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Introduction by Dr. Kevin W. Bruhn, Principal Investigator, Molecular Medicine, LABIOMED
(Medical Diploma) Szeged, Hungary, in 1987... He came to the United States in 1990,
spent 8 years at Ohio State, as a Research Scientist there... before coming to Harbor-UCLA in 1998...
He spent some time with Dr. Paul Lee, who is here today,
learning mass spec(trometry) and stable isotope applications
Dr. Boros is currently an Associate Professor working at UCLA...
And he is actually the Chief Scientific Officer at an LABIOMED spin-off company called SiDMAP.
SiDMAP is a pharmaceutical service provider for all phase metabolomics.
Laszlo is going to talk to us about some of his work, metabolomics, and beyond, and, so....
I turn it over to you..., thank you doctor!
And, it is a privilege, and thank you for the very kind introduction.
This is the last presentation of 2013, so I was trying to come up with a very interesting topic...
Which, I believe is a very.., truly.., a very intriguing topic...,
...just to talk about deuterium..., its depletion..., as part of cancer's epigenetic drivers...
This is a new theory, as well as a new model, that we have studied and now published (doi: 10.1371/journal.pone.0072179)
in the medical literature, in collaboration with the National Cancer Institute, who, actually...,
...provided the model for the study. So..., just to start...,
...let's refresh our memories about how cells,
in particular, how prokaryotic and eukaryotic cells, function?
Eukaryotic cells form nuclei, and these nuclei contain the DNA material, or...,
...the nucleic acid material, surrounded by membranes..., It's a more complex structure, with a larger size,
as far as the cell goes, and the nucleus has many more functions built into it...,
e.g.: ...to be able to differentiate, and to specialize in various functions...
And..., what is really interesting, and what captured me back in high school, and in..
..early college is that: If you (the cell) want to form nuclei contained with a membrane...,
you know.. the cell needs also all these other cellular particles...
And what we've got most interested in, is the mitochondria..., which means, that, really..., there is never an eukaryotic cell
with nucleic acid surrounded by a membrane, without mitochondria!
And..., there are no exceptions to this rule..., while cells, that do not have nuclear membranes,
or have no solid or eukaryotic-like nuclear membranes, never have mitochondria.
So.., the link between cell morphology, function and energy production, is where we started...
to look into mitochondria in more details..., and started using models...
Where the mitochondrion was not functioning, and cancer has..., cancer developed!
Just to review more background information..., the internal membrane system, which includes
the nuclear membrane and the Golgi apparatus, host a lot of functional proteins...
Which actually produce fatty acids and membranes, using specialized enzymes...
And the mitochondrion is the site of energy production, yet, it's really not only an energy producing cell,
...or cell organelle, if we review the evolution of mammalian cells!
Because, mitochondria first produce water..., which is one basic role of mitochondria...
And we will talk about this ..., a bit more, in details...
The cytoskeleton has structures, which include micro-tubes, actin and binding proteins and...,
...so on. So that, a cell, that is eukaryotic and differentiated,
can perform a number of additional functions, which also originate.., some of the cell's regulatory
elements originate from mitochondrial functions. A mitochondrion has an inner membrane, an outer membrane,
has cristae and also has the matrix!
And..., the matrix is where the citric acid cycle...
...takes place. The citric acid cycle, or Krebs cycle, is a set of
biochemical reactions that produces ... aaa ...
that produce reducing equivalent, NADH and the proton is actually carried through the cytochrome system
and, eventually, through cytochrome-c (complex-IV). The product of the proton released
from citric acid cycle intermediates is water..., if there is oxygen in the
mitochondria in order to produce water..., and this is how animals actually survive in the desert...,
where they do not drink! They use their mitochondria, instead, to actually produce
water! and ...aaa...
that's how the lizards, the snakes and sea fish, and the ... marine animals do not need to
drink water as they produce water in their mitochondria...
what the substrates are? I am going to talk about in more details.
There are two water molecules produced from..., when the Cycle is turned over and with
the addition of oxygen water production is enhanced.
Yet, if the cells need ATP, what they do is, they actually shovel the protons
to the inter-membrane matrix (space), and they actually use this complex-V, or ATP...
...synthase. And, as they actually channel protons back into the matrix, they also produce ATP...
...or energy. So the hydrogen comes from citric...
...acid cycle metabolites and intermediates, so they are very critical to drive the whole process of...
...water production, and also the proton channeling back and forth...
...into the matrix. So...,
Our hypothesis was from the very beginning, when I was preparing this talk and was getting...
