Deuterium depletion and mitochondrial NADPH production: The link for epigenetic control of oncogenesis

Science Friday Seminar December 13, 2013

Deuterium depletion and mitochondrial NADPH production: The link for epigenetic control of oncogenesis László G. Boros, M.D. Associate Professor of Pediatrics Harbor-UCLA Medical Center, Torrance, CA INVESTIGATOR - LABIOMED SIDMAP, LLC, Scientist

http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

Pro-/Eukaryotic Cell Structures

http://www.phschool.com/science/biology_place/biocoach/cells/common.html


Pro-/Eukaryotic Cell Structures

http://www.phschool.com/science/biology_place/biocoach/cells/common.html


Animal cell  Internal membrane (nuclear membrane, Golgi apparatus)  Mitochondria  Cytoskeletal structures  Microtubules  Actin and actin binding proteins, e.g., α-actinin, fimbrin, filamin  Microtubules, myosins provide dynamic character


Mitochondria 1) Inner membrane 2) Outer membrane 3) Crista 4) Matrix

http://en.wikipedia.org/wiki/Eukaryote


TCA, citric acid, Krebs cycle

http://high-fat-nutrition.blogspot.com/2012/07/protons-wheres-bias.html


http://en.wikipedia.org/wiki/File:Citric_acid_cycle_with_aconitate_2.svg


X



Hereditary leiomyomatosis and renal cell cancer syndrome – I.  Reed’s syndrome (familial leiomyomatosis cutis et uteri)  Rare inherited condition  Multiple cutaneous leiomyomas


Hereditary leiomyomatosis and renal cell cancer syndrome – II.

Cutaneous piloleiomyomata Gideon Smith MD PhD, Noushin Heidary MD, Rishi Patel MD, Karla Rosenman MD, Shane A Meehan MD, Hideko Kamino MD, Miguel Sanchez MD

Dermatology Online Journal 15 (8): 10


Clear cell renal cell carcinoma

http://en.wikipedia.org/wiki/File:Clear_cell_renal_cell_carcinoma_high_mag.jpg


Clear cell renal cell carcinoma  Cortical tumor  Malignant epithelial cells with clear cytoplasm  Compact-alveolar (nested)  Acinar growth pattern

 Intricate, arborizing vasculature  Granular eosinophilic cytoplasm may be present

http://emedicine.medscape.com/article/1612043-overview


Clear cell renal cell carcinoma

http://en.wikipedia.org/wiki/File:Clear_cell_renal_cell_carcinoma_high_mag.jpg


Hereditary leiomyomatosis and renal cell cancer syndrome  The syndrome is caused by a mutation in the fumarate hydratase gene  Accumulation of fumarate  Autosomal dominant disease


X



Restored fumarate hydratase  Restoring FH activity by inserting the fumarate hydratase gene into kidney tumor cells (UOK262WT) has been previously shown not to be tumorigenic  These cells fail to produce tumors in nude mice

Tong, et al. The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels. Cancer Cell. 2011 Sep 13;20(3):315-27. doi: 10.1016/j.ccr.2011.07.018 http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

X

http://biologyofcancer.org/Publications.html


Fumarate accumulation and epigenetic events – I.  High mitochondrial concentrations of fumarate may cross into the cytosol via dicarboxylate carriers to inhibit prolyl hydroxylases  These target HIF-1α for VHL-dependent degradation under normoxia, thus rendering HIF-1α constitutively active  Non-specific succination of cysteine residues of proteins  Elevated levels of superoxide and peroxide (ROS), which is another important mediator of HIF-1α stabilization Yang Y, et al. Metabolic reprogramming for producing energy and reducing power in fumarate hydratase null cells from hereditary leiomyomatosis renal cell carcinoma. PLoS One. 2013 Aug 15;8(8):e72179. doi: 10.1371/journal.pone.0072179 http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

The question asked by biochemists! What does fumarase do?


FH adds metabolic (matrix) water to TCA cycle intermediates



 1937 Nobel Prize in Physiology and Medicine for his studies of biological oxidations, fumarate hydratase and for discovering ascorbic acid in adrenal glands. http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

He proposed hydrogen-carrying catalytic role of succinate and fumarate in the 1920s

Malate



The cycle consumes net two molecules of water in mammals



Nature 150, 21-21 (04 July 1942) | doi:10.1038/150021a0 Metabolic Water and Desiccation KENNETH MELLANBY THE utilization by the body of ingested food substances and of tissue reserves yields among other things quantities of metabolic water. As the complete combustion of 100 gm. of fat produces about 110 gm. of metabolic water, whereas 100 gm. of carbohydrate yields only 55 gm. of water, fat reserves and fatty foods are believed to be particularly valuable as a protection against desiccation. This contention would appear to be supported by the fact that many animals which exist in deserts have large reserves of fat.


Natural fat with low deuterium

The cycle consumes net two molecules of water in mammals



Metabolic water with low deuterium content

Malate



Deuterium content in free water and food – I.  Measured using isotope-ratio mass spectrometry as VSMOW - Vienna Standard Mean Ocean Water (~155 ppm)  Flour ~150 ppm (parts per million)  Table sugar ~146 ppm  Cottege cheese (dry) ~136 ppm  Olive oil ~130 ppm  Butter ~124 ppm  Fatty acid animal fat ~118 ppm ER Kerstel, et al. Simultaneous determination of the (2)h/(1)h, (17)o/(16)o, and (18)o/(16)o isotope abundance ratios in water by means of laser spectrometry. Anal Chem. 1999 Dec 1;71(23):5297-303. doi: 10.1021/ac990621e http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

Deuterium content in free water and food – II.  Measured using isotope-ratio mass spectrometry as VSMOW - Vienna Standard Mean Ocean Water (~155 ppm)  Unsaturated fat ~110 ppm  Unsaturated plant fat at double bonds ~102 ppm

I Billault, et al. Quantitative deuterium isotopic profiling at natural abundance indicates mechanistic differences for delta 12-epoxidase and delta 12-desaturase in Vernonia galamensis. J Biol Chem. 2005 May 6;280(18):17645-51 http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB

Enzymes of photosynthesis and fat synthesis/desaturation drop deuterium Enzymes dropping deuterium

H

D http://mriclassroom.weebly.com/photosynthesis.html


Fig. 1 Fatty acids (~118 ppm)

Glucose - free water (150 ppm)

Glycolysis

Hexose/pentose cycling

Oxidative pentose cycle

CS and Aconitase

c)

G6PDH and 6PGD

NADP+D

a) Fumarate hydratase

b) Citrate

NADP+D (118 ppm)

NADP+ dependent IDH

FAS

S

c)

H2O 2

GSH-D (150 ppm)

X “Light” DNA and nuclear plasma membrane fatty acids (118 ppm)

(150 ppm)

X d)

What is wrong with deuterium?  It is twice as heavy as hydrogen  Chemically identical  Deuterium participates in reductive synthesis  Changes DNA methylation kinetics

 Alters gene expression  Likely induces aneuploidy


What is wrong with deuterium?

Hydrogen http://www.phoenixweightlifting.com/forums/viewtopic.php?t=1440


What is wrong with deuterium?

Deuterium http://listofsports.com/wp-content/uploads/2012/02/1859059.jpg


How does deuterium get into DNA?

http://thyroidcancersurvivor.wordpress.com/tag/abnormal-cells/


X

Tedeschi PM, et al. Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells. Nature - Cell Death Dis. 2013 Oct 24;4:e877. doi: 10.1038/cddis.2013.393.

[1,2-13C2]-glucose ATP

Pentose phosphate pathway

HK

ADP

NADP

NADPH TPI

glucose-6P PGI

TPI

6P-gluconolactone

TPI

xylulose-5P

ribulose-5P TK

ribose-5P

ADP

TK

fructose-1,6biP

Glycolysis

G6PDH

6P-gluconate TK

fructose-6P PFK

ATP

NADPH + 13CO2

NADP

FBPA DHAP

TPI NAD

TA

GA3P

erythrose-4P

GAPDH

Serine synthesis

NADH 1,3-biphosphoglycerate ADP

sedoheptulose-7P

PGK

PHGDH

NAD

3pg

PSAT

3php

ATP

SOG pathway

KG

glut

pser

PSPH

ser

PGK

Ham

SHMT

P

X NADH LDH

ATP

Hox NADH

One carbon cycle

NH3

C1ST

C1ST

mlthf Hred

PK

NADP

methf NADPH

NAD

pyruvate NAD

NADH + 13CO2

PDH

ATP + HCO3

[2,3-13C2]-Acetyl-CoA

AcCoA

CoA CS

NAD

TCA cycle

ADP

ACC

MalCoA

FAS

citrate ACO

oxaloacetate [13C2]-Lactate

GC

T

L

phosphoenolpyruvate

10fthf

2

glyc

2-phosphoglycerate ENO

ADP C1ST

thf 13CO

ADP

ATP + for

NADH

Fatty acid synthesis [13C]-palmitate

isocitrate NAD NADH + 13CO2

IDH KG

NADPH + NH3 GLDH

NAD [4,5-13C2]-glutamate



Glucose - free water (150 ppm)

Glycolysis



CS and Aconitase

c)

Fumarate hydratase

G6PDH and 6PGD

NADP+D

a)

X

c)

b)

H2O 2

Citrate

NADP+D (118 ppm)

(150 ppm)

GSH-D (150 ppm)

NADP+ dependent IDH

X FAS

S

X “Heavy/sticky” DNA and nuclear plasma membrane fatty acids (150 ppm)

d)

Glycolysis Tracer TCA RNA ribose Fatty acid

WT-pFH+262

EV-FH-262

FH-262

FH-268

R2

Correl

[7] - Glucose tracer consumption (mg/24h)

100.0

128.1

147.5

167.3

1

1

[12] - 13CO2 Glucose oxidation complete (D13C/12C)

100.0

66.3

59.1

76.4

0.3991

-0.6318

[17] - Lactate 13C labeled fraction (Sm)

100.0

107.3

107.8

107.2

0.6793

0.8242

[22] - G6PDH flux NADPH production (m1/m2)

100.0

104.9

105.6

113.6

0.8904

0.9436

[22B] - Lactate concentration (peak area)

100.0

123.2

137.0

126.2

0.6562

0.8101

[76] - Glutamate 13C labeled fraction (Sm)

100.0

47.9

42.9

34.4

0.8458

-0.9196

[77] - Glutamate 13C Content (Smn)

100.0

49.9

43.4

33.7

0.8720

-0.9338

[79] - Glutamate via PDH (m2/Sm)

100.0

82.4

78.6

60.4

0.9581

-0.9788

[80] - Glutamate via OA recycling (m3/Sm)

100.0

72.9

55.8

58.1

0.8740

-0.9349

[81] - Glutamate via PC and PDH (m4/Sm)

100.0

226.0

219.8

248.7

0.7986

0.8936

[87B] - Glutamate-concentration (peak area)

100.0

74.7

55.6

59.5

0.8614

-0.9281

[143] - Lignocerate (C24:0) 13C labeled fraction (Sm)

100.0

73.2

64.9

70.1

0.7310

-0.8550

[144] - Lignocerate (C24:0) 13C Content (Smn)

100.0

67.2

57.2

60.1

0.7962

-0.8923

[150B] - Lignocerate (C24:0) concentration (peak area)

100.0

176.1

258.8

136.1

0.1717

0.4143

100.0

91.8

92.1

74.3

0.8121

-0.9011

100.0

91.6

92.1

73.7

0.8069

-0.8983

[296] - RNA-ribose via G6PDH/NADPH (m1/Sm)

100.0

112.2

111.8

140.4

0.7991

0.8939

[297] - RNA-ribose via Transketolase (m2/Sm)

100.0

91.2

91.3

74.0

0.8318

-0.9120

[305B] - RNA-ribose concentration (peak area)

100.0

113.2

124.6

116.9

0.6701

0.8186

107 % - 120 %

121 % - 134 %

135 % - 149 %

[294] - RNA-ribose

13

C labeled fraction (Sm)

[295] - RNA-ribose

Percent of Control:

< 64 %

13

C content (Smn)

65 % - 78 %

79 % - 92 %

93 % - 106 %

> 150 %


Glucose - DDW water (100 ppm)

Glycolysis



CS and Aconitase

c)

Fumarate hydratase

G6PDH and 6PGD

NADP+D

a)

X

c)

b)

H2O 2

Citrate

NADP+D (118 ppm)

(100 ppm)

GSH-D (100 ppm)

NADP+ dependent IDH

X FAS

S

X “Light” DNA and nuclear plasma membrane fatty acids (100 ppm)

d)

labeled fatty acid fractions (% of total) 13C

120

De novo fatty acid and cholesterol synthesis

MIA-PaCa palmitate C:16 synthesis

100 80

MIA-PaCa lignocerate C:24 synthesis

MCF-7-Breast cholesterol synthesis

P<0.05

60 P<0.05

P<0.05

40 20 0 150 100 50 25

150 100 50 25 150 100 50 25 Deuterium (2H) in water (ppm)

http://www.hyd.hu/documents/deuterium-dw-somlyai-boros-june-25-06.pdf


What is lignocerate?  Saturated rigid phospholipid component of the nuclear membrane  Helps maintaining aneuploidy


Take-Home Capsule – I.  Impaired mitochondria are involved in cell transformation by limiting the low natural deuterium containing complete fatty acid oxidation product, metabolic water, to enter nuclear membranes and nucleotides via reductive synthesis


Take-Home Capsule – II.  In turn, “heavy” natural water and sugar dependent NADPH production taken over by the oxidative branch of the pentose cycle, as well as the SOGC-pathway, are deuterium loading “ticking time bombs” with a strong isotope effect and thus oncogenic epigenetic events that include unstable hydrogen bonds, aneuploidy in DNA structures and chemically altered methylation sites with severely disrupted gene expression patterns


Take-Home Capsule – III.  Nevertheless, extra-mitochondrial NADPH synthesis opens a therapeutic window for deuterium depleted water to maintain and/or restore normal cellular functions.


Take-Home Capsule – IV.  Our approach provides a novel mechanism regarding lipid based ketogenic diets in the presence of hyperbaric oxygen treatment to decrease metastasis formation by producing low deuterium metabolic water via complete oxidation to prevent DNA, histone and nuclear membrane deuteration during NADPH dependent reductive synthesis.


Acknowledgments 1 Dr. Gabor Somlyai – HYD, LLC Tracy Lagunero - SiDMAP Analyses Dr. W-N Paul Lee – UCLA Dr. W. Marston Linehan, Cris Rickets and Youfeng Yang – NCI


Acknowledgments 2 This work was supported by the Hirshberg Foundation for Pancreatic Cancer Research to LGB The UCLA Clinical & Translational Science Institute (UL1TR000124) and UCLA Center for Excellence in Pancreatic Diseases - Metabolomics Core (1-P01 AT003960-01A1).


"Research is to see what everybody else has seen and to think what nobody else has thought." -- Albert Szent-Györgyi http://youtu.be/GkYAjabGxJs - doi: 10.12918/SCIENCEFRDEC132014LGB - Teaching Evaluation - doi: 10.12918/SCIENCEFDEC132014EVAL

Science-Fridays-LABiomed02a-DDW_English 1 00:00:06,009 --> 00:00:11,000 Introduction by Dr. Kevin W. Bruhn, Principal Investigator, Molecular Medicine, LABIOMED 2 00:00:11,009 --> 00:00:18,000 (Medical Diploma) Szeged, Hungary, in 1987... He came to the United States in 1990, 3 00:00:18,009 --> 00:00:27,000 spent 8 years at Ohio State, as a Research Scientist there... before coming to Harbor-UCLA in 1998... 4 00:00:27,009 --> 00:00:32,000 He spent some time with Dr. Paul Lee, who is here today, 5 00:00:32,009 --> 00:00:36,000 learning mass spec(trometry) and stable isotope applications 6 00:00:36,009 --> 00:00:43,000 Dr. Boros is currently an Associate Professor working at UCLA... 7 00:00:43,009 --> 00:00:51,000 And he is actually the Chief Scientific Officer at an LABIOMED spin-off company called SiDMAP. 8 00:00:51,009 --> 00:00:58,000 SiDMAP is a pharmaceutical service provider for all phase metabolomics. 9 00:00:58,009 --> 00:01:04,000 Laszlo is going to talk to us about some of his work, metabolomics, and beyond, and, so.... 10 00:01:04,009 --> 00:01:07,000 I turn it over to you..., thank you doctor! 11 00:01:07,009 --> 00:01:11,000 And, it is a privilege, and thank you for the very kind introduction. 12 00:01:11,009 --> 00:01:20,000 This is the last presentation of 2013, so I was trying to come up with a very interesting topic... 13 00:01:20,009 --> 00:01:27,000 Which, I believe is a very.., truly.., a very intriguing topic..., Page 1

Science-Fridays-LABiomed02a-DDW_English 14 00:01:27,009 --> 00:01:38,000 ...just to talk about deuterium..., its depletion..., as part of cancer's epigenetic drivers... 15 00:01:38,009 --> 00:01:45,000 This is a new theory, as well as a new model, that we have studied and now published (doi: 10.1371/journal.pone.0072179) 16 00:01:45,009 --> 00:01:50,000 in the medical literature, in collaboration with the National Cancer Institute, who, actually..., 17 00:01:50,009 --> 00:01:59,000 ...provided the model for the study. So..., just to start..., 18 00:01:59,009 --> 00:02:03,000 ...let's refresh our memories about how cells, 19 00:02:03,009 --> 00:02:09,000 in particular, how prokaryotic and eukaryotic cells, function? 20 00:02:09,009 --> 00:02:18,000 Eukaryotic cells form nuclei, and these nuclei contain the DNA material, or..., 21 00:02:18,009 --> 00:02:27,000 ...the nucleic acid material, surrounded by membranes..., It's a more complex structure, with a larger size, 22 00:02:27,009 --> 00:02:32,000 as far as the cell goes, and the nucleus has many more functions built into it..., 23 00:02:32,009 --> 00:02:41,000 e.g.: ...to be able to differentiate, and to specialize in various functions... 24 00:02:41,009 --> 00:02:47,000 And..., what is really interesting, and what captured me back in high school, and in.. 25 00:02:47,009 --> 00:02:53,000 ..early college is that: If you (the cell) want to form nuclei contained with a membrane..., 26 00:02:53,009 --> 00:02:58,000 you know.. the cell needs also all these other cellular particles... 27 00:02:58,009 --> 00:03:07,000 And what we've got most interested in, is the mitochondria..., which means, that, really..., there is never an eukaryotic cell Page 2

