Characterization of the recalcitrant CYP1 phenotype found in Atlantic killifish (Fundulus heteroclitus) inhabiting a Superfund site on the Elizabeth River, VA

Abstract

Fundulus heteroclitus (Atlantic killifish) found at the Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in Portsmouth, VA (USA), have been shown to be resistant to the teratogenic effects of creosote-contaminated sediments found at this highly contaminated site. Many of the polycyclic aromatic hydrocarbons (PAHs) found at the ER are known to activate the aryl hydrocarbon receptor (AHR), and are thought to mediate their toxic effects through this pathway. Activation of the AHR results in the induction of several Phase I and II metabolic enzymes. It has been previously shown that the AHR of killifish from the ER are refractory to induction by AHR agonists. To more fully characterize this altered AHR response, we exposed embryos from the ER and from a reference site on King's Creek, VA (KC) to two PAHs, benzo[alpha]pyrene (BaP) and benzo[k]fluoranthene (BkF), and to the dioxin-like compound (DLC), 3,3',4,4',5-pentachlorobiphenyl (PCB126). We compared their developmental and molecular responses by screening the embryos for CYP1A enzyme activity, cardiac deformities, and mRNA expression of CYP1A, CYP1B1, CYP1C1, and AHR2. Basal gene expression of both CYP1A and CYP1B1 was 40% higher in the KC control embryos compared to those from the ER, while AHR2 and CYP1C1 were not significantly different between the populations. Exposure of KC embryos to BaP, BkF, and PCB126 induced CYP1A activity and cardiac deformities. In contrast, CYP1A activity was induced in ER embryos only in response to BkF exposure, although this induction in ER embryos was significantly lower than that observed in KC fish at comparable concentrations. ER embryos did not develop cardiac deformities in response to any of the chemicals tested. CYP1A, CYP1B1 and CYP1C1 mRNA were all significantly induced in the KC embryos after exposure to BaP, BkF and PCB126. Exposure to BaP and BkF in ER embryos resulted in a significant induction of CYP1A mRNA, albeit significantly lower than observed in KC fish. Interestingly, BaP exposure resulted in induction of CYP1B1 at comparable levels in embryos from both populations. CYP1s were not induced in ER embryos in response to PCB126, nor was CYP1C1 for any treatment examined. Additionally, AHR2 was not significantly induced for any of the treatment groups. This study further characterizes the AHR response in killifish, and provides greater insight into the adapted ER phenotype. The ER adaptation involves the suppression of normal AHR-inducible gene expression for all three CYP1 genes, and therefore is likely an alteration in AHR signaling or control.

Fig. 1

Dose response curve of CYP1 enzymatic activity as measured by the in ovo EROD assay (96 hpf) and cardiac deformities (144 hpf) for BaP, BkF, and PCB126. Main effects and the interaction of population and treatment (BaP, BkF, and PCB126 independently) on EROD induction were significant (p < 0.001). There was a significant increase in EROD activity compared to controls in KC embryos exposed to each concentration of BaP, BkF and PCB126 (p < 0.001). There was no significant increase in EROD activity in BaP- or PCB126- dosed ER embryos; however, there was a significant increase in ER embryos exposed to 1, 100, and 300 μg/L BkF (p < 0.001). Main effects and interaction of population and treatment (BaP, BkF, and PCB126 independently) on cardiac deformities were significant (p < 0.001). There was a significant increase in cardiac deformities in KC embryos exposed to 400 μg/L BaP, 100 and 300 μg/L BkF, and 1 μg/L PCB126, relative to controls (p < 0.001). There were no significant increases in cardiac deformities in ER embryos dosed with BaP, BkF, or PCB126. EROD data is represented as average percent induction of control ± SEM; n ≥ 10. Cardiac deformities represented as average deformity score ± SEM; n ≥ 10. “*” indicates a significant difference from control among EROD data. “‡” indicates a significant difference from control among deformity data. “#” indicates a significant difference between populations.

Fig. 2

AHR2 mRNA induction in KC and ER embryos exposed to BaP, BkF and PCB126. Statistical analysis was performed using an ANOVA and Dunnett's post hoc test to determine treatments that differed from controls. The main effects and interaction of population and treatment were not significant for BaP, BkF or PCB126. Data represented as average fold induction ± SEM; n ≥ 6 pools of 2 embryos.

Fig. 3

mRNA induction of the metabolic enzymes CYP1A, CYP1B1, and CYP1C1 in KC and ER embryos exposed to BaP. Statistical analysis was performed using an ANOVA and Dunnett's post hoc test to determine treatments that differed from controls. For CYP1A and CYP1C1 the main effects and interaction of population and BaP treatment were significant (p < 0.001). The main effect of treatment was significant for CYP1B1. CYP1A and CYP1C1 were induced at levels above control in KC embryos dosed with 10, 100, 200, and 400 μg/L BaP (p ≤ 0.01). CYP1B1 was induced at levels above control in KC and ER embryos dosed with 10, 100, 200, and 400 μg/L BaP (p < 0.05). CYP1A was induced in ER embryos exposed to 400 μg/L BaP (p < 0.01). CYP1C1 was not significantly induced in ER embryos for any of the BaP treatments examined. Data represented as average fold induction ± SEM; n ≥ 6 pools of 2 embryos. “*” indicates a significant difference from control among mRNA data. “#” indicates a significant difference between populations.

Fig. 4

mRNA induction of the metabolic enzymes CYP1A, CYP1B1, and CYP1C1 in KC and ER embryos exposed to BkF. Statistical analysis was performed using an ANOVA and Dunnett's post hoc test to determine treatments that differed from controls. For CYP1A, CYP1B1 and CYP1C1 the main effects and interaction of population and BkF treatment were significant (p < 0.001). CYP1A was induced at levels above control in KC embryos dosed with 0.1, 1, 100, and 300 μg/L BkF (p < 0.001) and in ER embryos dosed with 100 and 300 μg/L BkF (p < 0.001). CYP1B1 and CYP1C1 were induced at levels above control in KC embryos dosed with 100 and 300 μg/L BkF (p < 0.001). CYP1B1 and CYP1C1 were not significantly induced in ER embryos for any of the BkF treatments examined. Data represented as average fold induction ± SEM; n ≥ 6 pools of 2 embryos. “*” indicates a significant difference from control among mRNA data. “#” indicates a significant difference between populations.

Fig. 5

mRNA induction of the metabolic enzymes CYP1A, CYP1B1, and CYP1C1 in KC and ER embryos exposed to PCB126. Statistical analysis was performed using an ANOVA and Dunnett's post hoc test to determine treatments that differed from controls. For CYP1A, CYP1B1 and CYP1C1 the main effects and interaction of population and PCB126 treatment were significant (p < 0.001). CYP1A, CYP1B1 and CYP1C1 were induced at levels above control in KC embryos dosed with 0.1 and 1 μg/L PCB126 (p < 0.001). CYP1A, CYP1B1 and CYP1C1 were not significantly induced in ER embryos for any of the PCB126 treatments examined. Data represented as average fold induction ± SEM; n ≥ 6 pools of 2 embryos. “*” indicates a significant difference from control among mRNA data. “#” indicates a significant difference between populations.