The Mechanisms of DNA Damage with Prof Michael Fenech

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The Mechanisms of DNA Damage with Prof Michael Fenech

What dietary, environmental and lifestyle factors pose a risk to our DNA? 

Today we are joined by Professor Michael Fenech, an internationally recognised researcher in nutritional genomics and genetic toxicology. 

Prof Fenech shares with us the mechanisms of DNA damage and the role that the assessment of micronuclei may play in evaluating a person's genetic status regarding exposure to environmental genotoxins and dietary deficiencies. 

Covered in this episode

[00:26] Introducing Prof Michael Fenech
[01:35] The mechanisms of DNA damage
[10:22] Delicate balance: cellular adaptation
[13:34] Threats to our DNA
[19:31] How commonplace is damaged DNA?
[24:51] Micronuclei and disease
[36:57] Accessing a micronucleus assay
[49:31] The connection between methylation and micronuclei


Mark: Hi, everyone, and welcome. Professor Michael Fenech is my guest today. And he's recognised internationally for his research in nutritional genomics and genetic toxicology. He's also played a huge part in the careers of many geneticists, such as Denise Furness, well-known to many of our listeners. In the past 8 years, he's presented in over 55 international conferences. Google Scholar h-index is 84 and based on a staggering 31,000 total academic citations. He is a giant in the field of toxicology and genetics.

Michael co-founded the HUMN project on micronuclei in human populations in 1997, and now in 2018, he's established the Genome Health Foundation to promote education, research, and translation of knowledge on environmental and lifestyle factors that cause or prevent DNA damage. 

Today, we'll be talking with him about his work, especially in the area of micronuclei and the importance of his discoveries in clinical practice. Hi and welcome, Professor Michael Fenech.

Michael: I'm here. Thank you. Thanks for the invitation.

Mark: It's our pleasure. We've talked a lot to Denise Furness about nutrigenomics and areas of genomics and genetics in the past. What I'm keen to get from you as the kind of grandfather in this area, shall I say, of genetics and toxicogenomics is a bit of a take on the difference between toxic injury, DNA damage, chromosomal damage, SNPs… We're being lumped as practitioners with all of these ideas, and people use the term mutation and damage in very, very loose terms. So I'm trying just to talk with you about what kind of damage occurs from what kind of areas? What's the difference between SNPs and mutations? Are they the same? They're used interchangeably sometimes. So I just want to work through a few of those because of your background in nutrigenomics and toxicogenomics. 

I thought what we do is just start with what kind of things damage chromosomes and damage DNA? What kind of environmental, nutritional, and other factors are we talking about that can cause damage to our genetic structure?

Michael: So, let's first of all start by imagining a world where there aren't any toxins in the environment that could harm DNA.

Mark: That's a long time ago.

Michael: Okay. Well, probably it's never been.

Mark: There's probably never been, you're right. Oxygen has always been out there for a billion years, hasn't it?

Michael: That's right, but let's try to imagine back. Now, if that were the case, we still need...the cells in the body still need nutrients to make copies of DNA. Okay? And the building blocks of DNA are the bases, A, T, G, C, and we need nutrients to be able to synthesise those. 

Mark: Right. 

Michael: And some of the key nutrients include folate, vitamin B12, zinc, magnesium. The B vitamins, folate, folate in particular is needed to synthesize, to make the DNA bases and other co-factors such as zinc and magnesium are needed for the DNA polymerases to make copies of the DNA.

Mark: Right. 

Michael: So, if one is deficient in these nutrients, amongst others, and there are others nutrients needed, then the ability to make accurate copies of DNA is diminished. And as a consequence, mutations accumulate within the genome.

Mark: Just mistakes of copying rather than damage to anything?

Michael: Yes, that's the mistakes of copying or incomplete copying that leads to breaks in the DNA quite often. 

Mark: Right. 

Michael: It could also be mutations that are epigenetic. In other words, the inability to replicate the epigenetic marks properly or to erase them and so on. 

So, nutrition alone can cause a lot of mutations when there is deficiency but also when there is excess. So, that's the first thing to consider. That nutrition has to be optimal to minimise DNA damage due to defects in making good copies of DNA.

Mark: Because there are billions of base pairs. So, it is an extraordinary copying issue every time a cell divides, isn't it?

Michael: Well, that's happening all the time in our body. 

Mark: Yeah, yep. 

Michael: So, you start life as one cell, and that makes another copy of itself, and so on and so forth. So, from the very beginning, you can accumulate damage. You're effectively going to start aging from the very first division if there is malnutrition. 

Mark: Right. 

Michael: Now, that is just nutrition. Now, within the body itself, certain chemicals can be generated that are also genotoxic. Okay? Like hydroxyl radicals, for example. Or formaldehyde. These are generated endogenously, within the body and within cells.

Mark: In normal metabolism?

Michael: In normal metabolism. 

Mark: Okay. 

