Persistent Epigenetic Consequences of Critical-Window Exposures

"An old criticism of our work is that we used pharmacological doses for our experiments.
It is true, but it was necessary to understand that the epigenetic transgenerational process is
possible. Now that we know that exposure to compounds can induce epigenetic transgenerational
effects in the germ line the next obvious question is exactly that: what are the epigenetic
transgenerational effects of these compounds that we purposely take in pharmacological doses?"

Carlos Guerrero-Bosagna, PhD, a post-doctoral fellow at Linkoping University in Sweden, studies epigenetic consequences of early life exposures. He investigates how environmental exposures of a variety of sources, including toxicants, nutrition, and stress, can interfere with developmental and epigenetic mechanisms. He is also interested in the molecular processes that genetic responsivity to environmental cues may be relevant from an evolutionary perspective. 

While at Washington State University, Dr. Guerrero-Bosagna studied multigenerational epigenetic effects of various endocrine-disrupting toxicants in mice, and his name appears on several of the most-cited studies in this rapidly emerging field.

Interviewed by Jill Escher, May 2014

---

You have worked on some groundbreaking research related to multigenerational effects of pregnancy exposures.  How did you arrive at this area of reproductive biology?


I came from the world of evolutionary biology. I have always been interested in evolutionary theory but I was never convinced by the neo-Darwinian argument that environmental factors are not a big player in the generation of genetic changes.  On the other hand, I never understood the fierce dismissal and often mocking of the Lamarckian ideas in schools and universities; particularly, because Darwin himself never denied Lamarck’s ideas. In epigenetics I found the mechanisms that allow you to understand the action of environmental exposures on the genome.

Then, if you open your mind to the idea that environmental factors can indeed influence the genome and also inheritance, then reproductive biology becomes the discipline of excellence to study if and how these effects can propagate to future generations. I would go further; several disciplines have acted as main contributors of ideas to evolutionary biology at different times in science history, starting with taxonomy, then statistics, genetics and developmental biology. I think reproductive biology will now become the main discipline contributing ideas to evolutionary biology, because it will consider the integration between environmental exposures and inheritance. Epigenetic mechanisms will be the main tool used to explain this integration. 


Tell me about the molecular vulnerabilities of the germline. During what stages are the male and female germ cells most vulnerable to exposures?


Major vulnerabilities occur in DNA methylation during the development of the germ line. DNA methylation is the addition of methyl groups (i.e. CH3 molecules) to some cytosines in the genome. Both male and female germ line undergo major re-settings of DNA methylation at overlapping times during the development, starting at the time of migration of primordial germ cells, until the time of the establishment of these cells where the gonads will be. This resetting involves an initial massive demethylation event followed by a reestablishment of DNA methylation. The re-establishment of DNA methylation occurs a bit earlier in the male germ line than in the female germ line.

The whole DNA methylation re-setting process takes only a few days in vertebrates. During this period the germ cells become very susceptible to environmental influences, which can in turn interfere with this resetting in DNA methylation. Moreover interferences with this period are correlated with changes in DNA methylation that can be transmitted through several generations. Recently, however, studies have emerged showing that influences on not only on the developing germ line but on adult spermatogenesis could also produce both epigenetic and transgenerational effects. It remains to be seen if effects triggered during these two different developmental times are similar.


What types of exposures seem to have deleterious molecular effects on the germline?


From an epigenetic perspective several endocrine disruptors have shown to produce deleterious effects in the germ line. These include the fungicide vinclozolin, plastic components such as bisphenol A and phthalates, pesticides such as DEET and DDT, the compound of cosmetics parabens, and a few others.    


How is it that exposures to a fetus’s germline can have lasting consequences, all the way through maturation, decades later, of that individual’s gametes?

This occurs when DNA methylation patterns are affected. DNA methylation is an epigenetic modification of the DNA that depends on the availability of methyl groups in the diet and the action of enzymes, called DNA methyltransferases. There are different types of these enzymes and the act differently during the development. As a generalization, enzymes that define methylation patterns act very early in the development, while the enzymes that maintain the methylation patterns are active throughout life.

When the initial establishment of DNA methylation is disturbed, then altered patterns of DNA methylation are maintained in every cell division in the body, which at some point in life will generate an altered phenotype. However, if the altered DNA methylation occurs in the germ line, then this alteration can be propagated to the following generations, when reproduction takes place.

Germline vulnerability has been a relatively neglected part of toxicology. For example, the FDA has never assessed risk of pregnancy drugs on fetal germline. In your opinion, what actions should be taken to assess risks of chemicals and pharmaceuticals to germline?


