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Published in final edited form as: Mol Cell Endocrinol. 2014 Sep 3;398(0):36–41. doi: 10.1016/j.mce.2014.08.011

Dioxin Induction of Transgenerational Inheritance of Disease in Zebrafish

Tracie R Baker a, Tisha C King-Heiden b, Richard E Peterson a, Warren Heideman a
PMCID: PMC4262573  NIHMSID: NIHMS636633  PMID: 25194296

Abstract

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is an aryl hydrocarbon receptor (AHR) agonist, an endocrine disruptor, and a potent global pollutant. TCDD exposure is associated with diseases of almost every organ system, and its toxicity is highly conserved across vertebrates. While the acute developmental effects of dioxin exposure have been extensively studied, the ability of early sublethal exposure to produce toxicity in adulthood or subsequent generations is poorly understood. This type of question is difficult to study because of the time frame of the effects. With human subjects, such a study could span more than a lifetime. We have chosen zebrafish (Danio rerio) as a model because they are vertebrates with short generation times and consistent genetic backgrounds. Zebrafish have very modest housing needs, facilitating single and multigenerational studies with minimal time and expense. We have used this model to identify transgenerational effects of TCDD on skeletal development, sex ratio, and male-mediated decreases in reproductive capacity. Here we compare these findings with transgenerational effects described in laboratory rodent species. We propose that the zebrafish is a cost-effective model system for evaluating the transgenerational effects of toxic chemicals and their role in the fetal basis of adult disease.

Keywords: TCDD, AHR, zebrafish, rodent, transgenerational, epigenetic

Introduction

Mounting evidence suggests that environmental factors can alter developmental programming, resulting in the adult onset of latent diseases, including but not restricted to cancer, diabetes, cardiovascular disease and reproductive disorders (Gluckman and Hanson, 2004; Lau and Rogers, 2004; Heindel, 2005; Marczylo et al., 2012; Veenendaal et al., 2013). The etiology of some diseases is now linked to tissue- and developmental stage-specific epigenetic alterations in gene expression, resulting from nutritional deficits or exposure to contaminants in utero. Exposure to endocrine disruptors is of concern due to the roles that hormones play in regulating transient and irreversible developmental processes. Evidence is mounting that developmental exposure to chemicals, including endocrine disruptors, results in adult disease (Heindel, 2008; Corrales et al., 2014b).

TCDD is a toxic environmental contaminant that impacts growth and development in vertebrates and is associated with several diseases. It is the prototypical member of a family of dioxin-like compounds (DLCs), and is generally produced as a byproduct of industrial processes and waste incineration. TCDD is stable in the environment, highly lipophilic and bioaccumulative, and human exposure comes mostly through dietary sources. TCDD acts primarily through activation of the AHR/ARNT transcriptional regulator to alter gene expression, but cross talk with other signal transduction systems is suspected (Poland and Bradfield, 1992; Swanson and Bradfield, 1993; Schmidt and Bradfield, 1996; Puga et al., 2009). AHR activation by TCDD leads to altered expression of hormone receptors, receptor activators and repressors, metabolic enzymes needed for metabolism of xenobiotics and hormone synthesis and degradation, and other gene products required for normal development and endocrine function (Abbott et al., 1994; Gierthy et al., 1996; Safe et al., 1998; Massaad et al., 2002; Beischlag et al., 2008)

Diseases in humans that have been associated with exposure to TCDD include cancer as well as chloracne, porphyria, and defects in the cardiovascular, skeletal, immune, central nervous system, hepatic and reproductive systems (Eskenazi et al., 2000; Guo et al., 2000; Pelclova et al., 2006; Warner et al., 2007; NAS-IOM, 2011; Warner et al., 2011). Recent epidemiologic evaluation following a major industrial release of TCDD revealed that exposure to TCDD in utero leads to reduced sperm quality, feminized sex ratio, and altered thyroid function in the offspring (Mocarelli et al., 2000; Baccarelli et al., 2008; Mocarelli et al., 2011).