Science-Fridays-LABiomed02a-DDW_English 28 00:03:07,009 --> 00:03:16,000 with nucleic acid surrounded by a membrane, without mitochondria! 29 00:03:16,009 --> 00:03:23,000 And..., there are no exceptions to this rule..., while cells, that do not have nuclear membranes, 30 00:03:23,009 --> 00:03:28,000 or have no solid or eukaryotic-like nuclear membranes, never have mitochondria. 31 00:03:28,009 --> 00:03:35,000 So.., the link between cell morphology, function and energy production, is where we started... 32 00:03:35,009 --> 00:03:40,000 to look into mitochondria in more details..., and started using models... 33 00:03:40,009 --> 00:03:48,000 Where the mitochondrion was not functioning, and cancer has..., cancer developed! 34 00:03:48,009 --> 00:03:55,000 Just to review more background information..., the internal membrane system, which includes 35 00:03:55,009 --> 00:04:01,000 the nuclear membrane and the Golgi apparatus, host a lot of functional proteins... 36 00:04:01,009 --> 00:04:09,000 Which actually produce fatty acids and membranes, using specialized enzymes... 37 00:04:09,009 --> 00:04:15,000 And the mitochondrion is the site of energy production, yet, it's really not only an energy producing cell, 38 00:04:15,009 --> 00:04:21,000 ...or cell organelle, if we review the evolution of mammalian cells! 39 00:04:21,009 --> 00:04:27,000 Because, mitochondria first produce water..., which is one basic role of mitochondria... 40 00:04:27,009 --> 00:04:30,000 And we will talk about this ..., a bit more, in details... 41 00:04:30,009 --> 00:04:37,000 The cytoskeleton has structures, which include micro-tubes, actin and binding proteins and..., Page 3

Science-Fridays-LABiomed02a-DDW_English 42 00:04:37,009 --> 00:04:41,000 ...so on. So that, a cell, that is eukaryotic and differentiated, 43 00:04:41,009 --> 00:04:49,000 can perform a number of additional functions, which also originate.., some of the cell's regulatory 44 00:04:49,009 --> 00:04:56,000 elements originate from mitochondrial functions. A mitochondrion has an inner membrane, an outer membrane, 45 00:04:56,009 --> 00:05:00,000 has cristae and also has the matrix! 46 00:05:00,009 --> 00:05:05,000 And..., the matrix is where the citric acid cycle... 47 00:05:05,009 --> 00:05:11,000 ...takes place. The citric acid cycle, or Krebs cycle, is a set of 48 00:05:11,009 --> 00:05:18,000 biochemical reactions that produces ... aaa ... 49 00:05:18,009 --> 00:05:29,000 that produce reducing equivalent, NADH and the proton is actually carried through the cytochrome system 50 00:05:29,009 --> 00:05:36,000 and, eventually, through cytochrome-c (complex-IV). The product of the proton released 51 00:05:36,009 --> 00:05:42,000 from citric acid cycle intermediates is water..., if there is oxygen in the 52 00:05:42,009 --> 00:05:47,000 mitochondria in order to produce water..., and this is how animals actually survive in the desert..., 53 00:05:47,009 --> 00:05:51,000 where they do not drink! They use their mitochondria, instead, to actually produce 54 00:05:51,009 --> 00:05:53,000 water! and ...aaa... 55 00:05:53,009 --> 00:06:02,000 that's how the lizards, the snakes and sea fish, and the ... marine animals do not need to 56 Page 4

Science-Fridays-LABiomed02a-DDW_English 00:06:02,009 --> 00:06:06,000 drink water as they produce water in their mitochondria... 57 00:06:06,009 --> 00:06:10,000 what the substrates are? I am going to talk about in more details. 58 00:06:10,009 --> 00:06:18,000 There are two water molecules produced from..., when the Cycle is turned over and with 59 00:06:18,009 --> 00:06:22,000 the addition of oxygen water production is enhanced. 60 00:06:22,009 --> 00:06:29,000 Yet, if the cells need ATP, what they do is, they actually shovel the protons 61 00:06:29,009 --> 00:06:37,000 to the inter-membrane matrix (space), and they actually use this complex-V, or ATP... 62 00:06:37,009 --> 00:06:43,000 ...synthase. And, as they actually channel protons back into the matrix, they also produce ATP... 63 00:06:43,009 --> 00:06:48,000 ...or energy. So the hydrogen comes from citric... 64 00:06:48,009 --> 00:06:54,000 ...acid cycle metabolites and intermediates, so they are very critical to drive the whole process of... 65 00:06:54,009 --> 00:07:00,000 ...water production, and also the proton channeling back and forth... 66 00:07:00,009 --> 00:07:03,000 ...into the matrix. So..., 67 00:07:03,009 --> 00:07:10,000 Our hypothesis was from the very beginning, when I was preparing this talk and was getting... 68 00:07:10,009 --> 00:07:16,000 ...ready to present, was that..., how does the weight of hydrogen affects the... 69 00:07:16,009 --> 00:07:22,000 ...kinetics of these pumps, and, in overall, the biochemical reactions associated... 70 Page 5

Science-Fridays-LABiomed02a-DDW_English 00:07:22,009 --> 00:07:33,000 with mitochondrial failure, in cancer especially, and what're key components of this system, which are responsible... 71 00:07:33,009 --> 00:07:34,000 for epigenetic events! So... 72 00:07:34,009 --> 00:07:40,000 This is, in more details, the chain or set of reactions that happen in the mitochondria 73 00:07:40,009 --> 00:07:47,000 or the TCA cycle, and, as you can see, citrate is formed, which 74 00:07:47,009 --> 00:07:52,000 is why it is called the citrate cycle. And this citrate is formed from a two carbon metabolite..., 75 00:07:52,009 --> 00:07:56,000 ...which is acetyl-CoA and an oxaloacetate, which is in the Cycle. 76 00:07:56,009 --> 00:08:00,000 There is this six carbon citrate, which turns over..., 77 00:08:00,009 --> 00:08:07,000 ...two carbons are lost in the form of CO2, and as CO2 is produced, these hydrogens are... 78 00:08:07,009 --> 00:08:15,000 ...actually taken out from the intermediates of the TCA cycle... 79 00:08:15,009 --> 00:08:20,000 What is really interesting is that not identical carbons get oxidized in the first round, which... 80 00:08:20,009 --> 00:08:25,000 ...have been entering the Cycle! And it is also very interesting that there... 81 00:08:25,009 --> 00:08:30,000 ...is water consumed, while the cycle is spinning over... 82 00:08:30,009 --> 00:08:36,000 So..., it is kind of interesting, and, it is really intriguing to know, why water is consumed, when, in 83 00:08:36,009 --> 00:08:40,000 the meantime, oxygen is reduced, and why water is necessary, which is... 84 Page 6

Science-Fridays-LABiomed02a-DDW_English 00:08:40,009 --> 00:08:44,000 matrix water, the one that is produced by cytochrome-c (and complex-IV)..., 85 00:08:44,009 --> 00:08:51,000 why this water is reused in the mammalian system, instead of using it as a water source..., 86 00:08:51,009 --> 00:08:54,000 ...just like the animals in the desert would do! 87 00:08:54,009 --> 00:09:03,000 Obviously, using matrix water for reductive synthesis, is where I am going to lead the topic...! 88 00:09:03,009 --> 00:09:11,000 Yet, it's a very intriguing system, as finding out, how water is exchanged and how the hydrogen... 89 00:09:11,009 --> 00:09:15,000 ...is channeled into DNA, eventually..., which I am going to show you! 90 00:09:15,009 --> 00:09:23,000 Because, what is very interesting is that there is a cancer model, which we have studied in... 91 00:09:23,009 --> 00:09:29,000 ...collaboration with the National Cancer Institute, where one of the enzymes of the Cycle, called... 92 00:09:29,009 --> 00:09:36,000 ...fumarate hydratase, or fumarase, is mutated! So kidney cells that do not have this fumarate... 93 00:09:36,009 --> 00:09:44,000 ...hydratase or fumarase enzyme, are not able to use water to hydrate fumarate into malic acid! 94 00:09:44,009 --> 00:09:50,000 And, as the result of that, the Cycle would turn back around, and actually, through reductive... 95 00:09:50,009 --> 00:09:56,000 carboxylation, the Cycle would actually become the consumer of hydrogen of some other source..., 96 00:09:56,009 --> 00:09:59,000 ...other than water of the matrix! So..., 97 00:09:59,009 --> 00:10:06,000 with this total turning back around of TCA cycle metabolism, cancer readily develops... 98 00:10:06,009 --> 00:10:10,000 Page 7

Science-Fridays-LABiomed02a-DDW_English ...and, it is actually a very aggressive cancer, which is called the... 99 00:10:10,009 --> 00:10:19,000 ...clear renal cell carcinoma syndrome, which is associated with hereditary leiomyomatosis..., 100 00:10:19,009 --> 00:10:31,000 after 10-15 years of this fumarate hydratase deficiency, and also these skin bumps. These patients... 101 00:10:31,009 --> 00:10:38,000 ...will actually develop cancer! It's a rare disease, it's called the Reeds syndrome..., 102 00:10:38,009 --> 00:10:41,000 ...based on the investigator who described it. 103 00:10:41,009 --> 00:10:45,000 And, what we see in the beginning of the diseases is that there are these skin bumps..., 104 00:10:45,009 --> 00:10:54,000 ...histologically they look like a wide broad keratin layer between the sub-cutaneous... 105 00:10:54,009 --> 00:11:00,000 ...structures. And this is the pre-exposition state, or this the... 106 00:11:00,009 --> 00:11:07,000 ...preliminary stage before kidney cancer develops in these patients! 107 00:11:07,009 --> 00:11:13,000 When kidney cancer develops, the cancer is really unique in the sense that it is called... 108 00:11:13,009 --> 00:11:18,000 ...clear cell renal cell carcinoma... It used to be called a type of hypernephroma..., 109 00:11:18,009 --> 00:11:23,000 ...but it is actually developing from the kidney tissue! 110 00:11:23,009 --> 00:11:30,000 And, what you see here is that there are clear cells with nuclear morphology characteristic... 111 00:11:30,009 --> 00:11:33,000 ...of tumor cells! It is a very aggressive cancer -- and here... 112 00:11:33,009 --> 00:11:38,000 ...is the normal kidney tissue, and because of this characteristics..., with the accumulation... Page 8

Science-Fridays-LABiomed02a-DDW_English 113 00:11:38,009 --> 00:11:46,000 ...of this cystic material in the cells, the cells' nuclei are pushed to the side, and..., 114 00:11:46,009 --> 00:11:51,000 ...because of this pathological feature, they call this type of cancer... 115 00:11:51,009 --> 00:11:57,000 ...clear cell kidney (renal) cell carcinoma. And it develops based on this fumarate hydratase... 116 00:11:57,009 --> 00:12:00,000 ...mutation, which is in the TCA cycle! 117 00:12:00,009 --> 00:12:05,000 So..., the cancer is a cortical tumor... 118 00:12:05,009 --> 00:12:15,000 ...and it is a malignant epithelial cell - type of tumor, and it is clear, but I am not going to talk here about morphology..., 119 00:12:15,009 --> 00:12:17,000 ...in more details... But, yet..., just... 120 00:12:17,009 --> 00:12:24,000 ...let's go to the syndrome, which is actually caused by a mutation in... 121 00:12:24,009 --> 00:12:28,000 ...the fumarate hydratase gene, and as a result of that, there is an accumulation 122 00:12:28,009 --> 00:12:31,000 of water with fumaric acid in the TCA cycle and the mitochondria! 123 00:12:31,009 --> 00:12:39,000 And, it is an autosomal dominant disease. What we see here, is that once the mutation occurs..., 124 00:12:39,009 --> 00:12:44,000 ...over a longer period of time, there is fumaric acid buildup... 125 00:12:44,009 --> 00:12:52,000 ...and there is decreasing malic acid concentration... And the question is, how these changes actually... 126 00:12:52,009 --> 00:12:59,000 ...contribute to cancer development? Because, just looking at this scenario, you... Page 9

Science-Fridays-LABiomed02a-DDW_English 127 00:12:59,009 --> 00:13:08,000 ...would claim that this particular model is more..., would affects more intensely..., or more obviously, the... 128 00:13:08,009 --> 00:13:15,000 ...energy production system of the Cycle! But, actually..., over time, a very aggressive cancer... 129 00:13:15,009 --> 00:13:20,000 ...develops... Question 01: Sorry,... is the accumulation of fumaric acid... 130 00:13:20,009 --> 00:13:26,000 what causes the bloating of cells? And also succinic acid... 131 00:13:26,009 --> 00:13:30,000 ...so, actually there is a number of metabolites that accumulate in the mitochondria, fumaric acid... 132 00:13:30,009 --> 00:13:34,000 ...is one of them, and also succinic acid accumulates... 133 00:13:34,009 --> 00:13:39,000 ...so everything, that actually ...aaa... lead to malate formation, accumulate, (including matrix water)... 134 00:13:39,009 --> 00:13:45,000 ...and, actually..., there is very little malic acid concentrate in these tumor cells! 135 00:13:45,009 --> 00:13:49,000 (Dissolved) fumaric and succinic acid cause hydropic degeneration (of the mitochondria)... 136 00:13:49,009 --> 00:13:54,000 ...now, I want to mention that ... if you ...., not only fumarate hydratase, but succinate 137 00:13:54,009 --> 00:14:00,000 formation, or succinate dehydrogenase mutations, or succinyl-CoA synthetase abnormalities, also... 138 00:14:00,009 --> 00:14:04,000 ...cause cancer! Somewhat different kind of cancers, and there... 139 00:14:04,009 --> 00:14:12,000 ...is a literature on it and they find TCA cycle enzyme mutations in more and more tumor phenotypes! 140 00:14:12,009 --> 00:14:20,000 In fact, if you look at cancer as a disease, the first obvious metabolic event is decreased... Page 10

Science-Fridays-LABiomed02a-DDW_English 141 00:14:20,009 --> 00:14:24,000 ...mitochondrial function. This is..., this will lead to the Warburg effect..., 142 00:14:24,009 --> 00:14:29,000 where pyruvate can not enter the mitochondria, substrates can not be oxidized. 143 00:14:29,009 --> 00:14:37,000 So..., in general, either through hypoxia..., as age advances..., or through fumarate hydratase... 144 00:14:37,009 --> 00:14:43,000 and other enzyme deficiencies in the TCA cycle, one of the basic principles of cancer development... 145 00:14:43,009 --> 00:14:48,000 ...is mitochondrial function... decrease, for one reason, or for another. 146 00:14:48,009 --> 00:14:55,000 So..., it is generally accepted that the mitochondrion, per se, yet..., its specific enzyme mutations..., 147 00:14:55,009 --> 00:15:04,000 ...more specifically, contribute to cancer development, this is very common in cancer... 148 00:15:04,009 --> 00:15:07,000 So..., Question 02: I have a question, neither muscle, nor... 149 00:15:07,009 --> 00:15:09,000 fat tissue have defects? No... 150 00:15:09,009 --> 00:15:15,000 Do you expect muscle defects, in muscle mitochondria? That is not the leading.... 151 00:15:15,009 --> 00:15:21,000 ...Other organs are rather affected, including the skin with bumps, but the leading clinical symptoms are... 152 00:15:21,009 --> 00:15:27,000 ...leading to skin lesions and also kidney cancer development, in this particular mutation. [mammillary muscle of the nipple (genital leiomyoma), the smooth muscle of blood vessels (angioleiomyoma) may also be affected] 153 00:15:27,009 --> 00:15:29,000 So..., 154 00:15:29,009 --> 00:15:34,000 ...what is more interesting, and, this is really intriguing that... Page 11

Science-Fridays-LABiomed02a-DDW_English 155 00:15:34,009 --> 00:15:38,000 ...Once we restore fumarate hydratase functions in these tumor cells... 156 00:15:38,009 --> 00:15:43,000 ...they actually lose the ability to produce tumors in nude mice... 157 00:15:43,009 --> 00:15:50,000 ...so, actually, restoring this one single enzyme in the TCA cycle, reverses the disease itself... 158 00:15:50,009 --> 00:15:58,000 So..., the National Cancer Institute has developed... 159 00:15:58,009 --> 00:16:05,000 ...a cancer cell model, which is termed as UOK262. And there is a wild type, which has the insert... 160 00:16:05,009 --> 00:16:08,000 ...of the normal mitochondrial fumarate hydratase gene. 161 00:16:08,009 --> 00:16:13,000 And so they have a wild type normal fumarate hydratase activity, in these cells! 162 00:16:13,009 --> 00:16:19,000 They also have the empty vector phenotype. So, once you actually insert this enzyme back... 163 00:16:19,009 --> 00:16:24,000 ...into the mitochondria, then, they actually become non-tumorigenic! 164 00:16:24,009 --> 00:16:31,000 So, they can not really cause tumors any more. If you actually inject these tumor cells into... 165 00:16:31,009 --> 00:16:35,000 ...nude mice, as long as there is a fumarate hydratase mutation, there is going to be tumor formation... 166 00:16:35,009 --> 00:16:41,000 ...But once you restore fumarate hydratase function, then the mice are happy! 167 00:16:41,009 --> 00:16:49,000 So, there is no tumor formation. They have published this in a Cancer Cell biology... (doi: 10.1016/j.ccr.2011.07.018 - Figure 1) 168 00:16:49,009 --> 00:16:52,000 ...paper and also some of the publications come from Nature! Page 12

Science-Fridays-LABiomed02a-DDW_English (doi: 10.1038/nature10642) 169 00:16:52,009 --> 00:16:57,000 We have "pheno-typed" these cells for the National Cancer Institute... 170 00:16:57,009 --> 00:17:03,000 ...and we used our metabolic 13C tracer methodology in order to dissect the mechanism that are... 171 00:17:03,009 --> 00:17:08,000 ...clearly involved in the tumor formation process. And we studied two tumor phenotypes, which 172 00:17:08,009 --> 00:17:15,000 are fumarate hydratase deficient and also the fumarate hydratase restored cells. 173 00:17:15,009 --> 00:17:23,000 And we published together in the Public Library of Science (PLoS) ONE in August (2013), which I am going (doi: 10.1371/journal.pone.0072179) 174 00:17:23,009 --> 00:17:25,000 to mention in detail! So..., 175 00:17:25,009 --> 00:17:33,000 ...if you look at the literature, as far as explaining why mitochondrial..., 176 00:17:33,009 --> 00:17:41,000 ...increased mitochondrial concentration of fumarate cause cancer, you can actually see a number... 177 00:17:41,009 --> 00:17:45,000 ...of hypotheses! Obviously, there are a number of studies that... 178 00:17:45,009 --> 00:17:52,000 ...address this issue. And one possibility is that high fumarate concentrations... 179 00:17:52,009 --> 00:17:57,000 ...or fumarate intermediate concentration, with increased concentrations..., 180 00:17:57,009 --> 00:18:04,000 ...they actually go into the cytosol and they inhibit certain enzymes..., 181 00:18:04,009 --> 00:18:10,000 ...which activate signaling for cancer development. And there is a number of, for example, this... 182 00:18:10,009 --> 00:18:16,000 Page 13

Science-Fridays-LABiomed02a-DDW_English ...hypoxia induced factor, which is involved in the metabolic changes..., 183 00:18:16,009 --> 00:18:21,000 ...or epigenetic changes that are associated with the disease! 184 00:18:21,009 --> 00:18:27,000 And there is also a non-specific succinate binding to cysteine residues of proteins... 185 00:18:27,009 --> 00:18:35,000 And, also there are modifications in DNA -- protein binding, because of the increased availability... 186 00:18:35,009 --> 00:18:42,000 ...of these organic acids, coming from the Cycle, and elevated levels of superoxide, because... 187 00:18:42,009 --> 00:18:48,000 ...of the lack of mitochondrial functions are also involved in the explanations, also involved... 188 00:18:48,009 --> 00:18:51,000 ...in the mechanism. Now..., 189 00:18:51,009 --> 00:18:56,000 ...and they have published these in..., we have published these possibilities and scenarios..., 190 00:18:56,009 --> 00:19:00,000 ...in collaboration with the National Cancer Institute! 191 00:19:00,009 --> 00:19:05,000 Now, we as biochemists with Dr. Lee, what we would... 192 00:19:05,009 --> 00:19:09,000 ...ask is: what does, actually..., fumarate hydratase do? 193 00:19:09,009 --> 00:19:14,000 What is the enzyme's activity? What is the function of it? 194 00:19:14,009 --> 00:19:19,000 And I actually posed this question to a few molecular biologists, and asked them what the enzyme would do? The... 195 00:19:19,009 --> 00:19:21,000 ...answer was: they would not know, which is interesting, really! 196 00:19:21,009 --> 00:19:28,000 Page 14

