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.

Cigarette smoking, urinary acidity, and nicotine excretion under natural conditions.

For 40 males who smoked 20 cigarettes a day titratable acidity of the urine was significantly correlated with nicotine/cotinine excretion at several sample intervals, as was urinary pH, but not urinary acidity and daily cigarette consumption or serum COHB.

Effects of phenol and 2,6-dimethoxyphenol (syringol) on in vivo formation of N-nitrosomorpholine in rats.

We determined the effects of phenol and 2,6-dimethoxyphenol (syringol) on N-nitrosomorpholine (NMOR) formation in rats given morpholine and nitrite by gavage. At 30 min post-gavage the recovery (from the stomach, duodenum and blood) of 564 micrograms NMOR was six times higher when administered to rats by gavage with 2 g of semipurified diet (SPD) than when given without food. Rats were gavaged with 12 mg each of morpholine, one of the modifiers and nitrite and examined 30 min later. Syringol decreased the amount of NMOR in both the stomach and blood by 89%, while phenol had no effect. We compared these results with those obtained with ascorbic acid and thiocyanate. The effect of ascorbic acid was similar to that of syringol. However, thiocyanate increased the amount of NMOR in the stomach and blood 2.7- and 4-fold, respectively. When 2 g of SPD was administered to rats by gavage, together with the precursors, syringol and ascorbic acid blocked NMOR formation in the stomach by 58 and 45%, respectively, and thiocyanate enhanced the yield 1.5-fold. The effect of phenol was not significant for the stomach and blood and that of the other modifiers was not significant for blood. Administration of the reactants together with food decreased the NMOR level in blood 155-fold relative to controls (no food), suggesting that food decreased the absorption rate over a 30-min period. These results demonstrate the modifying effect of phenol and syringol on NMOR formation in vivo to be similar to that observed in a previous in vitro study, and show that the effect of food on NMOR levels in blood was more important than that of the modifiers.

Beta- to omega-hydroxylation of the esophageal carcinogen methyl-n-amylnitrosamine by the rat esophagus and related tissues.

When the esophageal carcinogen methyl-n-amylnitrosamine (MNAN; concentration, 3 mg/liter) was incubated in vitro with rat esophagi for 3 hr, five principal neutral metabolites (Metabolites 2 to 6; total yield, 3.0% of the MNAN per 100 mg tissue) were separated by gas chromatography, with detection by a thermal energy analyzer. Rat liver produced similar metabolites (total yield, 2.1% of the MNAN per 100 mg tissue). Metabolites 4 to 6 and a minor product, Metabolite 7, were tentatively identified as 2-, 3-, 4-, and 5-hydroxy-MNAN (HO-MNAN), respectively, from a comparison of their gas chromatography retention times with those of the synthesized compounds. Rat esophagus produced similar amounts of 3- and 4-HO-MNAN and lesser amounts of 2-HO-MNAN, whereas rat liver produced mainly 4-HO-MNAN. Rat nasal tissue metabolized 8.0% of the MNAN per 100 mg tissue, with a metabolite pattern like that of the esophagus. Rat lungs produced mostly 5-HO-MNAN. A comparison of yields from tissues of rats, hamsters, guinea pigs, and mice supported the view that the total production of neutral MNAN metabolites indicated the sensitivity of MNAN carcinogenesis, with some exceptions. MNAN injected i.p. was less carcinogenic for the esophagus and nasal cavity in Sprague-Dawley than in MRC-Wistar rats, perhaps because the livers of Sprague-Dawley rats metabolized more of the MNAN. The urine of MNAN-treated MRC-Wistar rats contained MNAN and metabolites provisionally identified as 2-, 3-, and (as the major product) 4-HO-MNAN. The identity of the urinary 4-HO-MNAN was confirmed by gas chromatography-mass spectrometry. We speculate that tissues like the esophagus, which (unlike the liver) produce significant proportions of 2- and 3-HO-MNAN, also produce significant amounts of the most likely proximal carcinogen, 1-HO-MNAN.