Laboratory studies confirm the potential for TCDD to cause disease later in life. Direct exposure to TCDD leads to infertility in many vertebrate species, including humans, and is associated with down-regulation of enzymes in the estrogen synthesis pathway, decreased egg release, increased number of atretic ovarian follicles, and decreased fertilization success (DeVito and Birnbaum, 1994; King-Heiden et al., 2006; Yoshizawa et al., 2009; King-Heiden et al., 2012; Baker et al., 2013). Toxicity in adults following TCDD exposure during early development suggests that physiologic systems are being mis-programmed and that exposure to TCDD can potentially initiate irreversible and permanent modifications in gene expression and cell lineages. However, the molecular mechanisms that underlie latent and transgenerational disease caused by developmental exposure to TCDD are not well understood.

Our recent work has focused on studying the latent and transgenerational effects of TCDD exposure during critical periods of development, using zebrafish (Danio rerio) as a model system. In this review, we compare our findings with effects observed in rodent studies to highlight the usefulness of this model system for evaluating the potential for chemicals to cause disease in adults and subsequent generations.

Zebrafish as a Model for Multigenerational Studies

To study transgenerational effects, we need a vertebrate model that has a short time to sexual maturity so that we can study successive generations. From this perspective, humans are not ideal subjects for study (Heindel, 2007; Skogen and Overland, 2012). In addition, the diverse genetics of the human population, confounded by individual variations in exposures make studies with human subjects difficult. The zebrafish is well established as a model for investigating human disease, especially as it pertains to altered development. Attributes that make the zebrafish outstanding in this arena are: short time to sexual maturity (about 3–4 months), transparent embryos that allow observation of organ development without disturbing the embryo, the ability to obtain large groups of synchronously developing embryos, low cost for exposure chemicals since volumes are small, and the ease of housing multiple generations of fish. This last point means that one can expose the first F0 generation and maintain offspring across many generations inexpensively and compactly.

While small rodent models are more common than small fish models for studying human disease, rodents have a number of disadvantages for studying the fetal basis of adult disease. Rats and mice for example have far fewer offspring per pair, and maintenance costs are considerably greater. While zebrafish reach sexual maturity in a similar timeframe to some rodents, their small size allows for the ability to house and maintain large groups of synchronously developing fish over multiple generations inexpensively and compactly. Similar to human populations, laboratory zebrafish are less isogenic than laboratory rodent strains, which decreases inbreeding effects when studying changes in the zebrafish genome/epigenome. Zebrafish developmental processes are well characterized, and many organs and cell types have been marked with fluorescent reporters in transgenic lines. Due to complete sequencing of the zebrafish genome, technologies that include specific antibodies, genetic/epigenetic markers, and high throughput sequencing also can be readily utilized. MicroRNAs may be involved in the transgenerational inheritance of disease (Wagner et al., 2008; Grandjean et al., 2009) and there is a rapidly growing microRNA literature in zebrafish. Finally, developing zebrafish are very small and transparent, so development can be readily followed with microscopy and automated screening techniques (Kaufman et al., 2009; Wittmann et al., 2012; Westhoff et al., 2013).

Even though zebrafish are oviparous, the reproductive system of fish and mammals is similar. The testis and ovary in zebrafish contain the same germ cells that are found in mammals, and hormonal regulation of spermatogenesis and oogenesis is highly conserved across vertebrates, occurring via the hypothalamic-pituitary-gonadal axis (Segner, 2009; Liu et al., 2011; Lohr and Hammerschmidt, 2011).

Defining Transgenerational Toxicity: Zebrafish vs. Rodents

Chemical exposures that affect subsequent generations are now well documented. An epigenetic mechanism is likely for cases of multigenerational disease, in which neither the parent nor the offspring have been directly exposed. This is a transgenerational effect because there is no direct connection to chemical exposure (Skinner, 2008). In rodent models, exposure during early development requires prenatal exposure in an F0 generation mother during pregnancy (Figure 1, left column). This leads to F1 offspring that developed in an exposed environment. The F2 offspring then develop in parents that were exposed in utero, so only effects in the F3 generation can be due to epigenetic alterations in gametes.

Figure 1. Comparison of the experimental design of a transgenerational study in the laboratory rodent and zebrafish.

Figure 1

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In the rodent model, in utero exposure to a chemical leads to direct exposure of the F0, F1 and F2 generations and the F3 generation is not exposed. In the zebrafish model, juvenile developmental exposure leads to direct exposure of the F0 and F1 generations to the chemical and the F2 generation is not exposed.

