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

Nephrotoxicity assessments of benzo(a)pyrene during zebrafish embryogenesis

[[abstract]]Benzo(a)pyrene is a chemical produced during the process of making fried, roasted, and smoked foods. It remains unclear whether benzo(a)pyrene affects the early development of human organs. In this study, we used the transgenic zebrafish line Tg(wt1b:GFP) as a model to assess benzo(a)pyrene-induced kidney malformation. By soaking zebrafish embryos in benzo(a)pyrene at various doses (2, 20, and 200 ppb), only a minor effect on the survival rate was detected (0 ppb: 97.8 ± 1.9 %; 2–200 ppb: 89.1 ± 5.8–91.5 ± 8.3 %). However, benzo(a)pyrene significantly affected the development of the kidney (malformation rates ranges from 50.0 ± 3.5 to 77.4 ± 5.3 %). Various abnormalities, such as unusual curving of pronephric tubes, swollen glomerulus, and incomplete development of pronephric ducts, were observed. This study provides a rapid and effective protocol for the evaluation of the notable effects of benzo(a)pyrene on embryonic kidney development.[[notice]]補正完

Novel cDNA sequences of aryl hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments

Reproductive changes have been observed in painted turtles from a site with known contamination located on Cape Cod, MA, USA. We hypothesize that these changes are caused by exposure to endocrine-disrupting compounds and that genes involved in reproduction are affected. The aryl hydrocarbon receptor (AHR) is an orphan receptor that is activated by environmental contaminants. AHR mRNA was measured in turtles exposed to soil collected from a contaminated site. Adult turtles were trapped from the study site (Moody Pond, MP) or a reference site and exposed to laboratory environments containing soil from either site. The red-eared slider was used to assess neonatal exposure to soil and water from the sites. The environmental exposures occurred over a 13-month period. Juveniles showed an age-dependent increase in brain AHR1. Juvenile turtles exposed to the MP environment had elevated gonadal AHR1. Adult turtles exposed to the MP environment showed significantly decreased brain AHR2. The painted turtle AHR is the first complete reptile AHR cDNA sequence. Phylogenetic analysis of the painted turtle AHR showed that it clusters with other AHR2s. Partial AHR1 and partial AHR2 cDNA sequences were cloned from the red-eared slider. MEME analysis identified 18 motifs in the turtle AHRs, showing high conservation between motifs that overlapped functional regions in both AHR isoforms

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

The teleost swimbladder is assumed a homolog of the tetrapod lung. Both swimbladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swimbladder fails to inflate with exposure to 3,3’,4,4’,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P4501 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swimbladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependance of the effect of PCB126 on swimbladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swimbladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swimbladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swimbladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swimbladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swimbladder. Our results indicate that PCB126 blocks swimbladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swimbladder cells