TOXICITA ULTRAJEMNÝCH ČÁSTIC V ŽIVOTNÍM PROSTŘEDÍ M. Machala Výzkumný ústav veterinárního lékařství, Brno
[email protected]
OBSAH PŘEDNÁŠKY
1. Co je nanotoxikologie? 2. Mechanismy toxicity PAHs a dalších chemických kontaminantů vázaných na vzduchové částice 3. Terénní studie: jsou nanočástice hlavními nosiči PAHs and jejich genotoxicity a dioxinové aktivity?
CO JE NANOTOXIKOLOGIE ?
INTRODUCTION I. Nanotoxicology and human toxicology of engineered nanoparticles II. Airborne ultrafine and nanoparticles – toxic effects of NPs as well as chemicals adsorbed on surface of particulate matter (PM), environmental health & human biomarkers of exposure III. Nanobiomaterials, nanomedicine, pharmacokinetics / particokinetics, adverse effects of biocompatible nanomaterials
Nano-bioanalytical sciences (determination of size, surface, and other physico-chemical characteristics, chemical composition etc.) Modes of action of NPs and chemicals adsorbed on NPs Exposure scenarios and risk assessment of nanomaterials, “nanosafety“
Studies on effects of engineered and environmental nanoparticles major biological models:
in vitro models;
ecotoxicology tests (bacteria Vibrio fischeri, Daphnia magna, Chlorella vulgaris, zebrafish etc.);
experimental animal studies (rodents);
human biomarkers (oxidative stress, inflammation and immunity parameters).
Studies on effects of engineered and environmental nanoparticles: major toxicological end-points
DNA damage, oxidative stress;
disruption of intercellular communication and cell adhesion, remodeling of membrane lipids and cytoskeleton and modulation of cell-surface mediated and intracellular signaling;
modulation of gene expression;
effects on cell populations (proliferation, cell death, differentiation).
Current limits of nanotoxicological studies:
We have only limited understanding of fate, transport and toxicity of ENPs and airborne PM.
The tools to study these interactions are being developed.
Several potentially important toxic modes of action are currently not covered within nanotoxicology.
Exposure protocols are based on classical toxicology paradigms and are often insufficient (NPs agregate, sorption, surface effects etc.....).
HLAVNÍ MECHANISMY TOXICITY LÁTEK ADSORBOVANÝCH NA ČÁSTICE
HLAVNÍ MECHANISMY TOXICITY XENOBIOTIK
„Přímé“ mutageny / genotoxiny; metabolická aktivace
promutagenů, adukty s proteiny a DNA, chromosomální aberace
AhR-dependentní dioxinová aktivita Další receptor-dependentní mechanismy endokrinní disrupce (např. estrogenita)
Oxidativní stres (tvorba ROS, peroxidace lipidů, oxidativní poškození DNA a proteinů), modulace signální transdukce pomocí ROS
Neurotoxicita, imunotoxicita, poruchy metabolismu endogenních látek atd.
GENOTOXICITA – METABOLICKÁ AKTIVACE POLYCYKLICKÝCH AROMATICKÝCH UHLOVODÍKŮ
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 1.
Metabolická aktivace – cytochromy P450 (CYP1A1, CYP1A2, CYP1B1) + alternativní dráha (AKR1C9, AKR1A1)
Oxidativní stres
Stabilní adukty DNA-PAH
Oxidativní poškození DNA (oxidace bazí, apurinová místa)
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 2. DETEKCE POŠKOZENÍ DNA (stabilní adukty DNA s metabolity PAHs) Metoda 32P-postlabeling: expozice buněk, izolace DNA, digesce a značení 32P, TLC, detekce radioakt. fosforu DBalP, DBaeP, BgChry, BaP – silně genotoxické PAHs; DBahA, BbF, Chry, BaA – převážně negenotoxické efekty (aktivace AhR atd.)
