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Grantee Research Project Results

NCER Grantee Research Project Results

Oxidation of Cysteine S-Conjugates by Rabbit Liver Microsomes and cDNA-Expressed Flavin-Containing Mono-oxygenases: Studies With S-(1,2-Dichlorovinyl)-L-cysteine, S-(1,2,2-Trichlorovinyl)-L-cysteine, S-Allyl-L-cysteine, and S-Benzyl-L-cysteine

EPA Grant Number: U914810
Title: Oxidation of Cysteine S-Conjugates by Rabbit Liver Microsomes and cDNA-Expressed Flavin-Containing Mono-oxygenases: Studies With S-(1,2-Dichlorovinyl)-L-cysteine, S-(1,2,2-Trichlorovinyl)-L-cysteine, S-Allyl-L-cysteine, and S-Benzyl-L-cysteine
Investigators: Ripp, Sharon L.
Institution: University of Wisconsin - Madison
EPA Project Officer: Broadway, Virginia
Project Period: January 1, 1995 through January 1, 1996
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1995)
Research Category: Fellowship - Health , Health Effects , Academic Fellowships

Description:

Objective:

The objective of this research project is to oxidize cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases.

Approach:

Rabbit liver microsomes catalyzed the highly stereoselective, NADPH- and time-dependent S-oxidation of S-benzyl-L-cysteine (SBC), S-ally-L-cysteine (SAC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) to their respective sulfoxides. Methimazole, a flavin-containing mono-oxygenase (FMO) substrate, inhibited S-oxidation of all four conjugates. The cytochrome P450 inhibitor 1-benzylimidazole did not affect SAC, SBC, or DCVC S-oxidation, but inhibited the S-oxidation of TCVC. Solubilization of microsomes also inhibited TCVC activity, whereas SBC, SAC, and DCVC activities were not affected. Because these results suggested that FMOs were the major catalysts of SBC, SAC, and DCVC sulfoxidations, the four conjugates were evaluated as substrates for cDNA-expressed rabbit FMO isoforms FMO1, FMO2, FMO3, and FMO5. At equimolar concentrations (10 mm), FMO1 S-oxidized SBC and SAC, but no sulfoxides were detected with DCVC or TCVC. FMO3 S-oxidized all four conjugates. Km values determined with FM03 were comparable to Km values from rabbit liver microsomes. S-Oxidation by FMO2 was detected only with SAC, and no sulfoxides were detected in incubations with FMO5. These results show that FMO isoforms can catalyze cysteine conjugate S-oxidation, and that the specific isoform involved depends on the structure of the cysteine conjugate. The cysteine conjugates with more nucleophilic sulfur atoms, SAC and SBC, were much better FMO substrates than those having the less nucleophilic sulfur atoms DCVC and TCVC. The sulfoxides of TCVC and DCVC were reactive toward glutathione, whereas the sulfoxides of SBC and SAC were not reactive. These results provide evidence for different chemical reactivities of these sulfoxides.

Supplemental Keywords:

fellowship, rabbit liver microsomes, cysteine conjugates, sulfoxides, GSH, reduced gluatathione, SAC, S-Allyl-L-cysteine, SCB, S-benzyl-L-cysteine, DCVC, S-(1,2-dichlorovinyl)-L-cysteine, TCVC, S-(1,2,2-trichlorovinyl)-L-cysteine, CAN, acetoritile, b-lyase, cysteine conjugate b-lyase, AOAA, aminooxyacetic acid, high performance liquid chromatography, HPLC, fast atom bombardment-mass spectrometry, FAB-MS, flavin-containing monooxygenase, FMO, trifluoroacetic acid, TFA., Health, Scientific Discipline, Risk Assessments, Environmental Microbiology, DNA probes, rabbit liver microsomes, animal model, genetic analysis, cysteine

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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