Introduction Zoxamide (3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl- 2-oxopropyl)-4-methylbenzamide) is a benzamide fungicide highly active against oomycetes on potatoes, grapevine, fruit, and vegetables (Young & Slawecki 2001). It causes mitotic arrest by speciically binding to β-tubulin, inhibiting tubuli polymerization and cell division (Young et al. 2006). Zoxamide is a registered fungicide and its toxicity proile has been evaluated with respect to all endpoints required for registration of a plant-protection product according to the current guidelines (for a brief sum- mary, see European Commission 2004). Interestingly, Xenobiotica, 2010; 40(1): 72–82 Address for Correspondence: Franz Oesch, Institute of Toxicology, University of Mainz, Rheinblick 21, Wackernheim D-55263, Germany. E-mail: oesch@ uni-mainz.de RESEARCH ARTICLE In vitro mammalian metabolism of the mitosis inhibitor zoxamide and the relationship to its in vitro toxicity F. Oesch 1 , M. Metzler 2 , E. Fabian 3 , H. Kamp 3 , T. Bernshausen 3 , G. Damm 2 , S. Triebel 4 , J. DÖhmer 5 , R. Landsiedel 3 , and B. Van Ravenzwaay 3 1 Institute of Toxicology, University of Mainz, Mainz, Germany, 2 University of Karlsruhe (TH), Karlsruhe, Germany, 3 Experimental Toxicology and Ecology, BASF AG, Ludwigshafen, Germany, 4 University of Kaiserslautern, Kaiserslautern, Germany, and 5 GenPharmTox BioTech AG, Martinsried/Planegg, Germany Abstract 1. The in vitro mammalian metabolism of the fungicide zoxamide is related to its in vitro mammalian toxicity. 2. After incubation of zoxamide with rat liver microsomes leading to practically 100% metabolism (mostly hydroxylated zoxamide), the cytotoxicity (methyl thiazole tetrazolium (MTT) test) and the mitosis-in- hibiting potential (shown by cell count and by cell cycle analysis) for V79 were not distinguishable from those of zoxamide, demonstrating that the hydroxylation of zoxamide did not change the cytotoxicity or mitosis-inhibiting potential as determined by these assays. After incubation of zoxamide with rat liver S9 predominantly leading to conjugation with glutathione, and after incubation of zoxamide with rat liver slices predominantly leading to the glucuronide of the hydroxylated zoxamide, these activities were eliminated demonstrating that the glutathione conjugate and the glucuronide had lost the activities in these assays due either to no intrinsic potential of these conjugates or to their inability to penetrate the plasma membrane of mammalian cells. 3. It is concluded that the metabolic hydroxylation of zoxamide did not change its activity in the assays used for investigating its inluence on cell proliferation, cell cycle and cytotoxicity, while the formation of conjugates with glutathione or glucuronic acid led to the apparent loss of these activities. 4. Thus, with zoxamide as a prototype, it was shown that, in principle, mammalian metabolism and its relationship to mammalian detoxication of fungicidal mitosis inhibitors may be reasonably anticipated from in vitro studies. In addition, the results provide a rational for the observed absence of typically mitosis inhibition-associated toxicities of zoxamide in mammals in vivo. Keywords: Zoxamide; cytochromes P450 (CYP); glutathione conjugation; glucuronidation; fungicide; mitosis inhibition; cytotoxicity; metabolic detoxication (Received 14 July 2009; revised 19 September 2009; accepted 21 September 2009) ISSN 0049-8254 print/ISSN 1366-5928 online © 2010 Informa UK Ltd DOI: 10.3109/00498250903353464 http://www.informahealthcare.com/xen Xenobiotica Downloaded from informahealthcare.com by ReprintsDesk on 04/30/12 For personal use only.