...ready to present, was that..., how does the weight of hydrogen affects the...
...kinetics of these pumps, and, in overall, the biochemical reactions associated...
with mitochondrial failure, in cancer especially, and what're key components of this system, which are responsible...
for epigenetic events! So...
This is, in more details, the chain or set of reactions that happen in the mitochondria
or the TCA cycle, and, as you can see, citrate is formed, which
is why it is called the citrate cycle. And this citrate is formed from a two carbon metabolite...,
...which is acetyl-CoA and an oxaloacetate, which is in the Cycle.
There is this six carbon citrate, which turns over...,
...two carbons are lost in the form of CO2, and as CO2 is produced, these hydrogens are...
...actually taken out from the intermediates of the TCA cycle...
What is really interesting is that not identical carbons get oxidized in the first round, which...
...have been entering the Cycle! And it is also very interesting that there...
...is water consumed, while the cycle is spinning over...
So..., it is kind of interesting, and, it is really intriguing to know, why water is consumed, when, in
the meantime, oxygen is reduced, and why water is necessary, which is...
matrix water, the one that is produced by cytochrome-c (and complex-IV)...,
why this water is reused in the mammalian system, instead of using it as a water source...,
...just like the animals in the desert would do!
Obviously, using matrix water for reductive synthesis, is where I am going to lead the topic...!
Yet, it's a very intriguing system, as finding out, how water is exchanged and how the hydrogen...
...is channeled into DNA, eventually..., which I am going to show you!
Because, what is very interesting is that there is a cancer model, which we have studied in...
...collaboration with the National Cancer Institute, where one of the enzymes of the Cycle, called...
...fumarate hydratase, or fumarase, is mutated! So kidney cells that do not have this fumarate...
...hydratase or fumarase enzyme, are not able to use water to hydrate fumarate into malic acid!
And, as the result of that, the Cycle would turn back around, and actually, through reductive...
carboxylation, the Cycle would actually become the consumer of hydrogen of some other source...,
...other than water of the matrix! So...,
with this total turning back around of TCA cycle metabolism, cancer readily develops...
...and, it is actually a very aggressive cancer, which is called the...
...clear renal cell carcinoma syndrome, which is associated with hereditary leiomyomatosis...,
after 10-15 years of this fumarate hydratase deficiency, and also these skin bumps. These patients...
...will actually develop cancer! It's a rare disease, it's called the Reeds syndrome...,
...based on the investigator who described it.
And, what we see in the beginning of the diseases is that there are these skin bumps...,
...histologically they look like a wide broad keratin layer between the sub-cutaneous...
...structures. And this is the pre-exposition state, or this the...
...preliminary stage before kidney cancer develops in these patients!
When kidney cancer develops, the cancer is really unique in the sense that it is called...
...clear cell renal cell carcinoma... It used to be called a type of hypernephroma...,
...but it is actually developing from the kidney tissue!
And, what you see here is that there are clear cells with nuclear morphology characteristic...
...of tumor cells! It is a very aggressive cancer -- and here...
...is the normal kidney tissue, and because of this characteristics..., with the accumulation...
...of this cystic material in the cells, the cells' nuclei are pushed to the side, and...,
...because of this pathological feature, they call this type of cancer...
...clear cell kidney (renal) cell carcinoma. And it develops based on this fumarate hydratase...
...mutation, which is in the TCA cycle!
So..., the cancer is a cortical tumor...
...and it is a malignant epithelial cell - type of tumor, and it is clear, but I am not going to talk here about morphology...,
...in more details... But, yet..., just...
...let's go to the syndrome, which is actually caused by a mutation in...
...the fumarate hydratase gene, and as a result of that, there is an accumulation
of water with fumaric acid in the TCA cycle and the mitochondria!
And, it is an autosomal dominant disease. What we see here, is that once the mutation occurs...,
...over a longer period of time, there is fumaric acid buildup...
...and there is decreasing malic acid concentration... And the question is, how these changes actually...
...contribute to cancer development? Because, just looking at this scenario, you...
...would claim that this particular model is more..., would affects more intensely..., or more obviously, the...
...energy production system of the Cycle! But, actually..., over time, a very aggressive cancer...
...develops... Question 01: Sorry,... is the accumulation of fumaric acid...
what causes the bloating of cells? And also succinic acid...
...so, actually there is a number of metabolites that accumulate in the mitochondria, fumaric acid...
...is one of them, and also succinic acid accumulates...
...so everything, that actually ...aaa... lead to malate formation, accumulate, (including matrix water)...
...and, actually..., there is very little malic acid concentrate in these tumor cells!
