title: Development of pyrethroid-like fluorescent substrates for glutathione S-transferase.
abstract: The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides, including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric preference (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers). A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized 1-chloro-2,4-dinitrobenezene (CDNB), a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (k(cat)/K(M) ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC, suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes.
authors.affiliation: Department of Entomology, University of California, Davis, CA 95616, USA.
authors.forename: Huazhang
authors.initials: H
authors.lastname: Huang
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yao
authors.forename: Jun-Yan
authors.initials: JY
authors.lastname: Liu
authors.forename: Aman I
authors.initials: AI
authors.lastname: Samra
authors.forename: Shizuo G
authors.initials: SG
authors.lastname: Kamita
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Cornel
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry; D003465:Culex / Q000201:enzymology; D005453:Fluorescence; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D011722:Pyrethrins / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D011722:Pyrethrins; C017160:cypermethrin; D006923:Hymecromone; D005982:Glutathione Transferase; D002265:Carboxylic Ester Hydrolases