Dosing time with ascorbic acid and nitrate, gum and tobacco chewing, fasting, and other factors affecting N-nitrosoproline formation in healthy subjects taking proline with a standard meal.

The N-nitrosoproline (NPRO) test measures the potential for intragastric formation of carcinogenic nitrosamines in humans. Nitrate and L-proline are administered to volunteers. Noncarcinogenic NPRO is produced by an acid-catalyzed reaction of proline (a model for ingested amines) with nitrate-derived nitrite in the stomach. It is then absorbed and excreted in the urine, which is analyzed for NPRO. We studied the effect of certain dietary and other factors on the levels of urinary NPRO. For (generally) 5 days, healthy adult subjects (mostly men) followed a diet low in preformed NPRO, nitrate, proline, and (on days 4 and 5) ascorbic acid. The tests were conducted on days 4 and 5. In the standard test, the subjects took 400 mg nitrate at 11 a.m., and at noon they ate a standard 700-calorie meal containing 500 mg proline. (In previous tests, proline was given 1 h after or between meals.) Urines were collected for 24 h, and samples were analyzed for NPRO by published methods. This standard test yielded 26 +/- 2 (mean +/- SE) nmol NPRO compared with 5 +/- 1 nmol NPRO when proline alone was taken. In variations of the standard test, NPRO yield was not significantly affected by the subjects' gender, the time at which the standard meal was eaten, the size of the meal, or the drinking of extra water after the meal. Doses of 100 and 200 mg nitrate had lesser effects on NPRO yield than did the dose of 400 mg nitrate. Nitrate (400 mg) produced the most NPRO when it was given 1 h before the meal. Fasting increased NPRO yield by 3-4 times compared to giving proline with a meal. One g of ASC given 5 or 2 h before, with, or 1 or 2 h after the meal with proline inhibited NPRO formation by mean values of 0, 71, 71, 67, and 19%, respectively. Chewing gum or tobacco for 2-3 h after the test meal did not increase NPRO formation or salivary nitrate levels, but salivary nitrite was not taken, chewing tobacco appeared to increase salivary nitrite and nitrate levels. The weak carcinogen N-nitrososarcosine (NSAR) was also detected in some tests, and the standard group showed 21 +/- 3 nmol NSAR. A high NSAR result (44 +/- 7 nmol) for women undergoing the standard test should be reexamined. We discuss applying these results to the conduct of future NPRO tests, as well as their implications for reducing the potential production of carcinogenic nitrosamines in the stomach.

N-nitrosoproline excretion by rural Nebraskans drinking water of varied nitrate content.

The N-nitrosoproline (NPRO) test for in vivo nitrosation was applied in a study of 44 rural Nebraska men drinking high- or low-nitrate water from private wells. The subjects followed diets low in NPRO and nitrate for 5 days. On days 4 and 5 they avoided ascorbate-rich foods. Urine was collected for 24 h on day 4 while the subjects followed normal activities and on day 5 after an overnight fast and taking 500 mg L-proline. We determined NPRO, nitrate, creatinine, and specific gravity in the urines, and nitrite and nitrate in single saliva specimens collected on days 4 and 5. Results for all variables were separated into those above and below the median values and were analyzed by univariate and multivariate consideration of the contingency tables. Nitrate concentration in drinking water (> or = or < 10 ppm nitrate-nitrogen) was significantly associated with both day 4 and day 5 NPRO (> or = or < 1.5 micrograms/day; P < 0.04); and with urine nitrate (> or = or < 1.5 mmol/day), saliva nitrite (> or = or < 5 mg/liter), and saliva nitrate (> or = or < 25 mg/liter) (P < or = 0.002). Urine nitrate was significantly (P < or = 0.03) associated with both day 4 and day 5 NPRO, with odds ratios of 4.2 and 5.4, respectively. Creatinine was positively associated with NPRO on day 4 (P = 0.04). These findings, like those of a recent study in Denmark, showed an association between nitrate intake in water and NPRO formation. Their significance for people drinking high-nitrate water remains to be determined.

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.

Hydroxy metabolites of methyl-n-amylnitrosamine produced by esophagus, stomach, liver, and other tissues of the neonatal to adult rat and hamster.

We measured the ability of neonatal to adult MRC-Wistar rat and Syrian hamster tissues to convert the esophageal carcinogen methyl-n-amylnitrosamine (MNAN) into the stable metabolites 2- to 5-hydroxy-MNAN and 3- and 4-oxo-MNAN. Slices or pieces of freshly removed tissues were incubated for 3 h with 23 microM MNAN and dichloromethane extracts were analyzed by gas chromatography-thermal energy analysis. The sum of the metabolites was expressed as percent metabolism of MNAN/100 mg tissue ("percent metabolism"). Tissues of animals from 1 day before birth to 56-70 days of age were examined. Metabolites in rat esophagus reached 12.6% at 6 days of age, three times the adult level, and that in hamster esophagus reached 13.1% at birth, 22 times the adult level. Forestomach metabolism was 1.9% in 3-day rats and 5.7% in 3-day hamsters, though the adult levels were less than 0.5%. Metabolism in rat, but not hamster, liver showed a peak at 9 days that was 3.6 times the adult level. Hamster, but not rat, skin showed about 1% metabolism. Total metabolism by glandular stomach, lung, and trachea of both species also showed changes with age. Ratios between 2-, 3-, 4-, and 5-hydroxy-MNAN were of three types: considerable 2-, 3-, and 4-hydroxy-MNAN, typical of esophagus; mainly 4-hydroxy-MNAN, typical of liver; and mainly 5- with some 4-hydroxy-MNAN, typical of rat lung. Incubation of adult rat liver and esophagus with varied MNAN concentrations showed apparent Km values of 150 (esophagus) and 300 (liver) microM. Metabolite yields after young and adult rat esophagus and liver were incubated with 23 microM MNAN for 1, 2, or 3 h indicated that differing in vitro stability of enzyme activities did not explain the age differences. The 2.9- to 3.6-fold differences in total metabolite yield between young and adult rat esophagus and liver, observed when these tissues were incubated with 23 microM MNAN, was in contrast to the 1.3- to 1.6-fold difference when these tissues were incubated with 300 or 600 microM MNAN, suggesting that much of the observed age difference was specific to low MNAN concentrations. MNAN hydroxylation could be used to indicate tissue susceptibility to MNAN carcinogenesis and the presence of enzymes (probably cytochrome P-450 isozymes) that catalyze each of the three types of MNAN metabolism.