Sarah H. Warren

Glutathione S-transferase-mediated induction of GC AT transitions by halomethanes in salmonella

Halomethanes are among the most common mutagenic and carcinogenic disinfection by‐products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH2Cl2) and bromodichloromethane (BrCHCl2) can be mediated by a theta‐class glutathione S‐transferase (GSTT1‐1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base‐substitution strain TA1535 (hisG46, rfa, δuvrB), into which has been cloned the GSTT1‐1gene from rat. In the present report, we have ex tended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr3) and chlorodibromomethane (ClCHBr2), are also mediated by GSTT1‐1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr3 ≈ ClCHBr2 > BrCHCl2 ≈ CH2Cl2. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by per forming colony probe hybridizations of ∼100 revertants induced by each compound. The majority (96–100%) of the mutations were GC → AT transitions, and 87–100% of these were at the second position of the CCC/GGG target. In contrast, only15% of mutants induced by CH2Cl2 were GC → AT transitions in the absence of the GSTT1‐1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1‐1 to mediate the mutagenicity of these di‐ and trihalomethanes and the induction of almost exclusively GC → AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST‐mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH‐DNAadducts. Environ. Mol. Mutagen. 30:440–447, 1997 Published 1997 Wiley‐Liss, Inc. This article is a US Government work and as such, is in the public domain in the United States of America.

Effects of operating variables on PAH emissions and mutagenicity of emissions from woodstoves

As part of the Integrated Air Cancer Project, the U.S. Environmental Protection Agency (EPA) has conducted field emission measurement programs in Raleigh, North Carolina, and Boise, Idaho, to identify the potential mutagenic impact of residential wood burning and motor vehicles on ambient and indoor air. These studies included the collection of emission samples from chimneys serving wood burning appliances. Parallel projects were undertaken in instrumented woodstove test laboratories to quantify woodstove emissions during operations typical of in-house usage but under more controlled conditions. Three woodstoves were operated in test laboratories over a range of burnrates, burning eastern oak, southern yellow pine, or western white pine. Two conventional stoves were tested at an altitude of 90 m. One of the conventional stoves and a catalytic stove were tested at an altitude of 825 m. Decreasing burnrate increased total particulate emissions from the conventional stoves while the catalytic stoves total particulate emissions were unaffected. There was no correlation of total particulate emissions with altitude whereas total polynuclear aromatic hydrocarbon (PAH) emissions were higher at the lower altitude. Mutagenicity of the catalytic stove emissions was higher than emissions from the conventional stove. Emissions from burning pine were more mutagenic than emissions from oak.

Integrated Disinfection By-Products Research: Salmonella Mutagenicity of Water Concentrates Disinfected by Chlorination and Ozonation/Postchlorination

Although chemical disinfection of drinking water is a highly protective public health practice, the disinfection process is known to produce toxic contaminants. Epidemiological studies associate chlorinated drinking water with quantitatively increased risks of rectal, kidney, and bladder cancer. One study found a significant exposure-response association between water mutagenicity and relative risk for bladder and kidney cancer. A number of studies found that several types of disinfection processes increase the level of mutagens detected by the Salmonella assay. As part of a comprehensive study to examine chlorinated and ozonated/postchlorinated drinking water for toxicological contaminants, the Salmonella mutagenicity assay was used to screen both volatile and nonvolatile organic components. The assay also compared the use of reverse osmosis and XAD resin procedures for concentrating the nonvolatile components. Companion papers provide the results from other toxicological assays and chemical analysis of the drinking water samples. The volatile components of the ozonated/postchlorinated and chlorinated water samples and a trihalomethane mixture were mutagenic to a Salmonella tester strain transfected with a rat theta-class glutathione S-transferase and predominantly nonmutagenic in the control strain. In this study, the nonvolatile XAD concentrate of the untreated water possessed a low level of mutagenic activity. However, compared to the levels of mutagenicity in the finished water XAD concentrates, the contribution from the settled source water was minimal. The mutagenicity seen in the reverse osmosis concentrates was < 50% of that seen in the XAD concentrates. Overall, mutagenic responses were similar to those observed in other North American studies and provide evidence that the pilot plant produced disinfection by-products similar to that seen in other studies.