Science-Fridays-LABiomed02a-DDW_English I think, as we are getting into physiology and System's Biology, this is also a very important... 197 00:19:28,009 --> 00:19:33,000 ...question, and one of the key questions! So, what it actually does..., fumarate hydratase... 198 00:19:33,009 --> 00:19:39,000 ...does, it just adds metabolic matrix water to TCA cycle intermediates! 199 00:19:39,009 --> 00:19:48,000 So, fumarate hydratase uses water and introduces it back into the TCA cycle! 200 00:19:48,009 --> 00:20:02,000 And as you can see..., the enzyme, itself, was described before the 1930s by Dr. Albert Szent-Györgyi..., 201 00:20:02,009 --> 00:20:08,000 ...who also discovered vitamin-C and he got the Nobel prize in medicine in 1937. 202 00:20:08,009 --> 00:20:13,000 What is really interesting is that my grandparents, on my Mom's side, they knew him personally... 203 00:20:13,009 --> 00:20:16,000 ...as they also lived in Szeged at that time, in those times. 204 00:20:16,009 --> 00:20:24,000 So, and my Grandma was talking about him, in fact they had the same boots, they had... 205 00:20:24,009 --> 00:20:27,000 ...season tickets to the Opera and they had the same boot! 206 00:20:27,009 --> 00:20:34,000 So, the two families were not friends, but they knew each other very well. 207 00:20:34,009 --> 00:20:42,000 And he (Dr. Szent-Györgyi) is the one, who suggested that there is this hydrogen carrying catalytic role for... 208 00:20:42,009 --> 00:20:45,000 ...succinate and fumarate, and it was back in the 1920s. 209 00:20:45,009 --> 00:20:52,000 So, succinate and fumarate and malate are actually involved in carrying protons through... 210 00:20:52,009 --> 00:20:56,000 ...the Cycle. And this was, at that time, a very intriguing idea! Page 15

Science-Fridays-LABiomed02a-DDW_English 211 00:20:56,009 --> 00:20:58,000 Because, the cycle was not yet closed..., 212 00:20:58,009 --> 00:21:02,000 ...Krebs' initial reactions had not really been clarified, yet..., 213 00:21:02,009 --> 00:21:09,000 ...yet, Dr. Szent-Györgyi already laid some of the reactions down, which are involved in... 214 00:21:09,009 --> 00:21:13,000 ...the citric acid cycle, with the use of hydrogen, and protons... 215 00:21:13,009 --> 00:21:17,000 ...and this was the main function of succinate and fumarate in muscle cells, to carry these... 216 00:21:17,009 --> 00:21:23,000 protons! So ..., as you can see, there are two places..., 217 00:21:23,009 --> 00:21:30,000 and there are actually two net water molecules consumed by the Cycle, as it turns over. 218 00:21:30,009 --> 00:21:39,000 One enters at aconitase, which actually provides the hydrogen for the first hydrogen- or proton-yielding... 219 00:21:39,009 --> 00:21:42,000 ...reaction. And the second one is at fumarate hydratase..., 220 00:21:42,009 --> 00:21:50,000 ...which actually, ...with the cycle turning over, actually, will provide the proton for NADPH synthesis..., 221 00:21:50,009 --> 00:21:54,000 ...which I am going to show in a minute, how it works. 222 00:21:54,009 --> 00:21:56,000 It seems that water, or metabolic or matrix water... 223 00:21:56,009 --> 00:22:05,000 ...enters the intermediate pool very quickly, as just to replace many of the hydrogens... 224 00:22:05,009 --> 00:22:10,000 ...and "do not bring" water into the cycle, because we have our own water in the cycle, that we... Page 16

Science-Fridays-LABiomed02a-DDW_English 225 00:22:10,009 --> 00:22:16,000 ...are going to use for turning the cycle over for reducing equivalent synthesis! 226 00:22:16,009 --> 00:22:23,000 Now ..., as I was mentioning, the original role of the TCA cycle, or the normal mitochondria..., 227 00:22:23,009 --> 00:22:29,000 ...is actually to produce water. And we know this, because when we look at physiology... 228 00:22:29,009 --> 00:22:37,000 ...for example, animals that live either in the ocean, and they can not really drink salt water... 229 00:22:37,009 --> 00:22:41,000 ...from the ocean, or they live in the desert, where there is no water... 230 00:22:41,009 --> 00:22:45,000 ...they use the mitochondria to burn fat! 231 00:22:45,009 --> 00:22:50,000 And this is why..., if you look at the camel, if you look at the scorpion, the rattle snake..., 232 00:22:50,009 --> 00:22:58,000 that they all have to have a fat pool, which they get from their prey, that synthesize fat from plants..., 233 00:22:58,009 --> 00:23:04,000 which live in the desert. And, surprisingly, from 100 grams of fat they... 234 00:23:04,009 --> 00:23:07,000 can actually produce one hundred ten grams of water! 235 00:23:07,009 --> 00:23:11,000 And this is because of the saturation, the hydrogen saturation of the fat so you just... 236 00:23:11,009 --> 00:23:14,000 ...need the oxygen! And if you talk to a physiologist about what... 237 00:23:14,009 --> 00:23:19,000 ...would be the best model for studying lungs, they'll always tell you they are looking at snake lungs. 238 00:23:19,009 --> 00:23:23,000 The snakes really have huge lungs just to supply... 239 Page 17

Science-Fridays-LABiomed02a-DDW_English 00:23:23,009 --> 00:23:27,000 ...oxygen for their water production in the Cycle. 240 00:23:27,009 --> 00:23:31,000 Question 03: Laszlo, is this why weight watchers tell you to drink a lot of water? 241 00:23:31,009 --> 00:23:39,000 No, actually it's for some other reason, because we do not use our own (metabolic) water... "as water" 242 00:23:39,009 --> 00:23:42,000 Our problem is..., if you look at the Cycle, ...and this is the... 243 00:23:42,009 --> 00:23:46,000 ...main difference between the rattle snakes and the mammals..., 244 00:23:46,009 --> 00:23:49,000 we invented the mitochondria to use it for energy production..., 245 00:23:49,009 --> 00:23:54,000 ...ATP synthesis, actually, instead of releasing (matrix) water, we reuse... 246 00:23:54,009 --> 00:24:00,000 ...95% of it for NADPH synthesis and for membrane turnover! 247 00:24:00,009 --> 00:24:06,000 We cannot produce net water! Migrating birds can..., and this is how it all developed: 248 00:24:06,009 --> 00:24:10,000 1) the rattle snake uses mitochondria for water production..., 249 00:24:10,009 --> 00:24:14,000 but the reptiles that actually "fly..." (migrate) to temperate seasons..., 250 00:24:14,009 --> 00:24:19,000 where there is water, so they can drink now..., so they invented the 251 00:24:19,009 --> 00:24:23,000 mitochondrial function of producing heat and ATP, so they can stay... (in the cold)! 252 00:24:23,009 --> 00:24:30,000 So..., it is an evolutionary impact on metabolism, eventually..., to use mitochondria for something else..., 253 00:24:30,009 --> 00:24:33,000 Page 18

Science-Fridays-LABiomed02a-DDW_English ...so..., even though we produce the same amount of matrix water..., 254 00:24:33,009 --> 00:24:38,000 we reuse it through fumarate hydratase, so matrix water is reintroduced into the Cycle... 255 00:24:38,009 --> 00:24:41,000 and, it is carried out from the cycle (as malate), which I am going to show you! 256 00:24:41,009 --> 00:24:48,000 This is why we have to drink water. If "rattle snakes exercise", they do... 257 00:24:48,009 --> 00:24:53,000 ...not have to drink water, because they just burn more fat, and..., use fat for water production! 258 00:24:53,009 --> 00:25:00,000 But, anyways, Dr. Mellanby was an ornithologist and he was... 259 00:25:00,009 --> 00:25:05,000 ...involved and interested in studying animals that live in the desert. 260 00:25:05,009 --> 00:25:15,000 Now, if you use carbohydrates as the source of NAD(P)H in the Cycle you only generate 55 grams... 261 00:25:15,009 --> 00:25:20,000 ...of water from carbohydrates. And this is simply because their hydrogen content... 262 00:25:20,009 --> 00:25:24,000 is not as high as that in fat, as saturated fat has the highest hydrogen content. 263 00:25:24,009 --> 00:25:31,000 So, they need oxygen to produce water and animals can live in the desert with that approach. 264 00:25:31,009 --> 00:25:38,000 So, we are using up matrix water, instead of producing it and leaving it as water. 265 00:25:38,009 --> 00:25:44,000 Obviously the rattle snake, because they cannot keep producing ATP by synthesis and recycling (of water).., 266 00:25:44,009 --> 00:25:49,000 ...as they use up matrix water for maintaining biological solvent functions..., 267 00:25:49,009 --> 00:25:52,000 ...snakes have to hibernate, because cannot really control body temperature..., Page 19

Science-Fridays-LABiomed02a-DDW_English 268 00:25:52,009 --> 00:26:00,000 ...snakes have to use and generate heat from sunlight, so that they can use matrix water for carrying... 269 00:26:00,009 --> 00:26:09,000 ...out functions that are involved in enzyme activities and to contain, maintain water content... 270 00:26:09,009 --> 00:26:13,000 ...in their bodies! Once we use matrix water up for malate and... 271 00:26:13,009 --> 00:26:18,000 ...eventually NADPH synthesis, as the reducing equivalent..., 272 00:26:18,009 --> 00:26:23,000 ...we are going to lose the ability to use matrix water to produce our own water, so we... 273 00:26:23,009 --> 00:26:28,000 have to drink water. So we have to replenish our water from other sources! 274 00:26:28,009 --> 00:26:34,000 So, the NADH source and the water in the matrix..., 275 00:26:34,009 --> 00:26:41,000 ...metabolic water, has a very unique characteristic. 276 00:26:41,009 --> 00:26:44,000 And that means, that.., it is actually low in deuterium! 277 00:26:44,009 --> 00:26:49,000 And this, actually, will take us to another story, which, I think, is very intriguing. 278 00:26:49,009 --> 00:26:53,000 As this metabolic water is different from the water that you drink. 279 00:26:53,009 --> 00:26:59,000 So, whatever you drink there, has higher deuterium to what each mitochondrion produces. 280 00:26:59,009 --> 00:27:02,000 And the reason for that is...., 281 00:27:02,009 --> 00:27:10,000 because..., in your food, your fat, in your long chain saturated fatty acids, the hydrogen content... Page 20

Science-Fridays-LABiomed02a-DDW_English 282 00:27:10,009 --> 00:27:16,000 ...of your fat has actually lower deuterium, than that of the water, what you drink! 283 00:27:16,009 --> 00:27:23,000 We measure deuterium content, in any substrate, including water, as the Vienna standard of... 284 00:27:23,009 --> 00:27:30,000 ...mean oceanic water, which has around 155 parts per million (ppm). 285 00:27:30,009 --> 00:27:37,000 If you look at your food source, looking at your flour, table sugar, they are actually... 286 00:27:37,009 --> 00:27:41,000 ...very close to the 150 ppm. And when you get to the cottage cheese, which... 287 00:27:41,009 --> 00:27:48,000 ...is a good mixture of fat and carbohydrates, you can see it is dropping to 136! 288 00:27:48,009 --> 00:27:54,000 If you look at olive oil, it is 130 ppm. Then, in butter it is 124! 289 00:27:54,009 --> 00:27:58,000 And, in fatty acid of animal fat, it is only 118 ppm. 290 00:27:58,009 --> 00:28:06,000 That means, as more fatty the food gets, less deuterium those fatty acids accumulate, or contain! 291 00:28:06,009 --> 00:28:08,000 More interestingly..., 292 00:28:08,009 --> 00:28:16,000 ...actually, if you look at unsaturated fat from plants, those are actually very low in deuterium. 293 00:28:16,009 --> 00:28:23,000 And, actually, if you look at double bonds in your unsaturated fat, it is only 102 ppm..., 294 00:28:23,009 --> 00:28:27,000 ...getting close to almost a 40% drop in deuterium content. 295 00:28:27,009 --> 00:28:37,000 So..., more fat or unsaturated fat you actually consume, your water going to have less and... 296 Page 21

Science-Fridays-LABiomed02a-DDW_English 00:28:37,009 --> 00:28:42,000 ...less deuterium and so is your matrix water, in mitochondria..., 297 00:28:42,009 --> 00:28:47,000 ...will have less and less deuterium. What's also very interesting is that, actually..., 298 00:28:47,009 --> 00:28:51,000 ...this occurs, or seems to be occurring, during photosynthesis. 299 00:28:51,009 --> 00:28:58,000 Light enters plant cells, you can see here the water from our environment..., 300 00:28:58,009 --> 00:29:05,000 ...or the free water, which has 155 ppm, which will actually be cleaved, while oxygen is being produced. 301 00:29:05,009 --> 00:29:12,000 And the reducing equivalent, which uses this hydrogen, enters this cycle, called the Calvin cycle..., 302 00:29:12,009 --> 00:29:15,000 ...which actually drop deuterium. 303 00:29:15,009 --> 00:29:20,000 So, there is discrimination against deuterium, when it comes to photosynthesis (and cleaving water). 304 00:29:20,009 --> 00:29:26,000 So because of photosynthesis, and the enzymes that perform photosynthesis, plants drop deuterium... 305 00:29:26,009 --> 00:29:31,000 ...during the photosynthesis process! The fat pool the plant will synthesize... 306 00:29:31,009 --> 00:29:36,000 ...has lower deuterium. So, when we consume fat and burn it, in mitochondria..., 307 00:29:36,009 --> 00:29:43,000 ...the NADPH pool, that we restore, is actually low in deuterium ... 308 00:29:43,009 --> 00:29:45,000 and its... Go ahead!... 309 00:29:45,009 --> 00:29:56,000 Question 04: Are those enzymes less efficient in using deuterium from water? Why is deuterium getting dropped? Because..., 310 Page 22

Science-Fridays-LABiomed02a-DDW_English 00:29:56,009 --> 00:30:00,000 ...deuterium is twice as heavy as hydrogen! I know, but are those enzymes less efficient... 311 00:30:00,009 --> 00:30:04,000 in carrying deuterium? Yes, those enzymes have actually less binding..., 312 00:30:04,009 --> 00:30:08,000 ...which is actually dropping, it is physically to drop deuterium, which is fascinating! 313 00:30:08,009 --> 00:30:12,000 Do we know which enzyme(s)? OK, there are a number of enzymes, which are, actually..., 314 00:30:12,009 --> 00:30:19,000 ...responsible for deuterium depletion, especially desaturases. 315 00:30:19,009 --> 00:30:31,000 Desaturases seem to drop deuterium like crazy. Thank you! Trying to go back... [deuterium bonds in water require more photons and sunlight to break, during photosynthesis] 316 00:30:31,009 --> 00:30:39,000 It is a French team, Dr. Billault, I hope I say it right, they actually measure deuterium content..., 317 00:30:39,009 --> 00:30:47,000 ...in a number of fat products, so they know what, actually, unsaturated carbon sites... 318 00:30:47,009 --> 00:30:52,000 ...are the lowest in deuterium, and what plants and fats those are! (doi: 10.1074/jbc.M500909200) 319 00:30:52,009 --> 00:30:59,000 They have had a huge facility, just to..., actually, just to measure that and they use these isotope... 320 00:30:59,009 --> 00:31:07,000 ...ratio mass spectrometers, so they actually combust these substrates to measure their... 321 00:31:07,009 --> 00:31:13,000 ...deuterium content. But, anyways... 322 00:31:13,009 --> 00:31:27,000 So, you can actually deplete deuterium in your cells, using NADPH, restored after photosynthesis..., 323 00:31:27,009 --> 00:31:33,000 ...or, if you live at high altitudes. Your water will have low deuterium because the rain falls out... Page 23

Science-Fridays-LABiomed02a-DDW_English 324 00:31:33,009 --> 00:31:37,000 ...with heavy water, first... (at lower altitudes). So..., 325 00:31:37,009 --> 00:31:42,000 ...and those are the people who live the longest. The Peruvian people, actually, they may... 326 00:31:42,009 --> 00:31:46,000 ...live, in Caucasia, the Himalayans, they may live over a 100 years! 327 00:31:46,009 --> 00:31:50,000 And, what is really interesting is that, they do not have their natural or authentic... 328 00:31:50,009 --> 00:31:53,000 ...words for cancer, meaning that, it is really unusual that they... 329 00:31:53,009 --> 00:31:56,000 ...would get cancer, at those high altitudes. 330 00:31:56,009 --> 00:32:00,000 And, actually, as they drink (mountain) water, they do not need... 331 00:32:00,009 --> 00:32:07,000 ...their lowered deuterium water..., plus the photosynthesis enzymes, they can really go low, as far as deuterium enrichment. 332 00:32:07,009 --> 00:32:15,000 Which is really interesting! So..., if we look at the model, when a normal... 333 00:32:15,009 --> 00:32:24,000 ...cell uses fatty acids for producing (matrix) water and using that water for NADPH production..., 334 00:32:24,009 --> 00:32:29,000 ...what you can see is, because of the low deuterium content in fat..., 335 00:32:29,009 --> 00:32:39,000 ...you are going to introduce in nucleic acid and DNA a low deuterium content, as far as its... 336 00:32:39,009 --> 00:32:47,000 ...structure goes. And your DNA will have more hydrogen than deuterium, if mitochondrial function is maintained. 337 00:32:47,009 --> 00:32:52,000 It is simply because your food source with mitochondrial... Page 24

Science-Fridays-LABiomed02a-DDW_English 338 00:32:52,009 --> 00:32:59,000 ...function is to restore and to maintain low deuterium water content in the matrix. 339 00:32:59,009 --> 00:33:02,000 And we know from stable isotope studies, that... 340 00:33:02,009 --> 00:33:10,000 ...actually, mitochondria oxidize about 90% of acetyl-CoA that come in to get oxidized from fatty acids. 341 00:33:10,009 --> 00:33:16,000 So, either dietary or fatty acids we internally re-produce from dietary fats. 342 00:33:16,009 --> 00:33:23,000 So, the mitochondria in the matrix always have, or try to decrease deuterium..., 343 00:33:23,009 --> 00:33:31,000 ...while the arm of high water intake, with high ppm deuterium water intake, through the pentose cycle..., 344 00:33:31,009 --> 00:33:38,000 ...or the peroxisomal beta oxidation process, cannot reach into the DNA, in normal resting cells. 345 00:33:38,009 --> 00:33:46,000 And, with normal oxygen supplies, NADPH production goes through the mitochondrial... 346 00:33:46,009 --> 00:33:50,000 ...cycle intermediates, that have low deuterium, of about 120 ppm. 347 00:33:50,009 --> 00:33:55,000 Actually, if you feed an individual with only a fat diet - like a ketogenic diet - and in cancer... 348 00:33:55,009 --> 00:34:03,000 ...it is very beneficial. With the ketogenic diet you can decrease body water deuterium down... 349 00:34:03,009 --> 00:34:11,000 ...to about 110 ppm, and simply, because fat has lower deuterium for the mitochondria to exchange water. 350 00:34:11,009 --> 00:34:18,000 So..., what is wrong with deuterium? So why... 351 00:34:18,009 --> 00:34:24,000 ...is deuterium so bad, as far as its clinical behavior, or its physical behavior goes? 352 Page 25