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 3. POSTGENOTOXICKÉ SIGNÁLY (sensory poškození, regulátory DNA „repairu“ a apoptózy) Roos, Kaina, 2006 Sensory genotoxicity, přenos post-genotoxických signálů a regulace: ATM, ATR, H2AX, ChK1, ChK2, p53
HLAVNÍ TYPY POŠKOZENÍ DNA („apurinic sites“ a oxidačně modifikované báze)
Oxidativní deaminace
Depurinace (nestabilní modifikované nukleotidy)
PRODUKTY OXIDATIVNÍHO POŠKOZENÍ DNA
Biomarker oxidativního poškození DNA (stanovení HPLC)
DIOXINOVÁ TOXICITA AhR activation: ligand
AhR hsp90 p23
XAP2 cytoplasm ARNT ??
nucleus
transcriptional coregulators
Xenobiotic response genes (modified from Kewley et al., 2004)
TNGCGTG
ENDOKRINNÍ DISRUPCE: AKTIVACE AhR A NUKLEÁRNÍCH RECEPTORŮ
Aktivace AhR (indukce CYP1A1/1A2/1B1; modulace bun. cyklu, apoptózy, imunosuprese, syndromy dioxinové toxicity, vývojová toxicita, metabolismus steroidních hormonů)
Xenoestrogeny a antiestrogeny (efekty na ERa, ERb a efekty na biosyntézu estrogenů)
Xenoandrogeny / antiandrogeny (efekty na AR) Xenobiotika modulující thyroidní funkce efekty (efekty na transport a metabolismus thyroidů, modulace TR )
CHEMICKÁ KARCINOGENEZE
CHEMICKÁ KARCINOGENEZE
1. GENOTOXICKÉ POŠKOZENÍ - INICIACE 2. PŘEŽÍVANÍ / PROLIFERACE GENOTOXICKY POŠKOZENÝCH BUNĚK
3. TRANSFORMACE VE VÍCE AGRESÍVNÍ, METASTAZUJÍCÍ KLON(Y)
ZÁKLADNÍ MECHANISMY NÁDOROVÉ PROMOCE Negenotoxické efekty cizorodých látek Endogenní látky (cytokiny, růstové faktory, signál. transdukce)
(Hannahan, Weinberg, 2000)
INTERCELULÁRNÍ SPOJENÍ GJIC
Základní stavební jednotkou jsou proteiny konexiny (connexin 32, 43 atd.), které tvoří hexamery (konexony); konexony sousedních buněk mohou tvořit společný kanál, kterým procházejí signální molekuly (cAMP, Ca2+ atd.)
INHIBICE GJIC (scrape-loading / dye transfer assay; prostup fluoreskující luciferové žluti monovrstvou buněk)
kontrola
expozice nádorovými promotery vede k inhibici GJIC
DISRUPCE ADHERENTNÍCH SPOJENÍ (ADHERENS JUNCTIONS)
DISRUPCE ADHERENTNÍCH SPOJENÍ (ADHERENS JUNCTIONS) signálování kateninu/cadherinu
biosensor density
kontrola bun. akumulace
ARE ULTRAFINE PARTICLES MAJOR CARRIERS OF c-PAHs AND THEIR GENOTOXICITY AND DIOXINLIKE ACTIVITY?
Aim of the study:
There is a wide range of organic and inorganic pollutants that become associated with NPs (including ultrafine/nanofraction of PM). There are two major goals – to assess toxicity of NPs and to assess toxicity of chemicals adsorbed on the surface of NPs (toxicological impact of organic pollutants such as PAHs, PCBs, dioxins). Focus on the major toxic modes of action of PM – genotoxicity and dioxin-like activity.
What is dioxin-like activity and how to determine it? 1) Aryl hydrocarbon receptor (AhR) is a key transcription factor of many developmental, metabolic and other processes. 2) Sustained AhR activation is associated with chemical carcinogenesis (including tumor progression), developmental perturbations and immunotoxicity. 3) Exposure to highly persistent TCDD and TCDD-like compounds as well as chronic exposure to PAHs leads to long-term activation of AhR and AhR-mediated toxicity.
