Ascorbate-nitrite reaction: possible means of blocking the formation of carcinogenic N-nitroso compounds.

The formation of carcinogenic N-nitroso compounds by the chemical reaction between nitrous acid and oxytetracycline, morpholine, piperazine, N-methylaniline, methylurea, and (in some experiments) dimethylamine was blocked by ascorbic acid. The extent of blocking depended on the compound nitrosated and on the experimental conditions. Urea and ammonium sulfamate were less effective as blocking agents. The possibility of in vivo formation of carcinogenic N-nitroso compounds from drugs could be lessened by the combination of such drugs with the ascorbic acid.

Metabolism of N-nitrosohexamethyleneimine to give 1,6-hexanediol bound to rat liver nucleic acids.

Rats were gavaged with the liver carcinogen H-nitrosohexamethyleneimine labeled with 3H or 14C and killed 16 hours later. The liver RNA and DNA were isolated and hydrolyzed with 1 M HCl at 100 degrees C. Chromatography of the 3H-labelled RNA hydrolysate on a cation exchange resin (NH4+ form), with water elution, separated five radioactive peaks, with peak "E" containing 27% of the bound 3H. There were no radioactive peaks in the 7-substituted guanine region. Hydrolysis of the 3H-labeled DNA gave a similar profile, but E contained only 5% of the 3H. The major component of E was identified as 1,6-hexanediol by its behavior and/or that of its benzoate derivative on cation exchange, anion exchange, thin-layer and gas-liquid chromatography, and by recrystallization of a mixture of the E and diol benzoates to constant specific radioactivity.

Induction of mouse lung adenomas by amines or ureas plus nitrite and by N-nitroso compounds: effect of ascorbate, gallic acid, thiocyanate, and caffeine.

Lung adenomas were induced in strain A mice by chronic treatment with N-nitroso compounds (given in drinking water) and with amines or ureas in food plus NaNO2 in drinking water. We studied the effects of varying the concentrations of three N-nitroso compounds and NaNO2 concentration in the morpholine plus NaNO2 and methylurea plus NaNO2 systems. Sodium ascorbate (NaASC) at the highest level tested (11.5 or 23 g/kg food) gave 89-98% inhibition of adenoma induction by the NaNO2 plus piperazine, morpholine, and methylurea systems. In 7 groups, NaASC produced increases of 15-59% in adenoma induction by nitrosomorpholine (NM) and mononitrosopiperazine (MNP), possibly because the mice consumed more of the nitrosamine solution. Adenoma induction by morpholine plus NaNO2 was strongly inhibited by gallic acid, moderately inhibited by caffeine, and unaffected by thiocyanate (all added to the food). Gallic acid inhibited or had no effect on the action of NM and MNP. We discussed the proposal that NaASC (or perhaps gallic acid) be administered with readily nitrosatable drugs.

Kinetics of nornicotine and anabasine nitrosation in relation to N'-nitrosonornicotine occurrence in tobacco and to tobacco-induced cancer.

The kinetics of nornicotine and anabasine nitrosation, studied in aqueous solution, obeyed equations typical for the nitrosation of aliphatic secondary amines, with third-order stoichiometric rate constants of 1.15 (nornicotine) and 0.86 (anabasine) M-2 sec-1. The similarity of the two rate constants suggested that the nitrosonornicotine occurring in tobacco arises from nicotine rather than nornicotine, because tobacco contains anabasine but apparently does not contain nitrosoanabasine. The high rate constants suggested that in vivo nitrosation of these secondary amines may constitute a hazard to tobacco smokers and chewers, in addition to that presented by preformed nitrosonornicotine.

Induction of malignant bone tumors in rats by 1-(2-hydroxyethyl)-1-nitrosourea.

Forty male MRC-W rats were treated chronically with 1-(2-hydroxyethyl)-1-nitrosourea in drinking water (total dose, 330 mg/rat). Fifteen rats (38%) developed osteogenic osteosarcomas or chondrosarcomas of the lower vertebrae. This was the first time that an orally administered organic compound induced a high incidence of these tumors.