Science-Fridays-LABiomed02a-DDW_English 00:34:24,009 --> 00:34:30,000 Actually, it is twice as heavy as hydrogen. Hydrogen has one proton, and deuterium has... 353 00:34:30,009 --> 00:34:35,000 ...one proton and one neutron, so it actually weighs twice as much as hydrogen. 354 00:34:35,009 --> 00:34:43,000 Yet, it is chemically identical, so it participates in every reaction that our body carries out... 355 00:34:43,009 --> 00:34:50,000 ...as far as reductive synthesis goes. And actually when deuterium gets into DNA, it occupies... 356 00:34:50,009 --> 00:34:57,000 ...these methylation sites, and when the methylation enzymes come, they can remove hydrogen very easily..., 357 00:34:57,009 --> 00:35:04,000 but when it comes to, when the site is occupied by this heavy deuterium, it really causes a trouble. 358 00:35:04,009 --> 00:35:09,000 And, because of this heavy deuterium incorporation into nucleic acid..., 359 00:35:09,009 --> 00:35:13,000 ...there is altered gene expression and methylation kinetics will change. 360 00:35:13,009 --> 00:35:21,000 And, likely because of the stronger mass induced resonance between hydrogen and deuterium bonds..., 361 00:35:21,009 --> 00:35:29,000 ...in each base pair with counterpart opposite bases, deuterium may be involved in aneuploidy. 362 00:35:29,009 --> 00:35:38,000 So..., hydrogen being light, half the mass of deuterium, it is easy to lift..., 363 00:35:38,009 --> 00:35:45,000 ...but when it comes to deuterium, it is going to be a huge effort for any enzyme to deal with it..., 364 00:35:45,009 --> 00:35:49,000 especially, when it is an enzyme, which is structurally incorporating it into DNA..., 365 00:35:49,009 --> 00:35:55,000 ...or the nucleic acid membranes that are participating in those bindings. Hydrogen bonds are really..., 366 00:35:55,009 --> 00:35:59,000 Page 26

Science-Fridays-LABiomed02a-DDW_English truly, very important, when it comes to DNA physiology (and nuclear stability) 367 00:35:59,009 --> 00:36:06,000 So..., in a normal cell, where mitochondria is active, we can actually keep a very decent... 368 00:36:06,009 --> 00:36:11,000 ...nuclear structure and DNA structure. When it comes to cancer and mitochondria... 369 00:36:11,009 --> 00:36:19,000 ...are dismantled, and they use the pentose cycle, as well as other reactions that actually load... 370 00:36:19,009 --> 00:36:23,000 ...your NADPH and reducing equivalent system with free water..., 371 00:36:23,009 --> 00:36:28,000 ...you are going to have morphological changes. And with that, we will have physiological changes..., 372 00:36:28,009 --> 00:36:38,000 ...morphological and biological changes in cell size, cell behavior, and the cell's regulatory mechanisms. 373 00:36:38,009 --> 00:36:43,000 So..., we believe that the first very important epigenetic event..., 374 00:36:43,009 --> 00:36:51,000 that is really (excuse me) to modify nuclear function, before methylation occurs..., 375 00:36:51,009 --> 00:36:55,000 ...is actually DNA deuteration! And because of its chemical and physical... 376 00:36:55,009 --> 00:37:00,000 ...impact and characteristics, this is a very important event in cancer development. 377 00:37:00,009 --> 00:37:09,000 Now, where can NADPH enter, or hydrogen or deuterium enter, metabolism, when the mitochondria... 378 00:37:09,009 --> 00:37:16,000 ...are not active, or in cancer, when there is no oxygen, or when there is an enzyme mutation... 379 00:37:16,009 --> 00:37:20,000 ...in the mitochondria? It is either through this serine oxidation glycine cleavage system..., 380 00:37:20,009 --> 00:37:27,000 ...which we have recently published in Nature. This is one of the sites where water, not matrix water..., Page 27

Science-Fridays-LABiomed02a-DDW_English (doi: 10.1038/cddis.2013.393). 381 00:37:27,009 --> 00:37:30,000 ...but free water, is the source of hydrogen/deuterium..., 382 00:37:30,009 --> 00:37:38,000 ...or through the pentose cycle, which actually uses glucose, or table sugar, or flour, which... 383 00:37:38,009 --> 00:37:41,000 ...we use for cooking, and it has higher (deuterium) ppm. 384 00:37:41,009 --> 00:37:47,000 It is interesting that the plant's fatty acid synthesis enzymes drop deuterium..., 385 00:37:47,009 --> 00:37:54,000 ...but actually in fruit plants when fruit sugar is generated (with intense sunlight), those photosynthesis enzymes can better hold deuterium. 386 00:37:54,009 --> 00:37:58,000 So the sugar, and also the water, which goes through... 387 00:37:58,009 --> 00:38:04,000 ...and generates the NADPH through the oxidative branch of the pentose cycle, actually, 388 00:38:04,009 --> 00:38:07,000 has high deuterium. And we have been publishing regarding the... 389 00:38:07,009 --> 00:38:17,000 ...role of NADPH synthesis in the oncogenic process through the oxidative branch of the pentose cycle. [PMID: 9488183] 390 00:38:17,009 --> 00:38:20,000 In fact, this is the set of reactions that Glivec controls! 391 00:38:20,009 --> 00:38:25,000 Glivec inhibits the oxidative branch of the pentose cycle, which we have shown in the Journal... 392 00:38:25,009 --> 00:38:32,000 ...of Biological Chemistry. So, in cancer we have to offer another model..., [doi: 10.1074/jbc.M105796200 - 10.1056/NEJM200207043470116] 393 00:38:32,009 --> 00:38:38,000 ...and that is simply when (mitochondrial) fumarate hydratase, or, when isocitrate dehydrogenase mutations... Page 28

Science-Fridays-LABiomed02a-DDW_English 394 00:38:38,009 --> 00:38:44,000 ...occur, the low deuterium containing matrix water cannot enter metabolism... 395 00:38:44,009 --> 00:38:53,000 And what the cells are left with are glycolysis, pentose cycling, (cytoplasmic fumarate hydratase), the SOGC pathway and, actually..., 396 00:38:53,009 --> 00:39:01,000 ...high deuterium is getting in from free water, or from substrates other than fatty acids, which would lower deuterium. 397 00:39:01,009 --> 00:39:04,000 So..., eventually, cancer cells will end up with a heavy sticky DNA..., 398 00:39:04,009 --> 00:39:08,000 ...and nuclear plasma membrane fatty acids, that are actually loaded (with deuterium)..., 399 00:39:08,009 --> 00:39:20,000 ...or, actually because of synthesis, new DNA synthesis via NADP deuterium or NADPD reducing equivalent, cancer cells will going... 400 00:39:20,009 --> 00:39:24,000 ...to have membranes, and also fatty acids, that are deuterium rich. 401 00:39:24,009 --> 00:39:28,000 And, this is what happens actually in these tumor cells. 402 00:39:28,009 --> 00:39:32,000 When you look at the fumarate hydratase wild type cells..., 403 00:39:32,009 --> 00:39:37,000 ...and you look at the empty vector, we are going to look at the fumarate hydratase mutant... 404 00:39:37,009 --> 00:39:42,000 ...through two different phenotypes, which actually lack the enzyme, one is a primary..., 405 00:39:42,009 --> 00:39:48,000 ...the other one is a metastatic tumor. If you look at, for example, the nuclear membrane... 406 00:39:48,009 --> 00:39:55,000 ...long chain saturated (lignocerate) formation, you are going to see..., as tumorigenesis advances..., 407 00:39:55,009 --> 00:40:00,000 that is how they going to incorporate more and more long chain saturated fatty acids... Page 29

Science-Fridays-LABiomed02a-DDW_English 408 00:40:00,009 --> 00:40:06,000 ...into their nuclear membranes. So..., they need these "double bags" with rigid... 409 00:40:06,009 --> 00:40:11,000 ...membranes to keep this aneuploidy and nuclear structure together. 410 00:40:11,009 --> 00:40:18,000 And this is the type of metabolic change, which is very important, when it comes to..., 411 00:40:18,009 --> 00:40:26,000 ...for example, a fumarate hydratase mutation, and also which comes with an aggressive tumorigenic... 412 00:40:26,009 --> 00:40:30,000 ...phenotype. And, if you remember, this guy, the one with the wild type fumarate hydratase gene... 413 00:40:30,009 --> 00:40:34,000 and reaction, after we restored it, these are not tumorigenic! 414 00:40:34,009 --> 00:40:37,000 But the other ones, are! So..., actually, for example, as far as a long chain... 415 00:40:37,009 --> 00:40:44,000 ...saturated lignoceric acid of nuclear membranes, it is responsive to fumarate hydratase activity... 416 00:40:44,009 --> 00:40:48,000 So..., Question 05: Excuse me, that means .. if you have wild type .... 417 00:40:48,009 --> 00:40:55,000 ...cells and you treat them with a carbon source that contain deuterium, high level... 418 00:40:55,009 --> 00:41:05,000 ...of deuterium, .. they still function properly, that means your starting model changes... 419 00:41:05,009 --> 00:41:08,000 That is right! Have you published this? 420 00:41:08,009 --> 00:41:12,000 We are publishing..., we have published the background... [doi: 10.1371/journal.pone.0072179] 421 00:41:12,009 --> 00:41:16,000 ....But in concept, it does not matter, in concept; you already have a mutation, so this is a mutation... Page 30

Science-Fridays-LABiomed02a-DDW_English 422 00:41:16,009 --> 00:41:20,000 ...you do not know if that mutation causes a secondary effect, that you see in increasing... 423 00:41:20,009 --> 00:41:28,000 ...deuterium, and these pose a concomitant increase that you see, and changes in chromatin. So, it does not... 424 00:41:28,009 --> 00:41:32,000 ...mean that it is going to be the deuterium! The only way to prove that the deuterium is doing... 425 00:41:32,009 --> 00:41:38,000 this to the system, if you just change deuterium, only deuterium, and if you also... 426 00:41:38,009 --> 00:41:43,000 ...going to see in the cells that deuterium changes the chromatin! 427 00:41:43,009 --> 00:41:49,000 This is a very good question! And this is exactly what we did. Let me show you the next study... 428 00:41:49,009 --> 00:41:55,000 ...we did, because what we did is we actually treated the tumor cells with deuterium depleted water. 429 00:41:55,009 --> 00:42:01,000 So, we actually left out the deuterium. Question(cont): But if the water needs to be produced by the... 430 00:42:01,009 --> 00:42:06,000 ...mitochondria, so that neither water nor deuterium is going to be precipitated into the DNA? 431 00:42:06,009 --> 00:42:12,000 It will, it would, through the oxidative branch of the pentose cycle (and cytoplasmic fumarate hydratase) Question(cont) ...the pentose cycle 432 00:42:12,009 --> 00:42:15,000 It is free water through the pentose cycle! This is the whole point...: 433 00:42:15,009 --> 00:42:22,000 If your mitochondria are not working you still have a mechanism to get low deuterium into... 434 00:42:22,009 --> 00:42:25,000 ...these cells and that is through the oxidative branch of the pentose cycle! 435 00:42:25,009 --> 00:42:29,000 So..., if you actually treat these cells, tumor cells..., Page 31

Science-Fridays-LABiomed02a-DDW_English 436 00:42:29,009 --> 00:42:35,000 ...with low deuterium water, let's see what happens! But let me go first through the system! 437 00:42:35,009 --> 00:42:41,000 So..., we have tumor cells that do not... 438 00:42:41,009 --> 00:42:47,000 ...use matrix water. And so the likelihood of low deuterium getting into... 439 00:42:47,009 --> 00:42:54,000 the DNA structures, through the oxidative branch of the pentose cycle, opens a window for us..., 440 00:42:54,009 --> 00:43:00,000 ...which can actually affect the system through water, simply, not matrix water, but next... 441 00:43:00,009 --> 00:43:03,000 ...treating these cells with deuterium depleted water. 442 00:43:03,009 --> 00:43:12,000 So..., and in fact this is what happens! Because, if we treat these cells with low deuterium water... 443 00:43:12,009 --> 00:43:15,000 ...and this is a poster which we presented at... 444 00:43:15,009 --> 00:43:25,000 ...the 2006 Metabolomics (Society) Conference. If you look at the 150 ppm, which is the control... (doi: 10.12918/HYDLLCHU2006POST) 445 00:43:25,009 --> 00:43:31,000 ...reference water, and at 100, 50 and 25 ppm, so we are decreasing the deuterium in drinking... 446 00:43:31,009 --> 00:43:37,000 ...water, or media water, in these tumor cells. As you can see, the same fatty acid, lignoceric... 447 00:43:37,009 --> 00:43:45,000 ...acid, which is the nuclear membrane fatty acid, (DDW) actually introduces a drop in new lignocerate... 448 00:43:45,009 --> 00:43:52,000 ...synthesis, in nuclear size and also in tumorigenicity of these cells. 449 00:43:52,009 --> 00:43:59,000 And this is the same, this is with MIA PaCa pancreatic adenocarcinoma cells. Page 32

Science-Fridays-LABiomed02a-DDW_English 450 00:43:59,009 --> 00:44:04,000 If you look at the palmitate synthesis, which... 451 00:44:04,009 --> 00:44:10,000 ...is the shorter chain fatty acid, only the highest, 25 parts per million deuterium depleted water... 452 00:44:10,009 --> 00:44:20,000 ...lowers the de novo fatty acid synthesis. If you look at MCF-7 breast cancer cells and... 453 00:44:20,009 --> 00:44:25,000 ...look at the nuclear membrane cholesterol synthesis, which is another important structural fatty acid... 454 00:44:25,009 --> 00:44:31,000 ...or sterol, what you can see is coming from 150 ppm, which is... 455 00:44:31,009 --> 00:44:38,000 the reference range, down to the 100, 50 and 25 ppm, you can see a decrease in cholesterol... 456 00:44:38,009 --> 00:44:44,000 ...synthesis and nuclear size. Now, besides that, if you look at c-myc expression, 457 00:44:44,009 --> 00:44:48,000 ...and oncogenic expressions, they also correlate with these changes (doi: 10.1080/01635581.2013.756533) 458 00:44:48,009 --> 00:44:54,000 If you look at the DNA structure, what you are going to see is the deuterium content... 459 00:44:54,009 --> 00:45:00,000 of the DNA, itself, especially at the cytosine's hydrogen group, where actually methylation... 460 00:45:00,009 --> 00:45:05,000 ...occur, may decrease also. So now..., even though your mitochondria is defective... 461 00:45:05,009 --> 00:45:11,000 ...you can still introduce low deuterium into the cells through the oxidative branch of... 462 00:45:11,009 --> 00:45:14,000 ...the pentose cycle through drinking water! So, eventually...., 463 00:45:14,009 --> 00:45:25,000 ...you can change, for example, lignocerate synthesis, which is a structural membrane fatty acid... Page 33

Science-Fridays-LABiomed02a-DDW_English 464 00:45:25,009 --> 00:45:30,000 ...synthesis. Thirty percent of nuclear membranes have this fatty acid in their phospholipids... 465 00:45:30,009 --> 00:45:38,000 ...which is a very rigid and a very important fatty acid species to actually synthesize... 466 00:45:38,009 --> 00:45:42,000 ...a rigid nuclear structure! Now..., interestingly..., plant cells, which... 467 00:45:42,009 --> 00:45:51,000 ...are eukaryote cells, they have to consume water, because that is what they use for photosynthesis [and can not oxidize fat]... 468 00:45:51,009 --> 00:45:58,000 ...obviously, to keep their (deuterium loaded) nuclear structures stable, they have to use proteoglycans and... 469 00:45:58,009 --> 00:46:03,000 ...they have to synthesize sugar components into their cell membranes... 470 00:46:03,009 --> 00:46:12,000 ...and, for example cellulose, which keeps those nuclear structures less vulnerable to aneuploid changes! 471 00:46:12,009 --> 00:46:15,000 This (nuclear stability) is what lignocerate does for mammalian cells... 472 00:46:15,009 --> 00:46:18,000 ... because we really cannot link those large sugar molecules up! 473 00:46:18,009 --> 00:46:24,000 So..., lignoceric acid is the long chain saturated fatty acid of the nuclear membrane, which is... 474 00:46:24,009 --> 00:46:32,000 ...involved in determining nuclear structure, in size and strength! Eventually, and this is what I'd like to ..., just 475 00:46:32,009 --> 00:46:39,000 ...to process the last few slides. And this is the set of take home messages. 476 00:46:39,009 --> 00:46:45,000 Impaired mitochondria are involved in cell transformation and this is by limiting the... 477 00:46:45,009 --> 00:46:51,000 ...low natural deuterium containing complete fatty acid oxidation product, which is metabolic water... Page 34

Science-Fridays-LABiomed02a-DDW_English 478 00:46:51,009 --> 00:46:57,000 ...to enter nuclear membranes and nucleotides through reductive synthesis... 479 00:46:57,009 --> 00:47:04,000 The other very important message is that, in turn, heavy or natural water and sugar... 480 00:47:04,009 --> 00:47:10,000 ...dependent NADPH production takes over by the oxidative branch of the pentose cycle..., 481 00:47:10,009 --> 00:47:17,000 ...as well as other-pathways that I showed you, which actually are ticking time bombs with a strong... 482 00:47:17,009 --> 00:47:21,000 ...isotope effect. And there is an oncogenic epigenetic event..., 483 00:47:21,009 --> 00:47:25,000 ...the likelihood of more frequent oncogenic and epigenetic events... 484 00:47:25,009 --> 00:47:30,000 ...with unstable hydrogen bonds and aneuploidy! 485 00:47:30,009 --> 00:47:36,000 Nevertheless, extra-mitochondrial NADPH synthesis opens a therapeutic window. 486 00:47:36,009 --> 00:47:42,000 So.., using the oxidative branch of the pentose cycle (and cytoplasmic fumarate hydratase), we can actually still introduce low... 487 00:47:42,009 --> 00:47:47,000 ...deuterium content into DNA. If you use water, or if you use a food source... 488 00:47:47,009 --> 00:47:52,000 ...that has low deuterium. And..., eventually... 489 00:47:52,009 --> 00:47:59,000 ...this approach can explain some of the recent findings that are entering the medical literature. 490 00:47:59,009 --> 00:48:02,000 For example..., if you feed your rats with a ketogenic diet..., 491 00:48:02,009 --> 00:48:09,000 ...which is only fat based, and you actually treat them with oxygen treatment, or hyperbaric... Page 35

