The etiology of bladder cancer among never smokers without occupational or environmental exposure to established urothelial carcinogens remains unclear. Urinary mutagenicity is an integrative measure that reflects recent exposure to genotoxic agents. Here, we investigated its potential association with bladder cancer in rural northern New England. We analyzed 156 bladder cancer cases and 247 cancer-free controls from a large population-based case-control study conducted in Maine, New Hampshire, and Vermont. Overnight urine samples were deconjugated enzymatically and the extracted organics were assessed for mutagenicity using the plate-incorporation Ames assay with the Salmonella frameshift strain YG1041 + S9. Logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) of bladder cancer in relation to having mutagenic versus nonmutagenic urine, adjusted for age, sex, and state, and stratified by smoking status (never, former, and current). We found evidence for an association between having mutagenic urine and increased bladder cancer risk among never smokers (OR = 3.8, 95% CI: 1.3-11.2) but not among former or current smokers. Risk could not be estimated among current smokers because nearly all cases and controls had mutagenic urine. Urinary mutagenicity among never-smoking controls could not be explained by recent exposure to established occupational and environmental mutagenic bladder carcinogens evaluated in our study. Our findings suggest that among never smokers, urinary mutagenicity potentially reflects genotoxic exposure profiles relevant to bladder carcinogenesis. Future studies are needed to replicate our findings and identify compounds and their sources that influence bladder cancer risk.

CONTEXT:Soy biodiesel is the predominant biodiesel in the USA, but there is little understanding of the classes of chemicals responsible for the mutagenicity of its emissions. OBJECTIVE:We determined some of the chemical classes responsible for the mutagenicity of the particulate matter (PM) of the emissions from petroleum diesel (B0) and biodiesel containing increasing concentrations of soy methyl esters (B20, B50, and B100). MATERIALS AND METHODS:We subjected organic extracts of the PM to bioassay-directed fractionation by sequential elution on silica gel with solvents of increasing polarity to produce four fractions per fuel. We injected these onto high performance liquid chromatography to produce 62 sub-fractions per fraction based on chemical polarity and evaluated all fractions and sub-fractions for mutagenicity in Salmonella. We correlated the results with the concentrations of 32 polycyclic aromatic hydrocarbons (PAHs) in the fractions. RESULTS:The mutagenicity-emission factors of the fractions generally decreased with increasing concentrations of soy in the fuel. Despite the different chemical compositions of the fuels, the extractable organics of all four emissions had similar features: ∼60% of the mass was nonpolar, non-mutagenic compounds; most of the PAHs were polar; and most of the mutagenicity was due to weakly polar and polar compounds. Some of the mutagenicity of B20 was due to highly polar compounds. CONCLUSIONS:The PM from soy biodiesel emissions was less mutagenic than that from petroleum diesel, and this reduction was associated with reduced concentrations of various weakly polar, polar, and highly polar mutagens, including PAHs, aromatic amines, nitroarenes, and oxy-PAHs.

Abstract Petroleum diesel exhaust is a complex mixture containing many probable and known carcinogens. The development of biodiesel and biodiesel blends offers a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel exhaust particles (DEP)from petroleum diesel and biodiesel. Two different petroleum diesel DEPs were examined (C-DEP and B0), as well as one biodiesel DEP (B100), and DEP resulting from combustion of a blend of 20% B100 and 80% B0 (B20), which is representative of commercially available biodiesel. Oxidative activation was performed in the presence of calf thymus DNA (ctDNA) using rat liver microsomal fractions with required cofactors (S9), and nitroreductive activation was performed using xanthine oxidase (XO) and hypoxanthine in the presence of ctDNA. The modified DNA was hydrolyzed and analyzed by 32P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with polycyclic aromatic hydrocarbon (PAH) and nitro-PAH adducts. The types and quantities of DNA adducts produced from two independent petroleum diesel DEPs were similar, with evidence of formation of both PAH- and nitroPAH- derived adducts. In contrast, the biodiesel DEP B100 induced higher total levels of DNA adducts in both activation systems. The lowest levels of DNA adduct formation were observed with B20 DEP. These results suggest that the DEP from available biodiesel blends (B20) pose lower risk for induction of DNA damage than petroleum diesel. This is an abstract or a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Jeffrey A. Ross, Garret B. Nelson, Esrta Mutlu, Sarah H. Warren, Matthews P. Peggy, M. Ian Gilmour, David M. DeMarini. DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts. [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 826. doi:10.1158/1538-7445.AM2015-826

Several types of diesel exhaust particles (DEPs) have been used for toxicology studies, including a high-organic automobile DEP (A-DEP) from Japan, and a low-organic forklift DEP developed by the National Institute of Standards and Technology (N-DEP). However, these DEPs were not characterized extensively for chemical composition or sub-fractionated and tested extensively for mutagenicity. We collected a compressor-generated DEP (C-DEP) and characterized it by conducting bioassay-directed fractionation of the extractable organics in Salmonella and correlating the results by hierarchical clustering with the concentrations of 32 polycyclic aromatic hydrocarbons (PAHs). Relative to A- and N-DEP, the mutagenic potency of C-DEP was intermediate in TA100 +S9 (PAH mutagenicity) but was lowest in TA98 -S9 (nitroarene mutagenicity). More than 50% of the mass of the extractable organics of C-DEP eluted in the nonpolar Fraction 1, and only ∼20% eluted in the moderately polar Fractions 2 and 3. However, most of the mutagenicity eluted in Fractions 2 and 3, similar to A-DEP but different from N-DEP. HPLC-derived mutagrams of 62 sub-fractions per fraction confirmed that most of the mutagenicity was due to moderately polar compounds. The diagnostic strains identified a strong role for PAHs, nitroarenes, aromatic amines, and oxy-PAHs in the mutagenicity of C-DEP. Hierarchical clustering confirmed the importance of oxy-PAHs but not that of nitroarenes. To our knowledge this is the first use of hierarchical clustering to correlate chemical composition with the mutagenicity of a complex mixture. The chemical analysis and mutagenicity of C-DEP described here makes C-DEP suitable for additional toxicological studies.

