Epigenetics for ecotoxicologists

Abstract

Expanding Ideas About Biological Inheritance Our genetic makeup is an intrinsic part of ourselves; we inherit it from our parents and carry it with us throughout life. This is a familiar concept, but less familiar is the idea that our parents' experiences as children might also be inherited in the form of chemical marks on our DNA. A team of researchers from McGill University in Montreal, Canada explored this idea in rats. They knew that maternal grooming and nursing behavior in the first weeks of life could affect how pups responded to stress in adulthood. In 2004, they showed that specific effects of maternal grooming and nursing were linked with methylation on a gene that is part of the stress response pathway. When pups born to high-grooming mothers were raised by low-grooming mothers, and vice versa, the pups took on the methylation marks and the associated stress response of the mother who raised them. This study shows that parental behavior early in life can be registered by the epigenome and profoundly affect behavior in adulthood. This, in turn, may cause the epigenetic marks to be passed on to the next generation 1. In classic genetics, genetic material is in the form of DNA and encodes all of the information necessary for life. This information is copied faithfully and passed down as cells divide. Every cell contains a complete copy of the DNA code, but the pattern of gene expression—that is, which genes are turned “on” (expressed) or “off” (nonexpressed)—determines the cell type and function. A skin cell on your arm contains the same DNA code as a nerve cell in your brain, but their extreme differences in structure and function are due to the particular set of genes that each is expressing. Epigenetics refers to an annotation in the form of chemical marks on top of the DNA code; the prefix “epi” comes from a Greek word meaning “over” or “above.” These chemical marks, which are discussed in detail below, affect which genes are expressed and at what levels. Epigenetic marks are highly influenced by the environment and can be inherited along with the genetic code as cells divide mitotically, and in some cases meiotically, from one generation to the next. Epigenetics is receiving significant attention in the field of biomedicine. A PubMed search for “epigenetic” revealed that more than 1,300 review articles have been written on the topic, of which more than 50% were published in the last three years alone. Epigenetics provides a mechanism for the Barker hypothesis, which postulates that nutrition and other environmental factors early in development can alter susceptibility to chronic diseases in adulthood. A classic example of this is that individuals who were conceived during the Dutch Winter Hunger (1944–1945) have persistent epigenetic alterations on a characteristic gestational marker, the IGF2 gene, as adults. Furthermore, individuals who experienced the Dutch Winter Hunger have higher rates of metabolic disorders and cardiovascular disease, the epigenetic mechanisms of which are the focus of ongoing studies. Nutritional deprivation may also have an impact on human longevity; indeed, lifespan is negatively correlated with food abundance available to our great-grandparents during their prepubertal growth. It is hypothesized that there is an epigenetic basis for this observation, although the mechanism remains unknown 2. Increasingly, researchers are recognizing the role of epigenetic gene regulation in cancer. Elucidating the role of epigenetic marks in susceptibility to cancer has spawned a new focus in which treatment targets not only the genome, but also the epigenome—a biomedical approach that is rapidly expanding to other diseases. Epigenetic marks serve as biomarkers for disease susceptibility, and make ideal targets for disease therapy and prevention. Epigenetics is a paradigm-shifting concept that is now gathering attention in ecological sciences 3, and more recently ecotoxicology 4. In this article, we discuss this emerging science in the context of ecotoxicological research, present examples of how epigenetics is applicable to ecotoxicology, and suggest exciting avenues for future research. DNA methylation: Methylation of cytosine (C) residues, one of the four nucleotides that make up the genetic code, limits access of the DNA sequence to key proteins that initiate gene expression. Methylation occurs primarily on C residues that are followed by a guanine (G) residue, referred to as CpG methylation. The degree of methylation on a particular gene can turn expression up or down; in general, heavily methylated genes are less likely to be expressed. Histone modification: Histones are proteins around which DNA is tightly wound into structures called nucleosomes. These structures allow DNA to be compacted inside the nucleus and also serve a regulatory purpose. Genes that lie within areas of tightly coiled DNA are not easily accessed nor expressed. Epigenetic marks in the form of chemical modifications on histone tails can affect expression of genes. Mechanisms of epigenetic inheritance 5. Reprinted by permission from Macmillan Publishers Ltd: Nature 441:143–145, © 2006. DNA methylation and histone structure are essential for establishing patterns of