The N-hydroxylation of carcinogenic arylamines represents an initial step in their metabolic activation. Animal studies have shown that this reaction is catalyzed by the cytochrome P450 (P450) enzymes P450 1A1 and P450 1A2. In this study, utilizing enzymes expressed in Escherichia coli (and purified) or in human B-lymphoblastoid cells, the catalytic activities of recombinant human P450 1A1, P450 1A2, and P450 3A4 for N-hydroxylation of several carcinogenic arylamines were determined. P450 1A2 from both expression systems catalyzed the N-hydroxylation of 4-aminobiphenyl and the heterocyclic amines, 2-amino-3-methylimidazo[4,5-f/quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rates were similar, with values of 1.1-7.8 nmol/min/nmol P450. In contrast, P450 1A1 catalyzed N-hydroxylation of only PhIP, and no activity was observed with P450 3A4. Further kinetic analysis with purified P450 1A2 showed similar Km and Vmax values for N-hydroxylation of the arylamines. Furafylline and fluvoxamine, inhibitors of P450 1A2 activity in human liver microsomes, were found to be inhibitory of the recombinant P450 1A2 N-hydroxylation activity. Results from this study are supportive of a major role for human P450 1A2 in the metabolic activation of arylamines.

1. The oxidative metabolism of [3-36C]chloropropan-1,2-diol (alpha-chlorohydrin, I) was studied in male rats. Two metabolites were isolated and identified as beta-chlorolactic acid (IV) and oxalic acid (V). 2. Neither alpha-chlorohydrin nor beta-chlorolactate was concentrated in any tissue. Traces of an intermediate metabolite, beta-chlorolactaldehyde (III) were detected in the urine within 4 h of administration. Studies in vitro indicated that the metabolic pathway is: alpha-chlorohydrin leads to beta-chlorolactaldehyde leads to beta-chlorolactic acid. 3. A comparative study of the metabolism of 36Cl- and 14C-beta-chlorolactate showed that oxalate was produced slowly and, as calcium oxalate, caused a type of renal glomerular nephritis. This pathological condition is responsible for the diuretic action of both alpha-chlorohydrin and beta-chlorolactate and, in higher doses, for their toxicities. 4. The role of oxalate, as a metabolite of alpha-chlorohydrin and of a number of related compounds, in inducing the formation of spermatocoeles in the male rat reproductive tract is discussed.

Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin-particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.

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