Michael: And when they occur in normal levels the cell can cope with that because it can easily detoxify them. But if there are any metabolic blocks that result in the accumulation of these toxic metabolites, then they too, will induce DNA damage within the body. 

Now, these metabolic blocks can happen, again, because of deficiencies in the co-factors and nutrients. Okay?

Mark: Yep. 

Michael: So if you're deficient in zinc or manganese, then superoxide dismutase may not function well. Or the DNA repair enzyme hOGG1 may not function well, which means it cannot repair oxidation to guanine, for example.

Mark: Right.

Michael: And for these reasons, again, nutrition can, therefore, disable the detoxifying potential of the cells in the body. 

Mark: Okay. 

Michael: So that's the nutritional side, very, very briefly. And on the environmental side, then of course, there are the environmental factors that we worry a lot about, that we are exposed to, either because these contaminants are in our environment. We're talking now about man-made contaminants. They could also be natural contaminants, let's say, from...produced by plants and other organisms. 

Mark: Right. 

Michael: And, of course, there's also the potential of genotoxic effects from ultraviolet radiation and ionizing radiation as well.

Now, again, there are detoxifying and DNA repair mechanisms that are built into the cells in the body. But these can be readily overwhelmed when the toxin, genotoxin level in the environment is above a certain threshold. And that will vary depending on each genotoxicant.

Mark: And I guess each individual as well because of that inability to repair the SOD levels or things like that. Very massively, I would guess, from person to person.

Michael: Absolutely. We know that there is variation between people. I mean, we've done tests where we've challenged, let's say, the blood cells of 10 different individuals and you can see the difference amongst them in terms of the DNA damage that was induced.

Usually, there are not huge difference amongst, you know, the general population. But there would be, let's say, 1 in 100 or 1 in 50 people that would be particularly sensitive to one or more genotoxicants, and they would really be very sensitive to that damage. 

One example that comes to mind as a common mutation in the gene ataxia-telangiectasia. It codes for the protein ATM. And ATM is one of those key proteins in cells that senses DNA damage, and then occludes the DNA repair machinery to repair that. And 1 in 100 of us are heterozygotes for mutation that disables this gene. And the DNA damage sensitivity, particularly to DNA strand breaks, is increased. And, of course, in the homozygotes, it's...

Mark: Massively increased.

Michael: ...particularly bad. Yeah. And that's just one DNA repair gene. And there's more than 120 of them, which affect different kinds of lesions on the DNA.

Mark: So the kind of toxicants that we're talking about there… I mean we have the endogenous ones, the formaldehyde, the superoxides, and the hydroxyl radicals we are producing in the trillions of molecules every second, I'm guessing. So, we have a balance system of repair versus the metabolic damage, the necessary metabolic damage of just oxidative phosphorylation.

When you exceed that, so I'm just thinking of someone, say, athletics. Athletes that are at a very high level of oxygen turnover, can you endogenously intoxicate yourself with metabolic processes or do our metabolic processes and repair mechanisms match up with each other? 

Michael: Okay, so it's a good question. So, a bit of stress is not bad because it can help up-regulate the defense mechanisms. 

Mark: Right. 

Michael: So, there is a capacity to adapt to the stress. And in athletes, athletes usually tend to have a strong capacity to adapt to the increased stress. Right?

Mark: Right. 

Michael: However, there is, again, a strong individual variation in the capacity to adapt, in other words, to up-regulate the defense mechanisms in response to stress. 

So, those who actually become high-performance athletes can do so because of this increased inherent capacity for adaptation to the stress.

Mark: Right. 

Michael: In other words, they have a phenotypic flexibility that is better than in other people. And, for example, in my case, I know there's a limit of physical exercise I can do, but I try to go beyond that. It actually doesn't do me any good.

Mark: I'm guessing that changes with age as well?

Michael: Oh, yeah, absolutely. Of course, it will change with age. That's right.

Mark: So is this the Nrf2 kind of process of moderate stressing, moderated oxidative stressing, inducing a protective response that's being talked about in all of our conferences now?

Michael: The adaptation can occur at many different levels, and the Nrf2 pathway is one of those that relates to the inflammation. 

Mark: Right. 

Michael: But this occurs at many different levels. 

Mark: Oh, okay. 

Michael: It can occur at the DNA repair level, it can occur at the antioxidant response level. It can occur also maybe at the level of the ability to absorb nutrients into the body when there is a slight nutritional deficiency. 

Mark: Right. 

Michael: So, yeah, it's not just one pathway. It would be to every possible pathways because of the network, metabolic network, the intricate metabolic network in the cells.

Mark: So they are interdependent, and nutrition or kind of foundation of good nutrition and availability of micronutrients is a kind of blanket of the necessary protective responses. But then there are overloads that are going to...there's almost going to be obligatory damage. I'm thinking of radiation exposure, those types of things. Genotoxic chemicals that our body is not prepared for, and not really inducible enzymes that we can protect ourselves with. 

