Acquired Resistance to Ah Receptor Agonists in a Population of Atlantic Killifish (Fundulus heteroclitus) Inhabiting a Marine Superfund Site: In Vivo and in Vitro Studies on the Inducibility of Xenobiotic Metabolizing Enzymes

Abstract

New Bedford Harbor (NBH), MA, is a federal Superfund site that is heavily contaminated with polychlorinated biphenyls (PCBs) and other halogenated aromatic hydrocarbons (HAHs), including some potent aryl hydrocarbon receptor (AhR) agonists. A population of Atlantic killifish (Fundulus heteroclitus) continues to inhabit this site, despite accumulating extraordinarily high concentrations of PCBs (272 microg/g dry weight). To determine if NBH killifish have developed resistance to HAHs that act through the AhR, we examined the inducibility of cytochrome P4501A1 (CYP1A1), UDP glucuronosyl transferase (UGT), and glutathione S-transferase (GST) in fish from NBH and a reference site, Scorton Creek (SC, Cape Cod, MA; PCB concentrations 0.177 microg/g dry weight). 2,3,7,8-Tetrachlorodibenzofuran (TCDF) induced CYP1A1 mRNA, protein, and activity in SC fish in all tissues examined (liver, heart, gut, gill, kidney, spleen, and gonad). In contrast, NBH fish expressed low levels of CYP1A1 and showed no induction of CYP1A1 mRNA, protein, or activity by TCDF, or induction that was lower in magnitude or required higher doses of inducer. p-Nitrophenol UGT activity was not induced by TCDF in either population, while GST activity with 1-chloro-2,4-dinitrobenzene as substrate was induced only in NBH fish in one experiment. Inducibility of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or beta-naphthoflavone (BNF) was measured in primary hepatocyte cultures prepared from SC and NBH fish. TCDD induced CYP1A1 activity (ethoxyresorufin O-deethylase) to the same degree in hepatocytes from both populations, demonstrating the functionality of the AhR signaling pathway in NBH fish. However, hepatocytes from NBH fish were 14-fold less sensitive to TCDD than were those from SC fish. The nonhalogenated AhR agonist BNF also induced CYP1A1 in cells from both populations, although with only a 3-fold difference in sensitivity (NBH < SC). These results indicate that chronic exposure to high levels of HAHs has led to a reduction in the sensitivity of NBH killifish to AhR agonists. The resistance is systemic and pretranslational, and exhibits compound-specific differences in magnitude. These findings suggest an alteration in the AhR signal transduction pathway in NBH fish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

“…Fundulus larvae and adults from other populations have a low basal level of CYP1A expression in proximal tubules of the kidney even in the control fish (Elskus et al, 1999;Bello et al, 2000). CYP1A is only active at low basal levels during the embryonic stages (Binder and Stegeman, 1984) and may not be detected using immunohistochemical techniques.…”

When evolution is the solution to pollution: Key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

“…However, it was soon discovered that embryonic, larval, and adult Atlantic killifish from polluted sites were refractory to the induction of the cytochrome P450 1A (CYP1A) response that is the hallmark biomarker of exposure to many DLCs and PAHs that were in such abundance at these sites (Bello et al., 2001; Elskus et al., 1999; Nacci et al., 1999; Powell, Bright, Bello, & Hahn, 2000; Prince & Cooper, 1995a; Van Veld & Westbrook, 1995). …”

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

“…They are tolerant of significant changes in many environmental conditions, including salinity (Griffith, 1974;Wood and Marshall, 1994;Nordlie, 2006), pH (Gonzalez et al, 1989), temperature (Umminger, 1971;Dunson et al, 1993;Smith and Able, 1994;Nordlie, 2006), and oxygen (Wannamaker and Rice, 2000;Smith and Able, 2003;Stierhoff et al, 2003;Nordlie, 2006). Specific killifish populations have adapted to a variety of anthropogenic stressors, including polycyclic aromatic hydrocarbons (PAH) (Meyer et al, 2002;Ownby et al, 2002;Meyer and Di Giulio, 2003), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Prince and Cooper, 1995a,b), polychlorinated biphenyls (PCB) (Nacci et al, 1999;Bello et al, 2001), and mercury (Weis et al, 1981). For these reasons, killifish have become one of the premier teleost environmental models, and certainly one of the most well-studied estuarine environmental models (Burnett et al, 2007).…”

“…Of the killifish populations known to have adapted to environmental contaminants, several have adapted specifically to chemicals that act via AhR-dependent mechanisms, and alterations to the AhR pathway seem to be a critical component of the adaptation (Prince and Cooper, 1995a,b;Nacci et al, 1999;Powell et al, 2000;Bello et al, 2001;Ownby et al, 2002;Bacanskas et al, 2004;Hahn et al, 2004). Specifically, all of these populations exhibit a marked reduction in CYP1A inducibility in response to a variety of AhR agonists (Prince and Cooper, 1995a;Elskus et al, 1999;Nacci et al, 1999;Bello et al, 2001;Arzuaga and Elskus, 2002;Meyer et al, 2002.…”