Endogenous Formation Of N-nitroso Compounds Research Articles

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Ingestion of Nitrate and Nitrite and Risk of Stomach and Other Digestive System Cancers in the Iowa Women's Health Study.

Nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOC) which are potent animal carcinogens for the organs of the digestive system. We evaluated dietary intakes of nitrate and nitrite, as well as nitrate ingestion from drinking water (public drinking water supplies (PWS)), in relation to the incidence (1986–2014) of cancers of the esophagus (n = 36), stomach (n = 84), small intestine (n = 32), liver (n = 31), gallbladder (n = 66), and bile duct (n = 58) in the Iowa Women’s Health Study (42,000 women aged from 50 to 75 in 1986). Dietary nitrate and nitrite were estimated using a food frequency questionnaire and a database of nitrate and nitrite levels in foods. Historical nitrate measurements from PWS were linked to the enrollment address by duration. We used Cox regression to compute hazard ratios (HR) and 95% confidence intervals (CI) for exposure quartiles (Q), tertiles (T), or medians, depending on the number of cancer cases. In adjusted models, nitrite intake from processed meats was associated with an increased risk of stomach cancer (HRQ4vsQ1 = 2.2, CI: 1.2–4.3). A high intake of total dietary nitrite was inversely associated with gallbladder cancer (HRQ4vsQ1 = 0.3, CI: 0.1–0.96), driven by an inverse association with plant sources of nitrite (HRQ4vsQ1 = 0.3, CI: 0.1–0.9). Additionally, small intestine cancer was inversely associated with a high intake of animal nitrite (HRT3vsT1 = 0.2, CI: 0.1–0.7). There were no other dietary associations. Nitrate concentrations in PWS (average, years ≥ 1/2 the maximum contaminant level) were not associated with cancer incidence. Our findings for stomach cancer are consistent with prior dietary studies, and we are the first to evaluate nitrate and nitrite ingestion for certain gastrointestinal cancers.

Ingestion of nitrate and nitrite from drinking water and diet and cancers of the gastrointestinal tract in Iowa women

Background: Nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOC), potent animal carcinogens in the gastrointestinal tract. Methods: We evaluated nitrate ingestion from public drinking water supplies (PWS), private well use, and dietary nitrate and nitrite intakes in relation to incidence (through 2014) of cancers of the esophagus (n=37), stomach (97), small intestine (47), liver (56), and gallbladder (66) in the Iowa Women’s Health Study (42,000 women ages 50-75 in 1986). Historical nitrate measurements from PWS were linked to the enrollment address by duration. We computed average concentrations and number of years >5 mg/L NO3-N (½ the maximum contaminant level [MCL]). Dietary nitrate and nitrite were estimated using a food frequency questionnaire. We used Cox proportional hazards models to compute hazard ratios (HR) and 95% confidence intervals (CI). Results: In adjusted models, liver cancer risk was elevated among private well users (HR=2.5, CI 0.8-8.5) and those with any years >5 mg/L NO3-N (HR=2.0, CI 0.8-4.9); there was no association with average concentrations (HRper1mg/L NO3-N =1.1, CI 0.7-1.7). Private well use and PWS nitrate concentrations were not associated with other cancers. Dietary nitrate and nitrite from plants was inversely associated with small intestine (HRQ4vsQ1=0.4, CI 0.1-0.9) and gall bladder cancer (HRQ4vsQ1=0.3, CI 0.1-0.8), respectively; there were no associations for the other cancers. Nitrite intake from processed meats was associated with increased risk of stomach (HRnitriteQ4vsQ1=1.7, CI 1.0-3.0) and elevated risk of liver cancer (HRnitriteQ4vsQ1=1.8, CI 0.8-4.0) but was inversely associated with small intestine cancer (HRnitriteQ4vsQ1=0.4, CI 0.1-1.0). Power was limited, but there was no evidence for effect modification by vitamin C and red meat, factors affecting nitrosation. Conclusion: We found novel suggestive evidence that private well use and PWS nitrate may increase liver cancer risk. These associations should be evaluated in studies with larger case numbers.

