1 1

SO DM

P-p53 (Ser15) β-aktin

Obr. 18: Fosforylace proteinu p53 na Ser15 po 24 hod expozici 10 µM MeBaA a BaA stanovená western blotem. DMBA byl testován v koncentracích 0,1; 1 a 10 µM, koncentrace 10 µM byla použita jako pozitivní kontrola na každém blotu. Detekce β-aktinu byla použita pro kontrolu rovnoměrnosti množství bílkovin. Uveden je reprezentativní obrázek ze tří nezávislých experimentů.

% fragmentovaných jader

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D

M SO D M BA

0

Obr. 19: Indukce pozdní apoptózy po 48 hod expozice buněk WB-F344 testovaným sloučeninám. Pozdní apoptóza byla stanovena jako procento buněk s fragmentovaným jádrem za použití barvení DAPI a mikroskopického hodnocení. Data představují průměry ± SD ze tří nezávislých experimentů provedených v duplikátech. Průměry jsou významně odlišné od kontroly při * p < 0.05 a ** p < 0.01.

44

Výsledky

DMSO

10-MeBaA 10 µM

DMBA 100 nM

R1: 6.7 % ± 0.7

R1: 18.2 % ± 3.2

R1: 38.1 % ± 4.9

R3: 5.8 % ± 1.17

R3: 25.7 % ± 2.3

R3: 19.7 % ± 0.7

Obr. 20: Příklad indukce apoptózy měřené průtokovou cytometrií. Region R1 představuje raně apoptotické buňky (Annexin-V pozitivní, PI negativní), region R2 živé buňky (Annexin-V a PI negativní) a region R3 pozdně apoptotické/nekrotické buňky (Annexin-V a PI pozitivní). Data představují průměry z minimálně tří nezávislých experimentů ± SD.

4.2.4. Tvorba DNA aduktů a oxidativního stresu U vybraných MeBaA, BaA a DMBA byla metodou 32P-postlabeling studována jejich schopnost vytvářet DNA adukty a schopnost vyvolávat oxidativní stres, detekovaná pomocí průtokové cytometrie (metoda neprezentována v této disertační práci, viz článek v příloze II.). Jak ukazuje tab. 2, DMBA vytvářel vysoké hladiny DNA aduktů, potvrzující jeho silnou genotoxicitu, zatímco 10-MeBaA indukoval velmi nízké množství aduktů podobně jako 12MeBaA, ale více než 7-MeBaA. DMBA i 10-MeBaA významně zvyšovaly tvorbu ROS, zatímco expozice neprezentována).

7-MeBaA

tento

efekt

v buňkách

WB-F344

nevyvolala

(data

Tab. 2: Hladiny DNA aduktů indukované v buňkách WB-F344 po 24 hod expozici vybraným MeBaA, BaA a DMBA. Data byla získána z minimálně dvou nezávislých experimentů prováděných v triplikátech a představují průměr ± SD. 8

BaA 10 µM 7-MeBaA 10 µM 10-MeBaA 10 µM 12-MeBaA 10 µM DMBA 1uM

DNA adukty / 10 nukleotidů 1,2 ± 0,38 0,4 ± 0,031 3,1 ± 1,4 2,8 ± 0,24 230 ± 77

45

Výsledky

4.3. Genotoxické a tumor-promoční efekty 7H-dibenzo[c,g]karbazolu a jeho syntetických derivátů v jaterních epiteliálních buňkách WB-F344 Uveřejněno v: Vondráček, J., Švihálková-Šindlerová, L., Pěnčíková, K., Krčmář, P., Andrysík, Z., Chramostová, K., Marvanová, S., Valovičová, Z., Kozubík, A., Gábelová, A. and Machala, M. (2006). 7H-dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stem-like’ cells. Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis 596, p. 43 - 56. Valovičová, Z., Marvanová, S., Mészárosová, M., Srančíková, A., Trilecová, L., Milcová, A., Líbalová, H., Vondráček, J., Machala, M., Topinka, J. and Gábelová, A. Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells. Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis, in press.

46

Výsledky

4.3.1. Aktivace metabolizačních enzymů Pro detekci aktivace metabolizačních enzymů v jaterních progenitorových buňkách WB-F344 po expozici 7H-dibenzo[c,g]karbazolu (DBC) a jeho syntetickým derivátům 5,9dimethyldibenzo[c,g]karbazolu (diMeDBC) a N-methyldibenzo[c,g]karbazolu (N-MeDBC) byla použita metoda RT-PCR. Jak ukazuje obr. 21, mRNA CYP1A1 byla indukována po expozici DBC a velmi silně po expozici diMeDBC. DiMeDBC také výrazně indukoval mRNA CYP1A2, zatímco DBC pouze velmi málo, nicméně hladiny mRNA CYP1A2 jsou v buňkách WB-F344 velmi nízké. Hladina mRNA CYP1B1 a AKR1C9 byla zvýšena po expozici DBC i diMeDBC na podobnou úroveň. N-MeDBC neindukoval (vůbec nebo pouze velmi slabě) zvýšenou expresi mRNA žádného z testovaných metabolizačních enzymů.

Obr. 21: Indukce mRNA CYP1A1, CYP1A2, CYP1B1 a AKR1C9 po 24 hod expozici 1 µM DBC, diMeDBC a N-MeDBC ve srovnání s modelovým AhR ligandem TCDD (1 nM). Výsledky byly získány metodou RT-PCR a jsou vyjádřeny jako průměry ze tří nezávislých experimentů ± SD.

4.3.2. Narušení buněčného cyklu a indukce proliferace Dále byl zkoumán vliv DBC a jeho derivátů na buněčný cyklus a proliferaci konfluentních buněk WB-F344, jelikož modulace buněčného cyklu a zvýšená proliferace patří 47

Výsledky k významným tumor-promočním mechanismům. DBC a diMeDBC indukovaly výraznou akumulaci buněk v S-fázi (nad 20 %) (obr. 22), podobně jako genotoxické PAU. Významně zvýšená proliferace byla detekována pouze u 1 µM diMeDBC (obr. 22). N-MeDBC výrazněji neovlivnil ani buněčný cyklus, ani proliferaci.

Obr. 22: Modulace buněčné proliferace vyjádřená pomocí procenta buněk v S-fázi a počtů buněk a buněčná smrt po 72 hod expozici DBC a jeho methylovaným derivátům. Procento buněk v S-fázi bylo měřeno průtokovou cytometrií, počet buněk byl stanoven na přístroji Coulter Counter. Procento buněk v pozdní apoptóze bylo stanoveno barvením DAPI a mikroskopickým hodnocením fragmentace jader. Jako pozitivní kontrola bylo použito 1 nM TCDD a 10 nM DBalP v případě stanovení apoptózy. Data jsou vyjádřena jako průměry ze tří nezávislých experimentů ± SD. Průměry jsou významně odlišné od kontroly při * p < 0,05 a ** p < 0,01.

48

Výsledky

4.3.3. Indukce apoptózy a fosforylace proteinu p53 DBC i diMeDBC indukovaly fragmentaci buněčných jader jako marker pozdní apoptózy zejména v koncentraci 10 µM (obr. 22). Indukce apoptózy v rané fázi těmito dvěma sloučeninami po 48 hod expozici byla potvrzena i pomocí dvojího barvení Annexinem-V a PI v koncentraci 10 µM a v případě DBC statisticky významně už při koncentraci 1 µM (data neprezentována). Také způsobovaly fosforylaci proteinu p53 (obr. 23) narozdíl od N-MeDBC, který neindukoval ani apoptózu, ani fosforylaci p53.

Obr. 23: Fosforylace proteinu p53 na Ser15 po 24 hod expozici DBC a jeho derivátům detekovaná metodou western blot.

4.3.4. Poškození DNA - tvorba stabilních DNA aduktů, zlomů a mikrojader V buňkách exponovaných DBC bylo metodou 32P-poslabeling nalezeno velké množství DNA aduktů, které při koncentraci 1 µM odpovídalo množství aduktů indukovaných BaP (použit jako pozitivní kontrola) (tab. 3). DiMeDBC překvapivě vyvolával velmi nízkou tvorbu DNA aduktů, téměř u detekčního limitu. N-MeDBC také indukoval velmi nízké hladiny DNA, nicméně ty byly 4 - 6 krát vyšší než po expozici diMeDBC. Aby se vyloučila možnost, že by diMeDBC vytvářel DNA adukty, které by mohly být rychle odstraňovány, byly exponované buňky WB-F344 sklízeny ve dvouhodinových intervalech během 24 hod a použity pro měření DNA aduktů. V žádném časovém bodě nebyly adukty nalezeny (data nejsou prezentována). Ke zjištění dalších typů poškození DNA byla použita gelová elektroforéza jednotlivých buněk (SCGE, comet assay) prováděná při hodnotě pH vyšší než 12,6. Za těchto podmínek jsou detekovány jedno- a dvouřetězcové zlomy, křížové vazby, místa nekompletní excizní opravy a alkali-labilní místa. Všechny tři testované látky - DBC, diMeDBC i N-MeDBC dávaly pozitivní odpověď v tomto testu v rozmezí koncentrací 0,1 – 20 µM po expozici 2 hod (obr. 24). 49

Výsledky Další známkou poškození DNA, resp. klastogenity, je tvorba mikrojader. Klastogenní aktivita dibenzokarbazolů byla hodnocena v koncentračním rozmezí 0,5 - 2,5 µM 24 a 48 hod po ukončení dvouhodinové expozice buněk WB-F344. Všechny dibenzokarbazoly indukovaly statisticky významnou tvorbu mikrojader ve srovnání s kontrolními buňkami (data nejsou prezentována). Tab. 3: Hladiny DNA aduktů indukované v buňkách WB-F344 po 24 hod expozici DBC, diMeDBC, N-MeDBC a BaP jako pozitivní kontrole. Data byla získána z minimálně dvou nezávislých experimentů prováděných v triplikátech a představují průměr ± SD.

Sloučenina

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Obr. 24: Detekce poškození DNA alkalickou SCGE v buňkách WB-F344 po 2 hod expozici DBC, diMeDBC a N-MeDBC v koncentracích 0,1 – 20 µM. Zlomy na DNA byly měřeny bezprostředně po ukončení expozice. Data představují průměry ± SD ze tří nezávislých experimentů prováděných v triplikátech sklíček a byla statisticky vyhodnocena Studentovým t-testem. Průměry jsou významně odlišné od kontroly při * p < 0,05; ** p < 0,01 a *** p < 0,001.

50

Výsledky

4.3.5. Kinetika oprav zlomů na DNA Kinetika oprav zlomů na DNA byla sledována pomocí metody SCGE v různých časových bodech až do 48 hod po ukončení dvouhodinové expozice dibenzokarbazoly (obr. 25). Poměrně rychlé odstranění zlomů bylo pozorováno v buňkách exponovaných diMeDBC - poškození DNA dosáhlo pozaďové hodnoty 16 hod po expozici. V buňkách exponovaných DBC bylo množství zlomů sníženo až 24 hod po expozici a významná hladina poškození v buňkách zůstávala i 48 hod po expozici. Poškození DNA N-MeDBC se snížilo už 16 hod po expozici a dosáhlo pozaďové hodnoty 48 hod po expozici.

40 35

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Obr. 25: Kinetika znovuspojování zlomů na DNA v buňkách WB-F344 exponovaných 2 hod 1 µM koncentraci DBC, diMeDBC a N-MeDBC. Hladiny zlomů na DNA byly měřeny v několika časových intervalech po skončení expozice. Data představují průměry ± SD ze tří nezávislých experimentů prováděných v triplikátech sklíček a byla statisticky vyhodnocena Studentovým t-testem. Průměry jsou významně odlišné od kontroly při * p < 0,05 a *** p < 0,001.

4.3.6. Oxidativní poškození DNA a oxidativní stres Pro detekci dalšího typu poškození DNA, kterým by mohl diMeDBC vyvolávat genotoxické efekty, byla použita modifikovaná metoda SCGE (Collins, 1995). Modifikace spočívá v inkubaci DNA s reparačně specifickou endonukleázou - proteinem Fpg, který štěpí DNA v místě bázové modifikace, fragmentovaných (FAPY-adukty) a oxidovaných (8-oxodG) purinů. Mírný, ale statisticky významný nárust v množství míst citlivých na Fpg byl pozorován po působení diMeDBC, zatímco DBC ani N-MeDBC tento typ poškození neindukovaly. Kinetika oprav Fpg-senzitivních míst byla pomalá, zlomy na DNA způsobené 51

Výsledky Fpg byly odstraněny 16 hod po skončení expozice (data neprezentována). Schopnost dibenzokarbazolů indukovat oxidativní stres a vytvářet reaktivní kyslíkové radikály (ROS), které mohou zapříčinit oxidativní poškození DNA, byla ověřena pomocí průtokové cytometrie po expozici 24 hod. Všechny tři dibenzokarbazoly indukovaly tvorbu ROS, nejsilněji diMeDBC (tab. 4) (metoda neprezentována v této disertační práci, viz článek v příloze IV).

Tab. 4: Relativní hodnoty reaktivních kyslíkových radikálů (ROS) produkovaných v buňkách WB-F344 po 24 hod expozici 1 µM koncentraci DBC, diMeDBC a N-MeDBC. Jako pozitivní kontrola byl použit 250 µM peroxid vodíku (H2O2) s délkou expozice 5 min. Data představují průměr ± SD z nejméně dvou nezávislých experimentů. Významně odlišné od kontroly (DMSO) při * p < 0,05; významně odlišné od diMeDBC při $

p < 0,05.

Sloučenina

Expozice

Intenzita fluorescence (%)

DMSO

24 h

100

DBC

24 h

diMeDBC

24 h

166 ± 12.0 * 295 ± 61.7 *

N-MeDBC

24 h

205 ± 32.3 *

H2O2

5 min

707 ± 77.3 *

,$

,$

52

Výsledky

4.4. Účinky komplexní směsi PAU vázaných na částice ovzduší SRM 1649a v jaterních progenitorových buňkách Rukopis v přípravě: Andrysík, Z., Marvanová, S., Ciganek, M., Neča, J., Pěnčíková, K., Vondráček, J., Mahadevan, B., Topinka, J., Baird, W.M., Kozubík, A. and Machala, M. Genotoxic and nongenotoxic effects of complex airborne PAH mixture SRM1649a in rat liver epithelial cells.

SRM (Standardní referenční materiál) 1649a představuje komplexní vzorek pevných částic ovzduší z městské oblasti (NIST, 1982; NIST, 2001) a je používán nejen pro hodnocení analytických metod, ale i pro výzkum svých biologických účinků. V této studii byly zkoumány genotoxické a tumor-promoční efekty SRM 1649a na potkaních jaterních epiteliálních buňkách WB-F344 s cílem zjistit mechanismy působení tohoto environmentálního vzorku. Hrubý extrakt SRM 1649a (označený jako CE), obsahující všechny organické sloučeniny přítomné ve vzorku, byl frakcionován a společně se svými chromatografickými frakcemi použit pro jednotlivé experimenty (postup frakcionace není v disertační práci popisován, bude součástí článku). Neutrální aromatická frakce (označená jako F1) obsahuje PAU, methylované PAU, polychlorované bifenyly a polychlorované dibenzodioxiny a dibenzofurany. Semipolární frakce (F2) obsahuje především nitrované PAU a polární frakce (F3) oxidované PAU.

53

Výsledky

4.4.1. Aktivace metabolizačních enzymů Metodou RT-PCR byla měřena indukce mRNA CYP1A1 a CYP1B1 po 24 hod expozici buněk WB-F344 extraktu SRM1649a (CE) a jeho neutrální frakci (F1). Jak ukazuje obr. 26, CE i F1 indukovaly mRNA CYP1A1 od koncentrace 0,1 mg/ml, F1 silněji než CE, a mRNA CYP1B1 od koncentrace 0,01 mg/ml. Pomocí metody western blotting byla také zjištěna zvýšená exprese proteinů CYP1A1 a CYP1B1 po působení CE, F1 a F3 (data neprezentována v disertační práci, viz rukopis v příloze V). CYP1A1 mRNA

CYP1B1 mRNA

50

násobky indukce

40

8

CE F1

CE F1

7

násobky indukce

45

35 30 25 20 15 10

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mg / ml

Obr. 26: Indukce mRNA CYP1A1 a CYP1B1 po 24 hod expozici hrubému extraktu SRM 1649a (CE) a jeho neutrální frakci F1. Jako pozitivní kontrola byl použit 1 nM TCDD. Výsledky byly získány metodou RT-PCR a jsou vyjádřeny jako průměry ze dvou nezávislých experimentů.

4.4.2. Tvorba DNA aduktů Metodou 33P-postlabeling a analýzou pomocí vysokoúčinné kapalinové chromatografie (HPLC) byly stanoveny DNA adukty, které vznikly po 24 hod expozici buněk WB-F344 BaP, DBalP a hrubému extraktu SRM 1649a. Jak ukazuje obr. 27, po expozici BaP byl detekován jeden hlavní pík eluovaný s retenčním časem 35 min, který odpovídal aduktu (+)-anti-BPDEdG (benzo[a]pyren-7,8-dihydrodiol-9,10-epoxid-deoxyguanosin). Po expozici DBalP byl detekován také jeden pík s retenčním časem 75 min, odpovídající aduktu (−)-anti-DBPDE-dA (dibenzo[a,l]pyren-11,12-dihydrodiol-13,14-epoxid-deoxyadenosin). Ovšem v DNA buněk exponovaných extraktem komplexního vzorku SRM 1649a byl nalezen pouze jeden hlavní pík, odpovídající aduktu benzo[a]pyrenu (+)-anti-BPDE-dG. Dalšími identifikovanými byly adukty DBalP-dihydrodiolepoxidu, které byly vytvořeny ale ve velmi malém množství. Kvantitativně bylo množství aduktu po působení extraktu SRM asi šestkrát nižší než po působení BaP (tab. 5). Analýza aduktů byla provedena v laboratoři dr. Bairda, Oregon State University, USA. 54

Výsledky

BaP

4

(+)-anti-BPDE-dG

(−)-anti-DBPDE-dA

DBalP

extrakt SRM 1649a (+)-anti-BPDE-dG (−)-anti-DBPDE-dA

Obr. 27: Eluční profily (získané pomocí HPLC) DNA aduktů značených 33P, vytvořených v buňkách WB-F344 po 24 hod expozici 10 µM BaP, 100 nM DBalP a hrubému extraktu SRM 1649a o koncentraci 2,5 mg/ml. Píky aduktů BaP-dihydrodiolepoxidu a DBalP-dihydrodiolepoxidu byly označeny podle elučních časů publikovaných v Mahadevan, 2005 a Buters, 2002.

Tab. 5: DNA adukty v buňkách WB-F344 po 24 hod expozici. Výsledky jsou ze dvou nezávislých experimentů provedených v triplikátech.

BaP

Koncentrace

DNA adukty PAU pmol / mg DNA

10 µM

3

DBalP

100 nM

29

Extrakt SRM 1649a

2,5 mg / ml

0,5

4.4.3. Fosforylace proteinu p53 a apoptóza Metodou western blotting byla sledována fosforylace tumor-supresorového proteinu p53. Jak ukazuje obr. 28, hrubý extrakt a polární frakce F3 vyvolávaly detekovatelnou fosforylaci p53, ale na hladiny celkové p53 neměly vliv. Raná a pozdní apoptóza/nekróza byla stanovena pomocí barvení buněk Annexinem-V a PI po expozici 48 hod. Jak ukazuje obr. 29, neutrální frakce F1 nevyvolala zvýšenou apoptózu 55

Výsledky ani nekrózu. Hrubý extrakt silně zvýšil procento buněk zejména v pozdní apoptóze/nekróze. Polární frakce F3 také vyvolávala zvýšenou buněčnou smrt, zejména pozdní apoptózu/nekrózu, ale v menší míře než hrubý extrakt. Apoptóza byla dále hodnocena mikroskopicky po barvení DAPI jako fragmentace jader po expozici 72 hod. Jak ukazuje obr. 30, zvýšenou fragmentaci jader vyvolával hrubý extrakt SRM a F3.

Obr. 28: Aktivace proteinu p53 v buňkách WB-F344 exponovaných 24 hod SRM 1649a. K expozici byla použita koncentrace 2,5 mg/ml hrubého extraktu (CE) a jeho frakcí (F1 - F3). Jako pozitivní kontrola byl použit 100 nM DBalP a 10 µM BaP. Obrázek představuje jeden ze tří nezávislých experimentů.

Obr. 29: Příklad indukce apoptózy v buňkách WB-F344 po 48 hod expozici hrubému extraktu (CE) SRM 1649a a jeho frakcím (F1 - F3). Buňky byly barveny Annexinem-V a PI, k detekci byla použita průtoková cytometrie. Region R1 představuje živé buňky, region R2 raně apoptotické buňky a region R3 pozdně apoptotické/nekrotické buňky. Data reprezentují jeden ze čtyř nezávisle provedených experimentů.

56

Výsledky

Obr. 30: Indukce pozdní apoptózy po 72 hod expozici buněk WB-F344 hrubému extraktu (CE) SRM a jeho frakcím. Jako pozitivní kontrola byl použit 1 µM BaP. Pozdní apoptóza byla stanovena jako procento buněk s fragmentovaným jádrem za použití barvení DAPI a mikroskopického hodnocení. Data představují průměry ± SD ze tří nezávislých experimentů. Průměry jsou významně odlišné od kontroly při * p < 0,05.

4.4.4. Vliv SRM na buněčnou proliferaci a cyklus Vliv hrubého extraktu SRM a jeho frakcí na proliferaci buněk WB-F344 po 72 hod expozici je ukázán na obr. 31. Hrubý extrakt a frakce F3 v nižších koncentracích nejprve vyvolávaly buněčnou proliferaci, ve vyšších koncentracích ale převážily jejich genotoxické účinky a počet buněk prudce klesl. To je v souladu s výše uvedenými výsledky měření apoptózy a aktivace proteinu p53. Neutrální aromatická frakce F1 už od nízkých koncentrací vyvolávala statisticky významnou proliferaci a ani v nejvyšší koncentraci se neprojevila apoptóza. Semipolární frakce F2 mírně, ale statisticky významně, zvyšovala proliferaci pouze ve dvou nejvyšších koncentracích. Pro detekci počtu buněk akumulovaných v S-fázi po expozici 48 hod byla vybrána koncentrace 0,5 mg/ml. Hrubý extrakt, F1 i F3 za těchto podmínek zvyšovaly akumulaci buněk v S-fázi na obdobné úrovni jako pozitivní kontrola TCDD, F2 neměla na počet buněk v S-fázi vliv (obr. 32).

57

Výsledky

Obr. 31: Modulace buněčné proliferace hodnocená jako počty buněk po 72 hod expozici hrubému extraktu (CE) SRM 1649a a jeho frakcím (F1 - F3). Jako modelový stimulátor proliferace buněk WB-F344 byl použit 1 nM TCDD. Data představují průměry ± SD ze tří nezávislých experimentů. Průměry jsou významně odlišné od kontroly při * p < 0,05.

Obr. 32: Procenta buněk akumulovaných v S-fázi buněčného cyklu po 48 hod expozici buněk WB-F344 hrubému extraktu (CE) a jeho frakcím (F1 - F3), stanovená průtokovou cytometrií za použití barvení propidium jodidem. Jako pozitivní kontrola byl použit 1 nM TCDD. Data představují průměry ± SD ze tří nezávislých experimentů. Průměry jsou významně odlišné od kontroly při * p < 0,05.

58

Diskuze a závěry

5.

Diskuze a závěry

Chemické karcinogeny mohou jako své cíle ovlivňovat i pluripotentní kmenové buňky a raně progenitorové buňky (Trosko, 2005). Přesto je většina studií zabývajících se účinky PAU dosud zaměřena na diferencované buňky nebo jejich deriváty. Progenitorové oválné buňky v játrech představují buněčnou populaci, která silně proliferuje v odpověď na působení různých chemických karcinogenů (Wu, 2007). Tyto oválné buňky jsou schopny diferencovat jak na hepatocyty, tak na epiteliální buňky žlučových kanálků a předpokládá se o nich, že hrají významnou roli v hepatokarcinogenezi (Roskams, 2006). Předchozí experimenty v modelové linii potkaních jaterních epiteliálních buněk WBF344, které sdílejí fenotypické vlastnosti s oválnými buňkami, ukázaly, že vybrané genotoxické PAU jsou schopny v těchto buňkách vyvolat aktivaci proteinu p53 a apoptózu (Chramostová, 2001; Andrysík, 2006). Tyto efekty, společně s indukcí oprav DNA a zastavením buněčného cyklu, mohou být iniciovány tvorbou stabilních DNA aduktů (Ramet, 1995; Binková, 2000; Guo, 2002). První studie Topinka, 2008 (kap. 4.1., příloha I) uvádí výsledky ukazující, že v jaterních buňkách WB-F344 jsou DBalP, BaP, BgChry a DBaeP schopny vyvolat tvorbu stabilních DNA aduktů s následnou aktivací proteinu p53 a apoptózou. Další testované karcinogenní PAU – Chry, BaA, BbF a DBahA (IARC, 1983) neindukovaly vůbec nebo pouze velmi slabě tvorbu DNA aduktů v buňkách WB-F344, s čímž byla ve shodě i skutečnost, že neaktivovaly protein p53 a významněji ani apoptózu. Lze předpokládat, že v případě těchto karcinogenních sloučenin hrají v působení na jaterní progenitorové buňky významnější roli negenotoxické efekty, jako jsou aktivace receptoru AhR, narušení mezibuněčné komunikace nebo buněčné proliferace (Bláha, 2002; Chramostová, 2004; Andrysík, 2007). Další závěr vyplývající z této studie souvisí se skutečností, že pro přeměnu PAU na reaktivní metabolity, které jsou schopny mimo jiné tvořit DNA adukty, je nutná aktivace cytochromy P450 podrodiny 1 (review viz Xue, 2005). Všechny testované PAU s vyjímkou DBalP účinně indukovaly expresi CYP1A1 a CYP1B1 v buňkách WB-F344. CYP1B1, který je kritickým enzymem pro genotoxicitu DBalP (Buters, 2002), je v buňkách WB-F344 konstitutivně exprimován (Švihálková-Šindlerová, 2007; Zatloukalová, 2007). Výsledky tedy ukazují, že tato konstitutivní hladina CYP1B1 je dostatečná pro tvorbu genotoxických reaktivních metabolitů DBalP, který je sám o sobě slabým ligandem AhR (Machala, 2001). Proto je možné uzavřít, že indukce bioaktivačních CYP1 enzymů není rozhodujícím faktorem pro genotoxicitu PAU. V prezentované studii byl dále zkoumán efekt inhibitorů CYP1B1 – fluoranthenu a TMS (Chun, 2005; Shimada, 2006) na apoptózu vyvolanou DBalP. Oba inhibitory byly schopny účinně zabránit apoptóze vyvolané DBalP, což potvrzuje, že CYP1B1 hraje jednu z klíčových rolí v genotoxických a cytotoxických procesech indukovaných PAU v buňkách WB-F344. 59

Diskuze a závěry Ve druhé studii Marvanová, 2008 (kap. 4.2., příloha II) byly zkoumány toxické účinky monomethylovaných derivátů benz[a]anthracenu v potkaních epiteliálních buňkách WB-F344. Pouze jedna sloučenina, 10-MeBaA, vykazovala mírně genotoxické účinky v buňkách WB-F344, a to mírnou tvorbu stabilních DNA aduktů, zvýšenou produkci ROS, fosforylaci proteinu p53, apoptózu a zvýšené procento buněk v S-fázi. Tyto efekty byly ovšem výrazně nižší než v případě DMBA, velmi silného genotoxinu ve WB-F344. Ostatní MeBaAs, včetně 7-MeBaA, u kterého byly v předchozích studiích nalezeny tumor-iniciační, karcinogenní a mutagenní účinky v hlodavčích kožních a bakteriálních modelech (Dunning and Curtis, 1960; Stevenson and von Haam, 1965; Glatt, 1981; Wislocki, 1982; Utesch, 1987), v jaterních buňkách WB-F344 nevyvolávaly genotoxické efekty, zato byly schopny významně aktivovat receptor AhR. Výsledky naznačují, že MeBaAs mohou významně přispívat k toxicitě environmentálních směsí PAU zejména prostřednictvím aktivity zprostředkované receptorem AhR, která je silnější než u jejich parentální sloučeniny BaA. Rozdílnost toxických efektů MeBaAs v kožních a bakteriálních modelech publikovaných ve starších studiích a jejich efektů v jaterních buňkách WB-F344 poukazuje na to, že by mělo být při hodnocení efektů PAU věnováno více pozornosti i buněčným modelům z jiných tkání než pouze kožním, zejména jaterním a plicním. Třetí studie Vondráček, 2006 (kap. 4.3., příloha III) se zabývala účinky látek patřících mezi N-heterocyklické aromatické uhlovodíky, a to environmentálního polutantu 7H-dibenzo[c,g]karbazolu (DBC) a jeho syntetických derivátů 5,9-dimethyldibenzo[c,g]karbazolu (diMeDBC) a N-methyldibenzo[c,g]karbazolu (N-MeDBC) v jaterních progenitorových buňkách WB-F344. Tyto sloučeniny byly původně syntetizovány za účelem studia vztahu mezi chemickou strukturou a biologickou aktivitou (Valéro, 1983; Périn, 1984). DiMeDBC vykazuje striktní hepatokarcinogenitu (Valéro, 1983; Renault, 1998), zatímco NMeDBC má sarkomagenní účinky (Périn, 1984; Schurdak, 1987). Jejich genotoxické efekty byly popsány v několika in vitro a in vivo studiích (Valéro, 1983; Warshawsky, 1996; PérinRoussel, 1997; Taras-Valéro, 2000; Gábelová, 2002; Gábelová, 2004), nicméně existuje málo informací o jejich tumor-promočních účincích. V prezentované studii byly nalezeny rozdíly v efektech zejména mezi DBC a diMeDBC na jedné straně a sarkomagenem N-MeDBC na straně druhé v buňkách WB-F344. DBC i diMeDBC byly schopny aktivovat metabolizační enzymy CYP1A1, 1A2, 1B1 a AKR1C9, po jejich působení došlo k výraznému nárustu počtu buněk akumulovaných v S-fázi, doprovázenému výraznou apoptózou a fosforylací tumorsupresorového proteinu p53. Významně zvýšená proliferace byla pozorována pouze u 1 µM diMeDBC. Tyto účinky ukazují obdobně jako u PAU na silnou genotoxicitu testovaných sloučenin (Chramostová, 2004; Andrysík, 2007; Švihálková-Šindlerová, 2007). Naproti tomu N-MeDBC nebyl schopen aktivovat zkoumané metabolizační enzymy, neindukoval ani apoptózu a fosforylaci p53, ani zvýšenou proliferaci. Mírně zvýšená akumulace buněk v Sfázi byla nalezena pouze v nejvyšší testované 10 µM koncentraci N-MeDBC. Nicméně i mezi 60

