The effect of haem in red and processed meat on the endogenous formation of N-nitroso compounds in the upper gastrointestinal tract

Red and processed meat (PM) consumption increases the risk of large bowel cancer and it has been demonstrated that haem in red meat (RM) stimulates the endogenous production of N-nitroso compounds (NOCs) within the human intestine. To investigate whether N-nitrosation occurs in the upper gastrointestinal tract, 27 ileostomists were fed diets containing no meat, or 240 g RM or 240 g PM in a randomly assigned crossover intervention design carried out in a volunteer suite. Endogenous NOC were assessed as apparent total N-nitroso compounds (ATNC) in the ileostomy output. ATNC concentration in the diets was 22 microg ATNC/kg (RM) and 37 microg ATNC/kg (PM), and 9 microg ATNC/kg in the no meat diet. Levels significantly increased to 1175 microg ATNC/kg SEM = 226 microg ATNC/kg) following the RM (P=0.001) and 1832 microg ATNC/kg (SEM=294 microg ATNC/kg) following PM (P<0.001) compared to the no meat diet (283 microg ATNC/kg, SEM=74 microg ATNC/kg). ATNC concentrations in the ileal output were equivalent to those measured in faeces in similarly designed feeding studies. Supplementation with either 1 g ascorbic acid or 400 IU alpha-tocopherol had no effect on the concentration of ATNC detected in the ileal output. In in vitro experiments, N-nitrosomorpholine (NMor) was formed in the presence of nitrosated haemoglobin, at pH 6.8 but not in the absence of nitrosated haemoglobin. These findings demonstrate that haem may facilitate the formation of NOC in the absence of colonic flora in the upper human gastrointestinal tract.     

The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice

The crypts of the gastrointestinal mucosa are highly structured and polarised organs with rapid cell proliferation and an hierarchical organisation with relatively few stem cells. These tend to be located at specific positions in the tissue—at the crypt base in the colon and about four cell positions from the base (above the Paneth cells) in the small intestine.A small but constant level of spontaneous cell death occurs in the crypt. The levels of cell death are elevated by small exposures to radiation or cytotoxic drugs. The morphology of the cell death is typical of apoptosis. The maximum yield of cell death following cytotoxic exposure is observed at about 3–6h after treatment and for many agents the death is characteristically located at the fourth (stem) cell position in the small intestine.The significance and implications of these observations are discussed in relation to the internal screening and programming within damage cells and with respect to tissue homeostatic mechanisms.     

Caffeine-derived N-nitroso compounds. V. Carcinogenicity of mononitrosocaffeidine and dinitrosocaffeidine in bd-ix rats

Mononitrosocaffeidine (MNC) and dinitrosocaffeidine (DNC) are new N- nitroso compounds obtained from in vitro nitrosation of caffeidine, a hydrolysis product of caffeine present in a typically made and widely consumed tea from Kashmir (India), a high incidence area of esophageal and stomach cancer. The chemical synthesis, in vitro metabolic studies and mutagenicity of the compounds has been previously reported. DNC, a nitrosamide is highly mutagenic both with and without metabolic activation whereas MNC, like several other aromatic asymmetric nitrosamines, does not exhibit genotoxic or mutagenic properties. We now report the results of the first carcinogenicity experiments on chronic oral administration of these compounds in BD-IX rats. The acute LD50 of MNC and DNC were about 1300 and 230 mg/kg b.w., respectively. Lung oedema and gastrointestinal haemorrhages were the first symptoms of intoxication observed after 2 days for both the compounds. All three dose groups of MNC treated rats showed localization of tumours in nasal cavity (93.9-100% of all malignant tumours). The tumours were histologically diagnosed as neuroepitheliomas of the olfactory epithelium (neuroblastoma of the bulbus olfactorii) and squamous cell carcinoma of the nasal cavity in the ratio of 3:1. No tumours of the nasal cavity were observed in the untreated controls. DNC, in contrast, induced squamous cell carcinoma of forestomach in 100% animals at low and high doses, of which nearly half the tumours metastasized predominantly into the peritoneum. No forestomach tumours were seen in the untreated controls. The data presented here clearly show the potential for induction of malignant tumours and distinct organ- specificity by MNC and DNC in rats, and support the postulate that a chronic exposure to these compounds may provide a carcinogenic risk for high incidence of gastrointestinal cancers in Kashmir.      

