The aryl hydrocarbon receptor and the cardiovascular system in zebrafish (Danio rerio)
Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized. Following method development, a series of experiments was performed on adult zebrafish (Danio rerio) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated. The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. 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 with several genes related to cardiac development and one primary gene linked to vascular development.
zebrafish, aryl hydrocarbon receptor, cytochrome P450, cyclooxygenase, cardiovascular
Master of Science (M.Sc.)