So, what are the threats from outside that have the biggest impact on whether chromosomal breakage or DNA repair shortfalls? What kind of things should we be looking for as clinicians, especially, to try and reduce that? 

Michael: Yep. I mean, first of all, one can start from those exogenous genotoxicants that we have control over and those that we don't. Right? So the ones we have control of are the ones that we put into our body through our mouth or putting on our skin, right? For whatever reasons. 

Now, in terms of the diet, we know that the way we prepare food can increase genotoxicants in the food. So every time when you brown food at high temperature, the browning reaction, the reason you see the browning is because you're creating new chemicals. So when we cook at high temperature, you know, and there’s protein and sugars that creates Maillard reaction products, such as glyoxal, which are genotoxic, potentially genotoxic, depending on the form. When you barbecue meat, when the fat goes on the charcoal and that is burnt, the smoke that is generated contains polycyclic aromatic hydrocarbons. When you pan fry meat at high temperature, the browning reaction generates heterocyclic aromatic amines, which are also genotoxins, and so on and so forth. 

So, it's not that you shouldn't absolutely...that you should avoid these things altogether. But of course, it's better to minimise exposure to genotoxicants, especially if you have control over that. 

Mark: Right. 

Michael: Alcohol is another example. So, there is this...well, alcohol is converted to acetaldehyde into the body. And acetaldehyde is a carcinogen...

Mark: I did not know that.

Michael: ...which is normally detoxified to acetate. However, the ability to do the detoxification varies greatly between people. Because there's a common polymorphism in the acetaldehyde hydrogenase enzyme. And this has already been shown many times that those people who drink alcohol and have this polymorphism accumulate more DNA damage in the body, compared to drinkers who don't have this problem of mutation.

Mark: Can you pick that by… acetaldehyde tends to leave the headaches and the problems, doesn't it? The people who are alcohol intolerant, can you pick that clinically or is that...?

Michael: Oh yes, you can. There is a questionnaire called the TAST, T-A-S-T questionnaire. If you don't want to do the genetic test, you can use this.

Mark: Right. 

Michael: And phenotype the patients or the clients in this manner. So it would be symptoms of headache, dizziness, and so on. There's about, I think, seven, six or seven different aspects to it.

Mark: So, for those people, alcohol is particularly genotoxic. I know the association of alcohol with nasopharyngeal carcinomas or oropharyngeal carcinomas. So, that is, I'm guessing, direct effect of alcohol on those areas, and then maybe the acetaldehyde remaining for a prolonged period. Is that the kind of concept about how that would happen? 

Michael: Yeah, that is most likely the mechanism.

Mark: For the mutation?

Michael: Yeah, that the... Well, alcohol gets metabolised quite rapidly to acetaldehyde. And then, if the acetaldehyde is not detoxified, it will then will link with DNA and protein, that might cause cross-links, DNA protein cross-links. That's what formaldehyde does, or acetaldehyde. They would probably act similarly.

Mark: So you've got rid of the barbecues and the beer.

Michael: I wouldn’t want to get rid of them all together for heaven's sake, no. But it's just to be aware that that is a risk and that people vary in their susceptibility. So you wouldn't know. So, in a social occasion, right?

Mark: Right. 

Michael: You wouldn't... Everybody's drinking the same amount, but the harm or benefit done will vary between people. That's all I can say at the moment. And the problem is, you don't feel DNA damage.

Mark: Yes. 

Michael: So you wouldn't know. Because the reason is the DNA damage will not kill you immediately. It's a cumulative thing. So it's not a life and death situation, which is why you don't feel it. But in the long run, it will get you in terms of the risks. We know very clearly that the risks for developmental and degenerative diseases are increased with DNA damage.

Mark: Right. 

Michael: Goes up, and that's unequivocal. That is definitely the case.

Mark: I remember Bruce Ames had that statement, which you need a mutagen and a mitogen. You not only have to damage DNA, you have to induce it to replicate if you're going to get a cancer. But I'm guessing some of these mutations result in replication anyway or a loss of control of replication.

Michael: Well, that's right. I mean, as you age, you get more and more cells accumulated that are initiated, that have DNA damage. Many cells. 

Mark: Yep. 

Michael: We're talking of more than a 1 in 1000 cells with serious chromosomal damage. 

Mark: Yep. 

Michael: In fact, it would be more likely 1 in 100 if we base it on the micronucleus frequency alone. 

Mark: Right. 

Michael: And so that's a lot. A lot of your body, if you think about it, a big chunk of your body being massively mutated.

Mark: That's quite an image, but thanks for that.

Michael: I'm sorry but that's the truth.

Mark: Wow, 1%. I did not appreciate that. I was thinking of the 1 in 100,000.

Michael:: And that's probably an underestimate. 

Mark: Right. 

Michael: So it's remarkable that we can still survive in that way. Ultimately what kills you with cancer is when the cancer grows and metastasises. 