Dose-response curves for liver DNA fragmentation induced in rats by sixteen N-nitroso compounds as measured by viscometric and alkaline elution analyses

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyze dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses of sixteen N-nitroso compounds. Statistically significant changes of DNA viscometric parameters, which are considered indicative of DNA fragmentation, were produced by N-nitrosodimethylamine (0.022 mg/kg), N-nitrosomethylethylamine (0.025 mg/kg), N-nitrosodiethylamine (0.067 mg/kg), N-nitrosodiethanolamine (1.03 mg/kg), N-nitrosodi-n-propylamine (0.31 mg/kg), N-nitrosodi-n-butylamine (0.083 mg/kg), N-nitroso-N-methylurea (0.56 mg/kg), N-nitroso-N-ethylurea (0.37 mg/kg), N-nitroso-N-butylurea (0.16 mg/kg), streptozotocin (20 mg/kg), N-nitrosomorpholine (0.4 mg/kg), N-nitrosopiperidine (2.22 mg/kg), N-nitrosopyrrolidine (5.0 mg/kg), 1-nitroso-2-imidazolidinone (0.31 mg/kg), and N-methyl-N'-nitro-N-nitrosoguanidine (5.57 mg/kg). The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed that in our experimental conditions higher doses are needed to produce a statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound-induced DNA fragmentation, but it does not exclude that the sensitivity of alkaline elution can be improved by appropriate modifications of the experimental procedure. With both techniques DNA damage was undetectable in liver of rats treated with 540 mg/kg of the non-hepatocarcinogen N-nitrosodiphenylamine. With the exception of N-nitrosodiethanolamine, that exhibited a plateau effect, all the other N-nitroso compounds examined displayed a linear dose-response curve over the entire wide range of doses tested. Consequently, a nonlinearity of the relationship between dose and tumor response cannot be attributed to a nonlinearity of the pharmacokinetic processes involved in the formation of DNA damage.     

N-nitroso compounds and Helicobacter pylori in the gastric remnant.

AIMS AND BACKGROUND: The aims of this study were to investigate the role of N-nitroso compounds (NOC) and Helicobacter pylori (H. pylori) in gastric stump carcinogenesis. METHODS AND STUDY DESIGN: Analyses of biochemical parameters such as pH and NOC concentration were carried out on 65 fasting gastric juice samples obtained at endoscopy from 45 patients previously submitted to partial gastrectomy for benign peptic ulcer disease (23 Billroth I, 22 Billroth II/Reichel-Polya) and 20 normal controls. Biopsy specimens were taken to determine histology and H. pylori status. RESULTS: Significantly higher mean pH values and NOC concentrations were found in partial gastrectomies compared to normal controls. In relation to surgical methods, higher mean pH values and NOC concentrations were observed in the gastric juice of patients with Billroth II compared to Billroth I gastrectomies. Independently of the type of surgical reconstruction, higher mean NOC levels were recorded in patients with more severe histological changes and H. pylori infection. CONCLUSIONS: All these data suggest that high levels of NOC in gastric juice and H. pylori infection could be cofactors in gastric stump carcinogenesis.     

Occurrence of and exposure to N-nitroso compounds in tobacco

The concentrations of 21 N-nitroso compounds in smokeless tobaccos are presented. Tobacco-specific nitrosamines accounted for 70-90% of the total identified N-nitroso compounds. Daily exposure of smokeless tobacco users to preformed N-nitroso compounds may exceed 200 micrograms/day in certain populations.     

