Nitric oxide and TNF-alpha trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice.

Recombinase-activating gene-2-deficient (Rag2(-/-)) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2(-/-) mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1(+) neutrophils and elevated tumor necrosis factor-alpha (TNF-alpha) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-alpha and iNOS expression and suppressed cancer. Anti-inflammatory CD4(+) regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-alpha expression, and elevated NO production in colon carcinogenesis.

Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

Nitrate synthesis in the germfree and conventional rat.

Metabolic balance studies show that germfree and conventional Sprague-Dawley rats synthesize nitrate. Equivalent results for germfree and conventional rats eliminate the microflora as obligatory components of nitrate production. Nitrate synthesis appears to be a mammalian process.

Formation of cyanamides from secondary amines in human saliva.

1-Morpholinocarbonitrile (1-cyanomorpholine) was formed from morpholine when this amine was incubated in whole human saliva. Several other secondary amines appeared to form analogous products, and this transformation may therefore represent a general metabolic pathway for amines in saliva.

Growth of a thermophilic bacterium on hydrocarbons: a new source of single-cell protein.

A species of the genus Bacillus, capable of growth on normal alkanes at 70 degrees C, was isolated by the method known as enrichment culture. Preliminary analyses of its amino acid composition indicate that it would be a good source of protein for human nutrition. Possible advantages of the use of such organisms for the production of single-cell protein include simplification of fermentor cooling and asepsis.

Nitrite and nitrate are formed by endogenous synthesis in the human intestine.

Studies of nitrate balance in humans and analyses of fecal and ileostomy samples indicate that nitrite and nitrate are formed de novo in the intestine, possibly by heterotrophic nitrification. These findings significantly alter our previous conceptions of human exposure to nitrite and suggest an even wider role for nitrite in the etiology of human cancer.

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.

Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Gastric cancer in Colombia. IV. Nitrite and other ions in gastric contents of residents from a high-risk region.

Samples of gastric contents from 2 groups of patients from a region of high risk for gastric cancer were analyzed for pH, nitrite, nitrate, thiocyanate, and chloride. In each group, the patients could be divided into 2 subgroups: those with a gastric pH of less than 5 and those with a gastric pH of greater than 5. Above pH 5, nitrite was correlated with nitrate. The pH greater than 5 subgroups had significantly higher (P less than 0.01) nitrite content (20- to 100-fold). Some high- and low-nitrite samples were also analyzed for macro and trace metal ions, but differences were not significant. This is the first report in which patients with diagnosed gastric pathology related to a precancerous state were shown to have high levels of a putative carcinogen precursor. The results were compatible with our original hypothesis of intragastric nitrite formation by bacterial reduction of nitrate and concomitant synthesis of carcinogenic N-nitroso compounds.

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.

Diet and chronic atrophic gastritis: a case-control study.

A hospital-based case-control study of gastric cancer precursor lesions was conducted in a high-risk black population in southern Louisiana. Ninety-three subjects with biopsy-proved advanced chronic atrophic gastritis were compared to two control series: a gastroscopy clinic series and a general hospital-admission series. Dietary case-control differences indicated a protective effect associated with fruit and vegetable intake and with dietary vitamin C and a risk elevation associated with milk consumption. The protective effect associated with consumption of fruits, vegetables, and vitamin C is consistent with findings for gastric cancer and with the etiologic hypothesis of intragastric nitrosation. A twofold increased risk was associated with cigarette smoking. Gastric juice pH, NO3-, and NO2- were determined for subjects undergoing gastroscopy, and comparisons were made between this high-risk U.S. group and a Colombian population with a much greater magnitude of risk; the latter had higher NO3- and NO2- levels. An increase in pH was associated with increasing severity of gastric lesions. Levels of pH and NO2- concentration were significantly correlated (P less than .0005); however, in Louisiana the large difference in NO2- concentration associated with pH elevation is not associated with histopathologic severity. Divergent trends with severity of lesions for NO3- concentration were seen in the two populations.

Relationship between environmental tobacco smoke exposure and carcinogen-hemoglobin adduct levels in nonsmokers.