A comparative assessment of Boise, Idaho, ambient air fine particle samples using the plate and microsuspension Salmonella mutagenicity assays

The primary objective of this study is to characterize the genotoxic potential of the ambient air aerosols collected within an air shed impacted primarily by wood smoke and automotive emissions. The study also examines the relative merits of a microsuspension assay and the standard plate assay for monitoring the presence of airborne particle-bound mutagens. Wintertime ambient air particulate samples collected from Boise, Idaho, USA, were shown to contain extractable organic matter that is mutagenic in the Salmonella typhimurium microsuspension and plate-incorporation assays. Differences in the results from the primary sites, auxiliary sites and the background site demonstrate that the particle-bound mutagens are not evenly distributed within the air shed and are more associated with the location of sampling than with the time of sampling or the type of bioassay used to evaluate the samples. This study also demonstrates that the bioassay protocol used in such studies should depend upon the characteristics of the air sheds mutagens and the purpose of the study. For example, the microsuspension assay gave somewhat more variable results between samples but was approximately threefold more sensitive than the plate assay. When strain TA98 was used in the microsuspension assay, the mutagenic response was greater without an exogenous activation system. The reverse was true for the plate assay in which the use of an exogenous activation system increased the mutagenicity response. TA100 in the microsuspension assay provided results comparable to those with TA98. This is important because TA100 can also be used to bioassay semivolatile and volatile organics associated with ambient air mutagenicity. This, in turn, allows a comparison of the mutagenicity of organics collected by differing methods due to their volatility. Future studies should be directed toward correlation of mutagenicity results with other analytical results in order to further develop methods for better characterization of the genotoxicity of ambient air.

Biomarkers of Exposure, Effect, and Susceptibility in Workers Exposed to Nitrotoluenes

Nitrotoluenes, such as 2-nitrotoluene, 2,4-dinitrotoluene (24DNT), and 26DNT, are carcinogenic in animal experiments. Humans are exposed to such chemicals in the workplace and in the environment. It is therefore important to develop methods to biomonitor people exposed to nitrotoluenes to prevent the potential harmful effects. For the present study, workers exposed to high levels of these chemicals were investigated. The external dose (air levels), the internal dose (urine metabolites), the biologically effective dose [hemoglobin (Hb) adducts and urine mutagenicity], and biological effects (chromosomal aberrations and health effects) were determined. Individual susceptibility was assessed by determining genetic polymorphisms of enzymes assumed to function in nitrotoluene metabolism, namely glutathione S-transferases (GSTM1, GSTT1, GSTP1), N-acetyltransferases (NAT1, NAT2), and sulfotransferases (SULT1A1, SULT1A2). The levels of urinary metabolites did not correlate with the air levels. The urinary mutagenicity levels determined in a subset of workers correlated with the levels of a benzylalcohol metabolite of DNT. The Hb-adducts correlated with the urine metabolites but not with the air levels. The frequency of chromosomal aberrations (gaps included) was increased (P < 0.05) in the exposed workers in comparison with a group of factory controls and correlated with the level of 24DNT Hb-adducts in young subjects (<31 years). The GSTM1-null genotype was significantly more prevalent in the controls than in the exposed group, which probably reflected an elevated susceptibility of the GSTM1-null genotype to adverse health effects of DNT exposure, such as nausea (odds ratio, 8.8; 95% confidence interval, 2.4-32.2). A statistically significant effect was seen for SULT1A2 genotype on a 24DNT Hb-adduct; GSTP1 genotype on a 2,4,6-trinitrotoluene Hb-adduct; and SULT1A1, SULT1A2, NAT1, GSTT1, and GSTP1 genotypes on chromosomal aberrations in the exposed workers. (Cancer Epidemiol Biomarkers Prev 2006;15(3):559–66)

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene

Abstract 2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.

Bioassay-directed fractionation and sub-fractionation for mutagenicity and chemical analysis of diesel exhaust particles.

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. Environ. Mol. Mutagen. 54:719–736, 2013.

Oral treatment of Fischer 344 rats with weathered crude oil and a dispersant influences intestinal metabolism and microbiota.