In contrast, zebrafish eggs are fertilized in water, embryos develop externally, and are subsequently exposed at the juvenile stage of development (Figure 1, right column). Thus, F0 fish are equivalent to F1 mice because they develop in an exposed environment. The F1 zebrafish generation originates from gametes produced by exposed fish, similar to the F2 mouse generation. The gametes producing the F2 zebrafish generation have not been exposed so the effects seen in F2 zebrafish are transgenerational. Thus, the F2 zebrafish is equivalent to the exposure-free F3 mouse.

Using Zebrafish to Identify Transgenerational Effects of TCDD

Sublethal TCDD exposure in utero and in early development leads to adverse health effects in adulthood and subsequent generations. Adverse effects have included increased congenital abnormalities, decreased survival, differences in sex ratios of offspring, and decreased reproductive function and fertility in both males and females (Wolf et al., 1999; Nomura et al., 2004; Ikeda et al., 2005a and b; King-Heiden et al., 2009; Ding et al., 2011). Transgenerational effects of TCDD exposure have now been observed in mice, rats and zebrafish (Bruner-Tran and Osteen, 2011; Manikkam et al., 2012a and b; Nilsson et al., 2012; Baker et al., 2014). The TCDD-induced transgenerational defects identified in these species involve skeletal development, sex ratio, ovary, and reproductive success and are summarized in Table 1.

Table 1.

Sex-Specific, Transgenerational Effects of TCDD in Rodents and Zebrafish

Species Sex Transgenerational Effect TCDD Exposure of F0 Generation Reference
Mice Female Decreased pregnancy rate
Increased preterm birth		 10 μg/kg, po, at E15.5 Bruner-Tran and Osteen, 2011
Rats Female Decreased follicle number
Decreased primordial follicles
Early puberty
Increased small cysts in ovary		 100 ng/kg/day, ip, E8 to E14 Nilsson et al., 2012
Manikkam et al., 2012a,b
Male Increased kidney disease
Zebrafish Female and Male Changed in sex ratio
Increased skeletal malformations		 50 pg/ml, 1 hr, static waterborne at 3 and 7 wpfa Baker et al., 2014
Maleb Decreased egg release
Decreased egg fertilization
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a

Weeks post fertilization

b

Decreased egg release and fertilization is observed in control, female zebrafish mated with TCDD lineage F2 males. It does not occur when TCDD lineage F2 females are mated with control males. Thus, the transgenerational effect of TCDD, in decreasing reproductive capability in zebrafish, is male-mediated.

Skeletal Development

Direct TCDD exposure causes skeletal, cartilage, and bone abnormalities in several animal models (Peterson et al., 1993; Hornung et al., 1999; Xiong et al., 2008; Bursian et al., 2013). Spina bifida, a developmental abnormality that is caused by incomplete closing of the neural tube and malformed vertebrae, occurs in human offspring following exposure to Agent Orange, a TCDD-contaminated herbicide (NAS-IOM, 2011). In mink, skeletal abnormalities were observed in F1 offspring of TCDD-exposed adults (Bursian et al., 2013). TCDD exposure during development altered craniofacial structures in adult zebrafish, and produced scoliosis-like kinks in the axial skeletons of adult F0 parents as well as in F1 and F2 offspring (Table 1; King-Heiden et al., 2009; Baker et al., 2013; Baker et al., 2014). Transgenerational skeletal abnormalities have not yet been reported in mammals. This response might be idiopathic to fish, but the effects may also be easier to observe in zebrafish. It is also possible that zebrafish are more sensitive to skeletal toxicity compared to mammals. If so, a zebrafish model may be useful in developing screens for transgenerational skeletal effects of chemical exposure.

Sex Ratio

One effect of human exposure to TCDD is a change in the sex ratio towards a higher percentage of girls born to parents exposed during an industrial accident in Seveso, Italy (Clapp and Ozonoff, 2000; Eskanazi et al., 2004; Mocarelli et al., 2000 and 2008). Some studies in rats and zebrafish have also produced a shift in the sex ratio of offspring toward females (Ikeda et al., 2005b; Baker et al., 2013). The shift towards females was also observed in the F1 and F2 offspring of F0 zebrafish exposed to TCDD during sexual differentiation and maturation (Table 1; Baker et al., 2014). While not as well understood in fish as in mammals, sex determination in zebrafish is primarily genetic, with environmental factors influencing sex secondarily (Liew and Orban, 2014). Although, zebrafish would be useful in screening for the ability of environmental contaminants to influence sex ratios, zebrafish do not have a pair of highly differentiated sex chromosomes. Until we know more about what determines male and female sex in zebrafish, mechanistic interpretation will be difficult.

