By Alma Laney and Alison Bernstein
This post is the first in a series about transgenerational inheritance, epigenetics, and glyphosate that address questions raised by the publication of the paper, Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology.
What is transgenerational inheritance?
Transgenerational inheritance is the concept that traits can be passed on from parent to great-grandchildren. In the context of toxicology, this hypothesis can be described as “ancestral environmental exposures to non-mutagenic agents can exert effects in unexposed descendants.” If you imagine a person being exposed to some substance, their reproductive cells are exposed so their children are also exposed (intergenerational inheritance). If that person is a pregnant female, the reproductive cells of their offspring are exposed so the grandchildren are also exposed (multigenerational inheritance). Thus, true transgenerational inheritance can only be observed in the great-grandchildren’s generation (transgenerational inheritance).
This graphic was originally published in a post on transgenerational exposure in the context of trauma and the Holocaust here.
What is transgenerational epigenetic inheritance?
Transgenerational inheritance can occur through epigenetic, ecological, or cultural mechanisms (See Figure 1 of the linked paper below).
Epigenetic inheritance
The focus of the paper under discussion is the epigenetic mechanisms through the germline, or transgenerational epigenetic inheritance. In any experiment of transgenerational inheritance, it is critical to use a careful study design to separate the epigenetic piece from the other mechanisms.
Epigenetics can be defined as: “the processes and marks on or around the DNA processes that control the activity of the genome and can be mitotically and/or meiotically inherited.” It encompasses a set of mechanisms that regulate gene expression and that can be inherited from cell to cell within an organism. Sometimes, if they occur in germline cells, these mechanisms may also be inherited from parent to offspring. Epigenetic mechanisms can also be sensitive to environmental inputs. Because they can be modified by the environment and may be inherited from parent to offspring, epigenetic mechanisms are a prime candidate for mediating transgenerational inheritance.
Epigenetics generally refers to four mechanisms.
- Cytosine modifications: These are direct covalent modifications of cytosines in the DNA sequences, including DNA methylation, which is measured in the paper under discussion.
- Histone modifications: Histones are proteins that, with DNA, form chromatin and make up chromosomes. Each histone has a tail that can be covalently modified.
- Non-coding RNAs: These functional RNAs that are not translated into protein and are involved in many cellular processes, including regulation of the epigenome.
- Long range chromatin interactions: This refers to the 3D arrangement of DNA and chromosomes within cells. In addition to the packing of DNA into chromosomes by histones, chromosomes interact with themselves and with other chromosomes to form functional domains.
These four mechanisms do not exist in isolation. They form a network of interacting mechanisms that all work together to affect gene expression. For an overview on these mechanisms of epigenetics, please visit the “Intro to Epigenetics” series at Mommy, PhD.
Transgenerational epigenetic inheritance is well documented in plants and the commonly used model organisms, such as C. elegans (roundworms) and D. melanogaster (fruit flies). However, whether transgenerational inheritance occurs in mammals is still unclear.
Does transgenerational epigenetic inheritance occur in humans?
The existence of transgenerational epigenetic inheritance remains unclear in mammals. There are a few reasons why this is hard to answer.
First, humans are complicated. When we have evidence of transgenerational inheritance of a trait in people, it is nearly impossible to separate the cultural and ecological effects from the epigenetic effects to definitively say if that inheritance occurs partly or exclusively through a biological mechanism. In humans, exposures are rarely isolated to the original generation only, making it extremely difficult to separate out true transgenerational effections. In addition, even when exposures are isolated, those exposures often produce differences that have their own effects. In the example of the Holocaust, it is difficult to separate the effects of trauma from living through the Holocaust on offspring from the effect of having a parent who lived through the Holocaust on offspring.
In order to determine if transgenerational inheritance occurs, scientists must stop the exposure in the original generation to isolate the exposure. While this can be done in model organisms in the lab, exposures are rarely isolated to a single generation in humans. Even when they are, the genetic, ecological and cultural confounders are so complex that it is extraordinarily difficult to conclusively identify transgenerational epigenetic inheritance in humans.
Second, experimental design is extremely complicated. We can use model organisms (such as mice or rats) to control for some of these factors to determine if transgenerational epigenetic inheritance occurs in mammals. However, when properly designed, these experiments are extremely complicated, expensive, and time-consuming, as described in A guide to designing germline-dependent epigenetic inheritance experiments in mammals. These experiments can be done, but at this point in time, very few studies are properly designed to actually be able to answer this question. We will discuss this in more detail below when we get to the methods of the paper under discussion.
Third, germline reprogramming clears (erases) many epigenetic marks twice during mammalian development. First, DNA methylation marks are cleared during germ-cell development. There is a second wave of demethylation following fertilization; the timing of this demethylation and the reestablishment of methylation patterns is different for maternal and paternal chromosomes. A subset of genes (mostly imprinted genes) do not undergo this second wave of demethylation and are more sensitive to environmental regulation. Thus, only a subset of the genome could be undergoing translational epigenetic inheritance. While research in the area is still evolving, it is clear that more of the genome than previously thought is protected from this second wave of demethylation. But transgenerational epigenetic inheritance seems unlikely to be a genome-wide phenomenon.
State of the transgenerational inheritance science
This 2018 state of the science report on transgenerational inheritance from the National Toxicology Program cites 21 papers from the lab that published the current glyphosate study. It summarizes the weaknesses of the existing evidence and underscores the need for well-designed studies.
“In conclusion, a broad range of exposures and outcomes have been reported to support transgenerational inheritance of health effects. Over 80 different agents have been tested in a transgenerational experimental design; and this state of the science review collected and categorized the literature into a systematic evidence map for transgenerational inheritance by broad health effect categories, exposures, and evidence streams. This scoping review and evidence map identifies serious limitations in the available bodies of evidence to support a systematic review for reaching hazard conclusions or even rating certainty in the bodies of evidence under evidence to decision frameworks such as the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach.”
This report includes assessments of potential bias in the studies that do exist. The images below show a summary of their assessment of bias in animals studies (top) and specifically in animal studies of vinclozolin (bottom). The top panel shows that the probability of bias is “probably high” for many papers on many measures, with more than half of papers showing a “definitely high” risk of bias for confidence in the exposure characterization. The bottom panel shows the risk of bias from individual studies.
You can see from these images that much of the risk of bias seems to arise from the failure to report specific aspects of the methods and results. Nine of the fifteen papers listed in this panel are from the lab that performed the study in question. The areas identified as being of high risk for bias are also problematic in the current study as we will go through in detail below. This doesn’t necessarily mean that the studies are flawed or the results are biased, but it does mean that the results cannot be accurately interpreted and it is not possible to determine if they are flawed or valid.
View the other parts of our series on transgenerational epigenetic inheritance:
- Transgenerational Epigenetic Inheritance and Glyphosate: Do we need to think of the grandchildren? (Introduction)
- Part 1: What is transgenerational inheritance? (this post)
- Part 2: Guidelines for studying epigenetic inheritance
- Part 3: Is the dose of glyphosate appropriate?
- Part 4: The problem of founder effects
- Part 5: Statistics
- Part 6: Methylation analysis
- Conclusions