Impacts of TCDD and MeHg on DNA methylation in zebrafish (Danio rerio) across two generations

Abstract

This study aimed to investigate whether dioxin (TCDD) and methylmercury (MeHg) pose a threat to offspring of fish exposed to elevated concentrations of these chemicals via epigenetic-based mechanisms. Adult female zebrafish were fed diets added either 20 μg/kg 2,3,7,8 TCDD or 10 mg/kg MeHg for 47 days, or 10 mg/kg 5-aza-2′-deoxycytidine (5-AZA), a hypomethylating agent, for 32 days, and bred with unexposed males in clean water to produce F1 and F2 offspring. Global DNA methylation, promoter CpG island methylation and target gene transcription in liver of adult females and in 3 days post fertilization (dpf) F1 and F2 embryos were determined with HPLC, a novel CpG island tiling array containing 54,933 different probes and RT-qPCR, respectively. The results showed that chemical treatment had no significant effect on global DNA methylation levels in F1 (MeHg and TCDD) and F2 (MeHg) embryos and only a limited number of genes were identified with altered methylation levels at their promoter regions. CYP1A1 transcription, an established marker of TCDD exposure, was elevated 27-fold in F1 embryos compared to the controls, matching the high levels of CYP1A1 expression observed in F0 TCDD-treated females. This suggests that maternal transfer of TCDD is a significant route of exposure for the F1 offspring. In conclusion, the selected doses of TCDD and MeHg, two chemicals often found in high concentrations in fish, appear to have only modest effects on DNA methylation in F1 (MeHg and TCDD) and F2 (MeHg) embryos of treated F0 females.

Introduction

Dioxins and methylmercury (MeHg) are contaminants often found in elevated levels in fish. Dioxins are highly toxic substances mainly released into the environment by industrial activities. Dioxins are fat-soluble, and concentrate in fatty tissues of fish. Fatty fish such as salmon can therefore contain relatively high concentrations of dioxins (Hites et al., 2004). TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a persistent environmental contaminant that acts as a reproductive toxicant and endocrine disruptor in nearly all vertebrates (Tillitt et al., 2008). TCDD toxicity in fish is mediated by the aryl hydrocarbon receptor (AHR) pathway. Mercury (Hg) may have natural or anthropogenic origin. In order for Hg to bioaccumulate in fish, it must first be transformed into a bioavailable form of organic mercury, i.e. MeHg. In fish, Hg is therefore predominantly present as MeHg, which often accounts for more than 80% of total Hg (Grieb et al., 1990, Bloom, 1992; Batchelar et al., 2013b). Hg resides in the entire body—including muscle, liver and kidney (Kidd and Batchelar, 2012). In mammals, MeHg primarily acts as a neurotoxin, while in fish the chemical may also disturb reproductive hormones and act as an endocrine disruptor, mediating development of smaller gonads, delaying spawning and affecting fecundity (Klaper et al., 2006, Klaper et al., 2008, Liu et al., 2013). Several studies have also shown that MeHg induces oxidative stress and apoptosis in fish (Gonzalez et al., 2005, Klaper et al., 2008, Cambier et al., 2010, Olsvik et al., 2011, Richter et al., 2011).

Juvenile and larval life stages of fish are highly susceptible to the toxic effects of TCDD (Peterson et al., 1993). While diet is the major source of exposure to TCDD (Nichols et al., 1998) and MeHg (Phillips and Buhler, 1978, Hall et al., 1997) in adult fish, maternal transfer has been shown to be a significant route of exposure to these substances for larval and juvenile fish (Russell et al., 1999, Latif et al., 2001, Heiden et al., 2005, Alvarez Mdel et al., 2006). According to Russell et al. (1999), the concentrations of organochlorine chemicals in eggs of oviparous organisms range between 25.1% and 56% of the maternal tissue concentration. Significant amounts of TCDD transferred to eggs can disrupt critical developmental events and can cause cardiovascular dysfunction, edema, hemorrhages, craniofacial malformations, growth arrest, and mortality (Peterson et al., 1993, Cook et al., 2003, Carney et al., 2004, Heiden et al., 2005, Heiden et al., 2008, Hill et al., 2005, Tillitt et al., 2008). Less information is available on the effects of MeHg on eggs and larvae of fish species. However, as MeHg is a known endocrine disruptor and a potent neurotoxin, it has the potential to cause variety of abnormalities in the offspring. In eggs of adult fathead minnows (Pimephales promelas) exposed to 8.5 mg/kg of MeHg, the concentrations of MeHg were 35% of the concentrations detected in adults (Hammerschmidt and Sandheinrich, 2005). This suggests that embryos are potentially exposed to about the same effective internal concentration as the maternal organisms from which the eggs originated.

Environmental chemicals can affect the expression of specific genes and pathways not only in the exposed individual but also in the subsequent generations through epigenetically mediated modulation (Skinner et al., 2010, Vandegehuchte and Janssen, 2011). The current study was designed to investigate if dioxin (TCDD) and MeHg can induce DNA methylation abnormalities in the directly exposed F0 female fish and F1 embryo and whether the induced DNA methylation changes can be transmitted to the F2 embryo. To investigate this a single-dose experiment was conducted in which adult female (F0) zebrafish (Danio rerio) were exposed to 20 μg/kg TCDD and 10 mg/kg MeHg for 47 days, or 10 to mg/kg 5-aza-2′-deoxycytidine (5-AZA), a hypomethylating agent, for 32 days. The treated F0 female fish were bred with non-exposed male fish to generate the F1 and F2 generations. As the treated females (F0) were transferred to clean water for breading, any transfer of epigenetic abnormalities to the F2 generations must be through the germline. The exposure concentrations were selected based on previous data (TCDD: Heiden et al., 2005, MeHg: Ellingsen, unpublished data, 5-AZA: Aniagu et al., 2008). To evaluate possible effects in the developing fish, three days post fertilization (dpf) embryos were selected for analysis of the F1 and F2 generations. Earlier studies in zebrafish have shown that DNA methylation reprogramming events associated with blastula stage have already been completed by this time and the overall percentage of DNA methylation is similar to adult zebrafish (Fang et al., 2013). Global DNA methylation was assessed with HPLC, genome-wide promoter DNA methylation with CpG island (CGI) tiling array, and transcription of selected genes with RT-qPCR. To our knowledge this is the first attempt to determine whether TCDD and MeHg, two environmental contaminants that accumulate in seafood, can exert effects via DNA methylation across generations in fish.