Mark: Right. 

Michael: Now, we now know in the past five years or so, some very interesting new knowledge has accumulated regarding micronuclei. In fact, it's become evident from a number of studies… So, maybe I should say something about micronuclei before I continue...

Mark: I think that would be great. What's a micronuclei? 

Michael: So, micronuclei were discovered more than 100 years ago by Howell and Jolly, who were haematologists. They observed these small nuclei in red blood cells, and they're known as Howell-Jolly bodies, actually. 

Mark: Right. 

Michael: So, probably, and through...during the medical degree, you might have heard about Howell-Jolly body…

Mark: Yes. 

Michael: As part of your hematology course.

Mark: I did.

Michael: And the Howell-Jolly bodies are micronucleus. At the time, it was not known what they were. But eventually, about 40, 50 years ago, it was observed that they increased in mice exposed to radiation and genotoxic chemicals. And it became evident that they must arise from chromosomal aberrations. 

What actually happens is that when a chromosome is broken, the fragment, the broken end of a chromosome, lacks the centromere needed to pull it to the poles of the cell during nuclear division.

Mark: So it floats free?

Michael: So it floats free. And therefore, and as a result, it's not incorporated in the two daughter nuclei during nuclear division, during mitosis. 

And then, as it floats free, it forms its own membrane around it and makes a micronucleus, that's how it happens. Now, in the red blood cells that happens in the bone marrow, in the normoblasts, the precursors of the red blood cells. What happens, in this case, is the nucleus is taken out of the cell as it would normally, but the micronucleus remained.

Mark: Right. 

Michael: Which is why see then the Howell-Jolly body or the micronucleus in the erythrocyte. And in fact this measurement of micronuclei and erythrocytes is one of the standard tests for genotoxic chemicals in the mouse model.

Mark: I think you were deeply involved in producing that test, were you not?

Michael: I was involved mainly in producing the test with lymphocytes. 

Mark: Right. 

Michael: The erythrocytes assay, you cannot really do in humans because the spleen removes the erythrocytes with micronuclei in them. So you don't see them.

Mark: Right. 

Michael: Unless you do some unique approach, which is laborious. But what I did was I developed an assay using human lymphocytes. Which has the advantage that you can study the DNA damage both in vitro as well as in vivo in humans.

Mark: Right. 

Michael: And this test is now a test endorsed by the OECD. So there's a protocol how to do this, and it's used worldwide. And it's also, the test is also used ex vivo in people to study the effects of exposure to environmental genotoxins and dietary deficiencies. 

Mark: Right.

Michael: And it's endorsed by the International Atomic Energy Agency to measure DNA damage following a radiation accident for biodosimetry purposes.

Mark: Is it more broadly applicable? Is this a test that can be used where there is clear nutritional deficiencies? Do you get an answer on micronuclei assessment if the DNA repair or chromosomal damage is high for other reasons?

Michael: Yeah.

Mark: You do?

Michael: So there has been lots of studies. I've contributed quite a lot to that, but so have many others. As has Bruce Ames as well. And so, you can study the effects of nutritional deficiencies in this test.

Mark: Right.

Michael: Both in vitro and in vivo. We showed...we published lots of papers on folate, and zinc, and selenium on this. Both in vitro and in vivo, and the in vitro models and in vivo studies tend to agree with each other.

Mark: Right. 

Michael: So yes, you can do it for nutritional purposes as well as for environmental exposure purposes. And when you model in vitro, we can also see that the susceptibility to the environment of genotoxins is increased if you are also deficient in nutrients needed for replication and repair.

Mark: Okay. Well, you've made sense of something that happened with a colleague of yours, Judy Ford, who worked with us. We had a clinic where people were exposed to high levels of pesticides back in the '90s. 

Michael: Yes. 

Mark: And I think she was using your assay. And the micronuclei, the increased micronuclei was quite dramatic in that group. But they were people who were sick, they were not healthy. 

Michael: Yes. 

Mark: They've been exposed to chemicals and somehow they have become sicker than most other people did. 

Michael: Yes. 

Mark: She found very high levels of the micronuclei on the assessments. She was criticised for it because, you know, the thought was this was radiation-only or you had to have high genotoxic exposure. But in sick people, I'm guessing they're a different population? They're vulnerable, to begin with. And so maybe the rules of what it takes are less.

Michael: Yes. So, in fact, there's a lot of work being done on micronuclei and disease. 

Mark: Right. 

Michael: In fact, we are organising a workshop in May next year in Rennes in France, just on this topic, on micronuclei and disease. Because the evidence is really strong that micronuclei are increased in disease. And the evidence that they predict the risk of diseases also increased.

Mark: Okay. Disease, generally, as in, across organ systems, across age groups or something more specific?

Michael: Yeah. Well, the initial studies were made in relation to cancer and to a cohort of about 7,000 people around the world, and via the HUMN, H-U-M-N project, we showed that the micronucleus predicts cancer risk. 

Mark: Right. 

Michael: So those were the medium to high level of micronuclei of about a 70% increased risk of cancer.

Mark: Okay.

Michael: There have also been other studies showing that the higher magnitude, as you can see, predicts cardiovascular disease mortality. We also did a study during pregnancy and Denise was involved in that study. She was a Ph.D. student during that. During that, those at 18 weeks gestation we took a blood sample from women who were pregnant and showed that the higher level of micronuclei predicted the risk for pre-eclampsia and IUGR.

Mark: Wow. 

Michael: And this is what you would expect because cells that have micronuclei in them have difficulty in dividing. The rate of division slows down because of cell cycle checkpoints. So the ability to develop, you know, the circulation in the placenta and so on, that develop the blood cells and grow the cells for that purpose gets diminished somewhat.

Mark: And it's the placenta that does a lot of the signaling that causes the pre-eclampsia problems. And it remains a mystery in the medical circles. We just say it's mysterious, but you're saying that there can be a component of just straightforward damage that is unrepaired that slows down cell replication?

Michael: Yeah, so damaged cells have difficulty in dividing. And the only way they can divide is to actually reduce the threshold of the checkpoint. But in doing so you then accumulate more damaged cells. 

Mark: Right. 

Michael: Now that in itself creates a problem. So I'll come back to these new developments that have occurred recently with regard to the micronucleus index.

So first, what's been discovered is, and these are all papers published in top journals like "Nature," and so on and so on. Is that when a chromosome gets... And before I do that, I also need to mention that the micronuclei can also originate from mal-segregation of whole chromosomes. So, the chromosomes don't necessarily have to be damaged. It could be that the lagging chromosome happens because there's a defect in the mitotic spindle, or in the centromere or in the kinetichore of chromosome. Now, if that happens the chromosome lags behind during anaphase, and is left swimming in between the two daughter nuclei, and eventually forms a micronucleus itself.

Now, the entrapment of the chromosome in a micronucleus causes...has many consequences. Now, the first one is that the entrapped chromosome cannot replicate its DNA properly. It's out of synchrony with the main nucleus. And also it doesn't have all the proteins needed to import the building blocks of the DNA. As a consequence, the chromosome gets replicated in patches but it's not ligated. In other words, it gets shattered. When the cell goes into the next cycle of division, that shattered chromosome, there are two things can happen. One of them, the cell may catch up and religate the fragments. But when it does that, they're ligated in a random manner. So that in one cell division cycle, you get a hypermutated chromosome. And this has been observed in cancer.

So that's the first thing. So micronuclei, the entrapment of chromosomes in a micronucleus, in itself, is a hypermutation event. Not only because of the imbalance in the genetic material in that cell, but also because that chromosome gets hypermutated.

Mark: Does it still code for proteins? Is there enough in the micronuclei for it to go on? Is it a bit of a garbage that's left behind or is it still metabolically active? 

Michael: It would still be metabolically active.

Mark: Even though it's outside the main nucleus?

Michael: It eventually can get reincorporated in the main nucleus.

Mark: Darn.

Michael: Right? That's the problem. So when the membranes dissolve, when it goes to the next cycle, that can happen. But quite often it tends to also get lost. It depends what happens. Anything you can imagine can happen, actually happens. 

Mark: Right. 

Michael: This has been followed by video imaging of the cells. 

Now, the other possibility that's been observed is that the shattered chromosome does not get repaired, and the DNA from the shattered chromosome leaks into the cytoplasm. Now, the presence of DNA in the cytoplasm is a trigger for inflammation. 

By that I mean, that there is an inflammatory pathway known as the cGAS-STING pathway that recognises DNA in the cytoplasm. This was evolved to detect viral DNA or foreign DNA. 

Mark: Oh, okay. 

Michael: It's also designed to detect mitochondrial leakage of DNA. But even the DNA from micronuclei can trigger this inflammatory pathway, and therefore create a pro-inflammatory process.

Mark: And the pro-inflammatory process is intracellular, at that point, but it's an intracellular process that signals?

Michael: Intracellular, but it also produces chemokines to bring to the immune system to try and take out that damaged cell.

Mark: Which I'm guessing is the job of the natural killer cells because this will otherwise appear in normal HLA description on a cell surface.

Michael: Yeah, that’s right. 

Mark: Okay. 

Michael: Yes. Now, and under normal circumstances, when your immune system is working well, that would be ideal. This is part of the senescence process.

Mark: Yep. 

Michael: To remove damaged cells. But the problem is that as we age, we have so many cells with micronuclei in them, and because the immune system is depressed, it doesn't get resolved. And you create this pro-inflammatory phenotype.

Mark: Right. 

Michael: Which is now thought to be part of what we call inflammaging.

Mark: Right. 