(Dissolved) fumaric and succinic acid cause hydropic degeneration (of the mitochondria)...
...now, I want to mention that ... if you ...., not only fumarate hydratase, but succinate
formation, or succinate dehydrogenase mutations, or succinyl-CoA synthetase abnormalities, also...
...cause cancer! Somewhat different kind of cancers, and there...
...is a literature on it and they find TCA cycle enzyme mutations in more and more tumor phenotypes!
In fact, if you look at cancer as a disease, the first obvious metabolic event is decreased...
...mitochondrial function. This is..., this will lead to the Warburg effect...,
where pyruvate can not enter the mitochondria, substrates can not be oxidized.
So..., in general, either through hypoxia..., as age advances..., or through fumarate hydratase...
and other enzyme deficiencies in the TCA cycle, one of the basic principles of cancer development...
...is mitochondrial function... decrease, for one reason, or for another.
So..., it is generally accepted that the mitochondrion, per se, yet..., its specific enzyme mutations...,
...more specifically, contribute to cancer development, this is very common in cancer...
So..., Question 02: I have a question, neither muscle, nor...
fat tissue have defects? No...
Do you expect muscle defects, in muscle mitochondria? That is not the leading....
...Other organs are rather affected, including the skin with bumps, but the leading clinical symptoms are...
...leading to skin lesions and also kidney cancer development, in this particular mutation. [mammillary muscle of the *** (genital leiomyoma), the smooth muscle of blood vessels (angioleiomyoma) may also be affected]
So...,
...what is more interesting, and, this is really intriguing that...
...Once we restore fumarate hydratase functions in these tumor cells...
...they actually lose the ability to produce tumors in nude mice...
...so, actually, restoring this one single enzyme in the TCA cycle, reverses the disease itself...
So..., the National Cancer Institute has developed...
...a cancer cell model, which is termed as UOK262. And there is a wild type, which has the insert...
...of the normal mitochondrial fumarate hydratase gene.
And so they have a wild type normal fumarate hydratase activity, in these cells!
They also have the empty vector phenotype. So, once you actually insert this enzyme back...
...into the mitochondria, then, they actually become non-tumorigenic!
So, they can not really cause tumors any more. If you actually inject these tumor cells into...
...nude mice, as long as there is a fumarate hydratase mutation, there is going to be tumor formation...
...But once you restore fumarate hydratase function, then the mice are happy!
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)
...paper and also some of the publications come from Nature! (doi: 10.1038/nature10642)
We have "pheno-typed" these cells for the National Cancer Institute...
...and we used our metabolic 13C tracer methodology in order to dissect the mechanism that are...
...clearly involved in the tumor formation process. And we studied two tumor phenotypes, which
are fumarate hydratase deficient and also the fumarate hydratase restored cells.
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)
to mention in detail! So...,
...if you look at the literature, as far as explaining why mitochondrial...,
...increased mitochondrial concentration of fumarate cause cancer, you can actually see a number...
...of hypotheses! Obviously, there are a number of studies that...
...address this issue. And one possibility is that high fumarate concentrations...
...or fumarate intermediate concentration, with increased concentrations...,
...they actually go into the cytosol and they inhibit certain enzymes...,
...which activate signaling for cancer development. And there is a number of, for example, this...
...hypoxia induced factor, which is involved in the metabolic changes...,
...or epigenetic changes that are associated with the disease!
And there is also a non-specific succinate binding to cysteine residues of proteins...
And, also there are modifications in DNA -- protein binding, because of the increased availability...
...of these organic acids, coming from the Cycle, and elevated levels of superoxide, because...
...of the lack of mitochondrial functions are also involved in the explanations, also involved...
...in the mechanism. Now...,
...and they have published these in..., we have published these possibilities and scenarios...,
...in collaboration with the National Cancer Institute!
Now, we as biochemists with Dr. Lee, what we would...
...ask is: what does, actually..., fumarate hydratase do?
What is the enzyme's activity? What is the function of it?
And I actually posed this question to a few molecular biologists, and asked them what the enzyme would do? The...
...answer was: they would not know, which is interesting, really!
I think, as we are getting into physiology and System's Biology, this is also a very important...
...question, and one of the key questions! So, what it actually does..., fumarate hydratase...
...does, it just adds metabolic matrix water to TCA cycle intermediates!
So, fumarate hydratase uses water and introduces it back into the TCA cycle!
And as you can see..., the enzyme, itself, was described before the 1930s by Dr. Albert Szent-Györgyi...,
...who also discovered vitamin-C and he got the Nobel prize in medicine in 1937.
