Michiel B. Vandegehuchte

Epigenetics and its implications for ecotoxicology

Epigenetics is the study of mitotically or meiotically heritable changes in gene function that occur without a change in the DNA sequence. Interestingly, epigenetic changes can be triggered by environmental factors. Environmental exposure to e.g. metals, persistent organic pollutants or endocrine disrupting chemicals has been shown to modulate epigenetic marks, not only in mammalian cells or rodents, but also in environmentally relevant species such as fish or water fleas. The associated changes in gene expression often lead to modifications in the affected organism’s phenotype. Epigenetic changes can in some cases be transferred to subsequent generations, even when these generations are no longer exposed to the external factor which induced the epigenetic change, as observed in a study with fungicide exposed rats. The possibility of this phenomenon in other species was demonstrated in water fleas exposed to the epigenetic drug 5-azacytidine. This way, populations can experience the effects of their ancestors’ exposure to chemicals, which has implications for environmental risk assessment. More basic research is needed to assess the potential phenotypic and population-level effects of epigenetic modifications in different species and to evaluate the persistence of chemical exposure-induced epigenetic effects in multiple subsequent generations.

Epigenetics in an ecotoxicological context

Epigenetics can play a role in interactions between chemicals and exposed species, between species and abiotic ecosystem components or between species of the same or another population in a community. Technological progress and advanced insights into epigenetic processes have led to the description of epigenetic features (mainly DNA methylation) in many ecologically relevant species: algae, plants, several invertebrates and fish. Epigenetic changes in plants, insects and cladocerans have been reported to be induced by various environmental stress factors including nutrition or water deficiency, grazing, light or temperature alterations, social environment, and dissolved organic matter concentrations. As regards chemicals, studies in rats and mice exposed to specific pesticides, hydrocarbons, dioxins, and endocrine disrupting chemicals demonstrated the induction of epigenetic changes, suggesting the need for further research with these substances in an ecotoxicological context. In fish and plants, exposure to polyaromatic hydrocarbons, metals, and soluble fractions of solid waste affected the epigenetic status. A novel concept in ecotoxicological epigenetics is the induction of transgenerational stress resistance upon chemical exposure, as demonstrated in rice exposed to metals. Evaluating epigenetics in ecotoxicological field studies is a second relatively new approach. A cryptic lineage of earthworms had developed arsenic tolerance in the field, concurrent with specific DNA methylation patterns. Flatfish caught in the framework of environmental monitoring had developed tumours, exhibiting specific DNA methylation patterns. Two main potential implications of epigenetics in an ecotoxicological context are (1) the possibility of transgenerationally inherited, chemical stress-induced epigenetic changes with associated phenotypes and (2) epigenetically induced adaptation to stress upon long-term chemical exposure. Key knowledge gaps are concerned with the causality of the relation between epigenetic and phenotypic changes, the persistence of transgenerational effects, the implications at population level and the costs of tolerance. Epigenetic changes following exposure to multiple stressors constitute another promising area of further research.

Assessment of marine debris on the Belgian Continental Shelf

A comprehensive assessment of marine litter in three environmental compartments of Belgian coastal waters was performed. Abundance, weight and composition of marine debris, including microplastics, was assessed by performing beach, sea surface and seafloor monitoring campaigns during two consecutive years. Plastic items were the dominant type of macrodebris recorded: over 95% of debris present in the three sampled marine compartments were plastic. In general, concentrations of macrodebris were quite high. Especially the number of beached debris reached very high levels: on average 6429±6767 items per 100 m were recorded. Microplastic concentrations were determined to assess overall abundance in the different marine compartments of the Belgian Continental Shelf. In terms of weight, macrodebris still dominates the pollution of beaches, but in the water column and in the seafloor microplastics appear to be of higher importance: here, microplastic weight is approximately 100 times and 400 times higher, respectively, than macrodebris weight.

