Endogenous Nitrosation Research Articles

439 The effect of oral L-tyrosine, vitamin E and vitamin C administration on perphenazine - induced catatonia in rat

Experiments were performed with rats to examine the sources and disposition of dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO), all potential substrates for in vivo nitrosation to form N-nitrosodimethylamine (NDMA), a potent carcinogen. When bolus doses of [14C]DMA or [14C]TMA were given ip, recovery of radioactivity in the urine was essentially complete, and respiratory excretion, fecal excretion, and accumulation in tissues of these amines or their metabolites were negligible. Urine analysis following doses of stable isotopes showed that DMA was not converted to TMA or TMAO. Varying amounts of TMA were oxidized to TMAO, the fraction oxidized decreasing at higher doses of TMA. Ingestion and excretion of naturally occurring methylamines were monitored over a 5-day period in separate groups of normal and germ-free rats. The results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes. The net intake or excretion of TMA and TMAO observed in normal and germ-free rats is consistent with bacterial synthesis of TMA followed by its almost complete oxidation to TMAO. Blood concentrations of DMA and TMA were measured in rats for 8 hr following <5, 100, or 1000 μmol bolus iv or ip doses of radioisotopes or stable isotopes. At any given dose of DMA or TMA, the decay in blood concentration was approximately monoexponential. At the lowest (most physiologic) dose the apparent volume of distribution (VD) for DMA was larger than that for TMA. Both values of VD greatly exceeded the size of the animals, suggesting that DMA and TMA are highly concentrated at one or more locations in the body. This was confirmed by measurements in tissue homogenates sampled 1 hr after a dose. The overall handling of methylamines by rats is generally consistent with observations in humans. The presence of high local concentrations of DMA and TMA at various extragastric sites merits further investigation in connection with the potential for endogenous nitrosation of these methylamines to form NDMA.

Nitrosation of dietary myosmine as risk factor of human cancer

The tobacco alkaloid myosmine was detected in nut and nut products [J. Agric. Food Chem. 46 (1998) 2703]. Upon nitrosation, myosmine yields 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and N-nitrosonornicotine (NNN) [J. Agric. Food Chem. 48 (2001) 392]. NNN is a strong oesophageal carcinogen in rats. Metabolic activation of NNN leads to formation of DNA and protein adducts which release HPB upon hydrolysis. In the present study the time, pH and dose-dependent nitrosation of myosmine and its covalent binding to DNA was investigated. [5- 3H]myosmine was incubated with nitrite for 1−24 h in buffer solutions adjusted to pH 1−6. At pH 2−4 myosmine was easily nitrosated and gave rise to two major products, HPB and NNN, and five minor not yet identified products. Maximal formation was achieved for HPB at pH 2 after 8 h (72% of total radioactivity) and for NNN at pH 3 after 8 h (16%). For DNA binding studies labeled myosmine was incubated under nitrosation conditions with calf thymus DNA. Within 3 h up to 0.1% of the radioactivity was covalently bound to DNA. Endogenous nitrosation of myosmine, present in nuts and other dietary components could constitute a significant risk factor for tumours in the upper intestinal tract such as oesophageal adenocarcinoma, which are unrelated to tobacco smoking and alcohol abuse.

N-nitrosoproline formation in rats fed enzyme resistant starch

The aim of this work was to study the influence of raw or retrograded enzyme resistant starch (enzyme resistant starch type 2 or 3) feeding to rats on the faecal and urinary excretion of N-nitrosoproline, which is a valuable index of endogenous nitrosation. Rats were either concurrently treated with 2,500 ppm dietary proline and 3,000 ppm nitrite in their drinking water or not treated. The diets contained 0% or 6% resistant starch type 2 or 3. No N-nitrosoproline was detected in the faeces and the urine of non-treated animals. Intake of raw and retrograded enzyme resistant starch by animals receiving nitrite and proline caused a decreased output of total N-nitrosoproline by 24% and 14%, respectively. On average, 69% of the N-nitrosoproline output was found in the urine and the remainder in the faeces. The observed reduction of the endogenous formation of N-nitrosoproline might be due to an increase of the viscosity of the contents of the stomach and the small intestine, thus lowering the formation and/or absorption of N-nitrosoproline. Whether the decrease of the caecal pH following enzyme resistant starch intake had any effect on the in vivo bacterial formation of N-nitrosoproline could not be demonstrated in this study. Further research is necessary in order to clarify this inhibitory effect of enzyme resistant starch feeding.