BACKGROUND: A potent bladder carcinogen for workers in the dye industry, 4-aminobiphenyl (4-ABP), is present in environmental tobacco smoke and has been shown to bond covalently with hemoglobin. PURPOSE: The goal of this study was to examine the relationship between exposure to environmental tobacco smoke and levels of 4-ABP-hemoglobin adducts in nonsmoking pregnant women and to compare adduct levels in those women with levels in smoking pregnant women. METHODS: A questionnaire on smoking and exposure to environmental tobacco smoke was administered to 15 pregnant women who smoked cigarettes and 40 who did not smoke. Exposure was quantified for 1 week with a personal diary and by air sampling with a monitor worn by each woman. The monitor collected nicotine by passive diffusion to a filter treated with sodium bisulfate, and the deposit on the filter was analyzed by gas chromatography. Aliquots of maternal blood and cord blood collected during delivery were analyzed for 4-ABP-hemoglobin adducts by gas chromatography with negative ion chemical ionization mass spectrometry. RESULTS: The mean adduct level in smokers (184 pg of 4-ABP per gram of hemoglobin) was substantially higher than that in nonsmokers (22 pg/g). This difference was statistically significant. Among nonsmokers, the levels of 4-ABP adducts increased significantly with increasing environmental tobacco smoke level (P = .009). Those in the lowest exposure category (< 0.5 micrograms/m3 weekly average nicotine) had median 4-ABP-hemoglobin adduct levels of 15 pg of 4-ABP per gram of hemoglobin, while those in the highest exposure category (> or = 2.0 micrograms/m3) had median levels of 26 pg/g. Nonsmokers in this study had a median adduct level of 20 pg/g, and smokers had a median level of 143 pg/g. CONCLUSIONS: 4-ABP-hemoglobin adduct levels in nonsmokers were 14% of the levels in smokers, which is consistent with findings of 20% in two other studies. Nonsmokers may receive a nontrivial dose of carcinogens from environmental tobacco smoke proportional to their exposure to environmental tobacco smoke. IMPLICATION: The relationship between environmental tobacco smoke exposure and 4-ABP-hemoglobin adduct levels supports epidemiologic evidence that environmental tobacco smoke is carcinogenic to passive smokers.

4-Aminobiphenyl hemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.

Maternal-fetal exchange of a potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in smoking (n = 14) and nonsmoking (n = 38) pregnant women. N-Hydroxy-4-aminobiphenyl, the active metabolite of 4-aminobiphenyl, forms chemical addition products (adducts) with hemoglobin. Levels of 4-aminobiphenyl hemoglobin adducts were measured in maternal-fetal paired blood samples obtained from smoking and nonsmoking women during labor and delivery. Carcinogen-hemoglobin adducts were detected in all maternal and fetal blood samples. Levels of such adducts were significantly higher (P less than .001) in maternal and fetal blood samples from smokers: the mean 4-aminobiphenyl hemoglobin adduct level was 92 +/- 54 pg/g of hemoglobin in blood samples from fetuses of smokers, and 17 +/- 13 pg/g of hemoglobin in blood samples from fetuses of nonsmokers; the mean maternal 4-aminobiphenyl hemoglobin adduct level was 183 +/- 108 pg/g of hemoglobin in smokers, and 22 +/- 8 pg/g of hemoglobin in nonsmokers. Fetal carcinogen-adduct levels were consistently lower than maternal levels: the mean maternal to fetal ratio was 2.4 +/- 1.1 in smokers and 1.9 +/- .98 in nonsmokers. Fetal 4-aminobiphenyl hemoglobin adduct levels were strongly associated (correlation coefficient [r2] = .51, P = .002) with maternal 4-aminobiphenyl hemoglobin adduct levels when paired samples from smoking mothers were analyzed. A measure of third-trimester tobacco smoke exposure based on number of cigarettes smoked per day, amount of each cigarette smoked, and depth of inhalation was associated (r2 = .59, P = .029) with maternal 4-aminobiphenyl levels but not with fetal 4-aminobiphenyl levels. This study demonstrates that a potent tobacco-related carcinogen, 4-aminobiphenyl, or its active metabolite, N-hydroxy-4-aminobiphenyl, crosses the human placenta and binds to fetal hemoglobin in concentrations that are significantly higher in smokers than in nonsmokers.

Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers.

In 100 healthy volunteers, we have studied the relationship between the type (air- or flue-cured) and number of cigarettes smoked and different biomarkers relevant to the risk of bladder cancer, including the levels of 4-aminobiphenyl (ABP) hemoglobin adduct (a marker of internal dose), urinary mutagenicity in Salmonella typhimurium TA98, and the N-acetylation phenotype (a marker of susceptibility). ABP is a potent bladder carcinogen that is N-acetylated as an overall detoxification step. Levels of the ABP hemoglobin adduct were higher in smokers of black tobacco (air-cured) than in smokers of blond tobacco (flue-cured), confirming our earlier study. In addition, "slow" acetylators had higher levels of the ABP hemoglobin adduct for the same type and quantity of cigarettes smoked. Urinary mutagenicity was also associated with quantity of cigarettes but not with the acetylation phenotype. Convex dose-response relationships were found between the amount smoked and ABP hemoglobin adduct levels or urinary mutagenicity. In 15 nonsmokers who reported exposure to environmental tobacco smoke, ABP hemoglobin adduct levels, unlike urinary mutagenicity, were found to be an aspecific exposure indicator.

