Determination of total N-nitroso compounds and their precursors in frankfurters, fresh meat, dried salted fish, sauces, tobacco, and tobacco smoke particulates.

Total N-nitroso compounds (NOC) and NOC precursors (NOCP) were determined in extracts of food and tobacco products. Following Walters' method, NOC were decomposed to NO with refluxing HBr/HCl/HOAc/EtOAc and NO was measured by chemiluminescence. NOC were determined after sulfamic acid treatment to destroy nitrite, and NOCP were determined after treatment with 110 mM nitrite and then sulfamic acid. Analysis without HBr gave results < or =20% of those with HBr. This NOC method was efficient for nitrosamines but not nitrosoureas. The standard nitrosation for determining NOCP gave high yields for readily nitrosated amines, including 1-deoxy-1-fructosylvaline, but not for simple amines, dipeptides, and alkylureas. Mean NOC and NOCP results were (respectively, in micromol/kg of product) 5.5 and 2700 for frankfurters, 0.5 and 660 for fresh meat, 5.8 and 5800 for salted, dried fish, and 660 and 2900 for chewing tobacco (all for aqueous extracts) and 220 and 20000 nmol/cigarette for MeCN extracts of cigarette smoke filter pads.

Depentylation of the rat esophageal carcinogen, methyl-n-pentylnitrosamine, by microsomes from various human and rat tissues and by cytochrome P450 2A3.

Methyl-n-pentylnitrosamine (MPN) is carcinogenic for the rat esophagus. To determine organ specificity for MPN activation by human tissues, microsomes isolated from human organs (snap-frozen <6 h after death or removed surgically) were incubated with [pentyl-(3)H]MPN, and [(3)H]pentaldehyde formation was measured by high-pressure liquid chromatography of its 2,4-dinitrophenylhydrazone using radioflow assay. With 100 microM MPN, mean depentylation rates were 6.6 (liver), 2.9 to 3.8 (kidney, stomach, small intestine, and colon), and 0.4 to 1.6 (esophagus, lung, and skin) pmol of pentaldehyde/mg of protein/min. Of 14 human esophagi, four showed relatively high depentylation rates of 3.3 to 4.1 pmol/mg/min. Apparent K(m) was 80 to 160 microM (V(max), 3-15 pmol/mg/min) for three esophagi, 90 to 130 (2 livers), and 1330 (1 kidney) microM. Rat tissues showed mean depentylation rates for 100 microM MPN of 24.9 (liver), 14.5 (esophagus), 7.0 (lung), and 0.0 to 2.7 (5 other tissues) pmol/mg/min. MPN depentylation by rat cytochrome P450 2A3 showed an apparent K(m) of 8 microM (V(max), 70 pmol/nmol of P450/min) and was competitively inhibited by the CYP2A inhibitor coumarin (apparent K(i), 4 microM). Coumarin (0.4 mM) inhibited microsomal depentylation of 100 microM MPN by 37 to 62% for human esophagus, liver, kidney, and colon and for rat esophagus but not for rat liver and lung. MPN depentylation by rat esophageal microsomes increased up to 90% on adding P450 reductase. The results indicate organ-specific MPN metabolism by rat but not human esophagus. Nevertheless, the relatively high activity of four human esophagi might indicate increased susceptibility of some individuals to carcinogenesis by unsymmetrical dialkylnitrosamines.

Nitrate and nitrite concentrations in human saliva for men and women at different ages and times of the day and their consistency over time.