What is really interesting is that my grandparents, on my Mom's side, they knew him personally...
...as they also lived in Szeged at that time, in those times.
So, and my Grandma was talking about him, in fact they had the same boots, they had...
...season tickets to the Opera and they had the same boot!
So, the two families were not friends, but they knew each other very well.
And he (Dr. Szent-Györgyi) is the one, who suggested that there is this hydrogen carrying catalytic role for...
...succinate and fumarate, and it was back in the 1920s.
So, succinate and fumarate and malate are actually involved in carrying protons through...
...the Cycle. And this was, at that time, a very intriguing idea!
Because, the cycle was not yet closed...,
...Krebs' initial reactions had not really been clarified, yet...,
...yet, Dr. Szent-Györgyi already laid some of the reactions down, which are involved in...
...the citric acid cycle, with the use of hydrogen, and protons...
...and this was the main function of succinate and fumarate in muscle cells, to carry these...
protons! So ..., as you can see, there are two places...,
and there are actually two net water molecules consumed by the Cycle, as it turns over.
One enters at aconitase, which actually provides the hydrogen for the first hydrogen- or proton-yielding...
...reaction. And the second one is at fumarate hydratase...,
...which actually, ...with the cycle turning over, actually, will provide the proton for NADPH synthesis...,
...which I am going to show in a minute, how it works.
It seems that water, or metabolic or matrix water...
...enters the intermediate pool very quickly, as just to replace many of the hydrogens...
...and "do not bring" water into the cycle, because we have our own water in the cycle, that we...
...are going to use for turning the cycle over for reducing equivalent synthesis!
Now ..., as I was mentioning, the original role of the TCA cycle, or the normal mitochondria...,
...is actually to produce water. And we know this, because when we look at physiology...
...for example, animals that live either in the ocean, and they can not really drink salt water...
...from the ocean, or they live in the desert, where there is no water...
...they use the mitochondria to burn fat!
And this is why..., if you look at the camel, if you look at the scorpion, the rattle snake...,
that they all have to have a fat pool, which they get from their prey, that synthesize fat from plants...,
which live in the desert. And, surprisingly, from 100 grams of fat they...
can actually produce one hundred ten grams of water!
And this is because of the saturation, the hydrogen saturation of the fat so you just...
...need the oxygen! And if you talk to a physiologist about what...
...would be the best model for studying lungs, they'll always tell you they are looking at snake lungs.
The snakes really have huge lungs just to supply...
...oxygen for their water production in the Cycle.
Question 03: Laszlo, is this why weight watchers tell you to drink a lot of water?
No, actually it's for some other reason, because we do not use our own (metabolic) water... "as water"
Our problem is..., if you look at the Cycle, ...and this is the...
...main difference between the rattle snakes and the mammals...,
we invented the mitochondria to use it for energy production...,
...ATP synthesis, actually, instead of releasing (matrix) water, we reuse...
...95% of it for NADPH synthesis and for membrane turnover!
We cannot produce net water! Migrating birds can..., and this is how it all developed:
1) the rattle snake uses mitochondria for water production...,
but the reptiles that actually "fly..." (migrate) to temperate seasons...,
where there is water, so they can drink now..., so they invented the
mitochondrial function of producing heat and ATP, so they can stay... (in the cold)!
So..., it is an evolutionary impact on metabolism, eventually..., to use mitochondria for something else...,
...so..., even though we produce the same amount of matrix water...,
we reuse it through fumarate hydratase, so matrix water is reintroduced into the Cycle...
and, it is carried out from the cycle (as malate), which I am going to show you!
This is why we have to drink water. If "rattle snakes exercise", they do...
...not have to drink water, because they just burn more fat, and..., use fat for water production!
But, anyways, Dr. Mellanby was an ornithologist and he was...
...involved and interested in studying animals that live in the desert.
Now, if you use carbohydrates as the source of ***(P)H in the Cycle you only generate 55 grams...
...of water from carbohydrates. And this is simply because their hydrogen content...
is not as high as that in fat, as saturated fat has the highest hydrogen content.
So, they need oxygen to produce water and animals can live in the desert with that approach.
So, we are using up matrix water, instead of producing it and leaving it as water.
Obviously the rattle snake, because they cannot keep producing ATP by synthesis and recycling (of water)..,
...as they use up matrix water for maintaining biological solvent functions...,
...snakes have to hibernate, because cannot really control body temperature...,
...snakes have to use and generate heat from sunlight, so that they can use matrix water for carrying...
