OH
O
Brx
Bry
Fengyan Liu1, Steve Wiseman1, Markus Hecker1,2, Yi Wan3, Jonathan Doering1, Paul Jones1,2, Michael Lam4, John Giesy1,4,5
1 Toxicology
Centre, University of Saskatchewan, Saskatoon, Canada 2 School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Canada 3 College of Urban and Environmental Science,
Peking University, Beijing, China 4 Department of Biology and Chemistry, and State Key Laboratory for Marine Pollution, City University of Hong Kong, Hong Kong, China 5 Department of Veterinary Biomedical
Sciences, University of Saskatchewan, Saskatoon, Canada
Introduction
 It has recently been shown hat naturally occurring methoxylated brominated
diphenyl ethers (MeO-BDEs), not synthetic polybrominated diphenyl ethers (PBDEs),
are the precursor of the hydroxylated brominated diphenyl ethers OH-BDEs (1,2).
Results – Study 1 (cont)
Influence of enzyme cofactors
Figure 2: Effect of co-factors on the
biotransformation of 6-MeO-BDE-47
to 6-OH-BDE-47 in rainbow trout
microsomes. BDL = below detection
limit. * p<0.05.
 OH-BDEs are more potent than PBDEs and MeO-BDEs.
 Natural origin of MeO-BDEs in the marine environment and great levels of MeOBDEs in some health products, may present risks to aquatic wildlife and
humans due to metabolic production of OH-BDEs.
 NADPH is required for the biotransformation of 6-MeO-BDE-47 to 6OH-BDE-47.
 The mechanism of biotransformation of MeO-BDEs to OH-BDEs is unknown.
Objectives
*
BDL
 Objective 1: Identify the enzyme(s) involved in biotransformation of 6-MeOBDE-47 to 6-OH-BDE-47 in fish.
 Objective 2: Investigate the in vitro hepatic metabolism of 6-MeO-BDE-47 to 6OH-BDE-47 in different fish species.
Involvement of CYP 1A1 and CYP 1A2
Figure 3: Effect of BNF exposure on
the biotransformation of 6-MeO-BDE47 to 6-OH-BDE-47 in rainbow trout
microsomes.
Methods
Study Design
 BNF stimulated greater EROD (Cyp1A1)
and MROD (Cyp1A2) activity.
Liver
Homogenization & centrifugation
 No effect of BNF on biotransformation
of 6-MeO-BDE-47 to 6-OH-BDE-47 in
microsomes from control and BNF
exposed trout.
S9 fraction
Ultracentrifugation
Microsomes
Cytosol
Study 1A
Phase Ⅱ
(DTT)
Phase Ⅰ
(NADPH)
Study1B
Study 1C
Study 1D
Study 2
Pharmacological approach
Candidate enzymes
Pharmacological approach
Figure 4: Concentration of 6-OHBDE-47 in microsomes incubated
with anti-Cyp1A or anti-Cyp-3A
antibodies, or inhibitors of Cyp1A,
Cyp2k or Cyp3A activity. * p<0.05.
 1A – Anti-Cyp1A antibody
 3A – Anti-Cyp3A antibody
 CL – Inhibitor of CYP 1A1, 2K1,
and 3A27
 BI – Inhibitor of CYP 2K1, 3A27
 GE – Inhibitor of CYP 3A27
 CL and BI significantly inhibited
biotransformation of 6-MeO-BDE-47
to 6-OH-BDE-47.
Enzyme kinetics in different species
 Study 1A: Cellular localization (microsomes, cytosol and S9 fraction) of the
biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47.
 Study 1B: Requirements of cofactors (NADPH and dithiothreitol [DTT]) for the
biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47
 Study 1C: Effects of cytochrome P450 enzyme inhibitors and antibodies on the
biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47
 Study 1D: Effect of aryl-hydrocarbon receptor activation (agonist BNaphthoflavone [BNF]) on biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47.
 Study 2: Species comparison of the biotransformation of 6-MeO-BDE-47 to 6-OH-BDE47 using microsomes from rainbow trout (Oncorhynchus mykiss), white sturgeon
(Acipenser transmontanus) and Goldfish (Carassius auratus auratus).
Results – Study 1
Localization of 6-OH-BDE-47 formation
Figure 1: Localization of the biotransformation of 6-MeO-BDE-47 to
6-OH-BDE-47 in Rainbow trout
microsomes. BDL: below detection
limit. * p<0.05.
*
 The greatest conversion of 6-MeOBDE-47 to 6-OH-BDE-47 occurred in
the microsomal fractions.
 6-OH-BDE-47 was not detected in
the cytosolic fraction.
References
1.
Wan, Y., Liu, F., Wiseman, S., Zhang, X., Chang, H., Hecker, M., Jones, P.D., Lam, M.H.W., Giesy, J.P., 2010. Interconversion of
hydroxylated and methoxylated polybrominated diphenyl ethers in Japanese medaka. Environ. Sci. Technol. 44, 8729–8735.
2.
Wan, Y., Wiseman, S., Chang, H., Zhang, X., Jones, P.D., Hecker, M., Kannan, K., Tanabe, S., Hu, J., Lam, M.H.W., Giesy, J.P., 2009.
Origin of hydroxylated brominated diphenyl ethers: natural compounds or man-made flame retardants? Environ. Sci. Technol. 43,
7536–7542.
3.
Liu F., Wiseman S., Wan Y., Doering J., Hecker M., Lam M.H.W., Giesy J.P. 2012. Multi-species comparison of the mechanism of
biotransformation of MeO-BDEs to OH-BDEs in fish. Aquat Toxicol 114-115: 182-188.
Acknowledgements
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Results – Study 2
Species differences in OH-BDE formation
Figure 5: Accumulation of 6-OHBDE-47 in microsomes from
rainbow trout, white sturgeon, and
goldfish.
 Formation of 6-OH-BDE-47
increased with incubation time.
 Significantly differences in the
biotransformation of 6-MeOBDE-47 to 6-OH-BDE-47 among
the three species.
Conclusions
 OH-BDEs formed in microsomes is catalyzed by NADPH dependent phase I enzymes.
 Biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47 is not catalyzed by CYP1A 1.
Cyp1A2 or CYP3A.
 CYP 2K is a likely candidate for catalyzing the biotransfromation 6-MeO-BDE-47 to
6-OH-BDE-47.
 Fish species differ in their capacity to biotransform 6-MeO-BDE-47 to 6-OH-BDE-47.
 Possible species differences in susceptibility to OH-BDE toxicity?
This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant [grant number 326415-07], and two Western Economic Diversification Canada grants (grant numbers 6971, 6807). The authors acknowledge the support of an
instrumentation grant from the Canada Foundation for Infrastructures. We acknowledge the support of the Aquatic Toxicology Research Facility (ARTF) at the Toxicology Centre, University of Saskatchewan. Special thanks to Ron Ek and the team at the Kootenay Trout Hatchery for supplying
white sturgeon embryos. Profs Giesy and Hecker were supported through the Canada Research Chair Program.