Diskuze a závěry účinky DBC a diMeDBC byly nalezeny rozdíly. DBC silně inhiboval mezibuněčnou komunikaci typu gap-junctions, zatímco diMeDBC byl silným agonistou receptoru AhR (data neprezentována v této doktorské práci, viz článek v příloze III), indukoval hladiny mRNA CYP1A1 a 1A2 silněji než DBC a stimuloval buněčnou proliferaci v jedné nižší koncentraci. Na tuto studii navazovala čtvrtá studie Valovičová, v tisku (kap. 4.3., příloha IV), zabývající se podrobněji genotoxickými účinky DBC a jeho dvou derivátů v buňkách WBF344. Výsledky této studie naznačují možné rozdílné mechanismy hepatokarcinogenity, kterými působí DBC a diMeDBC. DBC vytvářel stabilní DNA adukty, jejichž množství bylo po působení 1 µM DBC obdobné jako po působení 1 µM BaP. Poškození DNA způsobené DBC vyústilo ve tvorbu zlomů, mikrojader a ve značné zpoždění oprav poškozené DNA. Striktní karcinogen diMeDBC vyvolával v jaterních progenitorových buňkách překvapivě pouze zanedbatelné množství DNA aduktů. Měření aduktů v různých časových intervalech během 24 hod expozice vyloučilo možnost, že by DNA adukty vznikaly a byly rychle opraveny ještě před ukončením expozice. Navzdory absenci tvorby aduktů byly nalezeny významné hladiny zlomů na DNA a vznik mikrojader. Poškození DNA bylo narozdíl od DBC rychle opraveno; zlomy na DNA už nebyly detekovány 16 hod od ukončení expozice. Zvýšená migrace DNA z buněk ovlivněných diMeDBC po inkubaci s reparačně specifickým enzymem Fpg naznačuje, že příčinou genotoxicity diMeDBC by mohlo být oxidativní poškození (8-oxodG) nebo bázové modifikace (tzv. FAPY-DNA adukty). Tuto hypotézu podporuje také dva a půlkrát zvýšená hladina ROS v buňkách exponovaných diMeDBC ve srovnání s kontrolou. Sarkomagen N-MeDBC vyvolával v buňkách WB-F344 sice nízkou hladinu stabilních DNA aduktů, ale tato hladina byla pravděpodobně dostatečná pro vznik zlomů na DNA a mikrojader. Tato poškození byla nicméně účinně opravena a neprojevila se na buněčné úrovni, jak ukázala předchozí studie Vondráček, 2006. Pátá studie Andrysík et al., rukopis (kap. 4.4., příloha V) se zabývala genotoxickými a tumor-promočními účinky reálné environmentální směsi SRM 1649a - vzorku městského ovzduší (prachu) v buněčném modelu WB-F344. Vedle účinků hrubého extraktu SRM byly zkoumány také účinky jeho frakcí za účelem identifikace skupiny látek, které se na daných efektech podílejí. Po působení hrubého extraktu SRM vyvolával v buňkách WB-F344 tvorbu aduktů významně pouze BaP-dihydrodiolepoxid, dalšími identifikovanými byly adukty DBalPdihydrodiolepoxidu, které byly vytvořeny ale ve velmi malém množství. Adukty dalších PAU se nevytvářely ve významném množství a nebyly ani identifikovány. Další genotoxické účinky (apoptóza, aktivace p53 a snížení počtu buněk) byly pozorovány pouze ve vyšších koncentracích po působení hrubého extraktu a polární frakce F3. Tyto výsledky potvrzují již dříve pozorovaný jev, že genotoxicita PAU ve směsích není aditivní, ale její výsledný efekt je slabší. Např. také ve studii na lidských embryonálních plicních fibroblastech ovlivněných 61

Diskuze a závěry směsí osmi PAU byl jako dominantní identifikován adukt BaP-dihydrodiolepoxidu (Binková, 2004). Tento jev je pravděpodobně způsoben schopností některých PAU (např. ve směsích hojně zastoupeného fluoranthenu) inhibovat cytochromy P450 (Shimada, 2006), jak bylo ukázáno ve studii Topinka, 2008 (kap. 4.1., příloha I). Genotoxické efekty byly nalezeny jen při vysokých dávkách vzorku, genotoxicita není tedy hlavním mechanismem toxicity této komplexní směsi. Aktivace receptoru AhR detekovaná jako tzv. dioxinová aktivita (data neprezentována v této doktorské práci, viz rukopis v příloze V), AhR-dependentní proliferace, s ní spojená akumulace buněk v S-fázi, aktivace CYP1A1, CYP1B1 a cyklinu A (data neprezentována v této doktorské práci, viz rukopis v příloze V) byly pozorovány po působení hrubého extraktu, frakce F1 obsahující PAU a F3 obsahující oxidované PAU už od nízkých koncentrací. Semipolární frakce F2 obsahující především nitrované PAU nevyvolávala žádný ze studovaných efektů. Prezentované výsledky dále naznačují, že významným mechanismem působení environmentálních směsí jsou tumor-promoční efekty, především aktivace AhR, projevující se na rozdíl od genotoxicity v nižších koncentracích.

62

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Přehled publikací a odborných příspěvků

7.

Souhrnný přehled publikací a odborných příspěvků

Publikace v časopisech: Topinka, J., Marvanová, S., Vondráček, J., Sevastyanova, O., Nováková, Z., Krčmář, P., Pěnčíková, K., and Machala, M. (2008). DNA adducts formation and induction of apoptosis in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aromatic hydrocarbons. Mutation Research 638, p. 122-132. Impact Factor = 4.111 Marvanová, S., Vondráček, J., Pěnčíková, K., Trilecová, L., Krčmář, P., Topinka, J., Nováková, Z., Milcová, A. and Machala, M. (2008). Toxic effects of methylated benz[a]anthracenes in liver cells. Chemical Research in Toxicology 21, p. 503-512. Impact Factor = 3.508 Švihálková-Šindlerová, L., Machala, M., Pěnčíková, K., Marvanová, S., Neča, J., Topinka, J., Sevastyanova, O., Kozubík, A. and Vondráček, J. (2007). Dibenzanthracenes and benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver 'stem-like' cells. Toxicology 232, p. 147-159. Impact Factor = 2.919 Vondráček, J., Švihálková-Šindlerová, L., Pěnčíková, K., Marvanová, S., Krčmář, P., Ciganek, M., Neča, J., Trosko, J.E., Upham, B., Kozubík, A. and M. Machala. (2007). Concentrations of methylated naphtalenes, anthracenes, and phenanthrenes occuring in Czech river sediments and their effects on toxic events associated with carcinogenesis in rat liver cell lines. Environmental Toxicology and Chemistry 26, p. 2308-2316. Impact Factor = 2.202 Vondráček, J., Švihálková-Šindlerová, L., Pěnčíková, K., Krčmář, P., Andrysík, Z., Chramostová, K., Marvanová, S., Valovičová, Z., Kozubík, A., Gábelová, A. and Machala, M. (2006). 7H-dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stemlike’ cells. Mutation Research 596, p. 43-56. Impact Factor = 3.340 Valovičová, Z., Marvanová, S., Mészárosová, M., Srančíková, A., Trilecová, L., Milcová, A., Líbalová, H., Vondráček, J., Machala, M., Topinka, J. and Gábelová, A. (2009). Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells. Mutation Research, in press. Impact Factor = 4.159

Vybrané odborné příspěvky:

Marvanová, S., Hrubá, E., Krčmář, P., Nováková, Z., Milcová, A., Topinka, J., Souček, K., Vondráček, J. and Machala, M. Effects of environmental carcinogen benzo[a]pyrene in human androgen-dependent prostate adenocarcinoma cell line LNCaP. In Autumn Meeting – Genetic Toxicology and Cancer Prevention, Bratislava, Slovakia, 22.24.10.2007, p. 9-10. Přednáška. 73

Přehled publikací a odborných příspěvků Marvanová, S., Topinka, J., Sevastyanova, O., Vondráček, J., Nováková, Z., Milcová, A., Krčmář, P., Pěnčíková, K. and Machala, M. Genotoxic and non-genotoxic effects of carcinogenic PAHs in rat liver epithelial cells WB-F344. In Autumn Meeting – Genetic Toxicology and Cancer Prevention, Bratislava, Slovakia, 22.-24.10.2007, p. 45. Poster. Marvanová, S., Vondráček, J., Pěnčíková, K., Topinka, J. and Machala, M. Genotoxické procesy a metody detekce v modelech in vitro. In 30. pracovní dny České a slovenské společnosti pro mutagenezu zevním prostředím a Československé biologické společnosti: Aktuální problematika genetické toxikologie, Brno, 9.-11.5.2007, p. 17. ISBN 978-80-7013-454-2. Přednáška. Marvanová, S., Pěnčíková, K., Vondráček, J., Krčmář, P., Topinka, J., Neča, J. and Machala, M. Genotoxické a negenotoxické efekty methylovaných benz[a]anthracenů v potkaních jaterních epiteliálních buňkách WB-F344. In Jesenné pracovné dni – Genetická toxikológia a prevencia rakoviny, Bratislava, Slovensko, 23.-25.10.2006, str. 14-15. Přednáška. Marvanová, S., Košťálová, L., Ciganek, M., Černá, M., Vondráček, J., Machala, M. and Hansen, L.G. Prevalent and episodic PCB congeners in environmental and human milk samples and their provisional risk assessment based on dioxin-like and non-dioxin-like toxic modes of action. In The fourth PCB workshop: Recent advances in the environmental toxicology and health effects of PCBs, Zakopane, Poland, 6-10 September 2006, p. 99. ISBN 83-923451-1-8. Poster. Marvanová, S., Pěnčíková, K., Neča, J., Ciganek, M., Machala, M., Kočan, A. and Vondráček, J. In vitro evaluation of genotoxic and nongenotoxic effects and chemical analysis of river sediment extracts and their fractions. In 36th Annual Meeting of the European Environmental Mutagen Society, Prague, Czech Republic, 2-6 July 2006, p. 186. Poster. Andrysík, Z., Marvanová, S., Krčmář, P., Vondráček, J., Mahadevan, B., Baird, W.M., Pěnčíková, K., Neča, J., Ciganek, M., Kozubík, A. and Machala, M. Standard reference material SRM1649a may contribute to carcinogenesis through both genotoxic and nongenotoxic mechanisms of action. In 36th Annual Meeting of the European Environmental Mutagen Society, Prague, Czech Republic, 2-6 July 2006, p. 185. Poster. Marvanová, S., Valovičová, Z., Rybář, R., Krčmář, P., Vondráček, J., Gábelová, A. and M. Machala. Comparison of rat liver epithelial WB-F344 cells and hepatoma cell lines as in vitro models for PAH genotoxicity studies. In SETAC Europe 15th Annual Meeting, Lille, France, 22-26 May 2005, p. 276-277. Poster. Marvanová, S., Valovičová, Z., Krčmář, P., Švihálková-Šindlerová, L., Andrysík, Z., Vondráček, J., Gábelová, A. a M. Machala. Srovnání jaterních epiteliálních buněk potkana WB-F344 a hepatoma buněčných linií jako in vitro modelů pro studium genotoxicity PAHs. In XXIII. Xenobiochemické symposium, Valtice, 16.-19.5.2005, str. 54. Poster. Marvanová, S., Pěnčíková, K., Švihálková-Šindlerová, L., Chramostová, K., Valovičová, Z., Andrysík, Z., Plíšková, M., Suchý, J., Gábelová, A., Vondráček, J. a M. Machala. Genotoxické efekty a aktivace receptoru AhR vybranými skupinami polyaromatických uhlovodíků v jaterních epiteliálních buňkách. In Jesenné pracovné dni – Genetická toxikológia a prevencia rakoviny, Bratislava, Slovensko, 18.-20.10.2004, str. 6-7. ISBN 80-969136-9-7. Přednáška. 74

Příloha I

Topinka, J., Marvanová, S., Vondráček, J., Sevastyanova, O., Nováková, Z., Krčmář, P., Pěnčíková, K., and Machala, M. (2008). DNA adducts formation and induction of apoptosis in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aromatic hydrocarbons. Mutation Research 638, 122-132.

Available online at www.sciencedirect.com

Mutation Research 638 (2008) 122–132

DNA adducts formation and induction of apoptosis in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aromatic hydrocarbons Jan Topinka a , Soˇna Marvanová b , Jan Vondrácˇ ek b,c , Oksana Sevastyanova a , Zuzana Nováková a , Pavel Krˇcmáˇr b , Kateˇrina Pˇenˇc´ıková b , Miroslav Machala b,∗ a

Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, 142 20 Prague, Czech Republic b Veterinary Research Institute, 621 00 Brno, Czech Republic c Laboratory of Cytokinetics, Institute of Biophysics, AS CR, 612 65 Brno, Czech Republic Received 17 July 2007; received in revised form 6 September 2007; accepted 11 September 2007 Available online 16 September 2007

Abstract The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[a,l]pyrene, and partly also benzo[g]chrysene and benz[a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[a,l]pyrene, and to a lesser extent also benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[a]anthracene, chrysene, benzo[b]fluoranthene and dibenzo[a,h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3 ,5 -tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[a,l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[a,l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells. © 2007 Elsevier B.V. All rights reserved. Keywords: PAHs; Liver progenitor cells; Cytochromes P450; DNA adducts; p53 protein; Apoptosis

1. Introduction ∗

Corresponding author at: Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic. Tel.: +420 533331813; fax: +420 541211229. E-mail address: [email protected] (M. Machala). 0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.09.004

The liver is an organ with a remarkable regenerative capacity, which is under normal conditions provided by preexisting hepatocytes and cholangiocytes [1]. However, when the ability of hepatocytes to divide and

J. Topinka et al. / Mutation Research 638 (2008) 122–132

replace damaged tissue is compromised, a population of bipotent progenitor cells is activated, and it may give rise to both hepatocytes and biliary epithelial cells [2,3]. The function and nomenclature of stem cells in liver is still an area of dispute, nevertheless it is now generally agreed that liver oval cells are ‘stem-like’ cells descending from a population of progenitor cells involved in liver regeneration [2,3]. Importantly, hepatocellular carcinoma may arise from cells at various stages of differentiation, including oval cells [4,5]. Therefore, liver stem-like cells might play a significant role in hepatocarcinogenesis [4]. Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental carcinogens; nevertheless, the liver is not usually considered to be a primary target tissue of mutagenic PAHs. However, it has been shown that PAH exposure might enhance the hepatocarcinogenicity of chronic hepatitis B virus infection, suggesting that PAHs might play a role in etiology of hepatocellular carcinoma [6]. Until recently, almost all in vitro studies on mutagenicity of PAHs in liver cells, have focused on hepatocytes or hepatoma cells as model systems, despite the fact that their effects on immature liver cells might significantly differ from toxic events occurring in mature hepatocytes [7]. The rat liver epithelial stem-like WB-F344 cell line, isolated from the liver of an adult male Fischer 344 rat, is considered to be an in vitro model of bipotent oval cells as it shares their phenotype [8,9]. Our recent studies have shown that PAHs or heterocyclic aromatic compounds induce multifaceted responses in these cells, including inhibition of gap-junctional intercellular communication (GJIC), stimulation of cell proliferation or induction of programmed cell death [10–13]. This makes them a valuable model for studying the cellular processes involved in tumor promotion. It has been proposed that aryl hydrocarbon receptor (AhR) plays a dual role in both genotoxic and nongenotoxic events in rat liver epithelial WB-F344 cells, including the transcription control of gene expression of PAH-metabolizing cytochrome P450 (CYP) enzymes and AhR-dependent release from growth contact inhibition of the cells [10]. The WB-F344 cells have been previously shown to contain inducible cytochrome P4501A1 (CYP1A1), one of the key PAH-bioactivating enzymes, as well as CYP1-dependent 7-ethoxyresorufin O-deethylase activity [10,12]. This suggested that this model liver oval cell line is capable to activate PAHs and to form ultimate genotoxic metabolites. However, formation of DNA adducts as a principle genotoxic event observed after carcinogenic PAH exposure has not been investigated yet. The adverse effects of PAHs have been intensively studied during past decades in a number of in vivo

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and in vitro systems [14,15]. Their genotoxic, tumorpromoting and carcinogenic effects have been recognized to be largely structure-, cell- and tissue-specific [14]. For the present study, we selected chrysene (Chry), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), benzo[g]chrysene (BgChry), dibenzo[a,h]anthracene (DBahA), dibenzo[a,l]pyrene (DBalP), and dibenzo[a,e]pyrene (DBaeP), as model carcinogenic PAHs with diverse molecular weight, chemical structure and genotoxic/tumor-promoting potencies [11,12,16–20]. In our previous studies [12,21], some potent mutagens, including DBalP and BaP, have been found to induce increased levels and phosphorylation of p53 tumor suppressor protein, accumulation of cells in S-phase of cell cycle and/or apoptosis, which are all markers of PAH-induced genotoxic insult [21–24]. In contrast, other PAHs, which are also considered to be potent carcinogens, such as BaA and BbF, failed to activate p53 or to induce a significant level of cell death in WB-F344 cells [12,21]. This suggested significant differences in the ability of the selected compounds to elicit genotoxic damage. Therefore, in the present study, we investigated the DNA adduct formation in rat liver WB-F344 cells exposed to carcinogenic PAHs by 32 P-postlabeling technique. The amount of adducts was compared with the potency of individual compounds to induce CYP1A1 and CYP1B1 expression, phosphorylation of tumor suppressor protein p53 in response to substantial genotoxic damage, and induction of programmed cell death. Our results provide evidence that PAH exposure may lead to significant DNA adduct formation in a model of liver epithelial ‘stem-like’ cells, which is associated with further cellular events, including induction of apoptosis. Our data also suggest that genotoxicity of PAHs in this cellular model is primarily associated with CYP1B1 activity. 2. Materials and methods 2.1. Chemicals and biochemicals Spleen phosphodiesterase was purchased from ICN Biomedicals (Irvine, CA); ribonuclease A and T1, proteinase K, micrococcal nuclease, nuclease P1, and protein assay kit (No. 5656) were from Sigma–Aldrich (Prague, Czech Republic); polyethylene-imine cellulose TLC plates (0.1 mm) were from Macherey-Nagel (Düren, Germany); dibenzo[a,l]pyrene (CAS No. 191-30-0, purity 99.8%) was obtained from Midwest Research Institute (Kansas City, MO); benz[a]anthracene (CAS No. 86-73-7, purity 99.9%), chrysene (CAS No. 218-01-9, purity 99.9%), benzo[b]fluoranthene (CAS No. 205-99-2, purity 99.9%), benzo[a]pyrene (CAS No.

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50-32-8, purity 99.9%), dibenz[a,h]anthracene (CAS No. 5370-3, purity 99.9%), dibenzo[a,e]pyrene (CAS No. 192-65-4, purity 99.8%) and fluoranthene (CAS No. 206-44-0, purity 99.9%) were from Supelco (Bellefonte, PA); benzo[g]chrysene (CAS No. 196-78-1, purity 99.9%) was supplied by Chiron (Trondheim, Norway); benzo[c]phenanthrene (CAS No. 19519-7, purity 99.9%) was from Promochem GmbH (Wesel, Germany); 2,4,3 ,5 -tetramethoxystilbene was from Merck Chemicals Limited (Nottingham, Great Britain); T4 polynucleotide kinase was from USB (Cleveland, OH); and ␥-32 P-ATP (3000 Ci/mmol, 10 ␮Ci/␮l) from GE Healthcare (Little Chalfont, UK). All other chemicals and solvents were of HPLC or analytical grade.

of distinct adduct spots was measured by liquid scintillation counting. To determine the exact amount of DNA in each sample, aliquots of DNA enzymatic digest (1 ␮g of DNA hydrolysate) were analyzed for nucleotide content by reversephase HPLC with UV detection, which simultaneously allowed to check the purity of DNA. DNA adduct levels were expressed as adducts per 108 nucleotides. A BPDE-DNA adduct standard was run in triplicate in each postlabeling experiment to control for interassay variability and to normalize the calculated DNA adduct levels.

2.2. Cell culture

WB-F344 cells in confluent state (seeded at an initial density 25,000 cells/cm2 , grown for 72 h) were exposed for 24 h to test compounds and DMSO as the solvent control. Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel). The amplifications of the samples were carried out using QuantiTect Probe RT-PCR kit according to manufacturer’s specifications. The sequences of primers and probes have been published previously [13]. The primers were designed on the exon junction for amplification of cDNA only. The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the following program: reverse transcription at 50 ◦ C for 20 min and initial activation step at 95 ◦ C for 15 min, followed by 35 cycles at 95 ◦ C for 0 s and 60 ◦ C for 60 s. All PCR reactions were performed in triplicates and changes in gene expression were calculated using the comparative threshold cycle method [27].

WB-F344 rat liver epithelial cells [9], kindly provided by Dr. J.E. Trosko (Michigan State University, East Lansing, MI), were cultured in Dulbecco’s modified Eagle’s Medium (Invitrogene, Carlsbad, CA), supplemented with 25 mM bicarbonate and sodium pyruvate (110 mg/l), 10 mM HEPES, and 5% heat-inactivated fetal bovine serum (PAA Laboratories, Linz, Austria). Only the cells at passage levels 15–25 were used throughout the study. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 ◦ C. The cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. 2.3. Cell treatment and DNA isolation WB-F344 cells (seeded at an initial density 20,000 cells/cm2 in 60-cm2 plates, grown for 48 h) were exposed for 24 h to test compounds and DMSO as a solvent control (0.1%). After exposure, cells were washed with cold PBS, scraped into the Eppendorf tubes, centrifuged and the cell pellets were stored at −80 ◦ C. The cell pellets were homogenized in a solution of 10 mM Tris–HCl, 100 mM EDTA, and 0.5% SDS, pH 8.0. DNA was isolated using RNAses A and T1 and proteinase K treatment followed by phenol/chloroform/isoamylalcohol as previously described [22]. DNA concentration was estimated spectrophotometrically by measuring the UV absorbance at 260 nm. DNA samples were kept at −80 ◦ C until analysis. 2.4. DNA adduct analysis 32

P-postlabeling analysis was performed as previously described [25,26]. Briefly, DNA samples (6 ␮g) were digested by a mixture of micrococcal endonuclease and spleen phosphodiesterase for 4 h at 37 ◦ C. Nuclease P1 was used for adduct enrichment. The labeled DNA adducts were resolved by two-directional thin layer chromatography on 10 cm × 10 cm PEI-cellulose plates. Solvent systems used for TLC were the following: D-1: 1 M sodium phosphate, pH 6.8; D-2: 3.8 M lithium formate, 8.5 M urea, pH 3.5; D-3: 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0. Autoradiography was carried out at −80 ◦ C for 6, 24 and 92 h. The radioactivity

2.5. Real time-PCR detection of CYP1A1 and CYP1B1 mRNA after 24 h exposure

2.6. Western blot analysis of phosphorylated p53 protein In order to determine effects of PAHs on Ser-15 phosphorylation of p53 protein, confluent WB-F344 cells (seeded at an initial density 25,000 cells/cm2 , grown for 72 h) were exposed for 24 h to the test compounds or to 0.1% DMSO as vehicle control. Whole cell lysates were prepared by harvesting cells in lysis buffer (1% SDS, 100 mM Tris, 10% glycerol, protease inhibitor cocktail) and sonication. Equal amounts of total protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membrane Hybond-P (GE Healthcare). The blotted membranes were blocked overnight at 4 ◦ C and incubated with primary antibody against p53 phosphorylated on Ser-15 (Cell Signaling Technology, Beverly, MA) for 2 h at room temperature. Peroxidase-conjugated swine anti-rabbit immunoglobulin antiserum (Sevapharma, Prague, Czech Republic) was used as a secondary antibody. For detection of total p53, membranes were blocked for 1 h at room temperature, incubated with primary antibody p53 (R-19) (Santa Cruz Biotechnology, CA) overnight at 4 ◦ C, and peroxidase-conjugated rabbit antigoat immunoglobulin antiserum (Sigma–Aldrich) was used as a secondary antibody. Expression of ␤-actin was used to verify equal loading, using monoclonal anti-␤-actin antibody, clone AC-15 (Sigma–Aldrich), as a primary antibody and


peroxidase-conjugated anti-mouse antibody (Sigma–Aldrich) as a secondary antibody. To visualize peroxidase activity, ECL Plus reagent (GE Healthcare) was used according to manufacturer’s instructions.

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of CYP enzymes for 30 min and then 100 nM DBalP was added only for 24 h. The apoptosis/necrosis was measured as described above. 2.8. Statistical analysis

2.7. Detection of apoptosis by flow cytometry WB-F344 cells (seeded at an initial density 25,000 cells/cm2 , grown for 72 h) were exposed to the test compounds for 48 h (medium was changed after 24 h exposure). Early stages of apoptosis are characterized by a translocation of phosphatidylserine from the inner part of the plasma membrane to the outer layer. The presence of phosphatidylserine at the cell surface was determined by staining with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) in combination with propidium iodide (40 ␮g/ml), in order to distinguish the cells with permeabilized and intact plasma membrane. Cells were harvested and stained according the manufacturer’s protocol and analyzed by FACSCalibur with CellQuest software (Becton Dickinson) as described previously [21]. In the experiments with compounds inhibiting the metabolizing enzymes, the confluent WB-F344 cells were first exposed to the inhibitors

All experiments were performed independently at least three times and the data were quantitatively expressed as means ± S.D. The apoptosis data were analyzed by Student’s t-test and p < 0.05 was considered significant.

3. Results 3.1. PAH–DNA adduct formation The DNA adduct formation was studied in WBF344 cells after 24-h exposure to selected carcinogenic PAHs. This cell model has been widely used to study tumor-promoting effects of xenobiotics [11,12,28,29]; however, very little is known about the induction of genotoxic events in these cells. DBalP was found to be the

Fig. 1. Autoradiograms of 32 P-labeled DNA isolated from rat liver epithelial WB-F344 cells incubated with 0.1 ␮M DBalP (A); 10 ␮M DBaeP (B); 1 ␮M BgChry (C); 1 ␮M DBahA (D); 1 ␮M BaP (E); 1 ␮M BbF (F); 10 ␮M Chry (G); 10 ␮M BaA (H); or vehicle (0.1% DMSO) (I). Duration of screen enhanced autoradiography was 6 h (A, E), 24 h (C, D, F, I), 72 h (B) and 92 h (G, H) at −80 ◦ C.

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Fig. 2. Effects of PAHs on total and Ser-15-phosphorylated p53 protein levels in WB-F344 cells. Cells were treated for 24 h with 10 ␮M concentration of PAHs, with exception of DBalP (100 nM). Cell lysates were prepared and Western blotting was performed as described in Section 2. The results shown here are representative of three independent experiments. Table 1 DNA adduct levels induced after 24-h exposure of WB-F344 cells to selected PAH compounds Compound concentration DBalPa BaP BgChry DBaeP DBahA BbF BaA Chry

Total DNA adducts (RAL × 108 ) 0.1 ␮M

1 ␮M

10 ␮M

297 ± 90 6.7 ± 0.2 8.1 ± 0.3 0.5 ± 0.1 0.8 ± 0.1 0.9 ± 0.2 n.a. n.a.

n.a.b

n.a. 43.8 ± 7.9 11.4 ± 3.4 6.9 ± 2.3 0.8 ± 0.1 0.7 ± 0.2 1.2 ± 0.4 0.7 ± 0.1

68.0 ± 6.5 26.0 ± 2.1 1.2 ± 0.3 1.2 ± 0.1 1.1 ± 0 n.a. n.a.

Total DNA adduct levels are expressed as a number of adducts per 108 nucleotides. Data from at least three independent incubations are expressed as means ± S.D. a Dibenzo[a,l]pyrene induced DNA adducts at concentrations of 1 nM—10.2 adducts/108 nucleotides and 10 nM—54.5 adducts/108 nucleotides. b n.a.: not analyzed.

most potent inducer of DNA adducts formation, which was effective already at 1 nM concentration. Both BaP and BgChry induced high levels of DNA adducts from 100 nM concentration, with 10 ␮M being already less effective than 1 ␮M, most probably due to their cytotoxicity (Table 1). DBaeP was also a potent inducer of DNA adducts, however only at 10 ␮M concentrations. In contrast, both BbF and DBahA formed only low amounts of DNA adducts within the 100 nM–10 ␮M concentration range. BaA and Chry induced only low DNA adduct levels at 10 ␮M concentration (Table 1). Representative autoradiograms of PAH–DNA adducts for all test compounds are shown in Fig. 1.

3.2. Accumulation of Ser-15-phosphorylated p53 protein The p53 tumor suppressor protein is a potent transcription factor accumulating in cells in response to signals arising from PAHs [23,30]. It can be posttranslationally modified at various amino acid residues and a number of studies have shown that phosphorylation of p53 protein at Ser-15 might play a significant role in the stabilization, up-regulation, and functional activation of p53 during genotoxic stress [31]. Therefore, we examined phosphorylation of p53 at Ser-15 after 24-h exposure to PAHs at 10 ␮M concentration; only DBalP was used at 100 nM concentration. Both DBalP and BgChry were powerful inducers of Ser-15 phosphorylation of p53 (Fig. 2). As further outlined in Fig. 2, these compounds also induced a significant stabilization and accumulation of total p53 protein. Other tested PAHs induced either low levels of Ser-15 phosphorylation (BaP, DBaeP) or had no effect on levels of p53 phosphorylation at this amino acid residue (Fig. 2). 3.3. Induction of apoptosis in WB-F344 cells treated with PAHs The apoptosis of WB-F344 cells was determined by flow cytometric method using the Annexin-V staining of phosphatidylserine combined with propidium iodide staining of late apoptotic/necrotic cells. As outlined in Fig. 3, the three most potent genotoxins, DBalP, BgChry and BaP, which also increased phosphorylation of p53, induced high levels of apoptosis (maximum over 50%) in treated cells in a concentration-dependent manner.