Urinary excretion of 3-methyladenine after consumption of fish containing high levels of dimethylamine.

The urinary excretion of the DNA alkylation product, 3-methyladenine (3-MeAde), was measured in human volunteers who were on controlled diets and consumed fresh fish, or frozen-stored fish that contained 50-fold higher levels of dimethylamine (DMA), with or without ingested nitrate. DMA potentially could react with nitrosating agents in the diet or within the body, and produce the potent carcinogen N-nitrosodimethylamine (NDMA), which can then react with DNA to form several adducts including 3-MeAde. Our findings show that there was no increase in urinary levels of 3-MeAde after consumption of fish preserved by frozen storage relative to levels after consumption of fresh fish. Furthermore, consumption of 225 mg sodium nitrate (equal to the nitrate content in a large glass of beet juice) at 1 h prior to consumption of the frozen-stored fish did not increase urinary 3-MeAde levels as would be expected if nitrate enhanced endogenous nitrosation of DMA. In contrast, urinary excretion of 3-MeAde from a volunteer who was a moderate cigarette smoker (11 cigarettes per day) was approximately 3- to 8-fold higher than dietary 3-MeAde intake. These findings indicate that consumption of high levels of DMA in fish does not result in detectable levels of NDMA formation and genetic damage as measured by the urinary biomarker 3-MeAde.

Drinking water and cancer.

Epidemiologic evidence on the relation between contaminants in drinking water and cancer is reviewed. The reviewed studies cover exposure to: disinfection byproducts; nitrate; arsenic and other metals; volatiles and contaminants from hazardous waste sites; asbestiform fibers; radionuclides; and fluoride. Most investigations are ecologic, with some confirmation of elevated risk from individual-based studies. In the case of waterborne arsenic, and possibly chlorination byproducts, there is a consistent but small body of epidemiologic evidence of an association with one or more types of cancer. Nitrate in groundwater has increased greatly over the years, and the demonstration of endogenous nitrosation among highly exposed subjects raises concern of elevated cancer risk. However, the epidemiologic data are not yet sufficient to draw a conclusion. There is a diversity of studies among populations exposed to water contaminated with pesticides, volatile organics, or mixtures from hazardous waste sites. Studies of asbestiform fibers and radionuclides in water are not conclusive, but there are suggested elevations of several cancer sites in highly exposed populations. There is no suggestion that fluoride in drinking water is linked with elevated risk of cancer. As topics for epidemiologic evaluation, drinking water contaminants pose methodologic problems common to studies designed to detect relatively small elevations in risk, with the added challenge of assessing exposures for many years in the past. Nevertheless, epidemiologic assessment is valuable and clearly warranted, given the potential public health impact of small risk elevations among very large exposed populations, and the limitations of toxicologic experiments in assessing carcinogenic risk of complex mixtures or of compounds for which appropriate animal models are not available.

Dietary and other lifestyle factors of women with brain gliomas in Los Angeles County (California, USA)

A population-based interview study in Los Angeles County (California, USA) of 94 women with intracranial gliomas and 94 individually matched neighborhood controls investigated the relationship to various sources of exposure to N-nitroso compounds and their precursors and to vitamins which inhibit the endogenous formation of these compounds. The study offers some support for the hypothesis that dietary sources of nitroso exposure relate to risk. Risk increased with increasing consumption of cured meats, most notably of bacon (odds ratio [OR] for the third tertile of intake = 6.6, 95 percent confidence interval [CI] = 1.9-22.5, P trend < 0.001). Risk was reduced with increasing intake of vegetables such as bell peppers (OR for third tertile = 0.2, CI = 0.1-0.7, P trend < 0.01). In addition, use of vitamin supplements appeared protective, and there was some suggestion that eating cured meats in combination with foods which inhibit endogenous nitrosation mitigates risk. Other potential sources of nitroso exposure such as smoking, cosmetics, and drinking water did not relate to risk. Despite the limitations of data on usual adult diet, it appears that dietary sources of nitroso compounds may be important in the development of gliomas.