Carcinogenicity test of acetoxime in MRC-Wistar rats.

Acetoxime was tested for carcinogenicity by chronic administration in the drinking water to male and female outbred MRC-Wistar rats. The dose of 1.0 g/liter was administered 5 days/week for 18 months (total dose, 6.2--7.0 g/rat). The test compound induced benign hepatocellular adenomas in 80% of the males but did not produce tumors in the females. This is the first report that oximes, which are fairly widely used in industry, are tumorigenic.

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.

Study of the carcinogenicity of large doses of dimethylnitramine, N-nitroso-L-proline, and sodium nitrite administered in drinking water to rats.

Large doses of dimethylnitramine (DMNM), N-nitroso-L-proline (NPRO), and sodium nitrite were administered in the drinking water to MRC Wistar rats for at least 1 year, and the rats were maintained for life. DMNM (total dose, 20 g/kg) produced liver tumors in 25 (69%) of the 36 rats and nasal cavity tumors in 9 (25%) of the rats. NPRO (total dose, 36 g/kg) induced no tumors in 37 treated rats. In the group receiving NaNO2 (3.0 g/liter drinking water; total dose, 63 g/kg), 8 (18%) of 45 rats had forestomach squamous papillomas. The tumor incidence in the NaNO2-treated group was significantly greater than that of 2% in a control group started 11 months earlier, which suggested that the NaNO2 was tumorigenic in this experiment.

Test of catechol, tannic acid, Bidens pilosa, croton oil, and phorbol for cocarcinogenesis of esophageal tumors induced in rats by methyl-n-amylnitrosamine.

Catechol (CAS: 120-80-9), given in drinking water to rats, was the most effective of 5 phenols in enhancing [3H]thymidine incorporation [( 3H]dThd-l) into esophageal DNA. To test for esophageal cocarcinogenesis, groups of 30 male MRC-Wistar rats received 3 weekly ip injections of 25 mg methyl-n-amylnitrosamine [(MNAN) CAS: 13256-07-0]/kg. From the time of the first MNAN injection, each group also received catechol, tannic acid (CAS: 1401-55-4), dried leaves of Bidens pilosa L., or croton oil (CAS: 8001-28-3) (respectively, 2, 10, 50, and 2 g/kg semipurified diet), or were given 20 ip injections of 6 mg phorbol (CAS: 17673-25-5)/rat. The rats were killed after 20-45, 46-52, or 53-72 weeks (subgroups A, B, and C). In the group given MNAN alone, most esophageal papillomas developed during the first 45 weeks. Both catechol and B. pilosa significantly increased the esophageal papilloma multiplicity (No. of papillomas/rat) induced by MNAN, with a maximum tumor yield of 2.2 times that in the corresponding subgroup treated with MNAN alone. Papilloma multiplicity increased from subgroup A to subgroup C in the MNAN plus B. pilosa group but not in the MNAN plus catechol group. No tumors were induced by the test cocarcinogens given without MNAN. We concluded that a) an increased esophageal [3H]dThd-I indicates potential cocarcinogenicity and b) catechol and B. pilosa were weak esophageal cocarcinogens. These results support the view that catechol in cigarette smoke and B. pilosa as eaten in South Africa contribute to the etiology of human esophageal cancer.

Disappearance of nitrite from the rat stomach: contribution of emptying and other factors.