Fig. 3. Representative diagrams of Annexin-V/propidium iodide (PI) staining of cells after 48 h exposure to carcinogenic PAHs. Cells were stained as described in Section 2 and analyzed on FACSCalibur flow cytometer. Region R1 represents early apoptotic cells (Annexin-V positive, PI negative), region R2 viable cells (both Annexin-V and PI negative) and region R3 late apoptotic/necrotic cells (both Annexin-V and PI positive). Data represent means from a minimum of three independent experiments ± S.D. The data were statistically analyzed by Student’s t-test. The means are significantly different from control group at *p < 0.05 or **p < 0.01.


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Fig. 5. Inhibition of DBalP-induced apoptosis by 2,4,3 ,5 tetramethoxystilbene (TMS), benzo[c]phenanthrene (BcPhe) and fluoranthene (Fla). Cells were pre-treated with indicated concentrations of test compounds for 30 min, followed by 24-h exposure to DBalP. Cells were stained as described in Section 2 and analyzed on FACSCalibur flow cytometer. The results are expressed as means ± S.D. of at least three independent experiments. The data were analyzed by Student’s t-test. The means are significantly different from positive control (DBalP) at *p < 0.05 and **p < 0.01.

Fig. 4. Induction of CYP1A1 and CYP1B1 mRNA following 24-h treatment with the studied 1 ␮M PAHs, as compared to a prototypical AhR ligand, TCDD. Total RNA was isolated and quantitative realtime RT-PCR was performed as described in Section 2. The results are expressed as mean ± S.D. of three independent experiments. Inducibility was compared with maximal induction obtained by treatment with 1 nM TCDD.

The percentage of apoptotic cells corresponded with increased accumulation of DNA adducts and induction of p53 phosphorylation. Lower, but significant increase in apoptosis was found also in cells treated with DBaeP in the range from 0.1 to 10 ␮M. On the other hand, only a marginal increase of percentage of apoptotic cells were found in samples treated with DBahA, BbF, and Chry, and exposure to BaA was without effect. High doses of almost all tested PAHs (with exception of DBalP, DBaeP and BaA) also induced a limited necrosis, which might be related both to their cytotoxicity and progression of apoptosis into its later stages. 3.4. Induction of key enzymes of metabolic activation of PAHs The induction of CYP1A1 and CYP1B1 mRNA was determined in WB-F344 cells treated with PAHs under this study by RT-PCR (Fig. 4). With exception of DBalP, BgChry and BaA, all other PAHs induced maximum levels of CYP1A1 comparable to model AhR ligand TCDD. DBalP was only a very weak inducer of both

enzymes at 100 nM concentration which was the maximum concentration used for induction of DNA adduct formation. In contrast, both BgChry and BaA were potent inducers of CYP1B1 mRNA expression, comparable to other tested compounds. These results showed that induction of expression of major CYP1 enzymes, which are involved in metabolic activation of PAHs does not present a limiting factor of PAH genotoxicity. Importantly, even partial induction after DBalP treatment was sufficient to enable formation of high levels of DNA adducts. Interestingly, several low-molecular-weight PAHs, which are present at high levels in complex environmental mixtures, such as fluoranthene, have been recently reported to inhibit CYP1A1 and/or CYP1B1 enzymes [32]. It has been also suggested that DBalP is metabolized to active genotoxic metabolites principally through CYP1B1 activity [33]. Therefore, we investigated whether inhibition of CYP1B1 by its specific inhibitors 2,4,3 ,5 -tetramethoxystilbene (TMS) [34] or fluoranthene may prevent apoptosis induced by DBalP, the most potent genotoxin identified in WB-F344 cells. As outlined in Fig. 5, both TMS and fluoranthene inhibited, in a dose-dependent manner, induction of apoptosis by DBalP. Fluoranthene was able to fully inhibit DBalPinduced apoptosis at 10 ␮M concentration. In contrast, benzo[c]phenanthrene, which has been reported to primarily inhibit CYP1A1 [32], was able to only partly inhibit apoptosis induced by DBalP. The inhibitors we used may have partly overlapping specificities, such as


inhibiting also CYP1A2 (fluoranthene); however, we have previously found that both basal and induced levels of CYP1A2 in WB-F344 cells are very low as compared to other liver cell lines, suggesting that its impact on genotoxicity of DBalP would be negligible. Our data thus confirmed the prominent role of CYP1B1 in metabolic activation of DBalP in WB-F344 cells and suggested that WB-F344 cells are a suitable model to study effects of PAHs on CYP level and genotoxic endpoints. 4. Discussion Chemical carcinogens may affect both the pluripotent stem cells and the early progenitor or transit amplifying cells as their targets [35]. However, most of the studies on formation and accumulation of DNA adducts of environmental mutagens, such as PAHs, have so far concentrated on differentiated cells and/or their derivatives. Nevertheless, the ultimate genotoxic and cytotoxic metabolites might preferentially accumulate in rapidly dividing stem cells in target organs, such as epidermal stem cells located in the hair follicles [36]. The alterations in DNA repair functions during differentiation may also limit efficient removal of DNA adducts in proliferating progenitor cells, making them a sensitive target of genotoxic compounds [37]. In liver, the progenitor oval cells represent cell population which extensively proliferates in response to diverse chemical carcinogens [38]. These cells are able to develop into both hepatocytes and biliary epithelial cells, and they have been suggested to play a significant role in hepatocarcinogenesis [4]. Currently, there is only limited information about genotoxic events in these cells, although they might be, e.g. a target of PAHs contributing to an increased risk of hepatocellular carcinoma [6]. The formation and accumulation of PAH dihydrodiol epoxides might significantly affect both survival and proliferation of liver immature cells. Our previous findings suggested that model genotoxic PAHs may activate p53 protein and induce apoptosis in WB-F344 cells, a model rat liver cell line sharing phenotypic properties with oval cells [10,12,21]. Because these events might be linked to the formation of stable PAH–DNA adducts, a principle form of PAH-induced genotoxic insult, we investigated the formation of DNA adducts of environmental carcinogenic PAHs and related effects in WB-F344 cells. In the present study, we detected high levels of DNA adducts in rat liver epithelial ‘stem-like’ cells treated with DBalP, BaP, BgChry and DBaeP; their respective genotoxic potencies were DBalP BaP > BgChry > DBaeP (Table 1). These results are in accordance with DBalP being the most tumorigenic and mutagenic PAH

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identified to date [39]. Metabolic activation of DBalP, BaP and BgChry leading to an extensive formation of DNA adducts have been reported from several in vitro and in vivo model systems, including primary mouse and human cells [22,33,40]. As counterparts of the highly mutagenic PAHs, we selected four additional carcinogenic PAHs, Chry, BaA, BbF, and DBahA for the present study [41]. Previously, DBaeP, and partly also BbF and DBahA, have been shown to elicit relatively high mutagenic potencies in metabolically competent human lymphoblastoma cells [16,17]; however, both BbF and DBahA formed only low levels of DNA adducts in human diploid lung fibroblasts [30], HepG2 cells [42] or in precision-cut rat liver slices [43]. Chry and BaA are only moderately mutagenic in both bacterial and mammalian assays [16,19,30]. In the present study, only low but discernible levels of DNA adducts were observed after treatment with DBahA, BbF, BaA and Chry. These results are in accordance with our previous study, in which both DBalP and BaP, but not Chry, BaA, DBahA or BbF, induced apoptosis in confluent WB-F344 cells in post-confluent proliferation assay [12]. The formation of genotoxic metabolites is considered to represent a critical event in the PAH-induced tumor initiation [44]. The formation of DNA adducts by PAHs is known to increase p53 tumor suppressor levels as well as to activate further events associated with induction of DNA repair, cell cycle arrest and/or apoptosis [22,24,45]. The activation and stabilization of p53 in cells treated with PAHs is reflected by p53 phosphorylation at Ser-15, which is associated with induction of programmed cell death [21,46–48]. In the present study, the percentage of apoptotic cells corresponded with formation of DNA adducts, i.e. all four compounds inducing high levels of DNA adducts (DBalP, BgChry, BaP and DBaeP) were potent inducers of apoptosis (Fig. 3). Similar to that, only these four PAHs were able to induce increased p53 phosphorylation (Fig. 2). We did not observed any increase of p53 protein phosphorylation in WB-F344 cells incubated with DBahA, BbF, Chry and BaA, which formed only low DNA adduct levels (Fig. 2). On the other hand, with exception of BaA, these compounds induce a slight increase of apoptosis, which might be related either to their cytotoxicity or to low but observable DNA adducts formation. High sensitivity of progenitor cells to apoptosis induced by DNA damage has been previously suggested to be a mechanism, which eliminates fast-proliferating progenitor cells, once damaged, from organism [37]. These results suggest that nongenotoxic events elicited by these compounds, such as activation of AhR, disruption of cell-to-cell communication or cell proliferation might play a more prominent

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role in their carcinogenic effects in liver progenitor cells [10–12,14]. On the other hand, induction of cell death by genotoxic compounds might also contribute to hepatocarcinogenesis, as it has been previously reported that the severe hepatocellular toxicity observed in livers from mice treated with genotoxins is followed by the liver regeneration [49,50]. Thus the extensive apoptosis induced by genotoxic compounds in liver cells can lead to regenerative cell proliferation, which may include progenitor cells, which in turn might contribute to tumor promotion [51,52]. The genotoxic damage to liver cells might also lead to necrosis associated with a release of further mediators, such as inflammatory cytokines, which are known to significantly contribute to proliferation of specific liver cell populations, including oval cells [2,53]. In this context, it is perhaps interesting that we have recently observed that ligands of AhR (which include many PAHs) and cytokines might synergistically enhance cell proliferation of liver epithelial cell in vitro [54]. Finally the apoptosis might remove only those cells with a very high extent of DNA damage; other cells with modified DNA might survive to harbor mutations and to further expand during tumor promotion. Many liver diseases are also associated with a chronic production of inflammatory cytokines and growth factors, which may inhibit apoptosis. Hepatocellular carcinoma is related closely to chronic hepatitis and cirrhosis, implying that alterations in growth control mechanisms during regeneration may be involved in hepatic carcinogenesis [55]. Under such conditions, initiated cells could overcome anti-carcinogenic mechanisms, such as removal of DNA-damaged cells through apoptosis [56]. Therefore, it seems important to interpret the present in vitro findings also in the in vivo context of liver cancer development, where various liver disease states play a prominent role. Given that the level of binding of reactive PAH metabolites to DNA correlates with relative carcinogenic potency of PAHs [44], the fact that some potent carcinogens among PAHs, such as DBahA, produced only a very limited amount of DNA adducts in WB-F344 cells deserves further attention. A variety of reasons might be responsible for this, including poor induction of metabolizing enzymes or reported inhibition of CYP1B1 activity by some PAHs [32]. Indeed, the metabolic activation of PAHs by CYP1 enzymes is a necessary prerequisite for the production of reactive PAH metabolites and DNA adducts (reviewed in [57]). Nevertheless, we found that all compounds under study, with exception of DBalP, were efficient inducers of CYP1A1/CYP1B1 expression in WB-F344 cells (Fig. 4). Therefore, it could be concluded that CYP1A1/1B1 induction is not

a rate-limiting factor for genotoxicity of PAHs with low potency to form DNA adducts. We found that CYP1B1, which is a critical enzyme for DBalP genotoxicity [33], is constitutively expressed in WB-F344 cells [58,59]. Its levels seem to be therefore sufficient for the formation of the ultimate genotoxic metabolites of DBalP, which itself is a poor AhR ligand [18]. CYP1B1 was also highly induced by all other carcinogenic PAHs under study, suggesting its possible important role in metabolism of carcinogenic PAHs. Therefore, we investigated effects of CYP1B1 inhibitors fluoranthene and TMS [32,34] on apoptosis induced by DBalP. Both inhibitors efficiently prevented the DBalP-induced apoptosis, thus confirming the key role this enzyme plays in genotoxic/cytotoxic processes induced by PAHs in WB-F344 cells. In conclusion, the present study shows, for the first time, a significant DNA adduct formation induced by a series of carcinogenic PAHs in a model of liver immature epithelial cells, which are besides mature hepatocytes another possible target of chemical hepatocarcinogens. Unlike in mature hepatocytes, CYP1B1 could be the most important P450 isoenzyme contributing to metabolic activation of PAHs in this model of liver progenitor cells. Formation of covalent DNA adducts might be an important mode of action of some PAHs, such as DBalP, BgChry, BaP and DBaeP, in this subpopulation of liver cells. On the other hand, the exact role of low levels of DNA adducts formed by BbF, DBahA, Chry and BaA in liver progenitor cells remains to be elucidated, as the previously reported nongenotoxic effects of other PAHs under study often appear already at lower concentrations [10,58]. Acknowledgements This study was supported by the Czech Ministry of Environment (Grant No. VaV SL/5/160/05) and the Czech Science Foundation (Grant No. 524/06/0517). The institutional support was provided by the Academy of Sciences of the Czech Republic (Research Plans AV0Z50390512 and AV0Z50040702) and the Czech Ministry of Agriculture (MZE0002716201). References [1] R. Taub, Liver regeneration: from myth to mechanism, Nat. Rev. Mol. Cell Biol. 5 (2004) 836–847. [2] K.N. Lowes, E.J. Croager, J.K. Olynyk, L.J. Abraham, G.C. Yeoh, Oval cell-mediated liver regeneration: role of cytokines and growth factors, J. Gastroenterol. Hepatol. 18 (2003) 4–12. [3] M.H. Walkup, D.A. Gerber, Hepatic stem cells: in search of, Stem Cells 24 (2006) 1833–1840.

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Příloha II

Marvanová, S., Vondráček, J., Pěnčíková, K., Trilecová, L., Krčmář, P., Topinka, J., Nováková, Z., Milcová, A. and Machala, M. (2008). Toxic effects of methylated benz[a]anthracenes in liver cells. Chemical Research in Toxicology 21, 503-512.

Chem. Res. Toxicol. 2008, 21, 503–512

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Toxic Effects of Methylated Benz[a]anthracenes in Liver Cells Sonˇa Marvanová,† Jan Vondrácˇek,†,‡ Katerˇina Peˇncˇíková,† Lenka Trilecová,† Pavel Krcˇmárˇ,† Jan Topinka,§ Zuzana Nováková,§ Alena Milcová,§ and Miroslav Machala*,† Department of Chemistry and Toxicology, Veterinary Research Institute, HudcoVa 70, 62100 Brno, Department of Cytokinetics, Institute of Biophysics, AS CR, KráloVopolská 135, 61265 Brno, and Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, V.V.i., Vídenˇská 1083, 14220 Prague, Czech Republic ReceiVed August 28, 2007

Monomethylated benz[a]anthracenes (MeBaAs) are an important group of methylated derivatives of polycyclic aromatic hydrocarbons (PAHs). Although the methyl substitution reportedly affects their mutagenicity and tumor-initiating activity, little is known about the impact of methylation on the effects associated with activation of the aryl hydrocarbon receptor (AhR)-dependent gene expression and/or toxic events associated with tumor promotion. In the present study, we studied the effects of a series of MeBaAs on the above-mentioned end points in rat liver cell lines and compared them with the effects of benz[a]anthracene (BaA) and the potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Methyl substitution enhanced the AhR-mediated activity of BaA derivatives determined in a reporter gene assay, as the induction equivalency factors (IEFs) of all MeBaAs were higher than that of BaA. IEFs of 6-MeBaA and 9-MeBaA, two of the most potent MeBaAs, were more than two orders of magnitude higher than the IEF of BaA. Correspondingly, all MeBaAs induced higher levels of cytochrome P450 1A1 mRNA. Both BaA and MeBaAs had similar effects on the expression of cytochrome P450 1B1 or aldo-keto reductase 1C9 in rat liver epithelial WB-F344 cells. In contrast to genotoxic DMBA, MeBaAs induced low DNA adduct formation. Only 10-MeBaA induced apoptosis and accumulation of phosphorylated p53, which could be associated with the induction of oxidative stress, similar to DMBA. With the exception of 10-MeBaA, all MeBaAs induced cell proliferation in contact-inhibited WB-F344 cells, which corresponded with their ability to activate AhR. 1-, 2-, 8-, 10-, 11-, and 12-MeBaA inhibited gap junctional intercellular communication (GJIC) in WB-F344 cells. This mode of action, like disruption of cell proliferation control, might contribute to tumor promotion. Taken together, these data showed that the methyl substitution significantly influences those effects of MeBaAs associated with AhR activation or GJIC inhibition. Introduction 1

Monomethylated derivatives of benz[a]anthracene (MeBaAs) are an important group of methylated polyaromatic hydrocarbons (PAHs) occurring in the environment. They have been identified in various environmental compartments, such as in river sediments, where they can be found at varying concentrations of up to hundreds of nanograms per gram of sediment dry weight (1, 2). They are also significant cigarette smoke constituents (3). The parental compound of MeBaAs, benz[a]anthracene (BaA), is classified as a probable human carcinogen (4). Importantly, its dimethylated derivative 7,12-dimethylbenz[a]anthracene (DMBA) is one of the most potent mutagenic * To whom correspondence should be addressed. Tel: +420-533331813. Fax: +420-541211229. E-mail: [email protected]. † Veterinary Research Institute. ‡ Institute of Biophysics. § Institute of Experimental Medicine. 1 Abbreviations: AhR, aryl hydrocarbon receptor; AKR, aldo-keto reductase; BaA, benz[a]anthracene; BPDE, benz[a]pyrene dihydrodiol epoxide; DAPI, 4′-6-diamidine-2-phenyl indole; DBalP, dibenzo[a,l]pyrene; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; DMBA, 7,12-dimethylbenz[a]anthracene; DMSO, dimethylsulfoxide; DR-CALUX, dioxin responsive chemical-activated luciferase expression; DRE, dioxin responsive element; GJIC, gap junctional intercellular communication; IEF, induction equivalency factor; MeBaA, methylated benz[a]anthracene; PAH, polyaromatic hydrocarbon; REP, relative potency; ROS, reactive oxygen species; RT-PCR, reverse trancription polymerase chain reaction; TBS, Trisbuffered saline; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

and carcinogenic PAHs (5), which can be used as an experimental inducer of tumors in both skin and mammary tissues (6). DMBA is a highly potent experimental carcinogen, thus suggesting that methyl substitution can significantly modify the carcinogenicity of BaA. The position of the methyl group has been reported to strongly affect metabolic activation, mutagenicity, tumor-initiating ability, and carcinogenicity of MeBaAs, in both bacterial and rodent skin experimental models (7–11). In these studies, 7-, 6-, 8-, and 12-MeBaA have been identified as the most potent mutagenic and tumor-initiating derivatives of BaA. Major attention has been paid to 7-MeBaA, which shares one position of methyl substitution with DMBA. 7-MeBaA has been found to induce DNA adduct formation in adult mouse epidermis (12). Several early studies have indicated that MeBaAs are complete carcinogens in newborn or adult mice and rats (7, 9, 13). However, in contrast to tumor-initiating events, very little interest has been paid so far to the effects of methyl substitution on the tumor-promoting ability of BaA derivatives and related events. It has been hypothesized that at least some PAHs may have tumor-promoting, nongenotoxic effects, which could be induced by both parental compounds and their metabolites (14). One of the mechanisms possibly participating in tumor promotion induced by PAHs is the activation of the aryl hydrocarbon receptor (AhR) (15). A number of PAHs are AhR agonists (16–18), and this ligand-activated transcription factor regulates

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Figure 1. Molecular structure of benz[a]anthracene.

the expression of cytochromes P4501A1, 1A2, and P4501B1, metabolizing PAHs to reactive dihydrodiol epoxides, which are able to form adducts with DNA (18–20). Apart from being involved in the metabolization of PAHs to their ultimate genotoxic metabolites, AhR might also participate in other toxic effects of PAHs, such as deregulation of cell cycle control (21–24). Other mechanisms, which have been linked to tumorpromoting effects of PAHs, include the inhibition of gap junctional intercellular communication (GJIC) (25–27) and perturbation of cell signaling (28–30). Our previous studies have suggested that the ultimate impact of PAHs on cell fate may reflect both genotoxic and nongenotoxic events, leading to cytotoxicity and/or induction of apoptosis on one side and to an increased cell proliferation and/or disruption of cell-to-cell communication on the other side (21, 31). The BaA methylation might thus differentially affect this balance of genotoxic and nongenotoxic events. Given that the information about the effects of methyl substitution on toxic events associated with tumor promotion is very limited, we used an established model of rat liver progenitor cells, the rat liver epithelial WB-F344 cell line, to study both the genotoxic effects of MeBaAs (the formation of DNA adducts, phosphorylation of p53 tumor suppressor, and induction of apoptosis) and the toxic modes of action associated with tumor promotion (AhR activation, disruption of cell cycle control, and inhibition of GJIC), as well as additional mechanisms, which might contribute to both types of carcinogenic effects s induction of enzymes involved in metabolic activation of MeBaAs and generation of oxidative stress. Our data suggested that genotoxic effects play only a minor role in the toxicity of monomethylated BaAs to liver cells and that methyl substitution had a significant impact on both AhR-dependent and -independent nongenotoxic effects of MeBaAs.

Experimental Procedures Chemicals. Dibenzo[a,l]pyrene (DBalP) and DMBA were purchased from Promochem GmBH (Wesel, Germany); 2-, 11-, and 12-MeBaA were from Midwest Research Institute (Kansas City, MO); 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, and 10-MeBaA (Figure 11) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (Prague, Czech Republic); benz[a]anthracene was from Supelco (Bellefonte, PA). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Cambridge Isotope Laboratories (Andover, MA). Spleen phosphodiesterase was purchased from ICN Biomedicals, Inc. (Irvine, CA); 4′-6-diamidine-2-phenyl indole (DAPI), ribonuclease A and T1, proteinase K, micrococcal nuclease, nuclease P1, and protein assay kit were from Sigma-Aldrich; polyethylene-imine cellulose TLC plates (0.1 mm) were from Macherey-Nagel (Düren, Germany); T4 polynucleotide kinase was from USB (Cleveland, OH); and γ-32P-ATP (3000 Ci/mmol, 10 µCi/µL) was from GE Healthcare (Little Chalfont, United Kingdom); propidium iodide was from AppliChem GmbH (Darmstadt, Germany). Cells. WB-F344 rat liver epithelial cells (kindly provided by James E. Trosko, MSU, East Lansing) were grown in Dulbecco’s modified Eagle’s medium (Invitrogene, Carlsbad, CA) supplemented with 25 mM sodium bicarbonate, 10 mM HEPES, and 5% heatinactivated fetal bovine serum (PAA, Pasching, Austria). Only the cells at passages 15-24 were used throughout the study. The rat

MarVanoVá et al. hepatoma H4IIEGud.Luc1 cells, licensed from BioDetection Systems (Amsterdam, The Netherlands), were grown in Dulbecco’s modified Eagle’s medium (Invitrogene), supplemented with 10% of heat-inactivated fetal bovine serum. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. Cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. Detection of AhR-Mediated Activity. The rat hepatoma H4IIEGud.Luc1.1 cell line, stably transfected with a luciferase reporter gene under the control of dioxin responsive elements, was used to detect the AhR-mediated activity in the dioxin responsive chemical-activated luciferase expression (DR-CALUX) assay (32). The assays were performed in 96 well cell culture plates. The cells were grown for 24 h to 90–100% confluency and exposed to the test or reference compounds (TCDD) dissolved in DMSO (maximum concentration 0.4%, v/v) for 24 h. The medium was removed, cells were washed with PBS, and the luciferase was extracted with the low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2diamin cyclic hexane-N,N,N′,N′-tetraacetic acid, pH 7.8). The plates were frozen at -80 °C, and the luciferase expression was then measured on a microplate luminometer using the Luciferase Assay Kit (BioThema, Handen, Sweden). Detection of GJIC. The modified scrape loading/dye transfer assay was performed as described previously (25). The confluent WB-F344 cells, grown in 24 well plates, were exposed to test compounds (up to 50 µM concentration), 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, positive control), or DMSO (negative control) for 30 min. After the exposure, the cells were washed twice with 0.5 mL of PBS; fluorescent dye was added (Lucifer Yellow 0.05% w/v in PBS), and the cells were scraped using a surgical blade. After 4 min, the cells were washed twice by 0.5 mL of PBS and fixed with 4% formaldehyde (v/v), and the migration of the dye was evaluated using an epifluorescence microscope. The distance of the dye migration from a scrape line was measured at six randomly chosen spots per scrape, using Lucia image analysis software (Laboratory Imaging, Prague, Czech Republic). Three independent experiments were carried out in duplicate, and at least three scrapes per well were evaluated. Real-Time Reverse Trancription Polymerase Chain Reaction (RT-PCR). Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel). The amplifications of the samples were carried out using QuantiTect Probe RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s specifications. All probes were labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) on the 5′-end and with the Black Hole 1 (BH 1) fluorescent quencher dye on the 3′-end. The sequences of primers and probes have been published previously (33). The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the conditions described previously (33). Gene expression for each sample was expressed in terms of the threshold cycle (Ct), normalized to housekeeping gene porphobilinogen deaminase (∆Ct). ∆Ct values were then compared between control samples (0.1% DMSO) and samples treated with PAHs to calculate ∆∆Ct (∆Ct[control] ∆Ct[xPAH]). The final comparison of transcript ratios between samples is given as 2–∆∆Ct (34). Assessment of Cell Proliferation and Cell Cycle Distribution. The proliferative effects of BaA, 12 MeBaAs, and DMBA on confluent WB-F344 cells were determined as described previously (23). The confluent cells were exposed to test compounds dissolved in DMSO (maximal concentration, 0.1% v/v) for 72 h. The medium with test compounds was changed daily. Following the treatment, the medium was removed, and cells were harvested by trypsinization and counted with a Coulter Counter (model ZM, Coulter Electronics, Luton, United Kingdom). Cells were then washed with PBS and fixed in 70% ethanol at 4 °C overnight. Fixed cells were washed once with PBS and stained with propidium iodide as described previously (33). Cells were then analyzed on FACSCalibur, using 488 nm (15 mW) air-cooled argon-ion laser for propidium iodide excitation and CellQuest software ver. 5.1.1 for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15000 events

Toxic Effects of Methylated Benz[a]anthracenes was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME). Detection of PAH-DNA Adducts. WB-F344 cells in a nearly confluent state (seeded at an initial density 23000 cells/cm2 in 60 cm2 plates, grown for 48 h) were exposed for 24 h to test compounds and DMSO as a solvent control (0.1%). After exposure, cells were washed with cold PBS, scraped into the Eppendorf tubes, and centrifuged, and the cell pellets were stored at -80 °C. The cell pellets were homogenized in a solution of 10 mM Tris-HCl, 100 mM EDTA, and 0.5% SDS, pH 8.0. DNA was isolated using RNase A and T1 and proteinase K treatment followed by phenol/chloroform/isoamylalcohol procedure as previously described (35). The DNA concentration was estimated spectrophotometrically by measuring the UV absorbance at 260 nm. DNA samples were kept at -80 °C until analysis. 32Ppostlabeling analysis was performed as previously described (36, 37). Briefly, DNA samples (6 µg) were digested by a mixture of micrococcal endonuclease and spleen phosphodiesterase for 4 h at 37 °C. Nuclease P1 was used for adduct enrichment. The labeled DNA adducts were resolved by two-directional thin layer chromatography on 10 cm × 10 cm PEI-cellulose plates. Solvent systems used for TLC were as follows: D-1, 1 M sodium phosphate, pH 6.8; D-2, 3.8 M lithium formate and 8.5 M urea, pH 3.5; and D-3, 0.8 M lithium chloride, 0.5 M Tris, and 8.5 M urea, pH 8.0. Autoradiography was carried out at -80 °C for 1-24 h. The radioactivity of distinct adduct spots was measured by liquid scintillation counting. To determine the exact amount of DNA in each sample, aliquots of DNA enzymatic digest (1 µg of DNA hydrolysate) were analyzed for nucleotide content by reverse-phase HPLC with UV detection, which simultaneously allowed us to check the DNA purity. DNA adduct levels were expressed as adducts per 108 nucleotides. A benzo[a]pyrene dihydrodiol epoxide (BPDE)-DNA adduct standard was run in triplicate in each postlabeling experiment to check for interassay variability and to normalize the calculated DNA adduct levels. Detection of Phosphorylated Form of p53 Protein. WB-F344 cells in a confluent state (seeded at an initial density 30000 cells/ cm2, grown for 72 h) were exposed for 24 h to test compounds and maximum 0.5% DMSO as the solvent control. DBalP was used as a positive control. After the exposure, cells were harvested into the lysis buffer (1% SDS, 10% glycerol, 100 mM Tris, and protease inhibitors) and the lyzates were sonicated. Protein concentrations were determined using bicinchonic acid and copper sulfate (SigmaAldrich). For Western blot analyses, equal amounts of total protein lyzates were separated by SDS-polyacrylamide gel electrophoresis on 10% gel and electrotransferred onto a PVDF membrane Hybond-P (GE Healthcare). Prestained molecular weight markers (Bio-Rad, Hercules, CA) were run in parallel. The blotted membranes were blocked overnight at 4 °C and incubated with primary antibody against p53 phosphorylated on Ser15 for 2 h at room temperature (Cell Signaling Technology, Beverly, MA), diluted in 2.5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween 20. After the membranes were washed in TBS with 0.1% Tween 20, peroxidase-conjugated swine antirabbit immunoglobulin antisera (Sevapharma, Prague, Czech Republic) were used as a secondary antibody. Expression of β-actin was used to verify equal loading; monoclonal anti-β-actin antibody, clone AC-15 (Sigma-Aldrich), was diluted in 2.5% milk in TBS and incubated for 2 h at room temperature; peroxidase-conjugated antimouse antibody (SigmaAldrich) was used as a secondary antibody. To visualize peroxidase activity, ECL Plus reagents (GE Healthcare) were used according to the manufacturer’s instructions. Detection of Cell Death. Confluent WB-F344 cells were exposed to the test compounds for 48 h including change of the fresh medium and the compound after 24 h. Early stages of apoptosis were characterized by translocation of phosphatidylserine from the inner part of the plasma membrane to the outer layer. The presence of phosphatidylserine at the cell surface was determined by staining with Annexin-V-Fluos (Roche Diagnostics) in combination with propidium iodide (40 µg/mL), to distinguish the cells with permeabilized and intact plasma membranes. Cells were harvested and

Chem. Res. Toxicol., Vol. 21, No. 2, 2008 505 stained according the manufacturer’s protocol and analyzed by FACSCalibur with CellQuest software (Becton Dickinson). For DAPI staining, cells fixed in 70% ethanol were incubated with 1 µg/mL DAPI (final concentration) for 5 min at room temperature. After incubation, the cells were centrifuged and mixed with 10-20 µL of MOWIOL solution (10% MOWIOL 4-88 was prepared in 25% glycerol, 100 mM Tris-HCl, pH 8.5) and mounted for observation under a fluorescence microscope. A minimum of 300 nuclei were counted per sample. Detection of Reactive Oxygen Species (ROS). Confluent WBF344 cells were exposed to the test compounds for 24 h. Hydrogen peroxide (exposure 5 min) was used as a positive control. After the exposure, the cells were twice washed by PBS, trypsinised, centrifuged, and resuspended with Hank’s balanced salt solution (PANBioTech GmbH, Aidenbach, Germany) with 5% heatinactivated fetal bovine serum. The cell suspension was incubated for 15 min with the fluorescent probe dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich); the final concentration was 20 µM. The cells were washed once again, centrifuged, and cooled on ice (except hydrogen peroxide-exposed cells). The fluorescence of dichlorofluorescein (DCF) was analyzed by FACSCalibur with CellQuest software (Becton Dickinson) (38). Statistical Analysis. All experiments were performed independently at least three times, and the data were quantitatively expressed as means ( SD and analyzed by t test and ANOVA followed by Tukey’s range test. When the variances were not homogeneous, a nonparametric Mann–Whitney U test was used. A p value of less than 0.05 was considered significant.