Bladder freeze ulceration and sodium saccharin feeding in the rat: examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes.

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.

Determination of N-nitrosobis(2-hydroxypropyl)amine in environmental samples.

N-Nitrosobis(2-hydroxypropyl)amine (ND2HPA) is a potent pancreatic carcinogen in hamsters and induces gastrointestinal and respiratory tract cancer in rats. The precursor amines, diisopropanolamine (Di-PA) and triisopropanolamine (Ti-PA), are used in some manufacturing processes and in cosmetic preparations. We have found low levels of ND2HPA in commercial Ti-PA (21-270 ng/g) and in Di-PA (20-1 300 ng/g) and have demonstrated that ND2HPA is formed from Ti-PA and nitrite in a yield comparable to that observed for formation of N-nitrosodiethanolamine (NDELA) from triethanolamine under relatively mild conditions. After reaction for 4 h at 37 degrees C (10 mmol/L amine, 40 mmol/L nitrite, pH 3.0), the ND2HPA yield was 0.51%. The NDELA yield under the same conditions was 0.96%. ND2HPA was determined by gas chromatography-thermal energy analysis (GC-TEA) and GC-high-resolution mass spectrometry (GC-MS) selected ion monitoring of the tert-butyldimethylsilyl (t-BDMS) ether after extraction on a Celite 560 column. The t-BDMS ethers of ND2HPA and NDELA yielded intense, structurally significant peaks at m/z 333.2030 and 305.1716, respectively. The GC-MS procedure provides sensitivity and selectivity comparable to that of GC-TEA.

The nitrosating agent in mice exposed to nitrogen dioxide: improved extraction method and localization in the skin.

We reported previously that mice exposed to atmospheric NO2 contained a nitrosating agent (NSA) that reacted with morpholine in aqueous methanol homogenates of the mice to give N-nitrosomorpholine. We have now found that N-nitrosomorpholine was also produced by reacting morpholine with ether extracts of aqueous homogenates prepared from NO2-exposed mice. After exposure to NO2 for 4 hr, mice contained NSA (5.0 nmol/g tissue, corrected to 50 ppm NO2 and assuming that 1 mol NSA yields 1 mol N-nitrosomorpholine). This is 3.6 times the concentration observed by our previous method. Some NSA (0.6 nmol/g tissue) was also detected in untreated mice. The NSA in ether extracts was nonvolatile and stable on storage at -15 degrees or for short periods in the presence of water at pH 1 to 10, but it was decomposed by a pH 1 solution of nitrite scavengers. It reacted to similar extents with three different secondary amines. Eighty-eight % of the NSA occurred in the skin, one-third of which was in the hair. The high skin concentration occurred when the bodies but not the heads of mice were exposed to NO2, indicating that the major exposure route was the skin. The NSA might consist of alpha-nitro or other activated nitrite esters derived from unsaturated lipids.

Gas-liquid chromatographic-thermal energy analyzer determination of N-nitrosodimethylamine in beer: collaborative study.

The GLC/TEA method for N-nitrosodimethylamine (NDMA) in beer was studied collaboratively by 13 laboratories from 7 countries. Collaborators were asked to analyze a total of 10 randomly labeled samples of beer consisting of the following duplicates: a naturally contaminated commercial beer; a beer extremely low (ca 0.1 ppb) in NDMA; and the low NDMA beer spiked with 0.5, 1.9, and 5.0 ppb NDMA. The pooled repeatability and reproducibility coefficients of variation (CV) for all samples were 17% and 27%, respectively. However, when data from 2 laboratories (outliers) were omitted, the corresponding CV values improved considerably (11% and 15%, respectively). Variance analysis showed the presence of a significant laboratory-sample interaction when all data were used for analysis, but this interaction disappeared when data from the 2 outlying laboratories were excluded. The pooled percent recovery of the overall method (omitting outliers) was 101.4 +/- 3.5. All the laboratories detected NDMA in the low NDMA beer. The method was adopted official first action.

Monitoring exposure of personnel to volatile nitrosamines in the laboratory environment.