gene expression in the early embryo, and these patterns often persist throughout life. They help determine which of the approximately 20,000 genes found in human DNA are expressed in each cell type and at what levels (see sidebar, Epigenetic Analogies). Certain stressors, such as radiation and genotoxic chemicals, can alter patterns of gene expression by mutating DNA directly, but these events are relatively rare. It is far more common for chemicals and other environmental cues to disrupt gene expression by mechanisms other than mutation. The term epigenetic applies only in cases where changes to gene expression are heritable (either mitotically or meiotically; see definitions in sidebar, Coming to terms…). This characteristic suggests that transient exposures can have important consequences at later life stages and also potentially affect the health of offspring. The epigenome acts as an interface between the changing environment and the genome, which is by necessity very stable and resistant to environmental influences. Whereas typical environmental alterations to gene expression will generally cease once a stressor is removed, epigenetic effects can persist as cells divide and even into successive generations. Epigenetic mechanisms for altering gene expression share some characteristics with mutation and other characteristics with metabolic transcriptional changes (Table 1). The fact that epigenetic marks are both heritable and susceptible to environmental influences makes them interesting and highly relevant to ecotoxicology. Epigenetic Analogies Epigenetic marks are often described using metaphors, the most common of which is “beads on a string,” referring to DNA that is spooled around histone proteins to allow for compaction inside a cell's nucleus (Fig. 1). The importance of the epigenome has been further described in several common analogies. For example, if the genome is compared to the hardware in a computer, the epigenome is the software that directs the computer's operation. Imagine your laptop without the various programs on which we have come to rely (e.g., Word, Excel, Firefox, iTunes). Without the epigenome, the genome would not function properly, and vice versa; they are inextricably linked. Another popular analogy for epigenetic control of gene expression is the dimmer switch on a lamp; epigenetic marks such as DNA methylation can turn genes on or off, but can also simply tune them up or down. The epigenome is a dynamic interface between a cell's changing environment and the relatively fixed genome. As such, epigenetic marks can be highly influenced by the environment. Diet, stress, and behavior can all leave epigenetic marks as previously described (e.g., grooming behavior in rats, Dutch Winter Hunger in humans). However, chemicals can also cause epigenetic effects. Structurally diverse classes of environmental contaminants including metals, endocrine-disrupting chemicals, organohalogens, and solvents have been linked to epigenetic effects, some of which may be transgenerational 6. In some cases, stressor-induced epigenetic modifications have been associated with negative health outcomes. Epigenetic effects occur at a wide range of contaminant levels, including very low concentrations of chemicals (Fig. 2). Environmental factors such as toxins, nutrition, behavior, and stress can influence epigenetic marks in the developing embryo and throughout life. These marks can even be passed on to unexposed generations through epigenetic inheritance. Genetically Identical Agouti Mice The viable yellow Agouti mouse (Avy) is an exquisite epigenetic biosensor for whether maternal nutritional and/or environmental factors alter the offsprings' epigenome and subsequent disease susceptibility later in life. In a natural population of Avy mice, variable DNA methylation patterns (not genetic code) at the agouti gene result in widely different phenotypes. For example, unmethylated yellow mice grow up to be obese and prone to diabetes and cancer; conversely, methylated brown mice remain lean and cancer- and diabetes-free. This mouse model has been used as a biosensor for several maternal factors including nutritional components like methyl donors (folic acid, vitamin B12, and choline) and genistein, found in soy and soy products, as well as environmental exposures including ethanol and bisphenol A (BPA). Interestingly, maternal dietary supplementation with methyl donors or soy will shift the population distribution of Agouti mice toward the methylated lean brown coat color. On the other hand, maternal exposure to BPA, the endocrine-disrupting compound found in polycarbonate plastic, shifts the population toward the unmethylated yellow coat color. Moreover, the BPA-induced shift toward the yellow obese phenotype can be counteracted by maternal dietary nutritional supplementation with either methyl donors or soy. These findings demonstrate that simple dietary changes can protect against the deleterious effects of environmental toxicants on the fetal epigenome 7. The dynamic nature of the epigenome, and the example of how contaminants and diet can interact to affect epigenetic endpoints in the Agouti mouse, highlight the importance of studying the epigenetic effects of contaminants in the context of multiple