Gender- and smoking-related bladder cancer risk.

BACKGROUND: There is growing evidence that, when smoking habits are comparable, women incur a higher risk of lung cancer than men. Because smokers are also at risk for bladder cancer, we investigated possible sex differences in the susceptibility to bladder cancer among smokers. METHODS: A population-based, case--control study was conducted in Los Angeles, CA, involving 1514 case patients with bladder cancer and 1514 individually matched population control subjects. Information on tobacco use was collected through in-person interviews. Peripheral blood was collected from study participants to measure 3- and 4-aminobiphenyl (ABP)-hemoglobin adducts, a marker of arylamine exposure. Data were analyzed to determine whether the risk of bladder cancer differs between male and female smokers and whether female smokers exhibit higher levels of ABP-hemoglobin adducts than male smokers with comparable smoking habits. All statistical tests were two-sided. RESULTS: Cigarette smokers had a statistically significant 2.5-fold higher risk (95% confidence interval = 2.1 to 3.0) of bladder cancer than never smokers. Use of filtered versus nonfiltered cigarettes, low-tar versus higher tar cigarettes, or the pattern of inhalation did not modify the risk. The risk of bladder cancer in women who smoked was statistically significantly higher than that in men who smoked comparable numbers of cigarettes (P =.016 for sex-lifetime smoking interaction). Consistent with the sex difference in smoking-related bladder cancer risk, the slopes of the linear regression lines of the 3- and 4-ABP--hemoglobin adducts by cigarettes per day were statistically significantly steeper in women than in men (P values for sex differences <.001 and.006, respectively). CONCLUSION: The risk of bladder cancer may be higher in women than in men who smoked comparable amounts of cigarettes.

Acetylator phenotype, aminobiphenyl-hemoglobin adduct levels, and bladder cancer risk in white, black, and Asian men in Los Angeles, California.

BACKGROUND: There is a large body of epidemiologic and experimental data that have identified a number of arylamines as human bladder carcinogens. Metabolic activation is required to biotransform these arylamines into their carcinogenic forms, and N-hydroxylation, which is catalyzed by the hepatic cytochrome P4501A2 isoenzyme, is generally viewed as the first critical step. On the other hand, the N-acetylation reaction, catalyzed by the hepatic N-acetyltransferase enzyme, represents a detoxification pathway for such compounds. The N-acetyltransferase enzyme is coded by a single gene displaying two phenotypes, slow and rapid acetylators. In the United States, cigarette smoking is a major cause of bladder cancer in men, and carcinogenic arylamines present in cigarette smoke are believed to be responsible for inducing bladder cancer in smokers. PURPOSE: Our purpose was to test the differences in three ethnic/racial groups for the prevalence of acetylator phenotypes and to ascertain whether slow acetylators actually have higher levels of activated arylamines in comparison with rapid acetylators. METHODS: One hundred thirty-three male residents of Los Angeles County who were either white, black, or Asian (Chinese or Japanese) and over the age of 35 years were assessed for their acetylator phenotype and levels of 3- and 4-aminobiphenyl (ABP) hemoglobin adducts. Subjects were either lifetime nonsmokers (n = 72) or current cigarette smokers of varying intensity (n = 61). RESULTS: The proportion of slow acetylators was highest among whites (54%), intermediate among blacks (34%), and lowest among Asians (14%). Similarly, geometric mean levels of both 3- and 4-ABP-hemoglobin adducts were highest in whites (1.80 and 49.2 pg/g hemoglobin [Hb], respectively), intermediate in blacks (1.54 and 38.5 pg/g Hb), and lowest in Asians (0.73 and 36.0 pg/g Hb). As expected, cigarette smokers had significantly higher mean levels of both 3- and 4-ABP-hemoglobin adducts relative to nonsmokers, and the levels increased with the number of cigarettes smoked per day (P < .0005 for both adducts). Slow acetylators consistently exhibited higher mean levels of ABP-hemoglobin adducts relative to rapid acetylators, independent of race and level of smoking. CONCLUSION: The present cross-sectional survey supports acetylation phenotype as an important determinant of bladder cancer risk and a possible major factor in the varying bladder cancer risk among whites, blacks, and Asians.

Flow cytometric analysis of the effect of sodium chloride on gastric cancer risk in the rat.

