SULFHYDRYL-DEPENDENT BIOTRANSFORMATION AND MACROMOLECULAR BINDING OF 1,2-DIBROMO-2,4-DICYANOBUTANE IN BLOOD

1,2-Dibromo-2,4-dicyanobutane (BCB) is a broad-spectrum microbicide us ed commercially in consumer products. The objectives of this study wer e to elucidate the biotransformation of BCB, characterize its ability to covalently bind macromolecules, and predict the possible toxicologi cal ramifications of such events. After iv administration of [C-14]BCB to male Fischer 344 rats, C-14-equivalents were observed to bind grad ually to blood constituents. By 48 hr, approximately 12% of the total dose was covalently bound. At no time was parent compound detected in the blood. However, the debrominated BCB metabolite 2-methyleneglutaro nitrile (MGN) was observed, In vitro experiments revealed that BCB was extremely labile and was readily debrominated in fresh whole blood, e rythrocyte preparations, and buffered glutathione (GSH) solutions. In each case, the formation of MGN was inhibited by the alkylation of fre e sulfhydryls with N-ethylmaleimide (NEM). For every 1 mol of BCB conv erted to MGN, 2 mot of GSH were oxidized to glutathione disulfide (GSS G) (BCB + 2 GSH --> MGN + GSSG + 2 HBr), The oxidation of free sulfhyd ryls during the conversion of BCB to MGN caused erythrocyte hemolysis (EC50 similar to 1 mM) in isolated preparations, Hemolysis was increas ed by coincubation of BCB with NEM (EC50 similar to 0.3 mM) and was de creased by coincubation with GSH (EC50 > 3 mM). However, MGN did not c ause hemolysis of erythrocytes, even at concentrations 10-fold higher than the EC,, of BCB, In vitro experiments also demonstrated that incu bation with either BCB or MGN resulted in significant macromolecular b inding to the erythrocyte fraction of the blood (similar to 80%). Incu bation with NEM resulted in a significant decrease in binding for both BCB (11.3% bound) and MGN (29.5% bound). Because BCB is rapidly debro minated in whole blood, it appears that MGN is the reactive species re sponsible for macromolecular binding, From these studies, we conclude that the conversion of BCB to MGN is mediated by a free sulfhydryl-dep endent biotransformation pathway. Furthermore, BCB biotransformation i s required for erythrocyte binding, and the consumption of free sulfhy dryls associated with the biotransformation of BCB is responsible for hemolysis.