Nitrate contamination of drinking water: relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of N-nitrosamines.

We studied peripheral lymphocyte HPRT variant frequency and endogenous nitrosation in human populations exposed to various nitrate levels in their drinking water. Four test populations of women volunteers were compared. Low and medium tap water nitrate exposure groups (14 and 21 subjects) were using public water supplies with nitrate levels of 0.02 and 17.5 mg/l, respectively. Medium and high well water nitrate exposure groups (6 and 9 subjects) were using private water wells with mean nitrate levels of 25 and 135 mg/l, respectively. Higher nitrate intake by drinking water consumption resulted in a dose-dependent increase in 24-hr urinary nitrate excretion and in increased salivary nitrate and nitrite levels. The mean log variant frequency of peripheral lymphocytes was significantly higher in the medium well water exposure group than in the low and medium tap water exposure groups. An inverse correlation between peripheral lymphocyte labeling index and nitrate concentration of drinking water was observed. Analysis of N-nitrosamine in the urine of 22 subjects by gas chromatography-mass spectrometry revealed the presence of N-nitrosopyrrolidine in 18 subjects. Analysis of the mutagenicity of well water samples showed that a small number of the well water samples were mutagenic in the Ames Salmonella typhimurium test after concentration over XAD-2 resin. In conclusion, consumption of drinking water, especially well water, with high nitrate levels can imply a genotoxic risk for humans as indicated by increased HPRT variant frequencies and by endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite.

Endogenous formation of nitrosamines and oxidative DNA-damaging agents in tobacco users.

One-third of all cancers worldwide can be attributed to various tobacco habits. Both in tobacco smoke and smokeless tobacco, carcinogenic N-nitroso compounds (NOC) are implicated as DNA-damaging agents in cancers of the aerodigestive tract and the pancreas. The exposure from nitrosamines in certain types of tobacco use such as "toombak" in Sudan could be as high as a few milligrams per day. Using the N-nitrosoproline test, it has been shown that smoking contributes to endogenous nitrosation and likely increases NOC formation in vivo. Smokeless tobacco, most widely used in the form of chewing of betel quid (BQ) with tobacco, was shown to particularly enhance endogenous nitrosation in the oral cavity, a site where chewing habits are causally associated with cancer. Poor oral hygiene was found to contribute to the formation of nitrosamines in the oral cavity. The evidence so far accumulated demonstrates that tobacco habits increase endogenous NOC formation, thus adding to the burden of exposure by preformed carcinogenic NOC in tobacco products. In snuff dippers, the unexpected higher level of HPB released from hemoglobin, an exposure marker for carcinogenic tobacco-specific nitrosamines, has been attributed to the endogenous formation of these carcinogens. Recent studies have demonstrated that besides carcinogenic tobacco-specific nitrosamines, reactive oxygen species derived from BQ ingredients could also play a role in the etiology of oral cancer in chewers. Although the use of chemopreventive agents may block nitrosation reactions in vivo in tobacco users, cessation of tobacco habits is the only safe way for an efficient reduction of cancer risk, in view of the high exposure to other (preformed) tobacco-related carcinogens.

Thiocyanate-independent nitrosation in humans with carcinogenic parasite infection.