Dietary sodium chloride has been identified, both experimentally and epidemiologically, as a risk factor for gastric cancer. In order to elucidate the manner in which salt increases gastric tumor incidence in N-methyl-N'-nitro-N-nitrosoguanidine-treated animals, flow cytometric cell cycle analyses were performed on rats which had been treated with 1 ml of a solution of saturated NaCl by gavage and sacrificed 0, 1, 6, 12, 24, or 48 h after treatment. The gastric antra were excised, disaggregated, and stained with propidium iodide for cell cycle analysis. Results showed that there is a reduction in cell yield at early time points due to the toxicity of NaCl, followed by a net increase in the number of cells in the S phase of the cell cycle at 24 h. Treatment of rats with NaCl 24 h prior to a dose of 10 micrograms of 3H-labeled N-methyl-N'-nitro-N-nitrosoguanidine did not lead to an increase in alkylation of DNA isolated from mucosal cells. Therefore, the hypothesis that salt enhances gastric cancer risk from N-methyl-N'-nitro-N-nitrosoguanidine by disruption of the "mucosal barrier" leading to an increased effective dose to target cells is not supported by the results of these experiments. Several studies have shown that cells in S phase are the most susceptible to mutagenesis and that increasing the number of cycling cells in a target organ will increase tumor incidence (e.g., partial hepatectomy). Thus it is possible that NaCl increases gastric cancer risk through the mitogenesis which results from the damage caused to the mucosa by this agent.

Sulfotransferase-mediated activation of 4-hydroxy- and 3,4-dihydroxy-3,4-dihydrocyclopenta[c,d]pyrene, major metabolites of cyclopenta[c,d]pyrene.

Cyclopenta[c,d]pyrene, a ubiquitous environmental and occupational pollutant, has been reported to be metabolically activated through epoxidation at the 3,4 double bond in the cyclopenta ring to produce an electrophilic and mutagenic cyclopenta[c,d]pyrene-3,4-epoxide. 4-Hydroxy-3,4-dihydrocyclopenta[c,d]-pyrene (4-HDCPP) and 3,4-dihydroxy-3,4-dihydrocyclopenta[c,d]pyrene (3,4-DHDCPP) are known to be major metabolites of cyclopenta[c,d]pyrene, which appear to be derived from cyclopenta[c,d]pyrene-3,4-epoxide. The present study was undertaken to determine whether 4-HDCPP or 3,4-DHDCPP can be further activated via the formation of reactive benzylic sulfuric acid ester metabolites. Thus, when 4-HDCPP or 3,4-DHDCPP was incubated with calf thymus DNA in the presence of rodent liver cytosol and the sulfo group donor, 3'-phosphoadenosine-5'-phosphosulfate, a significant covalent DNA binding was observed. This cytosol- and 3'-phosphadenosine-5'-phosphosulfate-dependent DNA binding was inhibited by 2,6-dichloro-4-nitrophenol and dehydroepiandrosterone, suggesting the involvement of both phenol and hydroxysteroid sulfotransferases in the activation of 4-HDCPP and 3,4-DHDCPP. A gender difference was observed for the hepatic cytosolic sulfotransferase activity for 4-HDCPP in rats (i.e., male > female). Of the two isomers of 3,4-DHDCPP, the trans-diol produced DNA adducts to a much greater extent than did the cis counterpart by sulfotransferase. 4-HDCPP and 3,4-DHDCPP were also mutagenic toward bacteria in the presence of hepatic cytosol and 3'-phosphadenosine-5'-phosphosulfate. The chemically synthesized sulfuric acid ester 4-sulfooxy-3,4-DCPP was directly mutagenic without any activation system. The data from this study suggest that sulfotransferase plays an important role in the activation of those secondary benzylic hydroxyl metabolites derived from cyclopenta[c,d]pyrene-3,4-epoxide and, possibly, from epoxides of other aromatic hydrocarbons.

In vivo metabolism and whole-blood clearance of n-nitrosomethylbenzylamine in the rat.

The pharmacokinetics and metabolism of N-nitrosomethyl-benzylamine, N-nitroso[methyl-14C]benzylamine, and N-nitrosomethyl[benzyl-7-14C]amine were studied in male Sprague-Dawley rats, and a major urinary metabolite was identified. N-Nitrosomethylbenzylamine (4.7 mg/kg body weight i.p.) was distributed throughout extracellular water and cleared from the whole blood by metabolism with a half-life of 66 min. Less than 1% of the administered dose of N-nitrosomethylbenzylamine (4.7 mg/kg i.p. or 3.3 mg/kg intragastric intubation) was excreted and expired as the parent compound. In the 24-hr period following injection of N-nitroso[methyl-14C1benzylamine (3.4 mg, 1 mCi/kg i.p.), 46% of the radioactivity administered was expired with a half-life of 2.1 hr. In contrast, 81% of the radioactivity from a dose of N-nitrosomethyl[benzyl-7-14C1amine (2.4 mg, 1 mCi/kg i.p.) was excreted in the urine with a half-life of 4.2 hr. Hippuric acid accounted for 80% of the radioactivity recovered in the urine.