Salivary nitrite arises from nitrate and is the main source of gastric nitrite, a precursor of carcinogenic N-nitroso compounds. We examined nitrate and nitrite levels in unstimulated saliva from subjects consuming low-nitrate low-vitamin C diets. When saliva was collected from six men at nine times of the day (Experiment 1), night time nitrite levels were significantly higher than day time values and nitrite varied more than nitrate. When saliva was collected from 29 subjects aged 19-37 or 60-84 years at four times of the day during 1991-1993 (Experiment 2), all older subjects and older men had significantly higher nitrite levels than the corresponding younger subjects, night time nitrite levels in men were significantly raised, and nitrate and nitrite levels in the same samples were closely correlated. Saliva was collected at 6.00 a.m. on two successive days in 1997 from 16 subjects who had collected saliva in 1991-1993 (Experiment 3). Nitrate and nitrite levels on day 1 of experiment 3 were closely correlated with those on day 2. Nitrate and nitrite levels on days 1 and 2 of Experiment 3 were correlated with the corresponding parameters in Experiment 2 with P = 0.04 and 0.08 for day 1, and 0.10 and 0.28 for day 2, respectively. Hence, saliva nitrite levels rose at night and were higher in older people, especially older men, and saliva nitrate and nitrite levels varied little from day to day, but varied more after 4-6 years.

Metabolism of the hamster pancreatic carcinogen methyl-2-oxopropylnitrosamine by hamster liver and pancreas.

BACKGROUND: The mechanism whereby methyl-2-oxopropylnitrosamine (MOP) is activated remains unknown. To begin investigating this mechanism, we followed MOP disappearance during its incubation with liver and pancreatic slices and homogenates from Syrian hamsters and rats. METHODS: After the incubations, disappearance of 100 microM MOP and appearance of a metabolite was followed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. RESULTS: Disappearance rates were 1.2 nmol/mg protein/h for hamster liver slices; zero for hamster pancreatic slices, ducts and acini; zero for rat liver and pancreatic slices; and 11.8, 12.8, 1.3, and 2.3 nmol MOP/mg/h for hamster liver homogenate and cytosol, and hamster pancreas homogenate and microsomes, respectively. The principal MOP metabolite was identified as methyl-2-hydroxypropylnitrosamine (MHP) by its HPLC behavior and its 1H-NMR and mass spectra. MHP yields were generally similar to MOP consumption, but were zero for hamster pancreatic homogenate despite its ability to metabolize MOP. CONCLUSION: MOP is a pancreatic carcinogen in hamsters but not in rats. In metabolic studies, hamster liver slices and homogenate (especially the cytosol) produced MHP from MOP. This is probably an inactivation reaction. Hamster pancreas homogenate (especially the microsome fraction), but not rat pancreas homogenate, metabolized MOP without forming MHP, indicating another route of metabolism, perhaps activation to give the proximal carcinogen.

Inhibition of methyl-n-amylnitrosamine hydroxylation by diallyl sulfide and phenethylisothiocyanate in the rat.

Formation of the stable 2-, 3-, and 4-hydroxy derivatives of methyl-n-amylnitrosamine (MNAN) probably reflects cytochrome P-450-catalyzed activation of MNAN by 1-hydroxylation. Here we studied inhibition of the oxidation of MNAN to hydroxy-MNANs (HO-MNANs) by freshly excised tissues from MRC-Wistar rats treated with the vegetable-derived chemicals diallyl sulfide (DAS) and phenethylisothiocyanate (PEITC). Rats were gavaged with DAS (200 mg/kg), PEITC (163 mg/kg), or vehicle (corn oil) alone. After various times, the rats were killed, the esophagus, nasal mucosa, and liver were removed, and the tissues/tissue slices were incubated for two hours with 23 microM MNAN. HO-MNAN formation was measured by gas chromatography-thermal energy analysis. Significant (p < 0.01) 72-75%, 40%, and 44% inhibitions of total HO-MNAN formation were observed for nasal mucosa removed at 3-18 hours, for esophagus at 18 hours, and for liver at 3 hours, respectively, after gavage of DAS. Significant (p < 0.03) 46-75% inhibition of HO-MNAN formations was observed for the esophagus at 2-24 hours after gavage of PEITC. In disposition studies, rats were treated with DAS (200 mg/kg) in corn oil and sacrificed after various intervals. DAS was determined by gas chromatography of tissue homogenate extracts. After gavage of DAS, its total recovery from all tissues studied was 27% of the dose after 45 minutes and 15-19% after 90 and 180 minutes, with > 80% of the recovered DAS in the stomach contents. Up to 2% per tissue of the recovered DAS was found in the stomach wall, liver, and blood. After intraperitoneal injection of DAS, < or = 2% of the dose was recovered in the blood and < or = 0.7% in the liver. Hence, gavage of DAS and PEITC significantly inhibited HO-MNAN formation for up to 18 and 24 hours, respectively, whereas DAS was > 80% metabolized 90 minutes after its gavage. These findings suggest that long-lasting inhibitors or their metabolites, or inactivation of P-450 enzymes, were responsible for the persistence of inhibition of MNAN metabolism.