Infection with the liver fluke, Opisthorchis viverrini, is a causative agent of cholangiocarcinoma. One possible contributing factor in this carcinogenesis is the chronic, local generation of nitric oxide by inflammatory cells expressing inducible nitric oxide synthase and the production of N-nitroso compounds via the reaction between amines and nitrosating agents derived from nitric oxide. Our previous studies provided evidence that nitric oxide synthesis is elevated during human liver fluke infection. Here we present data on the same sample of men which definitively demonstrates increased nitrosation of proline and thioproline (thiazolidine-4-carboxylic acid) among infected men compared to uninfected control subjects on a low nitrate diet. This difference was specifically abolished by co-administration of ascorbic acid with proline and by elimination of parasites by praziquantel treatment. Multivariate statistical models demonstrate the importance of salivary thiocyanate levels to variation in the nitrosation of proline among uninfected individuals, but not among those with current fluke infection. This suggests that considerable generation of nitrosating agents (N203/N204) in infected people may be occurring via oxidation of arginine by nitric oxide synthase in inflamed tissue which is thiocyanate insensitive. Analyses revealed positive associations between N-nitrosoproline excretion and nitrate/nitrite levels in urine, plasma and saliva and with usual alcohol intake; with variation in these trends between groups. In conclusion, we have confirmed the relationship between O.viverrini infection and enhanced endogenous nitrosation, showing evidence of its extragastric site. New information is also provided on the determinants of N-nitrosamino acid excretion in men on a controlled low nitrate diet without smoking, conditions which reduce exogenous sources of nitrosating agents.

Occupational exposures and gastric cancer aetiology

Epidemiological studies have frequently reported that many occupations are associated with an increased risk of gastric cancer. However, the diversity of occupations and inconsistencies across studies have meant that positive findings are often explained by confounding dietary and life-style factors related to low socioeconomic status and manual work. Nevertheless, exposure to preformed nitrosamines and to precursors of endogenous nitrosation occurs via inhalation and dermal absorption in a number of occupations and industries. It is still unclear whether such exposures alone contribute to gastric cancer aetiology, or whether they act in combination with diet and life-style risk factors

Identification of nitrophenols as nitrosation products from capsaicin and red chillies.

The in vitro nitrosation of sun-dried red chillies (Capsicum annuum) and of its constituents capsaicin and dihydrocapsaicin was studied. The nitrophenols 4-nitroguaiacol, 4,6-dinitroguaiacol, nitrocapsaicin and nitrodihydrocapsaicin were detected as final products, whereas no formation of nitrosamides was observed though this is expected from the amide precursors capsaicin and dihydrocapsaicin. The nitrosation occurs readily with remarkable yields of nitrophenols even at physiological pH and at nitrite concentrations which are within the range of those found in the human stomach. Due to their toxicity, nitrophenols need to be included in total risk assessment through potential endogenous nitrosation of foodstuffs.

Metabolism and Excretion of Methylamines in Rats

Experiments were performed with rats to examine the sources and disposition of dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO), all potential substrates for in vivo nitrosation to form N-nitrosodimethylamine (NDMA), a potent carcinogen. When bolus doses of [14C]DMA or [14C]TMA were given ip, recovery of radioactivity in the urine was essentially complete, and respiratory excretion, fecal excretion, and accumulation in tissues of these amines or their metabolites were negligible. Urine analysis following doses of stable isotopes showed that DMA was not converted to TMA or TMAO. Varying amounts of TMA were oxidized to TMAO, the fraction oxidized decreasing at higher doses of TMA. Ingestion and excretion of naturally occurring methylamines were monitored over a 5-day period in separate groups of normal and germ-free rats. The results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes. The net intake or excretion of TMA and TMAO observed in normal and germ-free rats is consistent with bacterial synthesis of TMA followed by its almost complete oxidation to TMAO. Blood concentrations of DMA and TMA were measured in rats for 8 hr following <5, 100, or 1000 μmol bolus iv or ip doses of radioisotopes or stable isotopes. At any given dose of DMA or TMA, the decay in blood concentration was approximately monoexponential. At the lowest (most physiologic) dose the apparent volume of distribution (VD) for DMA was larger than that for TMA. Both values of VD greatly exceeded the size of the animals, suggesting that DMA and TMA are highly concentrated at one or more locations in the body. This was confirmed by measurements in tissue homogenates sampled 1 hr after a dose. The overall handling of methylamines by rats is generally consistent with observations in humans. The presence of high local concentrations of DMA and TMA at various extragastric sites merits further investigation in connection with the potential for endogenous nitrosation of these methylamines to form NDMA.

