Biotransformation of polybrominated diphenyl ethers and polychlorinated biphenyls in beluga whale (Delphinapterus leucas) and rat mammalian model using an in vitro hepatic microsomal assay

Abstract

Although polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE) flame retardants are important organic contaminants in the tissues of marine mammals, including those species from the Arctic, there is exceedingly little direct evidence on congener-specific biotransformation. We determined and compared the in vitro metabolism of environmentally relevant PCB (4,4′-di-CB15, 2,3′,5-tri-CB26, 2,4,5-tri-CB31, 2,2′,5,5′-tetra-CB52, 3,3′,4,4′-tetra-CB77, 2,2′,4,5,5′-penta-CB101, 2,3,3′,4,4′-penta-CB105 and 2,3′,4,4′,5-penta-CB118), and PBDE (4,4′-di-BDE15, 2,4,4′-tri-BDE28, 2,2′,4,4′-tetra-BDE47, 2,2′,4,5′-tetra-BDE49, 2,2′,4,4′,5-penta-BDE99, 2,2′,4,4′,6-penta-BDE100, 2,2′,4,4′,5,5′-hexa-BDE153, 2,2′,4,4′,5,6′-hexa-BDE154 and 2,2′,3,4,4′,5′,6-hepta-BDE183) congeners using hepatic microsomes of a beluga whale (Delphinapterus leucas) from the Arviat (western Hudson Bay) area of the Canadian Arctic. Ortho–meta bromine-unsubstituted BDE15, BDE28 and BDE47 were significantly metabolized (100%, 11% and 5% depleted, respectively) by beluga, whereas control rat microsomes (from pooled male Wistar Han rats) metabolized BDE28, BDE49, BDE99 and BDE154 (13%, 44%, 11% and 17% depleted, respectively). CB15 and CB77 (putative CYP1A substrates) were more rapidly metabolized (100% and 93% depleted, respectively) by male beluga than CB26 and CB31 (CYP1A/CYP2B-like) (25% and 29% depleted, respectively), which were more rapidly metabolized than CB52 (CYP2B-like) (13% depleted). Higher chlorinated CB101 and CB105 showed no depletion. Rat control microsomes metabolized CB15 to a lesser extent (32% depleted) than beluga, but much more rapidly transformed CB52 (51% depleted, respectively). Within the 90 min in vitro assay time frame, the preference was towards metabolism of ortho–meta unsubstituted congeners (for both PCBs and PBDEs) in beluga whale, whereas for rat controls, meta–para unsubstituted congeners also substantially metabolized. For both beluga whale and rat, metabolic rates were inversely associated with the degree of halogenation. For the rapidly biotransformed CB15 and BDE15, water-soluble OH-metabolites were detected after incubation. These results indicate that CYP-mediated oxidative hepatic biotransformation is a metabolic pathway in the toxicokinetics of both PCB and PBDE congeners in beluga whales and in the rat model. This may suggest that the formation of potentially toxic oxidative PCB and PBDE products (metabolites), in addition to the parent pollutants, may be contributing to contaminant-related stress effects on the health of beluga whale.

Introduction

Cetaceans are exposed to persistent, bioaccumulative and potentially toxic substances through their diets, which they accumulate in lipid-rich tissues (blubber) throughout their long life spans (Houde et al., 2005). Currently used as flame retardant additives in polymeric materials, polybrominated diphenyl ethers (PBDEs) have been reported in blubber, and to a lesser extent in hepatic, tissue of a limited number of marine mammal species and populations (Andersson and Wartanlan, 1992, Haglund et al., 1997, de Boer et al., 1998, Boon et al., 2002, Ikonomou et al., 2002a, Ikonomou et al., 2002b, Law et al., 2002, She et al., 2002, Wolkers et al., 2004, Houde et al., 2005). In odontocetes cetaceans, PBDEs have been reported in the blubber and/or liver tissues of pilot whales (Lindström et al., 1999), killer whales (Rayne et al., 2004), and beluga whales from the St. Lawrence Estuary (Lebeuf et al., 2004, McKinney et al., 2006) and various Arctic regions (Law et al., 2003, McKinney et al., 2006, Wolkers et al., 2004). PBDEs have also been reported in dolphin species from the Mediterranean Sea (Pettersson et al., 2004) and in deep-sea inhabiting, Atlantic sperm whales (de Boer et al., 1998). In these reports, BDE47 was generally found to be the dominant congener, with lesser amounts of BDE99, BDE100, BDE153, and BDE154.

Great Lakes and Pacific coast fish (Ikonomou et al., 2002a, Law et al., 2003), and beluga whales (both those inhabiting the St. Lawrence Estuary and the Canadian Arctic regions) show temporally increasing PBDE levels (Stern and Ikonomou, 2000, Law et al., 2003, Lebeuf et al., 2004). PBDE concentrations in tissue (liver and blubber) of beluga whales are lower, but at increasingly comparable levels relative to PCBs and organochlorine (OC) pesticides (Metcalfe et al., 1999, Letcher et al., 2000a, Hobbs et al., 2003, McKinney et al., 2006). Multiple pathologies exhibited in the threatened (COSEWIC, 2004) St. Lawrence beluga whale population have been associated with PCB and OC exposure (De Guise et al., 1995), however PBDE exposure may be contributing to these contaminant-related effects associations.