UNLABELLED:Dietary exposures implicated as reducing or causing risk for colorectal cancer may reduce or cause DNA damage in colon tissue; however, no one has assessed this hypothesis directly in humans. Thus, we enrolled 16 healthy volunteers in a 4-week controlled feeding study where 8 subjects were randomly assigned to dietary regimens containing meat cooked at either low (100°C) or high temperature (250°C), each for 2 weeks in a crossover design. The other 8 subjects were randomly assigned to dietary regimens containing the high-temperature meat diet alone or in combination with 3 putative mutagen inhibitors: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design. Subjects were nonsmokers, at least 18 years old, and not currently taking prescription drugs or antibiotics. We used the Salmonella assay to analyze the meat, urine, and feces for mutagenicity, and the comet assay to analyze rectal biopsies and peripheral blood lymphocytes for DNA damage. Low-temperature meat had undetectable levels of heterocyclic amines (HCAs) and was not mutagenic, whereas high-temperature meat had high HCA levels and was highly mutagenic. The high-temperature meat diet increased the mutagenicity of hydrolyzed urine and feces compared to the low-temperature meat diet. The mutagenicity of hydrolyzed urine was increased nearly twofold by the inhibitor diet, indicating that the inhibitors enhanced conjugation. Inhibitors decreased significantly the mutagenicity of un-hydrolyzed and hydrolyzed feces. The diets did not alter the levels of DNA damage in non-target white blood cells, but the inhibitor diet decreased nearly twofold the DNA damage in target colorectal cells. To our knowledge, this is the first demonstration that dietary factors can reduce DNA damage in the target tissue of fried-meat associated carcinogenesis.TRIAL REGISTRATION:ClinicalTrials.gov NCT00340743.

Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used for large, multi-site, and/or time series studies, for bioassay-directed fractionation studies, for identifying the presence of specific classes of mutagens, and for doing site- or source-comparisons for relative levels of airborne mutagens. Early research recognized that although carcinogenic PAHs were present in air samples they could not account for the majority of the mutagenic activity detected. The mutagenicity of airborne particulate organics is due to at least 500 identified compounds from varying chemical classes. Bioassay-directed fractionation studies for identifying toxicants are difficult to compare because they do not identify all of the mutagens present, and both the analytical and bioassay protocols vary from study to study. However, these studies show that the majority of mutagenicity is usually associated with moderately polar/highly polar classes of compounds that tend to contain nitroaromatic compounds, aromatic amines, and aromatic ketones. Smog chamber studies have shown that mutagenic aliphatic and aromatic nitrogen-containing compounds are produced in the atmosphere when organic compounds (even non-mutagenic compounds) are exposed to nitrogen oxides and sunlight. Reactions that occur in the atmosphere, therefore, can have a profound effect on the genotoxic burden of ambient air. This review illustrates that the mutagenesis protocol and tester strains should be selected based on the design and purpose of the study and that the correlation with animal cancer bioassay results depends upon chemical class. Future emphasis needs to be placed on volatile and semi-volatile genotoxicants, and on multi-national studies that identify, quantify, and apportion mutagenicity. Initial efforts at replacing the Salmonella assay for ambient air studies with some emerging technology should be initiated.

Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DBPs) in drinking water. They include chloronitromethane (CHN), dichloronitromethane (DCNM), trichloronitromethane (TCNM), bromonitromethane (BNM), dibromonitromethane (DBNM), tribromonitromethane (TBNM), bromochloronitromethane (BCNM),dibromochloronitromethane (DBCNM), and bromodichloronitromethane (BDCNM). Previous studies of TCNM, DCNM, CNM, and TBNM found that all four were mutagenic in bacteria, and a recent study showed that all nine induced DNA damage in CHO cells. Here, all nine HNMs were evaluated in the Salmonella plate-incorporation assay +/− S9 using strains TA98, TA100, TA104, TPT100, and the glutathione transferase theta (GSTT1-1)-expressing strain RSJ100. All were mutagenic, most with and without S9. In the absence of S9, six were mutagenic in TA98, six in TA100, and three in TA104; in the presence of S9, these numbers were five, seven, and three, respectively. Thus, the HNMs-induced base substitutions primarily at GC sites as well as frameshifts. Although five HNMs were activated to mutagens in RSJ100 −S9, they produced ≤2-fold increases in revertants and potencies <506rev/μmol. The rank order of the HNMs by mutagenic potency in TA100 +S9 was (BCNM DBNM) > (TBNM CNM > BNM DCNM BDCNM) > (TCNM=DBCNM). The mean rev/μmol for the three groupings, respectively, were 1423, 498, and 0, which classifies the HNMs as weak mutagens in Salmonella. Reaction of the dihalo and monohalo HNMs with GSH, possibly GSTT1-1, is a possible mechanism for formation of ultimate mutagenic products. Because the HNMs are mutagenic in Salmonella (present study) and potent clastogens in mammalian cells [Environ. Sci. Technol. 38 (2004) 62], their presence in drinking water warrants further research on their potential health effects.