Impact of high drinking water nitrate levels on the endogenous formation of apparent N-nitroso compounds in combination with meat intake in healthy volunteers

BackgroundNitrate is converted to nitrite in the human body and subsequently can react with amines and amides in the gastrointestinal tract to form N-nitroso compounds (NOCs), which are known to be carcinogenic in animals. Humans can be exposed to nitrate via consumption of drinking water and diet, especially green leafy vegetables and cured meat. The contribution of nitrate from drinking water in combination with meat intake has not been investigated thoroughly. Therefore, in the present pilot study, we examined the effect of nitrate from drinking water, and its interaction with the consumption of white and processed red meat, on the endogenous formation of NOCs, taking into account the intake of vitamin C, a nitrosation inhibitor.MethodsTwenty healthy subjects were randomly assigned to two groups consuming either 3.75 g/kg body weight (maximum 300 g per day) processed red meat or unprocessed white meat per day for two weeks. Drinking water nitrate levels were kept low during the first week (< 1.5 mg/L), whereas in week 2, nitrate levels in drinking water were adjusted to the acceptable daily intake level of 3.7 mg/kg bodyweight. At baseline, after 1 and 2 weeks, faeces and 24 h urine samples were collected for analyses of nitrate, apparent total N-nitroso compounds (ATNC), compliance markers, and genotoxic potential in human colonic Caco-2 cells.ResultsUrinary nitrate excretion was significantly increased during the high drinking water nitrate period for both meat types. Furthermore, levels of compliance markers for meat intake were significantly increased in urine from subjects consuming processed red meat (i.e. 1-Methylhistidine levels), or unprocessed white meat (i.e. 3-Methylhistidine). ATNC levels significantly increased during the high drinking water nitrate period, which was more pronounced in the processed red meat group. Genotoxicity in Caco-2 cells exposed to faecal water resulted in increased genotoxicity after the interventions, but results were only significant in the low drinking water nitrate period in subjects consuming processed red meat. Furthermore, a positive correlation was found between the ratio of nitrate/vitamin C intake (including drinking water) and the level of ATNC in faecal water of subjects in the processed red meat group, but this was not statistically significant.ConclusionsDrinking water nitrate significantly contributed to the endogenous formation of NOC, independent of the meat type consumed. This implies that drinking water nitrate levels should be taken into account when evaluating the effect of meat consumption on endogenous formation of NOC.Trial registrationDutch Trialregister: 29707. Registered 19th of October 2018. Retrospectively registered.

Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete’s health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

Ingested nitrate and nitrite, disinfection by-products, and pancreatic cancer risk in postmenopausal women.

Nitrate and nitrite are precursors of N-nitroso compounds (NOC), probable human carcinogens that cause pancreatic tumors in animals. Disinfection by-products (DBP) exposures have also been linked with digestive system cancers, but few studies have evaluated relationships with pancreatic cancer. We investigated the association of pancreatic cancer with these drinking water contaminants and dietary nitrate/nitrite in a cohort of postmenopausal women in Iowa (1986-2011). We used historical monitoring and treatment data to estimate levels of long-term average nitrate and total trihalomethanes (TTHM; the sum of the most prevalent DBP class) and the duration exceeding one-half the maximum contaminant level (>½ MCL; 5 mg/L nitrate-nitrogen, 40 µg/L TTHM) among participants on public water supplies (PWS) >10 years. We estimated dietary nitrate and nitrite intakes using a food frequency questionnaire. We computed hazard ratios (HR) and 95% confidence intervals (CI) using Cox regression and evaluated nitrate interactions with smoking and vitamin C intake. We identified 313 cases among 34,242 women, including 152 with >10 years PWS use (N = 15,710). Multivariable models of average nitrate showed no association with pancreatic cancer (HRp95vs. Q1 = 1.16, 95% CI: 0.51-2.64). Associations with average TTHM levels were also null (HRQ4vs. Q1 = 0.70, 95% CI:0.42-1.18). We observed no trend with increasing years of exposure to either contaminant at levels >½ MCL. Positive associations were suggested in the highest dietary nitrite intake from processed meat (HRp95vs. Q1 = 1.66, 95% CI 1.00-2.75;ptrend = 0.05). We found no interactions of nitrate with known modifiers of endogenous NOC formation. Our results suggest that nitrite intake from processed meat may be a risk factor for pancreatic cancer.

