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 used commercially in consumer products. The objectives of this study were to elucidate the biotransformation of BCB, characterize its ability to covalently bind macromolecules, and predict the possible toxicological ramifications of such events. After iv administration of [14C]BCB to male Fischer 344 rats, 14C-equivalents were observed to bind gradually 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-methyleneglutaronitrile (MGN) was observed. In vitro experiments revealed that BCB was extremely labile and was readily debrominated in fresh whole blood, erythrocyte preparations, and buffered glutathione (GSH) solutions. In each case, the formation of MGN was inhibited by the alkylation of free sulfhydryls with N-ethylmaleimide (NEM). For every 1 mol of BCB converted to MGN, 2 mol of GSH were oxidized to glutathione disulfide (GSSG) (BCB + 2 GSH --> MGN + GSSG + 2 HBr). The oxidation of free sulfhydryls during the conversion of BCB to MGN caused erythrocyte hemolysis (EC50 approximately 1 mM) in isolated preparations. Hemolysis was increased by coincubation of BCB with NEM (EC50 approximately 0.3 mM) and was decreased by coincubation with GSH (EC50 > 3 mM). However, MGN did not cause hemolysis of erythrocytes, even at concentrations 10-fold higher than the EC50 of BCB. In vitro experiments also demonstrated that incubation with either BCB or MGN resulted in significant macromolecular binding to the erythrocyte fraction of the blood (approximately 80%). Incubation with NEM resulted in a significant decrease in binding for both BCB (11.3% bound) and MGN (29.5% bound). Because BCB is rapidly debrominated in whole blood, it appears that MGN is the reactive species responsible for macromolecular binding. From these studies, we conclude that the conversion of BCB to MGN is mediated by a free sulfhydryl-dependent biotransformation pathway. Furthermore, BCB biotransformation is required for erythrocyte binding, and the consumption of free sulfhydryls associated with the biotransformation of BCB is responsible for hemolysis.
Department of Pharmacology and Toxicology and Center for Toxicology, The University of Arizona, Tucson, AZ 85721-0207, USA.
J
J
Bao
J M
JM
Sauer
R L
RL
Smith
R K
RK
Kuester
M J
MJ
Kattnig
I G
IG
Sipes
D000818:Animals; D000890:Anti-Infective Agents / Q000097:blood / Q000493:pharmacokinetics*; D001711:Biotransformation; D002470:Cell Survival; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005978:Glutathione; D008297:Male; D013058:Mass Spectrometry; D009570:Nitriles / Q000097:blood / Q000493:pharmacokinetics*; D051381:Rats; D011916:Rats, Inbred F344
D000890:Anti-Infective Agents; D009570:Nitriles; D005978:Glutathione; C039411:1,2-dibromo-2,4-dicyanobutane