Results Effects of MeBaAs on AhR Activation and Inhibition of GJIC. In our previous work, we determined induction equivalency factors (IEFs) of a large array of PAHs to activate AhR, using a single reporter gene assay using the H4IIEGud.Luc cell line, which is stably transfected with luciferase reporter gene under the control of dioxin responsive elements (DREs) (16, 39). These included both BaA and DMBA, which both had similar IEFs of 7.04 × 10-6 and 5.41 × 10-6, respectively (IEFs were derived from EC50 values of PAHs and the reference compound TCDD after 24 h of exposure). To determine whether a single methyl substitution may have an impact on AhR activation by MeBaAs, we used the same experimental settings to determine their IEF values. The results are summarized in Table 1. All 12 monomethylated BaA derivatives activated AhR, with EC50 values ranging from 2 to 229 nM. It should be stressed that these values were in each case lower than the EC50 of parental compound. The most potent AhR agonists were 6- and 9-MeBaA. Their IEFs were almost three orders of magnitude higher than those previously reported for BaA and DMBA. With the exception of 5-MeBaA, all MeBaAs with methyl groups at positions 4-9, including the K region, were the most potent AhR ligands. In contrast, both MeBaAs with methyl groups positioned within the bay region, 1- and 12-MeBaA, were the weakest AhR agonists among MeBaAs. It has been suggested that inhibition of GJIC is closely correlated with tumor-promoting activity, therefore being a suitable parameter reflecting possible promoting activity of studied chemicals (40). The closure of gap junctions by tumor-promoting chemicals may lead to a release of the cells, including genotoxically damaged cells, from the control of neighboring cells and consequently to the disruption of homeostasis and cell-to-cell communication (26). The rat liver epithelial WB-F344 cell line is an established model for the detection of effects of tumor-promoting agents on GJIC, where gap junctions are formed almost exclusively by


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Table 1. Values of EC50 and Relative Potencies of MeBaAs To Activate AhR (DR-CALUX Assay; Exposure 24 h) and To Inhibit GJIC in WB-F344 Cells (Exposure 30 min)a DR-CALUX 1-MeBaA 2-MeBaA 3-MeBaA 4-MeBaA 5-MeBaA 6-MeBaA 7-MeBaA 8-MeBaA 9-MeBaA 10-MeBaA 11-MeBaA 12-MeBaA BaA DMBA

inhibition of GJIC

EC50 (nM)

IEF (TCDD, 24 h)

IC50 (µM)

229 87 72 27 101 7 25 37 2 117 116 215 1551 2026

4.8 × 10-5 1.3 × 10-4 1.5 × 10-4 4.1 × 10-4 1.1 × 10-4 1.7 × 10-3 4.4 × 10-4 3.0 × 10-4 4.6 × 10-3 9.4 × 10-5 9.5 × 10-5 5.1 × 10-5 7.0 × 10-6 5.4 × 10-6

10 15 NIb NI NI NI NI 13 NI 25 14 10 NI 21

a The numbers represent induction equivalency factors (IEFs) calculated as the ratio between the 50% effective concentration (EC50) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the concentration of appropriate MeBaA inducing the 50% of maximum TCDD-induced luciferase activity. All experiments were performed independently three times in triplicate. b NI, not inhibiting GJIC up to 50 µM. All experiments were performed independently three times in triplicate.

connexin 43 protein (41). A number of PAHs have been shown to inhibit GJIC in this cell model. Importantly, methyl substitution might significantly affect the inhibitory activity of MeBaAs against GJIC, as DMBA has been found to be an inhibitor of GJIC, while BaA has not been able to fully suppress GJIC (25). Therefore, we next examined the effects of MeBaAs on GJIC, using a standardized scrape loading/ dye transfer assay. We found that 1-, 2-, 10-, 11-, and 12MeBaA, as well as 8-MeBaA, inhibited GJIC with similar IC50 values (Table 1), while other MeBaAs caused no inhibition of GJIC up to the 50 µM concentration. Interestingly, with the exception of 8-MeBaA, we observed an inverse relationship between AhR activation and GJIC inhibition, as the compounds inhibiting GJIC were less efficient AhR inducers. AhR-Dependent Activation of Xenobiotic-Metabolizing Enzymes, Induction of Cell Proliferation, and Cell Cycle Perturbations. Our previous studies have suggested that activation of AhR by PAHs might play a dual role in their effects on WB-F344 cells. Activated AhR may both induce expression of enzymes involved in metabolic activation and detoxification of PAHs and stimulate cell proliferation in contact-inhibited cells, that is, disrupt the cell cycle control (21, 23). As we observed differences in effects of MeBaAs on AhR activity, we next examined the ability of MeBaAs (i) to induce expression of P450 1A1, P450 1B1, and aldo-keto reductase 1C9 (AKR1C9); (ii) to change cell cycle distribution of contact-inhibited WBF344 cells; and (iii) to stimulate cell proliferation of contactinhibited WB-F344 cells. First, we employed real-time RT-PCR to determine the effects of MeBaAs, BaA, and DMBA on the expression of enzymes, which participate in the formation of either dihydrodiol epoxide metabolites forming stable DNA adducts or in the production of PAH quinones leading to oxidative stress and/or DNA damage (42, 43). As summarized in Figure 22, all MeBaAs were able to elicit maximal induction of P450 1A1 and P450 1B1 mRNAs, as well as to significantly increase AKR1C9 mRNA, when applied at a 1 µM concentration. MeBaAs induced higher levels of P450 1A1 mRNA than BaA, similar to their effects on the DRE-controlled luciferase reporter in H4IIE.Gud.Luc cells. The AhR-dependent gene expression in WB-F344 cells was observed from low nanomolar

Figure 2. Induction of P450 1A1, P450 1B1, and AKR1C9 mRNA determined by real-time PCR following 24 h of treatment with MeBaAs or BaA (1 µM), DMBA (100 nM), or with a prototypical AhR ligand, TCDD (1 nM). Data are representative for three independent experiments performed in duplicate (means ( SD).

concentrations of MeBaAs (data not shown), thus confirming the results of the DR-CALUX assay. Our previous studies suggest that a moderately increased percentage of S phase cells (5-10% increase) in contactinhibited WB-F344 cells is indicative of the proliferative effects of nongenotoxic AhR ligands, whereas strong genotoxins induce massive accumulation of cells in the S phase (21, 23, 39). In the present study, we observed a modest accumulation of cells in the S phase of the cell cycle after 72 h of exposure to MeBaAs at a micromolar range concentration similar to BaA with one notable exception. As outlined in Figure 3, 10-MeBaA induced a higher accumulation of cells in the S phase (>20% cells at 1 µM) than any other MeBaA. In Figure 3, the effects of 10MeBaA are compared with those of 7- and 12-MeBaA, which were selected as representative MeBaAs, based on their methyl groups being present in one of the positions substituted in DMBA and with DMBA itself. DMBA induced a much higher accumulation of cells in the S phase than any MeBaA, apparently due to its known genotoxicity. These results suggested that only 10-MeBaA might have genotoxic effects in WB-F344 cells. This was partially confirmed, when we found that all MeBaAs, with the exception of 10-MeBaA, significantly increased cell numbers upon 72 h of cultivation; the potent genotoxin DMBA caused a significant decrease in cell numbers (Figure 4). Effects of MeBaAs on DNA Adduct Formation, Oxidative Stress, and Apoptosis. The above data suggest that

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Figure 3. Percentage of cells in the S phase of the cell cycle in WB-F344 after 72 h of treatment by 7-, 10-, and 12-MeBaA and DMBA detected on a FACSCalibur flow cytometer using propidium iodide staining. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).

compounds found to be mutagenic in other models, such as 7-MeBaA (10, 11), are probably not genotoxic to liver epithelial WB-F344 cells, whereas 10-MeBaA, which is considered only a weak mutagen and tumor-initiating compound in skin or bacterial models (10, 11), had effects suggesting its possible genotoxicity in WB-F344 cells. Therefore, we next determined the DNA adduct formation in WB-F344 cells exposed to selected MeBaAs, BaA, or DMBA by 32P-postlabeling assay (Table 2). DMBA produced a high level of DNA adducts, confirming its strong genotoxic potency, whereas 10-MeBaA induced much lower number of DNA adducts, similar to 12MeBaA and higher than 7-MeBaA (Table 2). These results suggest that MeBaAs do not form stable DNA adducts in WBF344 cells, despite their ability to increase levels of enzymes involved in their metabolic activation. Genotoxic PAHs have been suggested to induce oxidative stress in target cells, which might contribute to the damage of DNA and other macromolecules (42). As the formation of DNA adducts could not fully explain the toxicity of 10-MeBaA, we determined the production of ROS by flow cytometry, using DCF as a probe (38). As outlined in Figure 5, 24 h of exposure to DMBA and 10-MeBaA resulted in a significant increase in ROS formation, whereas 7-MeBaA did not elicit a significant increase of ROS levels as compared to the control cells. Interestingly, 7-MeBaA was not able to induce either stable DNA adducts or oxidative damage to DNA in liver cells. The toxic effects of 10-MeBaA also corresponded with the induction of phosphorylation of tumor suppressor protein p53 on Ser15 residue after 24 h of exposure (Figure 6). No other MeBaA induced phosphorylation of this amino acid residue, which has been shown to play a significant role in p53 stabilization, up-regulation, and functional activation during stress induced by genotoxic insult, including some PAHs (44–46). These data also corresponded with the induction of apoptosis. All MeBaAs, which were found to increase cell

numbers, induced a slight increase in percentage of cells with fragmented nuclei after 48 h of exposure (Figure 7), as well as a slight increase of Annexin-V-positive cells, representing early apoptotic stages (data not shown). In contrast, 10-MeBaA induced a significantly higher number of Annexin-V-positive cells (Figure 8) and cells with fragmented nuclei (Figure 7), although lower than DMBA. DMBA induced both a high accumulation of phosphorylated p53 and more pronounced apoptotic effects than 10-MeBaA. The parental compound BaA had no effects on either accumulation of phosphorylated p53 (Figure 6) or on apoptosis (reported previously in ref 23).

Discussion Methyl substitution has been shown to exert remarkable effects on the carcinogenicity of PAHs, such as DMBA. Monomethylated BaA derivatives can be found at significant levels in the environment or as cigarette smoke constituents (1–3). MeBaAs substituted in position 6, 7, 8, or 12 have been reported to be carcinogenic in both rat and mouse skin assays (7, 9), while other derivatives had only weak or no activity. The tumor-initiating activity of all 12 monomethylated benz[a]anthracenes has also been examined in the mouse skin model; 7-MeBaA was the most potent derivative, while all other MeBaAs elicited lower but significant tumor-initiating activity (11, 47). However, to this date, the carcinogenic or tumorinitiating activities of MeBaAs were studied almost exclusively in skin (7, 9, 11, 47), and little is known about their toxic or carcinogenic effects in other tissues or cellular models. In the present study, only one compound, 10-MeBaA, was found to be moderately genotoxic in liver epithelial WB-F344 cells, which could be, in part, also associated with the induction of oxidative stress in target cells. Nevertheless, although 10MeBaA induced the formation of DNA adducts, increased production of ROS, phosphorylation of p53 protein, apoptosis,


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Figure 4. Modulation of cell proliferation in WB-F344 assessed by counting cell numbers after 72 h of treatment by test compounds. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).

Table 2. DNA Adducts per 108 Nucleotides for Selected MeBaAs and DMBA as Determined by 32P-Postlabeling Method after 24 h of Exposure DNA adducts/108 nucleotides BaA (10 µM) 7-MeBaA (10 µM) 10-MeBaA (10 µM) 12-MeBaA (10 µM) DMBA (1 µM)

mean

SD

1.2 0.4 3.1 2.8 230

0.38 0.031 1.4 0.24 77

and increased percentage of cells in the S phase, all of these effects were markedly lower than genotoxic effects of DMBA, which was found be a powerful genotoxin in WB-F344 cells. Interestingly, 7-MeBaA did not form significant levels of stable DNA adducts and did not induce ROS formation, p53 phos-

phorylation, or apoptosis. This is in contrast with data obtained in bacterial mutagenicity assays or in rodent skin two-stage initiation-promotion assay. 7-MeBaA has been found to be significantly mutagenic in the Ames test with metabolic activation (10). It has also been the most potent derivative inducing sister chromatid exchanges (48). These results suggest that toxic effects of MeBaAs might be significantly different in organs such as the liver or lung. This fact should be taken into account in the risk assessment of MeBaAs based on bacterial mutagenicity or skin carcinogenicity data. In marked contrast to mutagenic and tumor-initiating effects of MeBaAs, other modes of toxic action, such as those contributing to tumor promotion, have received very little attention. Many PAHs are known to act as complete carcinogens, producing tumors following their repeated application, which

Toxic Effects of Methylated Benz[a]anthracenes

Figure 5. Fluorescence of dichlorofluorescein (DCF) following 24 h of treatment with selected MeBaAs and DMBA (10 µM) was detected as median fluorescence intensity (MFI). Hydrogen peroxide (250 µM, 5 min exposure) was used as a positive control. The results are expressed as means ( SD of results of three independent experiments done in triplicate. The data were analyzed statistically by Student’s t test. *Significant difference between control (0.1% DMSO) and treated samples (p < 0.05); **significant difference between control (0.1% DMSO) and treated samples (p < 0.01).

Figure 6. Induction of p53 phosphorylation at Ser15 after 24 h of exposure to the 10 µM MeBaAs or BaA as detected by Western blotting. DMBA was tested in concentrations of 0.1, 1, and 10 µM; the 10 µM concentration was used as a positive control on each blot. The detection of β-actin was used to confirm the equal loading.

suggests that either PAHs or their further active metabolites may act also as tumor promoters (14, 28). Although the mechanisms responsible for the tumor-promoting effects of PAHs are largely unknown, PAHs might activate several signaling pathways involved in the control of cell proliferation, differentiation, or apoptosis (28–30, 49). In the present study, we used liver epithelial cells as a model to study the impact of methyl substitution on AhR activation, deregulation of cell proliferation/apoptosis, and inhibition of GJIC. It has been proposed that AhR affinity may reflect the promoting effect of PAHs and that “initiation and promotion are provoked by different chemical species: reactive metabolites and the parent hydrocarbons, respectively” (15). However, there is only limited information on AhR-inducing potencies of MeBaAs. The AhR-binding and induction of EROD activities of seven MeBaAs in rat hepatoma cells have been reported, suggesting that these compounds have similar potencies to the parental BaA (17). On the other hand, Brack et al. (1) have reported that the relative potency (REP) of 9-MeBaA to induce EROD activity in a fish liver cell line could be two orders of

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magnitude higher than the relative potency of BaA. This would suggest that MeBaAs could significantly contribute to the overall AhR-mediated activity of complex environmental mixtures of PAHs, despite being present at significantly lower levels than BaA itself (1, 2). Indeed, we found in the DR-CALUX assay that several MeBaAs were more potent AhR ligands than BaA. The most potent AhR ligands were those MeBaAs that do not possess methyl group within or in the vicinity of the bay region. 4-, 6-, 7-, 8-, and 9-MeBaA were efficient inducers of AhRmediated activity. This holds true especially for 6-MeBaA and 9-MeBaA, whose EC50 values were comparable to some of the most potent AhR agonists among PAHs, such as dibenzanthracenes or benzofluoranthenes (16). Correspondingly to AhR activation data, all MeBaAs were efficient inducers of expression of AhR-dependent enzymes, P450 A1 and 1B1, with induction of P450 1A1 being higher than in case of BaA, their parent compound. All MeBaAs also induced expression of AKR1C9 mRNA, which may also participate in the bioactivation of polyaromatic compounds (42). These data suggested that AhR activation by MeBaAs could play a significant role in the toxic effects of MeBaAs in liver cells. However, the potencies of MeBaAs to induce AhR-mediated activity in liver cells did not correspond either with their mutagenic activities in bacterial assays (7-, 12-, and 5-MeBaAs being the most potent mutagens) (10) or with their skin carcinogenicity (7-, 12-, 6-, and 8-methyl derivatives have been reported as the most effective) (11, 47). Our previous studies have suggested that activation of AhR by PAHs may lead to disruption of cell cycle control in contactinhibited cells, followed by enhanced cell proliferation (21, 23). This mode of action might contribute to tumor-promoting effects of PAHs. In contrast, we have found that strong genotoxins induce cell cycle arrest/delay in the S phase and apoptosis in liver epithelial cells, both events being linked to DNA damage (21, 23, 39). Therefore, we determined modulation of cell cycle, cell proliferation, and apoptosis, to discriminate between genotoxic and nongenotoxic effects of MeBaAs. In accordance with their AhR-mediated activities, all MeBaAs, with the exception of weakly genotoxic 10-MeBaA, induced cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. Disruption of contact inhibition has been suggested to participate in tumor promotion (50, 51), suggesting that deregulation of cell cycle control by MeBaAs might potentially contribute to carcinogenic effects of MeBaAs. In contrast to the AhR-dependent effects of MeBaAs, only the compounds with the methyl group located within or in the vicinity of bay region (1-, 2-, 10-, 11-, and 12-MeBaA) were effective inhibitors of GJIC. The exception from this rule was 8-MeBaA, which was the only compound that acted as a strong inhibitor of GJIC and potent AhR ligand. The IC50 values of GJIC inhibitory effect of MeBaAs were similar to those of the most potent inhibitors of GJIC among PAHs, such as fluoranthene (25). Importantly, these results also confirm that methyl substitution may significantly increase the GJIC inhibitory potency of BaA derivatives, as previously demonstrated for DMBA (25). As outlined above, methyl substitution significantly modulated both GJIC inhibition and AhR-mediated activity of MeBaAs. Various approaches have been previously employed to define structure–activity relationships for carcinogenic effects of PAHs. However, these are hampered by the fact that classical parameters, such as area/depth2, length/width, or electron density do not change significantly within a series of structurally similar MeBaAs (52, 53). Nevertheless, there are some indications of parameters that might define toxic activities of MeBaAs. It has


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Figure 7. Induction of late apoptosis following 48 h of treatment of WB-F344 cells to the test compounds. Late apoptosis was determined as a percentage of cells with fragmented nuclei using DAPI staining and microscopic evaluation. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).

Figure 8. Example of apoptosis induction measured on a FACSCalibur flow cytometer. R1, region 1 represents early apoptotic cells (annexin-V positive, propidium iodide negative); R2, region 2 represents viable cells (both annexin-V and propidium iodide negative); and R3, region 3 contains late apoptotic and necrotic cells (both annexin-V and propidium iodide positive).

been suggested that substitutions in the bay region lead to a distortion of planarity, whereas MeBaAs substituted in the K region and neighboring positions are nearly planar (54). As AhR binds preferentially planar ligands (55), this might explain why both 1- and 12-MeBaA were the least potent AhR agonists identified in the reporter gene assay. Taken together, the present data suggest that methyl substitution modulates both AhR-dependent and -independent toxic effects of BaA derivatives. The in vitro toxic potencies of MeBaAs determined in liver cells were different from tumorinitiating, carcinogenic, and mutagenic potencies found either in skin or in bacterial models. This indicates that more attention should be paid to the evaluation of toxic effects of MeBaAs and related compounds in cellular models derived from other tissues, such as liver or lung. Nongenotoxic modes of action of MeBaAs, particularly the AhR transactivation, appeared to be more important than their genotoxicity in liver cells. As the AhR-mediated activity of MeBaAs was mostly higher than that of BaA, these compounds might significantly contribute to the

toxicity of complex mixtures of PAHs, which are present in various environmental compartments. Acknowledgment. This work was supported by EU FP6 project MODELKEY (51112-GOCE), the Czech Ministry of Agriculture (Grant MZE0002716201), and the Academy of Sciences of the Czech Republic (Research Plan AV0Z50040702).

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TX700305X

Příloha III Vondráček, J., Švihálková-Šindlerová, L., Pěnčíková, K., Krčmář, P., Andrysík, Z., Chramostová, K., Marvanová, S., Valovičová, Z., Kozubík, A., Gábelová, A. and Machala, M. (2006). 7H-dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stem-like’ cells. Mutation Research 596, 43-56.

Mutation Research 596 (2006) 43–56

7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stem-like’ cells ˇ alková-Sindlerov´ ˇ Jan Vondrácˇ ek a,b , Lenka Svih´ a a , Kateˇrina Pˇenˇc´ıková b , Pavel Krˇcmáˇr b , a,b a Zdenˇek Andrys´ık , Kateˇrina Chramostová , Soˇna Marvanová b , Zuzana Valoviˇcová c , Alois Kozub´ık a , Alena Gábelová c , Miroslav Machala b,∗ a

Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic b Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic c Department of Mutagenesis and Carcinogenesis, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovak Republic Received 30 June 2005; received in revised form 22 September 2005; accepted 30 November 2005 Available online 9 January 2006

Abstract Immature liver progenitor cells have been suggested to be an important target of hepatotoxins and hepatocarcinogens. The goal of the present study was to assess the impact of 7H-dibenzo[c,g]carbazole (DBC) and its tissue-specific carcinogenic N-methyl (N-MeDBC) and 5,9-dimethyl (DiMeDBC) derivatives on rat liver epithelial WB-F344 cells, in vitro model of liver progenitor cells. We investigated the cellular events associated with both tumor initiation and promotion, such as activation of aryl hydrocarbon receptor (AhR), changes in expression of enzymes involved in metabolic activation of DBC and its derivatives, effects on cell cycle, cell proliferation/apoptosis and inhibition of gap junctional intercellular communication (GJIC). N-MeDBC, a tissue-specific sarcomagen, was only a weak inhibitor of GJIC or inducer of AhR-mediated activity, and it did not affect either cell proliferation or apoptosis. DBC was efficient GJIC inhibitor, while DiMeDBC manifested the strongest AhR inducing activity. Accordingly, DiMeDBC was also the most potent inducer of cytochrome P450 1A1 (CYP1A1) and CYP1A2 expression among the three compounds tested. Both DBC and DiMeDBC induced expression of CYP1B1 and aldo-keto reductase 1C9 (AKR1C9). N-MeDBC failed to significantly upregulate CYP1A1/2 and it only moderately increased CYP1B1 or AKR1C9. Only the potent liver carcinogens, DBC and DiMeDBC, caused a significant increase of p53 phosphorylation at Ser15, an increased accumulation of cells in S-phase and apoptosis at micromolar concentrations. In addition, DiMeDBC was found to stimulate cell proliferation of contact-inhibited WB-F344 cells at 1 ␮M concentration, which is a mode of action that might further contribute to its hepatocarcinogenicity. The present data seem to suggest that the AhR activation, induction of enzymes involved in metabolic activation, inhibition of GJIC or stimulation of cell proliferation might all contribute to the hepatocarcinogenic effects of DBC and DiMeDBC. © 2005 Elsevier B.V. All rights reserved. Keywords: Dibenzocarbazoles; Gap junctional intercellular communication (GJIC); AhR activation; Cell proliferation; Apoptosis; Cytochromes P450

∗

Corresponding author. Tel.: +420 533331813; fax: +420 541211229. E-mail address: [email protected] (M. Machala).

0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.11.005

44

J. Vondrácˇ ek et al. / Mutation Research 596 (2006) 43–56

1. Introduction 7H-dibenzo[c,g]carbazole (DBC), an ubiquitous environmental pollutant, is a common component of a variety of complex mixtures derived from the incomplete combustion of organic matter, such as cigarette smoke, soot, tars and diesel engine exhaust (reviewed in [1]). DBC is a potent multi-species and multi-site carcinogen in in vivo animal studies with both local and systemic effects [1,2]. Based on these facts, the International Agency for Research against Cancer (IARC) [3,4] listed DBC as a possible human carcinogen (Group 2B), although the extent of human exposure is unknown [5]. DBC is a member of an important class of environmental compounds known as N-heterocyclic aromatic compounds (NHAs). The chemical structure of DBC shares the features of polycyclic aromatic hydrocarbons (PAHs), such as the aromatic skeleton with a typical bay region, while the pyrrolic NH group has a chemical behavior similar to that of the arylamides (Fig. 1). Although NHAs represent a relatively minor fraction of the crude organic mixtures of environmental pollutants, they may pose a serious carcinogenic risk to human due their intrinsic biological activity. DBC, for example, was shown to be a more potent pulmonary tumorigen in hamsters than benzo[a]pyrene [6]. This agent is unusual in its carcinogenic response; it can produce significant numbers of skin and liver tumors when applied topically to the back skin of mice [7]. On the other hand, its methyl derivatives, N-methylDBC (N-MeDBC) and 5,9-dimethyl-DBC (DiMeDBC) manifested specific tropism for the skin and liver, respectively. N-MeDBC produces DNA adducts, tumors and mutations mainly in the skin and lacks hepatocarcinogenic potential [8,9], whereas the strict hepatocarcinogen DiMeDBC produces tumors, adducts and mutations

Fig. 1. Chemical structures.

in the liver, but it is devoid of any activity in the skin [10–12]. Like many other chemical carcinogens, DBC requires metabolic activation to electrophilic species before they can interact with DNA and other macromolecules and exert their mutagenic and carcinogenic effects. It has been suggested that formation of bay region dihydrodiol epoxides and o-quinones are the principle metabolic pathways for DBC [1,13], while formation of radical cation is not the major route of DBC metabolic activation [14]. Both types of metabolites, dihydrodiol epoxides and o-quinones, may contribute to PAH-induced carcinogenesis and a relative importance of each pathway depends on tissue type and the level and inducibility of activation enzyme(s) [13,15]. Cytochromes P450 1A1 (CYP1A1), CYP1A2 and CYP1B1 are key enzymes involved in formation of PAH dihydrodiol epoxides, whereas dihydrodiol dehydrogenases (aldo-keto reductases—AKR) are responsible for formation of o-quinones and induction of oxidative stress and reactive semiquinones [13,15]. The role of CYP1 enzymes in vivo is more complex, as they might contribute to both bioactivation and detoxification of PAHs and related compounds, depending on cell type- and tissue-specific context [16]. Although the relative importance of CYP1A1 and CYP1A2 for metabolic activation of DBC and its derivatives has been studied in some detail [17–19], the information on inducibility of individual enzymes in specific target cells or tissues is mostly missing. Upregulation of phases I and II xenobioticmetabolizing enzymes, which are involved in biotransformation of PAHs and NHAs, is largely mediated via binding of ligands to the aryl hydrocarbon receptor (AhR), and it is currently the most clearly understood process of AhR signaling. Although the capacity of DBC and its derivatives to activate AhR could significantly affect their genotoxicity, there is no comprehensive information about the AhR activation by DBC and its derivatives. Their potencies to activate AhR might also differ in various liver cell populations, as their genotoxic effects are also different, e.g. in parenchymal and nonparenchymal liver cells [20]. Nevertheless, apart from regulation of expression of metabolizing enzymes, the AhR plays also an important role in cell cycle regulation, mitogenesis, oxidative stress and apoptosis [21–23]; processes that might significantly affect the carcinogenic properties of genotoxins. There is currently little information on non-genotoxic modes of action that might contribute to carcinogenicity of DBC and its derivatives, including their potential tumor promoting effects, such as perturbation of tissue


homeostasis due to disruption of cell-to-cell communication or proliferation/apoptosis balance. Inhibition of gap-junctional intercellular communication (GJIC) plays an important role in these mechanisms [24] and in vitro determination of this effect has been found as a suitable tool for detection of many tumor promoters and carcinogens [24,25]. A number of low-molecularweight PAHs are relatively efficient transient inhibitors of GJIC, suggesting that inhibition of GJIC could be one of modes of action they exert in target tissues [26,27]. The induction of cell proliferation in contact-inhibited rat liver epithelial cells after exposure to a number of PAHs is another mechanism of action of PAHs possibly associated with tumor promotion, which has been documented recently [28]. Stimulation of cell proliferation has been shown to be a critical event for expression of mutations in the mouse liver after DBC and DiMeDBC exposure [29–31]. The severe hepatocellular toxicity observed in livers taken from DBC-treated mice [1] is followed by the liver regeneration [11], suggesting that the extensive apoptosis induced by genotoxic DBC in liver cells [32,33] can lead to regenerative cell proliferation. The hepatotoxicity and resulting liver injury might also lead to necrosis associated with a release of further mediators, such as inflammatory cytokines, which could also significantly contribute to proliferation of specific liver cell populations, including oval cells [34,35]. Therefore, the induction of cell proliferation in liver progenitor cells observed in vitro after exposure to various types of AhR ligands [28,36–38] could further contribute to proliferative effects of DBC and DiMeDBC in liver. The AhR-mediated activity of DBC and its derivatives could thus play significant roles both in regulation of enzymes participating in formation of ultimate genotoxic, apoptosis-inducing metabolites and in modulation of cell proliferation. The rat liver epithelial stem-like WB-F344 cell line, isolated from the liver of an adult male Fischer 344 rat, is considered to be an in vitro model of pluripotent oval cells, presumed liver progenitor cells [39]. Our recent studies have shown that different PAHs or heterocyclic aromatic compounds are inducing multifaceted responses in these cells, including inhibition of GJIC, stimulation of cell proliferation or induction of programmed cell death [27,28,40], which makes them an interesting model for studies on cellular processes involved in carcinogenesis. The liver oval cells can give rise to both hepatocytes and biliary epithelial cells, and they might play a significant role in hepatocarcinogenesis [41,42]. Therefore, these cells could represent a possible target for DBC and DiMeDBC, both being potent liver carcinogens. The goal of this study was to eval-

