The Need & Difficulties of Controlling Micronutrients in The Diet of Test Subjects When Dealing With Endocrine & Epigenetic Disrupting Compounds

Designing the perfect experiment to answer the questions that you set out to study can be extremely complicated. There could be something in your protocol that you didn’t even think would be a problem that might turn out to be your worst nightmare.  For us, controlling for nutrition and diet might be a necessary component to our study that might have been overlooked had it not been for the growing body of evidence in the combined fields of nutrition and epigenetics.

What are epigenetics?

Think about this for a minute: 99.5% of the human genome is similar among every human being on the planet[i]. However, when you look around, it looks like there is a lot more variation than just ½ a percent.  So, what’s really going on here?  In addition to that tiny little ½ a percent, what makes us look/act different from each other (our individual phenotypes) can be explained in part by epigenetics.

In simple terms, epigenetics basically relates to how your genes are read by the building blocks of your body.

In the beginning of a human, epigenetics is how your body knows that a heart cell is a heart cell and not a brain cell, or something that belongs in your spleen or lungs. After all, every cell in your body has exactly the same DNA sequence. Epigenetics controls which parts of a cell’s DNA gets expressed or suppressed in order for that cell to be what your body needs to to be.

Epigenetics acts at its most fundamental level during the development of the embryo and fetus. But it continues to work regardless of age by turning some genes on or off or causing them to be overactive or lazy.

Epigenetics can affect the “expression” of genes for a lifetime after a person is born. Not to turn brain cells into toenails, but to direct how a gene works.

Lots of substances can affect epigenetic changes – some for the good – like folates — and some for the worse: Bisphenol A (BPA).

And as we will see further along, a substance can have different effects — or none at all – depending on its concentration.

Enter: DNA Methylation

These differences in genetic expression are controlled epigenetically by a couple of mechanisms, one of which is called DNA methylation. In mammals, DNA methylation depends on a variety of factors, including requiring specific enzymes to jump start and continue the reactions, as well as the building blocks necessary to complete the tasks: methyl groups from nutritional sources. Specifically, these methyl group donors from nutritional sources are responsible for methionine and folate metabolism, both of which are required for proper DNA methylation and subsequent gene expression.

Not only are both required for proper DNA methylation, but the methionine cycle itself is dependent upon the folate cycle, so without one, you don’t have the other, and epigenetic problems can start to arise. In addition to folate, the methionine cycle (define) also depends upon a variety of vitamins within the diet, so finding the right balance to attain proper functioning DNA methylation and gene expression can get very complicated very fast.

Epigenetics can also affect gene expression by controlling whether a gene region in a chromosome is available for expression or closed for business. This involves little DNA spools called histones.

Why is this important to the Stealth Syndromes Human Study supported by CRECH?

The mention of Folates – Folic Acid on your vitamin supplement label – is a major hint. It contributes “Methyl Groups” to your body. <diagram of methyl group>

Adding methyl groups to your diet is how folates contribute to good nutrition … but like all good nutrition, too little or too much can be harmful.

As you will see a bit farther along, folates can affect the effects of BPA in your body. And that affects how the study must determine and control precisely how much folate (and other methyl contributors) are in the diets of our test subjects.

Case Study: Maternal Nutrition During Pregnancy; too little or too much of a good thing?

It is very well documented that maternal nutrition during pregnancy has a major impact on the health and well-being of the developing fetus. For example, during the Dutch Hunger Winter between 1944 and 1945, when famine struck the Netherlands and food and other resources were being rationed, there were reported increases in a variety of chronic diseases in children conceived during this time, including cardiovascular disease, hypertension, and obesity[ii].

As another example, it is well documented how folic acid intake by the pregnant mother is crucial in reducing the chances of the developing embryo having neural tube defects which can ultimately cause spina bifida, anencephaly, and other spine and brain defects.

As mentioned earlier, certain gene expression mechanisms like DNA methylation also rely heavily on the presence of certain compounds that come from nutritional sources, so maternal nutrition is critical for the health and epigenetic future of the developing embryo/fetus. Therefore, if the mother isn’t getting the right nutritional balance of vitamins and other compounds, it could have lifelong effects on her child, much of which is still yet to be fully understood.

All About The Dose

So, as a pregnant woman, you need to make sure you’re getting the proper diet of vitamins and other compounds necessary for healthy embryonic development of your child.  That doesn’t mean you can just pound them back like nobody’s business.  Studies have found that folic acid and other vitamins are necessary for embryonic health for a variety of reasons, but it is important to note that there is such a thing as “too much of a good thing” here.

