Study on the role of glutathione in the biotransformation and toxicity of furazolidone using pig hepatocytes.
Incubation of monolayer cultures of pig hepatocytes with furazolidone resulted in increased intracellular oxidized glutathione (GSSG) levels but no decreased reduced glutathione (GSH) levels. However, a clear decrease in GSH levels was observed at the higher drug concentrations when either the synthesis of GSH was blocked with buthionine-d,l-sulphoximine (BSO) or the reduction of GSSG by 1,3-bis(2-chloroethyl)-1-nitrosourea. Furthermore, when cells containing (35)S-labelled GSH were exposed to furazolidone, a dose-related loss of radiolabelled GSH was observed. The increased loss was balanced by elevated levels in the medium of GSSG and a second compound, probably the disulphide of cysteine and GSH. The biotransformation of (14)C-furazolidone by cells resulted partly in the formation of the cyano metabolite as well as a large number of more hydrophilic unknown metabolites. No evidence was obtained for the excretion by the cells of the glutathione conjugate that was previously detected in microsomal incubations. This could, however, be due to the unstable nature of the conjugate, as demonstrated by its rapid disappearance when added to the medium. In the presence but not the absence of cells, this partly resulted in the formation of the cyano metabolite, which on prolonged incubation was further metabolized into more polar metabolites. Increased LDH leakage at high drug concentrations could be observed only when GSH levels were kept at a low level with BSO. Contrary to previous observations with microsomes, decreased GSH levels did not result in increased formation of protein-bound metabolites of furazolidone and the cyano metabolite. In addition, GSH depletion did not result in increased inhibition of the pyruvate metabolism by furazolidone. No evidence was obtained for the presence of reactive thiol conjugates of furazolidone in the protein fraction of cells incubated with the drug. It is concluded that GSH has an active role in the protection of cells against the cytotoxic effects of furazolidone related to oxidative stress, but that no evidence could be obtained for the formation and excretion of reactive thiol conjugates.
Hoogenboom
L A
LA
State Institute for Quality Control of Agricultural Products (RIKILT), Bornsesteeg 45, 6708 PD Wageningen, The Netherlands.
van Kammen
M
M
Huveneers-Oorsprong
M B
MB
Kuiper
H A
HA