A convenient sampling method was developed for collection of volatile nitrosamines from large-volume air samples. Stainless steel tubes containing 0.3 g Tenax GC were employed to collect nitrosamines from 5-30 1 air samples. Nitrosamines were eluted from the sample tubes with diethylether to minimize formation of artifacts which were observed when thermal desorption was employed. Eluates were analysed directly by GC-TEA and nitrosamine identities were confirmed using high-resolution GC-MS with selected ion-monitoring. The detection limit was approximately 0.8 micrograms/m3 (0.3 ppb) for NDMA in 2 ml of diethylether extract. The laboratory operations studied included chemical synthesis, trace analysis, animal treatment, microbial mutagenesis tests and in vitro biochemical procedures. In most cases, nitrosamines were not detected in laboratory air, but levels of 200-800 micrograms/m3 (42 to 180 ppb) of N-nitrosomethyl-tert-butylamine were measured during animal treatment, 0.8-8.6 micrograms/m3 (0.3 to 2.8 ppb) of NDMA during mutagenesis assays, 12-22 micrograms/m3 (4-7 ppb) of NDMA during in vitro metabolism studies and 11 micrograms/m3 (3.6 ppb) of NDMA in a walk-in refrigerator. Appropriate corrective measures reduced all nitrosamine levels to below the detection limit. Hamsters and rats treated with NDAA (80 mg/kg, s.c.) excreted 4.4 and 12.9%, respectively, of the nitrosamine in expired air in 24 hr. This route of excretion may be metabolically significant and should be considered in the safe design of animal treatment and holding facilities.

Air-water and ether-water distribution of N-nitroso compounds: implications for laboratory safety, analytic methodology, and carcinogenicity for the rat esophagus, nose, and liver.

The air-water distribution ratio K1, ether-water distribution ratio K2, and solubility in water were measured for 17 nitrosamines, 3 nitrosamides, and 1 nitrosocyanamide. For K1, air was analysed either by UV absorption of ethanol extracts or by gas chromatography. K1 at 37 degrees C varied from less than 2 X 10(-6) to 4.5 X 10(-3), with 11 compounds showing K1 greater than 10(-4). A literature analysis provided data on the carcinogenicity of these N-nitroso (NNO) compounds toward the esophagus, nose, and liver of the rat. This compilation of data on physical properties and carcinogenicity of NNO compounds was then analysed in terms of: a) safety of workers handling either solutions of volatile NNO compounds or animals treated with these compounds, b) analytic methdology, and c) possible correlations between K1-, K2-, and solubility-characteristics, and carcinogenicity. Overall correlations were not observed. However, within each of five chemical groups, K1 and K2 tended to be associated positively with esophageal and nasal carcinogenicity and negatively with hepatic carcinogenicity. Water solubility showed the opposite associations.

Nitrite, nitrosamines, and cancer.

Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution.

Sex dependent toxicity of four chemicals.

Toxicity investigations were conducted with 4 chemicals: hydrazine, 1,2-dimethylhydrazine di HCl, phenylhydrazine HCl and Beta-N-(gamma-L(+)-glutamyl)-4-hydroxymethylphenylhydrazine in Swiss mice. Hydrazine and phenylhydrazine HCl were administered daily in drinking water, the former at 0.01% and the latter at 0.001% concentrations. The 1,2-dimethylhydrazine di HCl and Beta-N-(gamma-L(+)-glutamyl)-4-hydroxymethylphenylhydrazine were given as a single subcutaneous injection at 45 mug and 100 mug/gr. body weight basis, respectively. The findings clearly showed that all four chemicals exerted a stronger toxic effect in male than in female mice. It is, therefore, recommended in similar situations to use different doses of chemicals for each sex in the long-term tumorigenesis studies.

Dihydroxyphenylalanine in rat food containing wheat and oats.

Dopa has been identified in rat food by three different fluorimetric assays and paper chromatography. Incubation of the rat food with proteolytic enzymes dramatically increased the measurable free dopa. Analysis of samples of six individual protein-containing constituents of rat food revealed that both wheat and oats contain dopa.