When it comes to folic acid, new research is suggesting that while the compound is absolutely critical for reducing neural tube defects in the developing embryo, too much of it can cause significant damage in other areas of development.  Specifically, in an on-going and not yet published study from a team at Johns Hopkins University, they reported that taking too much folic acid in pregnancy may increase the risk of autism in the developing child[iii].

Another literature review paper published recently in the journal Genes & Nutrition postulated further that reduced nutritional folate consumption during pregnancy can potentially lead to childhood leukemia as well as potentially many other cancers. This is likely due to increased DNA damage and other chromosomal abnormalities that are known to be linked with cancer, which result from not enough folate in the diet[iv].

It becomes clear from this on-going research that the dose is incredibly important.  A small dose of folic acid is crucial for preventing neural tube defects in embryos, while a larger dose may increase the risk of autism in the developing child. What we are seeing here is evidence of non-monotonic behavior at low doses, a concept which previously has been criticized by outdated toxicological thinking. In other words, we see a therapeutic response not only at a high dose (increased autism risk due to too much folate), but also a response at a low dose (increase neural tube defects due to too little folate). While it is not yet understood exactly what the correct dose should be, it is clear that determining the right dose is paramount to keeping ourselves and future generations as healthy as possible.

How Nutrition Could Impact BPA Exposure Risks in Everybody

In 2007, Dolinoy, Huang, and Jirtle from Duke University[v] performed a study on BPA exposure during pregnancy and how that exposure is in a sense “protected” by maternal folate intake. While the study was performed in rats, the results were promising and could have profound implications for both human maternal nutrition as well as implications for everyone, not just those who are pregnant.

To sum up the study, the Duke researchers found that BPA exposure during pregnancy resulted in DNA hypomethylation, meaning that BPA exposure results in increased gene expression and potentially increased risk for a variety of diseases. However, when the pregnant rats were given folic acid or genistein in their diets, DNA hypomethylation did not occur after BPA exposure.

These results indicate that dietary supplementation of folic acid or genistein can potentially counteract the negative effects of BPA on DNA methylation, thus reducing the risk of diseases linked to BPA exposure.

Of course, folic acid isn’t just necessary for expectant mothers.  All people need folates, as it is a critical component for proper function of the body and for genetic health and stability[vi]. Therefore, while the Duke study results focused primarily on pregnant rats, one can posit that dietary supplementation with folates could potentially counteract DNA hypomethylation caused by BPA exposure in all beings.  Of course, this is just speculation that should be tested further.

Why Controlling Test Diet In Our Study Is Important

With the growing evidence that dietary folates, and other phytoestrogenic compounds like gentein, may counteract or otherwise “hide” the effects that BPA has on DNA methylation and other epigenetic mechanisms, it becomes very important that we be aware of this and control our test diet in such a way that we are able to see the real effects of BPA.

In other words, we need to make sure that we’re not eating something that is going to “mask” or “hide” BPA’s effects, otherwise, our results could be contaminated and won’t really be answering the questions we set out to ask.

Concluding Thoughts

Answering the big question of how we can rid our bodies of BPA and reduce its effects is an enormous one, and one that is not without potential complications. Understanding not only how BPA works but also how other things as simple as what you eat daily can affect the health of your DNA will be of utmost importance to us as we begin our journey through this new study.


[i] Levy, S., Sutton, G., Ng, P.C., Feuk, L., Halpern, A.L., Walenz, B.P., Axelrod, N., Huang, J., Kirkness, E.F., Denisov, G., Lin, Y., MacDonald, J.R., et al. 2007. The diploid genome sequence of an individual human. PLOS Biology 5(10): e254.

[ii] Chmurzynska, A. 2010. Fetal programming: link between early nutrition, DNA methylation, and complex diseases. Nutrition Reviews 68(2): 87-98.

[iii] Raghavan, R., Fallin, M.D., and Wang, X. 2016. Maternal plasma folate, vitamin B12 levels and multivitamin supplementation during pregnancy and risk of Autism Spectrum Disorder in the Boston Birth Cohort. The FASEB Journal 30(1): Supplement 151.6.

[iv] Cantarella, C.D., Ragusa, D., Giammanco, M., and Tosi, S. 2017. Folate deficiency as predisposing factor for childhood leukaemia: a review of the literature. Genes & Nutrition 12:14.

[v] Dolinoy, D.C., Huang, D., and Jirtle, R.L. 2007. Maternal nutrient supplementation counteract bisphenol A-induced DNA hypomethylation in early development. Proceedings of the National Academy of Sciences (PNAS) 104(32): 13056-13061.

[vi] Cantarella, C.D., Ragusa, D., Giammanco, M., and Tosi, S. 2017. Folate deficiency as predisposing factor for childhood leukaemia: a review of the literature. Genes & Nutrition 12:14.