Dataset Information

Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD.

ABSTRACT: Atlantic sturgeon and shortnose sturgeon co-occur in many estuaries along the Atlantic Coast of North America. Both species are protected under the U.S. Endangered Species Act and internationally on the IUCN Red list and by CITES. Early life-stages of both sturgeons may be exposed to persistent aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs which are at high levels in the sediments of impacted spawning rivers. Our objective was to compare the PCBs and TCDD sensitivities of both species with those of other fishes and to determine if environmental concentrations of these contaminants approach those that induce toxicity to their young life-stages under controlled laboratory conditions. Because our previous studies suggested that young life-stages of North American sturgeons are among the more sensitive of fishes to coplanar PCB and TCDD-induced toxicities, we were interested in identifying the molecular bases of this vulnerability. It is known that activation of the aryl hydrocarbon receptor 2 (AHR2) in fishes mediates most toxicities to these contaminants and transcriptional activation of xenobiotic metabolizing enzymes such as cytochrome P4501A (CYP1A). Previous studies demonstrated that structural and functional variations in AHRs are the bases for differing sensitivities of several vertebrate taxa to aromatic hydrocarbons. Therefore, in this study we characterized AHR2 and its expression in both sturgeons as an initial step in understanding the mechanistic bases of their sensitivities to these contaminants. We also used CYP1A expression as an endpoint to develop Toxicity Equivalency Factors (TEFs) for these sturgeons. We found that critical amino acid residues in the ligand binding domain of AHR2 in both sturgeons were identical to those of the aromatic hydrocarbon-sensitive white sturgeon, and differed from the less sensitive lake sturgeon. AHR2 expression was induced by TCDD (up to 6-fold) and by three of four tested coplanar PCB congeners (3-5-fold) in Atlantic sturgeon, but less so in shortnose sturgeon. We found that expression of AHR2 and CYP1A mRNA significantly covaried after exposure to TCDD and PCB77, PCB81, PCB126, but not PCB169 in both sturgeons. We also determined TEFs for the four coplanar PCBs in shortnose sturgeon based on comparison of CYP1A mRNA expression across all doses. Surprisingly, the TEFs for all four coplanar PCBs in shortnose sturgeon were much higher (6.4-162 times) than previously adopted for fishes by the WHO.

SUBMITTER: Roy NK

PROVIDER: S-EPMC5855079 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD.

Roy Nirmal K NK Candelmo Allison A DellaTorre Melissa M Chambers R Christopher RC Nádas Arthur A Wirgin Isaac I

Aquatic toxicology (Amsterdam, Netherlands) 20180202

Atlantic sturgeon and shortnose sturgeon co-occur in many estuaries along the Atlantic Coast of North America. Both species are protected under the U.S. Endangered Species Act and internationally on the IUCN Red list and by CITES. Early life-stages of both sturgeons may be exposed to persistent aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs which are at high levels in the sediments of impacted spawning rivers. Our objective was to compare the PCBs and TCDD sensitivities of both speci ...[more]

PMID: 29427830

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Project description: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.

Project description:Sturgeon species are imperiled world-wide by a variety of anthropogenic stressors including chemical contaminants. Atlantic sturgeon, Acipenser oxyrinchus, and shortnose sturgeon, Acipenser brevirostrum, are largely sympatric acipenserids whose young life-stages are often exposed to high levels of benthic-borne PCBs and PCDD/Fs in large estuaries along the Atlantic Coast of North America. In previous laboratory studies, we demonstrated that both sturgeon species are sensitive to early life-stage toxicities from exposure to environmentally relevant concentrations of coplanar PCBs and TCDD. The sensitivity of young life-stages of fishes to these contaminants varies among species by three orders of magnitude and often is due to variation in the structure and function of the aryl hydrocarbon receptor (AHR) pathway. Unlike mammals, fishes have two forms of AHR (AHR1 and AHR2) with AHR2 usually being more highly expressed across tissues and functional in mediating toxicities. Based on previous studies in white sturgeon, A. transmontanus, we hypothesized that sturgeon taxa are unusually sensitive to these contaminants because of higher levels of expression and functional activity of AHR1 than in other fish taxa. To address this possibility, we characterized AHR1 in both Atlantic Coast sturgeon species, evaluated its' in vivo expression in young life-stages and in multiple tissues of shortnose sturgeon, and tested its ability to drive reporter gene expression in AHR-deficient cells treated with graded doses of PCB126 and TCDD. Similar to white sturgeon and lake sturgeon, AHR1 amino acid sequences in Atlantic sturgeon and shortnose sturgeon were more similar to mammalian AHRs and avian AHR1s than to AHR1 in other fishes, suggesting their greater functionality in sturgeon species than in other fishes. Exposure to graded doses of coplanar PCBs and TCDD usually failed to significantly induce AHR1 expression in young life-stages or most tissues of shortnose sturgeon. However, in reporter gene assays, AHR1 drove higher levels of gene expression than AHR2 alone, but their binary combination failed to drive higher levels of expression than either AHR alone. In total, our results suggest that AHR1 may be more functional in sturgeon species than in other fishes, but probably does not explain their heightened sensitivity to these contaminants.

Project description:Larval shortnose sturgeon, reared at 17°C, were subjected to delayed feeding treatments of 0, 5, 10, 15, 18, and 23 days post-yolk absorption to examine effects of food deprivation on growth, survival, swimming activity, and escape capabilities. Starvation affected growth and survival but despite degree of starvation, larvae were able to resume growth and experience high survivorship following feeding. Specific growth rate based on larval dry weight for the period directly following first feeding was highest for the day 15 and 18 delayed feeding treatments. There were no differences in survival between the 0 and 5 day treatments, however survival was reduced to 71.2%, 45.4%, and 28.8% for 10, 15, and 18 day delayed feeding treatments, respectively. Shortnose sturgeon had a point-of-no-return (PNR; 55.7% initiated feeding) at ~19 days (or 42 days post-fertilization) following the full absorption of yolk. Mean percent swimming activity and swimming speeds showed an interaction between delayed feeding treatment and larval age, such that no differences were detected at 1 and 6 days post-yolk absorption, while these swimming behaviors generally increased or spiked as feeding was delayed for 10, 15, and 18 days post-yolk absorption. At 23 days post-yolk absorption, only swimming speed increased for larvae that were denied food for 18 days. While there was an interaction between delayed feeding treatments and age for proportion of larvae exhibiting an escape response, generally, larvae from all feeding treatments exhibited a positive escape response. There were also interactions between delayed feeding treatments and age post-yolk absorption for mean and maximum escape speeds, such that less aggressive escape responses were typically detected the longer larvae were denied food. Our research suggests that larval shortnose sturgeon increase physical activity during periods of starvation to find a food patch while remaining vigilant but maybe not as capable to defend against a predatory attack as fed individuals.

Project description:Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), N(ω)-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.

Dataset Information

Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD.

Publications

Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD.

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