Diffusion of dialkylnitrosamines into the rat esophagus as a factor in esophageal carcinogenesis.

To indicate how readily nitrosamines (NAms) diffuse into the esophagus, we measured diffusion rate (flux) through rat esophagus of dialkyl-NAms using side-by-side diffusion apparatuses. Mucosal and serosal flux at 37 degrees C of two NAms, each at 50 microM, was followed for 90 min by gas chromatography-thermal energy analysis of NAms in the receiver chamber. Mucosal flux of one or two NAms at a time gave identical results. Mucosal flux was highest for the strong esophageal carcinogens methyl-n-amyl-NAm (MNAN) and methylbenzyl-NAm. Mucosal esophageal flux of 11 NAms was 18-280 times faster and flux of two NAms through skin was 13-28 times faster than that predicted for skin from the molecular weights and octanol:water partition coefficients, which were also measured. Mucosal: serosal flux ratio was correlated (P < 0.05) with esophageal carcinogenicity and molecular weight. For seven NAms tested for carcinogenicity by Druckrey et al. [(1967) Z. Krebsforsch., 69, 103-201], mucosal flux was correlated with esophageal carcinogenicity with borderline significance (P = 0.07). The MNAN:dipropyl-NAm ratio for mucosal esophageal flux was unaffected when rats were treated with phenethylisothiocyanate and was similar to that for forestomach, indicating no involvement by cytochromes P450. Mucosal esophageal flux of MNAN and dimethyl-NAm was reduced by >90% on enzymic removal of the stratum corneum, was unaffected by 0.1 mM verapamil and was inhibited 67-94% by 1.0 mM KCN and 82-93% by 0.23% ethanol. NAm flux through rat skin and jejunum was 5-17% of that through esophagus. Flux through skin increased 5-13 times after enzymic or mechanical removal of the epidermis; the histology probably explained this difference from esophagus. Hence, NAms could be quite rapidly absorbed by human esophagus when NAm-containing foods or beverages are swallowed, the esophageal carcinogenicity of NAms may be partly determined by their esophageal flux and NAm flux probably occurs by passive diffusion.

Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms.

Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]MNAN and [3H-2,3-pentyl]-MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high-performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed Km values of 64, 610, and 170-330 microM, respectively (Vmax: 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 microM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (Ki, 120 microM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7-hydroxylation by RLM and CYP2A6 (Ki, 3000 and 320 microM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. Km for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 microM, respectively (Vmax: 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

Effect of duodenal components of the refluxate on development of esophageal neoplasia in rats.

When duodenal content is allowed to reflux into the esophagus of nitrosamine-treated rats, esophageal cancer is induced more rapidly and at higher frequency than after carcinogen treatment alone. The purpose of the present study was to identify the components of the duodenal content that are responsible for enhancing esophageal carcinogenesis. Eight-week-old Sprague-Dawley rats underwent one of four operations as follows: diversion of bile alone, pancreatic juice alone, both bile and pancreatic juice into the esophagus, or a control operation with no induced reflux. Two weeks after surgery, rats were treated with the esophageal carcinogen 2, 6-dimethylnitrosomorpholine (48 mg/kg [0.1 of LD50] intraperitoneally weekly for 20 weeks). The rats were killed at age 30 weeks. The esophagus was removed and full-length strips were examined under a microscope; separate segments were taken for flow cytometric evaluation. The prevalence of DNA aneuploidy and histologic esophageal papillomas or squamous cancer was increased in carcinogen-treated rats with pancreatic juice reflux (P <0.05 vs. control) and the combination of pancreatic and bile reflux (P <0.05 vs. control) but not in rats with bile reflux alone. We conclude that pancreatic juice is the most potent component of the duodenal refluxate in the promotion of esophageal carcinogenesis in rats.

