Mohan Manikkam, Carlos Guerrero-Bosagna, Rebecca Tracey, Md. M. Haque, and Michael K. Skinner, Transgenerational Actions of Environmental Compounds on Reproductive Disease and Identification of Epigenetic Biomarkers of Ancestral Exposures, PLoS ONE 7(2), doi:10.1371/journal.pone.0031901 (28 February 2012)
Explaining what this team discovered — but first, an explanation of basic epigenetic terminology
Epigenetic factors biochemically influence DNA strands, without changing the DNA building blocks of our chromosomes. As we understand the process so far, epigenetic factors affect which DNA sequences/codons are turned “on” or “off” and when.
Methylation is one kind of epigenetic chemical programming. A methyl group is comprised of a carbon atom bonded to 3 hydrogens. In epigenetic methylation, these groups are attached to specific places on a DNA strand.
Epigenetics explains a lot of mysterious inheritance effects that we previously could not account for. That’s why accounting for the characteristics of the epigenome is important.
The basic idea underlying epigenetic inheritance from one generation to another
Toxins (and nutritional agents) can affect both somatic (body) and germ (eggs and sperm) cells. Insofar as we know, epigenetic changes to somatic cells stay with the original animal. In contrast, epigenetic changes to reproductive sperm and egg cells can be transmitted to the animal’s descendants.
For example, according to the research team’s paper, previous research had exposed pregnant rats to a fungicide called “vinclozolin.”
The resulting epigenetic methylation of sperm (caused by the fungicide) created abnormal sperm epigenomes. These abnormalities eventually resulted in a significant number of illnesses in descendant rats, including “mammary tumors, prostate disease, kidney disease, testis abnormalities, and immune abnormalities at high (20–50%) frequencies.”
The process is called “epigenetic trans-generational inheritance.”
A note on generational jargon
Genetics usually refers to the different generations in a lineage by using the terms P (parent) or F0 (filial generation zero) for the originating animal.
Thus children are F1, grandchildren F2, great grandchildren F3, and great-great grandchildren F4.
Proving epigenetic transmission in females requires going at least to the F3 generation
This is due to the fact the F1 child of the toxin-exposed parent is directly exposed to the toxin in utero and (unlike males, who develop sperm afterward) her developing ovarian eggs are, too.
That means that non-epigenetic effects in a female F1’s eggs might account for whatever changes show up in her F2 children.
In contrast, altered DNA in an F1 male’s sperm — which was produced only long after his parents’ exposure to the toxin — cannot be responsible for changes in his F2 children.
Given these differences in the timing of the development of the two sexes’ respective germ cells, experimenters have to carry trans-generational epigenetic experiments out to the F3 generation. Doing this ensures that they are skirting the egg-exposure issue, which complicates F1 and F2 results.
What the new study did
The team wanted (a) to test the epigenetic effects of four sets of toxins and (b) see whether they could find biomarkers that would allow “us” to discover whether an animal had been exposed to one of these poisons.
They made up four batches of toxic substances:
They called the first combination, “plastics.” It was comprised of bisphenol A, bis 2-ethylhexyl phthalate, and dibutyl phthalate.
The fourth was the jet fuel known as JP-8.
Note — what LD-50 means
An LD-50 dose kills 50 percent of the animals tested, during a specified time frame. Giving only 1 percent of that exposure hypothetically should result in much more subtle effects than death.
The timing of these injections was important. The effects of toxin exposure during embryo formation varies, depending on which cells, tissues, and organs are developing at the time.
Here, the pregnant rats were injected at embryo formation days 8 through 14. This timing correlates with the period of gonadal development in rats.
To see whether negative effects were transmitted generationally downstream, the scientists mated the F1 generation (meaning the children of the toxin-injected parents). They avoided mating siblings and cousins, so as to avoid inbreeding.
Then they mated the F2 generation (meaning “kids” of the F1 generation). These were the test rats’ “grandchildren.” The offspring of these F2 matings constituted the F3 generation, where the experiment ended.
The experimenters saw no signs of obvious toxicity in any of the filial (meaning F1, F2, and F3) generations.
But there was a hodgepodge of more subtle effects.
For example, plastics exposure delayed puberty in F1 females — but plastics, dioxin, and jet fuel advanced it in F2 and F3 females.
Plastics and dioxin advanced puberty in F2 males, but they and jet fuel had no discernible effect on F3 males.
And all the toxin groups reduced F3 females’ ovarian primary follicle pool size.
Note — on “primary follicle pool size”
This just means that there were fewer immature follicles left to develop into mature eggs in the female rats’ ovaries
The researchers wondered whether this (assumedly) negative change would result in premature ovarian failure.
Hormonally, in F3 generation male rats — plastics, dioxin, and jet fuel (but not insecticide) reduced testosterone levels.
There was, however, no parallel downward effect on females’ progesterone levels.
Consequently, the team concluded that male rats were subject to a trans-generational endocrine (hormonal) effects, but females were not.
“So, what mechanism caused these changes?”
According to the study, the toxins epigenetically reprogram sperm during sex determination.
That change “then promotes all tissues developed from that sperm to have altered cell and tissue transcriptomes that can promote transgenerational disease.”
Caveats — without meaning to take anything away from these findings
I was disturbed that the paper makes no overt mention about the number of rats they started with. That’s a bit of statistically crucial information. My suspicion is that the sample size was so excruciatingly small as to be statistically questionable. The more factors one is testing, the larger the sample size needs to be to generate reliable information.
Second, the (very possibly contradictory) hodgepodge of results means to me that randomness played a noticeable part in the team’s findings. Just because something changes does not mean that a causative agent was involved. This is a reasoning flaw that people, including scientists, are highly prone to make.
The moral? — Slightly more evidence for the suspicion that commonly encountered chemicals cause problems down the generational time-line
I am looking forward to more from members of this team. Theirs is respect-worthy work.