Ovary

While sex determination is not well understood in zebrafish, the pathways and genes involved in gonad differentiation are conserved between zebrafish and other vertebrates (Wilkins, 1995; Marshall-Graves and Peichel, 2010). Zebrafish (F0) exposed to TCDD as embryos or adults show similar ovarian toxicities (King-Heiden et al., 2005 and 2006; Hutz et al., 2006; Daouk et al., 2011; Baker et al., 2013). A group of studies from Skinner and colleagues reported several transgenerational phenotypic changes in F3 female rats following in utero exposure of the F1 generation to TCDD, including: early onset of puberty, reduced numbers of total follicles, reduced numbers of primordial follicles, and increased numbers of small cysts within the ovary (Table 1; Manikkam et al., 2012a and b; Nilsson et al., 2012). As in the rodent studies, F1 female zebrafish had abnormal ovarian structure, with atretic follicles (King-Heiden et al., 2009; Baker et al., 2014). However, this effect diminished with time, and was not statistically significant by the F2 generation. This steady diminution of effects over time may help provide insight into the mechanism of transgenerational effects, and will be an important part of assessing the long-term impact of developmental exposure on human populations.

Reproductive Success

Studies with mice have demonstrated transgenerational effects of TCDD exposure on fertility. Offspring of TCDD exposed mice were less likely than control to become pregnant, and those that did become pregnant were less likely than control to deliver at full term (Table 1, Bruner-Tran and Osteen, 2011). We observed similar results with F0 zebrafish exposed to TCDD during sexual development in the F0 generation. Both F1 and F2 offspring showed decreased reproductive capacity, with significantly decreased egg release and reduced percentage of eggs fertilized (Table 1; Baker et al., 2013 and 2014). A simple explanation for decreased egg release could be adverse effects on ovarian development, but interestingly, transgenerational reduction in egg release was linked to males rather than females. Egg release during spawning involves both a female releasing the eggs and a male eliciting egg release. In spawnings of TCDD-lineage F0, F1, and F2 zebrafish males with control females, fewer eggs were produced compared to controls. Similar results have been reported with control females that avoid mating with vinclozolin-lineage F3 male rats (Crews et al., 2007). In fact, decreased reproductive capacity was observed in spawnings of TCDD-lineage F1 and F2 zebrafish males with control females (Table 1; Baker et al., 2014), suggesting that transgenerational reduction in fertility can be attributed to effects on males in the lineage. These findings indicate that some transgenerational effects can be sex-specific. Decreased sperm release could explain these fertility deficits, but as with rodent studies, the testes of TCDD-lineage zebrafish appeared normal on histological examination (Manikkam et al., 2012a and b; Baker et al., 2014). Exposure to TCDD or PCBs in utero also decreased masculine, while increasing feminine sexual behavior in rats (Mably et al., 1992; Colciago et al., 2009). Thus, TCDD does something transgenerationally to alter reproductive success in the male; however at present we are limited to trying to link what is known about AHR and what we know about reproductive function. Further studies are needed to elucidate whether alterations in male zebrafish spawning behavior, pheromone production, and/or other aspects of male reproductive biology are causing decreased fertility and what mechanisms are responsible.

Epigenetic Effects of Chemical Exposure

As we search for the mechanisms that allow a chemical exposure to have effects that persist through multiple generations, epigenetic changes via covalent DNA and chromatin modification come to the forefront. Epigenetic modifications can be carried in the gametes, ultimately modifying gene expression to produce phenotypic changes. Heritable natural epigenetic changes producing a phenotype have been documented in plants, worms and insects (Cubas et al., 1999; Manning et al., 2006; Ruden and Lu, 2008; Greer et al., 2011). Kuroki and colleagues (2013) discovered that mice lacking the H3K9 demethylase, regulating histone function in the chromatin, were subject to male to female sex reversal, demonstrating that changes in chromatin can play a pivotal role in sex determination.