Effect of ascorbic acid dose taken with a meal on nitrosoproline excretion in subjects ingesting nitrate and proline.

We determined the dose of ascorbic acid (ASC) given to subjects with a standard 400-calorie meal that inhibited N-nitrosoproline (NPRO) formation when we gave 400 mg of nitrate one hour before and 500 mg of L-proline with the standard meal. Volunteers consumed their normal US diets but restricted their intakes of nitrate, proline, NPRO, and ASC. NPRO and N-nitrososarcosine (NSAR) were determined in the 18-hour urines by methylation followed by gas chromatography-thermal energy analysis. Mean NPRO yields were 10.7, 41.9, 33.2, 22.3, and 23.1 nmol for groups of 9-25 subjects taking proline alone, proline + nitrate, and proline + nitrate + 120, 240, and 480 mg of ASC, respectively. There was a significant trend to lower NPRO yields as the ASC dose was raised. These results correspond to inhibitions by ASC of 28%, 62%, and 60%, respectively. Pairwise comparison showed that each group taking ASC formed significantly less NPRO than the group given only proline + nitrate. Mean NSAR yields were 9.0 nmol when proline alone was taken and 16.9-24.0 nmol when proline + nitrate + ASC was taken, with no trend to increase as the ASC dose was raised. However, NPRO and NSAR yields in individual urines were correlated with each other. We concluded that 120 mg of ASC taken with each meal (360 mg/day) would significantly reduce in vivo nitrosamine formation, similar to tests by Leaf and co-workers (Carcinogenesis 8, 791-795, 1987) in which the reactants were taken between meals. The inhibitory dose of ASC may be < 120 mg/meal when doses of nitrate and proline are not taken.

Development of esophageal metaplasia and adenocarcinoma in a rat surgical model without the use of a carcinogen.

In order to establish an animal model for studying the cause and prevention of esophageal adenocarcinoma (EAC) and its frequent precursor, Barrett's esophagus (BE), factors affecting the pathogenic processes were investigated in an esophagoduodenal anastomosis model with rats. Experiments by us and others have shown that surgical treatment produced reflux esophagitis with cell hyperproliferation, but not EAC. Additional treatment with a carcinogen has been shown to be necessary for the development of EAC, squamous cell carcinomas (SCC) or EAC/SCC mixtures. We found that the surgically treated animals developed anemia due possibly to reduced iron absorption. When the operated animals were supplemented with iron, EAC occurred at a high rate (73%) after 30 weeks, and treatment with N'-nitrosonornicotine did not enhance the rate of tumorigenesis. Treatment with carcinogen, however, induced SCC in the group of rats killed after 22 weeks. The results suggest that iron overload, which is known to cause oxidative damage, is an enhancing factor for adenocarcinogenesis. The pathogenesis of EAC in the iron-supplemented, non-carcinogen treated group resembles human esophageal adenocarcinogenesis in many features. All the BE was the specialized type with goblet cells (containing sialomucin or sulfomucin) and columnar cells (containing acid or neutral mucin) as well as an incompletely developed brush border. Almost all of the BE was located at the bottom of the esophagus and was continuous with the duodenal mucosa; dysplasia became more frequent at later time points. All of the cancers were well-differentiated mucinous EAC, and most of the EAC had an adjacent area of BE with dysplasia. The results are consistent with the proposed human sequence for pathogenic events of BE progression to 'BE with dysplasia' and then to EAC. Esophagoduodenal anastomosis and iron treatment in rats produces a high rate of BE and EAC which are morphologically similar to human BE and EAC; this may be a useful animal model to study the development and prevention of EAC in humans.

Studies on experimental animals involving surgical procedures and/or nitrosamine treatment related to the etiology of esophageal adenocarcinoma.