Nitrate from Drinking Water and Diet and Bladder Cancer Among Postmenopausal Women in Iowa.

Background:Nitrate is a drinking water contaminant arising from agricultural sources, and it is a precursor in the endogenous formation of N-nitroso compounds (NOC), which are possible bladder carcinogens.Objectives:We investigated the ingestion of nitrate and nitrite from drinking water and diet and bladder cancer risk in women.Methods:We identified incident bladder cancers among a cohort of 34,708 postmenopausal women in Iowa (1986–2010). Dietary nitrate and nitrite intakes were estimated from a baseline food frequency questionnaire. Drinking water source and duration were assessed in a 1989 follow-up. For women using public water supplies (PWS) > 10 years (n = 15,577), we estimated average nitrate (NO3-N) and total trihalomethane (TTHM) levels and the number of years exceeding one-half the maximum contaminant level (NO3-N: 5 mg/L, TTHM: 40 μg/mL) from historical monitoring data. We computed hazard ratios (HRs) and 95% confidence intervals (CIs), and assessed nitrate interactions with TTHM and with modifiers of NOC formation (smoking, vitamin C).Results:We identified 258 bladder cancer cases, including 130 among women > 10 years at their PWS. In multivariable-adjusted models, we observed nonsignificant associations among women in the highest versus lowest quartile of average drinking water nitrate concentration (HR = 1.48; 95% CI: 0.92, 2.40; ptrend = 0.11), and we found significant associations among those exposed ≥ 4 years to drinking water with > 5 mg/L NO3-N (HR = 1.62; 95% CI: 1.06, 2.47; ptrend = 0.03) compared with women having 0 years of comparable exposure. TTHM adjustment had little influence on associations, and we observed no modification by vitamin C intake. Relative to a common reference group of never smokers with the lowest nitrate exposures, associations were strongest for current smokers with the highest nitrate exposures (HR = 3.67; 95% CI: 1.43, 9.38 for average water NO3-N and HR = 3.48; 95% CI: 1.20, 10.06 and ≥ 4 years > 5 mg/L, respectively). Dietary nitrate and nitrite intakes were not associated with bladder cancer.Conclusions:Long-term ingestion of elevated nitrate in drinking water was associated with an increased risk of bladder cancer among postmenopausal women.Citation:Jones RR, Weyer PJ, DellaValle CT, Inoue-Choi M, Anderson KE, Cantor KP, Krasner S, Robien K, Beane Freeman LE, Silverman DT, Ward MH. 2016. Nitrate from drinking water and diet and bladder cancer among postmenopausal women in Iowa. Environ Health Perspect 124:1751–1758; http://dx.doi.org/10.1289/EHP191

Ingested Nitrate and Breast Cancer in the Spanish Multicase-Control Study on Cancer (MCC-Spain).