Science-Fridays-LABiomed02a-DDW_English 492 00:48:09,009 --> 00:48:11,000 ...oxygen treatment, then you decrease tumorigenicity by over 78 percent! 493 00:48:11,009 --> 00:48:17,000 And in ketogenic diet and oxygen..., oxygen treatment on its own does not do much... 494 00:48:17,009 --> 00:48:22,000 ...but if we combine it with ketogenic diet that...., ketogenic diet on its own decreases tumorigenicity... 495 00:48:22,009 --> 00:48:26,000 by over 20 (~65) percent, but when you combine it with oxygen treatment... 496 00:48:26,009 --> 00:48:33,000 ...that is over 78 percent decrease (in tumorigenicity) and this is in PLoS ONE from a Florida group (doi: DOI: 10.1371/journal.pone.0065522) 497 00:48:33,009 --> 00:48:38,000 ...and what is really interesting is that..., it is really truly metabolic water..., 498 00:48:38,009 --> 00:48:43,000 ...when ketogenic diet can actually have any physiological effect or biological effect... 499 00:48:43,009 --> 00:48:48,000 ...in the presence of oxygen treatment, because this is a cytochrome-c (complex-IV)... 500 00:48:48,009 --> 00:48:54,000 ...dependent reaction, no pentose cycle, no peroxisomes can be directly involved... 501 00:48:54,009 --> 00:48:57,000 ...in the process, because oxygen is the driving force. 502 00:48:57,009 --> 00:49:02,000 And there is another very important aspect of this... 503 00:49:02,009 --> 00:49:08,000 ...and this is practically the complete oxidation of these fatty acids, which will and is providing... 504 00:49:08,009 --> 00:49:16,000 ...the low deuterium metabolic water to prevent, eventually, DNA, histone and nuclear membrane... 505 00:49:16,009 --> 00:49:20,000 ...deuteration, which we believe is a very important force... Page 36

Science-Fridays-LABiomed02a-DDW_English 506 00:49:20,009 --> 00:49:28,000 ...in driving epigenetic events! I have to thank our collaborators from Europe... 507 00:49:28,009 --> 00:49:34,000 ...who provided the deuterium depleted water, Dr. Lee, my colleague and... 508 00:49:34,009 --> 00:49:37,000 ...Dr. Linehan, who is at the National Cancer Institute..., 509 00:49:37,009 --> 00:49:45,000 ...and they provided for us the kidney cancer cell line, which actually has this fumarate... 510 00:49:45,009 --> 00:49:49,000 ...hydratase mutation, and the model, where fumarate hydrates is reintroduced. 511 00:49:49,009 --> 00:49:56,000 And we had several funding sources; one is the Hirshberg Foundation for Pancreatic Cancer Research. 512 00:49:56,009 --> 00:50:00,000 And we are also funded by the UCLA Clinical... 513 00:50:00,009 --> 00:50:04,000 ...and Translational Science Institute. And with that, I just wanted to thank you for 514 00:50:04,009 --> 00:50:10,000 ...coming and listening to my talk! Please send a teaching evaluation: Slides (PDF) - doi: 10.12918/SCIENCEFRDEC132014LGB Teaching evaluation (PDF) - doi: 10.12918/SCIENCEFDEC132014EVAL

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Science-Fridays-LABiomed02a-DDW_Magyar 1 00:00:01,009 --> 00:00:11,000 Dr. Papp András fordítása alapján végleges szöveg: Dr. Boros G. László Lektorálta: Dr. Somlyai Gábor, Biológus Bemutatás: Dr. Kevin W. Bruhn, Kutató Fõmunkatárs, Los Angeles Orvosbiológiai Kutató Intézet 2 00:00:11,009 --> 00:00:18,000 Orvosi diplomáját 1987-ben szerezte Szegeden, Magyarországon, majd 1990-ben az Egyesült Államokba jött. 3 00:00:18,009 --> 00:00:27,000 Nyolc évet töltött kutató fõmunkatársként az Ohió-i Állami Egyetemen, majd 1998-ban a Kaliforniai Egyetem Kikötõi Kórházi Központjához jött dolgozni, 4 00:00:27,009 --> 00:00:32,000 ahol Dr. Paul Lee-nél, aki szintén itt van ma, 5 00:00:32,009 --> 00:00:36,000 tanult tömegspektrometriás módszerekkel végzett stabil izotópos alkalmazásokat. 6 00:00:36,009 --> 00:00:43,000 Dr. Boros jelenleg társprofesszorként dolgozik a Kalifornia Egyetemen, 7 00:00:43,009 --> 00:00:51,000 és egyben tudományos igazgató a Los Angeles Orvosbiológiai Kutató Intézetben függetlenül mûködõ SiDMAP cégnél. 8 00:00:51,009 --> 00:00:58,000 A SiDMAP a metabolomika minden fázisát mûvelõ gyógyszerfejlesztési szolgáltató cég. 9 00:00:58,009 --> 00:01:04,000 László ma munkájáról beszél, anyagcsere kutatásról (metabolomikáról), és ami még ahhoz tartozik. 10 00:01:04,009 --> 00:01:07,000 Uram, öné a szó, egyben köszönöm doktor Úr. 11 00:01:07,009 --> 00:01:11,000 Megtisztelve érzem magam, és köszönöm a kedves bevezetõt. 12 00:01:11,009 --> 00:01:20,000 2013-ban ez az utolsó elõadás 13 00:01:20,009 --> 00:01:27,000 így igyekeztem egy nagyon érdekes témát hozni. 14 00:01:27,009 --> 00:01:38,000 Page 1

Science-Fridays-LABiomed02a-DDW_Magyar Úgy vélem, a deutérium-csökkentés és a rákkeletkezés epigenetikai részleteirõl beszélni igazán érdekfeszítõ lesz ma. 15 00:01:38,009 --> 00:01:45,000 Elméletnek és modellnek is újszerû, (doi: 10.1371/journal.pone.0072179) 16 00:01:45,009 --> 00:01:50,000 amit orvosi szaklapokban is közlünk, együttmûködésben az USA Nemzeti Rákkutató Intézetével, 17 00:01:50,009 --> 00:01:59,000 ahonnan a sejt-modellt is kaptuk ezekhez a vizsgálatokhoz. Elõször is... 18 00:01:59,009 --> 00:02:03,000 ...frissítsük fel, amit a sejtek mûködésérõl tudunk 19 00:02:03,009 --> 00:02:09,000 fõképp amit a prokarióta és eukarióta sejtekrõl tanultunk! 20 00:02:09,009 --> 00:02:18,000 Az eukarióta sejtekben sejtmagot találunk, ahol membránokkal körülvéve a DNS-, 21 00:02:18,009 --> 00:02:27,000 vagy nukleinsav található. A sejt igen összetett, a magnélkülieknél nagyobb méretû szerkezet, 22 00:02:27,009 --> 00:02:32,000 ahol számos funkció van elrejtve számos különbözõ speciális feladat ellátására, 23 00:02:32,009 --> 00:02:41,000 ahová, például..., differenciálódhatnak. 24 00:02:41,009 --> 00:02:47,000 És ami igazán érdekes, ez már a középiskolában... 25 00:02:47,009 --> 00:02:53,000 illetve az egyetemi évek elején is megragadott: ha egy sejt membránnal körülvett sejtmagot akar fejleszteni, 26 00:02:53,009 --> 00:02:58,000 akkor szüksége van az összes többi sejten belüli struktúrára is, 27 00:02:58,009 --> 00:03:07,000 ezek közül legérdekesebbek a mitokondriumok, illetve az a tény, hogy nincs olyan eukarióta sejt, 28 00:03:07,009 --> 00:03:16,000 Page 2

Science-Fridays-LABiomed02a-DDW_Magyar amelynek maghártyával körülvett nukleinsav-anyaga lenne ... mitokondriumok nélkül 29 00:03:16,009 --> 00:03:23,000 és ..., ez alól a szabály alól nincs kivétel! Másrészrõl, a sejtmag-hártya, vagy tömör... 30 00:03:23,009 --> 00:03:28,000 eukarióta jellegû sajtmag nélküli sejtekben mitokondriumok soha nem találhatóak. 31 00:03:28,009 --> 00:03:35,000 Tehát a sejtszerkezet, mûködés és energiatermelés kapcsolata volt az, ahol elkezdtük 32 00:03:35,009 --> 00:03:40,000 a mitokondriumokat alaposabban vizsgálni és modelleket alkalmazni, 33 00:03:40,009 --> 00:03:48,000 Olyanokat, ahol a mitokondriumok nem mûködnek, és ez rák kialakulásához vezet! 34 00:03:48,009 --> 00:03:55,000 Hogy jobban megértsük, elmondanám, hogy a sejten belüli membránrendszer, 35 00:03:55,009 --> 00:04:01,000 a maghártyával és a Golgi-készülékkel együtt, egy egész sor funkcionális fehérjét tartalmaz, 36 00:04:01,009 --> 00:04:09,000 melyek speciális enzimeikkel maguk is zsírsavakat és membránokat állítanak elõ. 37 00:04:09,009 --> 00:04:15,000 A mitokondrium pedig az energiatermelés helye, 38 00:04:15,009 --> 00:04:21,000 de ha az emlõssejtek evolúcióját tekintjük, a mitokondrium igazából nem csak energiát termel, 39 00:04:21,009 --> 00:04:27,000 hanem egyben a víztermelés helye is..., ez a mitokondriumok másik fõ feladata... 40 00:04:27,009 --> 00:04:30,000 és ezt részletesebben is megvizsgáljuk! 41 00:04:30,009 --> 00:04:37,000 A sejt belsõ váza a mikrotubulusokat, aktint, kötõfehérjéket, 42 00:04:37,009 --> 00:04:41,000 ...stb. tartalmazza. Így, egy differenciált eukarióta sejt Page 3

Science-Fridays-LABiomed02a-DDW_Magyar 43 00:04:41,009 --> 00:04:49,000 sokféle feladatot elláthat, melyek némely szabályozó eleme 44 00:04:49,009 --> 00:04:56,000 a mitokondriumokból származik! A mitokondriumban van egy belsõ és egy külsõ membrán, 45 00:04:56,009 --> 00:05:00,000 vannak a kriszták, és a mátrix... 46 00:05:00,009 --> 00:05:05,000 és itt a mátrixban megy végbe a citrátkör... 47 00:05:05,009 --> 00:05:11,000 A citrátkör vagy Szent-Györgyi–Krebs ciklus, egy biokémiai folyamatsor, 48 00:05:11,009 --> 00:05:18,000 mint tudjuk, 49 00:05:18,009 --> 00:05:29,000 mely redukáló ekvivalenst, NADH-t termel. NADH-ból keletkezõ protonok kapcsolatba kerülnek és áthaladnak 50 00:05:29,009 --> 00:05:36,000 a citokróm-rendszeren, ahol a citokróm-C (IV. komplex) által 51 00:05:36,009 --> 00:05:42,000 a trikarbonsav ciklus szubsztrátjaiból víz termelõdik..., ha van elegendõ oxigén 52 00:05:42,009 --> 00:05:47,000 amit a mitokondrium felhasználhat víz termelésére, és ez az, ahogy az állatok túlélnek a sivatagban, 53 00:05:47,009 --> 00:05:51,000 ahol ivás helyett mitokondriumaikkal termelnek maguknak ... 54 00:05:51,009 --> 00:05:53,000 vizet! és így ..., tehát..., 55 00:05:53,009 --> 00:06:02,000 ez az, ahogy a gyíkok, kígyók, tengeri halak és emlõsök megélnek anélkül, 56 00:06:02,009 --> 00:06:06,000 hogy vizet innának, mert a víz termelését mitokondriumaikkal végeztetik. Page 4

Science-Fridays-LABiomed02a-DDW_Magyar 57 00:06:06,009 --> 00:06:10,000 Hogy mik itt a szubsztrátok, azt hamarosan elmondom részletesebben is! 58 00:06:10,009 --> 00:06:18,000 Két vízmolekula képzõdik, miközben a ciklus egyet fordul, 59 00:06:18,009 --> 00:06:22,000 az oxigén jelenléte segíti is, és egyben elengedhetetlen a víz keletkezéséhez. 60 00:06:22,009 --> 00:06:29,000 Miközben a sejtek ATP szükségletüket elégítik és a protonokat 61 00:06:29,009 --> 00:06:37,000 a membránközi térbe továbbítják, a mátrixba visszaáramló protonok által hajtott V. komplex, 62 00:06:37,009 --> 00:06:43,000 az ATP-szintetáz, energiát, azaz ATP-t termel. 63 00:06:43,009 --> 00:06:48,000 Energiatermeléshez tehát a hidrogén a citrátkörben szereplõ szubsztrátokból származik, 64 00:06:48,009 --> 00:06:54,000 ...mármint szubsztrátokból és azok köztes termékeibõl, így a citrátkörben résztvevõ anyagokból származó hidrogén kulcsszerepet játszik a 65 00:06:54,009 --> 00:07:00,000 teljes víztermelõ folyamat mûködésében, valamint a protonok ki- és visszaáramlásának fenntartásában 66 00:07:00,009 --> 00:07:03,000 a mátrix felé. Így hát..., 67 00:07:03,009 --> 00:07:10,000 Elõadásomra való felkészülésem közben azt tartottam szem elõtt, mint kiinduló kérdést, 68 00:07:10,009 --> 00:07:16,000 hogy miképp befolyásolja a hidrogén atom tömege 69 00:07:16,009 --> 00:07:22,000 az elõbbiekben említett biokémiai folyamatok és ion pumpák mûködését, 70 00:07:22,009 --> 00:07:33,000 mint mitokondriumok zavarával kapcsolatos biokémiai események, fõleg rákban, és hogy ezek milyen epigenetikai hatásokat készítenek elõ! Page 5

Science-Fridays-LABiomed02a-DDW_Magyar 71 00:07:33,009 --> 00:07:34,000 Így, tehát..., 72 00:07:34,009 --> 00:07:40,000 Itt vannak a mitokondriumban lejátszódó 73 00:07:40,009 --> 00:07:47,000 pontosabban, a citrátkört alkotó reakciók. Mint látható, elõször citromsav keletkezik, 74 00:07:47,009 --> 00:07:52,000 innen a körfolyamat neve, egy két szénatomos 75 00:07:52,009 --> 00:07:56,000 acetil-CoA-ból és a körfolyamatot záró oxálecetsavból. 76 00:07:56,009 --> 00:08:00,000 Ez a hat szénatomos citrát fordul körbe, 77 00:08:00,009 --> 00:08:07,000 a ciklus elveszít két szénatomot CO2 formájában, és ahogy a CO2 keletkezik, hidrogének lépnek ki 78 00:08:07,009 --> 00:08:15,000 a ciklus köztes termékeibõl.. 79 00:08:15,009 --> 00:08:20,000 Érdekes, hogy nem a ciklus elején belépett 80 00:08:20,009 --> 00:08:25,000 két szénatommal azonos szénatomok oxidálódnak az elsõ körben, illetve az is érdekes hogy a ciklus, 81 00:08:25,009 --> 00:08:30,000 a körfolyamat maga, vízmolekulákat fogyaszt. 82 00:08:30,009 --> 00:08:36,000 Valójában nagyon fontos lenne tudni, hogy miért használódik fel víz akkor, 83 00:08:36,009 --> 00:08:40,000 amikor oxigén redukciójával egyidejûleg víz is termelõdik, illetve miért szükséges az, hogy a víz, 84 00:08:40,009 --> 00:08:44,000 ami egyébként mátrix víz, az amit a citokróm-C (IV. komplex) maga termel, Page 6

Science-Fridays-LABiomed02a-DDW_Magyar 85 00:08:44,009 --> 00:08:51,000 és hogy az emlõs sejtben miért használódik fel újra ez a víz, ahelyett, hogy az állati sejt vízigényét fedezné, 86 00:08:51,009 --> 00:08:54,000 mint ahogy ez a sivatagi állatokban történik. 87 00:08:54,009 --> 00:09:03,000 Mint azt sejthetik, mai témánkat a mátrix víz reduktív szintézisben való direkt felhasználása felé fogom irányítani. 88 00:09:03,009 --> 00:09:11,000 Nagyon érdekes ugyanis, hogy mi történik a vízzel, 89 00:09:11,009 --> 00:09:15,000 és hogyan kerülnek a mátrix víz hidrogénjei a sejt DNS-ébe, ezt fogom hát bemutatni. 90 00:09:15,009 --> 00:09:23,000 Van ugyanis egy nagyon érdekes rák-modell, 91 00:09:23,009 --> 00:09:29,000 amit az USA Nemzeti Rákkutató Intézettel együttmûködve vizsgálunk, ahol a citrátkör vizet használó enzimje, 92 00:09:29,009 --> 00:09:36,000 a fumarát-hidratáz vagy fumaráz, mutálva van! Az enzimet nélkülözõ vesesejtek 93 00:09:36,009 --> 00:09:44,000 nem tudják víz felhasználásával a fumársavat almasavvá alakítani a mitokondriumban! 94 00:09:44,009 --> 00:09:50,000 Emiatt a ciklus megfordul, reduktív karboxilálás történik, 95 00:09:50,009 --> 00:09:56,000 egy új reakciósor indul be, ami a hidrogént más forrásból veszi, 96 00:09:56,009 --> 00:09:59,000 nem a mátrix vizébõl. 97 00:09:59,009 --> 00:10:06,000 A citrátkör ezen teljes megfordulásával, pontosabban azt követõen, rák alakul ki. 98 00:10:06,009 --> 00:10:10,000 Ez egy gyors lefolyású rák, 99 Page 7

Science-Fridays-LABiomed02a-DDW_Magyar 00:10:10,009 --> 00:10:19,000 világos-sejtes veseráknak nevezett betegség, ami öröklõdõ leiomyomatosis-hoz kapcsolódik, 100 00:10:19,009 --> 00:10:31,000 a fumarát-hidratáz elvesztése után 10-15 évvel lép fel, és dudoros bõrtünetekkel is jár. Ezen betegek idõvel sajnos mind rákot fejlesztenek! 101 00:10:31,009 --> 00:10:38,000 Egy ritka betegség, Reeds-szindrómának is nevezik, 102 00:10:38,009 --> 00:10:41,000 leírója után. 103 00:10:41,009 --> 00:10:45,000 A betegség kezdetén a bõrben dudorok jelennek meg. 104 00:10:45,009 --> 00:10:54,000 Szövettanilag egy széles keratin-réteg látszik a bõralatti (simaizom) struktúrák hypertrófiája mellett. 105 00:10:54,009 --> 00:11:00,000 Ez a rákot megelõzõ állapot, 106 00:11:00,009 --> 00:11:07,000 mielõtt a vesedaganat kialakulna a betegekben. 107 00:11:07,009 --> 00:11:13,000 A kialakuló vesedaganat nagyon sajátos. 108 00:11:13,009 --> 00:11:18,000 Világos-sejtes veseráknak nevezik. Hypernephroma-nak is nevezték, 109 00:11:18,009 --> 00:11:23,000 de a daganat maga veseszövetbõl indul ki! 110 00:11:23,009 --> 00:11:30,000 Itt láthatók a világos sejtek. A magjuk morfológiája 111 00:11:30,009 --> 00:11:33,000 ráksejtekre jellemzõ! Ez egy nagyon agresszív rák. 112 00:11:33,009 --> 00:11:38,000 Itt pedig az egészséges veseszövet látható. Jellegzetes, 113 Page 8