After a brief review of the epidemiology and etiology of lower esophageal adenocarcinoma (EAC), this paper describes long-term experiments on animals (mostly rats) demonstrating that reflux of duodenal contents into the stomach can induce gastric and pancreatic cancer, that gastric reflux into the esophagus can induce Barrett's esophagus; that esophagoduodenostomy to facilitate duodenal reflux into the esophagus, together with administration of carcinogenic nitrosamines, induces squamous cancer and EAC in the lower esophagus; that both pancreatic juice and bile are involved in this induction of EAC; that a high-fat diet increases EAC induction; and that esophagoduodenostomy with gastrectomy and nitrosamine treatment or esophagojejunostomy without a carcinogen can produce up to an 88% incidence of EAC. Short-term animal experiments are reviewed in which bile salts and trypsin have damaged the esophagus and duodenal reflux has produced lipid peroxidation in the lower esophagus. Finally, I review arguments mostly derived from the animal experiments that reflux of unacidified duodenal juice via the stomach into the lower esophagus may help cause Barrett's esophagus and EAC, that excessive use of acid blockers might contribute to EAC induction, and that EAC induction may be reduced by surgery to repair the lower esophageal sphincter or perhaps by taking non-steroidal anti-inflammatory drugs.

Carcinogenicity tests of methyl-n-amylnitrosamine (MNAN) administered to newborn and adult rats and hamsters and adult mice and of 2-oxo-MNAN administered to adult rats.

We examined the toxicity and carcinogenicity in rodents of methyl-n-amylnitrosamine (MNAN), multiple doses of which are known to induce esophageal and nasal tumors in rats. A single i.p. injection of 50-70 mg MNAN/kg into adult rats produced a 74% incidence of esophageal squamous carcinomas (mean latency, 63 weeks). Single doses of 3.0-12.5 mg/kg of MNAN injected into newborn and 3-day-old rats and hamsters were not carcinogenic in rats and only weakly carcinogenic in hamsters. The low doses (used because larger doses produced lethal interstitial pneumonia) probably explain the low carcinogenicity, despite previous findings of extensive formation of stable hydroxy-MNANs from MNAN by the esophagus of both species at these ages, which may indicate MNAN activation. One i.p. injection of 70-100 mg MNAN/kg into adult Syrian hamsters was weakly carcinogenic for the esophagus and forestomach. Six injections of 75 mg MNAN/kg into adult hamsters induced lung and nasal cavity tumors (65 and 43% incidences, respectively), but only a few esophageal tumors. Three injections of 15 mg MNAN/kg into adult Swiss mice induced lung adenomas and esophageal papillomas in 71 and 32% incidences, respectively. These results partially agreed with previous studies on hydroxy-MNAN formation by the esophagus of these species. Six s.c. injections of 75 mg 2-oxo-MNAN/kg into adult rats induced tumors of the nasal cavity, esophagus and soft tissue at the injection site in 68, 63, and 32% incidences, respectively. This does not support the view that 2-oxo-MNAN is an active metabolite of MNAN.

Gastric juice protects against the development of esophageal adenocarcinoma in the rat.