To help understand how intragastric nitrosation forms N-nitroso compounds, nitriet disappearance from the rat stomach was measured after food containing nitrite was given. In preliminary experiments, nitrite disappearance from buffered aqueous solutions became more rapid as the pH was lowered from 5 to 1 and, at a given pH, was more rapid in a slurry of commercial rat food. The disappearance of nitrite from buffer was little affected by the addition of pepsin, mucin, albumin, or rat gastric contents. When starved rats were given 5 g food with 1.82 mg nANO3/g, nitrate was not reduced to nitrite in the stomach. Five g food containing 154 mug NaNO2/g was administered similarly, and the total stomach (T) and glandular and nonglandular parts (G and NG) were analyzed after 1.5 hours. Weight and nitrite concentration of the stomach contents dropped linearly and the amount of nitrite dropped exponentially (with a half-life of 1.4 hr). Mean nitrite concentration in G was less than half that in NG. From similar experiments with phenol red, emptying accounted for 60% of nitrite loss from T. In G, nitrite concentration was reduced about 3 times due to dilution and 3 times due to other causes. Conditions in G, e.g., nitrite concentration, pH, and empyting time, were discussed in relation to carcinogenesis experiments with nitrite plus amines and amides.

In vitro and in vivo formation of 7-(2'-carboxyethyl)guanine from the liver carcinogen 1-nitroso-5,6-dihydrouracil and its reactions with water and methanol.

Base-catalyzed hydrolysis of the potent liver carcinogen 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8] afforded 3-hydroxypropionic acid and acrylic acid in 72 and 6% yield, respectively. The base-catalyzed methanolysis of NDHU gave quantitative yields of 3-methoxypropionic acid and methyl carbamate. These results indicate that base-catalyzed NDHU decomposition produces a 2-carboxyethyl carbonium-ion-type intermediate. When 3H-labeled NDHU was reacted at pH 8 with DNA and RNA, 7-(2'-carboxyethyl)guanine (CEG) was detected in the hydrolysate and was identified by its cochromatography with authentic CEG in five (for DNA) or three (for RNA) systems and by a reverse isotope-dilution procedure. Radioactively labeled CEG (identified as before) and five other labeled chromatographic fractions were present in liver DNA and RNA hydrolysates after [3H]NDHU was gavaged into MRC Wistar rats. These fractions persisted in the liver DNA for various times up to 33 days after the gavage. The CEG fraction was 94% of the radioactivity in DNA reacted in vitro, but it reached only 21% of the radioactivity in the liver DNA. The results are related to a study on single-strand breaks produced by NDHU in rat liver DNA.

Liver and forestomach tumors and other forestomach lesions in rats treated with morpholine and sodium nitrite, with and without sodium ascorbate.

Administration to rats of ascorbate with morpholine and nitrite was previously shown to inhibit the liver tumor production and to enhance the induction of forestomach tumors, as compared to treatment with morpholine and nitrite. In a repetition of this experiment, 10 g morpholine/kg in the diet and 2 g sodium nitrite/liter in the drinking water were administered for life to male MRC-Wistar rats without (group 1) or with (group 2) 22.7 g sodium ascorbate/kg in the diet. Group 3 was untreated. Group 2 showed a lower liver tumor incidence with a longer latency than group 1, indicating a 78% inhibition by ascorbate of in vivo N-nitrosomorpholine (NMOR) formation. The incidence of forestomach papillomas was 3% in group 1, 38% in group 2, and 8% in group 3. The difference between groups 1 and 2 was not significant due to the shorter life-span of group 1. Group 1 and especially group 2 had more forestomach hyperplasia and hyperkeratosis than group 3. Ascorbate might have enhanced induction of these lesions because of an action synergistic with that of NMOR. However, it is most likely that the lowered NMOR dose and concomitantly increased survival produced by the ascorbate were solely responsible for the increased incidence of forestomach papillomas and other lesions in group 2.

Carcinogenicity of 1-(2-hydroxyethyl)-1-nitrosourea and 3-nitroso-2-oxazolidinone administered in drinking water to male MRC-Wistar rats: induction of bone, hematopoietic, intestinal, and liver tumors.