stressors. The field of ecotoxicology has a lot to offer in this regard. Several model organisms common to ecotoxicology can be studied in the laboratory under controlled conditions, and also in the field under real-life exposure to multiple stressors. Most research on epigenetic effects of environmental chemicals has been done in vitro or in biomedical organisms (rat, mouse, human), but a growing body of research shows that epigenetic effects of stressors are also observed in ecologically relevant organisms. Epigenetics has now been studied in 10 different species of ecological relevance, including plants, nematodes, water fleas, and even polar bears 4. It is important to note that the underlying mechanisms of epigenetics have not been fully described for many ecologically relevant organisms. Mechanisms of epigenetic inheritance appear to be quite well conserved across vertebrates, but invertebrates are more variable. For example, the nematode worm (Caenorhabditis elegans) does not have CpG methylation, whereas honey bees (Apis mellifera) and wasps (Nasonia vitripennis) do 2, 4. Further, it was recently discovered that fruit flies (Drosophila melanogaster) exhibit modest levels of methylation (0.1–0.4%) that is not limited to adjacent C-G dinucleotides, but also occurs at cytosines followed by a thymine or adenine. Expanding our knowledge in this area may be a key component of future “eco”-epigenetic studies. Recent findings suggest that invertebrate epigenetics may be a promising avenue of area of research within ecotoxicology. For example, water fleas (Daphnia magna) show decreased DNA methylation when reared in the presence of sublethal concentrations of the fungicide vinclozolin, the pharmaceutical 5-azacytidine, and zinc 4. Early life nutrition has been shown to induce epigenetic changes that determine whether a honeybee will become a queen or worker bee 8. Additionally, DNA methylation with a potential role in regulating gene expression was recently reported in a mollusk 9, a phylum that is frequently used in biomonitoring. Only a handful of studies have addressed epigenetic endpoints in organisms exposed to chemicals in the natural environment rather than the laboratory, and these studies may be critical to understanding how a lifetime of exposure to multiple environmental stressors contributes to epigenetic marks. One study exposed caged mice to air pollution and found that sperm DNA was hypermethylated compared to control mice. Hypermethylation persisted even after the mice were removed from the contaminated area 10. We have observed previously that DNA methylation decreased with methylmercury exposure in brain stems of wild male polar bears 11. Epigenetic effects due to contaminants have also been documented in human populations. For example, declines in global DNA methylation levels were observed in Greenlandic Inuit exposed to high levels of persistent organic pollutants such as DDT and PCBs 12. Although there has been much speculation in the scientific literature, true examples of transgenerational epigenetic inheritance in animals are scarce. In one notable experiment, pregnant rats exposed to the fungicide vinclozolin or the pesticide methoxyclor produced male offspring with lower sperm number and viability. This effect persisted to the F4 generation, with no additional exposure, and was correlated with altered patterns of DNA methylation 13. Many more examples of transgenerational epigenetic inheritance exist in plants. Effects of stressors such as temperature, salt, and ultraviolet radiation have been shown to be transmissible to nonexposed populations in several plant species 4. An enormous amount of biomedical research has been devoted to epigenetics in recent years. This wave is now beginning to hit the field of ecotoxicology, and the potential is exciting. The concepts behind epigenetics have stirred up ideas in ecotoxicology from the basic understanding of mechanisms of action, to risk assessment and policy. Here, we highlight what we consider to be some of the most interesting and promising implications of epigenetics for ecotoxicology. Perhaps the most consequential concept of epigenetics as it relates to ecotoxicology is that epigenetic mechanisms can create a temporal disconnect between exposure and effect. Exposure to chemicals early in development can leave epigenetic marks that result in disease later in life. Similarly, chemically induced epigenetic modifications incurred in one generation may be passed down to future generations in the absence of the initial stressor. The cause (chemical exposure) and effect (a diseased state) need not occur at the same time, at the same life stage, or even in the same generation. Chemicals with an epigenetic mode of action can leave a lasting mark that alters cell function, even when the chemical is no longer present. The concept of a lasting epigenetic signal is highly relevant to biomonitoring and ecological risk assessment. Biomonitoring is used to establish exposure levels by directly measuring chemical concentrations in target tissues. Along with information about the chemical's hazard, these values can support estimates of risk. Unfortunately, species-specific hazard data are lacking for many chemicals, and the epigenetic hazards of chemicals