Brigitte Pignatelli

Inhibitors of endogenous nitrosation mechanisms and implications in human cancer prevention

Although the proof that N-nitroso compounds (NOC), a versatile class of carcinogens in animals, are also carcinogenic in man is lacking, humans are exposed through ingestion or inhalation to preformed NOC in the environment and through the endogenous nitrosation of amino precursors in the body. Activated macrophages can synthesize nitrate, nitrite and nitrosating agents that can form NOC. A number of bacterial strains isolated from human infections can produce NOC enzymatically from precursors at neutral pH. As a consequence endogenous nitrosation may occur at various sites of the body such as the oral cavity, stomach, urinary bladder, lungs, and at other sites of infection or inflammation. Since the demonstration by Mirvish et al. (1972) showing that ascorbate can reduce tumor formation in animals following feeding of nitrite plus amine, numerous substances to which humans are exposed have been identified and shown to inhibit formation of NOC in vitro, in animal models and in humans. Such inhibitors of nitrosation include vitamins C and E, phenolic compounds, and complex mixtures such as fruit and vegetable juices or other plant extracts. Nitrosation inhibitors normally destroy the nitrosating agents and thus act as competitors for the amino compound that serves as substrate for the nitrosating species. Independently, epidemiological studies have already established that fresh fruits and vegetables that are sources of vitamin C, other vitamins and polyphenols have a protective effect against cancers at various sites and in particular gastric cancer. Although the evidence that endogenously formed NOC are involved in human cancers is far from conclusive, it is suggestive and justifies preventive measures for reducing exposure to NOC. This article briefly reviews (i) the chemistry of NOC formation and inhibition, (ii) the studies in experimental animals which showed that inhibition of endogenous NOC synthesis leads to a reduction of toxic, mutagenic and carcinogenic effects, (iii) recent studies in humans where the degree of inhibition of endogenous NOC synthesis was directly quantified and lastly (iv) the contribution of nitrosation inhibitors to human cancer prevention.

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa

OBJECTIVE:Helicobacter pylori infection causes gastric diseases, but the responsible mechanisms are not completely understood. They can involve DNA and tissue damage induced by reactive oxygen and nitrogen species. Our aim is to investigate the effects of bacterial eradication on oxidative stress by measuring changes of relevant markers.METHODS:Antral biopsies were obtained from 34 patients with chronic atrophic gastritis and peptic ulcer disease before and after bacterial eradication. The expression of inducible nitric oxide synthase (iNOS) and levels of nitrotyrosine (NTYR) and 8-hydroxy-2′-deoxyguanosine were assessed immunohistochemically as markers of nitric oxide (NO) production and of damage to proteins and DNA, respectively.RESULTS:Before treatment, the percentages of patients with staining were: 56 for iNOS in inflammatory cells, 79 and 61 for NTYR and 8-hydroxy-2′-deoxyguanosine in foveolar cells, respectively, and 82 for 8-hydroxy-2′-deoxyguanosine in lymphoid follicles. NTYR staining was associated with the intensity of inflammation (p = 0.04) and gastritis activity (p = 0.07). The prevalence of 8-hydroxy-2′-deoxyguanosine tended to be associated with that of NTYR. After successful H. pylori eradication, the prevalence of iNOS and NTYR (in mild gastritis) staining decreased (p < 0.001 and p < 0.06, respectively). 8-Hydroxy-2′-deoxyguanosine staining disappeared in 24% of cases but appeared in 18% of previously negative cases despite eradication.CONCLUSION:Targets of oxidative stress associated with H. pylori infection are inflammatory and deep foveolar cells and lymphoid follicles. This is the first report of 8-hydroxy-2′-deoxyguanosine localization in gastric mucosa. Oxidative stress is reduced by bacterial eradication in the first stages of mild gastritis. Moderate-severe gastritis may be a step that is reversible for iNOS, but partly irreversible for NTYR and 8-hydroxy-2′-deoxyguanosine.

Exposure of humans to endogenous N-nitroso compounds : implications in cancer etiology

Two sensitive procedures to quantitate human exposure to endogenous N-nitroso compounds (NOC) and/or methylating agents have been developed. One, the NPRO test, is based on the excretion of N-nitrosoproline (NPRO) and other N-nitrosoamino acids in the urine, that are measured as an index of endogenous nitrosation, following ingestion of precursors. The NPRO test has been applied to human subjects in clinical and epidemiological studies, and the kinetics and dietary modifiers of endogenous nitrosation have been investigated. Results obtained after application of the NPRO test to subjects at high risk for cancers of the stomach, esophagus, oral cavity and urinary bladder are summarized. In most instances, higher exposures to endogenous NOC were found in high-risk subjects, but individual exposure was greatly affected by dietary modifiers or disease state. Vitamin C efficiently lowered the body burden of intragastrically formed NOC. In experimental animals 3-methyladenine (3-MeAde) is excreted in urine following exposure to methylating NOC. Humans normally excrete 3-MeAde, the origin of which remains unknown. Recently developed analytical methodology permits large numbers of human urine samples to be analyzed and a wide variation is observed. Preliminary results suggest a weak correlation between basal NPRO excretion and background 3-MeAde excretion. Taken together, the results point to an etiological role of endogenously formed NOC in certain human cancers, and provide an interpretation of epidemiological findings that have shown protective effects of fruits and vegetables against several malignancies.