Soil‐solution speciation of CD as affected by soil characteristics in unpolluted and polluted soils

Total metal content by itself is insufficient as a measure to indicate actual environmental risk. Understanding the mobility of heavy metals in the soil and their speciation in the soil solution is of great importance for accurately assessing environmental risks posed by these metals. In a first explorative study, the effects of general soil characteristics on Cd mobility were evaluated and expressed in the form of empirical formulations. The most important factors influencing mobility of Cd proved to be pH and total soil content. This may indicate that current legislation expressing the requirement for soil sanitation in Flanders (Belgium) as a function of total soil content, organic matter, and clay does not successfully reflect actual risks. Current legal frameworks focusing on total content, therefore, should be amended with criteria that are indicative of metal mobility and availability and are based on physicochemical soil properties. In addition, soil-solution speciation was performed using two independent software packages (Visual Minteq 2.23 and Windermere Humic Aqueous model VI [WHAM VI]). Both programs largely were in agreement in concern to Cd speciation in all 29 soils under study. Depending on soil type, free ion and the organically complexed forms were the most abundant species. Additional inorganic soluble species were sulfates and chlorides. Minor species in solution were in the form of nitrates, hydroxides, and carbonates, the relative importance of which was deemed insignificant in comparison to the four major species.

Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model

The notion that phenotypic traits emerging from environmental experiences are heritable remains under debate. However, the recent report of nonmendelian transgenerational epigenetic inheritance, i.e., the inheritance of traits not determined by the DNA sequence, might make such a phenomenon plausible. In our study, by carrying out common garden experiments, we could provide clear evidences that, on exposure to nonlethal heat shocks, a parental population of parthenogenetic (all female) Artemia (originating from one single female) experiences an increase in levels of Hsp70 production, tolerance toward lethal heat stress, and resistance against pathogenic Vibrio campbellii. Interestingly, these acquired phenotypic traits were transmitted to three successive generations, none of which were exposed to the parental stressor. This transgenerational inheritance of the acquired traits was associated with altered levels of global DNA methylation and acetylated histones H3 and H4 in the heat‐shocked group compared to the control group, where both the parental and successive generations were reared at standard temperature. These results indicated that epigenetic mechanisms, such as global DNA methylation and histones H3 and H4 acetylation, have particular dynamics that are crucial in the heritability of the acquired adaptive phenotypic traits across generations.—Norouzitallab, P., Baruah, K., Vandegehuchte, M., Van Stappen, G., Catania, F., Vanden Bussche, J., Vanhaecke, L., Sorgeloos, P., Bossier, P. Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model. FASEB J. 28, 3552–3563 (2014). www.fasebj.org

Gene transcription profiles, global DNA methylation and potential transgenerational epigenetic effects related to Zn exposure history in Daphnia magna

A reduced level of DNA methylation has recently been described in both Zn-exposed and non-exposed offspring of Daphnia magna exposed to Zn. The hypothesis examined in this study is that DNA hypomethylation has an effect on gene transcription. A second hypothesis is that accumulative epigenetic effects can affect gene transcription in non-exposed offspring from parents with an exposure history of more than one generation. Transcriptional gene regulation was studied with a cDNA microarray. In the exposed and non-exposed hypomethylated daphnids, a large proportion of common genes were similarly up- or down-regulated, indicating a possible effect of the DNA hypomethylation. Two of these genes can be mechanistically involved in DNA methylation reduction. The similar transcriptional regulation of two and three genes in the F0 and F1 exposed daphnids on one hand and their non-exposed offspring on the other hand, could be the result of a one-generation temporary transgenerational epigenetic effect, which was not accumulative.

Locust phase polyphenism: Does epigenetic precede endocrine regulation?

The morphological, physiological and behavioural differences between solitarious and gregarious desert locusts are so pronounced that one could easily mistake the two phases as belonging to different species, if one has no knowledge of the phenomenon of phenotypic plasticity. A number of phase-specific features are hormonally controlled. Juvenile hormone promotes several solitarious features, the green cuticular colour being the most obvious one. The neuropeptide corazonin elicits the dark cuticular colour that is typical for the gregarious phase, as well as particular gregarious behavioural characteristics. However, it had to be concluded, for multiple reasons, that the endocrine system is not the primary phase-determining system. Our observation that longevity gets imprinted in very early life by crowding of the young hatchlings, and that it cannot be changed thereafter, made us consider the possibility that, perhaps, epigenetic control of gene expression might be, if not the missing, a primary phase-determining mechanism. Imprinting is likely to involve DNA methylation and histone modification. Analysis of a Schistocerca EST database of nervous tissue identified the presence of several candidate genes that may be involved in epigenetic control, including two DNA methyltransferases (Dnmts). Dnmt1 and Dnmt2 are phase-specifically expressed in certain tissues. In the metathoracic ganglion, important in the serotonin pathway for sensing mechanostimulation, their expression is clearly affected by crowding. Our data urge for reconsidering the role of the endocrine system as being sandwiched in between genetics and epigenetics, involving complementary modes of action.