What is dioxin-like activity and how to determine it? TCDD and Related Compounds
+ AhR Src 1
ARNT
HSP90 HSP90
P
2
HSP90 HSP90
Nuclear Factors
Src
AhR
ARNT
DRE-Luc
“Activated”
P Membrane Proteins
Increased Protein Phosphorylation
Modulation of Gene Expression
P Cytosolic Proteins
Light
Luciferase Adapted from Blankenship (1994)
Materials and Methods
Air sampling in 4 sites by HiVol cascade impactor (ultrafine aerosol collected on PTFE coated Glass Micro Fiber Absolute filters) Extraction by DCM and HPLC/FD analysis of c-PAHs In vitro acellular assay with DNA adduct analysis by P32-postlabeling and TLC Determination of AhR-dependent reporter gene expression (“dioxin-like activity“) in H4IIE.GudLuc cells exposed to DCM extracts of subfractions (DR-CALUX assay)
Diesel engine PM agregates (carbonaceous nano-PM aggregates)
(Murr, Garza, 2009)
Example of formation of PM (soot formation from PAHs and other compounds)
PAHs, dioxins, ..... adsorbed on PM surface
Distribution of c-PAHs in extractable organic matter prepared from various size fractions of ambient-air PM (ng/mg PM)
Distribution of c-PAHs in extractable organic matter prepared from various size fractions of ambient-air PM (ng/m3)
DNA formation after exposure to isolated subfractions of ambient PM (acellular genotoxicity assay: a sample + calf thymus DNA +/- S9) dae:1 – 10 m
+S9
-S9
0.5 – 1 m
0.17 – 0.5 m
<0.17 m
DNA formation after exposure to isolated subfractions of ambient PM (acellular genotoxicity assay: a sample +calf thymus DNA + S9; expressed as DNA adducts/mg PM)
Normalized data from acellular genotoxicity assay (+S9 metabolic activation, DNA adducts/m3)
Dioxin-like activity of PM fractions (expressed as TCDD equivalents per m3) 80
TEQ - DR-Calux
70
pg TCDD/m3 pg TCDD/m3
60 50
Strip mine Highway City centre Background station
80
40 30 20
60 40
10
1 - 10
Strip mine
0.5 - 1
Highway
< 0.17
0.17 - 0.5
0.5 - 1
1 - 10
< 0.17
0.17 - 0.5
0.5 - 1
1 - 10
< 0.17
0.17 - 0.5
0.5 - 1
1 - 10
< 0.17
0.17 - 0.5
0
0.5 - 1
1 - 10
0
20
Background station 0.17 - 0.5 < 0.17
City centre
Chemická analýza velikostních frakcí (pouze PAHs, vzorek Praha 2010) ng/m3 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 11 12 14 22 24 26 27 28 31
Compound Name Phenanthrene Anthracene Fluoranthene Pyrene Benz[a ]anthracene Chrysene Benzo[b ]fluoranthene Benzo[k ]fluoranthene Benzo[a ]pyrene Dibenz[a,h ]anthtacene Benzo[g,h,i ]perylene Indeno[1,2,3-cd ]pyrene 4H-Cyclopenta[def]phenanthrene Benzo[c]phenanthrene Triphenylene Cyclopenta[cd]pyrene Benzo[a]fluoranthene Benzo[j]fluoranthene Benzo[e]pyrene Perylene Dibenz[a,c]anthracene Dibenz[a,j]anthracene Coronene Dibenzo[b,k ]fluoranthene Naphtho[2,1-a ]pyrene Naphtho[2,3-e ]pyrene Naphtho[1,2-b ]fluoranthene Naphtho[1,2-k ]fluoranthene Benzo[a ]coronene Sum of PAHs
1-10 µm
0.5-1.0 µm
0.17-0.5 µm
<0.17 µm
0.26 0.04 0.61 0.46 0.24 0.41 0.35 0.16 0.25 0.02 0.56 0.27 0.05 0.06 0.08 0.14 0.08 0.20 0.17 0.04 0.02 0.02 0.10 0.02 0.03 0.02 0.04 0.04 0.02 4.78
1.53 0.14 1.60 1.21 0.37 0.56 0.48 0.23 0.45 0.03 0.61 0.29 0.17 0.13 0.09 0.36 0.11 0.32 0.22 0.08 0.03 0.04 0.10 0.03 0.06 0.03 0.07 0.06 0.04 9.45
0.55 0.05 0.54 0.39 0.13 0.18 0.14 0.08 0.15 0.01 0.22 0.13 0.06 0.02 0.03 0.11 0.00 0.10 0.07 0.03 0.01 0.02 0.04 0.01 0.02 0.01 0.02 0.02 0.01 3.17