Increased Oxidative and Nitrative Stress in Human Stomach Associated with cagA+ Helicobacter pylori Infection and Inflammation

In order to study the role of Helicobacter pylori infection in gastric carcinogenesis, we have measured oxidized (carbonyls) and nitrated (nitrotyrosine-containing) proteins as markers for oxidative and nitrative stress in 216 human gastric biopsies using dot and western immunoblots and correlated the results with H. pylori, cagA status, expression of interleukin-8 and inducible nitric oxide synthase (iNOS) mRNAs, and gastric pathology. Higher levels of both oxidized and nitrated proteins were found in patients with either chronic gastritis or duodenal ulcer than in those with normal mucosa. The levels of modified proteins were significantly higher in inflamed samples infected with H. pylori, especially cagA+ strains, and in those with expression of interleukin-8 and iNOS mRNAs than in those negative for these parameters. These results indicate that infection with cagA+ H. pylori induces significant oxidative and nitrative stress in stomach mucosa, contributing to the pathogenesis of H. pylori-associated gastroduodenal diseases.

Coexpression of Interleukin-8 and Inducible Nitric Oxide Synthase in Gastric Mucosa Infected with cagA+ Helicobacter pylori

The cagA-positive Helicobacter pylori strains are thought to be able to induce interleukin-8 expression and to be associated with gastroduodenal diseases. Inducible nitric oxide synthase (iNOS) may be involved in inflammatory pathogenesis. Our aim was to investigate the interrelationships between cagA and the expression of interleukin-8 and iNOS messenger RNAs, and with the type and degree of inflammation in gastric mucosa. In biopsies from 108 Chinese patients, the cagA, 16S rRNA, interleukin-8, and iNOS mRNAs were analyzed using reverse-transcription polymerase chain reaction. Specimens infected with cagA-positive strains had significantly more severe infiltration by mononuclear and polymorphonuclear leukocytes and more frequently expressed interleukin-8 and iNOS mRNAs than those infected with cagA-negative strains. iNOS and interleukin-8 mRNAs were significantly more frequently expressed together in the specimens with moderate or severe inflammation than in those with normal mucosa or mild inflammation. Our data suggest that interleukin-8 and excess nitric oxide play important roles in the pathogenesis of H. pylori-associated gastroduodenal diseases.

Oxidative Stress in Gastric Mucosa of Asymptomatic Humans Infected with Helicobacter pylori: Effect of Bacterial Eradication

Background. Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL‐8) are positive in approximately 50% of Helicobacter pylori‐related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL‐8 production in H. pylori‐infected asymptomatic humans, and to investigate the effect of eradication of H. pylori.

Improved group determination of total N-nitroso compounds in human gastric juice by chemical denitrosation and thermal energy analysis

A procedure for the determination of total N-nitroso compounds (NOC) in human gastric juice was developed by modifying earlier methods. The gastric juice sample, treated with sulphamic acid to remove nitrite, is injected directly into refluxing ethyl acetate containing either acetic acid for determining thermo-and acetic acid labile thermal energy analyser (TEA) responsive compounds (TAC), or into hydrogen bromide for the determination of TAC and NOC. The nitric oxide (NO) levels released are measured using thermal energy analysis with chemiluminescence detection, and the difference between the two determinations represents the concentrations of NOC in gastric juice. This method is not affected by nitrate concentrations of up to 1000 µmol l–1.The method was found to be rapid, reproducible and sensitive (detection limit 0.02 µmol l–1 NOC), requiring only small volumes of gastric juice and no prior extraction. Because the difficulties arising from the system response to the denitrosating agent and variability of NO release by acetic acid from nitrite were eliminated, this improved method can more accurately distinguish NOC from most other TEA-responsive species. Suitable techniques for stabilising gastric juice samples from duodenal ulcer and atrophic gastritis patients and the influence of the time and storage conditions on NOC concentrations have been studied.

Levels of direct-acting mutagens, total N-Nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 mumoles/l, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40-50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1-2% and N-nitrosamino acids for 8-16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography.

Inhibition of Nitrosation

Humans are exposed through ingestion or inhalation to preformed N-nitroso compounds (NOC) in the environment and through the endogenous nitrosation of amino precursors in the body. Activated macrophages and bacterial strains isolated from human infections can enzymatically produce nitrosating agents and NOC from precursors at neutral pH. As a consequence, endogenous nitrosation may occur at various sites of the body, such as the oral cavity, stomach, urinary bladder, and at other sites of infection or inflammation. Numerous substances to which humans are exposed have been identified and shown to inhibit formation of NOC. Such inhibitors include vitamins C and E, certain phenolic compounds, and complex mixtures such as fruit and vegetable juices or other plant extracts. Nitrosation inhibitors normally destroy the nitrosating agents and, thus, act as competitors for the amino compound that serves as substrate for the nitrosating species. Independently, epidemiological studies have already established that fresh fruits and vegetables that are sources of vitamin C, other vitamins, and polyphenols have a protective effect against cancers at various sites and in particular gastric cancer. This article briefly reviews (a) the chemistry of NOC formation and inhibition; (b) the studies in experimental animals that showed that inhibition of endogenous NOC synthesis leads to a reduction of toxic, mutagenic, and carcinogenic effects; (c) recent studies in humans where the degree of inhibition of endogenous NOC synthesis was directly quantified; and (d) the possible contribution of nitrosation inhibitors to human cancer prevention.

Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n=18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (e.g., quercetin, isorhamnetin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells.