Hydroxylation and dealkylation of methyl-n-butylnitrosamine and role of certain cytochrome P-450 isozymes in these reactions.

We studied the metabolism of methyl-n-butyl-nitrosamine (MBN), a carcinogen for the rat esophagus and liver. The 2-, 3- and 4-hydroxy derivatives were identified as new metabolites of MBN. In studies on tissue slices freshly removed from MRC-Wistar rats, MBN metabolism resembled that of the previously studied methyl-amylnitrosamine in the esophagus catalyzed 2- and 3- hydroxylation; liver, omega-1 hydroxylation; and lung, omega-hydroxylation of both nitrosamines. Liver microsomes from Sprague-Dawley rats catalyzed 2-, 3- and 4-hydroxylation of MBN, as well as the previously studied activating reactions of demethylation and debutylation. Phenobarbital induced all five reactions of MBN bh rat liver microsomes, especially debutylation; 3-methylhol-anthrene induced 3-hydroxylation and debutylation and isoniazid induced demethylation and debutylation. Monoclonal antibodies that inhibit specific cytochrome P-450 isozymes were used to identify the isozymes involved in each reaction. Antibody 4-7-1 appeared more specific than the previously used antibody 2-66-3 for inhibiting P-450 2B1 and/or 2B2. For the metabolism of both MBN and methylamylnitrosamine by rat liver microsomes, the antibody results indicated that P-450 2C11 mainly catalyzed demethylation and omega-1 hydroxylation, P-450 1A1 or 1A2 catalyzed 3-hydroxylation and debutylation or depentylation, P-450 2E1 produced demethylation and P-450 2B1 or 2B2 produced omega-1 hydroxylation, demethylation and debutylation or depentylation.

Ketonitrosamines as metabolites of methyl-n-amylnitrosamine (MNAN) and its hydroxy derivatives in the rat.

In a previous study of the metabolism of methyl-n-amylnitrosamine (MNAN) in the rat, 2- to 5-hydroxy-MNAN (HO-MNAN) were provisionally identified as metabolites and the identity of 4-HO-MNAN was confirmed by mass spectrometry. We now describe syntheses and mass and other spectra for 2- to 5-oxo-MNAN. Two previously unidentified MNAN metabolites were shown to be 3- and 4-oxo-MNAN. In addition to 4-HO-MNAN, we confirmed 3-HO-, 4-oxo- and (less certainly) 2-HO-MNAN as urinary MNAN metabolites by GLC-MS of HPLC fractions. Analysis with and without beta-glucuronidase treatment showed that the urinary HO-MNANs occurred as their beta-glucuronides. MNAN (25 mg/kg injected i.p.) had a blood half-life of 21 min in adult male rats. The blood also contained 4-HO- and 4-oxo-MNAN, which showed maximum levels that were 13 and 26% respectively of that for MNAN, and were cleared more slowly than MNAN. On incubation for 3 h with MNAN, rat esophagus produced 3- and 4-oxo-MNAN in yields that were 5% of those for the corresponding HO-MNANs. For MNAN metabolism, the 4-oxo-/4-HO-MNAN ratio of metabolites was 5% for adult rat liver and was 22% for adult hamster liver and 9-day-old rat liver. On incubation with 4-HO-MNAN for 3 h, oxidation to 4-oxo-MNAN was 16-25% for adult hamster or 9-day-old rat liver slices and for adult hamster liver homogenate. Homogenate activity was concentrated in the microsomal fraction, for which NAD was a more effective co-factor than NADP. A bacterial alcohol dehydrogenase oxidized 4-HO- to 4-oxo-MNAN in 38% yield/3 h. None of these preparations oxidized 2-HO- to 2-oxo-MNAN. It was concluded that 3- and 4-oxo-MNAN were metabolites of MNAN, apparently (for 4-oxo-MNAN) via HO-MNAN oxidation by a microsomal NAD-dependent enzyme, that 4-HO- and 4-oxo-MNAN formation was a major route of MNAN metabolism, and that 4-oxo-MNAN might play a role in MNAN carcinogenesis.

Nitrosamine formation from amines applied to the skin of mice after and before exposure to nitrogen dioxide.

Skin lipids of mice exposed to NO2 contain lipid-soluble nitrosating agent(s) (NSA) that react in vitro with amines to produce nitrosamines. To test whether this reaction occurs in skin, we exposed mice to 50 ppm NO2 for 4 h and, 20 h later, applied 25 mg morpholine or N-methylaniline to the skin, which was then analyzed for the corresponding nitrosamine. When morpholine was applied, mean N-nitrosomorpholine yield was only 0.3 nmol/mouse (not significant). When N-methylaniline was applied and mice were killed after 10-40 min, N-nitroso-N-methylaniline yield in the skin was 13-21 nmol/mouse of which 87% occurred in the hair. NSA formation when mice were exposed to 6.5 ppm NO2 was only 0.15% of that for exposure to 50 ppm NO2. NSA occurred mostly in surface lipids of the skin and its in vitro reaction to give nitrosamines was not inhibited by alpha-tocopherol. When morpholine was painted and mice were then exposed to 55 ppm NO2 for 30 min, the skins contained 19 nmol N-nitrosomorpholine/mouse, attributed to a direct reaction between NO2 and the amine. We concluded that nitrosamine formation in skin by this direct reaction may be more important than the reaction of amines with NO2-derived NSA.

Lipidic nitrosating agents produced from atmospheric nitrogen dioxide and a nitrosamine produced in vivo from amyl nitrite.

In studies on nitrosating agent(s) formed in skin of mice exposed to nitrogen dioxide, we showed that: (i) N-nitrosomethylaniline was produced in skin of mice exposed to nitrogen dioxide and then painted with N-methylaniline; (ii) a nitrosating precursor in methyl linoleate is associated with peroxidation products; (iii) cholesterol is a major nitrosating precursor in mouse skin, probably because it produces the nitrosating agent, cholesteryl nitrite; (iv) cholesteryl nitrite enhances autoxidation of lipids in vivo and on mouse skin and, like sodium nitrite, catalyses the autoxidation of iodide; (v) N-nitrosomethylaniline was produced in mice injected intraperitoneally with methylaniline and gavaged with amyl nitrite; and (vi) nitrosating agents may occur normally in human skin lipids.

Identification of cholesterol as a mouse skin lipid that reacts with nitrogen dioxide to yield a nitrosating agent, and of cholesteryl nitrite as the nitrosating agent produced in a chemical system from cholesterol.

The skin lipids of mice exposed to nitrogen dioxide (NO2) and mouse skin lipids exposed in vitro to NO2 contain nitrosating agents (NSAs), that react with amines to produce nitrosamines. This situation represents a potential hazard of exposure to NO2. A principal NSA precursor in mouse skin lipids was purified by thin-layer and high-performance liquid chromatography. Each fraction was assayed by bubbling in NO2 and determining NSA. The precursor was identified as cholesterol on the basis of its chromatographic behavior and spectral properties. In a chemical system, cholesterol reacted with NO2 to give 13% yields of an NSA, which was identified from its spectral properties as the previously known compound, cholesteryl-3-beta-nitrite. These findings and the chromatographic behavior of a major NSA in the skin lipids of NO2-exposed mice suggested that this NSA was cholesteryl nitrite.