0.85 0.06 0.61 0.40 0.12 0.14 0.11 0.05 0.10 0.01 0.15 0.09 0.07 0.03 0.02 0.06 0.00 0.08 0.05 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.01 3.15
Relativní koncentrace PAU s MW 252 [%] 30
30
1-10 µm
25
25
20
20
15
15
10
10
5
5 0
0
BaF BbF BkF BjF BaP BeP Per
BaF BbF BkF BjF BaP BeP Per
30
0.5-1.0 µm
30
0.17-0.5 µm
<0.17 µm
25
25
20
20
15
15
10
10
5
5 0
0
BaF BbF BkF
BjF BaP BeP
Per
BaF BbF BkF BjF BaP BeP
Per
Využití chemických dat pro výpočet relativních toxických potencí
• Karcinogenita: CEQ [ng BaP eq./m3] =RF (RPF) x konc. [ng/m3] RPF (Relativní faktory karcinogenní potence): US EPA (2010): Development of a relative potency factor (RPF) approach for polycyclic aromatic hydrocarbon (PAH) mixtures • Mutagenita: MEQ [ng BaP eq./m3] = RMF x konc. [ng/m3] RMFs (Relativní mutagenní faktory): Durant et al., 1996; Durant et al., 1999. • Dioxinová aktivita: IEQ [pg TCDD eq./m3] = IEF x konc. [pg/m3] IEF (indukční ekvivalenční faktory): Machala et al., 2001; Švihálková et al., 2007; Marvanová et al., 2008 a další články (užito testu DR-CALUX).
Toxické efekty vypočtené z koncentrací toxikantů Karcinogenní potence Mutagenní potence 2.0
CEQs [ng BaP eq. /m3]
Dioxinová potence 4.0
MEQs [ng BaP eq. /m3] IEQs [pg TCDD/m3]
1.0 3.0 1.0 2.0 0.5 1.0 0.0 1-10 µm
0.5-1.0 µm 0.17-0.5 µm
<0.17 µm 0.0 1-10 µm
0.5-1.0 µm 0.17-0.5 µm
<0.17 µm 0.0 1-10 µm
0.5-1.0 µm 0.17-0.5 µm
<0.17 µm
Dioxin-like activity of c-PAHs (calculated from concentration data and induction equivalency factors of individual PAHs) 1,4
2,0
Strip mine calculated from chemical data IEQ of c-PAH 1,8
1-10 0.5-1 0.17-0.5 <0.17
1,2
1,6 1,4
1,0
1,2 1,0
0,8
0,8 0,6
0,6 0,4
Strip mine
Highway BaA
City Chrycenter
<0.17
0.17-0.5
0.5-1
1-10
<0.17
0.5-1
1-10
0.17-0.5
0,0
<0.17
0,2
0.5-1
1-10
<0.17
0.17-0.5
0.5-1
1-10
0,0
0.17-0.5
0,4
0,2
BbF Background BkF station BaP
DBahA
IPY
CONCLUSIONS
The upper accumulation fractions (0.5-1 um) of ambient-air aerosols are major carriers of PAHs and exhibit the highest production of DNA adducts (when normalized per m3). The fractions of ultrafine particles (1-170 nm) showed generally lower concentrations of PAHs and lower efficiency to form DNA adducts. Similarly, the most potent induction of AhRdependent gene expression (“dioxin-like activity“, assessed in DR-CALUX assay) was found for the upper accumulation fraction; ultrafine fractions elicit the lowest activity. Nevertheless, chemical contaminants associated with nanoparticles contribute significantly to the overall toxicity of airborne particulate matter. Toxicity of nanoparticles themselves?
FUTURE EXPERIMENTS:
Toxicity of ultrafine and nanoparticles themselves vs. toxicity of coarse, upper accumulation and lower accumulation particles in lung epithelial and bronchial cells. Studies on effects of NPs and adsorbed chemical contaminants associated with carcinogenic and tumor promoting processes. Risk assessment of environmental NPs.
Acknowledgements K. Pěnčíková, M. Ciganek, J, Neča, J. Turánek (Dept. Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic) J. Vondráček (Department of Cytokinetics, Institute of Biophysics, ASCR, Brno) A. Milcová, J. Schmuczerová, R. Šrám, J. Topinka (Laboratory of Genetic Toxicology, IEM, Prague) Supported by the Centre of Excelence CENATOX (Czech Science Foundation, project No. P503/12/G147)