45

uate effects of hepatocarcinogenic dibenzocarbazoles, DBC and DiMeDBC, and sarcomagenic DBC derivative, N-MeDBC, on WB-F344 cells, focusing principally on the effects that might promote the genotoxic potential of these compounds. Therefore, the impact of these agents on GJIC, induction of AhR-mediated activity, expression of enzymes involved in their metabolic activation, or on cell proliferation, cell cycle and apoptosis, were investigated in contact-inhibited WB-F344 cells. 2. Materials and methods 2.1. Chemicals DBC (CAS No. 194-59-2) and its methylated derivatives (Fig. 1) were kindly provided by Dr. F. Périn, Institute Curie, France. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, CAS No. 1746-01-6) was from Cambridge Isotope Laboratories (Andover, MA). Antibodies to connexin43 and Ser-15 phosphorylated p53 tumor suppressor were obtained from Sigma–Aldrich (Prague, Czech Republic) and Cell Signaling Technology (Andover, MA), respectively. Horseradish peroxidase-conjugated anti-immunoglobulins were from Sigma–Aldrich; polyvinylidene difluoride (PVDF) membrane Hybond-P, and chemiluminescence detection reagents (ECL + Plus) were purchased from Ammersham (Aylesbury, UK). 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Fluka (Buchs, Switzerland). All other chemicals were provided by Sigma–Aldrich. 2.2. Cells WB-F344 rat liver epithelial cells were grown in modified Eagle’s minimum essential medium (Sigma–Aldrich) with 50% increased concentrations of essential and nonessential amino acids, and supplemented with sodium pyruvate (110 mg/l), 10 mM HEPES and 5% heat-inactivated fetal bovine serum (Sigma–Aldrich). Only the cells at passages 15–22 were used throughout the study. The rat hepatoma H4IIEGud.Luc1 cells, licensed from BioDetection Systems (Amsterdam, The Netherlands), were grown in the alpha modification of minimal essential medium (Sigma–Aldrich), supplemented with 10% of heat-inactivated fetal bovine serum. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 ◦ C. Cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. 2.3. Detection of AhR-mediated activity The rat hepatoma H4IIEGud.Luc1.1 cell line, stably transfected with a luciferase reporter gene under the control of dioxin responsive elements, was used to detect for AhRmediated activity in the DR-CALUXTM assay [43,44]. The assays were performed in 96-well cell culture plates. Briefly, 24 h after seeding at split ratio 1:2, cells (at 90–100%

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confluency) were exposed to the tested or reference compounds (TCDD) dissolved in dimethyl sulfoxide (DMSO). Maximum concentrations of DMSO did not exceed 0.4% (v/v). Following 6 or 24-h exposure, the medium was aspirated, cells were washed with phosphate-buffered saline (PBS) and luciferase was extracted with the low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2-diamin cyclic hexane-N,N,N ,N -tetraacetic acid, pH 7.8). The plates were frozen at −80 ◦ C and luciferase expression was then measured on a microplate luminometer using the Luciferase Monitoring Kit (Labsystems, Helsinki, Finland).

mal cycler (Corbett Research, Sydney, Australia), using the following program: reverse transcription at 50 ◦ C for 30 min and initial activation step at 95 ◦ C for 15 min, followed by 40 cycles at 94 ◦ C for 15 s and 60 ◦ C for 60 s. Gene expression for each sample was expressed in terms of the threshold cycle (Ct ), normalized to housekeeping gene porphobilinogen deaminase (Ct ). Ct values were then compared between control samples (DMSO 0.1%) and samples treated with dibenzocarbazoles to calculate Ct (Ct [control] − Ct [xDBC]). The final comparison of transcript ratios between samples is given as 2−Ct [45].

2.4. Real-time RT-PCR

2.5. Assessment of cell proliferation and cell cycle distribution

The levels of CYP1A1, CYP1A2, CYP1B1 and AKR1C9 mRNAs were determined by real-time RT-PCR. The sequences of primers and probes are listed in Table 1. The primers were designed on the exon junction for amplification of cDNA only. Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA), including treatment with DNase I (Qiagen). The amplifications of the samples were carried out using QuantiTect Probe RT-PCR Kit (Qiagen) according to manufacturer’s specifications. All probes were labeled with the fluorescent reporter dye 6-carboxyfluorescein (6-FAM) on the 5 -end, and with the Black Hole 1 (BH 1) fluorescent quencher dye on the 3 -end. The amplifications for CYP1A1, CYP1B1 and AKR1C9 were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the following program: reverse transcription at 50 ◦ C for 20 min and initial activation step at 95 ◦ C for 15 min, followed by 40 cycles at 95 ◦ C for 0 s and 60 ◦ C for 60 s. As the levels of CYP1A2 mRNA in WB-F344 cells were very low and undetectable with LightCycler, this reaction was run on the Rotor-GeneTM ther-

The proliferative effects of DBC and its derivatives on confluent WB-F344 cells were determined as described previously [28]. Briefly, cells were seeded in 4-well cell-culture plates (Nunc, Roskilde, Denmark) and grown until they reached an approximate confluency. The cells were exposed to tested compounds dissolved in DMSO for 72 h. The final concentration of DMSO did not exceed 0.1% (v/v) in any of the samples. The medium with tested compounds was changed daily. Following the treatment, medium was removed, cells were harvested with trypsin and counted with a Coulter Counter (Model ZM, Coulter Electronics, Luton, UK). Cells were then washed with PBS, and fixed in 70% ethanol at 4 ◦ C overnight. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1 M Tris–HCl, pH 8.0; 0.1% Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 ␮g/ml; RNAse A 50 Kunitz units/ml) [46] and incubated at 37 ◦ C for 30 min. Cells were then analyzed on FACSCalibur, using 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and

Table 1 Primer and probe sequences for quantitative real-time RT-PCR Genes/accession no.

Primer and probe sequencesa

Product length (bp)

CYP1A1 NM 012540

F980 R1122 P1057

5 -ATGTCCAGCTCTCAGATGATAAGGTC-3

5 -ATCCCTGCCAATCACTGTGTCTAAC-3 5 -CCAGGTACATGAGGCTCCAAGAGATAGC-3

167

CYP1A2 NM 012541

F886 R957 P917

5 -GTGAGAACTACAAAGACAACGGTG-3 5 -GTGACTGTTTCAAATCCAGCTCC-3 5 -CCCTCAGGAGAAGATTGTCAACATTGTC-3

94

CYP1B1 NM 012940

F1398 R1491 P1463

5 -CTCATCCTCTTTACCAGATACCCG-3 5 -GACGTATGGTAAGTTGGGTTGGTC-3 5 -CTCATGCAGGGCAGGCGGTCCCTCCCC-3

117

AKR1C9 D17310

F809 R908 P880

5 -TCACCTTTATCTCAACCAGAGCAA-3 5 -CTGGACTTTTCTGATCCACCCAT-3 5 -TTTGTCTCGTGAACTTCCCAGCGTGC-3

122

PBG-D X06827

F480 R612 P512

5 -CCTCACCTGGAATTCAAGAGTATTCG-3 5 -TTCCTCTGGGTGCAAGATCTGGCC-3 5 -CCTCAACACCCGCCTTCGGAAGCT-3

156

a The primers and probes are identified by letters designating the forward (F), the reverse (R) primer or the probe (P), and a number corresponding to the position of the base at the 5 -end of the positive strand of the primer or probe in the gene reference sequences, according to number of accession.


CELLQuestTM software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT Version 2.0 software (Verity Software House, Topsham, ME). 2.6. Cell death detection Cell death induced by the tested compounds was determined using morphological criteria (fragmentation of nuclei). Cells were incubated with test compounds or vehicle as described above. After indicated time period, cells were harvested by trypsinization (including the floating cells) and prepared for DNA labeling with DAPI. For DAPI staining, 5 × 105 cells were resuspended with 50 ␮l of methanol containing 1 ␮g/ml DAPI (final concentration) and incubated for 30 min at room temperature. After incubation, the cells were centrifuged and mixed with 15 ␮l of MOWIOL (10% MOWIOL 4-88 was prepared in 25% glycerol, 100 mM Tris–HCl, pH 8.5) solution and mounted for observation under a fluorescence microscope. A minimum of 200 nuclei were counted per sample. 2.7. Western blotting In order to determine effects of DBC and its two derivatives on Ser-15 phosphorylation of p53 protein, confluent WB-F344 cells were exposed for 24 h to test compounds or to 0.1% DMSO as vehicle control. Effects of DBC and methylated derivatives on connexin43 hyperphosphorylation have been determined in cells exposed to 30 ␮M DBC, DiMeDBC, NMeDBC, 20 nM TPA (positive control) and DMSO (negative control) for 30 min. Whole cell lysates were prepared by harvesting cells in lysis buffer (1% SDS, TRIS, 10% glycerol, protease inhibitor cocktail), and protein concentrations were determined with DC Protein Assay (BioRad, Hercules, CA). For Western blot analyses, equal amounts of total protein were subjected to 10% SDS PAGE, electrotransferred onto HybondP membrane, immunodetected using appropriate primary and secondary antibodies, and visualized by ECL + Plus reagent according to manufacturer’s instructions. After immunodetection, each membrane was stained with amidoblack to confirm equal protein loading. 2.8. Detection of GJIC The modified scrape loading/dye transfer assay was performed as described previously [27]. The confluent WB-F344 cells, grown in 24-well plates, were exposed to test compounds (up to 50 ␮M concentration), 12-O-tetradecanoylphorbol-13acetate (TPA) (20 nM, positive control), or DMSO (negative control) for 30 min. After the exposure, the cells were washed twice with 0.5× PBS; fluorescent dye was added (lucifer yellow 0.05%, w/v in PBS) and the cells were scraped using a surgical blade. After 4 min, the cells were washed twice by 0.5× PBS and fixed with 4% formaldehyde (v/v) and the migration of the dye was evaluated using an epifluorescence microscope. The distance of dye migration from a scrape line was measured at six randomly chosen spots per scrape, using

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Lucia image analysis software (Laboratory Imaging, Prague, Czech Republic). Three independent experiments were carried out in duplicate and at least three scrapes per well were evaluated. In a separate set of experiments, effects of compounds on GJIC were investigated after 60 min and 24-h incubation, respectively. 2.9. Statistical analysis For each compound tested, relative AhR-inducing potencies were defined as the ability to induce luciferase activity using concentration-response curves. Their relative induction equivalency factors (IEFs) were estimated as described previously [44]. The ratio of GJIC inhibition related to the negative control was evaluated and expressed in percentage (fraction of control, FOC) [27]. Cell proliferation data were expressed as means ± standard deviation for at least three independent repeats. The data were analyzed by Student t-test, or by the nonparametric Mann–Whitney U-test and Kruskal–Wallis analysis of variance, where appropriate. A p-value of less than 0.05 was considered significant.

3. Results 3.1. DiMeDBC is a significantly more potent AhR agonist than its parental compound or N-MeDBC The induction of AhR-mediated activity is an important effect of potent liver tumor promoters such as TCDD [37,47], and it leads to induction of enzymes involved both in metabolic activation and in detoxification of PAHs. Therefore, we investigated effects of DBC and its derivatives on AhR activation, using rat hepatoma cell line stably transfected with luciferase reporter gene under control of dioxin responsive elements. The capacity of dibenzocarbazoles to activate the AhR was measured using concentrations ranging from 100 pM to 10 ␮M. TCCD, the most potent AhR ligand, was used as a positive control. As shown in Fig. 2 and Table 2, Table 2 Relative potencies of DBC and its methylated derivatives to activate AhR (DR-CALUXTM assay), and to inhibit GJIC in WB-F344 cells

TCDD TPA DBC DiMeDBC N-MeDBC a

AhR-IEFsa (6 h)

AhR-IEFs (24 h)

GJIC inhibition IC50 (30 min)

1 – 4.91E−06 2.46E−03 2.74E−06

1 – 2.64E−07 3.17E−05 8.99E−07

– 7.6 nM 11.9 ␮M 38.7 ␮M 40.9 ␮M

The numbers represent induction equivalency factors (IEFs) calculated as the ratio between the 25% effective concentration (EC25) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and concentration of selected compound inducing the 25% of maximum TCDD-induced luciferase activity.

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enzymes that have been suggested to participate in their metabolic activation in WB-F344 cells, using realtime quantitative RT-PCR. As summarized in Fig. 3A, DiMeDBC was the most potent inducer of CYP1A1 expression among the three compounds tested and 1 ␮M concentration induced levels of mRNA similar to the prototypical AhR ligand TCDD. DBC also induced CYP1A1 expression, however the mRNA levels were significantly lower than those induced by DiMeDBC. Compared to these two compounds, the capacity of NMeDBC to induce CYP1A1 mRNA was very low. The background levels of CYP1A2 in WB-F344 cells are very low compared to rat hepatoma cells and induction of CYP1A2 mRNA levels is limited (manuscript in preparation). We found that DiMeDBC was inducing a significant increase in CYP1A2 mRNA, whereas DBC induced only a minor increase of CYP1A2 mRNA level (Fig. 3B). N-MeDBC was only a poor inducer of CYP1B1 expression, while both DBC and DiMeDBC increased levels of CYP1B1 mRNA to a similar extent (Fig. 3C). Similarly, both DBC and DiMeDBC increased expression of AKR1C9 mRNA (Fig. 3D). N-MeDBC again did not significantly increase AKR1C9 expression. We found that 10 ␮M concentration of N-MeDBC induced minor increases of CYP1A1, CYP1B1 and AKR1C9 mRNAs expression, however these were still significantly lower that the levels induced by 1 ␮M doses of DiMeDBC or DBC (data not shown). Fig. 2. Aryl hydrocarbon receptor-mediated induction of luciferase reporter gene in H4IIEpGudLuc1.1 rat hepatoma cell line by TCDD, DBC, DiMeDBC and N-MeDBC after 6 and 24-h exposure. The data shown here are representative of three independent experiments; error bars represent standard deviation from the mean value.

DiMeDBC was a relatively potent inducer of AhRmediated activity following the 6-h incubation and its activity declined following the 24-h incubation period. Its relative potency, expressed as induction equivalency factor (IEF), was more than two orders of magnitude higher than relative potencies of DBC and N-MeDBC. Both DBC and N-MeDBC induced less than 50% of the maximum TCDD-induced AhR-mediated activity (Fig. 2, Table 2). 3.2. Induction of mRNA expression of enzymes participating in metabolic activation As the above results suggested that dibenzocarbazoles are AhR agonists, we next investigated the effects of DBC and its methylated derivatives on expression of

3.3. Effects of DBC and its derivatives on cell proliferation, cell cycle and cell death Our recent results suggested that PAHs may induce multifaceted responses in rat liver epithelial cells, such as the modulation of proliferation/apoptosis balance, in addition to previously reported acute inhibition of GJIC [27,28]. Therefore, we investigated impact of both DBC and its two methylated derivatives on cell proliferation, cell cycle and apoptosis in contact-inhibited WB-F344 cells. The confluent cells were treated with dibenzocarbazoles at concentrations ranging from 100 pM to 10 ␮M. As summarized in Fig. 4A, both DiMeDBC and DBC were found to induce a sharp increase in percentage of S-phase cells, suggesting that, similar to other genotoxic polyaromatics, these compounds are inducing accumulation of cells in S-phase at concentrations 100 nM–10 ␮M (DBC) and 1–10 ␮M (DiMeDBC). DBC was most efficient at a concentration of 1 ␮M (∼40% cells in S-phase) while DiMeDBC at a concentration 10 ␮M (∼60% cells in S-phase). A statistically significant increase in cell number was found


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Fig. 3. Induction of CYP1A1 (A), CYP1A2 (B), CYP1B1 (C) and AKR1C9 (D) mRNA following 24-h treatment with DBC or its two methylated derivatives (1 ␮M) as compared to a prototypical AhR ligand, TCDD (1 nM). Total RNA was isolated and quantitative real-time RT-PCR was performed as described in Section 2. The results are expressed as mean ± S.D. of three independent experiments.

after 1 ␮M DiMeDBC treatment (Fig. 4B), while both compounds also markedly increased apoptosis, detected as nuclear fragmentation, especially at 10 ␮M concentration (Fig. 4C). Unlike two hepatocarcinogenic compounds, N-MeDBC did not affect significantly the cell cycle, DNA fragmentation and cell proliferation. A minor increase in percentage of cells in S-phase was detected only at 10 ␮M concentration.

bazoles at concentrations ranging from 0.01 to 10 ␮M (Fig. 5). Dibenzo[a,l]pyrene, the strongest genotoxin among PAH, was used as a positive control in these experiments. Both DBC and diMeDBC induced Ser-15 phosphorylation of p53, although to a lower extent than dibenzo[a,l]pyrene. In contrast, no increase in Ser-15 phosphorylation of p53 was determined in cells exposed to tissue-specific sarcomagen N-MeDBC.

3.4. DBC and DiMeDBC induce phosphorylation of p53 at serine 15

3.5. DBC is a potent inhibitor of GJIC in WB-F344 cells

The p53 tumor suppressor protein is a potent transcription factor that accumulates in cells in response to signals arising from DNA-damaging agents, including DBC [32]. It can be post-translationally modified at various amino acid residues and a number of studies have shown that phosphorylation of p53 protein at serine 15 may play a critical role in the stabilization, up-regulation and functional activation of p53 during genotoxic stress [48]. Phosphorylation of p53 at Ser15 was measured after 24-h exposure to dibenzocar-

The capacity of DBC and its methyl derivatives to inhibit the GJIC was measured in concentrations ranging from 1 to 50 ␮M after 30 min, 60 min and 24 h of exposure. As shown in Fig. 6A and B and in Table 2, DBC was efficient inhibitor of GJIC in rat liver epithelial cells. The parent molecule decreased significantly dye transfer at concentration as low as 10 ␮M and its IC50 after 30-min incubation was 11.6 ␮M. Interestingly, unlike many other PAHs that inhibit GJIC [26,27], the effect of DBC was not transient and it lasted at

50


Fig. 4. Modulation of cell proliferation and cell death by DBC, DiMeDBC and N-MeDBC in WB-F344 cells assessed by measuring percentage of cells in S-phase of cell cycle (A), by counting cell numbers (B) and by detecting of percentage of cells with fragmented nuclei considered apoptotic (C). Cells were cultured for 72 h prior to exposition, and then treated for another 72 h with TCDD or test compounds in a daily applied fresh medium. Cells were then trypsinized and counted on a Coulter Counter (Coulter Electronics, Luton, UK). Cells were then fixed in ethanol for a subsequent flow cytometric analysis of DNA content. The cell-cycle analysis was performed on a FACSCalibur flow cytometer equipped with CellQuest and ModFit software (Becton Dickinson, San Jose, CA, USA). For detection of apoptosis, both floating and adherent cells were collected and stained with DAPI. A minimum of 200 cells were counted per each sample. Dibenzo[a,1]pyrene (DBalP; 100 nM) was used as a positive control for induction of apoptosis. All data are expressed as mean ± S.D. of three independent experiments run in duplicates. * Significant difference between control (0.1% DMSO) and treated samples (p < 0.05). ** Significant difference between control (0.1% DMSO) and treated samples (p < 0.01).


Fig. 5. Induction of p53 phosphorylation at Ser15 by DBC and DiMeDBC. WB-F344 cells were treated with the indicated concentrations of test compound for 24 h. Dibenzo[a,1]pyrene (DBalP; 100 nM) was used as a positive control. Cell lysates were prepared and Western blotting was performed as described in Section 2. The results shown here are representative of three independent experiments.

least for 24-h (Fig. 6C). The longer time intervals were not tested due to the extensive cell death induced by this compound. In contrast, both DiMeDBC and NMeDBC failed to produce a complete inhibition of GJIC, and their respective IC50 doses were 38.7 and 40.9 ␮M.

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One of the potential mechanisms of GJIC inhibition is the induction of hyperphosphorylation of connexin43, a major connexin species present in rat liver epithelial cells. It is characteristic for prototypical tumor promoters, such as TPA or growth factors [49,50]. Connexin43 hyperphosphorylation was assessed in WB-F344 cells treated with equimolar concentration (30 ␮M) of dibenzocarbazoles for 30 min. However, unlike TPA, none of the tested compounds induced connexin43 hyperphosphorylation in rat liver epithelial cells (Fig. 6D), suggesting a distinct mechanism of GJIC inhibition. 4. Discussion A hallmark of numerous chemical carcinogens is their strict organ- or tissue-specificity; however, the precise mechanism of this event is still unknown. It is supposed that the lack of drug-metabolizing enzymes responsible for production of ultimate mutagens in a target tissue could play an important role in this phenomenon. Covalent binding of a xenobiotic into DNA or another form of DNA damage represents an essential step in chemical

Fig. 6. Inhibition of gap junctional intercellular communication (GJIC) by DBC and its methylated derivatives in WB-F344 cells. (A and B) Inhibition of GJIC after 30 min exposure. (C) Inhibition of GJIC after 30 min, 60 min and 24 h of incubation with increasing concentrations of DBC. The effects of test compounds on GJIC were determined by the scrape loading/dye transfer method as described in Section 2. The results are expressed as fraction of control (FOC) and they represent means ± S.D. of three independent experiments carried out in triplicates. (D) DBC, DiMeDBC and N-MeDBC do not induce connexin43 hyperphosphorylation. WB-F344 cells were treated with 30 ␮M of test compounds for 30 min. DMSO was used as a solvent control, while TPA (20 nM) was used as a positive control. Cell lysates were prepared and Western blotting was performed as described in Section 2. The results shown here are representative of three independent experiments.

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carcinogenesis, although a number of studies suggest that the spontaneous mutations, arising from errors during turnover of undamaged DNA, might represent the crucial initiating events [51]. Nevertheless, other biological pathways have to be triggered in the target tissue, in order to promote and complete the process of neoplastic transformation. Removal of an initiated cell from a control of neighboring cells, activation of a mitogenic pathways or inhibition of programmed cell death represent some of the critical events in chemical carcinogenesis [24]. The genotoxic effects of DBC and its tissue and organ specific derivatives are well documented both in vivo and in vitro [1,10,20,52]. Nevertheless, there is currently little information on effects of DBC and its derivatives on activation of AhR, the major regulator of PAH-metabolizing enzymes, on regulation of cell proliferation/apoptosis or on intercellular communication. The target organ of DBC and DiMeDBC, the liver, consists not only of hepatocytes and supporting nonparenchymal cells, but it also contains a population of progenitor cells, which in case of serious liver injury and inhibition of hepatocyte regenerative capacity, can give rise to oval cells [53]. The stem cell hypothesis of cancer suggests that cancer may arise from maturation block in progenitor cells and that stem or ‘stem-like’ cells are likely to play an important role in carcinogenesis [54,55]. The existence of stem cells in liver has been a matter of long debate, nevertheless it is now generally agreed that oval cells of liver are ‘stem-like’ cells descending from a population of progenitor cells involved in liver regeneration [34,56,57]. The oval cells can develop into both hepatocytes and biliary epithelial cells, and they might play a significant role in hepatocarcinogenesis [41,42,58,59]. Therefore, in the present study, we investigated the effects of DBC and its methylated derivatives on cell proliferation, cell death and GJIC, as well as their impact on AhR activity and expression of metabolizing enzymes in rat liver epithelial WB-F344 cells, which are considered to represent an in vitro model of oval cells [39]. The outcome of PAH effects in rat liver epithelial cells seems to reflect both AhR activation and genotoxic effects, the latter being induced by PAH metabolites [28]. Although the metabolic activation due to activity of enzymes expressed at basal levels might be sufficient for formation of DNA adducts [60], many PAHs and related compounds enhance their own activation via binding to the Ah receptor, which mediates the selective overexpression of particular drug metabolizing enzymes [16]. Induction of AhR gene battery has been also reported to be associated with generation of

oxidative stress, which may further contribute to DNA damage [23]. We found that, in contrast to DBC and N-MeDBC, DiMeDBC is an efficient AhR ligand in rat hepatoma cells (Fig. 3, Table 2), with a relative potency close to that of benzo[a]pyrene [44]. DiMeDBC was the most potent inducer of CYP1A1 mRNA expression in WB-F344 cells among the tested compounds. Only DiMeDBC also induced a significant increase in CYP1A2 mRNA levels, comparable to TCDD in rat liver epithelial cells, although the levels of its mRNA were still significantly lower than, e.g. in rat hepatoma cells (manuscript in preparation). This is in agreement with the study showing that DiMeDBC induces CYP1A2 expression in the adult mice liver, whereas DBC is only a poor inducer of CYP1A1/2 [52]. N-MeDBC, a tissuespecific sarcomagen, does not induce overexpression of CYP1A subfamily isoforms [52]. We found that it did not increase CYP1A2 levels in WB-F344 cells significantly, and it was also a poor inducer of CYP1A1 mRNA in these cells, and only at 10 ␮M concentration (data not shown). Both DBC and DiMeDBC were efficient inducers of CYP1B1 mRNA expression. N-MeDBC is a substrate for both CYP1A1 and CYP1A2, and it induces the highest level of gene mutations and DNA adducts due to activation via CYP1A2, in comparison to the parent molecule and the strict hepatocarcinogen DiMeDBC [17–19,61]. Therefore, its failure to stimulate expression of these enzymes might be responsible for the lack of its effects on p53 phosphorylation, S-phase arrest/delay and apoptosis, observed in the present study. It should be noted, however, that no direct relationship was observed between the capacity of DBC and its methylated derivatives to induce CYP1A1/2 and the tissue specificity of carcinogenesis or DNA binding in liver [52]. This suggests that other enzymes, such as CYP1B1, are also involved in their metabolic activation. The role of CYP1B1 in metabolic activation of dibenzocarbazoles is presently unclear. Nevertheless, a number of studies suggest that both CYP1A1 and CYP1B1 are actively involved in formation of ultimate genotoxic metabolites of PAHs and NHAs [13]. We found that both DBC and DiMeDBC can increase CYP1B1 expression in rat liver epithelial cells, while N-MeDBC only partially upregulated the expression of this enzyme at 10 ␮M concentration. Although CYP1B1 is traditionally viewed as being present mostly in extrahepatic tissues, PAHs are known to increase its levels in rodent liver [62,63] and the present data suggest that it is induced by NHAs in the population of liver progenitor cells. Interestingly, it has been shown that some poor AhR ligands among PAHs, such as dibenzo[a,l]pyrene, can increase CYP1B1 in murine liver, suggesting that


also AhR-independent mechanisms might participate in CYP1B1 regulation [63]. This is supported by the finding that 3-methylcholanthrene increases CYP1B1 expression in liver of AhR knock-out mice [60]. This might explain why DBC and DiMeDBC induced similar levels of CYP1B1 mRNA, despite the latter one being far more potent AhR ligand. In contrast to formation of DNA adducts due to CYP1A1/2 and/or CYP1B1 activity, little is known about the role of dihydrodiol dehydrogenases in metabolic activation of DBC and its derivatives. These monomeric cytosolic NADP(H)-dependent oxidoreductases have been suggested to play a significant role in the metabolism of xenobiotics, including PAHs [64]. Rat AKR1C9 catalyzes oxidation of PAH trans-dihydrodiols to reactive PAH o-quinones, and the formation of reactive and redox active o-quinones has been suggested to contribute to PAH-induced carcinogenesis [15]. A metabolite of DBC, DBC-3,4-dione, has been synthesized and it has been shown to form adducts with nucleic bases or nucleosides in vitro [65]. A liver DNA adduct has been identified in mice treated with DBC-3,4-dione, which is identical to one of the adducts identified in liver of mouse treated with DBC. This seems to suggest that AKR1C9 might participate in DBC metabolic activation. In the present study, both DBC and DiMeDBC were found to increase the expression of AKR1C9 mRNA in rat liver epithelial cells (Fig. 3D). To our knowledge, this is the first evidence that DBC or its derivative can increase AKR1C9 expression in liver cells, indicating that the formation of o-quinones might contribute to cytotoxicity and mutagenicity of DBC and DiMeDBC in liver. The induction of AKR1C9 expression by DBC and its liver-specific derivative, DiMeDBC, suggests that induction of oxidative stress might also contribute to their liver-specific carcinogenicity. It has been suggested that the formation of reactive and redox active o-quinones might lead to formation of reactive oxygen species, such as hydrogen peroxide, superoxide or hydroxyl radical, through redox cycling [15]. The oxidative stress might further contribute both to tumor initiation through inflicting oxidative DNA damage and to tumor promotion by activating specific cell signaling pathways associated, e.g. with regulation of cell proliferation [15]. PAHs and some other chemical carcinogens fail to induce G1 arrest, which has been suggested to increase the likelihood of fixation of mutations [66]. The activation of S-phase checkpoint could be an integral part of cellular response to compounds forming bulky DNA adducts, such as PAH dihydrodiol epoxides [67], while elimination of potential DNA lesions by removing damaged cells through apoptosis is a further option for