Gene transcription and higher-level effects of multigenerational Zn exposure in Daphnia magna.

Zn exposure of Daphnia magna during one generation has been shown to modulate gene transcription differently in Zn exposed organisms compared to their non-exposed offspring. Here we studied the transcriptional gene regulation with a cDNA microarray in D.magna exposed to Zn for three generations (F0-F2). For the first time molecular effects of multigeneration toxicant exposure in D. magna are described. Out of 73 differentially transcribed genes in the F1Zn exposed generation (compared to the F1 control), only seven genes were also differentially transcribed in the same direction in the F0Zn exposed daphnids (up or down, compared to the F0 control). The majority of the differentially transcribed unigenes in F1Zn exposed daphnids (78%) were not differentially transcribed in the F0Zn exposed organisms. This indicates that Zn exposure affected other molecular pathways in the second exposed generation, although a reduced reproduction and a reduction in juvenile growth were observed in both Zn exposed generations, compared to the respective controls. In the third Zn exposed generation (F2), no reduction in growth or reproduction compared to the control was observed. This acclimation was reflected in a significantly lower number of differentially transcribed genes, compared to the Zn exposed F0 and F1 generations.

Can metal stress induce transferable changes in gene transcription in Daphnia magna

DNA methylation has recently been reported in Daphnia magna, which indicates the possible presence of epigenetic mechanisms regulating gene expression in this species. As such, effects of transient chemical exposure could be transferred through epigenetic inheritance to non-exposed generations. In this study, in the Zn-exposed daphnids, a large number of genes were found to be differentially transcribed, amongst which transcription and translation related genes (downregulated), genes associated with oxidative stress (upregulated) and different types of metabolism-related genes (mostly upregulated). In the two subsequent generations of non-exposed daphnids, a considerable number of differentially regulated genes were observed, indicating an effect of Zn-exposure in the non-exposed progeny. However, none of the differentially transcribed genes observed in the Zn-exposed generation were regulated in the same direction in both non-exposed subsequent generations. The exposure of D. magna to a sublethal Zn concentration for one generation did not result in a stable transgenerational epigenetic effect with consequences for reproductive output nor was a stably epigenetically inheritable effect observed on the transcription of any of the studied genes. An important observation was the large number of genes that were differentially transcribed between different control generations with no pre-exposure history. These genes were not considered in the analysis of the effect of Zn exposure on gene transcription. This differential regulation between subsequent control generations was attributed to possible differences in synchronization of the molting and reproductive cycle of the daphnids in the different generations. This finding is of major importance for the interpretation and design of future microarray experiments with adult Daphnia.

Epigenetic alterations and decreasing insecticide sensitivity of the Asian tiger mosquito Aedes albopictus.

A range of environmental factors, including chemicals, can affect epigenetic processes in organisms leading to variations in phenotype. Thus, epigenetics displays an important environmentally responsive element. The transgenerational impact of environmental stressors on DNA methylation and phenotype was the focus of this study. The influence of two known DNA methylation-changing agents, the phytoestrogen genistein and the fungicide vinclozolin, on the overall DNA methylation level in the Asian tiger mosquito Aedes albopictus was investigated. The experiment comprised four generations in a full life-cycle design with an exposed parental generation and three consecutive non-exposed offspring generations. Application of the methylation agents to the parental generation of the study led to an alteration of the global DNA methylation level of the exposed individuals and those in two subsequent generations. The phenotypic variability of the offspring generations was assessed by examining their insecticide sensitivity. Here, a significant decrease in sensitivity (p<0.01) towards the model insecticide imidacloprid revealed alterations of the mosquitos phenotype in two subsequent generations. Thus, the evaluation of A. albopictus from an epigenetic perspective can contribute important information to the study of the high adaptability of this invasive disease vector to new environments, and its underlying mechanisms.