Testicular Toxicity of Molinate in the Rat: Metabolic Activation via Sulfoxidation 1 William T. Jewell, Rex A. Hess,* and Marion G. Miller Department of Environmental Toxicology, University of California, Davis, California 95616; and *Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61801 Received August 29, 1997; accepted January 15, 1998 Testicular Toxicity of Molinate in the Rat:Metabolic Activation via Sulfoxidation. Jewell, W. T., Hess, R. A., and Miller, M. G. (1998). Toxicol. Appl. Pharmacol. 149, 159 –166. Molinate is a thiocarbamate herbicide widely used in rice cul- ture. Studies conducted for regulatory purposes have indicated that molinate exposure causes male reproductive damage in rats. The present study describes the testicular lesion after administra- tion of single doses of molinate. The hypothesis that a metabolite of molinate is responsible for testicular toxicity was also investi- gated. Testicular damage was evaluated histopathologically in Sprague–Dawley rats 48 h and 1, 2, and 3 weeks after adminis- tration of molinate (100 – 400 mg/kg ip). No testicular damage was seen at any time point at the 100 mg/kg dose level. Damage was first seen 1 week after 200 mg/kg and 48 h after 400 mg/kg. The lesion was characterized by Sertoli cell vacuolation, failed spermi- ation, and phagocytosis of spermatids particularly evident at Stages X and XI. With increasing time, damage progressed until disorganization of the seminiferous epithelium was extensive, multinucleated giant cells were numerous, and neither spermato- zoa nor late step spermatids were present. At 3 weeks after ad- ministration of the two higher-dose levels, germ cells in the sem- iniferous tubules were almost completely absent. Administration of the sulfoxide metabolite of molinate (200 mg/kg ip) caused testicular damage similar in severity to that seen at the 400 mg/kg dose level for the parent compound, indicating that it was more potent as a testicular toxicant. In vitro metabolism studies using liver and testis microsomes found that the major metabolite in both preparations was molinate sulfoxide. Testis microsomes pro- duced only slightly less sulfoxide when compared with liver mi- crosomes. Molinate was also metabolized via ring hydroxylation to form small amounts of hydroxymolinate. The amount of hy- droxymolinate was substantially less in testis microsomes. Overall, these data indicate that sulfoxidation of molinate plays a role in molinate-induced testiculartoxicity. Moreover, molinate is metab- olized readily by both liver and testis microsomal enzymes, sug- gesting that the molinate toxic metabolite could be formed in the testis in close proximity to its site of action. © 1998 Academic Press Molinate (Ordram) is a thiocarbamate herbicide widely used in rice growing and of considerable economic importance to the rice industry. There are numerous regulatory reports, but none in the peer-reviewed literature, which indicate that ad- ministration of molinate results in male reproductive toxicity, while the only other effects noted were minor indications of neurotoxicity (CAL-EPA, 1996). Long-term, low-dose, chronic toxicity studies as well as some acute studies have been carried out in various species. Toxicological end points used were primarily fertility and changes in sperm parameters. When testicular damage was assessed histopathologically ‘‘ne- crosis of germinal cells’’ was reported in rats but not mice (CAL-EPA, 1996). It has been proposed that metabolic activation is involved in molinate-induced testicular toxicity (Krieger et al., 1992). Me- tabolism studies in rats have shown that molinate undergoes oxidative metabolism to form either molinate sulfoxide or ring-hydroxylated hydroxymolinate (DeBaun et al., 1978) (Fig. 1). The sulfoxidation of molinate represents a metabolic pathway where reactive electrophilic intermediates are gener- ated and sulfoxides of thiocarbamates are more potent car- bamylating agents (Casida et al., 1974). It has been demon- strated that molinate sulfoxide can covalently bind chick embryo proteins (Faiman et al., 1991) as well as covalently bind and inhibit aldehyde dehydrogenase (Hart and Faiman, 1995). Because of this chemical reactivity, the present study has investigated the possibility that sulfoxidation of molinate is responsible for generation of a metabolite capable of causing testicular damage. Molinate sulfoxide can either undergo glutathione conjuga- tion or be oxidized to the sulfone, which also can be conjugated by glutathione (DeBaun et al., 1978). The molinate glutathione conjugate is further metabolized and excreted in the urine as a mercapturate. In the rat, the mercapturate has been reported to make up 35.4% of the dose when molinate was administered at a dose level of 72 mg/kg po (DeBaun et al., 1978). In a human study, conducted at much lower dose levels (0.03– 0.7 mg/kg po), the mercapturate represented only 1–2% of the dose (Krieger et al., 1992). From the human data, it was suggested that metabolic differences between rats and humans could form 1 Supported by California Rice Research Board (RP-7) and by National Institute of Environmental Health Sciences Training Grant 5-T32-EOS-7059. Presented in part at the Annual Meeting of the Society of Toxicology, Los Angeles, California, 1996. TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 159 –166 (1998) ARTICLE NO. TO988380 159 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.