Characterization and cDNa cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster

Kiyoko Taniai, Ahmet B. Inceoglu, Kenji Yukuhiro, Bruce D. Hammock

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 melangaster 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.

Original languageEnglish (US)
Pages (from-to)4696-4705
Number of pages10
JournalEuropean Journal of Biochemistry
Volume270
Issue number23
DOIs
StatePublished - Dec 2003

Fingerprint

Epoxide Hydrolases
Clofibrate
Cloning
Drosophila melanogaster
Organism Cloning
Xenobiotics
Biotransformation
Phenoxyacetates
Larva
Insects
Sf9 Cells
Juvenile Hormones
Mammals
DNA Primers
Baculoviridae
Transcription
Microsomes
Glutathione Transferase
Gene Library
Viruses

Keywords

  • Detoxification
  • Drosophila melanogaster
  • Epoxide hydrolase
  • Induction
  • Insect

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization and cDNa cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster. / Taniai, Kiyoko; Inceoglu, Ahmet B.; Yukuhiro, Kenji; Hammock, Bruce D.

In: European Journal of Biochemistry, Vol. 270, No. 23, 12.2003, p. 4696-4705.

Research output: Contribution to journalArticle

Taniai, Kiyoko ; Inceoglu, Ahmet B. ; Yukuhiro, Kenji ; Hammock, Bruce D. / Characterization and cDNa cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster. In: European Journal of Biochemistry. 2003 ; Vol. 270, No. 23. pp. 4696-4705.
@article{ff28e181aacd474f9c3781544577aef6,
title = "Characterization and cDNa cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster",
abstract = "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 melangaster 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.",
keywords = "Detoxification, Drosophila melanogaster, Epoxide hydrolase, Induction, Insect",
author = "Kiyoko Taniai and Inceoglu, {Ahmet B.} and Kenji Yukuhiro and Hammock, {Bruce D.}",
year = "2003",
month = "12",
doi = "10.1046/j.1432-1033.2003.03868.x",
language = "English (US)",
volume = "270",
pages = "4696--4705",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "23",

}

TY - JOUR

T1 - Characterization and cDNa cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster

AU - Taniai, Kiyoko

AU - Inceoglu, Ahmet B.

AU - Yukuhiro, Kenji

AU - Hammock, Bruce D.

PY - 2003/12

Y1 - 2003/12

N2 - 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 melangaster 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.

AB - 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 melangaster 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.

KW - Detoxification

KW - Drosophila melanogaster

KW - Epoxide hydrolase

KW - Induction

KW - Insect

UR - http://www.scopus.com/inward/record.url?scp=0345688025&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0345688025&partnerID=8YFLogxK

U2 - 10.1046/j.1432-1033.2003.03868.x

DO - 10.1046/j.1432-1033.2003.03868.x

M3 - Article

VL - 270

SP - 4696

EP - 4705

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 23

ER -