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
The overall objective of this project is to understand the mechanisms underlying differential sensitivity to the developmental toxicity of dioxins. Project investigators are addressing this question by elucidating the differences in the expression and function of two distinct aryl hydrocarbon receptors (AHR1 and AHR2) and an AHR repressor during development, using a fish model of dioxin sensitivity and resistance. Previously, a population of the estuarine teleost Fundulus heteroclitus from New Bedford Harbor (NBH) has been characterized. The researchers showed that these fish were approximately 15-fold less sensitive to TCDD than F. heteroclitus from a reference site, Scorton Creek, Sandwich, MA (SC). To investigate the role of the aryl hydrocarbon receptor (AHR) signal transduction pathway in this dioxin resistance, scientists measured the relative levels of AHR1, AHR2, and ARNT2 mRNA in tissues of adult fish from SC and NBH. Adult SC fish expressed AHR2 and ARNT2 mRNA in all tissues examined, while AHR1 was expressed predominantly in brain, heart, and gonad. In contrast, AHR1 mRNA was widely expressed in NBH fish, appearing with unusual abundance in gill, gut, kidney, liver, and spleen. This AHR1 expression pattern was not observed in the lab-reared progeny of NBH fish, demonstrating that constitutive AHR1 expression in gill, gut, kidney, liver, and spleen is not a heritable phenotype. Furthermore, widespread AHR1 expression was not induced in reference-site fish by TCDD or PCB mixtures, suggesting that aberrant AHR1 expression is not simply a normal physiological response to contaminant exposure.
Evidence for altered regulation of AHR1 in New Bedford fish prompted the investigators to sequence multiple clones from the NBH and SC populations. These studies identified an AHR1 polymorphism in Fundulus, involving at least two AHR1 alleles that differ at 8 amino acids (of 942). A survey of the two populations showed that the two AHR1 alleles are present at approximately equal frequencies in the reference population, whereas one allele predominates (95%) in NBH fish. When the two AHR1 alleles were expressed in vitro, they did not differ in their TCDD-binding affinity. Current efforts are underway to explore possible allele-specific differences in other functions.
Another possible mechanism for the dioxin resistance of NBH fish is the enhanced expression of a repressor of AHR function. One such repressor, denoted AHR repressor (AHRR), has been identified in mice (Mimura et al., 1999, Genes Dev.). Using RT-PCR with degenerate primers, we cloned and sequenced an AHRR homolog from Fundulus (FhAHRR). Preliminary RT-PCR analyses indicate that FhAHRR expression is not altered in the adult resistant fish, on a whole tissue basis. However, FhAHRR expression is induced by TCDD treatment of reference fish. Future studies will examine the interaction of FhAHRR and AHR1 and AHR2 using transfection of mammalian cells.