Superfund Research Program
Mechanisms and Impacts of PCB Resistant Fish
Project Leader: Mark E. Hahn (Woods Hole Oceanographic Institution)
Co-Investigators: Sibel I. Karchner (Woods Hole Oceanographic Institution), Neelakanteswar Aluru (Woods Hole Oceanographic Institution)
Grant Number: P42ES007381
Funding Period: 1995-2020
Heritability of dioxin resistance in Fundulus from New Bedford Harbor:
Project investigators raised offspring (F1 fish) from fish collected at New Bedford and the reference site, Scorton Creek (SC). These F1 fish (adults) were exposed to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and project investigators subsequently measured [3H]-TCDD uptake and CYP1A expression. CYP1A1 was induced in livers of SC offspring, but not NBH offspring, despite similar uptake in the two populations. Thus, the dioxins resistance observed earlier is heritable.
Inducer-dependent differences in magnitude of resistance:
To investigate the magnitude of this resistance and to compare CYP1A1 inducibility by halogenated versus non-halogenated AHR agonists in these fish, CYP1A1 expression (as EROD activity) was measured in primary cultures of Fundulus hepatocytes treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or -naphthoflavone (BNF). Hepatocytes from NBH fish (EC50: 0.604 nm) were 14-fold less sensitive to TCDD than SC fish (EC50: 0.043 nm), but only 3-fold less sensitive to BNF (NBH EC50: 350 nm; SC EC50: 113 nm).
Functional characterization of two Ah receptors in Fundulus:
The full-length sequences of both AHR1 and AHR2 were obtained. Fundulus AHR1 encodes a 942-amino acid protein with a predicted molecular mass of 103.6 kDa. Fundulus AHR2 is 905-amino acids and 99.9 kDa. To begin to establish the functional characteristics of the two Fundulus AHR proteins, cDNAs encoding fAHR2 and fAHR1 were cloned into expression vectors and expressed using an in vitro transcription and translation (TnT) system. Velocity sedimentation analysis of the expressed proteins demonstrated that both fAHR1 and fAHR2 exhibit specific, high-affinity binding of [3H]-TCDD. Thus, both fAHR1 and fAHR2 encode bone fide "Ah receptors"; Fundulus is the first species shown to possess two AHR forms.