Metabolism of nitrosoureas and formamides : a probe into their toxicity as precursors of reactive isocyanates

The focus of this research was to investigate the biotransformation of nitrosoureas and formamides to isocyanates as a mechanism by which these compounds could elicit their toxicity. The metabolism of 1 -(2-chloroethyl)-3-cyclohexyl- 1 -nitrosourea (CCNU) and 1,3- bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied in rats and in patients on chemotherapy; the biotransformation of N-formyl amphetamine (NFA) was examined in rats and in rat hepatic subcellular fractions; and carbamoyl thiol conjugates were investigated with respect to their reactivity and toxicity toward mitochondrial enzymatic processes. CCNU is hydroxylated in vivo to 4-hydroxy and 3-hydroxy CCNU which, along with the parent compound, decompose to the corresponding isocyanates, namely, 4- hydroxycyclohexyl, 3-hydroxycyclohexyl, and cyclohexylisocyanate. Evidence for the formation of these reactive electrophiles in vivo was inferred from the LCJMS identification of their glutathione (GSH) and N-acetylcysteine (NAC) conjugates which were excreted in the bile and urine, respectively, of dosed rats, and as NAC conjugates in the urine of patients on chemotherapy. This GSH-dependent pathway of metabolism contributed substantially to metabolism of CCNU in rats, accounting for 14.3 ± 2.9 % of the dose of CCNU excreted in urine as carbamoylated NAC conjugates in 24 h. BCNU decomposes in vivo to 2-chioroethyl isocyanate (CEIC) which conjugates with GSH. In support of this contention, GSH and NAC conjugates of CEIC were identified as metabolites in the bile and urine, respectively, of BCNU-dosed rats. Quantitative analysis of the urine of five patients on BCNU therapy revealed that concentrations of the NAC conjugate of CEIC varied from 5.0 to 13.6 nmol/mL. Bioactivation of NFA to 1-methyl-2-phenylethyl isocyanate (MPIC) was investigated in rats by screening bile and urine for conjugates downstream of the phase I event. NFA was administered to rats as a mixture of protio and pentadeuteriophenyl labelled analogues to characterize the carbamoylating activity of MPIC by LCIMS contour formatting. This LCIMS technique facilitated the identification of metabolites by presenting chromatographic and mass spectral data together as a two-dimensional array. Glutathione, cysteinylgIyciri, cysteine and NAC conjugates of the isocyanate MPIC were identified as biliary metabolites, whereas only the NAC conjugate was excreted in urine. The excretion of all metabolites of the mercapturate pathway in bile is a novel finding for formaniides. The catalytic activity of rat hepatic microsomes and mitochondria in the conversion of NFA to MPIC was investigated by performing incubations in the presence of GSH to trap MPIC in the form of S-[( 1-methyl-2-phenylethyl)carbamoyljglutathione (SMPG), which was detected by LCIMS/MS. NFA was converted by microsomes to SMPG in a manner suggestive of cytochrome P450 catalysis. SMPG formation was marginally elevated (25 %) in microsomes from rats treated with acetone and phenobarbital, inducers of P450 2E1 and 2B l/2B2, respectively. In addition, microsomal conversion of NFA to SMPG was marginally affected by diethyldithiocarbamate (DEDTC) and orphenadrine, mechanism-based inhibitors of P450 2E1 and 2B 112B2, respectively. Taken together, these data suggest that neither P450 2E1 nor 2B1/2B2 play a major role in the metabolism of NFA to MPIC. Intact mitochondria also performed the biotransformation of NFA to SMPG. We sought to demonstrate the involvement of mitochondrial P450 in this process by using sonicated mitoplasts supplemented with NADPH. Although SMPG was detected as a mitoplast product of NFA, low but significant levels of microsomal contamination did not allow the involvement of mitochondrial P450 to be unequivocally proven. The GSH and NAC conjugates of CEIC were examined with respect to their stability, reactivity and inhibitory properties toward mitochondrial enzyme activities. S-[(2- chloroethyl)carbamoyl]glutathione (SCEG) exhibited a half-life of 5.0 h in solution. In the presence of NAC, the SCEG concentration declined more rapidly (t[sub ½] 44 mm) by reaction with the free thiol to form N-acetyl-S-[(2-chloroethyl)carbamoyl]cysteine (NCEC). In the converse situation, NCEC appeared more stable than the GSH conjugate, exhibiting a half-life of 3.7 h when it reacted with GSH to form SCEG. When NCEC was administered to rats, SCEG was identified as a biliary metabolite by LCIMS/MS. This result was considered reflective of an exchange of CEIC between NCEC and endogenous GSH. Mitochondrial glutathione reductase (GR) was inhibited by SCEG to approximately 10 % of control within 7 mm, with the activity remaining depressed at 6.5 h. S (methylcarbamoyl)glutathione (SMG), the GSH conjugate of methyl isocyanate, impaired the performance of mitochondrial respiration. State Ill oxidative phosphorylation, apparent in control mitochondria with ADP/O ratios of 2.4 to 3.2 (glutamate/malate as substrate) and respiratory control ratios of 3.0 to 7.2, was absent in mitochondria exposed to SMG. Taken together, these results provide evidence for the toxicity of conjugated thiol metabolites.

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