...out functions that are involved in enzyme activities and to contain, maintain water content...
...in their bodies! Once we use matrix water up for malate and...
...eventually NADPH synthesis, as the reducing equivalent...,
...we are going to lose the ability to use matrix water to produce our own water, so we...
have to drink water. So we have to replenish our water from other sources!
So, the NADH source and the water in the matrix...,
...metabolic water, has a very unique characteristic.
And that means, that.., it is actually low in deuterium!
And this, actually, will take us to another story, which, I think, is very intriguing.
As this metabolic water is different from the water that you drink.
So, whatever you drink there, has higher deuterium to what each mitochondrion produces.
And the reason for that is....,
because..., in your food, your fat, in your long chain saturated fatty acids, the hydrogen content...
...of your fat has actually lower deuterium, than that of the water, what you drink!
We measure deuterium content, in any substrate, including water, as the Vienna standard of...
...mean oceanic water, which has around 155 parts per million (ppm).
If you look at your food source, looking at your flour, table sugar, they are actually...
...very close to the 150 ppm. And when you get to the cottage cheese, which...
...is a good mixture of fat and carbohydrates, you can see it is dropping to 136!
If you look at olive oil, it is 130 ppm. Then, in butter it is 124!
And, in fatty acid of animal fat, it is only 118 ppm.
That means, as more fatty the food gets, less deuterium those fatty acids accumulate, or contain!
More interestingly...,
...actually, if you look at unsaturated fat from plants, those are actually very low in deuterium.
And, actually, if you look at double bonds in your unsaturated fat, it is only 102 ppm...,
...getting close to almost a 40% drop in deuterium content.
So..., more fat or unsaturated fat you actually consume, your water going to have less and...
...less deuterium and so is your matrix water, in mitochondria...,
...will have less and less deuterium. What's also very interesting is that, actually...,
...this occurs, or seems to be occurring, during photosynthesis.
Light enters plant cells, you can see here the water from our environment...,
...or the free water, which has 155 ppm, which will actually be cleaved, while oxygen is being produced.
And the reducing equivalent, which uses this hydrogen, enters this cycle, called the Calvin cycle...,
...which actually drop deuterium.
So, there is discrimination against deuterium, when it comes to photosynthesis (and cleaving water).
So because of photosynthesis, and the enzymes that perform photosynthesis, plants drop deuterium...
...during the photosynthesis process! The fat pool the plant will synthesize...
...has lower deuterium. So, when we consume fat and burn it, in mitochondria...,
...the NADPH pool, that we restore, is actually low in deuterium ...
and its... Go ahead!...
Question 04: Are those enzymes less efficient in using deuterium from water? Why is deuterium getting dropped? Because...,
...deuterium is twice as heavy as hydrogen! I know, but are those enzymes less efficient...
in carrying deuterium? Yes, those enzymes have actually less binding...,
...which is actually dropping, it is physically to drop deuterium, which is fascinating!
Do we know which enzyme(s)? OK, there are a number of enzymes, which are, actually...,
...responsible for deuterium depletion, especially desaturases.
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]
It is a French team, Dr. Billault, I hope I say it right, they actually measure deuterium content...,
...in a number of fat products, so they know what, actually, unsaturated carbon sites...
...are the lowest in deuterium, and what plants and fats those are! (doi: 10.1074/jbc.M500909200)
They have had a huge facility, just to..., actually, just to measure that and they use these isotope...
...ratio mass spectrometers, so they actually combust these substrates to measure their...
...deuterium content. But, anyways...
So, you can actually deplete deuterium in your cells, using NADPH, restored after photosynthesis...,
...or, if you live at high altitudes. Your water will have low deuterium because the rain falls out...
...with heavy water, first... (at lower altitudes). So...,
...and those are the people who live the longest. The Peruvian people, actually, they may...
...live, in Caucasia, the Himalayans, they may live over a 100 years!
And, what is really interesting is that, they do not have their natural or authentic...
...words for cancer, meaning that, it is really unusual that they...
...would get cancer, at those high altitudes.
And, actually, as they drink (mountain) water, they do not need...
...their lowered deuterium water..., plus the photosynthesis enzymes, they can really go low, as far as deuterium enrichment.
Which is really interesting! So..., if we look at the model, when a normal...
...cell uses fatty acids for producing (matrix) water and using that water for NADPH production...,
...what you can see is, because of the low deuterium content in fat...,
...you are going to introduce in nucleic acid and DNA a low deuterium content, as far as its...
...structure goes. And your DNA will have more hydrogen than deuterium, if mitochondrial function is maintained.
