Sensitivity to Dioxin Decreases as Zebrafish Mature

Agonists for the aryl hydrocarbon receptor (AHR) include halogenated aromatic hydrocarbons (HAHs), a family of chemicals that includes polychlorinated dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls (Abel and Haarmann-Stemmann, 2010; Hankinson, 1995; Ramadoss et al., 2005; White and Birnbaum, 2009). These widespread environmental contaminants cause a range of toxic effects, including carcinogenesis, developmental and reproductive defects, and death (Nguyen and Bradfield, 2008). Salmonids are especially sensitive to HAHs: lake trout (Salvelinus namaycush) at early life stages are currently among the most sensitive vertebrates known (Elonen et al., 1998; Tanguay et al., 1999; Van den Berg et al., 1998; Walker and Peterson, 1994; Walker et al., 1996). The prototype HAH used to study toxicity is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD binding causes AHR to form a heterodimer with aryl hydrocarbon receptor nuclear transporter (ARNT) in the nucleus, where it regulates gene transcription (Jones et al., 1986; Nguyen and Bradfield, 2008). AHR and ARNT are both necessary for TCDD toxicity (Prasch et al., 2006; Tanguay et al., 2003). The zebrafish (Danio rerio) has proven to be a useful model for studying TCDD toxicity (Carney, et al., 2006b). In zebrafish, TCDD causes a spectrum of toxic effects that matches that observed in lake trout: pericardial and yolk sac edema, heart malformation, and craniofacial and skeletal defects (Henry et al., 1997). Although the molecular mechanism remains unclear, the cardiotoxicity produced by TCDD has been extensively characterized in the zebrafish embryo (Antkiewicz et al., 2005, 2006; Canga et al., 1988; Heideman et al., 2005). TCDD exposure leads to decreased cardiac output, a reduction in peripheral blood flow, pericardial edema, and unlooping of the two chambered zebrafish heart (Antkiewicz et al., 2005; Carney, et al., 2006b; Henry et al., 1997). AHR activation by TCDD or other agonists also disrupts atrioventricular and bulboventricular valve formation and halts bulbus arteriosus growth (Grimes et al., 2008; Mehta et al., 2008). At the molecular level, TCDD has been shown to downregulate transcripts associated with cell proliferation. This effect on gene expression precedes an actual reduction in cardiomyocyte proliferation and number (Carney, et al., 2006b). Ultimately, toxicity progresses to complete heart failure with ventricular standstill and cessation of blood flow. All of these effects can be observed within the first 4 days post-fertilization (dpf). Work with trout and other fish species indicates that adults are considerably less sensitive to the toxic effects of TCDD than early life stage embryos (Peterson et al., 1993; Walker and Peterson, 1994). It is possible that AHR activation disrupts processes important for development that are no longer critical as organogenesis is completed. The difficulty in following the growth and development of species such as trout has made it difficult to examine this in detail, and we know little about how sensitivity to TCDD changes during growth and development. To address this, we took advantage of the zebrafish model to systematically document changes in TCDD sensitivity throughout the developmental process. In this report, we demonstrate that the acute cardiotoxic effects produced by TCDD in embryos are completely absent in fish exposed as juveniles. The zebrafish heart is most sensitive to TCDD at approximately 48–96 hpf and then becomes increasingly resistant to acute cardiotoxicity as the fish moves through the larva stage. This period of sensitivity to TCDD cardiotoxicity does not coincide with the initial development of the heart: the heart is formed prior to 48 hpf. Analysis of heart morphology and peripheral blood flow shows that sensitivity to cardiotoxicity ends at a point that coincides with larval metamorphosis. This metamorphosis alters fins, pigmentation, and muscle to transform the larval shape into the adult body plan. The onset of metamorphosis occurs when the larva is approximately 5 mm in length (Brown, 1997; Parichy et al., 2009). Under our rearing conditions, this takes place at approximately 2 weeks post-fertilization. Because AHR is a transcriptional regulator, changes in messenger RNA produced by TCDD are likely to be important in controlling cellular responses. Microarray experiments comparing transcript levels in embryonic and juvenile hearts reveal drastic differences, with only minor overlaps, in the transcriptional responses to TCDD. This indicates a fundamental shift during development in the way that heart cells respond to TCDD activation of AHR.