Superfund Research Program
Mechanisms of Dioxin Sensitivity and Acquired Resistance
Experimental toxicology and epidemiology have shown that species, populations, sexes, and developmental stages differ in sensitivity to chemical-induced disease. Differences in sensitivity to the reproductive, immunotoxic, and carcinogenic effects of chlorinated dioxins, polychlorinated biphenyls (PCBs) and related planar halogenated aromatic hydrocarbons (PHAHs) are well known. In addition, long term exposure to PHAH in the environment can lead to acquired resistance in highly exposed populations such as those inhabiting Superfund sites. Such differential sensitivity complicates evaluation of the risks that these compounds pose to human and ecological health. An understanding of the biological mechanisms underlying this differential sensitivity could help resolve some of the questions surrounding the health risks from PHAHs.
As part of the Boston University Superfund Basic Research Program, scientists at the Woods Hole Oceanographic Institution (WHOI) are conducting research that seeks to understand the mechanisms of animal resistance to PHAHs, especially resistance that develops in natural populations of animals after long-term exposure associated with hazardous waste sites. Recent studies have been conducted using a PHAH-resistant population of killifish (Fundulus heteroclitus) that resides in the Acushnet River Estuary near New Bedford Harbor, Massachusetts, a site highly contaminated with PCBs.
Induction of cytochrome P4501A1 (CYP1A1) can serve as a useful marker for exposure to and effects of PHAHs. Unlike fish inhabiting other sites contaminated with PHAHs, however, Fundulus from New Bedford Harbor do not exhibit elevated levels of CYP1A1 protein or catalytic activity, despite accumulating high concentrations of PCBs. The WHOI researchers compared the "dioxin" responsiveness of New Bedford Harbor fish to that of Fundulus collected at a reference site, Scorton Creek, MA. Scorton Creek fish showed strong induction of hepatic CYP1A mRNA, protein, and activity after treatment with the model PHAH 2,3,7,8-tetrachlorodibenzofuran (TCDF). In contrast, no induction of hepatic CYP1A1 was observed in New Bedford Harbor fish. The same difference in responsiveness was also seen in extrahepatic tissues (heart, gill, kidney) from these fish. Experiments using primary cultures of hepatocytes from Scorton Creek and New Bedford Harbor fish showed that New Bedford Harbor fish were approximately 14-fold less sensitive to CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results suggest that long term exposure to high levels of PHAH can alter the responsiveness of some fish populations to these compounds, and that Fundulus heteroclitus can serve as a single-species model of both dioxin sensitivity and resistance.
The WHOI researchers have also been characterizing the mechanism of dioxin resistance in killifish by studying proteins - namely, the aryl hydrocarbon (Ah) receptor and Ah receptor nuclear translocator (ARNT) - through which dioxins and coplanar PCBs act to cause toxicity. While studying the mechanism of resistance, investigators recently discovered that there are two Ah receptor genes in killifish, unlike the single form identified so far in humans. Both killifish Ah receptor forms (designated AHR1 and AHR2) have been cloned and their amino acid sequences determined. The researchers found evidence for alternatively spliced forms of both Ah receptors in some tissues. The sequence of the Fundulus ARNT protein has also been determined. Future research in this area will determine if AHR1, AHR2, ARNT or their alternatively spliced forms are involved in the PHAH resistance in this population of killifish.
In related studies, a Fundulus CYP1A1 was cloned and sequenced. CYP1A1 proteins have multiple roles in dioxin toxicity, including the generation of reactive oxygen species (ROS), a process enhanced by interaction with poorly metabolized substrates such as certain PHAH. The potential roles of CYP1A1 and ROS in dioxin toxicity and resistance in Fundulus are also being examined.
These studies of dioxin resistance in Fundulus are being coordinated in part with researchers at the EPA laboratory in Narragansett, Rhode Island, who are studying the resistance of these fish to the embryo-larval toxicity of dioxins and its implications for an ecological risk assessment being carried out at the New Bedford Harbor site.
In addition to its utility in a specific ecological risk assessment, this research is significant in a broader context in that it is providing a fundamental understanding of the mechanisms underlying PHAH resistance. A comparison of what is learned in this natural population of fish to what is known from studies with laboratory rodents will provide an evolutionary perspective on the mechanism of dioxin action and its conservation in diverse vertebrate species. This knowledge will contribute to understanding similar aspects of dioxin toxicity in humans, including inter-individual variability in susceptibility.
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To learn more about this research, please refer to the following sources:
- Bello SM, Franks DG, Stegeman JJ, Hahn ME. 1998. Dioxin Resistance in Killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund Site: In Vivo and In Vitro studies of CYP1A1 Inducibility. In: Proceedings of the Society of Environmental Toxicology and Chemistry (SETC) 19th Annual Meeting. Charlotte, NC. pp.168.
- Hahn ME. 1998. Mechanisms of innate and acquired resistance to dioxin-like compounds. Reviews in Toxicology 2:395-443.
- Hahn ME. 1998. The aryl hydrocarbon receptor: a comparative perspective. Comp Biochem Physiol 121(1-3):23-53. PMID:9972449
- Morrison HG, Weil EJ, Karchner SI, Sogin ML, Stegeman JJ. 1998. Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1A genes: Update with new sequences. Comp Biochem Physiol 121C:231-240. PMID:9972465
- Powell WH, Karchner SI, Hahn ME. 1998. Aryl Hydrocarbon Signaling Components in Fundulus heteroclitus: A Phylogenetic Perspective. In: Proceedings of the Society of Environmental Toxicology and Chemistry (SETC) 19th Annual Meeting. Charlotte, NC.
- Schlezinger JJ, Struntz WD, Stegeman JJ. 1998. Cytochrome P4501A (CYP1A) and planar halogenated hydrocarbons (pHAH): Oxidative inactivation and reactive oxygen production. In: Proceedings of the 37th Society of Toxicology Annual Meeting, March 1-5. Seattle, WA.
- Bello SM, Stegeman JJ, Hahn ME. 1997. Lack of cytochrome P450-1A inducibility in Fundulus heteroclitus from New Bedford Harbor: A case of dioxin resistance?. In: Proceedings of the Society for Environmental Toxicology and Chemistry Annual Meeting. San Francisco, CA.
- Hahn ME, Karchner SI, Shapiro MA, Perera SA. 1997. Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family. Proc Natl Acad Sci U S A 94(25):13743-13748. PMID:9391097 PMCID:PMC28377
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