It is simply because your food source with mitochondrial...
...function is to restore and to maintain low deuterium water content in the matrix.
And we know from stable isotope studies, that...
...actually, mitochondria oxidize about 90% of acetyl-CoA that come in to get oxidized from fatty acids.
So, either dietary or fatty acids we internally re-produce from dietary fats.
So, the mitochondria in the matrix always have, or try to decrease deuterium...,
...while the arm of high water intake, with high ppm deuterium water intake, through the pentose cycle...,
...or the peroxisomal beta oxidation process, cannot reach into the DNA, in normal resting cells.
And, with normal oxygen supplies, NADPH production goes through the mitochondrial...
...cycle intermediates, that have low deuterium, of about 120 ppm.
Actually, if you feed an individual with only a fat diet - like a ketogenic diet - and in cancer...
...it is very beneficial. With the ketogenic diet you can decrease body water deuterium down...
...to about 110 ppm, and simply, because fat has lower deuterium for the mitochondria to exchange water.
So..., what is wrong with deuterium? So why...
...is deuterium so bad, as far as its clinical behavior, or its physical behavior goes?
Actually, it is twice as heavy as hydrogen. Hydrogen has one proton, and deuterium has...
...one proton and one neutron, so it actually weighs twice as much as hydrogen.
Yet, it is chemically identical, so it participates in every reaction that our body carries out...
...as far as reductive synthesis goes. And actually when deuterium gets into DNA, it occupies...
...these methylation sites, and when the methylation enzymes come, they can remove hydrogen very easily...,
but when it comes to, when the site is occupied by this heavy deuterium, it really causes a trouble.
And, because of this heavy deuterium incorporation into nucleic acid...,
...there is altered gene expression and methylation kinetics will change.
And, likely because of the stronger mass induced resonance between hydrogen and deuterium bonds...,
...in each base pair with counterpart opposite bases, deuterium may be involved in aneuploidy.
So..., hydrogen being light, half the mass of deuterium, it is easy to lift...,
...but when it comes to deuterium, it is going to be a huge effort for any enzyme to deal with it...,
especially, when it is an enzyme, which is structurally incorporating it into DNA...,
...or the nucleic acid membranes that are participating in those bindings. Hydrogen bonds are really...,
truly, very important, when it comes to DNA physiology (and nuclear stability)
So..., in a normal cell, where mitochondria is active, we can actually keep a very decent...
...nuclear structure and DNA structure. When it comes to cancer and mitochondria...
...are dismantled, and they use the pentose cycle, as well as other reactions that actually load...
...your NADPH and reducing equivalent system with free water...,
...you are going to have morphological changes. And with that, we will have physiological changes...,
...morphological and biological changes in cell size, cell behavior, and the cell's regulatory mechanisms.
So..., we believe that the first very important epigenetic event...,
that is really (excuse me) to modify nuclear function, before methylation occurs...,
...is actually DNA deuteration! And because of its chemical and physical...
...impact and characteristics, this is a very important event in cancer development.
Now, where can NADPH enter, or hydrogen or deuterium enter, metabolism, when the mitochondria...
...are not active, or in cancer, when there is no oxygen, or when there is an enzyme mutation...
...in the mitochondria? It is either through this serine oxidation glycine cleavage system...,
...which we have recently published in Nature. This is one of the sites where water, not matrix water..., (doi: 10.1038/cddis.2013.393).
...but free water, is the source of hydrogen/deuterium...,
...or through the pentose cycle, which actually uses glucose, or table sugar, or flour, which...
...we use for cooking, and it has higher (deuterium) ppm.
It is interesting that the plant's fatty acid synthesis enzymes drop deuterium...,
...but actually in fruit plants when fruit sugar is generated (with intense sunlight), those photosynthesis enzymes can better hold deuterium.
So the sugar, and also the water, which goes through...
...and generates the NADPH through the oxidative branch of the pentose cycle, actually,
has high deuterium. And we have been publishing regarding the...
...role of NADPH synthesis in the oncogenic process through the oxidative branch of the pentose cycle. [PMID: 9488183]
In fact, this is the set of reactions that Glivec controls!
Glivec inhibits the oxidative branch of the pentose cycle, which we have shown in the Journal...
...of Biological Chemistry. So, in cancer we have to offer another model..., [doi: 10.1074/jbc.M105796200 - 10.1056/NEJM200207043470116]
...and that is simply when (mitochondrial) fumarate hydratase, or, when isocitrate dehydrogenase mutations...
...occur, the low deuterium containing matrix water cannot enter metabolism...