53

maintenance of genetic integrity of multicellular organisms [68]. Induction of S-phase arrest and/or apoptosis is observed with a number of genotoxic PAHs, as well as with their proximate carcinogenic metabolites [28,66,69–72]. Both DBC and DiMeDBC induced apoptosis and accumulation of cells in S-phase of cell cycle especially at concentrations 1–10 ␮M after 72 h of treatment (Fig. 5). In contrast, induction of apoptosis or accummulation of cells in S-phase was not observed with N-MeDBC, again suggesting that this compound was not inducing massive DNA damage in WB-F344 cells. In rat liver epithelial cells, both PAHs and heterocyclic aromatic compounds can disrupt cell proliferation/apoptosis balance and stimulate cell proliferation in contact-inhibited cells [28,40], a mode of action typically induced by some tumor promoters [73]. In this study, DiMeDBC significantly increased cell numbers at 1 ␮M concentration, although to a lower extent than TCDD, a perstistent AhR ligand. However, higher concentrations of DiMeDBC failed to potentiate cell proliferation, most probably due to induction of apoptosis. The induction of cell proliferation at lower concentration and induction of apoptosis at higher concentrations of DiMeDBC, might reflect the fact that its high levels saturate DNA repair mechanisms, which consequently leads to accumulation of high levels of DNA adducts [30,74] and activation of apoptosis due to DNA damage. In vivo, the extensive cell death through apoptosis or necrosis may lead to regenerative cell proliferation in the liver, which seems to be a critical event in DiMeDBC-mediated mutagenicity in mice [29–31]. The stimulation of cell proliferation observed at lower dose might thus further contribute to hepatocarcinogenicity of DiMeDBC. Induction of high amounts of DNA adducts can lead to upregulation of p53 tumor suppressor levels and its activity by PAHs [72,75] and DBC [32]. The p53 protein can be post-translationally modified at various amino acid residues. Various studies have shown that phosphorylation of p53 protein at serine 15 may is important for the stabilization and functional activation of p53 during cellular stress [48,70]. We found that both DBC and DiMeDBC but not N-MeDBC significantly increased p53 phosphorylation at serine 15 (Fig. 5). These results strongly support the hypothesis that DBC and DiMeDBC are efficiently metabolized in WB-F344 cells to their proximate genotoxic metabolites, which can then lead to p53 activation, as well as accumulation of cells in S-phase and extensive apoptosis. Many environmentally important PAHs, especially those with lower molecular mass, are potent in vitro inhibitors of GJIC in WB-F344 rat liver epithelial cell line [27]. Disruption of GJIC is supposed to be involved

54


in tumor promotion, as the reduced GJIC is a characteristic feature of tumor cells and a vast majority of tumor promoters inhibit GJIC [24,25]. The parent compound DBC was a powerful inhibitor of GJIC in WB-F344 cells. Its IC50 value approached the efficiency of fluoranthene, the most potent inhibitor of GJIC identified among PAHs so far [26,27]. Unlike other PAHs, which are only transient inhibitors of GJIC, DBC induced a permanent downregulation of GJIC, without signs of recovery for up to 24 h. Nevertheless, this long-term effect might also be associated with induction of apoptosis by this compound. On the other hand, neither DBC nor N-MeDBC were able to fully suppress GJIC; their IC50 values were considerably higher in comparison to DiMeDBC (Table 2). The inhibition of GJIC was not associated with induction of connexin43 hyperphosphorylation. This suggested that the mode of action of DBC differs from that of TPA, a model tumor promoter, which is known to induce connexin43 phosphorylation through activation of protein kinase C and mitogen-activated protein kinases ERK1/2 [49,50]. Taken together, significant differences among the effects of DBC and its two tissue-specific methylated derivatives on rat liver epithelial cells were observed in the present study. The sarcomagenic N-MeDBC was a weak inhibitor of GJIC and a weak inducer of the AhR-mediated activity, which failed to induce expression of enzymes involved in formation of its proximate genotoxic metabolites. It did not induce either apoptosis or a significant accumulation of cells in S-phase, nor did it increase p53 phosphorylation. In contrast, both liver carcinogens, DBC and DiMeDBC, induced p53 phosphorylation, S-phase arrest/delay and apoptosis in rat liver epithelial ‘stem-like’ cells. Nevertheless, there were significant differences among their effects. DBC was an effective GJIC inhibitor, while DiMeDBC was a potent AhR agonist, which induced significantly higher levels of CYP1A1/2 mRNAs and stimulated cell proliferation. Both DBC and DiMeDBC increased expression of CYP1B1 and AKR1C9, which might contribute to their metabolic activation and their hepatocarcinogenicity. Thus, both compounds elicit multiple toxic events associated with carcinogenicity, in this in vitro model of liver progenitor cells. However, it remains to be established, whether the pluripotent oval cells are a target for DBC and DiMeDBC in vivo. Acknowledgments The authors thank Dr. F. Périn, Department of Genotoxicity and Carcinogenicity, Institute Curie, France for providing DBC derivatives. This study was supported

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[63] T. Shimada, K. Inoue, Y. Suzuki, T. Kawai, E. Azuma, T. Nakajima, M. Shindo, K. Kurose, A. Sugie, Y. Yamagishi, Y. FujiiKuriyama, M. Hashimoto, Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice, Carcinogenesis 23 (2002) 1199–1207. [64] M.E. Burczynski, H.K. Lin, T.M. Penning, Isoform-specific induction of a human aldo-keto reductase by polycyclic aromatic hydrocarbons (PAHs), electrophiles, and oxidative stress: implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenase, Cancer Res. 59 (1999) 607–614. [65] W. Xue, A. Siner, M. Rance, K. Jayasimhulu, G. Talaska, D. Warshawsky, A metabolic activation mechanism of 7Hdibenzo[c,g]carbazole via o-quinone. Part 2: covalent adducts of 7H-dibenzo[c,g]carbazole-3,4-dione with nucleic acid bases and nucleosides, Chem. Res. Toxicol. 15 (2002) 915–921. [66] Q.A. Khan, A. Dipple, Diverse chemical carcinogens fail to induce G(1) arrest in MCF-7 cells, Carcinogenesis 21 (2000) 1611–1618. [67] R.S. Weiss, P. Leder, C. Vaziri, Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint, Mol. Cell Biol. 23 (2003) 791–803. [68] C.J. Norbury, B. Zhivotovsky, DNA damage-induced apoptosis, Oncogene 23 (2004) 2797–2808. [69] B.D. Jeffy, E.U. Schultz, O. Selmin, J.M. Gudas, G.T. Bowden, D. Romagnolo, Inhibition of BRCA-1 expression by benzo[a]pyrene and its diol epoxide, Mol. Carcinogenesis 26 (1999) 100– 118. [70] Y.W. Kwon, S. Ueda, M. Ueno, J. Yodoi, H. Masutani, Mechanism of p53-dependent apoptosis induced by 3-methylcholanthrene: involvement of p53 phosphorylation and p38 MAPK, J. Biol. Chem. 277 (2002) 1837–1844. [71] S. Chen, N. Nguyen, K. Tamura, M. Karin, R.H. Tukey, The role of the Ah receptor and p38 in benzo[a]pyrene-7,8dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxideinduced apoptosis, J. Biol. Chem. 278 (2003) 19526–19533. ˇ am, [72] B. Binková, Y. Giguere, P. Rössner Jr., M. Dostál, R.J. Sr´ The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution, Mutat. Res. 471 (2000) 57–70. [73] F. Oesch, A. Schafer, R.J. Wieser, 12-O-tetradecanoylphorbol13-acetate releases human diploid fibroblasts from contactdependent inhibition of growth, Carcinogenesis 9 (1988) 1319–1322. [74] D. Renault, D. Brault, Y. Lossouarn, O. Périn-Roussel, D. TarasValéro, F. Périn, V. Thybaud, Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole, Carcinogenesis 21 (2000) 289–294. [75] M. Ramet, K. Castren, K. Jarvinen, K. Pekkala, T. TurpeenniemiHujanen, Y. Soini, P. Paakko, K. Vahakangas, p53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines, Carcinogenesis 16 (1995) 2117– 2124.

Příloha IV. Valovičová, Z., Marvanová, S., Mészárosová, M., Srančíková, A., Trilecová, L., Milcová, A., Líbalová, H., Vondráček, J., Machala, M., Topinka, J. and Gábelová, A. (2009). Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells. Mutation Research, in press.

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Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells ˇ Marvanová b, Monika Mészárosová a, Annamária Sranˇcíková a, Lenka Trilecová b Zuzana Valoviˇcová a, Sona c , Alena Milcová , Helena Líbalová c, Jan Vondráˇcek b,d, Miroslav Machala b, Jan Topinka c, Alena Gábelová a,∗

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Laboratory of Mutagenesis and Carcinogenesis, Cancer Reserach Institute, Vlárska 7, SAS, 833 91 Bratislava, Slovakia Department of Chemistry and Toxicology, Veterinary Research Institute, 621 00 Brno, Czech Republic c Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, v.v.i. 142 20 Prague, Czech Republic d Laboratory of Cytokinetics, Institute of Biophysics, AS CR, 612 65 Brno, Czech Republic

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Keywords: DNA-adducts DNA strand breaks Oxidative stress Kinetics of DNA repair

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Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC), induced several cellular events that may contribute to tumor promotion in WB-F344 cells, an in vitro model of liver oval cells [J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z. Andrysik, K. Chramostova, S. Marvanova, Z. Valovicova, A. Kozubik, A. Gabelova, M. Machala, 7HDibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stem-like’ cells, Mutat. Res. Fundam. Mol. Mech. Mutagen. 596 (2006) 43–56]. In this study, we focused on the genotoxic effects generated by these dibenzocarbazoles in WB-F344 cells to better understand the cellular and molecular mechanisms involved in hepatocarcinogenesis. Lower IC50 values determined for DBC and DiMeDBC, as compared with N-MeDBC, indicated a higher sensitivity of WB-F344 cells towards hepatocarcinogens. Accordingly, DBC produced a dose-dependent DNA-adduct formation resulting in substantial inhibition of DNA replication and transcription. In contrast, DNA-adduct number detected in DiMeDBC-exposed cells was almost negligible, whereas N-MeDBC produced a low level of DNA-adducts. Although all dibenzocarbazoles significantly increased the level of strand breaks (p < 0.05) and micronuclei (p < 0.001) after 2-h treatment, differences in the kinetics of strand break rejoining were found. The strand break level in DiMeDBC- and N-MeDBCexposed cells returned to near the background level within 24 h after treatment, whereas a relatively high DNA damage level was detected in DBC-treated cells up to 48 h after exposure. Additional breaks detected after incubation of DiMeDBC-exposed WB-F344 cells with a repair-specific endonuclease, along with a nearly 2.5-fold higher level of reactive oxygen species found in these cells as compared with control, suggest a possible role of oxidative stress in DiMeDBC genotoxicity. We demonstrated qualitative differences in the DNA damage profiles produced by hepatocarcinogens DBC and DiMeDBC in WB-F344 cells. Different lesions may trigger distinct cellular pathways involved in hepatocarcinogenesis. The low amount of DNA damage, together with an efficient repair, may explain the lack of hepatocarcinogenicity of N-MeDBC. © 2009 Published by Elsevier B.V.

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Abbreviations: AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; DBC, 7Hdibenzo[c,g]carbazole; DiMeDBC, 5,9-dimethyldibenzo[c,g]carbazole; Fpg, formamidopyrimidine-DNA glycosylase/AP endonuclease; ␥H2AX, histone H2AX phosphorylation; MI, mitotic index; MNi, micronuclei; MTT, methyl thiazolyl blue tetrazolium bromide; N-MeDBC, N-methyldibenzo[c,g]carbazole; ROS, reactive oxygen species; SCGE, single-cell gel electrophoresis. ∗ Corresponding author. E-mail address: [email protected] (A. Gábelová).

1. Introduction

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7H-Dibenzo[c,g]carbazole (DBC) (Fig. 1), a potential human carcinogen (Group 2B) [1,2], is a ubiquitous environmental pollutant. This agent has been found in various complex mixtures of organic compounds resulting from incomplete combustion of organic materials such as soot and tars [3], diesel engine exhaust [4], synthetic fuel material [5], coal and oil processing [6], and tobacco smoke [7]. DBC has been shown to be a potent multi-species and

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0027-5107/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2009.02.014

Please cite this article in press as: Z. Valoviˇcová, et al., Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2009), doi:10.1016/j.mrfmmm.2009.02.014

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DBC (CAS No. 194-59-2), DiMeDBC and MeDBC were kindly provided by Dr. Francois Périn (Institute Curie, France). Benzo[a]pyrene (B[a]P, CAS No. 50-32-8), methyl thiazolyl blue tetrazolium bromide (MTT, CAS No. 298-93-1), and 4 ,6diamidino-2-phenylindole (DAPI, CAS No. 28718-90-30), anti-␤-actin, Clone AC-15, anti-Mouse IgG, spleen phosphodiesterase, RNases A and T1, proteinase K, micrococcal nuclease and nuclease P1 were purchased from Sigma–Aldrich (Deisenhofen, Germany). T4 polynucleotide kinase was from USB (Cleveland, OH, USA); ␥-32 PATP (3000 Ci/mmol, 10 ␮Ci/␮L) from GE Healthcare (Little Chalfont, UK); and 0.1 mm polyethylene-imine cellulose thin-layer chromatography (TLC) plates from Macherey-Nagel (Düren, Germany). P-H2AX (Ser 139) was from Upstate (Lake Placid, NY, USA), peroxidase-conjugated swine antirabbit immunoglobulin antisera was from Sevapharma (Czech Republic), ECL Plus reagent was from GE Healthcare (Little Chalfont, UK), and aflatoxin B1 (AFB1, CAS No. 1162-65-8) from Serva (BioTech, Slovakia). Stock solutions of DBC, MeDBC, DiMeDBC, B[a]P and AFB1 in dimethyl sulfoxide (DMSO; 2 mM) were kept at −20 ◦ C and diluted immediately before use in DMSO. Media, fetal calf serum (FCS) and other chemicals used for cell cultivation were purchased from Gibco (KRD Limited, Slovakia). All other chemicals and solvents were of high-performance liquid chromatography (HPLC) or analytical grade.

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2.2. Cell culture

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WB-F344 cells were kindly provided by Dr. J. E. Trosko (MSU, East Lansing, MI, USA). They were cultivated in modified Eagle’s minimum essential medium (MEM) with 50% increased concentrations of essential and non-essential amino acids, and supplemented with sodium pyruvate (110 mg/L), 10 mM HEPES, 10% FCS and antibiotics (penicillin 200 U/mL, streptomycin and kanamycin 100 ␮g/mL). Cells were cultured in a humidified atmosphere of 5% CO2 at 37 ◦ C.

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2.3. Treatment of cells

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Exponentially growing WB-F344 cells were exposed to carcinogens for 2 h and 24 h depending on the endpoint. Stock solutions (2 mM) of DBC, N-MeDBC, DiMeDBC, AFB1 and B[a]P in DMSO were further diluted to reach the final concentrations (DBC, MeDBC, DiMeDBC, B[a]P: 0.1–100 ␮M, and AFB1 0.001–1 ␮M). The final concentration of vehicle (DMSO) never exceeded 0.5% (v/v) in any of the samples; control cells (negative control) were therefore exposed to 0.5% DMSO. At treatment end, cells were washed twice with culture medium and then processed immediately, or incubated in fresh medium for different time intervals, and then processed.

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Viability determination was carried out in plastic 96-well cell culture cluster plates at 5 × 103 cells/well and photometric evaluation (at 540 nm excitation and 690 nm emission wavelengths) using the Multiskan Multisoft plate reader (Labsystems, Finland) and Genesis software provided by the producer. IC50 values were calculated from the dose–response curves using CalcuSyn software (Biosoft, Cambridge, UK).

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multi-site carcinogen, with local and systemic effects (reviewed in [8]). DBC carcinogenicity was demonstrated in different species (mouse, rat, hamster, and dog) and tissues (skin, lung, forestomach, and urinary bladder), with the liver being the prime target organ after DBC administration via different routes. In addition to hepatocellular adenoma and carcinoma, severe hepatocellular toxicity, including biliary hyperplasia, has been observed in livers taken from DBC-treated mice [9–11]. The synthetic methyl derivative of DBC and 5,9dimethyldibenzo[c,g]carbazole (DiMeDBC), is one of the most efficient strictly organ-specific carcinogens. This derivative is devoid of activity in the skin, but produces multiple malignant liver tumors with lung metastases in 100% of animals after subcutaneous injection or skin application [10,12,13]. In contrast to DBC, sex dimorphism in response to DiMeDBC treatment was observed in mice; males were much less sensitive than females to this derivative [14]. The strict hepatocarcinogen DiMeDBC is remarkably less hepatotoxic than DBC; no histologically detectable toxicity has been found in the liver at lower (10 mg/kg) concentration [14]. At high concentration (90 mg/kg), an early hepatocellular degeneration/necrosis was observed, mostly in centrilobular areas [15]. In the liver, DBC and DiMeDBC produce tumors, DNA adducts and gene mutations [13,16]. At equimolar concentrations, DiMeDBC induces fewer mutations and DNA adducts than the parent compound [17], which is probably due to a reduced access to the NH group for drug metabolizing enzymes, caused by a steric hindrance of two methyl groups at the C5 and C9 positions [16]. The heterocyclic nitrogen is supposed to have an important role in liver carcinogenicity and strongly affects the biological activity of DBC. Substitution of methyl groups at the C6 and C8 positions or directly at the heterocyclic nitrogen resulted in the loss of activity in the liver [16,18]. The synthetic methyl derivative Nmethyldibenzo[c,g]carbazole (N-MeDBC) induces sarcomas, respiratory tumors and papillomas, but lacks hepatocarcinogenic potential [19,20]. Analogs of DBC bearing sulphur, oxygen or carbon in place of nitrogen also do not demonstrate carcinogenic activity [21]. Cell lines established from a particular tissue are a valuable tool for better understanding of cellular and molecular mechanisms which may underlie the tissue specificity of chemical carcinogens. The diploid rat epithelial cells WB-F344 are considered to be an in vitro model of liver oval cells [22]. The latter can proliferate when regenerative hepatocyte proliferation is compromised, and can differentiate into hepatocytes [23] and biliary epithelial cells [24]. There is increased evidence that the small epithelial oval-shaped (hepatic progenitor) cells are, in addition to hepatocytes, a possible target cell population for hepatocarcinogenesis [25,26]. These cells have been observed in various models of rodent experimental carcinogenesis and human liver diseases associated with an increased incidence of hepatocellular carcinoma or cholangiocarcinoma [27]. Experiments with polycyclic aromatic and heterocyclic aromatic hydrocarbons have shown that the WB-F344 cell line is a useful tool for analysis of cellular and molecular mechanisms involved in toxic effects of environmental carcinogens [28–31].

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Fig. 1. Chemical structure of 7H-dibenzo[c,g]carbazole.

In our recent study, the liver carcinogens DBC and DiMeDBC significantly affected cellular pathways associated with tumor promotion in WB-F3444 cells [32]. DBC was an efficient inhibitor of gap-junctional intercellular communication (GJIC), and DiMeDBC manifested the strongest aryl hydrocarbon receptor (AhR)mediated activity. The tissue-specific sarcomagen N-MeDBC failed to substantially affect these cellular events associated with tumor promotion. DBC and DiMeDBC, in contrast to N-MeDBC, induced p53 phosphorylation, S-phase delay of cell cycle and apoptosis [32]. The differences in cell response to DBC and DiMeDBC treatment suggested that these liver carcinogens may produce qualitatively (or at least quantitatively) different DNA lesions in WB-F344 cells. Because the genotoxic potential of DBC, DiMeDBC and N-MeDBC has not been studied in liver progenitor cells or in their models such as WB-F344 cells, the primary objective of this work was to assess the DNA damage profile generated by individual dibenzocarbazoles in this liver “stem-cell like” cell line. In addition to clastogenic effects (DNA adducts, DNA breakage, micronuclei), cellular responses (DNA repair kinetics, induction of oxidative stress, histone H2AX phosphorylation) were investigated to obtain a more comprehensive picture of the genotoxic effects generated by the tissue-specific dibenzocarbazole derivatives in the WB-F344 cell line.

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DNA was isolated using RNases A and T1 and proteinase K treatment followed by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation [33]. DNA concentrations were estimated spectrophotometrically by measuring UV absorbance at 260 nm. DNA samples were stored at −80 ◦ C until analysis. 32 P-Postlabelling analyses were done as previously described [34]. Briefly, DNA samples (exact amount of DNA was 6 ␮g) were digested by a mixture of micrococcal nuclease and spleen phosphodiesterase for 4 h at 37 ◦ C. The nuclease P1 procedure was used for adduct enrichment. Adducted nucleotides were enzymatically labelled using ␥-32 P-ATP and T4 polynucleotide kinase, and separated by multidirectional polyethylenimine–cellulose thin-layer chromatography (TLC). The solvents used were: D1, 1 M sodium phosphate, pH 6.8; D2, 3.54 M lithium formate, 8.5 M urea, pH 3.5; D3, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0; D4 = D1, same direction as D3. After screen-enhanced autoradiography at −80 ◦ C, the distinct DNA adduct spots were cut out and evaluated by measuring 32 P-radioactivity using liquid scintillation spectroscopy. To determine the exact amount of DNA in each sample, aliquots of the enzymatic DNA digests were analyzed for nucleotide content by reverse-phase HPLC with UV detection, which simultaneously allowed for controlling DNA purity. DNA adduct levels were expressed as adducts per 108 nucleotides. A BPDE-derived DNA adduct standard derived from the liver of rats treated with benzo[a]pyrene (100 mg, i.p.) was run in triplicate to control inter-assay variability and to normalize calculated DNA adduct levels. Data are mean values of total DNA adducts derived from at least three biological replicates.

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Exponentially growing WB-F344 cells were exposed to dibenzocarbazoles (1–10 ␮M) or positive controls for 2 h. After treatment, fresh medium containing [14 C]thymidine (1 ␮Ci/mL) or [14 C]uridine (1 ␮Ci/mL) or [14 C]-l-leucine (0.2 ␮M/mL) was added to all dishes. At different time intervals (30 min, 60 min, 90 min, 120 min, 180 min, and 300 min), cells were rinsed with SSC buffer (0.15 M sodium chloride, 0.015 M sodium citrate) and ice-cold 5% trichloroacetic acid (TCA) was added. The next day, cells were harvested and filtered through a membrane (0.45 ␮m pore), washed and dried. Radioactivity was measured on a liquid scintillation counter Beckman LS 1801.

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The procedure of Singh et al. [35], modified by Collins et al. [36] and Gabelova et al. [37] was followed. In brief, liver cells embedded in 0.75% LMP agarose and spread on a base layer of 1% NMP agarose in PBS buffer (Ca2+ and Mg2+ free) were placed in a lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, pH 10 and 1% Triton X-100) at 4 ◦ C for 1 h. In experiments focused on detection of oxidative DNA damage, slides were washed three times for 5 min in endonuclease buffer (40 mM HEPES–KOH, 0.1 M KCl, 0.5 mM EDTA, pH 8.0) and incubated with formamidopyrimidine–DNA glycosylase/AP nuclease (Fpg; 30 min) at 37 ◦ C. Slides were transferred to an electrophoretic box and immersed in an alkaline solution (300 mM NaOH, 1 mM Na2 EDTA, pH > 13). After 40 min unwinding time, a voltage of 25 V (300 mA) was applied for 30 min at 4 ◦ C. Slides were neutralized with 3× 5 min washes with Tris–HCl (0.4 M, pH 7.4), and stained with 20 ␮L of ethidium bromide (EtBr, 10 ␮g/mL). EtBr-stained nucleoides were examined with an Olympus BX51 fluorescence microscope by image analysis using Komet 5.0 (Kinetic Imaging, Ltd., Liverpool, UK) software. The percentage of DNA in the tail (% tail DNA) was used as a parameter for measurement of DNA damage (DNA strand breaks). One hundred nucleoids were scored per each sample in one electrophoretic run.

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Confluent WB-F344 cells were exposed to test compounds for 24 h. Hydrogen peroxide (exposure, 5 min) was the positive control. After exposure, cells were washed twice with PBS, trypsinized, centrifuged, and resuspended with Hank’s balanced salt solution with 5% heat-inactivated fetal bovine serum (FBS). The cell suspension was incubated for 15 min with the fluorescent probe 2 ,7 -dichlorofluorescein diacetate (DCFH-DA, 20 ␮M). Cells were washed again, centrifuged, and cooled on ice (except hydrogen peroxide-exposed cells). The fluorescence of dichlorofluorescein (DCF) was analyzed on FACSCalibur (at 505 nm excitation and 535 nm emission wavelengths) and analyzed with CellQuest software (Becton Dickinson, San Jose, CA, USA).

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Two sampling time intervals, 24 h and 48 h after treatment, were used to determine the number of micronuclei (MNi) induced by individual dibenzocarbazole derivatives after cell exposure for 2 h. WB-F344 cells were washed with 0.9% NaCl, incubated in mild hypotonic solution (0.075 M KCl/0.9% NaCl, 1:19) for 10 min at 37 ◦ C, fixed with methanol–glacial acetic acid (3:1) for 15 min at 37 ◦ C, rinsed with distilled water and air dried. Fixed cells were stained with DAPI (2 ␮g/mL) for 30 min in the dark at room temperature, rinsed with McIlvaine’s buffer and distilled water, and dried and mounted with glycerol. MNi were identified based on the criteria

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specified by Miller et al. [38]. Proliferation status (mitotic index, MI) of WB-F344 cells was measured according to Eckl and Raffelsberger [39]; cell death (apoptosis and necrosis) was determined using morphological criteria (fragmentation of nuclei) after Oberhammer et al. [40]. Two thousand cells per dish were analyzed using the fluorescence microscope Olympus BX51. Data are mean ± S.D. of at least two parallel dishes per one experiment from three independent experiments.

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Confluent WB-F344 cells were exposed for 24 h to test compounds or to 0.1% DMSO as vehicle control. The effects of test compounds on histone H2AX phosphorylation were determined in whole-cell lysates prepared by harvesting cells in lysis buffer (1% sodium dodecyl sulphate (SDS), TRIS, 10% glycerol, protease inhibitor cocktail). Total protein concentrations were determined with DC Protein Assay (BioRad, Hercules, CA, USA). For Western blot analyses, equal amounts of total protein were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), electrotransferred onto Hybond-P, immunodetected using appropriate primary and secondary antibodies, and visualized by ECL Plus reagent according to manufacturer’s instructions. Determination of actin level was used to confirm equal protein loading. Densitometric analyses using ImageJ 1.38e software (http://rsb.info.nih.gov/ij/) was employed to measure the intensity of bands corresponding to the phosphorylated H2AX (␥H2AX) form. The fold of ␥H2AX expression was estimated semi-quantitatively as the ratio of induced to control level of band density.

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Data are given as mean values with ±S.D. The differences between treated samples and untreated control were evaluated by the Student’s t-test. The threshold of statistical significance was set at p < 0.05.

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3.1. Cytotoxicity of DBC and its derivatives in the rat liver progenitor WB-F344 cells

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Cell viability after exposure to dibenzocarbazoles and the positive controls B[a]P and AFB1 was measured after short-term (2 h) and long-term (24 h) cell exposure. Equimolar concentrations ranging from 5 ␮M to 100 ␮M were used for dibenzocarbazole and B[a]P, whereas 0.05–10 ␮M were chosen for AFB1. The parameter IC50 was applied to compare the cytotoxicity of chemicals under study. Substantial differences in IC50 values between hepatocarcinogenic and sarcomagenic carcinogens have been assessed (Table 1). Considerable lower IC50 values were determined for the liver carcinogens

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Table 1 The IC50 values of DBC, DiMeDBC, N-MeDBC, B[a]P and AFB1 after 2 h and 24 h cell treatment. IC50 values were calculated using the CalcuSyn software.

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Table 2 The relative DNA adduct levels detected in WB-F344 cells exposed to DBC, N-MeDBC, DiMeDBC and B[a]P for 24 h. Compounds

Conc. [␮M]

DNA adducts/108 nucleotides

DBC

1 10

29.6 ± 10.6 56.3 ± 10.4

DiMeDBC

1 10

0.2 ± 0.4 0.5 ± 0.3

N-MeDBC

1 10

0.8 ± 0.4 3.0 ± 1.8

1

27.6 ± 1.5

B[a]P

Data represent the mean ± S.D. from at least two independent experiments on each triplicate of sheets.


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Fig. 2. Representative autoradiograms of 32 P-labeled DNA isolated from rat liver epithelial WB-F344 cells incubated with vehicle (0.5% DMSO) (A), 1 ␮M B[a]P (B), 1 ␮M and 10 ␮M DBC (C and D), 1 ␮M and 10 ␮M DiMeDBC (E and F), and 1 ␮M and 10 ␮M N-MeDBC (G and H). Chromatographic conditions (see Section 2) were the same for all compounds. Film exposure was followed: control, B[a]P and DBC 24 h, DiMeDBC and N-MeDBC 72 h.

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DBC and DiMeDBC, as well as the positive control AFB1 (IC50 : 48 ␮M, 30 ␮M, and 2.2 ␮M, respectively) compared with B[a]P and tissue-specific sarcomagen N-MeDBC (IC50 > 100 ␮M for both agents) after 2 h exposure. Although long-term treatment (24 h)

increased the cytotoxicity of all carcinogens under study, N-MeDBC was less cytotoxic (IC50 > 50 ␮M) even after 24 h treatment, as compared with DBC and DiMeDBC (IC50 : 11 ␮M and 9 ␮M, respectively). These preliminary experiments suggested a higher sensitivity of


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the liver progenitor cells towards both hepatocarcinogens, DBC and DiMeDBC.