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.     

Induction of gastrointestinal tract tumors in rats with N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]

Tumors of the gastrointestinal tract were induced in white non-inbred rats exposed to MNNG in various doses. Gastric tumors appeared in the dosage of 153 mg, with its 2 and 3.3 times increase no change in the frequency of gastric tumors was noted. The frequency of jejunal tumors was higher with increased MNNG dosage.     

Induction of gastrointestinal tumors in mice fed the fungicide folpet: possible mechanisms

Dietary administration of the fungicide folpet, N-(trichloromethylthio) phthalimide, to B6C3F1 mice at dose levels of 1,000, 5,000 and 10,000 ppm induced a dose-related appearance of duodenal atypical hyperplasia, adenomas and adenocarcinomas. The appearance in some of these animals of gastric papillomas and gastric squamous cell carcinomas was correlated in many instances to the presence of duodenal obstructions. It is suggested that the gastric lesions appeared subsequent to, and as an indirect result of, these partial lumenal duodenal obstructions. We suggest that the presence of duodenal obstructions is consistent with the notion that reflux of folpet, bile acids and pancreatic enzymes into the stomach may have acted to irritate and consequently stimulate local neoplastic proliferation. In addition, the duodenal obstructions may have resulted in delayed emptying time of the stomach contents with consequential stagnation. This would cause high concentrations of folpet to act locally on the gastric mucosa.     

Analysis and occurrence of total N-nitroso compounds in the Japanese diet

A quantitative technique for differentiating the total N-nitrosamides from the total N-nitroso compounds has been devised. The principle of this method is based on the chemiluminescence response of a nitroso compound under different denitrosating conditions. We also examined the effect of nitrite scavengers on the stability of N-nitroso compounds and found that both ammonium sulfamate and hydrazine sulfate can be satisfactorily used for the detection of chemiluminescence response with hydrogen bromide-acetic acid reagent. A survey of the occurrence of total N-nitroso compounds, total N-nitrosamides, volatile N-nitrosamines, nitrates and nitrites in the Japanese diet has been conducted. Fairly high levels of total N-nitroso compounds were detected in salt-fermented vegetables, the highest being 2 500 micrograms/kg in hakusai-zuke (salt-fermented Chinese cabbage), followed by 253 micrograms/kg in takuan (salt-fermented radish roots). It is noteworthy that the amounts of total N-nitroso compounds in these products almost coincided with those of total N-nitrosamides, although no appreciable amounts, or only trace quantities, of volatile nitrosamines could be detected in these vegetable products.     

食物中N-亚硝基化合物与肿瘤关系的研究进展

迄今已研究过的300多种亚硝基化合物中,＞90%对动物有不同程度的致癌性和具有明显的亲器官性,引起了人们的高度重视和很多学者的深入研究.从其理化性质、食物中的来源、致癌作用和预防措施4个方面进行了总结论述.N-亚硝基化合物分为N-亚硝胺和N-亚硝酰胺两大类,后者化学性质较活泼.食物中N-亚硝基化合物主要来源于鱼、肉制品、乳制品、蔬菜水果和发酵食品,人体也能合成一定量的N-亚硝基化合物.N-亚硝基化合物没有直接致癌作用,主要是其化学性质活泼易生成烷基偶氮羟基化合物和氨氮化合物而呈现致癌活性.其致癌机制研究显示,亚硝胺可引起食管上皮细胞相关癌基因抑癌基因发生改变,大大促进癌变.DNA碱稀释过滤法证明,N-亚硝基化合物含氮杂环化借诱发DNA互补碱对之间的交联而启动细胞的癌变.目前尚缺少N-亚硝基化合物对人类直接致癌的资料.流行病学调查资料表明,与人类紧密相关的肿瘤主要是食管癌和胃癌等消化系统肿瘤.预防N-亚硝基化合物对人体健康的危害主要是:减少亚硝胺及其前体物硝酸盐及亚硝酸盐的摄入和阻断亚硝胺在体内的合成.     

