Synergistic embryotoxicity of polycyclic aromatic hydrocarbon aryl hydrocarbon receptor agonists with cytochrome P4501A inhibitors in Fundulus heteroclitus

Abstract

Widespread contamination of aquatic systems with polycyclic aromatic hydrocarbons (PAHs) has led to concern about effects of PAHs on aquatic life. Some PAHs have been shown to cause deformities in early life stages of fish that resemble those elicited by planar halogenated aromatic hydrocarbons (pHAHs) that are agonists for the aryl hydrocarbon receptor (AHR). Previous studies have suggested that activity of cytochrome P4501A, a member of the AHR gene battery, is important to the toxicity of pHAHs, and inhibition of CYP1A can reduce the early-life-stage toxicity of pHAHs. In light of the effects of CYP1A inhibition on pHAH-derived toxicity, we explored the impact of both model and environmentally relevant CYP1A inhibitors on PAH-derived embryotoxicity. We exposed Fundulus heteroclitus embryos to two PAH-type AHR agonists, ss-naphthoflavone and benzo(a)pyrene, and one pHAH-type AHR agonist, 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB-126), alone and in combination with several CYP1A inhibitors. In agreement with previous studies, coexposure of embryos to PCB-126 with the AHR antagonist and CYP1A inhibitor alpha-naphthoflavone decreased frequency and severity of deformities compared with embryos exposed to PCB-126 alone. In contrast, embryos coexposed to the PAHs with each of the CYP1A inhibitors tested were deformed with increased severity and frequency compared with embryos dosed with PAH alone. The mechanism by which inhibition of CYP1A increased embryotoxicity of the PAHs tested is not understood, but these results may be helpful in elucidating mechanisms by which PAHs are embryotoxic. Additionally, these results call into question additive models of PAH embryotoxicity for environmental PAH mixtures that contain both AHR agonists and CYP1A inhibitors.

Figure 1. Dose–response curves showing percent control in ovo EROD induction and deformity index in embryos exposed to (A) BNF or (B) ANF. EROD values are missing for the 1,000, 5,000, and 10,000 μg/L concentrations because embryos from these treatment groups were too deformed to score for in ovo EROD. For the BNF control group, n = 20; for all other BNF treatments, n = 9 or 10. For each ANF treatment group, n = 8–10. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.

Figure 2. Effects of BNF with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index. The EROD value is missing for the 110 μg/L BNF + ANF treatment group because embryos in this treatment group were too deformed to score for in ovo EROD; n = 8 or 9 for each treatment group, except for EROD measurement in the 1.1 μg/L BNF + ANF (n = 6) and 11 μg/L BNF + ANF (n = 2) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.

Figure 3. Effects of BNF with and without 1 or 9 mg/L PBO cotreatment on in ovo EROD and deformity index; n = 7–10 for each treatment group, except for EROD measurements in the 50 μg/L BNF + 9 mg/L PBO (n = 5) and 100 μg/L BNF + PBO (n = 6) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.

Figure 4. Effects of BaP with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index; n = 9 or 10 for each treatment group, except for EROD measurement in the 5 μg/L BaP + ANF (n = 7), 10 μg/L BaP + ANF (n = 7), and 100 μg/L BaP + ANF (n = 3) treatment groups, because the remainder of embryos were too deformed to score for in ovo EROD. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.

Figure 5. Effects of FL (A) and AA (B) with and without 1 μg/L BNF cotreatment on in ovo EROD and deformity index. For (A), n = 9 or 10 for each treatment group, except for EROD measurement in the 1 μg/L BNF + 1,000 μg/L FL treatment group (n = 4), because the remainder of embryos were too deformed to score for in ovo EROD. For (B), n = 16 and 19 for control and BNF-alone treatment groups, respectively; for other treatment groups, n = 6–10. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.

Figure 6. Effects of PCB-126 with and without 100 μg/L ANF cotreatment on in ovo EROD and deformity index; n = 9 or 10 for all treatment groups. EROD values are mean ± SEM. See “Results” for explanation of statistical differences.