CYP1B1 knockdown does not alter synergistic developmental toxicity of polycyclic aromatic hydrocarbons in zebrafish (Danio rerio)

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are contaminants increasing in the environment largely due to burning of fossil fuels. Our previous work identified a synergistic toxicity interaction in zebrafish embryos occurring when PAHs that are agonists for the aryl hydrocarbon receptor (AHR) co-occur with PAHs that are CYP1A inhibitors. This toxicity is mediated by the AHR2, and morpholino knockdown of CYP1A exacerbated toxicity. This study tested two hypotheses: (1) in the absence of functional CYP1A, metabolism of PAHs is shunted towards CYP1B1, which has been shown in mammals to produce more reactive metabolites of PAHs; alternatively, (2) CYP1B1 serves a protective role similar to CYP1A. We used a morpholino approach to knockdown CYP1B1 alone and in co-knockdown with CYP1A to determine whether we could alter deformities caused by synergistic toxicity of PAHs. CYP1B1 knockdown was not different from non-injected controls; nor were CYP1B1+CYP1A co-knockdown deformities different from CYP1A knockdown alone. These data suggest that CYP1B1 is not a significant factor in causing synergistic toxicity of PAHs, nor, in contrast to CYP1A, in providing protection.

Fig. 1

Quantification at 96 hours post fertilization of pericardial edema (a), jaw-eye gap length (b), and representative images (c) of non-injected (NI; i.e. no morpholino), CYP1B1-MO, CYP1A-MO, and CYP1A+1B1-MOs exposed to the AHR agonist BNF, CYP1A inhibitor ANF, alone and in combination, and DMSO vehicle control. A two-factor ANOVA for injection and treatment was significant for both factors and interaction term (p≤0.008). Fisher’s PLSD was used as a posthoc test. Both deformity measures show that CYP1B1-MO is indistinguishable from NI (edema p=0.2, jaw p=0.9) and that CYP1A+1B1-MOs are not different from CYP1A alone (edema p=0.7, jaw p= 0.6). Knockdown of CYP1A with and without CYP1B1-MO exacerbates toxicity at the coexposure dose of 1µg/L BNF+ 50µg/L ANF, a dose not toxic to NI or CYP1B1-MO larvae (p≤ 0.01). All groups showed similar toxicity levels at the dose of 1µg/L BNF+ 100µg/L ANF, which were significantly elevated above single treatments and vehicle controls (p< 0.0001). Images of deformed larvae are outlined in black.

Fig. 2

Immunolocalization of CYP1B1 (530 nm excitation and 586 nm emission filter; 100X, f500 microscopy). (A) Control-morpholino embryo exposed to PCB 126 showing induction of CYP1B1 indicated by strong fluorescence in epithelial cells. (B) Control-morpholino embryo exposed to DMSO showing less CYP1B1 induction, as indicated by limited fluorescence compared to (A). (C) CYP1B1 morpholino embryo exposed to DMSO and (D) CYP1B1 morpholino embryo exposed to PCB 126 showing less CYP1B1 induction as indicated by limited fluorescence compared to (A).