Hyperplastic nodules of the liver induced in rats fed bracken diet

Hyperplastic nodules (HN) of the liver were induced in Charles River Sprague-Dawley rats (CD rats) and ACI rats fed a diet containing 30% bracken for 260 and 180 days, respectively. HN were also induced in high incidence in CD rats fed a diet containing the carcinogenic fractions of bracken extract.     

32P-post-labelling analysis of DNA adducts formed in the upper gastrointestinal tissue of mice fed bracken extract or bracken spores.

Bracken toxicity to both domestic and laboratory animals is well established and tumours are formed when rodents are treated with either bracken extracts or bracken spores. In this study we have administered bracken spores and extract to mice in order to investigate whether such exposure leads to the formation of DNA adducts. DNA, isolated from the upper gastrointestinal tract and liver, was digested to 3''-nucleotides. Adducts were extracted with butanol, 32P-post-labelled, separated by thin layer chromatography (TLC) and visualised and quantified using storage-phosphor technology. A cluster of adducts was clearly seen in the DNA of the upper gastrointestinal tract, but not liver, 5 and 24 h after treatment with bracken extract or bracken spores. These adducts were not observed in DNA extracted from vehicle-treated animals. Whereas, after 5 h adduct levels in extract and spore-treated animals were similar, after 24 h adduct levels in the extract-treated animals had diminished by > 75%, but levels in spore-treated animals remained similar to those found after 5 h. This suggests that the DNA-reactive compounds were being released slowly from the spores, even though the spores had been sonicated before administration. Adducts were also quantified after the addition of an internal standard (deoxyinosine 3''-monophosphate) by comparing the amount of label incorporated into the adducts with that found in a known amount of the internal standard. Adduct levels using this internal standard approach were similar to those found by direct measurement of radioactivity incorporated into the adduct, indicating that the labelling of adducts was quantitative. We have tried, unsuccessfully, to synthesise ptaquiloside, the principal carcinogenic component present within bracken. However, similar patterns of adducts were observed when two other compounds, (1-(4-chlorophenyl sulphonyl)-l-cyclopropane carbonitrile and 3-cyclopropylindeno [1,2-c] pyrazol-4-(O-methyl)oxime), which both contain a cyclopropyl ring, were administered to mice. The adducts detected in bracken-treated animals may, thus, have arisen from ptaquiloside but, whether these adducts arise directly from the compounds and bracken spores/extract themselves or via an indirect mechanism, remains to be determined. As bracken-induced DNA adducts are detectable in rodent tissues by a 32P-post-labelling procedure commonly employed to investigate DNA damage in human populations, it may prove possible to apply such approaches to determine human exposure.     

Altered tRNA methylation in rats and mice fed lipotrope-deficient diets

A diet that is deficient in methionine, choline, folic acidand vitamin B₁₂ has been found to induce alterations rapidlyin liver tRNA methylation in male Fischer rats. In vitro assaysindicated that activity of N2-guanine tRNA methyltransferaseII (NMG2) was increased to 150% of controls levels in 1 weekand 300% of control levels after 2 weeks or longer on this diet.Incompletely methylated tRNA was isolated from livers of thesesame animals, indicating that there was impairment of methylationin vivo. The effects on liver tRNA methylation of this methyl-deficientdiet were thus seen to mimic those of the liver carcinogen,ethionine, which also causes production of hypomethylated tRNAand increased activity of NMG2. The effect of the same dieton liver tRNA methyltransferase activity of C57BL/6J and C3H/HeJinbred mice were also studied. Intake of the lipotrope-deficientdiet Induced elevation in activity of liver N2-guanine tRNAmethyltransferase II activity in C57BL/6J mice, similar to thatseen in rats. In contrast, the methyl-deficient diet had verylittle effect on the same enzyme activity in C3H/HeJ animals.