And what the cells are left with are glycolysis, pentose cycling, (cytoplasmic fumarate hydratase), the SOGC pathway and, actually...,
...high deuterium is getting in from free water, or from substrates other than fatty acids, which would lower deuterium.
So..., eventually, cancer cells will end up with a heavy sticky DNA...,
...and nuclear plasma membrane fatty acids, that are actually loaded (with deuterium)...,
...or, actually because of synthesis, new DNA synthesis via NADP deuterium or NADPD reducing equivalent, cancer cells will going...
...to have membranes, and also fatty acids, that are deuterium rich.
And, this is what happens actually in these tumor cells.
When you look at the fumarate hydratase wild type cells...,
...and you look at the empty vector, we are going to look at the fumarate hydratase mutant...
...through two different phenotypes, which actually lack the enzyme, one is a primary...,
...the other one is a metastatic tumor. If you look at, for example, the nuclear membrane...
...long chain saturated (lignocerate) formation, you are going to see..., as tumorigenesis advances...,
that is how they going to incorporate more and more long chain saturated fatty acids...
...into their nuclear membranes. So..., they need these "double bags" with rigid...
...membranes to keep this aneuploidy and nuclear structure together.
And this is the type of metabolic change, which is very important, when it comes to...,
...for example, a fumarate hydratase mutation, and also which comes with an aggressive tumorigenic...
...phenotype. And, if you remember, this guy, the one with the wild type fumarate hydratase gene...
and reaction, after we restored it, these are not tumorigenic!
But the other ones, are! So..., actually, for example, as far as a long chain...
...saturated lignoceric acid of nuclear membranes, it is responsive to fumarate hydratase activity...
So..., Question 05: Excuse me, that means .. if you have wild type ....
...cells and you treat them with a carbon source that contain deuterium, high level...
...of deuterium, .. they still function properly, that means your starting model changes...
That is right! Have you published this?
We are publishing..., we have published the background... [doi: 10.1371/journal.pone.0072179]
....But in concept, it does not matter, in concept; you already have a mutation, so this is a mutation...
...you do not know if that mutation causes a secondary effect, that you see in increasing...
...deuterium, and these pose a concomitant increase that you see, and changes in chromatin. So, it does not...
...mean that it is going to be the deuterium! The only way to prove that the deuterium is doing...
this to the system, if you just change deuterium, only deuterium, and if you also...
...going to see in the cells that deuterium changes the chromatin!
This is a very good question! And this is exactly what we did. Let me show you the next study...
...we did, because what we did is we actually treated the tumor cells with deuterium depleted water.
So, we actually left out the deuterium. Question(cont): But if the water needs to be produced by the...
...mitochondria, so that neither water nor deuterium is going to be precipitated into the DNA?
It will, it would, through the oxidative branch of the pentose cycle (and cytoplasmic fumarate hydratase) Question(cont) ...the pentose cycle
It is free water through the pentose cycle! This is the whole point...:
If your mitochondria are not working you still have a mechanism to get low deuterium into...
...these cells and that is through the oxidative branch of the pentose cycle!
So..., if you actually treat these cells, tumor cells...,
...with low deuterium water, let's see what happens! But let me go first through the system!
So..., we have tumor cells that do not...
...use matrix water. And so the likelihood of low deuterium getting into...
the DNA structures, through the oxidative branch of the pentose cycle, opens a window for us...,
...which can actually affect the system through water, simply, not matrix water, but next...
...treating these cells with deuterium depleted water.
So..., and in fact this is what happens! Because, if we treat these cells with low deuterium water...
...and this is a poster which we presented at...
...the 2006 Metabolomics (Society) Conference. If you look at the 150 ppm, which is the control... (doi: 10.12918/HYDLLCHU2006POST)
...reference water, and at 100, 50 and 25 ppm, so we are decreasing the deuterium in drinking...
...water, or media water, in these tumor cells. As you can see, the same fatty acid, lignoceric...
...acid, which is the nuclear membrane fatty acid, (DDW) actually introduces a drop in new lignocerate...
...synthesis, in nuclear size and also in tumorigenicity of these cells.
And this is the same, this is with MIA PaCa pancreatic adenocarcinoma cells.
If you look at the palmitate synthesis, which...
...is the shorter chain fatty acid, only the highest, 25 parts per million deuterium depleted water...
...lowers the de novo fatty acid synthesis. If you look at MCF-7 breast cancer cells and...
...look at the nuclear membrane cholesterol synthesis, which is another important structural fatty acid...
