Characterization and profiling of hepatic cytochromes P450 and phase II xenobiotic-metabolizing enzymes in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary and the Canadian Arctic
Introduction
Marine mammals are particularly susceptible to contamination by organohalogens due to their large lipid reserves, relatively long life spans and elevated positions in the aquatic food web. A small, isolated and endangered population of beluga whales (Delphinapterus leucas) inhabits the St. Lawrence (SL) Estuary in Quebec, Canada, an area downstream of many sources of industrial and agricultural contaminants. Polychlorinated biphenyls (PCBs), organochlorine pesticides and other organohalogen contaminants have been detected in SL beluga tissues (e.g. Martineau et al., 1988, Muir et al., 1996, Gauthier et al., 1998, Metcalfe et al., 1999, Hickie et al., 2000, Hobbs et al., 2003) not only at elevated levels in comparison to Canadian Arctic (CA) belugas (Norstrom and Muir, 1994, Metcalfe et al., 1999), but also at levels known to elicit toxic effects in laboratory animals (Béland et al., 1993). A link has been suggested between tissue organochlorine levels and the health of the SL population (Martineau et al., 1994). Reproductive and immunological impairment and a high incidence of neoplasms are all strongly associated with pollutant exposure (De Guise et al., 1995).
Metabolic capacity is an important determinant in the bioaccumulation, biomagnification, toxicokinetics and potential toxicity of lipophilic organohalogen contaminants. The cytochrome P450 monooxygenases (CYPs) play a central role in the oxidative biotransformation (phase I) of a wide range of xenobiotic and endogenous compounds (Goksøyr and Forlin, 1992, Stegeman and Hahn, 1994, Lewis et al., 1998). Products from phase I metabolism are conjugated to larger endogenous molecules via catalytic mediation by phase II enzymes such as glutathione-S-tranferase (GST) and uridine diphosphoglucuronosyl transferase (UDPGT) (George, 1994, Wolkers et al., 1998). Phase I and II enzymes, most abundant in hepatic tissues, may transform lipophilic compounds into either detoxified or bioactivated forms. As there are qualitative and quantitative differences in the levels and inducibility of individual CYP isozymes among species and populations of species (Boobis et al., 1990, Smith, 1991), it is important to determine the ability of exposed animals to metabolically influence the toxicokinetics and fate of accumulated anthropogenic contaminants. There is limited documentation on the metabolic capacity of marine mammals towards xenobiotic compounds, and most reports are from seal populations of various species (Boon et al., 1992, Nyman et al., 2001). However, immunochemical characterization of CYP isozymes has been reported for some whale species and populations, mainly for families from the odontocete suborder. White et al., 1994, White et al., 2000 found homologues of CYP1A, CYP2B and CYP2E in the liver of CA beluga, and a CYP1A isoform has been characterized in various tissues of SL beluga (Wilson et al., 2000). CYP1B, CYP3A-like and CYP4A isozymes have been discovered in striped dolphin (Stenella coeruleoalba), pilot whale (Globicephala melas), minke whale (Balaenoptera acutorostrata) and/or sperm whale (Physeter macrocephalus) (Goksøyr et al., 1988, Goksøyr et al., 1989, Goksøyr, 1995, Celander et al., 2000, Godard et al., 2000, Boon et al., 2001). In beluga, catalytic characterization has been limited to CYP1A-mediated EROD, PROD, MROD (7-ethoxy-, pentoxy- and methoxyresorufin O-deethylase, respectively) and aryl hydrocarbon hydroxylase (AHH) activities (White et al., 1994, White et al., 2000, Addison et al., 1998). These studies rely on enzymatically-active hepatic tissues, but in contrast to laboratory specimens, it is often difficult to obtain well-preserved, enzymatically-viable tissues from stranded free-ranging animals. Therefore, catalytic activity as a quantitative indicator of metabolic potential is questionable in situations where liver preservation cannot be assured. Immunological profiling is a more robust technique to measure phase I and II enzymes, providing suitably cross-reactive Ab interactions are found. On the other hand, immunochemical characterization is not necessarily fully representative of existing protein profiles as identification depends on the constitutive and induced levels, and on the cross-reactivity of the selected antibodies for specific microsomal proteins (Goksøyr, 1995, Letcher et al., 1996, Lewis, 2000).
To more completely determine the metabolic potential of beluga whale, a more comprehensive assessment is required of hepatic xenobiotic-metabolizing enzymes in individuals from populations of contrasting exposure to contaminants that are capable of enzyme induction or suppression. In the present study, major phase I and II isozymes, CYP1A, CYP2B, CYP3A, CYP2E, EH and UDPGT, known to be involved in xenobiotic metabolism, were immunologically and catalytically characterized in the hepatic microsomes of belugas from the SL (tissue collected from stranded individuals) and from the Arviat region of western Hudson Bay in the Canadian Arctic (fresh tissue collected as part of native subsistence hunting). The results were used to evaluate the use of immunologic expression and catalytic activity of xenobiotic-metabolizing enzymes in the optimally preserved liver of beluga from a CA population as a model of metabolic potential in SL and other beluga populations.
Section snippets
Reagents
Bovine serum albumin (BSA), Coomassie Brilliant Blue G-250, potassium chloride, sodium dithionite, l-ascorbic acid, phenazine methosulfate, 7-ethoxyresorufin, resorufin, sodium dodecyl sulfate (SDS), bromophenol blue, 4-chloro-1-naphthol, Brij-58, 1-naphthol, 1-naphthyl β-d-glucuronide sodium salt and uridine 5′-diphosphoglucuronic acid trisodium salt were purchased from Sigma (St. Louis, MO, USA). Disodium ethylenediaminetetraacetate (Na2EDTA), acrylamide/bisacrylamide solution (30%, w/v,
Microsomal protein and total CYP content
Microsomal protein and total CYP contents of the beluga liver samples are given in Table 2. Dithionite difference spectra of SL1 to SL5 showed a peak at 420 nm, but not at 450 nm, even when treated with ascorbic acid and phenazine methosulfate (PMS) to remove the interfering CO-hemoglobin peak at 420 nm. Spectra of SL6 and the CA individuals showed a large 420 nm peak and no detectable 450 nm peak using the conditions of Omura and Sato (1964). However, when the SL6 and CA1 to CA10 microsomes were
Discussion
Although biotransformation is often a major factor in the toxicokinetics and fate of organic contaminants, little is known regarding characterization and catalytic activity of phase I and II xenobiotic-metabolizing enzymes in marine mammals. A greater understanding of metabolic potential is particularly important in marine mammals that are exposed to high levels of contaminants, such as those living in the St. Lawrence River Estuary. In this study, antibodies against different phase I and II
Conclusions
We report on the detailed immunochemical and catalytic profiling of the enzymes mediating xenobiotic-metabolism in beluga whales from more than one distinct population. Immunochemical results indicated that profiles of several major CYP enzymes, EH and UDPGT are similar between animals from the two populations, and for certain enzymes are comparable with previous hepatic enzyme characterization in beluga and other odontocete species. For the first time, proteins homologous to CYP3A, EH and
Acknowledgements
We would like to thank the World Wildlife Fund Canada (Endangered Species Recovery Fund) (to R.J. Letcher and P. Béland), the St. Lawrence National Institute of Ecotoxicology and the Department of Fisheries and Oceans Canada for financial support and/or assistance with sampling of St. Lawrence belugas, and Richard Plante and Carl Guimont from Filmar for recovering and transporting carcasses to our facility over the last 17 years. We would also like to thank the Arviat Hunters and Trappers
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