Background:Ingested nitrate leads to endogenous formation of N-nitroso compounds that are breast carcinogens in animals, but human evidence is limited.Objective:We evaluated ingested nitrate as a risk factor for breast cancer (BC) in a multicase–control study.Methods:Hospital-based incident BC cases and population-based controls were recruited in eight Spanish regions in 2008–2013; participants provided residential and water consumption from 18 years of age and information on known BC risk factors. Long-term nitrate levels (1940–2010) were estimated and linked with residential histories and water consumption to calculate waterborne ingested nitrate (milligrams/day). Dietary ingested nitrate (milligrams/day) was calculated using food frequency questionnaires and published dietary nitrate contents. Interactions with endogenous nitrosation factors and other variables were evaluated. A total of 1,245 cases and 1,520 controls were included in the statistical analysis.Results:Among the study regions, average ± SD waterborne ingested nitrate ranged from 2.9 ± 1.9 to 13.5 ± 7.5 mg/day, and dietary ingested nitrate ranged from 88.5 ± 48.7 to 154 ± 87.8 mg/day. Waterborne ingested nitrate was not associated with BC overall, but among postmenopausal women, those with both high nitrate (> 6 vs. < 2.6 mg/day) and high red meat intake (≥ 20 vs. < 20 g/day) were more likely to be cases than women with low nitrate and low red meat intake (adjusted odds ratio = 1.64; 95% confidence interval: 1.08, 2.49; overall interaction p-value = 0.17). No association was found with dietary nitrate.Conclusions:Waterborne ingested nitrate was associated with BC only among postmenopausal women with high red meat consumption. Dietary nitrate was not associated with BC regardless of the animal or vegetable source or of menopausal status.Citation:Espejo-Herrera N, Gracia-Lavedan E, Pollan M, Aragonés N, Boldo E, Perez-Gomez B, Altzibar JM, Amiano P, Zabala AJ, Ardanaz E, Guevara M, Molina AJ, Barrio JP, Gómez-Acebo I, Tardón A, Peiró R, Chirlaque MD, Palau M, Muñoz M, Font-Ribera L, Castaño-Vinyals G, Kogevinas M, Villanueva CM. 2016. Ingested nitrate and breast cancer in the Spanish Multicase-Control Study on Cancer (MCC-Spain). Environ Health Perspect 124:1042–1049; http://dx.doi.org/10.1289/ehp.1510334

Waterborne Ingested Nitrate And Breast Cancer Risk In The Spanish Multicase-Control Study On Cancer (MCC-Spain)

Introduction Ingested nitrate leads to endogenous formation of N-nitroso compounds that induce breast cancer in animal models, but limited evidence is available in humans. We aimed to evaluate wate...

Abstract 1883: Dietary nitrate and nitrite, micronutrients, and postmenopausal breast cancer risk in the NIH-AARP Diet and Health Study

Abstract Background: Ingested nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOCs), potential human carcinogens. Vitamin C inhibits and heme iron enhances NOC formation. To date, interactions between dietary nitrate and nitrite intakes and these micronutrients have not been evaluated in relation to the risk for breast cancer in prospective cohort studies. Methods: We followed 191,926 postmenopausal women in the NIH-AARP Diet and Health Study for incident breast cancer. Dietary intake was assessed using a food frequency questionnaire at baseline (1995). Daily nitrate and nitrite intakes were computed for total, animal, and processed meat sources, and adjusted for energy intake using a density method. We computed multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) by quintiles of nitrate and nitrite for all breast cancers, by estrogen/progesterone receptor (ER/PR) status, and by stage (localized, regional/distant) using Cox proportional hazards regression. We stratified analyses by low or high (≤ or &amp;gt; median) heme iron (138 mg/1,000kcal) and total vitamin C (273 mg/d) intakes. Results: We identified 7,508 incident invasive breast cancers (1995-2006). On average, 95% of nitrate and 70% of nitrite intakes were from plant sources. Red processed meat intake was not associated with risk of all breast cancer but was positively associated with risk of localized breast cancer (adjusted HR = 1.35 for Q5, CI = 1.11-1.42, p-trend&amp;lt;0.001). Heme iron intake was positively associated with risk of all breast cancer (HR = 1.12 for Q5, CI = 1.04-1.21, p-trend = 0.004). Total nitrate intake was associated with higher risk of all breast cancer, particularly among women with high heme iron intake (HR = 1.15, CI = 1.01-1.30 for Q5, p-trend = 0.02), but not among women with low heme iron intake (p-trend = 0.63, p-interaction = 0.17); similar associations were observed when evaluating total nitrate intake in relation to hormone receptor positive cancers (p-interaction = 0.09 for total nitrate x heme iron in relation to ER+/PR+ positive cancer). Total nitrite intake was not associated with overall breast cancer risk. Nitrite intake from processed red meats was associated with higher risk of localized (HR = 1.23, 95%CI = 1.09-1.39 for Q5, p-trend&amp;lt;0.001) but not regional/distant breast cancers (p-trend = 0.41). Vitamin C intake did not interact with nitrate or nitrite intake in relation to breast cancer risk. Conclusions: Our findings suggest that high nitrate intake and nitrite intake from processed meats may increase postmenopausal women's risk of breast cancer, especially for those with high heme iron intake. Citation Format: Maki Inoue-Choi, Rashmi Sinha, Gretchen L. Gierach, Mary H. Ward. Dietary nitrate and nitrite, micronutrients, and postmenopausal breast cancer risk in the NIH-AARP Diet and Health Study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1883. doi:10.1158/1538-7445.AM2015-1883

