Environmental nitroso compounds: reaction of nitrite with creatine and creatinine.

Creatine reacts with nitrite under acid conditions to produce first sarcosine and then N-nitrososarcosine, which is a weak carcinogen in the rat. Creatinine reacts with acidified nitrite to produce either creatinine-5-oxime or 1-methylhydantoin-5-oxime, depending on reaction conditions. The toxicity and environmental significance of these compounds is not yet known.

Daily rhythm in human urinary melatonin.

The melatonin in urine samples from six healthy adult volunteers was concentrated on Amberlite XAD-2 resin, eluted with organic solvents, and quantitated by use of a bioassay technique (the dermal melanaphore response of larval anurans to melatonin in their bathing medium). The melatonin content of samples collected between 11 p.m. and 7 a.m. was, in each case, several times higher than that of samples collected between 7 a.m. and 3 p.m. or between 3 p.m. and 11 p.m.

Kinetics of nitrosamine formation in the presence of micelle-forming surfactants.

The nitrosation of dihexylamine by nitrite was catalyzed in the presence of micelles of the cationic surfactant decyltrimethylammonium bromide. Rate enhancements up to 800-fold were observed. The magnitude of the rate enhancements was dependent on the amine structure, particularly the alkyl chain length. Rate enhancements for nitrosation were also observed in the presence of micelles of phosphatidylcholine and Triton X-100. We present a mechanistic interpretation of these catalytic effects.

Deuterium isotope effect in the microsomal metabolism of dimethylnitrosamine.

The rates of N-demethylation of dimethylnitrosamine and dimethylnitrosamine-d6 by rat liver microsomes were studied by monitoring formaldehyde production. Deuterium isotope effects of 1.6 and 3.8 were found for the Michaelis constant and the maximum velocity, respectively.

N-nitrosation of proline in smokers and nonsmokers.

Endogenous nitrosation of proline was investigated in smokers and nonsmokers. Volunteers consumed a volume of beet juice equivalent to 325 mg nitrate, and 1 hour later they consumed 500 mg proline. In separate experiments volunteers ingested proline alone. Twenty-four-hour urine samples were collected and analyzed for N-nitrosoproline. When proline alone was ingested, there was no significant difference in urinary nitrosoproline excretion between smokers and nonsmokers. When beet juice and proline were consumed, however, smokers produced approximately 2.5 times as much N-nitrosoproline as nonsmokers. Salivary thiocyanate levels were approximately 3.2 times higher in smokers compared to those in nonsmokers. Salivary nitrite levels of smokers and nonsmokers, either before or after beet juice consumption, were not different. Salivary nitrate concentrations, however, were higher in nonsmokers than in smokers after beet juice consumption but not before. Our results suggest that the higher level of salivary thiocyanate in smokers is responsible for the increased rate of endogenous nitrosation of proline in this group compared to the rate in nonsmokers. Nitrosating agents in cigarette smoke do not appear to play a significant role.

Induction and suppression of N-nitrosomethylbenzylamine activation by microsomes from rat liver and esophagus.

Male Sprague-Dawley rats were pretreated with various chemicals to determine their effects on the microsomal activation of the esophageal carcinogen N-nitrosomethylbenzylamine [( NMBzA ) CAS: 937-40-6; N-methyl-N- nitrosobenzylamine ] in the rat esophagus and, for comparative purposes, in the rat liver. When rats were pretreated with NMBzA , little change in hepatic NMBzA - debenzylase activity was observed. In contrast, NMBzA metabolism in the esophagus was significantly (60-65%) reduced. Similarly, pretreatment of rats with disulfiram [CAS: 97-77-8; bis( diethylthiocarbamoyl )disulfide] caused a 40% decrease in esophageal metabolism, but it had no significant effect in the liver. Pretreatments with the methylenedioxybenzenes safrole [CAS: 94-59-7; 4-allyl-1,2-(methylenedioxy)benzene], isosafrole [CAS: 120-58-1; 1,2-(methylenedioxy)-4-propenylbenzene], and dihydrosafrole (CAS: 94-58-6; 1,2-(methylenedioxy)-4- propylbenzene ) caused a marked induction (twofold to fivefold) of the hepatic metabolism of NMBzA , but again esophageal metabolism was suppressed. The results indicate that esophageal metabolism of NMBzA is either unchanged or suppressed by the various chemical pretreatments, but hepatic metabolism of the nitrosamine is induced by the methylenedioxybenzenes .