Phase I cytochrome P450 (CYP) and phase II conjugative enzymes, mainly operating in the liver, catalyze the metabolism of many endogenous and xenobiotic compounds in organisms including marine mammals (Goksøyr and Förlin, 1992, Stegeman and Hahn, 1994, Lewis et al., 1998). A variety of CYPs, as well as epoxide hydrolase (EH) and UDP glucuronosyl transferase (UDPGT) enzymes, that have been found in beluga whale (White et al., 1994, White et al., 2000, McKinney et al., 2004), may influence the capacity of these cetaceans to detoxify and eliminate contaminants. As well, these catalytic processes can result in the formation of toxic, retained and/or persistent metabolites, e.g. methyl sulfonyl- (MeSO₂-) and hydroxylated- (OH-) PCBs (Letcher et al., 2000b, McKinney et al., 2006, Verreault et al., 2005a). Exposure to OH-PCB metabolites has resulted in thyroidogenic effects, altered vitamin A levels, and inhibition of phase II sulfation and glucuronidation in experimental organisms (Brouwer et al., 1986, Schuur et al., 1998, van den Hurk et al., 2002). Therefore, PCB biotransformation is potentially a toxifying process in exposed organisms.

In the case of PCBs, congener patterns in tissues from prey and predator species in relation to the expression and activities of CYP enzymes has given rise to a metabolic classification scheme for marine mammals (Boon et al., 1997). In beluga whale from Canadian populations, oxidative metabolism of PCBs has also been evidenced by the finding of OH- and MeSO₂-PCBs in the liver and/or adipose tissues (Letcher et al., 2000a, McKinney et al., 2006).

In general, the toxicokinetics of PBDEs and other BFRs of environmental importance is not nearly as well understood as that of PCBs in wildlife and in particular in marine mammals (Hakk and Letcher, 2003). Still, possible metabolites of PBDEs, the OH-PBDEs, have been found in the liver tissues of beluga whales (McKinney et al., 2006), in the blood plasma of a killer whale (Bennett et al., 2002), and in some fish and bird species (Asplund et al., 1999, Marsh et al., 2004, Valters et al., 2005, Verreault et al., 2005b). In addition, MeO-PBDEs have been reported in ringed seal, beluga whale and fish (Haglund et al., 1997, Asplund et al., 1999, Marsh et al., 2004, Wolkers et al., 2004). Both OH-PBDEs and MeO-PBDEs may be of metabolic or natural origins (Malmvärn et al., 2005, Teuten et al., 2005).

Direct evidence of PBDE metabolism to OH-PBDE metabolites was reported in laboratory rodents after oral administration of BDE47, BDE99, BDE100 and BDE209 (Örn and Klasson-Wehler, 1998, Hakk et al., 2002, Mörck et al., 2003). Catalytic debromination of higher brominated to lower brominated BDE congeners has also been recently reported. In the gut of diet-exposed common carp (Cyprinus carpio), within 2 h of exposure, debromination of BDE209 to octabromo- and nonabromo-BDE congeners, BDE183 to BDE154, and BDE99 to BDE47 was shown (Stapleton et al., 2004a, Stapleton et al., 2004b, Stapleton et al., 2004c). OH-PBDEs have shown estrogenic and thyroidogenic activities in experimental organisms (Meerts et al., 2000, Meerts et al., 2001). It has yet to be definitively demonstrated for beluga whale, or any Arctic marine mammal, that OH-PBDE and OH-PCB congener residues found in tissue are formed in whole or part due to the metabolism of PBDEs and PCBs that have accumulated in tissues, as opposed to the direct ingestion of these metabolites.

Ethical rationale generally prevents organohalogen dosing studies in marine mammals, but in vitro assays provide a viable alternative. The contaminant(s) of interest can be incubated with prepared sub-cellular liver fractions and metabolic activity can be examined by monitoring depletion of the parent compound(s) and/or formation of metabolites (Murk et al., 1994, Boon et al., 1998, de Boer et al., 1998, Letcher et al., 1998, White et al., 2000, van Hezik et al., 2001, Li et al., 2003a). In vitro assays will not provide information on the metabolism of slowly transformed congeners, due to the time constraints of the assay. In addition, these assays may not be representative of what actually happens in vivo as, for instance, only one process (in this case, CYP-mediated oxidative biotransformation) can be studied, when there are many processes (e.g. reductive reactions, phase II reactions) that may be involved in the potential biotransformation of a single congener. However, when studying organisms for which dosing is not an ethically sound option, in vitro assays are a useful tool to provide information as to the metabolic potential towards the contaminant of interest. In the current study, we examine and compare the in vitro metabolism of PBDE and PCB congeners of environmental significance in order to determine whether OH-PBDEs and OH-PCBs previously detected in beluga whale (McKinney et al., 2006, Wolkers et al., 2004) result from the metabolism of parent PCB and PBDE congeners present in beluga tissues. We address the hypothesis that biotransformation plays a role in the fate and bioaccumulation of PCBs and PBDEs in beluga whale via (1) CYP-mediated oxidative biotransformation resulting in the formation of OH-PCB and OH-PBDE metabolites, and (2) catalytic debromination of higher brominated to lower brominated PBDE congeners.