Characterization and expression of cytochrome P4501A in Atlantic sturgeon and shortnose sturgeon experimentally exposed to coplanar PCB 126 and TCDD

Abstract

The AHR pathway activates transcription of CYP1A and mediates most toxic responses from exposure to halogenated aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs. Therefore, expression of CYP1A is predictive of most higher-level toxic responses from these chemicals. To date, no study had developed an assay to quantify CYP1A expression in any sturgeon species. We addressed this deficiency by partially characterizing CYP1A in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and shortnose sturgeon (Acipenser brevirostrum) and then used derived sturgeon sequences to develop reverse transcriptase (RT)-PCR assays to quantify CYP1A mRNA expression in TCDD and PCB126 treated early life-stages of both species. Phylogenetic analysis of CYP1A, CYP1B, CYP1C and CYP3A deduced amino acid sequences from other fishes and sturgeons revealed that our putative Atlantic sturgeon and shortnose sturgeon CYP1A sequences most closely clustered with previously derived CYP1A sequences. We then used semi-quantitative and real-time RT-PCR to measure CYP1A mRNA levels in newly hatched Atlantic sturgeon and shortnose sturgeon larvae that were exposed to graded doses of waterborne PCB126 (0.01–1000 parts per billion (ppb)) and TCDD (0.001–10 ppb). We initially observed significant induction of CYP1A mRNA compared to vehicle control at the lowest doses of PCB126 and TCDD used, 0.01 ppb and 0.001 ppb, respectively. Significant induction was observed at all doses of both chemicals although lower expression was seen at the highest doses. We also compared CYP1A expression among tissues of i.p. injected shortnose sturgeon and found significant inducibility in heart, intestine, and liver, but not in blood, gill, or pectoral fin clips. For the first time, our results indicate that young life-stages of sturgeons are sensitive to AHR ligands at environmentally relevant concentrations, however, it is yet to be determined if induction of CYP1A can be used as a biomarker in environmental biomonitoring.

Developmental toxicity, EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

“…At 96 hpf, ∼70% of the embryos exposed to PCB126 exhibited pericardial edema at the higher concentrations, and CYP1A mRNA expression was induced 6.17‐, 16.46‐, 46.98‐, and 41.72‐fold on exposure to 16, 32, 64, and 128 μg L −1 PCB126 concentrations, respectively. Notably, among the four key developmental stages studied (24, 72, 96, and 132 hpf), CYP1A mRNA expression showed the maximal induction at 132 hpf at all doses and this was associated with major developmental malformations, similar to a recent study of exposure of sturgeon species to PCB126 and TCDD where significant induction of CYP1A mRNA was observed even at the lowest doses but a lower expression was observed at the highest dose (Roy et al, ) like in our study. Exposure of zebrafish embryos to TCDD has been shown to increase CYP1A levels preceding the developmental toxicity endpoints (Andreasen et al, ; Prasch et al, ).…”

“…It is widely accepted that PAH toxicity is also largely AhR mediated, with activation of transcription of the CYP1A gene following exposure to several AhR ligands. In the embryos of many fish types, including killifish (Nacci et al, ; Wassenberg and Di Giulio, ; Arzuaga et al, ; Wills et al, 2009), medaka (Carney et al, ), Atlantic sturgeon ( Acipenser oxyrinchus oxyrinchus ) and shortnose sturgeon ( Acipenser brevirostrum ) (Roy et al, ), dab ( Limanda limanda ) (Kammann et al, ), salmon ( Salmo salar ) (Mortensen and Arukwe, ) and zebrafish and larvae; (Billiard et al, ; Timme‐Laragy et al, 2007; Otte et al, ), CYP1A‐encoded EROD enzyme activity, CYP1A protein and/or CYP1A mRNA expression are highly sensitive and dose responsive to induction on exposure to PCDD/Fs, PCBs, PAH, or TCDD.…”

Toxic effects of PCB126 and TCDD on shortnose sturgeon and Atlantic sturgeon

“…The AHRs have yet to be identified in sturgeon, but we recently demonstrated that AHR-mediated CYP1A expression is inducible in early life stages of shortnose sturgeon and Atlantic sturgeon by their exposure to environmentally relevant graded doses of TCDD and PCB126 [30]. Our results are consistent with the expectation that AHR-mediated toxic effects are conserved in sturgeon.…”

“…We have previously shown that CYP1A mRNA in both sturgeon is inducible and dose responsive to PCB126 and TCDD at environmentally relevant embryo burdens (minimal nominal water concentrations of 0.010 ppb for PCB126 and 0.001 ppb for TCDD) [30]. These results are indicative of activation of the AHR pathway and suggest that some early-life-stage toxicities mediated by AHR are possibly cooccurring.…”

A Dramatic Difference in Global Gene Expression between TCDD-Treated Atlantic Tomcod Larvae from the Resistant Hudson River and a Nearby Sensitive Population

“…Thus, RNA was analyzed for gene expression from 4 to 6 pools of tomcod larvae for each of three doses of TCDD and acetone control from each population. Total RNA concentrations for samples were measured in a NanoDrop2 spectrophotometer (Thermo Fisher Scientific) and used as a template for first strand cDNA synthesis using M-MLV Reverse Transcriptase (Promega, Madison, WI) as described in Roy etal. (2011) .…”