are practically unknown. To assess risk in the absence of detailed knowledge about hazard, ecotoxicologists often employ correlative approaches, linking chemical exposures with predictors of population health such as reproductive success or biomarker responses. Such connections may be difficult to establish in the presence of epigenetic modes of action, because the exposure and the adverse health outcome do not necessarily occur at the same time or even in the same generation. For example, elevated levels of a chemical detected in a population today may leave epigenetic marks that are not manifested for years or even decades. An improved understanding of the mechanisms behind toxicant-induced epigenetic marks and their consequences is critical for assessing the epigenetic risks of environmental chemicals. An epigenetic approach may also benefit ecotoxicology with respect to the field of biomarker research. Epigenetic marks on particular genes could serve as biomarkers for exposure to specific stressors or toxic outcomes. More general marks, such as global DNA methylation, might prove to be broad indicators of accumulated stress throughout an organism's lifetime. Epigenetic mechanisms may provide explanations for unresolved observations in the field of ecotoxicology. For example, epigenetic inheritance could explain why a population is slow to recover after remediation of contaminated sediments; the potentially transgenerational nature of epigenetic alteration means that benefits resulting from contaminant removal may not appear for generations. Conversely, epigenetics may be a mechanism for rapid adaptation to local contamination, and could explain how certain populations are able to thrive in very contaminated areas. Epigenetics may also explain some of the variability observed in studies relating contaminants and toxic effects in natural populations. We know that nutritional supplements can reverse the epigenetic effects of contaminants (see sidebar, Genetically Identical Agouti Mice). Nonchemical stressors, such as limited abundance of food, climate, and disease, may also modulate epigenetic responses and result in variability between individuals or even study years. Ecotoxicologists have long been concerned with subtle effects of chronic exposure to contaminants that may ultimately have an enormous impact on population health. Epigenetic biomarkers may provide early warnings for adverse outcomes associated with early and/or lifelong exposures to chemicals and other environmental stressors. Apical endpoints of ecotoxicological relevance, such as growth, development, and reproduction, are known to be under epigenetic regulation. With further research, the study of epigenetic marks—particularly on specific genes—may lead to an increased understanding of mechanisms of action of contaminants, and ultimately risk. Given the dynamic nature of the epigenome and the potential for the reversal of epigenetic marks, we wonder whether these molecular pathways will one day become targets for remediation. Coming to terms… Adenine (A)—One of the four nucleotide residues that make up the genetic code. Chromatin—A complex of genetic material and proteins (mostly histones) that condense to form chromosomes during cell division. Cytosine (C)—One of the four nucleotide residues that make up the genetic code. Deoxyribonucleic acid (DNA)—The genetic material of the cell. DNA is made up of strings of nucleotide residues. Epigenetics—The study of heritable changes in gene expression that do not involve changes in DNA sequence. Epigenome—The combination of epigenetic modifications across a whole genome. Gene expression—Transcription of the information encoded within a gene into mRNA and potentially into protein. The process of turning genes “on” or “off.” Genome—An organism's entire set of hereditary information. A genome can consist of DNA, for most living organisms, or riboneucleic acid (RNA), in the case of viruses. Guanine (G)—One of the four nucleotide residues that make up the genetic code. Heritable—In the context of epigenetics, heritable refers to traits that are passed on either mitotically as cells divide within an individual, or meiotically into future generations. Histones—Proteins around which DNA is coiled into space-saving and regulatory structures called nucleosomes. Meiotically—Referring to meiosis, a type of cell division in sexually reproducing organisms that results in cells with half the genetic material of the parent. These gametic cells can eventually give rise to offspring. Methylation (of DNA)—The addition of a methyl group, consisting of one carbon bonded to three hydrogens (CH3), to DNA. In eukaryotes, methylation generally occurs at cytosine residues (C) that are followed by a guanine (G) (called CpG methylation). DNA methylation can affect the level at which a gene is expressed. Mitotically—Referring to mitosis, the process by which a cell divides into two daughter cells, each containing a complete set of genetic material. Nucleotide residue—The basic structural unit of DNA. Transgenerational—In the context of epigenetics, transgenerational effects refer to effects that are passed on to the first unexposed generation. Thymine (T)—One of the four nucleotide residues that make up the genetic code.