CHLOROTHIOKETENE, THE ULTIMATE REACTIVE INTERMEDIATE FORMED BY CYSTEINE CONJUGATE BETA-LYASE-MEDIATED CLEAVAGE OF THE TRICHLOROETHENE METABOLITE S-(1,2-DICHLOROVINYL)-L-CYSTEINE, FORMS CYTOSINE ADDUCTS IN ORGANIC-SOLVENTS, BUT NOT IN AQUEOUS-SOLUTION

Chlorothioketene has been suggested as a reactive intermediate formed by the cysteine conjugate beta-lyase-mediated cleavage of S-(1,2-dichl orovinyl)-L-cysteine, a minor metabolite of trichloroethene. Halothiok etenes are highly reactive, and their intermediate formation may be co nfirmed by reactions such as cycloadditions and thioacylations of nucl eophiles. A precursor of chlorothioketene, S-( 1,2-dichlorovinyl)thioa cetate, is readly accessible by the reaction of dichloroethyne with th ioacetic acid. In presence of base, S-(1,2-dichlorovinyl)thioacetate i s cleaved to chlorothioketene. Chlorothioketene is not stable at room temperature and was characterized after transformation to stable produ cts by reaction with compounds such as cyclopentadiene, N,N-diethylami ne, and ethanol. In organic solvents, the cleavage of S-(1,2-dichlorov inyl)thioacetate in the presence of cytosine results in N-4-acetylcyto sine, N-4-(chlorothioacetyl)cytosine, and small amounts of 3-(N-4-thio acetyl)cytosine. No reaction products were seen with guanosine, adenos ine, and thymidine under identical conditions. When cytosine was react ed with S-(1,2-dichlorovinyl)thioacetate in aqueous solutions, only N- 4-acetylcytosine was formed. N-4-(Chlorothioacetyl)cytosine and 3-(N-4 -thioacetyl)cytosine were not detected even when using a very sensitiv e method, derivatization with pentafluorobenzyl bromide and electron c apture mass spectrometry with a detection limit of 50 fmol/mu L of inj ection volume. Aqueous solutions of DNA cleave S-(1,2-dichlorovinyl)th ioacetate to give N-4-acetyldeoxycyticline in DNA, but chlorothioketen e adducts of deoxynucleosides were also not detected in these experime nts. These results confirm the electrophilic reactivity of chlorothiok etene toward nucleophilic groups of DNA constituents in inert solvents but also demonstrate that the formation of DNA adducts under physiolo gical conditions likely is not efficient. Therefore, DNA adducts may n ot represent useful biomarkers of exposure and biochemical effects for trichloroethene.