Modeling groundwater nitrate concentrations in private wells in Iowa

Contamination of drinking water by nitrate is a growing problem in many agricultural areas of the country. Ingested nitrate can lead to the endogenous formation of N-nitroso compounds, potent carcinogens. We developed a predictive model for nitrate concentrations in private wells in Iowa. Using 34,084 measurements of nitrate in private wells, we trained and tested random forest models to predict log nitrate levels by systematically assessing the predictive performance of 179 variables in 36 thematic groups (well depth, distance to sinkholes, location, land use, soil characteristics, nitrogen inputs, meteorology, and other factors). The final model contained 66 variables in 17 groups. Some of the most important variables were well depth, slope length within 1km of the well, year of sample, and distance to nearest animal feeding operation. The correlation between observed and estimated nitrate concentrations was excellent in the training set (r-square=0.77) and was acceptable in the testing set (r-square=0.38). The random forest model had substantially better predictive performance than a traditional linear regression model or a regression tree. Our model will be used to investigate the association between nitrate levels in drinking water and cancer risk in the Iowa participants of the Agricultural Health Study cohort.

Selecting spatial scale of covariates in regression models of environmental exposures.

Environmental factors or socioeconomic status variables used in regression models to explain environmental chemical exposures or health outcomes are often in practice modeled at the same buffer distance or spatial scale. In this paper, we present four model selection algorithms that select the best spatial scale for each buffer-based or area-level covariate. Contamination of drinking water by nitrate is a growing problem in agricultural areas of the United States, as ingested nitrate can lead to the endogenous formation of N-nitroso compounds, which are potent carcinogens. We applied our methods to model nitrate levels in private wells in Iowa. We found that environmental variables were selected at different spatial scales and that a model allowing spatial scale to vary across covariates provided the best goodness of fit. Our methods can be applied to investigate the association between environmental risk factors available at multiple spatial scales or buffer distances and measures of disease, including cancers.

Influence of dietary carbohydrate and protein on colonic fermentation and endogenous formation of N-nitroso compounds

Influence of dietary carbohydrate and protein on colonic fermentation and endogenous formation of N-nitroso compounds

Nitrate and nitrite ingestion and risk of ovarian cancer among postmenopausal women in Iowa.

Nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOC), potential human carcinogens. We evaluated the association of nitrate and nitrite ingestion with postmenopausal ovarian cancer risk in the Iowa Women's Health Study. Among 28,555 postmenopausal women, we identified 315 incident epithelial ovarian cancers from 1986 to 2010. Dietary nitrate and nitrite intakes were assessed at baseline using food frequency questionnaire data. Drinking water source at home was obtained in a 1989 follow-up survey. Nitrate-nitrogen (NO3 -N) and total trihalomethane (TTHM) levels for Iowa public water utilities were linked to residences and average levels were computed based on each woman's duration at the residence. We computed multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards regression. We tested interactions of nitrate with TTHMs and dietary factors known to influence NOC formation. Ovarian cancer risk was 2.03 times higher (CI = 1.22-3.38, ptrend = 0.003) in the highest quartile (≥2.98 mg/L) compared with the lowest quartile (≤0.47 mg/L; reference) of NO3 -N in public water, regardless of TTHM levels. Risk among private well users was also elevated (HR = 1.53, CI = 0.93-2.54) compared with the same reference group. Associations were stronger when vitamin C intake was <median (pinteraction = 0.01 and 0.33 for private well and public supplies, respectively). Dietary nitrate was inversely associated with ovarian cancer risk (ptrend = 0.02); whereas, dietary nitrite from processed meats was positively associated with the risk (ptrend = 0.04). Our findings indicate that high nitrate levels in public drinking water and private well use may increase ovarian cancer risk among postmenopausal women.

