544 Introduction: BEN is an alkylating agent proposed for clinical studies based on its in vitro & in vivo activity against renal cell carcinoma. However, its metabolism is incompletely characterized, & its stability in tissue culture medium is unknown. Previous in vivo pharmacokinetic studies documented a number of metabolites of BEN (m/z 380) including: a carboxylic acid (m/z396) produced by aldehyde dehydrogenase 1; a mono-N-dealkylated metabolite (m/z 258); & 4 glucuronides. Because these metabolites & BEN hydrolysis products are likely to be less active than BEN, we characterized: 1) the enzyme responsible for BEN N-dealkylation; & 2) the stability of BEN in tissue culture medium. Materials & Methods: BEN metabolism by mouse & human hepatic fractions was characterized using NADH & NADPH as cofactors & in aerobic, anaerobic, & CO-saturated environments. Inhibition of BEN dealkylation by human microsomes was evaluated with CYP isoform-specific chemical inhibitors & isoform-specific antisera. Finally, cloned human CYP isoforms were studied for their ability to dealkylate BEN. Also, the rate & products of BEN decomposition were characterized in RPMI 1640 medium, with 20% heat-inactivated fetal calf serum, incubated at 37 °C & 5% CO2. Results: BEN was metabolized to products with m/z of 396 & 258 by hepatic S9 fractions. Metabolism of BEN to the m/z 396 product was confined to cytosol, whereas metabolism to the m/z 258 product was restricted to microsomes. Metabolism of BEN to the m/z 258 product by mouse & human microsomes was > 2 times faster with NADPH than with NADH, 2-8 times faster under aerobic conditions than under anaerobic conditions, & eliminated by CO. Human microsomal metabolism of BEN to the m/z 258 product was inhibited by the CYP3A inhibitor ketoconazole but not by chemical inhibitors of CYP2D6 (quinidine), 2C9 (tolbutamide), or 2E1 (disulfuram). Metabolism of BEN to the m/z 258 product was inhibited by anti-CYP3A antiserum but not by antisera to CYPs 2C8, 2C9, 2C19, or 2D6. BEN was metabolized to the m/z 258 product by cloned human CYP3A4, but not by cloned human CYPs 2C8, 2C9, 2C19, 2D6, 1A2, or 2E1. The di-dealkylated metabolite of BEN (m/z 136) was not observed. BEN decomposed in tissue culture medium with a t1/2 of ∼8 h. BEN decomposed sequentially to products with m/z 302 & 224, compatible with sequential hydrolysis of methane sulfonate groups resulting in mono- & di-hydroxy products. No products with m/z 302 or 224 were observed in microsomal or CYP incubations. Conclusions: CYP3A metabolizes BEN to a probably less active N-dealkylated metabolite. The decomposition of BEN, in tissue culture medium, to less active or inactive mono- & dihydroxy products should be considered when extrapolating in vitro data to in vivo &, possibly, clinical studies. Enzymes responsible for glucuronidation of BEN and metabolites remain to be characterized. Support: A Carnegie Mellon Small Undergraduate Research Grant & NCI-N01CM07106