Increased nitrosamine and nitrate biosynthesis mediated by nitric oxide synthase induced in hamsters infected with liver fluke (Opisthorchis viverrini).

We previously reported that increased endogenous nitrosation in human subjects infected with the liver fluke Opisthorchis viverrini in north-east Thailand could be a risk factor for the development of cholangiocarcinoma. In the present study we examined our hypothesis that this increased endogenous nitrosation is mediated by nitric oxide (NO) synthase induced by O. viverrini infestation. Syrian golden hamsters experimentally infected with O. viverrini liver fluke excreted in the urine significantly greater amounts of nitrate, a stable oxidization product of NO, than untreated hamsters (3.64 +/- 0.86 versus 2.64 +/- 0.60 mumol/hamster/day, P < 0.001). When the rapidly nitrosatable thiazolidine 4-carboxylic acid was administered orally, the infected hamsters also excreted significantly elevated levels of N-nitrosothiazolidine 4-carboxylic acid than untreated hamsters (4.27 +/- 2.20 versus 2.33 +/- 1.13 nmol/hamster/day, P < 0.01), indicating that endogenous nitrosation is elevated in the animals with liver fluke. NO synthase activity measured in liver cytosol was about twice as high in the infected hamsters as in untreated animals. The enzyme, whose biochemical characteristics were similar to that induced in activated murine macrophages, was immunohistochemically localized in the cytoplasm of macrophages and eosinophils in the inflammation zone surrounding the parasite-containing bile ducts. These results support our hypothesis that, in fluke-infected subjects, NO synthase induction leads to excess production of NO and the observed elevated endogenous nitrosation. Since high concentrations of NO exert cytotoxic and mutagenic effects per se, excess NO produced in chronically infected/inflamed tissues may also play a role in initiation and subsequent modulation stages of cholangiocarcinoma development.

In vivo nitrosation of methimazole in humans

Monitoring the excretion of the non-carcinogenic nitroso-methylaminoantipyrine (NMAA) after peroral applications of the analgesic drug metamizole is a useful procedure for evaluating endogenous nitrosation, also in man. The outstanding nitrosatibility of this drug led to high amounts of excreted NMAA, easily detected without derivatisation by HPLC. By nitrate ingestion considered as normal dietary intake the nitrosation is scarcely modified by ascorbic acid, sodium-bicarbonate or sodium-bicarbonate plus metronidazole. These results are an other indication of a third, cell-mediated path of endogeneous nitrosation in man.

O6-methylguanine in blood leucocyte DNA: an association with the geographic prevalence of gastric cancer and with low levels of serum pepsinogen A, a marker of severe chronic atrophic gastritis. The EUROGAST Study Group.

Using a competitive repair assay, 407 samples of peripheral blood leucocyte DNA from randomly selected subjects in 17 populations were tested for the presence of the adduct O6-methylguanine. With a limit of assay sensitivity of 0.05 fmol/micrograms DNA, the adduct was detected in 21 samples (5%). Sixteen positive samples came from 102 tested (16%) from two populations, in Japan and Portugal, with extremely high gastric cancer rates. This was significantly higher (P < 0.001) than the three positive samples out of 216 tested (1%) from populations with low or intermediate rates of gastric cancer. There was also an association between presence of the adduct and having a low (< 25 ng/ml) level of serum pepsinogen A, a marker of severe chronic atrophic gastritis. These results are consistent with the general involvement of methylating agents in the pathogenesis of gastric cancer and with the model proposing formation of such compounds by endogenous nitrosation in the hypochlorhydric stomach.