Kinetic studies on the hepatic microsomal metabolism of dimethylnitrosamine, diethylnitrosamine, and methylethylnitrosamine in the rat.

The oxidative dealkylation of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), and methylethylnitrosamine (MEN) by Sprague-Dawley rat liver microsomes was studied by a colorimetric assay for the simultaneous analysis of formaldehyde and acetaldehyde. Dealkylation in each instance followed Michaelis-Menten kinetics, but the Michaelis constant (Km) for DEN was an order of magnitude smaller than the Km for DMN. Km values for demethylation and deethylation of MEN were intermediate between those for DMN and DEN. DMN inhibited-deethylase, and DEN inhibited DMN-demethylase activity. The inhibition was of a mixed type in both instances.

Cellular toxicity of pancreatic carcinogens.

Azaserine (CAS: 115-02-6), streptozocin (CAS: 18883-66-4; streptozotocin), and N-nitrosobis(2-oxopropyl)amine [(BOP) CAS: 60599-38-4] produce different types of pancreatic tumors in rodents. We have investigated the toxic effects of these compounds on pancreatic tissues from Wistar rats and Syrian hamsters. Inhibition of protein synthesis was used as a measure of toxicity. Pancreatic islets and acinar cells from rat and hamster were labeled with [3H]leucine for 60 minutes in vitro in the presence of the various carcinogens. Azaserine, which produces acinar cell tumors in the rat, inhibited synthesis by all tissues; rat acinar cells, however, were most sensitive. Glutamine, but not serine, provided some protection against azaserine toxicity. Streptozocin inhibited synthesis by islets of both species and acinar cells from hamster; islets were the most sensitive. BOP and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, which induce ductal tumors in the hamster, had no effect on any of the tissues examined. These results indicate that the specificities of the cellular toxicities of the pancreatic carcinogens parallel, to some degree, their tumorigenic effects.

Nitrosamine formation in human saliva.

Nitrosamines formed when secondary amines were added to normal human saliva. Fractionation of saliva into cells and supernatant showed that factors that accelerated and retarded the nitrosation reaction were both present. Acidification of saliva greatly increased the nitrosamine yield, but differences in nitrosamine yield among saliva fractions were still observable.

Reaction of nitrite with ascorbate and its relation to nitrosamine formation.

In this study of the nitrosation of morpholine in the presence of ascorbic acid, the amount of ascorbate required to inhibit completely the formation of nitrosomorpholine depended on whether oxygen was present in the system. The nitric oxide (produced during the oxidation of ascorbate by nitrous acid) might have reacted with oxygen to yield additional oxidizing equivalents, or oxygen might have directly oxidized the ascorbate semiquinone intermediate produced in the initial step of oxidation reaction. A pH- dependent induction period observed during the nitrosation of morpholine in the presence of ascorbate and excess nitrite was accounted for by the kinetics of the reactions.

Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein.

BACKGROUND: We have previously shown that thermolyzed protein (casein) cooked with fat in the diet of the rat promotes the growth of aberrant crypt foci (putative precursors of colon cancer) assessed at 100 days. PURPOSE: To determine how thermolysis affects this promotion, we examined thermolysis conditions, quantity of thermolyzed protein in the diet, and duration of thermolysis. To determine whether the previous finding of promotion of aberrant crypt foci corresponds to promotion of cancers assessed much later, we carried out promotion studies until colon cancers appeared. METHODS: F344 rats were given an initiating dose of azoxymethane and were then randomly allocated to groups receiving diets differing in their quantity and quality of casein. The groups were examined for aberrant crypt foci and tumors in the colon. RESULTS: Aberrant crypt foci were promoted by diets containing thermolyzed casein (180 degrees C, 2 hours). Promotion increased with increasing level of thermolyzed casein in the diet (to 20%) and with increasing thermolysis time (to 4 hours). The number of animals with polyps and cancers was higher in the animals receiving thermolyzed protein (2 hours), 16/23 versus 9/26 (P less than .05) and 10/26 versus 3/27 (P less than .05), respectively. The number of aberrant crypts per focus and the number of large aberrant crypt foci were higher in the tumor-bearing animals. CONCLUSIONS: Thermolyzed casein promotes early colonic precursor lesions in a dose-dependent and thermolysis time-dependent manner; thermolyzed casein also promotes colon cancer. IMPLICATIONS: The promoter formed on thermolysis could be involved in colon cancers associated with diets cooked at elevated temperatures, such as can occur with high-fat diets.

Esophageal and hepatic microsomal metabolism of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in the rat.

The metabolism of the rat esophageal carcinogen N-nitrosomethylbenzylamine (NMBzA) was studied using microsomes prepared from liver and esophageal mucosa of untreated male Sprague-Dawley rats. NMBzA was extensively metabolized to benzaldehyde, benzyl alcohol, and formaldehyde by hepatic microsomes. The rate of metabolism at the benzyl moiety was 10-fold higher than that at the methyl moiety. Mucosal microsomes metabolized NMBzA to benzaldehyde and formaldehyde at rates one-fifth and one-sixtieth of those in the liver, respectively; benzyl alcohol formation was undetectable. Esophageal metabolism of NMBzA was exclusively located in the mucosa, preferentially in the microsomal fraction, was reduced nicotinamide adenine dinucleotide phosphate dependent, and was inhibited by CO and 2-diethylaminoethyl-2,2-diphenylvalerate. A low level of cytochrome P-450 was detected in the mucosal microsomes. Whereas hepatic metabolism of NMBzA was inducible by phenobarbitone pretreatment, mucosal metabolism was not altered by either phenobarbitone or 3-methylcholanthrene pretreatment. The hepatocarcinogen N-nitrosodimethylamine was extensively metabolized by hepatic microsomes to formaldehyde, but demethylation was not detected in the microsomes from esophageal mucosa, a nontarget tissue. The results indicate that rat esophageal mucosa contains an enzyme system which metabolizes NMBzA at a high rate and exhibits properties typical of cytochrome P-450. This enzyme may play a role in determining which compounds induce tumors in rat esophagus.

Regulation of mevalonate synthesis in rat mammary glands by dietary n-3 and n-6 polyunsaturated fatty acids.

It is well established that dietary n-6 polyunsaturated fatty acids (PU-FAs) enhance rat mammary tumor development whereas n-3 PUFAs inhibit it, yet the mechanisms are unclear. The objective of this study was to investigate a mechanism by which n-3 and n-6 PUFAs could modulate mammary carcinogenesis. Female Sprague Dawley rats were fed diets containing either menhaden (n-3) or safflower oil (n-6) in a 7% fat diet for 1 week. In comparison to the n-6 diet, the n-3 diet significantly reduced the activity and levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase in mammary glands, thereby suppressing the formation of mevalonate. In addition to being essential for cholesterol biosynthesis, mevalonate is also required for DNA synthesis and may be involved in malignant transformation. Serum cholesterol was lower in the n-3 group than in the n-6 group (1.91 +/- 0.18 versus 2.61 +/- 0.37 mM; P < 0.01). Extrahepatic tissues meet most of their cholesterol requirements from circulating cholesterol, and the internalized cholesterol down-regulates HMG-CoA reductase. Thus, the concomitant decrease in serum cholesterol and mammary gland HMG-CoA reductase levels suggests that changes in circulating cholesterol levels do not solely determine the activity of extrahepatic reductase. We conclude that the mevalonate pathway may be a mechanism through which different types of dietary fat modulate breast cancer development.

Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats.

The Copenhagen rat is completely resistant to mammary cancer induction by N-methyl-N-nitrosourea (MNU) when the carcinogen is administered during sexual development, a period when other strains of rats are normally susceptible to mammary gland carcinogenesis. Here we administered 30 mg/kg MNU i.p. to two groups of neonatal (2-3-day-old) Copenhagen rats. One group (group B, 18 animals) received no further treatment, while the other group (group C, 17 animals) received a second dose of 30 mg/kg MNU via the tail vein at 50 days of age. About 30% of the rats in group B and about 70% of those in group C developed mammary carcinomas before they were 1 year of age. About one-half of the tumors in both groups were cribriform adenocarcinomas and one-half were adenosquamous carcinomas. The latter tumor type has not been observed previously in susceptible rat strains. The ability to induce these mammary tumors in the Copenhagen rat suggests that the putative mammary carcinoma suppressor gene is functionally inactive in neonatal animals or is inactivated when these animals are treated with MNU.

Absence of volatile nitrosamines in human feces.

Using a method for nitrosamine analysis that gives high recovery values and that is free from artifactual synthesis of nitrosamines, we have shown that human feces do not contain volatile nitrosamines (detection limit, 0.1 to 0.5 microgram/kg). We also showed that nitrosation reactions are not catalyzed by fecal organisms. Following a 2-day anaerobic incubation of feces with either a secondary amine (dimethylamine, dipropylamine, or morpholine) or nitrite, no nitrosamine was formed. When the amine and nitrite were added together, nitrosamine was formed, but at a level of 2 to 20% of that formed in autoclaved feces under the same conditions. Nitrosamines were stable following anaerobic incubation with feces for up to 4 days. These results suggest that fecal organisms inhibit the chemical formation of nitrosamines instead of catalyzing it. When morphine and nitrate were added together, nitrosomorpholine was formed. Morpholine nitrosates so rapidly that it intercepts nitrite formed by the action of nitrate reductase before the nitrite can be further reduced. However, very high concentrations of morphine and nitrate, which are far from the conditions in normal feces, were required to form measurable nitrosomorpholine. We may conclude that N-nitroso compounds are unlikely to be formed in any significant amounts in the human colon.

Organ specificity in the microsomal activation and toxicity of N-nitrosomethylbenzylamine in various species.

The microsomal metabolism of the rat esophageal carcinogen N-nitrosomethylbenzylamine (NMBZA) at the methylene carbon atom to yield benzaldehyde was studied in various organs of a number of species to determine the role of metabolic activation in the carcinogenicity or toxicity of the nitrosamine. In the Sprague-Dawley rat, NMBZA was metabolized by microsomes from liver, lung, and esophageal mucosa. In the F344 rat and rabbit, metabolic activity was present in both liver and esophageal mucosa, the only tissues studied in these species. In contrast, in the Syrian hamster and BALB/cByJ mouse, NMBZA debenzylation was undetectable in the esophagus but occurred at relatively high rates in liver, lung, and kidney. The forestomach mucosa exhibited undetectable levels of activity in the Sprague-Dawley rat and BALB/cByJ mouse, although in the hamster, it was present at a very low level. Administration of a dose of NMBZA acutely toxic to the rat (18 mg/kg i.p.) resulted in significant cellular damage only to the rat esophageal mucosa, no other tissues examined in the rat, hamster, or mouse being affected. These observations, together with the available data on carcinogenicity of the nitrosamine in the rat and rabbit, suggest that in the esophagus, at least, metabolic activation of NMBZA is necessary to elicit its toxic and/or carcinogenic effect. However, NMBZA is also metabolized at a high rate in the liver of all species but is not toxic or carcinogenic in this tissue, suggesting that other factors besides metabolic activation must be involved in the resistance of hepatocytes to the effects of the nitrosamine. Microsomes prepared from human esophageal mucosa from six patients metabolized NMBZA at rates that were either undetectable or approximately 70 times lower than in the Sprague-Dawley rat.

Carcinogenicity of nitrosation products of ephedrine, sarcosine, folic acid, and creatinine.

Carcinogenic activity of several synthetic N-nitroso compounds was evaluated in C57BL/6J X C3HeB/FeJ F1 mice. Test substances, suspended in trioctanoin, were injected i.p. in three equal doses given on Days 1, 4, and 7 after birth and animals were held without further treatment for up to 85 weeks. Nitrosoephedrine at a total dose of 600 mg/kg induced metastasizing liver cell carcinomas in 28 of 30 animals. Nitrososarcosine (225 mg/kg) induced similar tumors in 8 of 14 animals. Nitrosofolic acid (375 mg/kg) induced lung adenocarcinomas in 4 of 28 mice. Creatinine-5-oxime (600 mg/kg) showed no evidence of carcinogenic activity. Diethylnitrosamine (12 mg/kg given in four doses), included as a positive control, caused metastasizing liver cell tumors in 23 of 25 animals.

Promotion of colonic microadenoma growth in mice and rats fed cooked sugar or cooked casein and fat.

We studied the effect of cooked food components on the promotion of microadenoma growth in the colons of mice and rats. CF1 mice and Fisher 344 rats were initiated with azoxymethane, with 152 mice receiving four weekly i.p. injections of 5 mg/kg, 59 rats receiving a single injection of 20 mg/kg, and 24 rats receiving 30 mg/kg. A week after the last injection, the animals were randomly assigned to one of eight diets with identical ingredients, but the three components, sucrose, casein, and beef tallow, either uncooked or cooked. Control animals were given diets with uncooked ingredients. Experimental animals were fed diets in which one, two, or three of the components were cooked in an oven at 180 degrees C until golden brown before they were added to the diet. After 100 days on the diets, the colons were fixed, stained with methylene blue, and scored for microadenomas. The mice and the rats fed cooked sucrose, or casein and beef tallow cooked together, had three to five times more large microadenomas than did the controls (P ranging from 0.02 to 0.0001). No significant increase was observed with the five other cooked diets. Two rats fed the casein and beef tallow cooked together had adenocarcinomas. Thus, a diet containing 20% of cooked sucrose, or 40% of casein and beef tallow cooked together, promotes the growth of colonic microadenomas in initiated mice and rats, and would appear to contain promoters for colon cancer.

Reevaluation of nitrate and nitrite levels in the human intestine.

Analyses of human fecal and ileostomy samples by a method that is insensitive and free from interferences indicate that nitrate and nitrite levels in the intestine are lower than reported previously. Fecal nitrate and nitrite concentrations ranged from 0 to 14 mumol/kg (0 to 0.9 ppm) and 5 to 19 mumol/kg (0.3 to 0.9 ppm), respectively. Ileostomy samples contained from 0 to 7 mumol/kg (0 to 0.4 ppm) and 0 to 15 mumol/kg (0 to 0.7 ppm) for nitrate and nitrite, respectively. We also showed that, when deliberately added to feces samples, nitrate and nitrite were destroyed during a two-hr incubation period in a reaction that depended on the presence of microorganisms. The results suggest that conditions in the lower gastrointestinal tract favor denitrification, not nitrification as had been proposed previously.

Metabolism of N-nitrosomethylbenzylamine by microsomes from perivenous and periportal hepatocytes.

Immunohistochemical studies have shown the presence of O6-methylguanine in the perivenous area but not the periportal area of the liver lobule following administration of N-nitrosomethylbenzylamine (NMBzA) to rats. This study was designed to investigate the hypothesis that hepatic heterogeneity of NMBzA metabolism determines the distribution of O6-methylguanine in the liver. Using microsomes prepared from purified perivenous and periportal hepatocytes, we showed that NMBzA debenzylase and demethylase activities were 2-fold and 1.5-fold higher, respectively, in perivenous than in periportal microsomes. Our results suggest that the combined effect of a higher rate of formation and lower rate of repair of O6-methylguanine in perivenous than in periportal hepatocytes could explain the localization of this base in zone 3 of the liver lobule following NMBzA treatment.