When oil is spilled into aquatic systems, chemical dispersants frequently are applied to enhance emulsification and biological availability. In this study, a mammalian model system was used to determine the effect of Bonnie Light Nigerian crude oil, weathered for 2 d with continuous spraying and recirculation, and a widely used dispersant, Corexit (Cx) 9527, on intestinal microbial metabolism and associated populations. To determine the subchronic dose, concentrated or diluted (1:2, 1:5, 1:10, 1:20) Cx9527 or oil was administered by gavage to Fischer 344 rats and the effect on body weight was determined. Next, rats were treated for 5 wk with oil, dispersant, or dispersant + oil. Body and tissue weights, urine mutagenicity, and the impact on the intestinal microflora and three microbial intestinal enzymes linked to bioactivation were determined in the small and large intestines and cecum. Two tested dispersants, Cx9527 and Cx9500, were toxic in vitro (1:1,000 dilution), and oil was not mutagenic in strains TA98 and TA100(+/-S9). None of the treated rats produced urine mutagens detected by TA98 or TA100. Undiluted dispersant was lethal to rats, and weight changes were observed depending on the dilution, whereas oil generally was not toxic. In the 5-wk study, body and tissue weights were unaffected at the doses administered. Small-intestinal levels of azoreductase (AR), beta-glucuronidase (BG), and nitroreductase (NR) were considerably lower than cecal and large-intestinal activities at the same time point. A temporal increase in AR activity was observed in control animals in the 3 tissues examined, and large-intestinal BG activity was elevated in 3-wk controls. No significant changes in cecal BG activity were observed. Oil- or dispersant-treated rats had mixed results with reduced activity at 3 wk and elevated activity at 5 wk compared to controls. However, when the dispersant was combined with oil at 3 wk, a reduction in activity was observed that was similar to that of dispersant alone. One-week nitroreductase activity in the small intestine and cecum was unaffected in the three treatment groups, but elevated activity was observed in the large intestines of animals treated with oil or dispersant. The effect of the combination dose was not significantly different from the control value. Due to experimental error, no 3- or 5-wk NR data were available. By 5 wk of treatment, enterobacteria and enterococci were eliminated from ceca of oil-treated rats. When oil was administered in combination with dispersant, an apparent protective effect was observed on the enterococci and lactose-fermenting and nonfermenting enterobacteria. A more detailed analysis at the species level revealed qualitative differences dependent on the treatment. This study suggests that prolonged exposure of mammals to oil, dispersant, or in combination impacts intestinal metabolism, which ultimately could lead to altered detoxification of oil constituents and coexposed toxicants.

Selection of a suitable extraction method for mutagenic activity from woodsmoke-impacted air particles

Abstract Extraction methods were evaluated for recovery of mutagenic activity from woodsmoke-impacted air particles. Soxhlet and sonication techniques were utilized with a variety of solvents to ascertain the effect of solvent choice, extraction methods, or dissolved gases in extraction solvents on the recovery of mutagenicity. Sonication extraction gave slightly less mass recovery than the Soxhlet method. Methanol extracted more mass than the other solvents with dichloromethane recovering the least. Dissolved gases were not found to have any effect, while mutagenicity was shown to be dependent upon solvent and extraction method. Soxhlet extraction with acetone and toluene/ethanol yielded the highest recovery of mutagenic activity, however, results indicated a solvent/solute interaction which chemically altered one or

Mutagenicity in Salmonella of hazardous wastes and urine from rats fed these wastes

15 hazardous industrial waste samples were evaluated for mutagenicity in the Salmonella plate-incorporation assay using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat liver S9. Dichloromethane/methanol extracts of the crude wastes were also evaluated. 7 of the crude wastes were mutagenic, but only 2 of the extracts of these 7 wastes were mutagenic; extracts of 2 additional wastes also were mutagenic. In addition, 10 of the crude wastes were administered by gavage to F-344 rats, and 24-h urine samples were collected. Of the 10 raw urines evaluated, 3 were mutagenic in strain TA98 in the presence of S9 and beta-glucuronidase. The 3 crude wastes that produced these 3 mutagenic urines were, themselves, mutagenic. Adequate volumes of 6 of the 10 raw urines were available for extraction/concentration. These 6 urines were incubated with beta-glucuronidase and eluted through Sep-Pak C18 columns; the methanol eluates of 3 of the urines were mutagenic, and these were the same 3 whose raw urines also were mutagenic. In general, the C18/methanol extraction procedure reduced the cytotoxicity and increased the mutagenic potency of the urines. To our knowledge, this is the first report of the mutagenicity of urine from rodents exposed to hazardous wastes. Based on the present results, the use of only strain TA98 in the presence of S9 might be adequate for general screening of hazardous wastes or waste extracts for genotoxicity. The urinary mutagenesis assay does not appear to be a useful adjunct to the Salmonella assay for screening hazardous wastes. The problems associated with chemically fractionating diverse types of hazardous wastes for bioassay are also discussed.