Predicting Nitrate Levels in Private Wells in Iowa for the Agricultural Health Study

Background: Contamination of drinking water by nitrate is a growing problem in many agricultural areas of the country. Ingested nitrate can lead to the endogenous formation of N-nitroso compounds (NOC), which are potent animal carcinogens. Objective: The goal was to develop a predictive model for nitrate levels in private wells in Iowa for the Agricultural Health Study. Methods: We obtained 34,084 measurements of nitrate in private wells, along with well depth and location. We created a training set of approximately 30% of the observations (n = 11,940) with sampling stratified on decade of sample (1980, 1990, 2000) and bedrock status (in or above). We built random forest models to predict log nitrate levels in the training set by systematically assessing the predictive performance of 179 variables in 36 thematic groups (well depth, land use, soil characteristics, nitrogen inputs, meteorology, and other factors). Results: The final model contained 17 variable groups and 66 variables. The most important variable was well depth. Other important variables included slope length within 1 km of the well, year of sample, distance to nearest animal feeding operation, and fertilizer applications with 1 km of the well in 1978. The mean squared error in the validation samples in the training set was 2.56 and the pseudo R-square of the estimated log nitrate in the training set was 0.81. We predicted nitrate levels in the testing set (n = 22,153) with good agreement (r = 0.62) between reported nitrate levels and model predictions. The random forest model had substantially better predictive performance than a traditional linear regression model or a classification tree. Conclusion: Our results demonstrate that a random forest is useful for predicting nitrate levels in private wells. The random forest model predictions of log nitrate will be used to investigate the association between nitrate levels in drinking water and cancer risk in the Agricultural Health Study cohort.

Dietary nitrate and nitrite intake and risk of colorectal cancer in the Shanghai Women's Health Study

Nitrate and nitrite are precursors of endogenously formed N-nitroso compounds (NOC), known animal carcinogens. Nitrosation reactions forming NOCs can be inhibited by vitamin C and other antioxidants. We prospectively investigated the association between dietary nitrate and nitrite intake and risk of colorectal cancer in the Shanghai Women's Health Study, a cohort of 73,118 women ages 40-70 residing in Shanghai. We evaluated effect modification by factors that affect endogenous formation of NOCs: vitamin C (at or above/below median) and red meat intake (at or above/below median). Nitrate, nitrite and other dietary intakes were estimated from a 77-item food frequency questionnaire administered at baseline. Over a mean of 11 years of follow-up, we identified 619 colorectal cancer cases (n = 383, colon; n = 236, rectum). Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazard regression. Overall, nitrate intake was not associated with colorectal cancer risk (HR = 1.08; 95% CI: 0.73-1.59). However, among women with vitamin C intake below the median (83.9 mg day(-1) ) and hence higher potential exposure to NOCs, risk of colorectal cancer increased with increasing quintiles of nitrate intake (highest vs. lowest quintile HR = 2.45; 95% CI: 1.15-5.18; p trend = 0.02). There was no association among women with higher vitamin C intake. We found no association between nitrite intake and risk of colorectal cancer overall or by intake level of vitamin C. Our findings suggest that high dietary nitrate intake among subgroups expected to have higher exposure to endogenously formed NOCs increases risk of colorectal cancer.

Development of a liquid chromatography/tandem mass spectrometry method to investigate the presence of biomarkers of DNA damage in urine related to red meat consumption and risk of colorectal cancer

The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence red meat, has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O(6)-carboxymethylguanine (O(6)CMG) and O(6)-carboxymethyl-2'-deoxyguanosine (O(6)CMdG) in urine in relation to red meat intake using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The presence of the adduct in urine samples either as the free base or as 2'-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large-scale analyses in the population and cancer prevention dietary strategies. An LC/MS/MS method for the quantitation of O(6)CMG and O(6)CMdG was developed. Urine samples collected from healthy volunteers on red meat and vegetarian diets were analysed either by direct injection or after purification by solid-phase extraction (SPE). A separate LC/MS/MS method for O(6)-methylguanine (O(6)MeG) and O(6)-methyl-2'-deoxyguanosine (O(6)MedG), which are possible hydrolysis products forming during the sample pre-treatment, was also developed. The developed LC/MS/MS method allowed the simultaneous measurement of O(6)CMG and O(6)CMdG. The limits of detection (LODs) were 0.38 ng/mL for O(6)CMG and 0.18 ng/mL for O(6)CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts in clinical samples were still found to be below the LOD. Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O(6)CMG and O(6)CMdG, and of the possible formation of O(6)MeG and O(6)MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.