Michael: So we get into a vicious cycle of DNA damage, micronucleus deformation, triggering of the cGAS-STING pathway and also the SASP pathway because this is senescent-associated, secretory pheynotype pathway. Which then triggers inflammation itself. And that process itself could generate oxidative stress and generate more chromosomal damage and micronuclei. 

Mark: Okay. 

Michael: So that's where we're at, you basically, once you have a rate of DNA damage, that it's too high you get into this vicious cycle that you cannot break out of.

Mark: I have to ask, why would there not be an apoptotic signal rather than an inflammatory one? A cell, if that damage is happening at that rate, I would have just thought biologically we would do better to program the cell to extinguish itself rather than...?

Michael: Yeah. That's true. And some of the damaged cells are taken out by apoptosis. Okay?

Mark: Yeah. 

Michael: But some of the cells do not go down that path, because they might have also accumulated a mutation in the p53 gene.

Mark: Right. 

Michael: Involved in apoptosis. So there are many things happening simultaneously. What we know for certain is that the micronucleus index and the chromosome aberration index increases with age. 

Mark: Okay. 

Michael: And also the evidence indicates that those who live longer are tracking at a lower rate of DNA damage than those who are...that then what you would project from the data from younger age groups.

Mark: So, one question which will be obvious to any of our listeners is, is this test, is the micronucleus assay, is it a micronucleus assay or micronuclei assay?

Michael: I prefer to call it micronucleus assay.

Mark: Right. Is it available? I mean, it sounds like it's going to become one of those indicators of who you focus on as the people who, given a certain exposure or nutrition, are at risk of a bad outcome. Is that in the clinical area yet or are we still years out from that? 

Michael: Okay. So, first of all, micronuclei assays are already in the clinical area in terms of the Howell-Jolly body. 

Mark: Right, yes. 

Michael: Test, right? But, the test, we have since developed these other tests. The lymphocyte assay, which is known also as the cytokinesis block micronucleus assay. The reason is that, to the cells that can express micronuclei have to complete one nuclear division. And then this test, which I developed a long time ago, we overcame that problem because, as you know, when you stimulate cells to divide, not all divide, and that varies between people, and it's less with age. So the test was not accurate at that point in time when I got involved as a Ph.D. student.

So what I did was I solved that problem by developing a method to block the cells that are stimulated, that complete nuclear division at the binucleated stage using a cytokinesis block, using a natural chemical called cytochalasin. And then what you do is you score the micronuclei specifically in the binucleated cells.

Mark: Okay, which is a light microscopy scoring, is that right?

Michael: Which is light microscopy-based but it's now fully automated. Because there's so much interest in this, and because it's used by big pharma for screening all their chemicals for their genotoxicity. It's been automated. There are many options for automation by in its cytometry or even image flow cytometry now, which is the latest development in this.

Mark: So is that accessible to healthcare practitioners, doctors, and the like? 

Michael: Yeah, so this is our focus is now to make this test accessible. And also, to make it relevant, to make people aware of its relevance in the clinical setting, which is why we're holding this workshop on micronuclei and disease.

Mark: Wherefore which we'll all be traveling to France just after the ski season is finished.

Michael: Well, yeah. If you prefer to do that, you're welcome. Okay, so it will be in Rennes, which is not far away from Paris and Normandy. 

Mark: Right. 

Michael: So the test is becoming available, and that's our goal. 

Mark: Okay. 

Michael: So one of this is... To support this, I set up a foundation called the Genome Health Foundation, and one of the aspects of that, as well as the HUMN project, which we've been doing for 20 years, is to now focus on this aspect of translating the use of this test into practice.

Mark: I would think most of our listeners could see the value of this straight away. I mean, the potential for the testing that looks at the damage, the unrepaired damage and the triggers for inflammation, is so high on the agenda of practitioners right now.

We've become far more aware of the role of inflammation in aging and damage. And if, you know, 100 years ago, if we had our wish it was we want antibiotics to kill the bugs and to stop infection and inflammation that way, and having won those battles, what we're left with now is long-term inflammatory responses that we are struggling to control and the drugs are not doing a great job. So we're looking for new ways to determine who's set for a bad outcome versus who's set for, you know, really resilient, and is probably going to make it through with less effort. 

So we could see it being a very valuable thing. Just practically, it's not available. You can't write a pathology form right now. So, I'm guessing this is something that you're moving to with this project to make it accessible to healthcare practitioners?

Michael: Yeah, that's right. So the first thing we did was developed, as part of the HUMN project, we first of all developed a harmonised protocol for this test. And we've also published OECD guidelines for it, at least for its use in vitro.

Mark: Right. 

Michael: And also an ISO standard for its use in the situation of exposure to ionising radiation. The protocol is already published in the International Atomic Energy Agency manual.

And, of course, we also publish reviews on this topic. So, my expectation is that from the workshop we have in May, we will then follow up with a special issue in one of the key journals on the relevance of micronuclei in disease.

Mark: And specifically the CBMN assay to identify that? So, is it focused on a practical...or your practical test is, you can quantify it?