Science-Fridays-LABiomed02a-DDW_Magyar 00:11:38,009 --> 00:11:46,000 ahogy a sejtekben cisztás állomány gyûlik fel, ami a sejtmagot az egyik szélre szorítja. 114 00:11:46,009 --> 00:11:51,000 Ez az a patológiai jellegzetesség, amirõl világos-sejtes 115 00:11:51,009 --> 00:11:57,000 veseráknak nevezték el, ami a citrátkörben szereplõ fumarát-hidratáz 116 00:11:57,009 --> 00:12:00,000 mutációja, vagy hibája miatt alakul ki. 117 00:12:00,009 --> 00:12:05,000 Tehát ez egy vese-kéregállományi daganat, 118 00:12:05,009 --> 00:12:15,000 rosszindulatú hámsejt-burjánzás. Világos sejtek láthatók, vannak még egyéb patológiai és morfológiai jellemzõk... 119 00:12:15,009 --> 00:12:17,000 is, ha a részleteket nézzük 120 00:12:17,009 --> 00:12:24,000 De lássuk magát a betegséget. Ezt ténylegesen a fumarát-hidratáz 121 00:12:24,009 --> 00:12:28,000 gén mutációja okozza, és emiatt a mitokondriumon belül 122 00:12:28,009 --> 00:12:31,000 a citrátkörben felhalmozódik mind a víz, mind a fumársav! 123 00:12:31,009 --> 00:12:39,000 Ez egy autoszomális domináns betegség. Azt látjuk, hogy miután a mutáció megtörténik, 124 00:12:39,009 --> 00:12:44,000 egy hosszabb idõtartam alatt fumársav halmozódik fel, 125 00:12:44,009 --> 00:12:52,000 miközben az almasav szintje csökken. A kérdés az, hogy ezek a változások 126 00:12:52,009 --> 00:12:59,000 hogyan járulnak a rák kialakulásához? Ugyanis, a folyamatokat nézve úgy tûnik, 127 00:12:59,009 --> 00:13:08,000 Page 9

Science-Fridays-LABiomed02a-DDW_Magyar hogy ez a modell inkább a citrátkört és az attól függõ energiatermelést érinti! 128 00:13:08,009 --> 00:13:15,000 De valójában az idõ múlásával egy nagyon agresszív daganat 129 00:13:15,009 --> 00:13:20,000 jelenik meg. Kérdés 01: Elnézést ... a sejtek a fumársav 130 00:13:20,009 --> 00:13:26,000 felhalmozódása miatt duzzadnak fel? És a borostyánkõsav miatt. 131 00:13:26,009 --> 00:13:30,000 A mitokondriumokban több metabolit is felhalmozódik, 132 00:13:30,009 --> 00:13:34,000 közöttük van a víz, a fumársav és a borostyánkõsav is. 133 00:13:34,009 --> 00:13:39,000 Tehát minden, amibõl a fumársav keletkezik, felhalmozódhat. 134 00:13:39,009 --> 00:13:45,000 Ezekben a sejtekben nagyon kevés az almasav, 135 00:13:45,009 --> 00:13:49,000 a duzzadásos degenerációt pedig az oldott fumársav és borostyánkõsav okozza. 136 00:13:49,009 --> 00:13:54,000 Amit meg szeretnék még említeni, hogy nem csak a fumarát-hidratáz, 137 00:13:54,009 --> 00:14:00,000 hanem a borostyánkõsav keletkezése, a szukcinát-dehidrogenáz vagy –szintetáz enzimek mutációja is 138 00:14:00,009 --> 00:14:04,000 tumort okoz! Ezek kissé másfajta rákok, 139 00:14:04,009 --> 00:14:12,000 de egyre több tumor-fenotípusban fedezik fel a citrátkör enzimeinek a mutációit! 140 00:14:12,009 --> 00:14:20,000 Az igazság pedig az, ha a rákbetegséget tekintjük, a legelsõ nyilvánvaló anyagcsere-változás 141 00:14:20,009 --> 00:14:24,000 Page 10

Science-Fridays-LABiomed02a-DDW_Magyar a mitokondriumok csökkent mûködése. Ez azután a Warburg-hatáshoz vezet, 142 00:14:24,009 --> 00:14:29,000 amikor is a piruvát nem tud belépni a mitokondriumba, így zsír és szubsztrátok nem tudnak eloxidálódni. 143 00:14:29,009 --> 00:14:37,000 Tehát, általánosságban, akár az életkor elõrehaladtával fellépõ (szöveti) hipoxiát nézzük, 144 00:14:37,009 --> 00:14:43,000 akár a fumarát-hidratáz, vagy a citrátkör más enzimeinek csökkent mûködését, a rákkeletkezés alapja 145 00:14:43,009 --> 00:14:48,000 a mitokondriumok ezen okok valamelyikére visszavezethetõ csökkent funkciója! 146 00:14:48,009 --> 00:14:55,000 Általánosan elfogadott, hogy a mitokondrium mint olyan, 147 00:14:55,009 --> 00:15:04,000 illetve enzimeinek mutációja, folyamatainak elégtelensége, mind hozzájárul a rák kialakulásához. 148 00:15:04,009 --> 00:15:07,000 Kérdés 02: Kérdésem van: van zavar az izom149 00:15:07,009 --> 00:15:09,000 vagy zsírszövetben? Nincs... 150 00:15:09,009 --> 00:15:15,000 Várható-e káros hatás az izmokra, az izom-mitokondriumokra...? 151 00:15:15,009 --> 00:15:21,000 Más szervek inkább érintettek, említettem a bõrdudorokat. 152 00:15:21,009 --> 00:15:27,000 Ennél a mutációnál a vezetõ klinikai jelenségek: a bõrelváltozások és a vesetumor kialakulása. [lásd még: genitalis leiomyoma, angioleiomyoma] 153 00:15:27,009 --> 00:15:29,000 Így hát..., 154 00:15:29,009 --> 00:15:34,000 Ami még igazán nagyon érdekes, 155 00:15:34,009 --> 00:15:38,000 Page 11

Science-Fridays-LABiomed02a-DDW_Magyar hogy amint helyreállítjuk a fumarát-hidratáz mûködését a tumorsejtekben, 156 00:15:38,009 --> 00:15:43,000 azok többé nem okoznak daganatot csupasz egérbe ültetés után. 157 00:15:43,009 --> 00:15:50,000 Azaz, a citrátkör egyetlen enzimjének helyreállítása visszafordítja magát a rákbetegséget. 158 00:15:50,009 --> 00:15:58,000 Az USA Nemzeti Rákkutató Intézetében létrehozták az UOK262 jelû ráksejt-modellt. 159 00:15:58,009 --> 00:16:05,000 Ennek van egy kijavított, úgynevezett "vad" típusú ráksejt párja, melybe bejuttatták a normális (mitokondriális) 160 00:16:05,009 --> 00:16:08,000 fumarát-hidratáz enzim gént, 161 00:16:08,009 --> 00:16:13,000 amivel a fumarát-hidratáz aktivitás a normálisra visszaállt! 162 00:16:13,009 --> 00:16:19,000 És van az „üres vektorral” génhiányosan hagyott fenotípus is. Tehát, amint az enzimet visszajuttatjuk 163 00:16:19,009 --> 00:16:24,000 ..a mitokondriumba, a sejtek elvesztik tumorképzõ jellegüket! 164 00:16:24,009 --> 00:16:31,000 Képtelenek arra, hogy daganatos sejtként mûködjenek. Így például, ha ezeket a tumorsejteket 165 00:16:31,009 --> 00:16:35,000 csupasz egérbe injektáljuk, mindig daganat alakul ki, mindaddig amíg a fumarát-hidratáz mutált! 166 00:16:35,009 --> 00:16:41,000 Amint a fumarát-hidratáz aktivitást helyreállítjuk, az egerek vidáman élnek, 167 00:16:41,009 --> 00:16:49,000 nincs daganatképzõdés bennük. Ezt a Cancer Cell biology-ban közölték. (doi: 10.1016/j.ccr.2011.07.018 - Figure 1) 168 00:16:49,009 --> 00:16:52,000 és a Nature-ben is van közleményük! (doi: 10.1038/nature10642) 169 Page 12

Science-Fridays-LABiomed02a-DDW_Magyar 00:16:52,009 --> 00:16:57,000 A sejtek pontos anyagcsere fenotípusát a Nemzeti Rákkutató Intézettel együtt állapítottuk meg. 170 00:16:57,009 --> 00:17:03,000 Saját 13C metabolikus nyomjelzõ módszerünket használtuk, 171 00:17:03,009 --> 00:17:08,000 hogy részletesen tisztázzuk mely folyamatok vesznek részt a daganat keletkezésében. Két tumoros fenotípust vizsgáltunk: 172 00:17:08,009 --> 00:17:15,000 1/ a fumarát-hidratáz hiányos, és 2/ a helyreállított fumarát-hidratázt hordozó sejteket. 173 00:17:15,009 --> 00:17:23,000 Ezt a PLoS ONE-ban közöltük augusztusban (2013), és az eredményeket most (doi: 10.1371/journal.pone.0072179) 174 00:17:23,009 --> 00:17:25,000 részletesen ismertetem. 175 00:17:25,009 --> 00:17:33,000 Az irodalomban számos elképzelés (hipotézis) jelent már meg arról, miért okoz rákot 176 00:17:33,009 --> 00:17:41,000 a mitokondriumokban megemelkedett fumársav-koncentráció! 177 00:17:41,009 --> 00:17:45,000 Számos kísérletben próbálták 178 00:17:45,009 --> 00:17:52,000 már tisztázni az okokat. Az egyik lehetõség, hogy a felhalmozódott fumársav, 179 00:17:52,009 --> 00:17:57,000 vagy annak anyagcsere termékei, 180 00:17:57,009 --> 00:18:04,000 kijutnak a citoszolba és ott bizonyos enzimeket gátolnak, 181 00:18:04,009 --> 00:18:10,000 ami beindítja a rákkeletkezéshez vezetõ jelátvivõ eseménysort. És sok adat van 182 00:18:10,009 --> 00:18:16,000 a hipoxia által indukált faktorra vonatkozóan, mely szerepet játszik az anyagcsere-változásokban, 183 Page 13

Science-Fridays-LABiomed02a-DDW_Magyar 00:18:16,009 --> 00:18:21,000 illetve a rákbetegséghez társuló epigegetikai változásokban! 184 00:18:21,009 --> 00:18:27,000 A citrátkörbõl származó szerves savak fokozott hozzáférhetõsége miatt fellép továbbá a szukcinát nem-specifikus kötõdése a fehérjék cisztein-oldalláncához, 185 00:18:27,009 --> 00:18:35,000 és a DNS-fehérje kötõdések megváltozása. 186 00:18:35,009 --> 00:18:42,000 A magyarázatnak része a szuperoxid ion felszaporodása is, 187 00:18:42,009 --> 00:18:48,000 a hiányzó mitokondriális mûködés miatt. 188 00:18:48,009 --> 00:18:51,000 kiesõ mitokondriális mûködés mint mechanizmus. Így néz ki a dolog..., 189 00:18:51,009 --> 00:18:56,000 Ezeket a lehetséges folyamatokat már közöltük. 190 00:18:56,009 --> 00:19:00,000 az USA Nemzeti Rákkutató Intézettel együtt! 191 00:19:00,009 --> 00:19:05,000 Namost...., orvos biokémikusok, mint például Dr. Lee és jómagam, kérdezzük azt: mit csinál valójában a fumarát-hidratáz? 192 00:19:05,009 --> 00:19:09,000 Mi az enzim mûködése, 193 00:19:09,009 --> 00:19:14,000 illetve annak pontos feladata? 194 00:19:14,009 --> 00:19:19,000 Valójában feltettem már ezt a kérdést néhány molekuláris biológus kollégának is, és a válasz mindig az volt, hogy nem tudják. 195 00:19:19,009 --> 00:19:21,000 Ez igazán különös! 196 00:19:21,009 --> 00:19:28,000 Úgy gondolom, ez élettani és rendszer-biológiai szempontból is igen fontos kérdés, egy lényegi kulcskérdés! Page 14

Science-Fridays-LABiomed02a-DDW_Magyar 197 00:19:28,009 --> 00:19:33,000 A fumarát-hidratáz pusztán azt csinálja, 198 00:19:33,009 --> 00:19:39,000 hogy metabolikus eredetû mátrix-vizet kapcsol a citrátkör köztitermékéhez, a fumársavhoz, 199 00:19:39,009 --> 00:19:48,000 ezzel fogja a mátrix vizet, és visszavezeti azt a citrátkörbe! 200 00:19:48,009 --> 00:20:02,000 Mint látják, magát az enzimet 1930 elõtt írta le Szent-Györgyi Albert, 201 00:20:02,009 --> 00:20:08,000 aki emellett a C-vitamint is felfedezte, és 1937-ben orvosi Nobel-díjat kapott. 202 00:20:08,009 --> 00:20:13,000 Érdekesség, hogy az anyai nagyszüleim személyesen ismerték Õt, 203 00:20:13,009 --> 00:20:16,000 mivel abban az idõben Szegeden laktak õk is. 204 00:20:16,009 --> 00:20:24,000 Nagymamám többször beszélt Róla, mivel a két családnak 205 00:20:24,009 --> 00:20:27,000 volt külön operabérlete ugyanabba a páholyba! 206 00:20:27,009 --> 00:20:34,000 Barátságban ugyan nem volt a két család, nem jártak össze, de jól ismerték egymást. 207 00:20:34,009 --> 00:20:42,000 Õ volt az, aki még a 20-as években feltételezte, hogy a szukcinátnak és 208 00:20:42,009 --> 00:20:45,000 a fumarátnak hidrogén-szállító katalitikus szerepe van, 209 00:20:45,009 --> 00:20:52,000 Azaz a borostyánkõsav, fumársav és almasav szerepe, a protonok szállítása 210 00:20:52,009 --> 00:20:56,000 a cikluson keresztül. Ez akkor nagyon fontos ismeret volt, Page 15

Science-Fridays-LABiomed02a-DDW_Magyar 211 00:20:56,009 --> 00:20:58,000 mert a ciklus még nem volt körbezárva. 212 00:20:58,009 --> 00:21:02,000 Az eredeti Krebs-féle reakciókat még nem tisztázták. 213 00:21:02,009 --> 00:21:09,000 Ennek ellenére Szent-Györgyi már leírta a citrátkört 214 00:21:09,009 --> 00:21:13,000 alkotó reakciók egy részét hirdogének/protonok szállítására, 215 00:21:13,009 --> 00:21:17,000 hogy az izomsejtekben a szukcinát és a fumarát fõ funkciója a protonok tovább szállítása. 216 00:21:17,009 --> 00:21:23,000 Tehát, amint látható, a ciklus körbefordulásakor 217 00:21:23,009 --> 00:21:30,000 két helyen összesen két vízmolekula használódik fel. 218 00:21:30,009 --> 00:21:39,000 Egyik az akonitáznál lép be, és adja a hidrogént az elsõ hidrogén-, illetve protonszolgáltató 219 00:21:39,009 --> 00:21:42,000 reakcióhoz. A második pedig a fumarát-hidratáznál, 220 00:21:42,009 --> 00:21:50,000 mely, ahogy a ciklus körbefordul, az NADPH-szintézishez szolgáltat majd protont. 221 00:21:50,009 --> 00:21:54,000 Hogy hogyan, azt mindjárt mutatom is. 222 00:21:54,009 --> 00:21:56,000 Biztosan mondhatjuk, hogy a víz, azaz a metabolikus ill. mátrix-víz, 223 00:21:56,009 --> 00:22:05,000 nagyon gyorsan belép a köztianyagcserébe, hogy visszajuttassa a hidrogéneket, 224 00:22:05,009 --> 00:22:10,000 ami azt mutatja, hogy nem kell a citrátkörbe hidrogént bevinni, mert megvan a cikluson belül a saját vizünk, 225 Page 16

Science-Fridays-LABiomed02a-DDW_Magyar 00:22:10,009 --> 00:22:16,000 amivel a ciklus körforgást mûködtetjük, miközben redukáló ekvivalenseket szintetizálunk! 226 00:22:16,009 --> 00:22:23,000 Namost..., mint már mondtam, a citrátkör és a normális mitokondriumok eredeti szerepe 227 00:22:23,009 --> 00:22:29,000 a víz elõállítása. Ezt az élettanból tudhatjuk, 228 00:22:29,009 --> 00:22:37,000 mert például a tengeri állatok, melyek nem isszák a sós tengervizet, 229 00:22:37,009 --> 00:22:41,000 vagy a vizet nélkülözni kényszerülõ sivatagi állatok, 230 00:22:41,009 --> 00:22:45,000 a mitokondriumaikban zsírt oxidálnak! 231 00:22:45,009 --> 00:22:50,000 Ha megnézzük a púpos tevét, a skorpiót, vagy a csörgõkígyót, 232 00:22:50,009 --> 00:22:58,000 mindnek kell legyen egy zsírraktára, amit a zsákmányukból szereznek, ezzel a sivatagi növények 233 00:22:58,009 --> 00:23:04,000 elfogyasztásából termelt zsírt égetik. 100g zsírból az ilyen állatokban, meglepõ módon, 234 00:23:04,009 --> 00:23:07,000 110g víz keletkezhet, 235 00:23:07,009 --> 00:23:11,000 mivel a zsírjuk telítve van hidrogénnel, 236 00:23:11,009 --> 00:23:14,000 amihez oxigén kell már csak a vízképzõdéshez! A fiziológusok szerint a tüdõ tanulmányozásához 237 00:23:14,009 --> 00:23:19,000 legjobb modellt a kígyók adják, 238 00:23:19,009 --> 00:23:23,000 nekik ugyanis óriási tüdejük van, 239 00:23:23,009 --> 00:23:27,000 Page 17

Science-Fridays-LABiomed02a-DDW_Magyar hogy oxigénnel láthassák el a vizet termelõ citrátkört. 240 00:23:27,009 --> 00:23:31,000 Kérdés 03: László, ezért mondják a fogyókúrás szakemberek, hogy sok vizet igyunk? 241 00:23:31,009 --> 00:23:39,000 Nem, ez más okból van. Mi nem használjuk (párologatjuk) el a saját anyagcsere vizünket. 242 00:23:39,009 --> 00:23:42,000 Ha megnézzük a citrátkört, és itt van 243 00:23:42,009 --> 00:23:46,000 a fõ különbség a csörgõkígyó és az emlõsök között, 244 00:23:46,009 --> 00:23:49,000 mi a mitokondriumainkkal energiát termelünk, 245 00:23:49,009 --> 00:23:54,000 ATP-t szintetizálunk. Víz termelése helyett a 246 00:23:54,009 --> 00:24:00,000 hidrogén 95%-át NADPH szintézisére és a membrán-termelésre használjuk, 247 00:24:00,009 --> 00:24:06,000 nettó vízkeletkezés bennünk nincs! A vándormadarakban keletkezik víz és fejlõdéstörténetileg így áll a dolog: 248 00:24:06,009 --> 00:24:10,000 1) A csörgõkígyó vizet termel a mitokondriumaival. 249 00:24:10,009 --> 00:24:14,000 A kedvezõbb éghajlatra, vizes helyekre vándorló hüllõk 250 00:24:14,009 --> 00:24:19,000 tudtak inni, ezért kifejlõdött bennük a 251 00:24:19,009 --> 00:24:23,000 mitokondriális hõ- és ATP-termelés, és így ott maradhattak a hidegben is. 252 00:24:23,009 --> 00:24:30,000 A mitokondriumok ilyen "újszerû" felhasználása tehát az evolúció anyagcserére tett hatását mutatja. 253 00:24:30,009 --> 00:24:33,000 Annak ellenére, hogy mi is ugyanannyi vizet termelünk, Page 18

