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.

Characterization of AHR1 and its functional activity in Atlantic sturgeon and shortnose sturgeon

“…RT-PCR was used to evaluate expression of AHR1 in shortnose sturgeon larvae that were water-borne exposed for 24 h to graded doses of four coplanar PCBs, TCDD, and an environmentally relevant Aroclor mixture and compared its expression to that previously quantified for AHR2 in the same specimens (Roy et al 2018). Unlike results for AHR2, we found no evidence of significantly induced AHR1 expression in shortnose sturgeon larvae for any of the six chemicals tested (Fig.…”

“…To empirically address this hypothesis, we cloned and characterized AHR1 in both Atlantic Coast sturgeon species in this study and AHR2 previously (Roy et al 2018). We demonstrated previously that AHR2 expression was significantly induced in larvae of both Atlantic Coast sturgeon species by exposure to TCDD, four different coplanar PCBs, and an Aroclor mixture (Roy et al 2018) and in some tissues of shortnose sturgeon by PCB126 (Roy et al 2011). Here, primers were also developed to quantify AHR1 expression in vivo in shortnose sturgeon larvae and tissues in RT-PCR assays.…”

“…Here, primers were also developed to quantify AHR1 expression in vivo in shortnose sturgeon larvae and tissues in RT-PCR assays. Furthermore, characterization of AHR1 here and AHR2 previously (Roy et al 2018) allowed us to insert the full length AHR1 and AHR2 coding sequences of both sturgeon species into expression vectors to evaluate their in vitro functional activities.…”

“…Eco RI– Eco RV fragments from 5’ AHR2 and Eco RV- Kpn I fragments from 3’ AHR2 were cloned into the linearized expression vector pcDNA3.1/Zeo(−). The cloned fragments in pAT-AHR2 and pSS-AHR2 were sequenced to ensure that they were identical to the Atlantic sturgeon and shortnose sturgeon AHR2 sequences previously determined (Roy et al 2018).…”

“…We have previously demonstrated that CYP1A mRNA expression in both Atlantic sturgeon and shortnose sturgeon was dose-responsive and significantly inducible at environmentally relevant concentrations of four coplanar PCBs (Roy et al 2018) and TCDD (Roy et al 2011). For example, significant CYP1A induction was observed in Atlantic sturgeon and shortnose sturgeon larvae that were waterborne exposed to TCDD at the lowest level tested, 0.001 parts per billion (ppb).…”