Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin-dependent monooxygenase 3.

Human flavin-dependent monooxygenase (FMO) isoforms 1 and 3 were expressed by retroviral gene transfer in mouse C3H/10T1/2 cells. FMO function was determined by the sulfoxidation of p-tolylmethylsulfide (TMS). Enzyme activity ranged from 4 to 30 nmol p-tolylmethylsulfoxide (TMSO)/30 min/mg cell protein for FMO 3 clones; for FMO 1 clones, the range was 1-6 nmol TMSO/30 min/mg. Cytotoxicity in these clones after exposure to thiocarbamate compounds was assessed by clonogenic assay. Thiourea (TU), phenylthiourea (PTU), and alpha-naphthylthiourea (ANTU) were toxic to FMO 3 cells but not to parental and FMO 1 clones; 50% toxicity was attained at 1x10(-4) M TU, 5x10(-6) M PTU, and 1x10(-6) M ANTU. Toxicity was observed after a minimum exposure time of 6 hr. Parental cells were resistant to toxicity for exposure times spanning the entire clonogenic assay period (10 days). Ethylene thiourea (ETU) was not toxic to FMO 3 cells, but preincubation with 1x10(-3) M ETU blocked TU toxicity. Reducing GSH levels by preincubation with 1x10(-5) M buthionine sulfoxime (BSO) increased TU sensitivity in FMO 3 cells from 1x10(-4) to 1x10(-6) M to achieve 50% toxicity. BSO also increased the sensitivity of "low expressor" FMO 3 clones to TU, but did not alter the refractoriness of either parental or FMO 1 expressing cells to TU. N-Acetylcysteine afforded modest protection to TU toxicity by shifting 50% cytotoxicity for TU from 5x10(-5) to 1x10(-3) M. TU mutagenicity was assayed by the development of ouabain resistance in parental and FMO 3 C3H/10T1/2 cells. Exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, direct acting mutagen) and TU was executed with and without prior sensitization with BSO. The mutation frequency for MNNG was 76/1x10(6) surviving cells, whereas no mutants were observed for TU-exposed cultures. The results of this study show that, in isolation, the major human hepatic form of FMO is capable of promoting thiocarbamate toxicity. Consistent with the known reactivity of thiocarbamate intermediates with GSH, treatments that alter GSH levels also altered toxicity in either the protective or sensitizing direction. These cell lines expressing variable levels of FMO 3 and TU sensitivity should prove useful as in vitro systems for dissecting the thiocarbamate toxicity pathway.