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3.2. Formation of DNA adduct by DBC and its derivatives

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3.4. DNA strand breaks and the kinetics of DNA repair

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DNA strand breaks are readily detected as the cellular response to exposure; therefore DNA strand break formation has been proposed to be a standard biomarker of DNA damage. The capacity of dibenzocarbazoles to generate DNA breaks was measured within the concentration range 0.1–20 ␮M (Fig. 4). All dibenzocarbazoles, regardless of their tissue specificities, induced significant levels of strand breaks in exposed cells (p < 0.05 to p < 0.001). Although DBC appeared to be the most efficient producer of DNA strand breaks, the level of breaks induced by this agent was not significantly different from DiMeDBC or N-MeDBC. In contrast, substantial differences in the kinetics of DNA strand break rejoining were determined in cells exposed to dibenzocarbazoles (Fig. 5). Relatively fast removal of strand breaks was observed in DiMeDBC-treated cells; the level of strand breaks reached the steady-state level within 16 h after treatment. A significant delay in DNA strand break rejoining was detected in DBC-treated cells; a substantial number of strand breaks was observed even 48 h after treatment. The level of breaks produced by N-MeDBC reached the background level within 24 h after exposure.

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3.5. Clastogenic activity of DBC and its derivatives

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Micronucleus formation has become an important endpoint in genotoxicity studies because a positive correlation exists between

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DNA adducts resulting from covalent binding of chemicals to DNA are a critical event in the initiation of cancer [41]. Therefore the ability of DBC, DiMeDBC and N-MeDBC to produce DNA adducts in the rat liver oval WB-F344 cells was evaluated at 1 ␮M and 10 ␮M. Based on our previous study [42], long-term (24 h) cell treatment was applied to assess the level of stable DNA adducts in exposed cells. Representative autoradiograms of DNA adducts formed in WB-F344 cells treated with individual compounds under study are shown in Fig. 2. Substantial differences in DNA adduct patterns were determined among individual dibenzocarbazoles. The DBC “fingerprint” was difficult to analyze due to close marked spots (Fig. 2C and D), while only two weak (but distinct) spots were found in cells exposed to the organ-specific hepatocarcinogen DiMeDBC (Fig. 2E and F). In contrast, four clear spots were detected in N-MeDBC chromatograms (Fig. 2G and H). B[a]P, the positive control, produced one dominant spot representing a well-known BPDE-DNA adduct (Fig. 2B) and no DNA adducts were found in control cells (Fig. 2A). At equimolar (1 ␮M) concentrations, DBC induced a comparable levels of DNA adducts as the reference compound B[a]P, whereas the DNA-adduct level generated by DiMeDBC at 1 ␮M and 10 ␮M was very low, close to the detection limit (Table 2). The DNA adduct level produced by N-MeDBC was also very low, nearly 40-fold lower than that of DBC. To elucidate and verify the inability of DiMeDBC to produce DNA adducts in WB-F344 cells, a time-course study was undertaken. DiMeDBC-treated cells were harvested at 2 h time intervals during exposure for 24 h, and DNA-adducts measured by 32 P-postlabelling. No DiMeDBC-related adducts were found at early time points, suggesting that the lack of DNA adducts in DiMeDBCexposed cells was not due to rapid elimination of DNA bulky adducts during 24-h treatment (data not shown). Because the cytotoxic effects of DiMeDBC were comparable with those of DBC, further experiments were undertaken to clarify the mechanism(s) which may underlie the toxic activity of DiMeDBC in WB-F344 cells.

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(Fig. 3A and B), while proteosynthesis was less affected (Fig. 3C). In contrast, neither DiMeDBC nor N-MeDBC, as well as the positive controls B[a]P and AFB1, significantly influenced the course of synthesis of macromolecules under identical treatment conditions (data not shown).

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3.3. The effect of DBC and its derivatives on the replication and transcription of DNA and proteosynthesis

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Fig. 3. The course of synthesis of DNA (A), RNA (B) and proteins (C) in WB-F344 cells after treatment with DBC. Cells were exposed to DBC at 1–10 ␮M for 2 h. The intensity of synthesis of macromolecules was analyzed at several time intervals after treatment. Each point represents the mean from at least two independent experiments.

Based on our previous study [42] and MTT assay results (Table 1), the short-term (2 h) treatment interval was chosen to evaluate the genotoxic effects of dibenzocarbazoles induced in WB-F344 cells. The effect of dibenzocarbazoles on replication (DNA synthesis), transcription (RNA synthesis) and proteosynthesis was assessed at concentrations 1–10 ␮M after 2 h cell exposure. DBC caused a substantial dose-dependent delay of DNA and mainly RNA syntheses during 5 h post-cultivation of WB-F344 cells in fresh medium

Fig. 4. Detection of DNA damage in WB-F344 cells exposed to DBC, DiMeDBC and N-MeDBC for 2 h by the alkaline SCGE. Cells were treated with equimolar concentrations 0.1–20 ␮M and DNA breakage was analyzed immediately after treatment. The bars represent the mean ± S.D. from three independent experiments on each triplicate of slides. The data were analyzed statistically by Student’s t-test. Significantly different from the control, *p < 0.05; **p < 0.01; ***p < 0.001.


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Table 3 The frequency of micronuclei induced by DBC, N-MeDBC, DiMeDBC, AFB1 and B[a]P in WB-F344 cells after 2 h treatment and the mitotic index of exposed cells determined 24 h and 48 h after cell exposure. Compound

Conc. [␮M]

Micronuclei (%)

Mitotic index (%)

24 h

48 h

24 h

48 h

3.8 ± 0.7

4.1 ± 1.4

4.4 ± 1.0

***

***

2.7 ± 0.3

DBC

0.5 1 2.5

6.3 ± 1.8 8.0 ± 1.5*** 8.4 ± 1.1***

8.2 ± 1.1 8.8 ± 0.9*** 11.0 ± 1.1***

4.1 ± 1.4 5.8 ± 1.5 5.4 ± 1.8

6.1 ± 2.0 7.0 ± 2.0 7.7 ± 1.3

DiMeDBC

0.5 1 2.5

6.6 ± 1.4*** 6.2 ± 1.1*** 6.9 ± 1.7***

8.3 ± 1.4*** 8.8 ± 1.0*** 8.5 ± 1.5***

6.4 ± 1.6 5.6 ± 0.2 6.2 ± 2.2

5.9 ± 1.1 8.0 ± 1.0 7.0 ± 2.0

N-MeDBC

0.5 1 2.5

6.1 ± 1.2*** 7.7 ± 1.1*** 6.5 ± 1.3***

7.3 ± 2.0** 9.3 ± 1.7*** 8.2 ± 1.4***

6.0 ± 2.0 6.9 ± 1.5 5.6 ± 1.4

5.7 ± 1.8 7.9 ± 2.3 6.6 ± 1.2

B[a]P

0.5 1 2.5

10 ± 0.9*** 10.7 ± 1.3*** 13.1 ± 0.9***

9.3 ± 1.1*** 9.2 ± 1.3*** 11.3 ± 1.9***

7.0 ± 0.8 6.4 ± 1.4 6.0 ± 2.0

4.2 ± 1.9 5.1 ± 1.7 5.4 ± 1.3

AFB1

0.1 0.5 1

4.4 ± 0.7*** 5.0 ± 1.0*** 5.3 ± 0.5***

6.0 ± 0.8*** 6.0 ± 0.6*** 6.6 ± 0.5***

5.1 ± 1.3 4.8 ± 1.1 5.3 ± 1.7

4.5 ± 1.16 4.9 ± 2.2 5.9 ± 1.6

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Data represent the mean ± S.D. of at least three independent experiments on each of two slides. ** Significantly different from control group at p < 0.01. *** Significantly different from control group at p < 0.001.

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3.6. Oxidative DNA damage and oxidative stress

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carcinogenicity and clastogenicity of chemical agents [43]. The clastogenic activities of dibenzocarbazoles and the reference compound B[a]P were assessed in concentration range 0.5–2.5 ␮M, whereas concentrations 0.1–1 ␮M were used for AFB1. WB-F344 cells were exposed to dibenzocarbazoles and positive controls for 2 h. The frequency of MNi and MI were assessed in WB-F344 cells at 24 h and 48 h after treatment (Table 3). In agreement with experiments focusing on the strand break formation, all dibenzocarbazoles induced a significant level of MNi compared with control cells. A dose-dependent increase of MNi was detected in DBC-treated cells at both sampling times (r = 0.802 and 0.880, respectively), but the rise in MNi generated by the strict hepatocarcinogen DiMeDBC and the tissue-specific sarcomagen N-MeDBC were less significant (DiMeDBC: r = 0.687 and 0.624; N-MeDBC: r = 0.576 and 0.629, respectively). B[a]P, the positive control, was the most potent inducer of MNi in WB-F344 cells. Under these treatment conditions, no substantial variation in the frequency of apoptotic and necrotic cells was determined in WB-F344 cells due to exposure to chemicals under study (data not shown). A trend towards increased MI that did not reach a statistical significance was found in exposed cells (Table 3).

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Fig. 5. The kinetics of DNA strand break rejoining in WB-F344 cells exposed to equimolar (1 ␮M) DBC, DiMeDBC and N-MeDBC for 2 h. DNA strand break level was measured at several time intervals after treatment. The columns represent the mean ± S.D. from three independent experiments on each of triplicate of slides. Data were analyzed by Student’s t-test. Significantly different from the control, *p < 0.05; ***p < 0.001.

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It is generally accepted that accumulation of oxidative damage to DNA is implicated in various human diseases, including cancer and aging [44]. In chemical carcinogenesis, highly redoxactive molecules produced during biotransformation of organic compounds can contribute to oxidative stress generation resulting in oxidative damage to DNA. To evaluate the mechanism by which the strict hepatocarcinogen DiMeDBC could mediate genotoxic effects (strand breaks, MNi) in WB-F344 cells, the modified SCGE assay was utilized [36]. This modification involves incubation of DNA with specific DNA repair endonuclease, the Fpg protein, which cleaves DNA in the site of base modifications, i.e., fragmented (FAPY-adducts) and oxidized (8-oxodG) purines. A slight but statistically significant level of Fpg-sensitive sites was determined in DiMeDBC-treated cells (Fig. 6A). Neither DBC nor N-MeDBC produced a significant level of base modifications in exposed cells. The kinetics of Fpg-sensitive sites removal produced by DiMeDBC was relatively slow; a detectable level of DNA breaks was determined up to 14 h after treatment (Fig. 6B). To verify the capacity of dibenzocarbazoles to induce oxidative stress and generate ROS which may cause oxidative DNA lesions, flow cytometric analysis of ROS production was carried out (see Section 2). WB-F344 cells were exposed to dibenzocarbazoles for 24 h; hydrogen peroxide (5 min, on ice) was the positive control in these experiments. At equimolar concentration (1 ␮M), DiMeDBC was the most potent producer of ROS compared with DBC and N-MeDBC (Table 4). Although the ROS level generated by DiMeDBC was lower than that produced by the positive control, it might still be sufficient to induce oxidative DNA damage in exposed cells.

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3.7. Phosphorylation of histone H2AX by DBC and its derivatives

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Phosphorylation of histone H2AX (termed ␥H2AX) represents one of an early events occurring in cells due to genotoxic stress [45]. ␥H2AX accumulation was measured after 24 h exposure to dibenzocarbazoles at 0.1–10 ␮M (Fig. 7). Dibenzo[a,l]pyrene, the strongest known genotoxin among polycyclic aromatic hydrocarbons (PAHs) was the positive control in these experiments. Exposure of WBF344 cells to the liver carcinogens DBC and DiMeDBC resulted in


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Treatment

[%]

DMSO DBC DiMeDBC N-MeDBC H2 O2

24 h 24 h 24 h 24 h 5 min

100 166 ± 12.0* , a 295 ± 61.7* 205 ± 32.3* 707 ± 77.3* , a

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Compounds

Data represent the mean ± S.D. of three independent experiments. a Significantly different from DiMeDBC at p < 0.05. * Significantly different from control (DMSO) at p < 0.05.

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a significant induction of histone H2AX phosphorylation; up to fivefold increase of ␥H2AX was determined at the highest concentration (Fig. 7). Only a weak increase in ␥H2AX level was detected in cells exposed to the tissue-specific sarcomagen N-MeDBC.

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4. Discussion

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This study is a follow-up of a previous investigation which suggested that the stem cell-like rat liver epithelial cells may be a potential target cell population for the liver carcinogens DBC and DiMeDBC [32]. Variability in cellular processes triggered by these agents in WB-F344 cells suggested differences in DNA lesions produced by DBC and DiMeDBC. Because the genotoxic effects of dibenzocarbazole derivatives have not been studied in this type of liver cells, this work was initiated as part of ongoing research devoted to understanding of molecular and cellular mechanisms involved in chemical hepatocarcinogenesis. A higher susceptibility of WB-F344 cells towards the liver carcinogens, as compared with sarcomagens, was observed after 2 h treatment (Table 1). The 24-h exposure to the systemic carcinogen DBC resulted in a significant, dose-dependent increase of DNA adducts (Fig. 2C and D). At equimolar (1 ␮M) concentration, the DNA adduct level was comparable with that of B[a]P (Table 2). The DNA adduct pattern produced by DBC in WB-F344 cells was similar to that found in human hepatoma HepG2 cells [46], but distinct from the one detected in mouse liver cells in vivo [8,16,18]. These observations seem to imply that additional drug-metabolizing enzymes expressed in the liver may be involved in DBC metabolism. Based on the differences in tissue distribution patterns of DBC-DNA-adducts detected in vivo [16,18] and in vitro [18,47,48], two metabolic pathways have been proposed for DBC: (i) activation involving the ring-carbon atoms, as is the case of PAHs, and (ii) metabolism at the pyrrolic NH group [8,19,49]. Although stable DNA adducts are supposed to be major DNA lesions responsible for the carcinogenicity of this agent in vivo [50], DBC, which has a relatively low ionization potential [51], can also form unstable, depurinating DNA adducts through one-electron oxidation mediated by radical cation pathway [52]. Aldo–keto reductases (AKR), which compete with cytochrome P450 (CYP) enzymes, can play a significant part in DBC metabolism [53,54]. Phenols are the major DBC metabolites found in vitro using mouse and rat liver microsomes [55–57] as well as human CYP1 enzymes [58]. The proximate carcinogens 3-OH-/4-OH-DBC [59] may be further activated to yield DBC-o-quinones via the AKR pathway, which can form stable and unstable DNA adducts. o-Quinones were also identified as a minor component of DBC metabolites in vitro [55]. DBC-3,4-dione is supposed to be the ultimate DBC metabolite [53]. DNA lesions produced by DBC resulted in a substan-

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Table 4 The relative level of reactive oxygen species (ROS) generated by equimolar (1 ␮M) DBC, DiMeDBC and N-MeDBC concentration in WB-F344 cells after 24 h treatment. Hydrogen peroxide (250 ␮M, 5 min treatment) was used as the positive control.

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Fig. 6. The level of oxidative DNA damage detected in WB-F344 cells exposed to equimolar (1 ␮M) DBC, DiMeDBC and N-MeDBC concentration for 2 h (A) and the kinetics of oxidative DNA damage repair (B). Opened portions of the bars represent DNA strand breaks (strand breaks and alkali-labile sites) detected immediately after treatment in the absence of Fpg endonuclease, the filled portion of the bar represent additional DNA strand breaks detected in the presence of Fpg endonuclease (Fpg-sensitive sites). The columns represent the mean ± S.D. from at least two independent experiments on each triplicate of slides. The kinetics of Fpg-sensitive site removal induced by DiMeDBC in WB-F344 cells was analyzed at several time intervals after treatment. Points represent the mean ± S.D. from at least two independent experiments on each triplicate of slides. Data were analyzed statistically by Student’s t-test. Significantly different from the control, *p < 0.05; ***p < 0.001; significantly different from DiMeDBC-treated cells in the absence of Fpg endonuclease; a p < 0.01.

Fig. 7. Histone H2AX phosphorylation (␥H2AX) detected in WB-F344 cells exposed to equimolar (0.1 ␮M, 1 ␮M and 10 ␮M) DBC, DiMeDBC, N-MeDBC, 0.1 ␮M DB[a,l]P (positive control), and 0.1% DMSO (negative control) for 24 h. For Western blot analyses, equal amounts of total protein were subjected to 10% SDS PAGE mini gel. Actin was used as loading control. For immunodetection, appropriate primary and secondary antibodies were utilized. RI—the relative band intensity, the ratio of induced to control level of band density. All RI values were checked and corrected relative to actin levels.


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cause a shift in the cellular redox balance, resulting in cellular oxidative stress [68,69]. Oxidative DNA damage produced by DiMeDBC may lead to a replication fork collision resulting in histone H2AX phosphorylation (Fig. 7). H2AX, a variant of histone H2A, is a critical factor for cellular protection [70]. It is rapidly phosphorylated at serine 139 in response to DNA double strand breaks [71], replication arrest [72], apoptosis [73], transcription inhibition [74], and oxidative stress [75]. Although the level of stable adducts determined in N-MeDBCtreated WB-F344 cells was low (Fig. 2G and H, Table 2), it was probably sufficient to produce DNA strand breaks and MNi (Fig. 4, Table 3). DNA strand break formation has been proposed to be a standard biomarker of DNA damage; however, this parameter is probably not specific enough to identify differences in tissue specificity of chemical compounds. DNA strand breaks are formed as a consequence of various events; they are induced directly by the agent, produced due to DNA damage removal in the process of DNA repair or can result spontaneously by release of unstable DNA adducts leading in alkali labile sites. DNA lesions generated by NMeDBC were efficiently repaired; no DNA strand breaks were found in N-MeDBC-treated cells 24 h after exposure (Fig. 5). Efficient elimination of N-MeDBC metabolites via conjugation reactions cannot be excluded, as, e.g., Perin et al. [49] showed that high N-MeDBC mutagenicity in vitro due to activation by pure microsomes was significantly reduced in the presence of cytosolic fraction. Despite the lack of biological activity of this tissue-specific sarcomagen in the liver [76]; a low level of DNA adducts (a level ∼300-fold lower than that produced by DBC) was detected in mouse liver exposed to N-MeDBC [20]. Contrary to DBC and DiMeDBC, N-MeDBC did not exhibit cytotoxicity (Table 1), cell-cycle arrest or apoptosis in WB-F344 cells [32]. No significant histone H2AX (Fig. 7) and p53 phosphorylation was found in N-MeDBC-treated WB-F344 cells [32]. In conclusion, the present study clearly demonstrated that different mechanisms may underlie genotoxic and non-genotoxic effects of liver carcinogens DBC and DiMeDBC in the WB-F344 cell line, a promising in vitro model of rat liver oval cells. DNA damage induced by the tissue-specific sarcomagen N-MeDBC may result in DNA strand breaks and MNi. The extent of these genotoxic effects was insufficient to produce additional cellular events related to hepatocarcinogenesis. Stable DNA adducts may mediate DBC genotoxicity in WB-F344 cells, but oxidative stress resulting in oxidative damage to DNA is likely to be the causal factor in DiMeDBC genotoxicity. Different DNA lesions may trigger distinct cellular pathways, resulting in diverse cell responses detected in WB-F344 cells. The previous study also showed that the strict hepatocarcinogen DiMeDBC is a relatively potent agonist of AhR, which is an important player in carcinogenesis. High levels of apparently active AhR characterize various tumors, even in the absence of exogenous ligands [77–79]. AhR can interact via molecular cross-talk with multiple signalling pathways involved in cellular growth, differentiation, and regulation of cell adhesion and migration [80,81]. Future studies should establish the significance of oxidative damage induced by DiMeDBC under in vivo conditions, as well as its relevance to hepatocarcinogenesis.

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tial dose-dependent inhibition of DNA and RNA syntheses (Fig. 3), caused DNA breakage (Fig. 4) and micronucleus formation (Table 3). DBC-bulky adducts generate serious distortion in DNA, resulting in a considerable delay in DNA damage removal (Fig. 5); even 48 h after exposure, a significant level of DNA strand breaks was detected in DBC-treated cells. Significant phosphorylation of histone H2AX (Fig. 7) and p53 protein [32] were determined in DBC-treated cells. Surprisingly, the strict hepatocarcinogen DiMeDBC produced an almost negligible level of stable DNA adducts under identical treatment conditions (Fig. 2E and F; Table 2). A time-course study aimed at the kinetics of DNA-adduct formation in cells exposed to DiMeDBC excluded a loss of bulky adducts already during cell treatment owing to rapid DNA repair. No specific DNA adduct spots were detected after the exposure of WB F344 cells by DiMeDBC for 2 h, 4 h, 6 h, and 12 h. DBC and DiMeDBC are potent liver carcinogens, but quantitative and qualitative differences in their biological effects were detected in vivo. At equimolar concentrations, DiMeDBC induced fewer mutations and DNA adducts even with different chromatographic mobility than the parent compound DBC [10,13,16,18]. DBC hepatocarcinogenicity is associated with strong hepatotoxicity whereas, under identical treatment conditions, no histologically detectable toxicity has been found in the liver of DiMeDBC-treated mice [14]. Despite the lack of stable DNA adducts, a significant level of strand breaks and MNi were ascertained in DiMeDBC-exposed cells (Fig. 4, Table 3). In contrast with DBC, the increase in strand breaks was not concentration dependent. One of the reasons of such phenomenon might be a rapid repair of DNA damage at these low concentrations as suggested, e.g., by Doak et al. [60]. Indeed the results depicted in Fig. 5 seem to suggest that the repair of breaks induced by DiMeDBC is more rapid, when compared with DBC; no DNA strand breaks were determined in cells 16 h after exposure. An explanation might be induction of oxidative stress. It is supposed that ROS generated by various agents are eliminated to some degree by inherent cellular defence mechanisms as presented, e.g., by Jenkins and colleagues [61]. DiMeDBC (Table 4) induced a significant level of ROS, as compared with both control and DBC. The modified comet assay was used to analyze the DNA-damage profiles induced by individual dibenzocarbazoles to evaluate the mechanism of DiMeDBC genotoxicity in WB-F344 cells. A significant rise in strand breaks due to incubation of DiMeDBC-exposed cells with Fpg protein, a repair-specific endonuclease, suggested that base modifications such as oxidized damage (e.g., 8-oxodG) or ringopened DNA adducts (Fapy-DNA adducts) may be responsible for DiMeDBC genotoxicity in these cells (Fig. 6A). In addition, similarity in the kinetics of DNA damage removal in the presence or absence of Fpg protein was found in DiMeDBC-exposed cells (Figs. 5 and 6B). No DiMeDBC-DNA adducts were detected also in V79MZh1A2 cells stably expressing human CYP1A2 despite a significant level of mutations and micronuclei detected in exposed cells [48]. In agreement with our present study, further experiments revealed possible role of oxidative DNA damage or unstable DNA adducts in DiMeDBC genotoxicity in V79MZh1A2 cells. DiMeDBC is a potent aryl hydrocarbon receptor (AhR)-agonist and accordingly increased significantly the expression of AhRmediated genes in WB-F344 cells, mainly CYP1A1 [32], although it is poorly metabolized by this cytochrome P450. No mutations, DNA strand breaks, micronuclei or DNA adducts were detected in V79MZh1A1 cells stably expressing CYP1A1 [48,62–64]. Based on these data, we hypothesized that DiMeDBC might cause a release of ROS due to uncoupling of the catalytic cycle of CYP1A1 similarly as the planar halogenated hydrocarbons. Both events, oxidative damage and CYP1A1 induction were detected in cells exposed to dioxin (TCDD) or polychlorinated biphenyl (PCB) congeners, which are AhR-agonists but are, at the same time, poorly metabolized by CYP1A1 [65–67]. On the other hand, AhR activation alone may also

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The authors wish to thank Professor F. Périn, Department of Genotoxicity and Carcinogenicity, Institute Curie, France, who provided the dibenzocarbazole derivatives; and Professor J. E. Trosko,

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MSU, East Lansing, MI, USA, who kindly offered the rat liver oval WB-F344 cells. The authors express their appreciation to Mrs. A. Vokáliková for excellent technical assistance. This study was supported by the grants awarded by the Scientific Grant Agency of SAS (No. 2/6063/26), Czech Ministry of Education (No.2B08005), and Czech Ministry of Agriculture (MZE0002716201). Zuzana Valoviˇcová, M.Sc. was a fellow of the European Social Fund Project (13120200038).

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Příloha V Andrysík, Z., Marvanová, S., Ciganek, M., Neča, J., Pěnčíková, K., Vondráček, J., Mahadevan, B., Topinka, J., Baird, W.M., Kozubík, A. and Machala, M. Genotoxic and nongenotoxic effects of complex airborne PAH mixture SRM1649a in rat liver epithelial cells. Rukopis v přípravě.

Genotoxic and nongenotoxic effects of complex airborne PAH mixture SRM1649a in rat liver epithelial cells Zdeněk Andrysíka,b, Soňa Marvanováa, Miroslav Ciganeka, Jiří Nečaa, Kateřina Pěnčíkováa, Jan Vondráčeka,b, Brinda Mahadevanc, Jan Topinkad, William M. Bairdc, Alois Kozubíkb and Miroslav Machalaa* a

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic

b c

Laboratory of Cytokinetics, Institute of Biophysics, ASCR, Brno, Czech Republic

Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis,

OR, USA d

Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, ASCR, Prague,

Czech Republic

*corresponding author

Introduction Among many effects of air pollution on human health increased risk of cancer is the most serious, as reported in large epidemiological studies from Europe and U.S. (Vineis, 2005). Standard Reference Material (SRM) 1649a represents a complex mixture of air pollutants collected in an urban area. SRM1649a contains a broad spectrum of polycylic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCB) congeners, chlorinated pesticides and many other compounds and elements (Wise, 2000). PAHs are a group of compounds whose contribution to carcinogenic potential of polluted air is well-known. A range of individual PAHs, mixtures containing PAHs (e.g. tobacco smoke, coal tars, mineral oils, soots, wood dust, diesel exhaust etc.) and industrial processess, during which PAHs are released (e.g. coke production, coal gasification, aluminium production etc.), are classified according to IARC as carcinogenic or probably carcinogenic, belonging to Group 1 or 2 (IARC 1983, 1984, 1987, 1989a, 1989b). Carcinogenicity, respectively tumor initiation by PAHs is supposed to be associated with genotoxic events, especially with the formation of DNA adducts and increased frequency of mutations both in vitro and in vivo (Baird, 2005, Mahadevan, 2003, Mahadevan, 2004, Hakura, 1998). Besides mostly stressed genotoxic effects of PAHs, various important nongenotoxic, tumor-promoting effects (Trosko, 1998), including cell cycle deregulation and proliferation induction (Chramostova, 2004, Andrysik, 2007), differentiation (Tai, 2007) or inhibition of gapjunctional intercellular communication (GJIC) (Blaha, 2002, Riverdal 2003) can contribute to carcinogenicity of PAHs. Loss of GJIC is a frequent event in carcinogenesis (Mesnil, 2005) suggesting importance of gap-junction structures in regulation of cell cycle (Moorby, 2001) and acquired autonomy of the cells with blocked GJIC on tissue regulatory signals (Trosko, 2005). Majority of effects of PAHs and their metabolites are mediated by aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. Activated by a wide plethora of xenobiotics and inducing transcription of many metabolizing enzymes (especially CYP1, Phase I biotransformation enzymes), AhR seems to have a primal role in metabolic activation and detoxification of PAHs (Nebert, 2004). Additional roles for AhR in development, maintaining of tissue homeostasis and proliferation control were suggested by studies exploring ahr-/- mouse model (Schmidt, 1996, Gonzales 1998) and by works focused on function of AhR without external activation (Ma, 1996, Puga 2002). Direct implication of AhR in PAHs-induced acute inhibition of GJIC is questionable since the most potent inhibitor of GJIC from PAH group, fluoranthene (Blaha, 2002), is only a poor AhR ligand (Machala, 2001). On the other hand, modulation of proliferation induced by PAHs is at least in rat liver cellular models AhR-dependent (Andrysik, 2007, Köhle, 2008). The role of AhR in proliferation control is controversial. Whereas presence of exogenous ligand of AhR in growing cells leads to inhibition of cell cycle progression (Weiss, 1996, Iseki, 2005, Marlowe, 2004, Elferink, 2003), in quiescent or contact-inhibited cell populations AhR stimulation may result in proliferation induction (Köhle, 1999, Dietrich, 2002, Vondráček, 2005). PAHs occur in the environment in mixtures and thus influence living organisms, but rarely as individual compounds. In addition to PAHs, a variety of PAH derivatives including methylated, chlorinated and oxygenated PAHs occurs in environment and there is only limited information concerning their participation on the overall carcinogenic effect of the complex mixture. Since effects of PAHs can differ considerably when applied as individual compounds or in mixtures (Binková, 2004, Musafia-Jeknic, 2005, Staal, 2007), it is of major importance to study effects of PAHs in mixtures and in case of environmental mixtures to define contribution of particular components to toxicity of the complex mixture.

Since many of these compounds are AhR ligands, the principal aim of our study was to evaluate AhR-mediated effects (gene expression of xenobiotics-metabolizing enzymes, changes in cell proliferation and cell cycle distribution) and inhibition of GJIC induced by SRM1649a in rat liver epithelial cells WB-F344 as well as to define a potential involvement of PAHs in these effects. AhR activation was in parallel determined using a rat hepatoma cell line stably transfected with AhR-responsive luciferase reporter gene. In order to discriminate between effects of PAHs, persistent chlorinated compounds present in SRM1649a and polar PAH-derived compounds, the crude extract of SRM1649a was fractionated and each of fractions was subjected to in vitro testing. Additional aim was to compare sensitivity of liver epithelial cells to genotoxic events resulting in stable PAH-DNA adduct formation and apoptosis with AhR transactivation.