Science-Fridays-LABiomed02a-DDW_Magyar 254 00:24:33,009 --> 00:24:38,000 azt a fumarát-hidratáz segítségével teljesen fel is használjuk, a víz tehát visszakerül a körfolyamatba, 255 00:24:38,009 --> 00:24:41,000 és onnan ki is kerül (mint almasav), ahogy azt mindjárt mutatom! 256 00:24:41,009 --> 00:24:48,000 Ezért kell tehát vizet innunk. Ha a "csörgõkígyó edz" és izommunkát végez, 257 00:24:48,009 --> 00:24:53,000 nem kellene innia, mivel több zsírt éget, és így abból vízhez is jut. 258 00:24:53,009 --> 00:25:00,000 Komolyra fordítva a dolgot, Dr. Mellanby egy ornitológus is volt, 259 00:25:00,009 --> 00:25:05,000 sivatagban élõ állatokat tanulmányozott. 260 00:25:05,009 --> 00:25:15,000 Így tudjuk, hogy amennyiben a citrátkörben szénhidrátokat használunk NADH termelésére, 261 00:25:15,009 --> 00:25:20,000 akkor 100g szénhidrátból csak 55g víz jön létre, mert kisebb bennük a hidrogén-tartalom, 262 00:25:20,009 --> 00:25:24,000 mint a zsírokban. A telített zsírok a leg hidrogén-dúsabbak, 263 00:25:24,009 --> 00:25:31,000 csak oxigén kell, és vizet produkálnak a Szent-Györgyi ciklusban, hogy a sivatagban maradhassanak ezzel a megoldással. 264 00:25:31,009 --> 00:25:38,000 Ezzel szemben mi felhasználjuk a mátrix vizet, ahelyett, hogy vízként használnánk azt! 265 00:25:38,009 --> 00:25:44,000 A dologban egy a bökkenõ, a csörgõkígyó nem tud ATP-t termelni a víz visszaforgatásával, 266 00:25:44,009 --> 00:25:49,000 mert azt biológiai oldószer funkciójában hasznosítja... 267 00:25:49,009 --> 00:25:52,000 így viszont nem tudja szabályozni a testhõmérsékletét, tehát hibernálódnia kell. Page 19

Science-Fridays-LABiomed02a-DDW_Magyar 268 00:25:52,009 --> 00:26:00,000 Napfénybõl kell hõt nyernie, viszont a saját maga termelte vizet felhasználhatja 269 00:26:00,009 --> 00:26:09,000 a test víztartalmának, enzimei mûködésének fenntartására, illetve, hogy ne száradjon ki! 270 00:26:09,009 --> 00:26:13,000 Mihelyt egy állat a mátrix-vizet almasav, 271 00:26:13,009 --> 00:26:18,000 illetve, NADPH redukáló ekvivalens szintézisére használja fel, 272 00:26:18,009 --> 00:26:23,000 többé nem tudja vízzel ellátni magát, 273 00:26:23,009 --> 00:26:28,000 azt más forrásból, például ivással kell fedezze. 274 00:26:28,009 --> 00:26:34,000 Namármost..., a metabolikus víz, 275 00:26:34,009 --> 00:26:41,000 ami a mátrix vize és NADH-forrás, nagyon különleges, ami azt jelenti, hogy alacsony 276 00:26:41,009 --> 00:26:44,000 a deutériumtartalma! 277 00:26:44,009 --> 00:26:49,000 De ez már egy másik, ugyancsak érdekfeszítõ történet, 278 00:26:49,009 --> 00:26:53,000 mivel az anyagcsere-víz más attól, mint amit megiszunk. 279 00:26:53,009 --> 00:26:59,000 Más szóval, a víz amit most is isznak, magasabb deutériumtartalmú, mint a mitokondriumokban keletkezõ víz. 280 00:26:59,009 --> 00:27:02,000 Ez azért van, 281 00:27:02,009 --> 00:27:10,000 mert az étel, benne a zsírok, a hosszú láncú telített zsírsavak, 282 Page 20

Science-Fridays-LABiomed02a-DDW_Magyar 00:27:10,009 --> 00:27:16,000 kevesebb deutériumot tartalmaznak, mint ami a megivott vízben található. 283 00:27:16,009 --> 00:27:23,000 Az anyagok deutériumtartalmát a 284 00:27:23,009 --> 00:27:30,000 "bécsi standard közepes tengervíz”-hez hasonlítjuk, mely durván 155 milliomod rész (ppm) deutériumot tartalmaz. 285 00:27:30,009 --> 00:27:37,000 Az olyan alaptáplálékok, mint a liszt vagy a cukor, 286 00:27:37,009 --> 00:27:41,000 szintén közel 150 ppm-et tartalmaznak. 287 00:27:41,009 --> 00:27:48,000 A túróban, ami jó elegye a zsíroknak és szénhidrátoknak, már csak 136 ppm deutérium van. 288 00:27:48,009 --> 00:27:54,000 Az olivaolajban 130 ppm, a vajban 124, 289 00:27:54,009 --> 00:27:58,000 az állati zsírok zsírsavjaiban pedig csupán 118. 290 00:27:58,009 --> 00:28:06,000 Tehát, minél zsírosabb a táplálék, annál alacsonyabb a deutérium tartalma. 291 00:28:06,009 --> 00:28:08,000 Még érdekesebb, 292 00:28:08,009 --> 00:28:16,000 hogy a telítetlen növényi zsírok deutériumtartalma nagyon alacsony, 293 00:28:16,009 --> 00:28:23,000 ahol a kettõs kötéses modelleknél ez mindössze 102 ppm, 294 00:28:23,009 --> 00:28:27,000 ami majdnem egy 40% közeli csökkenés deutériumtartalom tekintetében. 295 00:28:27,009 --> 00:28:37,000 Tehát, minél több zsírt, fõleg telítetlen zsírt fogyasztunk, 296 00:28:37,009 --> 00:28:42,000 Page 21

Science-Fridays-LABiomed02a-DDW_Magyar annál alacsonyabb lesz a mitokondriális mátrix vizének, 297 00:28:42,009 --> 00:28:47,000 és ezzel szervezetünknek a deutériumtartalma. 298 00:28:47,009 --> 00:28:51,000 Még érdekesebb, hogy mindez a fotoszintézis során dõl el. 299 00:28:51,009 --> 00:28:58,000 A fény belép a növényi sejtekbe, 300 00:28:58,009 --> 00:29:05,000 amivel a környezetbõl származó, 155 ppm deutériumtartalmú víz oxigén keletkezése mellett szétbomlik. 301 00:29:05,009 --> 00:29:12,000 A hidrogént megkötõ redukáló ekvivalens belép a Calvin-ciklusnak nevezett körfolyamatba, 302 00:29:12,009 --> 00:29:15,000 de közben a sejt a deutériumos vizet úgyszólván elejti. 303 00:29:15,009 --> 00:29:20,000 A fotoszintézis során tehát a deutériummal szemben kizárás és megkülönböztetés érvényesül, 304 00:29:20,009 --> 00:29:26,000 mert a résztvevõ enzimek a fotoszintézis során elejtik a deutériumot. 305 00:29:26,009 --> 00:29:31,000 A növényben keletkezõ zsírok tehát deutériumszegények lesznek. 306 00:29:31,009 --> 00:29:36,000 Ha ezt elfogyasztjuk és a mitokondriumokban elégetjük, 307 00:29:36,009 --> 00:29:43,000 a keletkezõ NADPH is deutériumszegény lesz. 308 00:29:43,009 --> 00:29:45,000 Igen, kérdezne? Megálljak? 309 00:29:45,009 --> 00:29:56,000 Kérdés 04: Miért ejti el az enzim a deutériumot? - Hogy miért? Mert a deutérium kétszer olyan nehéz, mint a hidrogén! 310 00:29:56,009 --> 00:30:00,000 Tudom, igaz lehet hogy ezek az enzimek kevésbé hatékonyak, Page 22

Science-Fridays-LABiomed02a-DDW_Magyar 311 00:30:00,009 --> 00:30:04,000 mikor deutériumot szállítanak? - Igen, ezeknek az enzimeknek a kötõhelyei ténylegesen, 312 00:30:04,009 --> 00:30:08,000 fizikai értelemben, elejtik a deutériumot, ami önmagában is elképesztõen érdekes! 313 00:30:08,009 --> 00:30:12,000 Tudják, hogy melyek ezek az enzimek? 314 00:30:12,009 --> 00:30:19,000 Számos ilyen enzim van, amelyek felelõsek a deutérium elhagyásáért, 315 00:30:19,009 --> 00:30:31,000 de fõleg a deszaturázok. Azok egyenesen ledobják magukról a deutériumot. Köszönöm szépen! 316 00:30:31,009 --> 00:30:39,000 Egy francia munkacsoport, Dr. Billault vezetésével, 317 00:30:39,009 --> 00:30:47,000 számos zsiradékban megmérték a deutérium-tartalmat. Innen tudjuk, [doi: 10.1074/jbc.M500909200] 318 00:30:47,009 --> 00:30:52,000 hogy melyik telítetlen szénatomoknál a legalacsonyabb a deutériumszint, és hogy milyen növényekben és zsírokban van ez. 319 00:30:52,009 --> 00:30:59,000 Tömegspektrometriával határozzák meg az izotóp-arányt, úgy, 320 00:30:59,009 --> 00:31:07,000 hogy a mintákat elégetik és így elemeire oxidálják 321 00:31:07,009 --> 00:31:13,000 a deutérium kihajtásáért. Tehát, 322 00:31:13,009 --> 00:31:27,000 csökkenthetõ a sejtek deutériumtartalma a fotoszintézisbõl származó NADPH felhasználásával. 323 00:31:27,009 --> 00:31:33,000 Vagy, aki magas hegységben lakik, az deutériumszegény vízhez jut, 324 Page 23

Science-Fridays-LABiomed02a-DDW_Magyar 00:31:33,009 --> 00:31:37,000 mivel a nehezebb vízmolekulák hamarabb kihullnak az esõvel. 325 00:31:37,009 --> 00:31:42,000 És ezek az emberek élnek a legtovább: Peruban, 326 00:31:42,009 --> 00:31:46,000 Kaukázusban, a Himalájában, akár 100 évnél is tovább élhetnek! 327 00:31:46,009 --> 00:31:50,000 Érdekes, hogy nincs is a nyelvükben saját szó a rákbetegségre, 328 00:31:50,009 --> 00:31:53,000 ami arra utal, 329 00:31:53,009 --> 00:31:56,000 hogy az, ott fenn náluk nagyon ritka. 330 00:31:56,009 --> 00:32:00,000 Annál a tengerfeletti magasságnál õk deutériumszegény vizet isznak, 331 00:32:00,009 --> 00:32:07,000 és a fotoszintézis enzimeinek köszönhetõen valóban alacsony a deutérium felvételük. 332 00:32:07,009 --> 00:32:15,000 Tehát a modellben, ha a sejt, zsírsavakból képezi a mátrix-vizet, 333 00:32:15,009 --> 00:32:24,000 és azt NADPH termelésre használja fel, 334 00:32:24,009 --> 00:32:29,000 akkor, a zsír alacsony deutériumtartalma miatt, 335 00:32:29,009 --> 00:32:39,000 a nukleinsavakban és a DNS-ben is alacsony lesz a deutériumszint. 336 00:32:39,009 --> 00:32:47,000 Ha a mitokondriumok jól mûködnek, a DNS-ben több hidrogén lesz, mint deutérium, 337 00:32:47,009 --> 00:32:52,000 egyszerûen mert a táplálékaink ilyenek, 338 00:32:52,009 --> 00:32:59,000 Page 24

Science-Fridays-LABiomed02a-DDW_Magyar és mert a mitokondrium mûködése során a mátrixba deutériumszegény víz kerül. 339 00:32:59,009 --> 00:33:02,000 Azt stabil izotópos kísérletekbõl tudjuk, 340 00:33:02,009 --> 00:33:10,000 hogy a táplálékkal felvett, vagy saját eredetû zsírsavakból származó acetil-CoA 90%-át 341 00:33:10,009 --> 00:33:16,000 a mitokondriumok oxidálják el. 342 00:33:16,009 --> 00:33:23,000 Tehát a mitokondrium, illetve a mátrix, mindig a deutériumszegény állapotra törekszik. 343 00:33:23,009 --> 00:33:31,000 Sem a kívülrõl felvett magas deutériumtartalmú víz, 344 00:33:31,009 --> 00:33:38,000 sem a pentóz-foszfát ciklusban vagy a mikroszómális béta-oxidációban keletkezõ magas deutériumtartalmú víz nem kerül be a DNS-be normál, nyugvó sejtekben, 345 00:33:38,009 --> 00:33:46,000 illetve, ha az oxigén-ellátás rendben van, és az NADPH-t a mitokondrium termeli 346 00:33:46,009 --> 00:33:50,000 a citrátkör köztitermékeibõl, melyek deutériumtartalma 120 ppm körül van. 347 00:33:50,009 --> 00:33:55,000 Ha csak zsírokkal táplálkozunk, az ilyen ketogén diéta rák esetén 348 00:33:55,009 --> 00:34:03,000 nagyon hasznos, mert 110 ppm körülire csökkenti 349 00:34:03,009 --> 00:34:11,000 a test vizének deutériumtartalmát, mivel a mitokondriumok az alacsonyabb deutériumtartalmú zsírokból termelnek vizet. 350 00:34:11,009 --> 00:34:18,000 De mi is a baj a deutériummal, 351 00:34:18,009 --> 00:34:24,000 klinikai vagy fizikai szempontból? 352 Page 25

Science-Fridays-LABiomed02a-DDW_Magyar 00:34:24,009 --> 00:34:30,000 A hidrogénben egyetlen proton van, a deutériumban 353 00:34:30,009 --> 00:34:35,000 egy proton és még egy neutron, tehát az kétszer nehezebb, mint a hidrogén. 354 00:34:35,009 --> 00:34:43,000 Kémiailag viszont azonos, ezért részt vesz a szervezet minden reduktív szintézises reakciójában. 355 00:34:43,009 --> 00:34:50,000 Bekerül a DNS-be is, és elfoglalja a metilációs helyeket. 356 00:34:50,009 --> 00:34:57,000 A metilezõ enzimek a hidrogént könnyen leszakítják, 357 00:34:57,009 --> 00:35:04,000 de ha deutériumot találnak ott, akkor gond van. 358 00:35:04,009 --> 00:35:09,000 A nehéz deutérium beépülése a nukleinsavakba 359 00:35:09,009 --> 00:35:13,000 megváltoztatja a génkifejezõdést, mert más lesz a metiláció kinetikája, 360 00:35:13,009 --> 00:35:21,000 és mert a nagyobb tömeg miatti más rezonancia megváltoztatja 361 00:35:21,009 --> 00:35:29,000 a szemben fekvõ bázisok közötti kötéseket, ami akár aneuploidiát is okozhat. 362 00:35:29,009 --> 00:35:38,000 A hidrogén kétszer könnyebb, így könnyen mozgatható. 363 00:35:38,009 --> 00:35:45,000 A deutériummal viszont minden enzim nehezen boldogul, 364 00:35:45,009 --> 00:35:49,000 fõleg ha azt a DNS, vagy 365 00:35:49,009 --> 00:35:55,000 a maghártya szerkezetébe kell beépítenie. 366 00:35:55,009 --> 00:35:59,000 Page 26

Science-Fridays-LABiomed02a-DDW_Magyar És a DNS mûködésében a hidrogénhidak nagyon fontosak. 367 00:35:59,009 --> 00:36:06,000 Normális sejtben, mûködõ mitokondriumok mellett, 368 00:36:06,009 --> 00:36:11,000 a sejtmag és a DNS szerkezete ép. Ráksejtek, ahol a mitokondriumok mindig leépülnek, 369 00:36:11,009 --> 00:36:19,000 a pentóz-foszfát ciklusból és más reakciókból szerzik 370 00:36:19,009 --> 00:36:23,000 az NADPH-t és más redukáló ekvivalenseket, szabad víz felhasználásával. 371 00:36:23,009 --> 00:36:28,000 Morfológiai, emiatt pedig fiziológiai változások alakulnak ki 372 00:36:28,009 --> 00:36:38,000 a sejt méretében, viselkedésében, és a szabályozó mechanizmusokban. 373 00:36:38,009 --> 00:36:43,000 Nézetünk szerint az elsõ fontos epigenetikai esemény, 374 00:36:43,009 --> 00:36:51,000 ami megváltoztatja a sejtmag mûködését, 375 00:36:51,009 --> 00:36:55,000 az a DNS deuterálódása. Ez, a DNS kémiai és fizikai jellemzõi miatt, 376 00:36:55,009 --> 00:37:00,000 a rákkeletkezés igen fontos lépése. 377 00:37:00,009 --> 00:37:09,000 De honnan tud belépni az NADPH, 378 00:37:09,009 --> 00:37:16,000 azaz a hidrogén, vagy deutérium, az anyagcserébe, ha a mitokondriumok nem mûködnek, 379 00:37:16,009 --> 00:37:20,000 vagy ahol nincs oxigén, vagy a mitokondrium enzimei mutáltak? 380 00:37:20,009 --> 00:37:27,000 Például a szerin-oxidációs glicin-hasításos rendszeren át, amint azt nemrég a Nature-ben közöltük. Ez is azon helyek Page 27

Science-Fridays-LABiomed02a-DDW_Magyar egyike, ahol nem a mátrix-víz, [doi: 10.1038/cddis.2013.393] 381 00:37:27,009 --> 00:37:30,000 hanem szabad víz a hidrogén-forrás. 382 00:37:30,009 --> 00:37:38,000 Vagy a pentóz-foszfát ciklusból, ami glükózt használ 383 00:37:38,009 --> 00:37:41,000 ami az ételekhez használt cukorból, lisztbõl, stb. származik, és magasabb a deutériumtartalma, 384 00:37:41,009 --> 00:37:47,000 mivel furcsa módon, a növények zsírsav-szintetizáló enzimei a deutériumot nem építik be, 385 00:37:47,009 --> 00:37:54,000 míg a fotoszintézis során keletkezõ cukrokba bekerül a Deutérium. 386 00:37:54,009 --> 00:37:58,000 Tehát a víz és a cukor is, amibõl a pentóz-foszfát ciklus oxidáló szakaszában 387 00:37:58,009 --> 00:38:04,000 az NADPH keletkezik, 388 00:38:04,009 --> 00:38:07,000 magas deutériumtartalmú. 389 00:38:07,009 --> 00:38:17,000 Régóta tudjuk, hogy a pentóz-foszfát ciklus oxidáló szakasza a rákban fontos, [PMID: 9488183] 390 00:38:17,009 --> 00:38:20,000 többek között a Glivec is pont ezt a reakciósort gátolja, a glükóz direkt oxidációját a pentóz-ciklusban. 391 00:38:20,009 --> 00:38:25,000 Ezt közöltük is a Biológiai Kémiai újságban. [doi: 10.1074/jbc.M105796200 - 10.1056/NEJM200207043470116] 392 00:38:25,009 --> 00:38:32,000 A ráksejtek tehát eltérõen mûködnek, 393 00:38:32,009 --> 00:38:38,000 azaz amikor a fumarát-hidratáz, vagy az izocitrát-dehidrogenáz is mutálva van, Page 28