Michael: Well, the micronucleus assay can actually be performed not just in lymphocytes, but also in epithelial cells as well.

Mark: Oh, right. 

Michael: And it will be fair. For example, in cervical cancer, it's been shown that counting micronuclei actually is of prognostic value in the disease, in cervical cancer. 

Mark: Okay.

Michael: But that information currently is not captured with the way that the cervical smears are done. And buccal cells, it is also indicative of higher risk of oropharyngeal cancer, for example.

Mark: Okay. 

Michael: So the lymphocyte assay, for example, we know it's increased in cancers. For example, at the Anderson Cancer Institute, Randa El Zein showed that the micronucleus cytome, I just...I've got a little bit to the side here. When you look at the binucleated cells in the lymphocyte assay, you can measure not only micronuclei, but also you have the advantage, because you've got the two nuclei, you can also see these bridges between the two nuclei. These bridges occur because of misrepair in DNA breaks, which generate dicentric chromosomes. Or it could be dicentric chromosomes due to telomere infusions. So that tells you even more information about the chromosomal instability. 

Mark: Right. 

Michael: And you can also measure these buds, which is a way that the cell tries to eliminate amplified DNA. There's a lot more to that story. 

But to cut the long story short, what they showed was that... That, this was a study in smokers, what they showed was that the assay could identify very accurately those smokers who are likely to have lung cancer by their frequency of micronuclei, nuclear plasmic bridges and bugs. And the fact, they have a patent on it. And they replicated their study at Harvard University. So it was at Anderson and at Harvard.

Mark: Now, are these cells you need to get from the lung or are these lymphocytes assays?

Michael: No, these are the lymphocytes.

Mark: Right. Okay. So you can predict end organ damage from the lymphocytes, okay.

Michael: Yeah, so the patients who got the cancer or are very likely to have the cancer and therefore could be triaged in this manner, have got the higher frequencies of micronuclei, and they have a patent on it.

Mark: That can be bad news, can't it? Because they can make the cost of the tests extreme.

Michael: It depends how it happens. But generally speaking, that's why you do this. So when you look at the literature, that those who develop cancer have got a higher micronucleus frequencies, generally speaking. 

But it's also relevant, for example, in chronic kidney disease, micronuclei increase. So, those who are getting homocysteine goes up, as you know, and those kidney disease homocysteine increases, and how homocysteine is associated with higher micronuclei. 

Mark: Right. 

Michael: We observed that long quiet a few years ago.

Mark: How they link...the high micronuclei induces or blocks the homocysteine from conversation to...? What's the link between those?

Michael: Well, there are two possibilities. One of them the kidney, with kidney disease, you lose the ability to reabsorb vitamin B12 from the kidney. 

Mark: Oh, of course. 

Michael: And therefore the patients could become B12 deficient. And B12 deficiency causes chromosome breaks because it disables the ability of cells to hang on to folate because you cannot convert 5-methyltetrahydrofolate to tetrahydrofolate, which is the form that is polyglutamated and stored. And therefore, the folate just goes in and out of the cells.

Mark: Right. 

Michael: And therefore they simply can't utilize folate, so that's one possibility. The other possibility is that on the homocysteine itself could be toxic. Whether they are genotoxic or not is not so clear.

Mark: We tend to think of it as not good for you anyway, irrespective of that with the high homocysteines and their link to inflammatory, cardiovascular, and other diseases. 

Michael: Yes. 

Mark: It does seem to be a difficult job for some people to bring homocysteine under control. And maybe we should be thinking more of chronic renal disease in those patients?

Michael: And there could be a number of reasons for that, as well. It could be that the oxidative stress in the body is not allowing cobalamin to be at oxidation stage one to function. So there are a number... So micronuclei associated with disease for different reasons depending on the disease, and you can also do this assay in different tissues that are...

Mark: If you can get samples of the tissue, would you say that it is's going to be more accurate or can we rely on the lymphocytes as a kind of surrogate of disease susceptibility?

Michael: Yeah. Actually the micronuclei predict cancer risk across all sites. 

Mark: Right. 

Michael: And some sites more than others. But they... So yeah, it's not...what you are seeing, and this has also been shown with DNA methylation. That DNA methylation in the blood cells reflects DNA methylation status in other organs in the body. So it's telling you about...I mean, lymphocytes travel throughout the body, so they're experiencing the metabolic environment.

Mark: Everywhere.

Michael: There's something everywhere. And because the majority of lymphocytes are quiescent, right, they're not diving in the blood, which is how they should be. And they are long-lived. They accumulate the lesions as they swim around in the body. So they kind of integrate the damage. And then when we collect them, what we do is we get them to express that damage by making them divide ex vivo, in vitro in a test tube and then you can see the damage.

Mark: And then you simply count the micronuclei? Is that ultimately what you're doing?

Michael: That's right.

Mark: And the automated counting?