Somehow I understand why the FDA has not done it yet. We are in the midst of a paradigm shift that starts from the scientific community but that will eventually reach the FDA and influence their decisions. This is a whole new dimension not only for the FDA but for all the field of toxicology. Toxicological analyses are already very complicated by several factors, including how to determine hazardous levels?  How different compounds interact and produce sometimes augmented effects? Are in vitro results applicable to in vivo? How efficiently are compounds metabolized?

So, in addition to all these challenges, now developmental, germ line and transgenerational effects have to be considered. It will be very challenging to develop screening tools for these effects and it might take years, but it is absolutely needed. Given the difficulties posed by this new dimension of analysis, in my opinion the FDA should start developing these tools as soon as possible. Epigenetic changes in the germ line would be a good way to start since it would indicate both past exposures and future propensity to diseases.


Tell us about your research into DDT.  Why do you think the gestational exposure may have had an obesogenic effect on later generations?

Yes, I am absolutely sure that past developmental exposures to DDT and other compounds have contributed to the current high incidence of obesity in the US and other developed countries. One of the main reasons this is my belief is because we have observed this transgenerational tendency to obesity not only with DDT but with other compounds as well, including plasticizers. So, my thinking is that the DDT is only one component responsible for this transgenerational obesogenic effect.

What is interesting, and scary, is that people have been exposed to several of these compounds at the same time, and they generate somehow repeatable phenotypes, the strongest being obesity and ovary abnormalities such as polycystic ovary.  Just as obesity, polycystic ovary incidence has experienced a tremendous increase in humans in the past decades.



How can endocrine disruptors affect the molecular architecture of the germline?

This topic is definitely a black box and is still work in progress. We have a paper that has been just accepted that shed some lights on patterns of genomic regions that could be specifically affected by environmental exposures. However, in vitro work should be performed in the future to try to understand the exact mechanism through which endocrine disruptors finally alters epigenetic patterns in a transgenerational manner in the germ line. It could be either a direct action or a cascade of events finally triggering other factors that might be involved in targeting specific genomic regions leading to alteration in DNA methylation patterns.



Do you do work in the area of genes that are monoallelically expressed? Are these genes, or perhaps other classes of genes, particularly susceptible to environmental exposures? If yes, which ones, and why?


I have not focused particularly on monoallelic expressed genes. In regards to classes of genes being more susceptible to environmental exposures, the answer is somehow overlapping with the previous answer. I think that there are regions in the genome that are more susceptible to become epigenetically altered in transgenerational manner. This needs more research, but I think that certain patterns of genomic sequences could ‘draw more attention’ to enzymes or transcription factors that are involved in the process of DNA methylation. Moreover, I think specific families of environmental compounds would act on also specific genomic regions, leading to differential establishment of DNA methylation patterns.


Many common pharmaceuticals are endocrine disruptors, but are taken every day by countless millions of patients. Do you think some drugs could have long-term epigenetic effects that have not yet been addressed?

This is a topic that scares me a lot. An old criticism of our work is that we used pharmacological doses for our experiments. It is true, but it was necessary to understand that the epigenetic transgenerational process is possible. Now that we know that exposure to compounds can induce epigenetic transgenerational effects in the germ line the next obvious question is exactly that: what are the epigenetic transgenerational effects of these compounds that we purposely take in pharmacological doses? And it gets even scarier when a quick search on pub med shows that antidepressants or compounds taken as antidepressants such as Saint John’s Wort have effects on spermatogenesis. Are these effects only detrimental on fertility or would they also produce and epigenetic transgenerational effect? I would think both.

What is your lab studying now?

Now I have moved to a laboratory that aims at studying epigenetic modifications as tools for welfare assessment in farm animals. I intend to determine how common environmental conditions to which farm animals are subjected, could influence epigenetic patterns in them. These conditions range from exposure to stressful confinement to the use of pharmacological compounds. Farm animals are an excellent model to test the use of epigenetic modifications as biomarkers. Our lab also has a great expertise in behavioral research; therefore, this opens the possibility of using farm animals as models with application in humans, to study the consequences of detrimental environmental conditions on the development of behavioral patterns, and the role of epigenetics in this.

Is there anything else you would like to add?

It has been a very exciting journey to be a researcher in environmental epigenetics. I feel that in this field I can conciliate the inherent fun of doing science with the generation of knowledge that can be directly applicable to improve the life conditions of humans and non-human animals.