...or sterol, what you can see is coming from 150 ppm, which is...
the reference range, down to the 100, 50 and 25 ppm, you can see a decrease in cholesterol...
...synthesis and nuclear size. Now, besides that, if you look at c-myc expression,
...and oncogenic expressions, they also correlate with these changes (doi: 10.1080/01635581.2013.756533)
If you look at the DNA structure, what you are going to see is the deuterium content...
of the DNA, itself, especially at the cytosine's hydrogen group, where actually methylation...
...occur, may decrease also. So now..., even though your mitochondria is defective...
...you can still introduce low deuterium into the cells through the oxidative branch of...
...the pentose cycle through drinking water! So, eventually....,
...you can change, for example, lignocerate synthesis, which is a structural membrane fatty acid...
...synthesis. Thirty percent of nuclear membranes have this fatty acid in their phospholipids...
...which is a very rigid and a very important fatty acid species to actually synthesize...
...a rigid nuclear structure! Now..., interestingly..., plant cells, which...
...are eukaryote cells, they have to consume water, because that is what they use for photosynthesis [and can not oxidize fat]...
...obviously, to keep their (deuterium loaded) nuclear structures stable, they have to use proteoglycans and...
...they have to synthesize sugar components into their cell membranes...
...and, for example cellulose, which keeps those nuclear structures less vulnerable to aneuploid changes!
This (nuclear stability) is what lignocerate does for mammalian cells...
... because we really cannot link those large sugar molecules up!
So..., lignoceric acid is the long chain saturated fatty acid of the nuclear membrane, which is...
...involved in determining nuclear structure, in size and strength! Eventually, and this is what I'd like to ..., just
...to process the last few slides. And this is the set of take home messages.
Impaired mitochondria are involved in cell transformation and this is by limiting the...
...low natural deuterium containing complete fatty acid oxidation product, which is metabolic water...
...to enter nuclear membranes and nucleotides through reductive synthesis...
The other very important message is that, in turn, heavy or natural water and sugar...
...dependent NADPH production takes over by the oxidative branch of the pentose cycle...,
...as well as other-pathways that I showed you, which actually are ticking time bombs with a strong...
...isotope effect. And there is an oncogenic epigenetic event...,
...the likelihood of more frequent oncogenic and epigenetic events...
...with unstable hydrogen bonds and aneuploidy!
Nevertheless, extra-mitochondrial NADPH synthesis opens a therapeutic window.
So.., using the oxidative branch of the pentose cycle (and cytoplasmic fumarate hydratase), we can actually still introduce low...
...deuterium content into DNA. If you use water, or if you use a food source...
...that has low deuterium. And..., eventually...
...this approach can explain some of the recent findings that are entering the medical literature.
For example..., if you feed your rats with a ketogenic diet...,
...which is only fat based, and you actually treat them with oxygen treatment, or hyperbaric...
...oxygen treatment, then you decrease tumorigenicity by over 78 percent!
And in ketogenic diet and oxygen..., oxygen treatment on its own does not do much...
...but if we combine it with ketogenic diet that...., ketogenic diet on its own decreases tumorigenicity...
by over 20 (~65) percent, but when you combine it with oxygen treatment...
...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)
...and what is really interesting is that..., it is really truly metabolic water...,
...when ketogenic diet can actually have any physiological effect or biological effect...
...in the presence of oxygen treatment, because this is a cytochrome-c (complex-IV)...
...dependent reaction, no pentose cycle, no peroxisomes can be directly involved...
...in the process, because oxygen is the driving force.
And there is another very important aspect of this...
...and this is practically the complete oxidation of these fatty acids, which will and is providing...
...the low deuterium metabolic water to prevent, eventually, DNA, histone and nuclear membrane...
...deuteration, which we believe is a very important force...
...in driving epigenetic events! I have to thank our collaborators from Europe...
...who provided the deuterium depleted water, Dr. Lee, my colleague and...
...Dr. Linehan, who is at the National Cancer Institute...,
...and they provided for us the kidney cancer cell line, which actually has this fumarate...
...hydratase mutation, and the model, where fumarate hydrates is reintroduced.
And we had several funding sources; one is the Hirshberg Foundation for Pancreatic Cancer Research.
And we are also funded by the UCLA Clinical...
...and Translational Science Institute. And with that, I just wanted to thank you for
...coming and listening to my talk! Please send a teaching evaluation: Slides (PDF) - doi: 10.12918/SCIENCEFRDEC132014LGB Teaching evaluation (PDF) - doi: 10.12918/SCIENCEFDEC132014EVAL