Michael: Yeah. You then wait for about two days until the cells complete nuclear division in the test tube and then you count the micronuclei in them.

Mark: I started this discussion today with plans to ask you a dozen more questions. That has been a very, very exciting discovery for me, I've got to say. The micronuclei that Judy Ford brought to our clinic years ago seemed to be quite significantly related to the severity of illness. That the more micronuclei that she was reporting, the sicker their patients tended to be. But we did what clinicians do, didn't take it any further at the time. Let it drop and now, 30 years, 25 years later, it's back again in a new form, which is the science seems settled, the arguments about the significance of it seemed to be settling and it's moving towards clinical application.

Michael: Yeah, that's right. Those were early days. I mean, when Judy Ford was doing that I had only just, it would have been only what? Three years after I developed the CBMN assay in lymphocytes. 

Mark: Yeah. 

Michael: So she pioneered its practical application.

Mark: Well, she also helped many, many people who are chronically sick understand more about their illness. I think she went much more into pregnancy and successful pregnancy that comes after that.

Michael: Oh yes, that's right. That’s right. 

Mark: But it is fascinating that the damage that we see that accumulates, that you can actually stare down the microscope, see it. It's not mythical, it's not a number on a machine, it's real damage to real chromosomes, and the process of inflammation that occurs afterwards, which is clinically really relevant.

Michael: And that micronucleus doesn't disappear. It has been shown in embryos that the phenotype of having a micronucleus gets passed on.

Mark: Wow. Across generations or just is that damage to the mother passed on to the...?

Michael: Within the lineage of that embryo.

Mark: Right. 

Michael: Throughout its life. So these events can start very early and they have a health influence. Regarding pregnancy and so on, it's also known that infertile couples have got higher micronuclei than fertile ones. And it's also increased in miscarriage and so on and so. 

Mark: Right. 

Michael: So there's other differences as well.

Mark: Okay. I mean, a miscarriage, you also have that issue of those with raised homocysteine and methylation disorders have a very high miscarriage rates. 

Michael: Oh yeah. 

Mark: And so I would guess that susceptibility, well, the concept of genetic susceptibility and ease of damage would make sense in evolutionary terms across generations. 

Michael: That’s right. 

Mark: That's how genes get selected for and against, isn't it?

Michael: Yes. Well, hypo-methylation itself increases micronuclei. And the reason for that is that the centromeres, the DNA around the centromeres needs to be hyper-methylated to be structurally intact. 

Mark: Right. 

Michael: If the methylation level is low in the pericentromeric region, that part of the chromosome de-spiralises, and cannot assemble the kinetochore. Probably it'd be segregated, it may also break. So that's why methylation is also related to micronuclei and methylation. I mean, if you put a demethylating agent in a test tube with lymphocytes, you will cause massive amounts of micronuclei.

Mark: Right. Professor Michael Fenech, I had a whole agenda to talk with you about. But, like those rabbit holes, you go down them, and that is in the most fascinating discussion about something that I thought I knew about, had discarded, and now I see it back on the agenda.

I can only hope that in May [2019] that this moves very quickly towards clinical applications so that we can identify, as clinicians, origins of inflammation, and also be able to triage those patients that we see that are on a course to a bad outcome, and separate them from the many that may not be. So, I want to thank you very much for today. And hopefully, I'll be able to talk with you again about the other dozen items that I had in mind.

Michael: Sure, but we probably need a break now.

Mark: We do need a break now, Michael, and thank you very, very much for today.

Michael: Okay, thanks. Thanks for the interesting discussion.

Mark: Thank you, Michael. I'm Dr. Mark Donohoe, and thank you for joining us today on FX Omics.


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Prof Michael Fenech

Professor Michael Fenech is recognised internationally for his research in nutritional genomics and genetic toxicology. The lymphocyte cytokinesis-block micronucleus (CBMN) assay he developed is endorsed by the International Atomic Energy Agency for in vivo radiation biodosimetry and by the OECD for in vitro testing of chemical genotoxicity. He further developed the lymphocyte CBMN assay into a 'cytome' assay of six biomarkers of DNA damage and cytotoxicity and, in collaboration, established a similar method for buccal epithelial cells; both published in Nature Protocols. In 2003 Dr Fenech proposed the Genome Health Clinic concept, a novel ageing/disease prevention strategy based on personalised diagnosis and prevention of DNA damage by nutrition/lifestyle intervention. His research also explores DNA damage effects of psychological stress and personalised nutrition for dementia prevention and cancer growth control. He co-founded the HUMN project on micronuclei in human populations in 1997 and the Asia-Pacific Nutrigenomics Nutrigenetics Organisation in 2014. In 2018 he established the Genome Health Foundation to promote education, research and translation of knowledge on environmental and life-style factors that cause or prevent DNA damage. Since 2010 he has been invited speaker at 55 international conferences (9 plenary lectures). His Google Scholar H-index is 84 based on 31,000 total career citations.