Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus
Introduction
The aryl hydrocarbon receptor (AHR) is part of the basic helix–loop–helix Per–ARNT–Sim (bHLH-PAS) protein superfamily of environmental sensors, developmental regulators, and co-activators (Crews, 1998, Gu et al., 2000, Ledent and Vervoort, 2001). Some of the most ubiquitous toxic compounds in the environment, including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated biphenyls (PCBs), and some polycyclic aromatic hydrocarbons (PAHs), elicit biochemical and toxic responses through the AHR signaling pathway. Following activation by a ligand such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the AHR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) (Reyes et al., 1992, Whitelaw et al., 1993), and interacts with AHR response elements (AHREs, also known as XREs or DREs) to regulate expression of genes encoding biotransformation enzymes (Negishi and Nebert, 1979, Nebert and Gonzalez, 1987, Telakowski-Hopkins et al., 1988). The AHR–ARNT complex also regulates numerous other genes involved in a variety of physiological processes (Puga et al., 2000). A negative regulatory loop in this pathway involves the related AHR repressor (AHRR), which represses AHR mediated gene transcription (Mimura et al., 1999, Karchner et al., 2002).
Gene and genome duplication events have expanded the vertebrate AHR gene family to include multiple AHR and AHRR genes. The number of AHR genes varies among taxa. For example, one AHR gene (Burbach et al., 1992) and one AHRR gene (Watanabe et al., 2001) have been identified in mammals, whereas up to six AHR paralogs (Hansson et al., 2004, Karchner and Hahn, 2004) and two AHRR paralogs (Evans et al., 2005) are present in bony fishes. The structural and functional diversity of AHR proteins may confer species- and strain-specific differences in the sensitivity to toxic AHR ligands (Hahn et al., 2005) and it is possible that numerous, possibly diverse, physiological roles are partitioned among multiple AHRs and AHRRs.
The Atlantic killifish Fundulus heteroclitus, distributed in shallow water habitats from Newfoundland to Florida (Able, 2002), is a versatile toxicological model used in field and laboratory research to elucidate the impacts and molecular mechanisms of chemical toxicity. Widespread and abundant, Atlantic killifish inhabit highly polluted areas where they are exposed to high concentrations of PCBs and PAHs (Lake et al., 1995, Bello, 1999, Nacci et al., 1999, Nacci et al., 2001). This has provided the opportunity to use killifish as a model to study environmental carcinogenesis (Vogelbein et al., 1990, Stine et al., 2004), endocrine disruption (Dube and MacLatchy, 2001, Sharpe et al., 2004, Boudreau et al., 2005, Greytak et al., 2005), PAH toxicity (Willett et al., 2001, Wassenberg and Di Giulio, 2004), and chemically mediated changes in gene expression (Peterson and Bain, 2004, Meyer et al., 2005). Killifish also have been used to investigate evolutionary adaptations to environmental change (Schulte, 2001, Cohen, 2002, Kraemer and Schulte, 2004, Oleksiak et al., 2005). Several geographically isolated populations of Atlantic killifish have independently developed heritable resistance to the toxic effects of contaminants in the environment (Prince and Cooper, 1995, Van Veld and Westbrook, 1995, Elskus et al., 1999, Nacci et al., 1999, Bello et al., 2001). The mechanisms underlying this resistance are not yet well understood, but in most populations, if not all, the AHR signaling pathway is involved (Wirgin and Waldman, 2004).
Several killifish genes in the AHR signaling pathway have been characterized, including CYP1A (Morrison et al., 1998), ARNT2 (Powell et al., 1999), AHR1 and AHR2 (Hahn et al., 1997, Karchner et al., 1999, Hahn et al., 2004), and AHRR (Karchner et al., 2002). Both killifish AHR1 and AHR2 bind TCDD and other AHR ligands in vitro and stimulate transactivation of reporter genes regulated by the CYP1A promoter in cultured mammalian cells. The killifish AHRR does not bind AHR ligands, but negatively regulates killifish AHR1 and AHR2 (Karchner et al., 2002).
Despite the intensity of research to characterize the AHR signaling pathway in killifish, a major limitation to identifying the physiological roles of the AHR and molecular mechanisms of resistance in this species is the lack of sensitive, specific antibodies to each AHR protein. Here we describe the development and screening of rabbit polyclonal antisera that distinguish among AHR family members from the Atlantic killifish and we examine the utility of these antibodies in AHR research.
Section snippets
Chemicals
2,3,7,8-Tetrachloro[1,6-3H]dibenzo-p-dioxin ([3H]TCDD; 35 Ci/mmol, 95% radiochemical purity) was purchased from Chemsyn Science Laboratories (Lenexa, KS), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were from Ultra Scientific (Hope, RI), l-[35S]methionine (> 1000 Ci/mmol, in vivo cell labeling grade) was from Amersham Biosciences (Piscataway, NJ). [14C]Ovalbumin was from NEN Research Products DuPont (Wilmington, DE) and [14C]catalase was synthesized and
Synthesis and purification of recombinant AHR1, AHR2, and AHRR proteins
To generate antibodies to killifish AHR1, AHR2, and AHRR, two constructs of each target protein, an N-terminal half and a C-terminal half (Fig. 1), were prepared for expression in E. coli. The N-terminal halves of the AHRs are comprised of the bHLH and PAS domains, the domains that are the most highly conserved among taxa. The intent of targeting the N-terminal halves of the AHR and AHRR proteins was to generate antibodies specific to each killifish protein, but with the possibility that they
Discussion
Investigations of the aryl hydrocarbon receptor signaling pathway in Atlantic killifish and other bony fish models have been hampered by the lack of suitable protein detection reagents. We have developed specific and sensitive polyclonal antisera against Atlantic killifish AHR1, AHR2 and AHRR. Our experiments demonstrate that these antibodies recognize in vitro-synthesized full-length proteins, proteins expressed by a mammalian cell line transfected with killifish AHR or AHRR constructs, and
Acknowledgements
We thank Drs. Richard Pollenz and Charles Rice for helpful technical discussions. Funding for this research was provided by National Institutes of Health, National Research Service Award (F32 ES05935) from the National Institute of Environmental Health Sciences (RRM), NIEHS Superfund Basic Research Program Grant P42 ES007381 at Boston University (MEH), and the Oliver S. and Jennie R. Donaldson Charitable Trust (MEH and RRM).
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2016, Aquatic ToxicologyCitation Excerpt :The expression plasmid for recombinant protein was isolated using a GeneJet Plasmid Miniprep Kit (Thermofisher), and used to transform DE3 E. coli (Arctic Express system, Agilent) using directions provided by the manufacturer. The transformed cells were then prepared for growth and induction of recombinant proteins as previously described (Merson et al., 2006). The culture was then centrifuged at 4000g for 20 min at 4 °C to obtain cell pellets containing recombinant proteins.
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