A previous report was made on the carcinogenicity of 1-(2-hydroxyethyl)-1-nitrosourea [(HENU) CAS: 13743-07-2] in rats. Because the cyclic nitrosocarbamate 3-nitroso-2-oxazolidinone (NOZ) is readily produced during the synthesis of HENU and can be confused with HENU, HENU was retested and NOZ was tested for carcinogenicity. Improved syntheses of both compounds are described. They were administered in drinking water to male MRC-Wistar rats for 1 year, starting at 3 or 9 weeks of age. The HENU-treated rats showed incidences of 48% for bone tumors, 32% for intestinal tumors (mostly duodenal adenocarcinomas), and 53% for lymphoma-leukemia. Of the bone tumors, which were evaluated microscopically and radiologically, 68% were osteosarcomas and 32% were osteoblastomas. The skeletal distribution of these tumors was similar to that of human osteosarcoma, with the tumors occurring most frequently in the lower limbs near the knees. Of the hematopoietic tumors, the majority were lymphoblastic lymphoma-leukemia, which showed a diffuse organ distribution resembling human B-cell (Burkitt's-like) lymphoblastic lymphoma-leukemia, and differed from the usual type of convoluted T-cell lymphoma-leukemia induced by other nitrosoureas in rats and mice. NOZ induced intestinal tumors (mostly duodenal adenocarcinomas) in 80% and liver tumors (mostly hepatocellular adenomas) in 53% of the rats.

Persistence of DNA single-strand breaks and other tests as indicators of the liver carcinogenicity of 1-nitroso-5,6-dihydrouracil and the noncarcinogenicity of 1-nitroso-5,6-dihydrothymine.

The cyclic nitrosourea 1-nitroso-5,6-dihydrothymine [(NDHT) 1-nitrosodihydrothymine] was not significantly carcinogenic when it was administered for 1 year in drinking water (206 mg/liter) to MRC-Wistar rats. In acute toxicity tests, ip injection of saline solutions of 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8; 1-nitrosohydrouracil], a strong liver carcinogen in rats, produced only mild liver toxicity but marked focal degeneration of myocardial fibers. NDHU injected ip in water solution produced subcapsular liver damage. NDHU, but not NDHT, induced unscheduled DNA synthesis in hepatocyte primary cultures. NDHU, NDHT, and methylnitrosourea [(MNU) CAS: 684-93-5; N-methyl-N-nitrosourea], a liver carcinogen only under special conditions, were tested for their ability, when injected ip into rats, to produce liver DNA damage measured as strand breaks by alkaline sucrose gradient centrifugation. The three nitrosoureas produced similar maximum DNA damage of 2.2-3.2 strand breaks/10(8) daltons. Eighty percent of the damage due to NDHU persisted for 7 days, and the damage at that time was significantly greater than that produced by NDHT and MNU. The varying persistence of liver DNA damage may explain why NDHU, but not NDHT, is a liver carcinogen.

Carcinogenicity test of six nitrosamides and a nitrosocyanamide administered orally to rats.

Six nitrosamides [ethylnitrosourea (ENU), 2-hydroxyethylnitrosourea (HENU), carboxymethylnitrosourea, 1-nitroso-5,6-dihydrouracil (NDHU), 1-nitrosohydantoin, and N-methyl-N-nitrosobenzamide (MNB)] and ethylnitrosocyanamide (ENC) were administered chronically in sodium citrate-buffered drinking water to MRC Wistar rats. ENU induced tumors of the reticuloendothelial system (RES) (50% incidence), mammary glands, and large intestine. NDHU in drinking water produced hepatocellular carcinomas (96% incidence), but NDHU injected ip caused mostly tumors at the injection sites (54% incidence). HENU produced bone tumors (38% incidence) and RES tumors (28% incidence). ENC produced nasal cavity tumors (36% incidence). Papillomas and/or carcinomas of the forestomach, tongue, and pharynx were induced by most of the compounds, with the highest incidence in the forestomach (47% for MNB); these tumors were attributed to local action when the compounds were ingested. Carcinogenicity was not quantitatively correlated with direct mutagenicity for Salmonella typhimurium TA1535.