Determination of 4-(methylnitrosamino)-4-(3-pyridyl)-butyric acid in tobacco, tobacco smoke and the urine of rats and smokers

The potential endogenous nitrosation of nicotine and/or nicotine metabolites has led to speculation on the possible formation of 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC) in smokers. A gas chromatographic method with thermal energy analytical detection is described for the determination of iso-NNAC in tobacco, tobacco smoke and urine. Sample pre-concentration is performed using C 18 extraction cartridges prior to esterification of iso-NNAC using ethereal diazomethane solution. Sample clean-up includes chromatography on aluminum and silica, and fractionation using high-performance liquid chromatography. The detection limits for iso-NNAC in tobacco, tobacco smoke and urine are 2 ng/g tobacco, 0.1 ng/cigarette and 20 ng/l urine, respectively.

Noninvasive methods for measuring DNA alkylation in experimental animals and humans.

Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the basis of noninvasive methods to measure DNA alkylation in vivo. In the case of methyl adducts, such as 7-methylguanine (7-MeGua), natural backgrounds exist due to RNA turnover. However, deuterated (d3) methylating agents or precursors give rise to d3-7-MeGua and d3-3-methyladenine (3-MeAde), which can be readily quantitated using gas chromatography-mass spectrometry (GC-MS). A deuterated probe drug, such as d6-aminopyrine, can be used to measure endogenous nitrosation levels in experimental animals. In contrast, for higher alkyl homologues of alkylpurines, natural backgrounds are low or nonexistent and can be directly measured by GC-MS using stable isotope labeled internal standards. For example, increased levels of urinary 3-ethyladenine were observed in cigarette smokers. Due to recent advances in analytical methodology, notably immunoaffinity cleanup of urine, measurements of excreted DNA adducts can be used in studies in human populations exposed to low levels of alkylating carcinogens.

Effects of fruit juices, processed vegetable juice, orange peel and green tea on endogenous formation of N-nitrosoproline in subjects from a high-risk area for gastric cancer in Moping County, China.

The effects of four fruit juices, processed vegetable juice, orange peel, green tea and low dose vitamin C on endogenous N-nitrosation in 86 subjects from a high-risk area for gastric cancer in Moping County, China were studied using urinary excretion of N-nitrosoproline (NPRO) as an indicator. After ingestion of 300 mg L-proline, urinary excretion of NPRO was significantly increased from a baseline of 2.5 +/- 1.6 micrograms/day to 8.7 +/- 6.2 micrograms/day. (P < 0.001). Vitamin C (75 mg) administration significantly reduced NPRO formation (62.3%, P < 0.002) although NPRO excretion remained higher than the baseline level (4.2 +/- 1.3 vs 2.2 +/- 1.2 micrograms/day, P < 0.001). Intake of fruit juices and green tea extracts (containing 75 mg vitamin C) or of orange peel powder (containing 3 mg vitamin C) together with 300 mg L-proline inhibited NPRO formation effectively to the baseline level or to levels significantly lower than the baseline level (P < 0.05-0.005). A processed juice of a number of vegetables (300 ml) significantly catalysed endogenous nitrosation (14.7 +/- 11.8 vs 9.4 +/- 4.7 micrograms/day, P < 0.05). Endogenous N-nitrosation was unaffected by the presence of intragastric lesions. The present study shows that endogenous nitrosation in this population is profoundly affected by environmental factors and that inhibitors, such as vitamin C, alpha-tocopherol and other non-nutritive compounds in the foods do inhibit endogenous nitrosation either synergistically or in an additive manner. The significance of fruits and vegetables in prevention of human cancers is discussed.

Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation

Naturally occurring dipeptides, cholecystokinine (CKK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10–30 min with 40 m m-nitrite (pH 3.5, 37°C), and the resultant products examined for mutagenicity in Salmonella typhimurlum TA100. Specific mutagenicities (net revertants per μmol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/μmol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/μmol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/μmol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/μmol, respectively), The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/μmol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.