Some polybrominated diphenyl ether (PBDE) flame retardants with wide environmental distribution inhibit TCDD-induced EROD activity in primary cultured carp (Cyprinus carpio) hepatocytes.
Ethoxyresorufin-O-deethylase (EROD) activity, a catalytic function of the cytochrome P450 1A (CYP1A) microsomal oxygenase subfamily, is a popular biomarker for exposure to xenobiotics, polyhalogenated aromatic hydrocarbons (PHAHs) in particular. It has found wide use in aquatic pollution assessment both in vivo and in vitro. In such studies, subjects are often exposed to complex mixtures where various constituents can interfere with EROD-activity, possibly resulting in inadequate estimation of toxic hazard or biological response. The present study investigates the effects of polybrominated diphenyl ethers (PBDEs), a relatively new and increasingly detected group of environmental contaminants, on the validity of EROD activity as exposure marker in carp (Cyprinus carpio) hepatocytes. Freshly isolated hepatocytes of a genetically uniform strain of male carp were co-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations of 0, 1, 3, 10, 30, and 100 pM, and one of the highly purified PBDE/PCB congeners (at concentrations of 0, 0.25, and 2.5 microM) or cleaned-up and untreated DE-71 samples (0, 0.1, and 1 microM). PBDEs were selected from the 209 possible congeners based on their relative abundance in environmental samples: BDE-47, BDE-99, BDE-100, and BDE-153. A tentative metabolite of BDE-47, 6OH-BDE-47, was also included. In addition, a commercial pentabrominated dipenylether mixture (DE-71) was tested for interference with EROD activity both with and without clean-up by carbon fractionating which removed possible planar contaminants. Polychlorinated biphenyl (PCB)-153, a reported inhibitor of EROD activity in flounder, was included for comparison. Cells were cultured for a total period of 8 days; exposure started at day 3 after cell isolation. After 5 days of exposure, cell pellets were frozen before EROD activity was determined. Upon exposure to TCDD, the cells responded with increased EROD activity as expected. Significant reduction of TCDD-induced EROD activity was found in the presence of BDE-47, BDE-99, and BDE-153, but not with BDE-100 and 6-hydroxylated BDE-47. Of these PBDE congeners, the most abundant congener in environmental samples, BDE-47, exhibited the strongest inhibition (down to 6% of the TCDD control value). The cleaned-up fraction of commercial penta-BDE (DE-71) mixture proved an even more potent inhibitor, resulting in reduction of EROD activity to 4% of the control values observed at 1.0 microM. BDE-47 and BDE-153 did not reduce TCDD-induced EROD activity when added shortly prior to measurement, suggesting possible interaction with TCDD at the level of CYP1A biosynthesis. PCB-153 did not show significant effects on EROD activity in carp in this study. The present results indicate that environmentally relevant PBDEs can interfere with determination of EROD activity in vitro, at levels reported earlier for PCBs. The observation that detected PBDE levels are rising, stresses the need for caution when interpreting EROD data on environmental samples.
Kuiper
R V
RV
Institute for Risk Assessment Sciences (IRAS), Utrecht University, PO Box 80176, 3508 TD Utrecht, The Netherlands. r.v.kuiper@vet.uu.nl
Bergman
A
A
Vos
J G
JG
van den Berg
M
M
D000704:Analysis of Variance; D000818:Animals; D015415:Biomarkers; D002347:Carps / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D005411:Flame Retardants / Q000633:toxicity*; D055768:Halogenated Diphenyl Ethers; D022781:Hepatocytes / Q000378:metabolism*; D006842:Hydrocarbons, Brominated / Q000633:toxicity*; D008297:Male; D010647:Phenyl Ethers / Q000633:toxicity*; D011075:Polybrominated Biphenyls; D011078:Polychlorinated Biphenyls; D000072317:Polychlorinated Dibenzodioxins; D013997:Time Factors
D015415:Biomarkers; D005411:Flame Retardants; D055768:Halogenated Diphenyl Ethers; D006842:Hydrocarbons, Brominated; D010647:Phenyl Ethers; D011075:Polybrominated Biphenyls; D000072317:Polychlorinated Dibenzodioxins; C086401:pentabromodiphenyl ether; D011078:Polychlorinated Biphenyls; D019363:Cytochrome P-450 CYP1A1; C014024:2,4,5,2',4',5'-hexachlorobiphenyl