OBJECTIVE: The authors investigate the effects of gastric juice on tumorigenesis in a rat model of esophageal adenocarcinoma. SUMMARY BACKGROUND DATA: In rats treated with the carcinogen methyl-n-amyl nitrosamine, squamous cancer of the esophagus develops in a time- and dose-dependent manner. When methyl-n-amyl nitrosamine treatment is preceded by an operation to induce reflux of duodenal and gastric juice into the esophagus, there is an increased yield of esophageal tumors, many of which are adenocarcinomas. When only gastric juice refluxes into the esophagus, the tumor yield is less and adenocarcinomas are not found. METHODS: Two hundred seventy 8-week old Sprague-Dawley rats were studied. Twenty unoperated rats served as controls. The remaining rats underwent the following operations: esophagoduodenostomy with gastric and vagal preservation to induce duodenogastroesophageal reflux (n = 48); esophagoduodenostomy with antrectomy and Billroth 1 reconstruction to produce reflux of duodenogastric juice with the exclusion of the antrum (n = 53); esophagoduodenostomy with proximal gastrectomy to induce hypergastrinemia and reflux of duodenogastric juice with exclusion of the body and forestomach (n = 51); esophagoduodenostomy plus total gastrectomy to produce reflux of duodenal juice alone (n = 50); and esophagoduodenostomy with vagal and gastric preservation but with division of the duodenum just beyond the pylorus and reimplantation into the jejunum, 13 cm distal to the esophagoduodenostomy. This produced reflux of duodenal juice with gastric juice diverted downstream, (n = 48). At 10 weeks of age, all rats were given 4 weekly doses of carcinogen (methyl-n-amyl nitrosamine, 25 mg/kg intraperitoneally), and survivors were killed at 36 weeks of age. RESULTS: The prevalence rate of esophageal adenocarcinoma was 30% in rats with duodenogastroesophageal reflux and 87% in rats with reflux of duodenal juice alone. Fifty-six percent of rats with reflux of duodenogastric juice with exclusion of the antrum and 72% of rats with reflux of duodenogastric juice with the exclusion of the body and forestomach developed adenocarcinoma, showing a progression increase in the prevalence of adenocarcinoma as less gastric juice was permitted to reflux with duodenal juice into the esophagus. CONCLUSION: In this rat model, the presence of gastric juice in refluxed duodenal juice against the development of esophageal adenocarcinoma. The protective effect appears to be due to acid secretion from the stomach. Continuous profound acid suppression therapy may be detrimental by encouraging esophageal metaplasia and tumorigenesis in patients with duodenogastroesophageal reflux.

Carcinogenesis by methylbenzylnitrosamine near the squamocolumnar junction and methylamylnitrosamine metabolism in the mouse forestomach.

We repeated and extended a 1973 study by Sander and Schweinsberg on forestomach tumorigenesis in mice by methylbenzylnitrosamine (MBZN). Groups of 80 adult CD-1 mice of both sexes received 96 mg/kg of MBZN subdivided into 24 doses of 4 mg/kg, 12 doses of 8 mg/kg or 6 doses of 16 mg/kg (groups 1-3, respectively). The mice were injected i.p. twice weekly with MBZN in 30% dimethylsulfoxide and 6-8 mice/group were killed every 4 weeks up to 40 weeks. Ten untreated control mice did not develop forestomach tumors. Forestomach papillomas occurred in 35-53% of the treated mice, with the highest incidence and shortest latency (mostly <24 weeks) in group 3. Squamous carcinomas of the forestomach were found in 31% of group 1 and 4-6% of groups 2 and 3. Ninety-two percent of the carcinomas and 94% of the papillomas in the 8-mm wide forestomach occurred < or = 1 mm from the squamocolumnar junction (SCJ) with the glandular stomach. This is interesting in view of the rising incidence of human adenocarcinoma near the gastroesophageal SCJ. Methyl-n-amylnitrosamine (MNAN) yields 2-, 3- and 4-hydroxy-MNAN (HO-MNAN) in a 1:3:2 ratio when incubated with rodent tissues for which MNAN is carcinogenic. This metabolism may be due to cytochrome P450 isoform believed responsible for MNAN and, probably, MBZN activation. When freshly excised mouse forestomach and esophagus were incubated for 2 h with 23 microM MNAN, total HO-MNAN yields were 0.79 +/- 0.05 and 1.81 +/-0.08 nmol/100 mg tissue per h (mean +/- SE), respectively, with about 1:3:2 ratios between 2-,3- and 4-HO-MNAN. This compares with published mean HO-MNAN yields in nmol/100 mg per h of 1.2 for rat esophagus (where MNAN and MBZN are strongly carcinogenic) and <0.1 for rat forestomach. These findings may explain why MNAN and MBZN induce forestomach tumors in mice but not in rats and why MNAN induces esophageal tumors in mice, but does not explain why MBZN given i.p. fails to induce esophageal tumors in mice. Three sections of the mouse forestomach (closest-to to furthest-from the SCG) showed total HO-MNAN yields from MNAN of 0.61+/-0.05, 0.38+/-0.03 and 0.48+/-0.02 nmol/100 mg per h (mean +/-SE), respectively. This may help explain why the MBZN-induced forestomach tumors were non-centrated near the SCJ.

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.

Use of monoclonal antibodies to cytochrome P450s to indicate the critical dealkylation and the P450s involved in methyl-n-amylnitrosamine mutagenicity in the presence of induced rat liver microsomes.

The mutagenicity for Salmonella typhimurium TA 1535 of the carcinogen methyl-n-amylnitrosamine (MNAN) was examined in the presence of rat liver microsomes from uninduced and induced rats. The number of mutations followed the order phenobarbital- and Aroclor-induced > 3-methylcholanthrene- and isoniazid-induced > uninduced microsomes. The MNAN metabolite 4-hydroxy-MNAN was not mutagenic. Using each type of induced liver microsomes, we examined the effect on MNAN mutagenicity of four monoclonal antibodies (MAbs) that inhibit cytochrome P450s. The MAbs inhibited MNAN mutagenicity in seven MAb-microsome combinations by up to 49%. Taken together, these results indicated that CYP (P450) 2B1/2B2 was responsible for one half and CYP 2C11 for one quarter of MNAN mutagenicity with phenobarbital-induced microsomes, CYP 1A1/1A2 accounted for about 40% of the mutagenicity with 3-methylcholanthrene-induced microsomes, CYP 2B1/2B2 accounted for half and CYP 1A1/1A2 and 2C11 for smaller proportions of the mutagenicity with Aroclor-induced microsomes, and CYP 1A1/1A2 accounted for about 30% of the mutagenicity with isoniazid-induced microsomes. With isoniazid-induced microsomes, MAb 2-66-3 to CYP 2B1/2B1 caused an unexpected 219% increase and MAb 1-68-11 caused a moderate increase in MNAN mutagenicity. The test MAbs also inhibited the microsome-catalyzed demethylation and depentylation of MNAN by up to 83%, confirming previous results. Four comparisons between individual mutagenic and metabolic results supported the view that depentylation of MNAN was more critical for its mutagenicity than was demethylation, e.g., with 3-methylcholanthrene- and Aroclor-induced microsomes, MAb 1-7-1 to CYP 1A1/1A2 inhibited mutagenesis and depentylation, but did not affect demethylation.

Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC.

The questions of whether and how N-nitroso compounds (NOC) may be inducing cancer in humans are discussed. The principal subjects covered include nitrite-derived alkylating agents that are not NOC, reasons for the wide tissue specificity of carcinogenesis by NOC, the acute toxicity of nitrosamines in humans, mechanisms of in vivo formation of NOC by chemical and bacterial nitrosation in the stomach and via nitric oxide (NO) formation during inflammation, studies on nitrite esters, use of the nitrosoproline test to follow human gastric nitrosation, correlations of nitrate in food and water with in vivo nitrosation and the inhibition of gastric nitrosation by vitamin C and polyphenols. Evidence that specific cancers are caused by NOC is reviewed for cancer of the stomach, esophagus, nasopharynx, urinary bladder in bilharzia and colon. I review the occurrence of nitrosamines in tobacco products, nitrite-cured meat (which might be linked with childhood leukemia and brain cancer) and other foods, and in drugs and industrial situations. Finally, I discuss clues from mutations in ras and p53 genes in human tumors about whether NOC are etiologic agents and draw some general conclusions.

Effect of gastroduodenal juice and dietary fat on the development of Barrett's esophagus and esophageal neoplasia: an experimental rat model.

BACKGROUND: Reflux of duodenal content into the lower esophagus of rats enhances the formation of nitrosamine-induced esophageal cancer and results in the induction of adenocarcinoma. We investigated the extent of the mucosal injury that was produced when the lower esophagus of rats was exposed to the reflux of gastroduodenal juice in the presence or absence of a carcinogen and tested the hypothesis that induction of esophageal cancer in this model would be influenced by the intake of dietary fat. METHODS: Esophagoduodenostomy with gastric preservation was performed in 165 Sprague-Dawley rats in order to expose the lower esophagus to the reflux of gastroduodenal juice. Postoperatively selected groups of rats were treated with the carcinogen methyl-n-amylnitrosamine (MNAN). Subsequently, rats were fed diets of differing fat and calorie content for 20 weeks until they were put to death. RESULTS: Refluxed gastroduodenal juice, in the absence of MNAN, induced esophageal inflammatory changes (diffuse papillomatosis and hyperkeratosis) in 38 of 39 rats (97%), specialized columnar metaplasia (Barrett's esophagus) in four of 39 (10%), dysplasia in three of 39 (8%), and squamous cell carcinoma in one of 39 (3%). Diet did not influence the incidence of neoplasia in the absence of MNAN treatment. In rats treated with MNAN, refluxed gastroduodenal juice induced inflammation in 110 of 111 rats (99%), columnar metaplasia in 14 of 111 (13%), and cancer in 63 of 111 (57%). Fifty-eight percent of esophageal tumors were squamous cell carcinoma and 42% were adenocarcinoma. The highest incidence of tumors was observed in rats fed the semipurified high-fat diet (24 of 29; 83%) compared with rats fed the semipurified control diet (13 of 29; 45%), semipurified, calorie-restricted diet (15 of 27; 55%), and chow diet (11 of 26; 42%), p < 0.05. CONCLUSIONS: Reflux of gastroduodenal content into the lower esophagus of rats can induce both Barrett's metaplasia and neoplasia. Addition of a carcinogen increases the tumor yield and results in a proportion of the lesions being adenocarcinoma. This carcinogenic process is promoted by a diet with a high fat content.

Effect of catechol and ethanol with and without methylamylnitrosamine on esophageal carcinogenesis in the rat.

Alcohol consumption and cigarette smoking are synergistic etiologic factors for squamous cell carcinoma of the esophagus in Western countries. Catechol, a constituent of cigarette smoke, was previously found to be a co-carcinogen with methyl-n-amylnitrosamine (MNAN) for esophageal tumors in rats, when it was given in the diet. Here we tested whether the inclusion of ethanol in a similar system had an additional promoting effect on esophageal carcinogenesis. Male MRC - Wistar rats were injected three times i.p. with 25 mg MNAN/kg starting from 7 weeks of age. A second group of rats was injected similarly with MNAN and treated for life with 10% ethanol and 0.2% catechol in the drinking water, starting at 6 weeks of age. One or more test chemicals were omitted in other groups. The rats were maintained until they died and were necropsied. The number of esophageal papillomas/rat was 2.18 +/- 0.36, 4.27 +/- 0.53, 2.54 +/- 0.48 and 3.21 +/- 0.52 (mean +/- SE) in groups treated with MNAN alone, MNAN + ethanol + catechol, MNAN + ethanol and MNAN + catechol, respectively. Esophageal carcinomas showed a similar trend, with the number of carcinomas/rat equal to 0.23 +/- 0.08 in the MNAN alone group and 0.50 +/- 0.14 in the MNAN + ethanol + catechol group. Tumor multiplicities for the esophageal papillomas and carcinomas were significantly (P < 0.05) greater in the MNAN + ethanol + catechol group than in the MNAN group. These findings indicate that, in the esophagus, catechol alone was not significantly co-carcinogenic with MNAN when it was given in the drinking water (unlike when given in the diet in our previous study), but that ethanol + catechol given in the water was co-carcinogenic with MNAN. Seven of 19 rats given ethanol + catechol without MNAN developed esophageal papillomas, as compared to zero incidence in untreated controls (P = 0.06). Forestomach papillomas occurred in 22% of all rats given catechol. Hence, for esophageal tumor induction, ethanol and catechol were co-carcinogenic with MNAN and appeared to be tumorigenic when given without MNAN. Ethanol and catechol could have increased the carcinogenicity because they affected MNAN metabolism. As a partial test of this possibility, the effect of feeding these compounds for 5-7 weeks separately or together was examined on 2-, 3-, 4-and 5-hydroxy-MNAN (HO-MNAN) production from MNAN by the esophagus and liver slices from freshly killed rats.(ABSTRACT TRUNCATED AT 400 WORDS)