Experimental evidence for inhibition of N-nitroso compound formation as a factor in the negative correlation between vitamin C consumption and the incidence of certain cancers.

Ascorbic acid (ASC) consumption is negatively correlated with the incidence of certain cancers. This is a review and update of the theory, which has recently been neglected, that this negative correlation is due to ASC inhibition of in vivo nitrosation. The review covers the older literature on ASC inhibition of carcinogenesis by nitrite administered with amines or amides and more recent studies on ASC inhibition of nitrosation by bacteria, nitrogen oxides, and activated macrophages; the role of oxygen in ASC inhibition of gastric nitrosation; ASC inhibition of N-nitrosoproline formation in subjects from areas with high incidences of certain cancers; dose and temporal relationships between ASC and in vivo nitrosation in humans; the role of substances other than ASC in the inhibition of nitrosation by vegetables and fruits; and the active secretion of ASC into the human stomach.

Carcinogenesis in rat esophagus by intraperitoneal injection of different doses of methyl-n-amylnitrosamine.

The carcinogenicity of methyl-n-amylnitrosamine in MRC-Wistar rats was determined after i.p. injection at a variety of dose schedules. After 6 weekly methyl-n-amylnitrosamine injections of 25 mg/kg or 12 weekly injections of either 12.5 or 25 mg/kg, the incidence of esophageal squamous cell papillomas was 85 to 100% and that of esophageal squamous cell carcinomas was 40 to 65%. With 12 injections, the mean survival time was 25 to 31 weeks. Treatment with 1 or 2 doses of 50 mg/kg produced a lesser incidence (less than 20%) of esophageal tumors, with a longer survival time of 67 to 77 weeks. One 85-mg/kg injection caused esophageal carcinomas in 5 of 7 rats. The treated groups also had squamous cell papillomas and carcinomas in the nasal cavity (up to 50% incidence) and trachea (up to 30% incidence). Hence, a 6- or 12-week treatment schedule was adequate for inducing esophageal tumors and could be used for studies on agents modifying esophageal tumor induction by methyl-n-amylnitrosamine.

Effects of nine N-nitroso compounds on the specific radioactivity of liver proteins after injection of [14C]leucine into rats.

We compared the effect of nine N-nitroso compounds, given by gavage to adult rats, on specific radioactivity of the trichloroacetic acid-precipitable liver proteins, 1 hr after the injection of [14C]leucine. The specific radioactivity was decreased by dimethylnitrosamine, diethylnitrosamine, methyl-n-butylnitrosamine, and nitrosomorpholine 5 to 10 hr after their administration; was increased by nitrosopiperidine, dinitrosopiperazine, and methylnitrosourea 5 to 24 hr after gavage; and was unaffected by nitrososarcosine and nitrosodihydrouracil. With dimethylnitrosamine, specific radioactivity was decreased by 10 but not 5 mg/kg. In control rats and rats given injections of either of two nitrosamines, protein specific radioactivity at 60 min after the [14C]leucine injection was 76 to 87% of that at 30 min, indicating some degradation of the proteins at 60 min. The liver:blood ratio of [14C]cycloleucine concentration was unaffected by four nitrosamines, indicating no effect on leucine transport. The effect of the nine compounds was examined on total pool size of free leucine in the liver, at times close to those for the maximum specific radioactivity effect. For these data, we calculated "corrected specific radioactivity," adjusted for changes in pool size. This adjustment is only a first approximation since, for example, the free leucine pool is not uniform with respect to protein synthesis. The four N-nitroso compounds that decreased specific radioactivity also decreased corrected specific radioactivity, even though they enlarged the leucine pool. Of the remaining compounds, two enlarged the leucine pool and three increased corrected specific radioactivity. For all nine compounds, the decrease in specific and correlated with the ability to cause acute liver necrosis. When nitrosodihydrouracil was excluded, the decrease in specific and corrected specific radioactivity was significantly correlated with the reported liver carcinogenicity.

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.

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.