Materials and Methods SRM 1649a extraction and fractionation. The standard reference material of urban airborne particles SRM 1649a was extracted and fractionated as previously reported (Ciganek, 2004). Briefly, SRM 1649a was extracted with dichloromethane in a Soxhlet apparatus for 8 h and fractionated to aliphatic, neutral aromatic, slightly polar and polar fractions on a silica gel column eluted by hexane, hexane/dichloromethane (1:1, v/v), dichloromethane and methanol. Additionally, an aliquot of crude extract was treated with H2SO4-activated silica gel to obtain a fraction of persistent organic pollutants (POPs) containing polychlorinated biphenyls, dibenzo-pdioxins, dibenzofurans and other dioxin-like compounds but not less persistent PAHs. Chemical analysis of PAH and PAH derivatives was performed in HPLC/DAD and GC/MS systems as reported previously (Ciganek, 2004). Cell culture and treatment. The rat liver epithelial WB-F344 cells (kindly provided by James E. Trosko, MSU, East Lansing) were cultivated in DMEM/F12 (1:1) supplemented with 5% heat-inactivated fetal bovine serum (PAA Laboratories, Linz, Austria). For experimental procedures cells were plated on 6- or 4-well plates with initial density of 3 x 104 cells per cm2. During 72 hours of cultivation cell population formed a monolayer of contact inhibited cells. Subsequently, cultivation medium was replaced for a fresh one and cells were subjected to the treatment with extract of SRM 1649a or its fractions. Respecting fast metabolization of some components of the mixture, in cells treated for prolonged periods cultivation medium was changed daily in order to keep constant concentrations of all compounds present in the mixture. Only cells within the 16th and 22nd passage were used throughout the study and the final concentration of solvent (dimethyl sulfoxide, DMSO) did not exceed 0.1% (v/v). AhR-mediated activity. The rat hepatoma H4IIEGud.Luc1.1 cell line, stably transfected with a luciferase reporter gene under the control of dioxin responsive elements, was used to detect the AhR-mediated activity in the DR-CALUX® assay (Sanderson, 1996). The assays were performed in 96-well cell culture plates. The cells were grown 24 h to 90-100% confluency and exposed to the test or reference compounds (TCDD) dissolved in DMSO (maximum concentration 0.4%, v/v) for 6 h or 24 h. The medium was removed, cells were washed with PBS and the luciferase was extracted with the low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2 diamin cyclic hexane-N,N,N´,N´ tetraacetic acid, pH 7.8). The plates were frozen at –80°C

and the luciferase expression was then measured on a microplate luminometer using the Luciferase Assay Kit (BioThema, Handen, Sweden). Real-time RT-PCR. Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel). The amplifications of the samples were carried out using QuantiTect Probe RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s specifications. All probes were labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) on the 5´-end, and with the Black Hole 1 (BH 1) fluorescent quencher dye on the 3´-end. The sequences of primers and probes have been published previously (Vondráček, 2006). The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the conditions described previously (Vondráček, 2006). Gene expression for each sample was expressed in terms of the threshold cycle (Ct), normalized to housekeeping gene porphobilinogen deaminase (∆Ct). ∆Ct values were then compared between control samples (DMSO 0.1 %) and samples treated with PAHs to calculate ∆∆Ct (∆Ct [control] - ∆Ct [xPAH]). The final comparison of transcript ratios between samples is given as 2-∆∆Ct (Livak, 2001). DNA isolation and 33P-Postlabeling of PAH-DNA adducts. A standard DNA isolation protocol was used (Luch, 1998). Briefly, cell pellets were homogenized with sterile plastic pestles in 200µL of SDS-EDTA homogenization buffer [100mM Tris, 100mM EDTA, 250 mM NaCl, 1% SDS (w/v)]. After homogenization the samples were treated with 20µL RNase A (10 mg/ml) at 37°C for one hour followed by treatment with proteinase K (500 µg/ml) (Sigma, St. Louis, MO) at 37°C for 1 h. The DNA was extracted with 1:1 volume of Tris-equilibrated phenol and chloroform:isoamyl alcohol (24:1) and then with equal volumes of chloroform:isoamyl alcohol (24:1). Both extractions were performed in Gel-Lock tubes (Eppendorf, Westbury, NY). The aqueous layer was treated with 1/10 volume of 5 M NaCl and twice the volume of cold 100% ethanol to precipitate the DNA, which was then dissolved in double-distilled water. The concentration was determined by UV absorbance at 260 nm. Postlabeling was carried out as described previously (Ralstone, 1997). Briefly, 10 µg DNA isolated from treated WB-F344 cells were digested with nuclease P1 and prostatic acid phosphatase, postlabeled with [γ-33P]ATP (3,000 Ci/mmol), cleaved to adducted mononucleotides with snake venom phosphodiesterase I, and pre-purified with a Sep-Pak C18 cartridge (Waters Corp., Milford, MA). DNA adducts were analyzed by reversed-phase high performance liquid chromotography (HPLC) utilizing a 5 µm Symmerty® C18 cartridge column (4.6 mm x 250 mm; Waters, Milford MA). The BP-DNA adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC grade methanol (solvent B). The elution gradient was as follows: 44-55% solvent B over 5 min, 55-60% solvent B over 5 min, 60-90% solvent B over 20 min, 90-100% solvent B over 5 min and 100-44% solvent B over 5 min. DBP-DNA adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 50% HPLC grade methanol/50% acetonitrile (solvent B). The elution gradient was as follows: 20-44% solvent B over 20 min, 44-60% solvent B over 40 min, 60-80% solvent B over 15 min and 80-20% solvent B over 1 min. DNA adducts formed from SRM 1649a treatment were resolved by elution at 1 ml/min 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC grade methanol (solvent B). The elution gradient was as follows: 44-60% solvent B over 40 min, 60-80% solvent B over 10 min, isocratic elution at 80% solvent B over 10 min and 80-44% solvent B over 5 min. The radiolabeled nucleotides were detected by an on-line radioisotope flow detector (Packard Instruments, Downers Grove,

IL) and the level of DNA binding was calculated based on the labeling efficiency of a [3H]BP7,8-diol 9,10-epoxide standard (Lau, 1991). At least 3 independent sets of the postlabeling reactions were carried out for each sample treated, in order to determine the total PAH-DNA adduct levels. Apoptosis detection. Phosphatidylserine (PS) translocation from the inner part of plasma membrane to the outer layer is a marker of early stages of apoptosis. The presence of PS at the cell surface was determined by staining with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) in combination with propidium iodide (PI, 20 mg/ml). PI positivity reflected disintegration of cytoplasmic membrane characteristic for late apoptotic and necrotic cells. Confluent cells treated for 48 hours with SRM 1649a mixture or its fractions were harvested and stained following the manufacturer's protocol and analyzed by flow cytometry. For apoptosis estimation based on morphological criteria WB-F344 cells were exposed to studied mixture for 72 hours. Harvested cells were fixed in ethanol and their nuclei were stained with DAPI (0.5 µg/ml) diluted in Mowiol/DABCO (Calbiochem, Merck KGaA, Darmstadt, Germany) mounting medium and observed under a fluorescence microscope. Morphology of 200 nuclei was assessed per sample. For flow cytometry as well as microscopical analysis both floating and attached cells were collected and analyzed together as one sample. Western blotting. Samples for protein analysis were prepared as described previously (Andrysík, 2006). Briefly, 24 hours treated cells were washed twice with PBSa and lysed in SDS sample buffer. Boiled (10 minutes at 90°C) and sonicated protein samples were resolved using standard SDS-PAGE in 10% gels and transferred on a PVDF membrane. The proteins of interest were detected with following antibodies: anti-CYP1A1 #299124 (Daiichi Sankyo, Tokyo, Japan), anti-cyclin A sc-751 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p53 (Ser15) antibody #9284 (Cell Signaling Technology, Inc.; Beverly, MA, USA), anti-p53 sc-1313 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), HRP-conjugated antibodies were purchased from GE Healthcare (Little Chalfont, UK). Proliferation assessment, cell cycle distribution and BrdU incorporation analyses. The experimental design for proliferation analysis in WB-F344 cells was described in detail in a previous study (Chramostová, 2004). Confluent cells cultivated in 4-well cell culture plates (Nunc, Roskilde, Denmark) were exposed to various concentrations of SRM1649a extract and/or its fractions for 72 hours. Both floating and attached cells were collected and counted in a Coulter Counter (Beckman Coulter, Brea, CA, USA). Cell cycle distribution was assessed in 48 h treated cells. Following the exposure, cells were trypsinized and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed with PBS and stained with Vindelov solution (1M Tris-HCl - pH 8.0; 0.1 % Triton X-100, v/v; 10 mM NaCl; propidium iodide (PI) 50 µg/ml; RNAse A 50 Kunitz units/ml) (Vindelov, 1977) at 37°C for 30 min. Stained cells were analyzed using FACSCalibur flow cytometer equipped with CELLQuestPro™ software for data acquisition (Becton Dickinson, San Jose, CA, USA). At least 15,000 cells were collected per sample. Acquired data were analyzed using ModFit LT version 3.0 software (Verity Software House, Topsham, ME, USA). In BrdU incorporation experiments, 1 mg/ml of BrdU was added to cultivation medium 1 h before cell harvesting. Cells were detached by trypsinization and fixed in 70% ethanol. Fixed cells were washed with PBSa, resuspended in 1 ml of 2 M HCl with 0.5% Triton X-100 and

incubated for 1 h at 37°C. Following incubation, samples were centrifuged and pellets were resuspended in 1 ml of 0.1 M NaB4O7 (pH 8.5), left for 10 min at room temperature, pelleted and resuspended in PBSa with 1% bovine serum albumin and 0.5% Triton X-100. After 12 h incubation at 4°C with FITC-conjugated mouse anti-BrdU antibody or isotype control FITClabeled antibody (both from BD Pharmingen, Becton Dickinson, San Jose, CA), cells were washed with PBSa and stained with PI (10 µg/ml) for a minimum of 1 h at 4 °C in the dark. For quantification of BrdU incorporating cells, multiple events were excluded from the analyses, and the voltage of photomultiplyer was set separately with respect to background fluorescence of the treated sample for each individual compound, based on the G0/G1 population fluorescence and the control isotype antibody staining. GJIC inhibition analysis. The modified scrape loading/dye transfer assay was performed as described previously (Bůáha, 2002). The confluent WB-F344 cells, grown in 24-well plates, were exposed to test compounds (up to 50 µM concentration), 12-O-tetradecanoylphorbol-13acetate (TPA) (20 nM, positive control), or DMSO (negative control) for 30 min. After the exposure, the cells were washed twice with 0.5 ml PBS; fluorescent dye was added (Lucifer Yellow 0.05% w/v in PBS) and the cells were scraped using a surgical blade. After 4 min, the cells were washed twice by 0.5 ml PBS and fixed with 4% formaldehyde (v/v) and the migration of the dye was evaluated using an epifluorescence microscope (Nikon, Inc., Japan). The distance of the dye migration from a scrape line was measured at eight randomly chosen spots per scrape, using Lucia image analysis software (Laboratory Imaging, Prague, Czech Republic). Three independent experiments were carried out and at least three scrapes per well were evaluated. Statistical analysis. Presented data were expressed as mean values ± S.D. For the data analysis Student t-test (cell numbers) or nonparametric Mann-Whitney U-test (% of cells in Sphase, AhR activity) were applied. P values less than 0.05 were considered statistically significant.

Results Chemical analysis of SRM 1649a. Concentrations of PAHs were determined in neutral aromatic F1 fraction and compared with certified concentration data on PAHs in SRM 1649a (Wise, 2000). Differences in individual concentration data were less than 20 % (data not shown) suggesting that extraction and fractionation steps were performed accordingly and that various classes of contaminants were properly distributed in the fractions. AhR-mediated activity. Activation of AhR is a crucial step in cellular response to exposure to various environmental pollutants. Since SRM1649a contains various compounds known to be effective as AhR activators (NIST, 1982; NIST, 2001), we analyzed the potential of the mixture and/or its fractions to activate AhR-mediated response. SRM1649a fractionation based on different molecule polarity was performed in order to separate some of the groups of compounds important for AhR activation. Three fractions under in vitro study contained among others PAHs, methylated PAHs, PCBs, PCDD/Fs (F1 fraction), nitrated PAHs (F2 fraction), and oxygenated derivatives of PAHs (F3 fraction). Aliquot of the fraction F1 was further treated with sulphuric acid in order to destroy PAHs, so that the resulting fraction contained only POPs as PCBs and PCDD/Fs (H2SO4/silica fraction). Aliphatic F0 fraction was not investigated. The effect of SRM1649a on activation of AhR-dependent transcription was studied using rat hepatoma H4IIE cells stably transfected with luciferase reporter gene controlled by dioxinresponse elements (DR-CALUX® assay). This bioassay was selected for assessment of AhRmediated activity due to the fact that relative potencies of a large series of individual PAHs and methyl-PAHs developed in this assay have been reported previously (Machala, 2001, ŠvihálkováŠindlerová, 2007, Machala, 2008, Marvanová, 2008). As shown in Fig. 1, crude extract of SRM1649a as well as fractions F1 and F3 showed high potential to induce AhR-mediated response. Sulphuric acid-treated fraction was significantly less potent to transactivate AhR, whereas cell exposure to F2 fraction caused only weak activation of AhR.

Figure 1. AhR-mediated activity assessment. Rat hepatoma H4IIE cells were subjected to treatment with SRM1649a and/or its fractions for 24 h. Toxic equivalents (TEQs) are expressed relative to TCDD. The data represent average values from three independent experiments.

Induction of CYP1A1 and CYP1B1 expression. As members of gene family participating in detoxification process CYP1A1 and CYP1B1 genes are under transcriptional control of AhR, the potential of SRM1649 to induce AhR-mediated response in rat hepatoma cells was further investigated in rat liver epithelial cell line WB-F344. When exposed to F1 fraction in concentration 1 mg/ml or higher, WB-F344 cells expressed similar level of CYP1A1 mRNA as in cells treated with 1 nM TCDD (Fig. 2). Both crude extract of SRM1649a and F3 fraction were found to be potent inducers of CYP1A1 with mRNA expression at levels ranging from 30 to 60 % of the TCDD response. The highest levels of CYP1B1 mRNA were induced after exposure to fraction F1 especially in concentration 0.5 mg/ml and higher; these levels were similar to that induced by 1 nM TCDD (Fig. 2). Both crude extract and F3 fraction appeared as potent inducers of CYP1B1, although in lesser extent than F1 fraction. Importantly, CYP1A1 and CYP1B1 mRNA were induced significantly at low doses (from 0.1 mg/ml). Similar elevations of both CYP1A1 and CYP1B1 expressions were found on the protein level (Fig. 3). 2.5 mg/ml of crude extract of the SRM1649a as well as F1 and F2 fractions induced CYP1A1 with the same efficiency as 1 nM TCDD, whereas the level of CYP1B1 in cells treated with crude extract and/or fractions of SRM1649a was slightly lower. CYP1B1 mRNA

CYP1A1 mRNA 8 7

40 35

CE F1 F3

30 25 20 15 10

fold induction

fold induction

50 45

6 5

CE F1 F3

4 3 2 1

5 0

0

DMSO

TCDD

0.01

0.1 mg / ml

0.5

1

1.5

DMSO

TCDD

0.01

0.1

0.5

1

1.5

mg / ml

Figure 2. Induction of CYP1A1 mRNA expression in WB-F344 cells treated with SRM1649a and its fractions. Confluent cells were exposed to various concentration of crude extract (CE) of SRM1649a and/or fractions of the studied mixture (F1 - F3) for 24 h. The data are representative of three independent experiments.

Figure 3. Protein levels of both CYP1A1 and CYP1B1 were elevated in WB-F344 cells exposed to 2.5 mg/ml of crude extract (CE) and/or fractions (F1 - F3) of SRM1649a for 24 h. In order to document the equal protein loading, expression of β-actin was assessed. Presented data are representative from three independent experiments.

PAH-DNA adducts in WB-F344 cells in culture. The PAH-DNA adduct formation in WB-F344 cells exposed 24 h to potent carcinogenic PAH BaP and DBalP as well as to SRM 1649a extract was determined by 33P-postlabeling and analyzed by HPLC. Representative HPLC elution profiles of the PAH-DNA adducts are depicted in Fig. 4. In WB-F344 cells treated with BaP one major peak eluting at a retention time of 35 min was identified and corresponded with the (+)-anti-BPDE-dG adduct. The cells treated with DBalP exhibited one major peak with a retention time of 75 min which corresponded with the (−)-anti-DBPDE-dA adduct. However, the HPLC profiles of cells treated with SRM 1649a extract revealed only one major peak eluting at a retention time of 35 min, which corresponded with the (+)-anti-BPDE-dG adduct peak of BaP. The peak of (−)-anti-DBPDE-dA adduct was also identified, but in very small amount. The average levels of PAH-DNA adducts formed in WB-F344 cells treated with BaP, DBalP or SRM 1649a extract are tabulated in Table 1. The maximum level of DNA adducts were formed in the DBalP treatment group (29 pmol/mg of DNA) in comparison to BaP (3 pmol/mg of DNA) or SRM 1649a extract (0.5 pmol/mg of DNA).

BaP

4 (+)-anti-BPDE-dG

(−)-anti-DBPDE-dA

DBalP

extract SRM 1649a (+)-anti-BPDE-dG

(−)-anti-DBPDE-dA

Figure 4. HPLC elution profiles of 33P-postlabeled DNA adducts formed in WB-F344 cells in culture after 24 h of treatment to BaP (10 µM), DBalP (100 nM) or SRM 1649a extract (2.5 mg/ml). In comparison with elution times reported by Mahadevan, 2005 and Buters, 2002, the BaP and DBalP peaks were labeled. Peak 4 resulted from reactions of (−)-anti diol epoxide with dA.

Table 1. PAH-DNA binding in WB-F344 cells treated with BP, DBP or SRM 1649a extract

__________________________________________________________________ Treatment Concentration PAH-DNA adducts pmol/mg DNA _______________________________________________________________________________ BaP 10 µM 3 DBalP 100 nM 29 SRM 1649a extract 2.5 mg/ml 0.5 _______________________________________________________________________________ Results indicate the PAH-DNA adducts formed from 2 different experiments averaging over at least three replicate measurements of postlabeling reactions.

Western blotting. Induction of level of p53 protein, especially its phosphorylated form is considered the markers of DNA damage response and induction of cell cycle, leading to either DNA repair or apoptosis of damaged cells (Luch, 1999, Binková, 2004). Phosphorylation of tumor-supressor protein p53 on Ser15 and accumulation of total p53 was detected using Western blot analysis after 24 h exposure of confluent WB-F344 cells at concentration 2.5 mg/ml. As shown in Fig. 5, crude extract of SRM 1649a (CE) and polar fraction (F3) induced detectable phoshorylation of p53, indicating DNA damage responses, but they had no effect on the level of total p53. Fraction F1 elicited no induction of p53 phosphorylation (Fig. 5).

Figure 5. Phosphorylation of p53 on Ser15 and accumulation of total p53 in WB-F344 cells treated for 24 h with SRM1649a and its fractions using concentration 2.5 mg/ml. DBalP (100 nM) and BaP (10 µM) were used as positive control compounds. The results shown here are representative of three independent experiments.

Apoptosis detection in cells exposed to SRM1649a and its fractions. Presence of phosphatidylserine on the cell surface is a characteristic event of early stages of apoptosis (Fadok, 1992). Double staining of cells with Annexin V and PI allows us to discriminate between early apoptotic (Annexin V positive) cells and late apoptotic/necrotic (positive for both Annexin V and PI). As documented in Fig. 6, 48 h of treatment with F1 fraction of SRM1649a did not affect cell surveillance. In contrast to F1 fraction crude extract of SRM1649a induced massive increase of late apoptotic/necrotic cell numbers, whereas cell exposition to F3 fraction led to moderate increase of cell death. Although the overall potential of F3 fraction induce cell death was less pronounced when measured as a ratio of Annexin V/PI positive cells the values were doubled compared to vehicle-treated population. Programmed cell death in WB-F344 cells treated with SRM1649a was further assessed by morphological criteria (nuclei fragmentation) As shown in Fig. 7, a significant increase in numbers of apoptotic cells was detected in cell populations exposed for 72 h to high concentrations of crude extract of SRM1649a (1.5 and 2.5 mg/ml) and F3 (2.5 mg/ml). On the

other hand, exposure to 2.5 mg/ml of F1 containing PAHs and other nonpolar compounds did not cause a significant increase of apoptosis in WB-F344 cells, although some of PAHs present in SRM1649a are well-known apoptosis inducers in various in vitro models (Chramostová, 2004, Hardin, 1992, Pliskova, 2005). BaP was used as a positive control demonstrating potency of WBF344 cells to respond with apoptosis to the treatment with genotoxic PAHs.

Figure 6. Cell death in induced by SRM1649a and its fractions detected by flow cytometry following Annexin VFITC staining. WB-F344 cells reached confluency during 72 h of cultivation. Following subsequent 48 h exposition to the studied mixtures cells were stained and analyzed as described in materials and methods. Presented data are representative of four independent experiments.

Figure 7. Apoptosis detection in WB-F344 cell line exposed to SRM1649a. Confluent cells were treated with tested mixtures for 72 h. Fixed cells were stained with DAPI and observed under a fluorescence microscope as described in Materials and methods. Cells with characteristic nuclear morphology were counted and the data are expressed as mean values from three independent experiments. An asterisk denotes significant difference as compared with the group treated with DMSO (0.1%) as a vehicle.

Proliferation induction by SRM1649a. As results presented above showed AhRdependent gene transactivation and induction of AhR target genes in cells treated with crude extract and/or fractions F1 and F3 of SRM1649a we further studied effect of the mixture on WBF344 cell proliferation. We and others reported previously that AhR ligands induce cell proliferation in confluent WB-F344 cells (Dietrich, 2003, Chramostová, 2004) and this proliferation is AhR-dependent (Andrysik, 2007). As outlined in Fig. 8, crude extract and fractions F1 and F3 induced cell proliferation with the same potency as they activated AhRdependent transcription. Starting from the dose of 0.1 mg/ml and reaching maximum at 0.5 mg/ml proliferation induction was documented by WB-F344 cell numbers increase following 72 h of incubation. In contrast to complete mixture and/or its fractions F1 and F3, the effect of F2 fraction was very weak and observable only in highest concentrations applied. Cell numbers decrease in populations treated with 1.5 and/or 2.5 mg/ml of crude extract or F3 fraction respectively, was associated with cell death (Fig. 6, 7). On the other hand, cell proliferation induced by PAHs-containing fraction F1 was sustained even in high concentration where no significant increase of cell death was reported.

Figure 8. Effect of SRM1649a on cell proliferation in WB-F344 cells. Confluent cells were treated with crude extract of SRM1649a and/or its fractions for 72 h. Following treatment period, cells were harvested and counted on a Coulter Counter. 0.1% DMSO was used as a vehicle control and 1 nM TCDD as a model stimulator of WB-F344 cells proliferation. Data represent mean values ± S.D. from three independent experiments. Asterisk denotes significant difference compared to untreated control (p≤0.05).

Effect of SRM1649a on cell cycle distribution. To further document the potential of SRM1649a to induce proliferation of WB-F344 cell line, we analyzed distribution of the cell population in cell cycle phases. As illustrated in Fig. 9, cell number increase following treatment with 0.5 mg/ml of crude extract and/or fractions F1 and F3 was associated with increase of ratio of cells in S phase of the cell cycle (Fig. 9A) and cyclin A expression (Fig. 9B), whereas F2 fraction lacked similar effects. Accumulation of cells in S phase can be consequence of both intensive proliferation and cell cycle arrest (Andrysík, 2007). To discriminate between these two events analysis of DNA synthesis via incorporation of BrdU was used. As depicted in Fig. 10, exposure of WB-F344 cells to 2.5 mg/ml of F1 fraction of SRM1649a led to increase of ratio of cells in S phase synthesizing

DNA, since application 2.5 mg/ml of F3 fraction resulted in partial inhibition of DNA synthesis in cells accumulated in S phase of the cell cycle. Maximal S phase arrest was observed in cells exposed to 1 µM of genotoxic BaP. Finally, cell treatment with 2.5 mg/ml of crude extract of SRM1649a resulted in inhibition of DNA synthesis and accumulation of cells in G0/G1 phase. A

B

Figure 9. Distribution of WB-F344 cells in cell cycle (A) and cyclin A (B) expression following treatment with SRM1649a. Confluent population of rat liver epithelial cells was exposed to 0.5 mg/ml of crude extract (CE) of SRM1649a and/or its fractions for 48 h. Following fixation in ethanol cells were stained with PI and analyzed on a flow cytometer for DNA content. Shown data represent mean values ± S.D. from three independent experiments. Asterisk denotes significant difference compared to untreated control (p≤0.05). For cyclin A expression detection similarly cultivated and treated WB-F344 cells were lysed in SDS-buffer and examined for protein expression by western blotting. Equal protein loading was documented by assessment of β-actin expression. Western blotting data are representative of three independent experiments.

Figure 10. BrdU incorporation documents alternations in cell cycle distribution in cells treated with SRM1649a. Confluent cells were subjected for 48 h to treatment with crude extract (CE) of SRM1649a and/or its fractions. Cells synthesizing DNA and incorporating BrdU were visualized by anti-BrdU FITC-labeled antibody. The data are representative of three independent experiments.

Gap-junctional intercellular communication is inhibited by SRM1649a. Loss of gapjunctions belongs to major hallmarks of neoplastic tissues and a decrease in GJIC is believed to be an important step in carcinogenesis (Trosko, 2005). In present study we used a scrape-loading dye-transfer assay to analyze the functionality of gap-junctions in a confluent culture of WBF344 cells exposed to tested mixture. 30 min of exposure to both crude extract and F3 fraction of SRM1649a in concentration 1 mg/ml and higher caused inhibition of GJIC (Fig. 11). In contrast to crude extract and F3 fraction, there was no significant effect of F1 on GJIC up to very high concentrations (25 mg/ml - data not shown).

Figure 11. Inhibition of gap-junctional intercellular communication by SRM1649a. Confluent WB-F344 cells were exposed for 30 min to crude extract (CE) of SRM1649a and/or its fractions. Following the incubation period gapjunctional intercellular communication was analyzed by scrape-loading dye-transfer assay. Presented data are mean values ± S.D. from three independent experiments.

Discussion Urban air pollution, largely derived from various sources of combustion causes a range of health problems including cancer (Schoket, 1999, Okona-Mensah, 2005). SRM1649a represents a standardized mixture of particulate material collected in urban area containing broad spectra of pollutants (Wise, 2000). Several studies showed mutagenic potential of respirable airborne particles in bacterial assays (Pedersen, 2004, Umbuzeiro, 2005) or mammalian cellular models (Mahadevan, 2005, Sevastyanova, 2007). PAHs, their derivatives and metabolites contained in airborne particulate matter appear to contribute significantly to overall carcinogenic potential of urban air pollution. Besides the most frequently mentioned genotoxic modes of action of PAHs, consisting in generation of DNA adducts, double strand breaks and mutations, various nongenotoxic effects of PAHs and their derivatives have been reported. PAHs via specific receptor activation (Machala, 2001), disturbance of steroid hormone signaling (Vondracek, 2002, Tsai, 2004) or mitogen-activated protein kinases (Andrysík, 2006) may alter gene expression, what can lead to deregulated cell proliferation (Chramostova, 2004, Andrysik, 2007, Köhle, 2008) or inhibition of apoptosis (Pääjärvi, 2008). These effects were frequently associated with AhR activation. AhR is responding on exposure to ligands from PAH group with high sensitivity; the

concentrations required for AhR-mediated transactivation were considerably lower compared to those necessary for DNA adducts formation, what was demonstrated previously for individual compounds (Machala 2001, Topinka, 2008) and this work describes such difference in responses for standardized environmental mixture of PAHs. Although a limited sensitivity of methods used for detection of DNA adducts should be considered, data on induction of apoptosis or necrosis only at high doses of SRM 1649a support the findings that AhR activation, induction of CYP1 enzymes and other AhR-dependent events associated with tumor promotion, such as release of the cells from contact inhibition, occur at remarkable lower exposure doses than DNA damage formation and apoptosis/necrosis. AhR-mediated activity of SRM1649a assessed by DR-CALUX assay (Fig.1) showed high values comparable with activity of river sediments (Vondracek, 2001) or indoor dust samples (Suzuki 2007). Interestingly, relatively high induction potency of polar aromatic F3 fraction was determined. Although the major part of AhR-mediated activity of SRM1649a is probably mediated by PAHs and polar polyaromatic compounds, the contribution of POPs such are PCBs or PCDD/Fs to overall activity of the sample seems to be relevant too. Induction of CYP1A1 and CYP1B1 mRNA and AhR-dependent release from contact inhibition in rat liver epithelial WBF344 cells was found at similar concentration range as in DR-CALUX, the assay using rat hepatoma cell line, suggesting relevance of screening DR-CALUX assay for assessment of AhRinducing potencies of xenobiotics. Although the induction of mRNA of CYP1 enzymes occured at low levels, comparable with other AhR-mediated effects, the 7-ethoxyresorufin O-deethylase activity, reflecting capacity of bioactivating CYP1 enzymes, was elevated significantly only at high doses (data not shown here). This suggests the hypothesis, that some components of the environmental mixture inhibited CYP1-dependent metabolic activation of PAHs and other progenotoxins. The same conclusion was suggested previously in the study on SRM 1649a effects in mammary carcinoma MCF-7 cells (Musafia-Jeknic, 2005). Inhibition of CYP1 enzymatic activity by fluoranthene and other individual PAHs have been reported as well (Shimada, 2006); inhibitory potency of fluoranthene and benzo[c]phenanthrene has been described also in WB-F344 cells (Topinka, 2008). Taken together, both genotoxic and nongenotoxic events have been determined in rat liver epithelial WB-F344 cells, the model of liver progenitor cells with metabolic activation competence, functional p53 protein and other DNA damage response pathways. Nongenotoxic effects associated with AhR activation appeared to be the most important processes occuring after low dose exposure to crude SRM1649a extract, neutral aromatic and polar aromatic fractions. PAHs contributed to these effects more significantly than persistent dioxin-like compounds. Acknowledgement. This work was supported by the Czech Ministry of Agriculture, grant No. 0002716202.

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