Several attempts have been made to identify epigenetic changes in DNA and chromatin in individuals displaying transgenerational effects of toxic chemicals. Skinner and colleagues have shown that DNA methylation is altered in many places throughout the genome in the affected generations compared to controls (Anway et al., 2005; Manikkam et al., 2012a and b). In other cases this group has also focused on altered gene expression patterns as biomarkers of the exposure (Nilsson et al., 2012). Dolinoy and colleagues (2006) showed an effect of genistein on coat color in mice that was associated with altered methylation upstream of the agouti gene, a regulator of coat color.

Studies on transgenerational effects of AHR agonists in zebrafish have just begun. Although no specific epigenetic change has been shown to produce the transgenerational effects caused by a toxicant, it appears likely that epigenetic changes play a role in producing and transmitting these effects through generations. Changes in DNA methylation and gene expression patterns have been identified in F0 generation zebrafish following exposure to benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene (Mirbahai et al., 2011; Fang et al., 2013; Corrales et al., 2014a and b). The transgenerational phenotypic effects identified in TCDD lineage zebrafish (Table 1, Baker et al., 2014) are similar to TCDD effects observed in the F0 generation. Whether the transgenerationally altered reproductive and skeletal phenotypes are due to epigenetic modifications in the regulation of AHR-ARNT signaling in these tissues will require further research.

In addition to clarifying mechanism, it will be important to assess the stability of the toxic effects across generations. While the effects on egg release persisted through the F2 generation in our zebrafish experiments, the effects on ovarian structure waned with each generation such that it was no longer observed in the F2 generation. The mechanism for such waning effects may be similar to the evolved multi-generational resistance to dioxin-like compound toxicity in wild fish populations (Wirgin et al., 2011). How long these effects last are vitally important. In the past we have been concerned about the persistence and chemical stability of the environmental contaminants themselves. However, if contaminants are capable of producing adverse effects that can be passed across generations, we also will want to know if these effects are reversible and how many generations will be affected.

Conclusion

Transgenerational toxicity due to TCDD exposure has been observed in mice, rats and zebrafish (Bruner-Tran and Osteen, 2011; Manikkam et al., 2012a and b; Baker et al., 2014). Remarkably, several of the phenotypic effects are similar across vertebrate classes, especially the reduction of reproductive capacity in unexposed TCDD-lineages. In zebrafish, unexposed TCDD-lineage F2 offspring have reproductive, skeletal, and sex ratio abnormalities. More specifically, the decrease in fertility and egg release in control female zebrafish is due to the unexposed, TCDD-lineage F2 male zebrafish. Thus, ancestral TCDD exposure reduces reproductive success of male zebrafish across multiple generations. This is most likely an epigenetic effect since TCDD has been shown not to be mutagenic (Poland and Glover, 1979). Epigenetic changes provide an avenue for better understanding how these heritable changes occur. Zebrafish are a promising model because many generations can be produced and studied in relatively little time and space. Also, reproduction and development are easy to assess in zebrafish, and this model is broadly available.

Highlights.

  • We review transgenerational effects of dioxin in fish and other vertebrate species

  • Zebrafish model is ideal for investigating multigenerational effects of chemicals

  • Dioxin induces transgenerational skeletal and reproductive phenoytpes in zebrafish

Acknowledgments

Funding

National Institutes of Health (NIH) (R01 ES012716) from the National Institute of Environmental Health Sciences (NIEHS) (to W.H. and R.E.P.); the University of Wisconsin Sea Grant Institute, National Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Commerce (NA 16RG2257), Sea Grant Project R/BT-22 and 25 (to W.H. and R.E.P.) and NIEHS support from T32 ES007015 and NCATS support from K01 OD010462 (to T.B.).

We would like to thank Dorothy Nesbit, Carrie Zellmer, and Bridget Baker for technical assistance, and advice. The contents are solely the responsibility of the authors and do not necessarily represent the official view of the National Institute of Environmental Health Sciences, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abbreviations

TCDD or dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin

DLC

dioxin-like compound

AHR

aryl hydrocarbon receptor

ARNT

aryl hydrocarbon receptor nuclear translocator

Footnotes

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