Science-Fridays-LABiomed02a-DDW_Magyar 394 00:38:38,009 --> 00:38:44,000 a deutériumszegény mátrix-víz nem tud belépni az anyagcserébe. 395 00:38:44,009 --> 00:38:53,000 A sejtek számára csak a glikolízis, a pentóz-foszfát ciklus és a szerin-oxidációs glicin-hasításos reakció marad meg, a cytoplazma fumarát-hidratáz enzimje mellett, 396 00:38:53,009 --> 00:39:01,000 és így az anyagcserébe bekerül a szabad víz, vagy a zsírsavtól eltérõ más tápanyagok magasabb deutériumtartalma, oxidációjuk során, mitokondriumok nélkül! 397 00:39:01,009 --> 00:39:04,000 Végül tehát nehéz, ragadós DNS-t kapunk, 398 00:39:04,009 --> 00:39:08,000 és olyan maghártyát, amiben a zsírsavak 399 00:39:08,009 --> 00:39:20,000 a magas deutérium (D)-tartalmú NADPH általi szintézisbõl eredõen 400 00:39:20,009 --> 00:39:24,000 maguk is magasabb deutériumtartalmúak. 401 00:39:24,009 --> 00:39:28,000 Ezekben a tumorsejtekben valóban így történik. 402 00:39:28,009 --> 00:39:32,000 Nézzük meg a fumarát-hidratázra vad típusú 403 00:39:32,009 --> 00:39:37,000 és enzimhiányos sejteket. Van kétféle fenotipusunk, 404 00:39:37,009 --> 00:39:42,000 az egyik primer tumor, 405 00:39:42,009 --> 00:39:48,000 a másik áttét. Ha a maghártya telített hosszúláncú zsírsavait tekintjük, 406 00:39:48,009 --> 00:39:55,000 ahogy ez az agresszív tumor kiterjed, 407 00:39:55,009 --> 00:40:00,000 egyre több hosszú szénláncú (C22:0) erõs és tartós lignocerin zsírsav épül be a maghártyákba, Page 29

Science-Fridays-LABiomed02a-DDW_Magyar 408 00:40:00,009 --> 00:40:06,000 mivel egyre több merev, kettõs falú "dupla bevásárló szatyor" kell, 409 00:40:06,009 --> 00:40:11,000 hogy egyben tarthassa a daganatos sejt az aneuploid sejtmagjait. 410 00:40:11,009 --> 00:40:18,000 Ez egy nagyon fontos változás a sejtanyagcserében, 411 00:40:18,009 --> 00:40:26,000 a fumarát-hidratáz mutációja szempontjából, és mert agresszíven terjedõ 412 00:40:26,009 --> 00:40:30,000 fenotípust eredményez. Emlékezzünk rá: az a sejt, ahol a fumarát-hidratáz vad típusú, 413 00:40:30,009 --> 00:40:34,000 mivel helyreállítottuk aktivitását, a sejt már nem képez rákot, 414 00:40:34,009 --> 00:40:37,000 míg a másik kettõ igen! Tehát a maghártya 415 00:40:37,009 --> 00:40:44,000 hosszú láncú telített és erõs zsírsavai reagálnak a fumarát-hidratáz aktivitás kiesésére. 416 00:40:44,009 --> 00:40:48,000 Kérdés 06: Elnézést… ezek szerint, 417 00:40:48,009 --> 00:40:55,000 ha vannak olyan sejtek, amelyeknek olyan szénforrást adunk, ami deutériumban gazdag, nagyon gazdag, 418 00:40:55,009 --> 00:41:05,000 és azok továbbra is normálisan mûködnek, ez azt jelenti, hogy a maga kiinduló modellje nem reagál, vagy változik! 419 00:41:05,009 --> 00:41:08,000 Igen..., ?? Kérdés 06 (folyt.) Vannak ezzel kapcsolatban irodalmi közleményei? 420 00:41:08,009 --> 00:41:12,000 Most vannak közlés alatt, a modellt és annak hátterét már közöltük. [doi: 10.1371/journal.pone.0072179] 421 00:41:12,009 --> 00:41:16,000 Page 30

Science-Fridays-LABiomed02a-DDW_Magyar Kérdés 06 (folyt.) Amit közölt az egy modell, az itt nem számít. 422 00:41:16,009 --> 00:41:20,000 Kérdés 06 (folyt.) Elméletileg a mutáció már megvan, de nem tudjuk, 423 00:41:20,009 --> 00:41:28,000 hogy annak a magasabb deutérium szinthez mi köze, mi a mutáció másodlagos következménye, nem biztos, hogy a deutérium az oka bárminek is itt! 424 00:41:28,009 --> 00:41:32,000 Kérdés 06 (folyt.) Ezt csak úgy lehetne eldönteni, ha egymagában, 425 00:41:32,009 --> 00:41:38,000 deutériumot adnának csak a rendszerhez, és figyelnék a változásokat 426 00:41:38,009 --> 00:41:43,000 a sejtben, a sejtmag hártyájában és a kromatinban. 427 00:41:43,009 --> 00:41:49,000 Ez egy nagyon jó kérdés! Had mondjam el a következõ kísérletet, 428 00:41:49,009 --> 00:41:55,000 amit csináltunk. Ez az volt, hogy deutériumszegény vízzel kezeltünk tumorsejteket, 429 00:41:55,009 --> 00:42:01,000 azaz kihagytuk a rendszerbõl a Deutériumot. Kérdés 06 (folyt.): Az imént mondta, hogy a mitokondriumoknak 430 00:42:01,009 --> 00:42:06,000 kell a vizet megtermelniük, akkor így viszont, sem a szabad víz, sem annak a deutérium hatása nem jut el a DNS-ig! 431 00:42:06,009 --> 00:42:12,000 De igen, a pentóz-foszfát ciklus oxidatív szakaszán keresztül eljut, és a citoplazmában lévõ fumarát-hidratázon keresztül, azok szabad vizet használnak... 432 00:42:12,009 --> 00:42:15,000 Ez a lényeg! 433 00:42:15,009 --> 00:42:22,000 Ha a mitokondriumok nem mûködnek, még mindig lehetséges deutériumszegény állapotot létrehozni a ráksejtben, 434 00:42:22,009 --> 00:42:25,000 azok magjában és a DNS-ben, például a pentóz-foszfát ciklus oxidatív szakaszán keresztül! 435 Page 31

Science-Fridays-LABiomed02a-DDW_Magyar 00:42:25,009 --> 00:42:29,000 Mindjárt mutatom, mi történik az anyagcserében amikor ráksejteket 436 00:42:29,009 --> 00:42:35,000 deutériumszegény vízzel kezelünk, de elõbb tekintsük át a rendszert. 437 00:42:35,009 --> 00:42:41,000 Ráksejtek vagy nem termelnek, vagy nem használnak fel mátrix-vizet, de inkább mindkettõ igaz. 438 00:42:41,009 --> 00:42:47,000 Az, hogy a pentóz-foszfát ciklus oxidatív szakaszán keresztül deutérium juthat 439 00:42:47,009 --> 00:42:54,000 a DNS szerkezetébe, megnyit számunkra egy utat, [doi: 10.1038/nrd3504 - 150-es hivatkozás] 440 00:42:54,009 --> 00:43:00,000 hogy egyszerûen a szabad víz által 441 00:43:00,009 --> 00:43:03,000 befolyásoljuk a rendszert, mátrix víz nélkül is. 442 00:43:03,009 --> 00:43:12,000 És pontosan ezt mutattuk ki anyagcsere utakban amikor deutériumszegény vízzel kezeltünk daganatos sejteket. 443 00:43:12,009 --> 00:43:15,000 Ezt a posztert 2006-ban, a Metabolomikai Társaság konferenciáján mutattuk be. [Boston, Harvard Egyetemen - doi: 10.12918/HYDLLCHU2006POST] 444 00:43:15,009 --> 00:43:25,000 Ha megnézi a 150 ppm vizet, mint kontrol, és összehasonlítja annak hatásait 445 00:43:25,009 --> 00:43:31,000 a 100, 50 és a 25 ppm csökkentett deutérium tartalmú vízzel, 446 00:43:31,009 --> 00:43:37,000 amit a sejtkultúrás folyadékhoz adunk, itt látjuk, hogy a lignocerinsav, 447 00:43:37,009 --> 00:43:45,000 a sejtmaghártya zsírsava, amit az imént mutattam be 448 00:43:45,009 --> 00:43:52,000 csökkentett mértékben keletkezik, és ez a kezelt sejtek magjának méretében és a tumorképzõ képességben is jelentõs csökkenést okoz. Page 32

Science-Fridays-LABiomed02a-DDW_Magyar 449 00:43:52,009 --> 00:43:59,000 Itt ugyanaz látható, MIA PaCa pankreász-adenokarcinóma sejtekkel. 450 00:43:59,009 --> 00:44:04,000 A palmitinsav, 451 00:44:04,009 --> 00:44:10,000 egy rövidebb szénláncú zsírsav, szintézisét csak a legjobban csökkentett, 452 00:44:10,009 --> 00:44:20,000 25 ppm deutériumtartalmú víz csökkenti. MCF-7 emlõrák-sejtekben, 453 00:44:20,009 --> 00:44:25,000 a sejtmaghártyához szükséges koleszterol - egy másik fontos strukturális lipid 454 00:44:25,009 --> 00:44:31,000 szterol termelése, és a sejtmag mérete, lecsökken, 455 00:44:31,009 --> 00:44:38,000 ha 150 ppm-rõl 100, 50 vagy 25 ppm-re visszük le a sejtkultúrás folyadék deutériumtartalmát. 456 00:44:38,009 --> 00:44:44,000 Ezen felül a c-myc és más onkogének kifejezõdése is 457 00:44:44,009 --> 00:44:48,000 párhuzamot mutat az elõbbi változásokkal. [doi: 10.1080/01635581.2013.756533] 458 00:44:48,009 --> 00:44:54,000 A DNS szerkezetét tekintve, ott is tovább csökkenhet a deutériumtartalom, 459 00:44:54,009 --> 00:45:00,000 különösen a citozin hidrogénjeinél 460 00:45:00,009 --> 00:45:05,000 ott, ahol a metiláció történik. Így pedig..., annak ellenére, hogy a mitokondriumok nem mûködnek, 461 00:45:05,009 --> 00:45:11,000 további lehetõségeink vannak alacsony deutérium bevitelére a sejtek nukleinsav- és annak membrán állományába a pentóz ciklus oxidatív karján keresztül, (vagy a citoplazma fumársav hidratáz enzimét használva), 462 00:45:11,009 --> 00:45:14,000 Page 33

Science-Fridays-LABiomed02a-DDW_Magyar szabad vízbõl is! Ezt mutatja az is, 463 00:45:14,009 --> 00:45:25,000 hogy például megváltoztatható a lignocerinsav szintézise. 464 00:45:25,009 --> 00:45:30,000 Ez egy fontos mag membránszerkezeti zsírsav: a maghártyák 30%-ában van jelen a foszfolipidekben ez a sav, 465 00:45:30,009 --> 00:45:38,000 mely nagyon merev, erõs is, és ezért fontos szerepe van a sejtmag szilárd szerkezetének megtartásában, 466 00:45:38,009 --> 00:45:42,000 fõleg a nagy és több magvú rákos sejtekben! 467 00:45:42,009 --> 00:45:51,000 Érdekes módon a növényi sejtek, melyek eukarióták maguk is, csak szabad vizet fogyaszthatnak a fotoszintézis miatt, (zsírsavak oxidációja nélkül) 468 00:45:51,009 --> 00:45:58,000 a sejtmag szerkezetének stabilizálását többek között proteinekkel és glikánokkal kell hogy megoldják. 469 00:45:58,009 --> 00:46:03,000 A sejthártyáikban cukor-eredetû komponensek vannak, 470 00:46:03,009 --> 00:46:12,000 a sejtmag aneuploidiával szemben stabilizált szerkezetét cellulózzal érik el! 471 00:46:12,009 --> 00:46:15,000 Ugyanezt az emlõsökben a lignocerinsav biztosítja, 472 00:46:15,009 --> 00:46:18,000 mert mi nem tudjuk összeállítani ezeket a nagy cukor-polimereket! 473 00:46:18,009 --> 00:46:24,000 Tehát a lignocerinsav a sejtmaghártyában található hosszú láncú telített zsírsav, 474 00:46:24,009 --> 00:46:32,000 mely a sejtmag méretét meghatározza és szilárdságát biztosítja! Errõl lesz szó az utolsó néhány dián is, 475 00:46:32,009 --> 00:46:39,000 és ezek lesznek a záró gondolataink. Kérem, hogy a következõ üzeneteket vigyék magukkal haza. 476 Page 34

Science-Fridays-LABiomed02a-DDW_Magyar 00:46:39,009 --> 00:46:45,000 A sérült mitokondriumoknak szerepük van a sejtek transzformálódásában azáltal, 477 00:46:45,009 --> 00:46:51,000 hogy lecsökken a természettõl fogva deutérium-szegény zsírsavak teljes eloxidálásából származó mátrix-víz 478 00:46:51,009 --> 00:46:57,000 reduktív szintézis útján való beépülése a sejtmaghártyába és a nukleotidokba. 479 00:46:57,009 --> 00:47:04,000 Azt is jegyezzük meg, hogy a közönséges víz, valamint a pentóz-foszfát ciklus oxidatív szakaszában történõ 480 00:47:04,009 --> 00:47:10,000 cukor-alapú NADPH-szintézis, 481 00:47:10,009 --> 00:47:17,000 és még az egyéb bemutatott anyagcsereutak, valójában erõs izotóphatást mutató, állandóan ketyegõ idõzített bombák. 482 00:47:17,009 --> 00:47:21,000 Ez egy tumorkeltõ epigenetikai hatás, 483 00:47:21,009 --> 00:47:25,000 az instabil hidrogén-kötések miatt gyakoribbak ugyanis 484 00:47:25,009 --> 00:47:30,000 a rák irányába ható epigenetikus események, és az aneuploidia! 485 00:47:30,009 --> 00:47:36,000 De ez nem feltétlenül baj, ugyanis a mitokondriumokon kívüli NADPH-szintézis 486 00:47:36,009 --> 00:47:42,000 egy új terápiás lehetõséget is megnyit: 487 00:47:42,009 --> 00:47:47,000 ez pedig a pentóz-foszfát ciklus oxidatív szakaszának felhasználása (valamint citoplazma fumarát-hidratázzal is), 488 00:47:47,009 --> 00:47:52,000 van lehetõségünk a DNS deutérium-szintjét csökkenteni, deutériumszegény ivóvíz vagy táplálék fogyasztása által. 489 00:47:52,009 --> 00:47:59,000 Legvégül, így gondolkozva megérthetõ néhány, az orvosi szakirodalomban nemrég megjelent eredmény. 490 00:47:59,009 --> 00:48:02,000 Page 35

Science-Fridays-LABiomed02a-DDW_Magyar Itt van például az, hogy ha patkányokat kizárólag 491 00:48:02,009 --> 00:48:09,000 zsíron alapuló, ketogén étrenden tartunk, 492 00:48:09,009 --> 00:48:11,000 és (túlnyomásos) oxigén-kezelést is adunk nekik, akkor bennük a tumorgyakoriság 70%-kal (~78%) csökken. 493 00:48:11,009 --> 00:48:17,000 Az oxigén-kezelés egymagában nem hat ilyen erõsen, 494 00:48:17,009 --> 00:48:22,000 de ha ketogén diétával kombináljuk, mely önmagában nagyobb mint 20 %-os (~60%) csökkenést okoz a rákkeletkezésben, 495 00:48:22,009 --> 00:48:26,000 akkor a csökkenés nagyobb mint 70%-ot (~78%) ér el. 496 00:48:26,009 --> 00:48:33,000 Ezt egy floridai csoport közölte le a PLoS ONE-ban. [doi: DOI: 10.1371/journal.pone.0065522] 497 00:48:33,009 --> 00:48:38,000 Ebben az a leginkább érdekes, 498 00:48:38,009 --> 00:48:43,000 hogy amennyiben a ketogén diétának van bármiféle élettani vagy biológiai hatása 499 00:48:43,009 --> 00:48:48,000 oxigén jelenlétében, az az oxidációs víznek köszönhetõ, 500 00:48:48,009 --> 00:48:54,000 lévén hogy a mitokondriumban egyesülhetnek csak zsírsav hidrogénjei oxigénnel, 501 00:48:54,009 --> 00:48:57,000 ahol a metabolikus víz a citokróm-C által (komplex IV) keletkezik, így lehet az oxigén hajtóerõ. 502 00:48:57,009 --> 00:49:02,000 Itt nem lehet ugyanis beleszólás sem a pentóz-foszfát ciklus, sem a citoplazmás fumarát-hidratáz, sem a peroxiszómák részérõl, mivel oxigén a folyamat hajtóereje. 503 00:49:02,009 --> 00:49:08,000 A zsírsavak teljes oxidációja az, ami a deutériumszegény anyagcsere-vizet szolgáltatja a DNS, Page 36

Science-Fridays-LABiomed02a-DDW_Magyar 504 00:49:08,009 --> 00:49:16,000 a hisztonok és a maghártya deutériumszegény állapotban való megõrzéséhez. 505 00:49:16,009 --> 00:49:20,000 Ez, véleményünk szerint, az epigenetikus folyamatoknak egyben fontos szabályozója is. 506 00:49:20,009 --> 00:49:28,000 Köszönetet kell mondjak a magyarországi es európai munkatársainknak, akik a deutériumszegény vizet szolgáltatták, 507 00:49:28,009 --> 00:49:34,000 kollégáimnak, dr. Lee-nek, 508 00:49:34,009 --> 00:49:37,000 és dr. Linehannak, 509 00:49:37,009 --> 00:49:45,000 az USA Nemzeti Rákkutató Intézetbõl. Az õ intézetébõl kaptuk azokat a veserák-sejtvonal anyagcsere mintákat, 510 00:49:45,009 --> 00:49:49,000 amelyekben a fumarát-hidratáz mutált, 511 00:49:49,009 --> 00:49:56,000 és amelyikben az helyre lett állítva. Finanszírozást több forrásból kaptunk, így a Hirshberg Hasnyálmirigyrák-Kutatási Alapítványtól, 512 00:49:56,009 --> 00:50:00,000 és a UCLA Klinikai és Transzlációs Tudományos Intézetétõl. 513 00:50:00,009 --> 00:50:04,000 Egyúttal köszönöm azt is, hogy eljöttek, meghallgattak, 514 00:50:04,009 --> 00:50:10,000 és ezzel elõadásomat is megtisztelték! [email protected] Elõadás diák (PDF) - doi: 10.12918/SCIENCEFRDEC132014LGB Elõadás értékelõ (PDF) - doi: 10.12918/SCIENCEFDEC132014EVAL

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Deuterium depletion and mitochondrial NADPH production: The link for epigenetic control of oncogenesis

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