Characterization and cDNA cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster.
In order to understand the roles of the epoxide hydrolases (EHs) in xenobiotic biotransformation in insects, we examined the induction of EHs by exogenous compounds in Drosophila melanogaster third instar larvae. Among the chemicals tested, clofibrate, a phenoxyacetate hypolipidermics drug, increased EH activity towards cis-stilbene oxide approximately twofold in larval whole-body homogenates. The same dose of clofibrate also induced glutathione S-transferase activity. The effect of clofibrate on EH induction was dose-dependent and the highest activity occurred with a 10% clofibrate application. Three other substrates conventionally used in EH assays (trans-stilbene oxide, trans-diphenylpropene oxide and juvenile hormone III) were poorly hydrolysed by larval homogenates, with or without clofibrate administration. Because the increased EH activity was localized predominantly in the microsomal fraction, we synthesized degenerate oligonucleotide primers with sequences corresponding to conserved regions of known microsome EHs from mammals and insects in order to isolate the gene. The 1597 bp putative cDNA of D. melanogaster microsomal EH (DmEH) obtained from a larval cDNA library encoded 463 amino acids in an open reading frame. Northern blot analysis showed that the transcription of DmEH was increased in larvae within 5 h of clofibrate treatment. Recombinant DmEH expressed in baculovirus hydrolysed cis-stilbene oxide (23 nmol.min-1.mg protein-1) and was located mainly in the microsomal fraction of virus-infected Sf9 cells. There was no detectable EH activity toward juvenile hormone III. These observations suggest that DmEH is involved in xenobiotic biotransformation, but not in juvenile hormone metabolism, in D. melanogaster.
Taniai
Kiyoko
K
Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, Tsukuba, Japan. taniai@affrc.go.jp
Inceoglu
Ahmet B
AB
Yukuhiro
Kenji
K
Hammock
Bruce D
BD
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D002994:Clofibrate / Q000494:pharmacology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger; D004851:Epoxide Hydrolases; D002994:Clofibrate