Phenotypic anchoring of gene expression after developmental exposure to aryl hydrocarbon receptor ligands in...

The genes mediating developmental aryl hydrocarbon (AhR)-induced cardiovascular deformities remain unclear, with many cytochrome P450 monooxygenase (CYP)-1 isoforms known to be AhR-responsive and now a cyclo-oxygenase (COX) isoform suspected to contribute developmental toxicity. More importantly, no previous study has examined the role of these genes in producing deformities using low enough concentrations of AhR agonists to permit survival beyond early larval stages. Zebrafish (Danio rerio) eggs were aqueously exposed to a variety of agents that had multiple modes of action, but all of which are reported to be AhR ligands; benzo-a-pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with resveratrol or alpha-naphthoflavone (ANF). Whole larvae CYP (subtypes 1A, 1B1, 1C1, and 1C2) and COX (subtypes 1, 2a, and 2b) mRNA expression was quantified at 5 and 10 dpf and correlated with developmental phenotype. Both TCDD and BaP caused dose-dependent AhR-associated deformities and mortalities by 10 dpf, while BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at 5 dpf with all of these larvae having died at the highest rate by 10 dpf. Furthermore, BaP/ANF co-exposure caused the most marked alterations in cardiac and vascular morphology at 10 dpf at the concentrations used, namely decreased ventricular length and chamber width with increased ventricular wall thickness, as well as increased blood vessel luminal diameter. Exposure to TCDD, BaP and ANF alone all significantly increased CYP1A mRNA expression, while only TCDD consistently increased CYP1C1 expression. In contrast, TCDD transiently decreased CYP1C2 expression. BaP alone had no effect on CYP1C1 expression, but decreased COX2b expression when alone or in combination with ANF. In fact, ANF exhibited additive agonistic effects on expression of CYP1A and CYP1C1 with both BaP and TCDD, although additive or potentiating effects of ANF on CYP1C2 and COX2b were observed with only BaP. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf. Principal components analysis suggests that cardiac deformities and blood vessel dilation were related positively to CYP1A and negatively to COX-2b gene expression. These relationships were separate from the one gene, CYP1C1, that was negatively associated with increased vascular wall thickness. However, further experiments are needed to confirm this difference and to determine whether the relationship is causative or merely associative.