Dietary Nitrates and Nitrites—Harmful? Helpful? Or Paradox?

Dietary Nitrates and Nitrites—Harmful? Helpful? Or Paradox?

Maternal dietary intake of nitrates, nitrites and nitrosamines and selected birth defects in offspring: a case-control study

BackgroundDietary intake of nitrates, nitrites, and nitrosamines can increase the endogenous formation of N-nitroso compounds in the stomach. Results from animal studies suggest that these compounds might be teratogenic. We examined the relationship between maternal dietary intake of nitrates, nitrites (including plant and animal sources as separate groups), and nitrosamines and several types of birth defects in offspring.MethodsFor this population-based case–control study, data from a 58-question food frequency questionnaire, adapted from the short Willett Food Frequency Questionnaire and administered as part of the National Birth Defects Prevention Study (NBDPS), were used to estimate daily intake of dietary nitrates, nitrites, and nitrosamines in a sample of 6544 mothers of infants with neural tube defects (NTD)s, oral clefts (OC)s, or limb deficiencies (LD)s and 6807 mothers of unaffected control infants. Total daily intake of these compounds was divided into quartiles based on the control mother distributions. Odds ratios (OR)s and 95% confidence intervals (CI)s were estimated using logistic regression; estimates were adjusted for maternal daily caloric intake, maternal race-ethnicity, education, dietary folate intake, high fat diet (> 30% of calories from fat), and state of residence.ResultsWhile some unadjusted ORs for NTDS had 95% (CI)s that excluded the null value, none remained significant after adjustment for covariates, and the effect sizes were small (adjusted odds ratios [aOR] <1.12). Similar results were found for OCs and LDs with the exception of animal nitrites and cleft lip with/without cleft palate (aORs and CIs for quartile 4 compared to quartile 1 =1.24; CI=1.05-1.48), animal nitrites and cleft lip (4th quartile aOR=1.32; CI=1.01-1.72), and total nitrite and intercalary LD (4th quartile aOR=4.70; CI=1.23-17.93).ConclusionsOverall, odds of NTDs, OCs or LDs did not appear to be significantly associated with estimated dietary intake of nitrate, nitrite, and nitrosamines.

Diet Composition Is Associated with Endogenous Formation of N-Nitroso Compounds in Obese Men1–3

Endogenous formation of carcinogenic N-nitroso compounds (NOC) occurs in the human gut. Red meat is considered the most important dietary component linked to NOC formation, although nitrate and vitamin C (VitC) also contribute. We previously showed that high-protein weight-loss diets increased fecal NOC and this was enhanced by simultaneous carbohydrate restriction. Although previous studies have focused on the effect of either 1 or 2 dietary components on endogenous NOC formation, no study to date has investigated the combined contribution of various dietary components. The current study therefore assessed the joint impact of several known dietary contributors to the endogenous formation of NOC in obese men. It also aimed to identify further novel contributors and investigate their role in explaining shifts in endogenous formation of NOC. Three dietary trials were conducted in obese men consuming body weight maintenance or weight-loss diets, with NOC measured in fecal samples. Consumption of meat-based weight-loss diets increased (P < 0.001) fecal NOC. Red meat intake was positively correlated with the fecal log NOC concentration (r = 0.60; P < 0.001). Dietary carbohydrate and sugar were negatively correlated with the fecal log NOC concentration (r = -0.66 for both; P < 0.001). Multiple regression analysis identified several dietary components that drive endogenous NOC formation, namely, red meat, nitrate, VitC, total energy, and nonstarch polysaccharides. We present a regression model that predicts endogenous NOC formation in obese men based on their dietary intakes. This model could improve the estimation of endogenous NOC formation, currently used in epidemiological studies into diet and cancer.