Advanced Collaborative Emissions Study Auxiliary Findings on 2007-Compliant Diesel Engines: A Comparison With Diesel Exhaust Genotoxicity Effects Prior to 2007.

Since its beginning, more than 117 years ago, the compression-ignition engine, or diesel engine, has grown to become a critically important part of industry and transportation. Public concerns over the health effects from diesel emissions have driven the growth of regulatory development, implementation, and technological advances in emission controls. In 2001, the United States Environmental Protection Agency and California Air Resources Board issued new diesel fuel and emission standards for heavy-duty engines. To meet these stringent standards, manufacturers used new emission after-treatment technology, and modified fuel formulations, to bring about reductions in particulate matter and nitrogen oxides within the exhaust. To illustrate the impact of that technological transition, a brief overview of pre-2007 diesel engine exhaust biomarkers of genotoxicity and health-related concerns is provided, to set the context for the results of our research findings, as part of the Advanced Collaborative Emissions Study (ACES), in which the effects of a 2007-compliant diesel engine were examined. In agreement with ACES findings reported in other tissues, we observed a lack of measurable 2007-compliant diesel treatment-associated DNA damage, in lung tissue (comet assay), blood serum (8-hydroxy-2'-deoxyguanosine [8-OHdG] assay), and hippocampus (lipid peroxidation assay), across diesel exhaust exposure levels. A time-dependent assessment of 8-OHdG and lipid peroxidation also suggested no differences in responses across diesel exhaust exposure levels more than 24 months of exposure. These results indicated that the 2007-compliant diesel engine reduced measurable reactive oxygen species-associated tissue derangements and suggested that the 2007 standards-based mitigation approaches were effective.

Part 3. Assessment of genotoxicity and oxidative damage in rats after chronic exposure to new-technology diesel exhaust in the ACES bioassay.

In 2001, the U.S. Environmental Protection Agency (EPA*) and the California Air Resources Board (CARB) adopted new standards for diesel fuel and emissions from heavy-duty diesel engines. By 2007, diesel engines were required to meet these new standards for particulate matter (PM), with other standards to follow. Through a combination of advanced compression-ignition engine technology, development of exhaust aftertreatment systems, and reformulated fuels, stringent standards were introduced. Before the 2007 standards were put in place by the EPA, human health effects linked to diesel exhaust (DE) exposure had been associated with diesel-fuel solvent and combustion components. In earlier research, diesel engine exhaust components were, in turn, linked to increased mutagenicity in cultures of Salmonella typhimurium and mammalian cells (Tokiwa and Ohnishi 1986). In addition, DE was shown to increase both the incidence of tumors and the induction of 8-hydroxy-deoxyguanosine (8-OHdG) adducts in rodents (Ichinose et al. 1997) and total DNA adducts in rats (Bond et al. 1990). Furthermore, DE is composed of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) and particulates. One such PAH, 3-nitrobenzanthrone (3-NBA), is also found in urban air. 3-NBA has been observed to induce micronucleus formation in the DNA of human hepatoma cells (Lamy et al. 2004). The current study is part of the Advanced Collaborative Emissions Study (ACES), a multidisciplinary program carried out by the Health Effects Institute and the Coordinating Research Council. Its purpose was to determine whether recent improvements in the engineering of heavy-duty diesel engines reduce the toxicity associated with exposure to DE components. To this end, we evaluated potential genotoxicity and induction of oxidative stress in bioassays of serum and tissues from Wistar Han rats chronically exposed--for up to 24 months--to DE from a 2007-compliant diesel engine (new-technology diesel exhaust, or NTDE). Genotoxicity was measured as DNA strand breaks in lung tissue, using an alkaline-modified comet assay. As a correlate of possible DNA damage evaluated in the comet assay, concentrations of the free DNA adduct 8-OHdG were evaluated in serum by a competitive enzyme-linked immunosorbent assay (ELISA). The 8-OHdG fragment found in the serum is a specific biomarker for the repair of oxidative DNA damage. In addition, an assay for thiobarbituric acid reactive substances (TBARS) was used to assess oxidative stress and damage in the form of lipid peroxidation in the hippocampus region of the brains of the DE-exposed animals. These endpoints were evaluated at 1, 3, 12, and 24 months of exposure to DE or to a control atmosphere (filtered air). At the concentrations of DE evaluated, there were no significant effects of exposure in male or female rats after 1, 3, 12, or 24 months in any measure of DNA damage in the comet assay (%DNA in tail, tail length, tail moment, or olive moment). The comparison of exposure groups versus control and the comparison of groups by sex for 1 and 3 months of exposure showed no significant differences in serum 8-OHdG concentrations (P > 0.05). The concentrations of 8-OHdG in all exposure groups at 3 months were higher than those in exposure groups at any other time point (P < 0.05). Looking at the levels of 8-OHdG in serum in the 12-month and 24-month groups, we saw a significant difference from control in the 12-month group at the mid and high levels (P < 0.05), as well as some other scattered changes. Sex differences were noted in the 12-month high-level group (P < 0.05). However, these differences did not follow an exposure-dependent pattern. All other comparisons were not significant (P > 0.05). Hippocampal concentrations of TBARs, measured as malondialdehyde (MDA), showed some small and scattered changes in groups exposed to different levels of DE and at different time points, but we did not consider these to be exposure-related. We concluded that exposure to DE in these rats did not produce any significant increase in oxidative damage to lipids or damage to DNA in the form of strand breaks.

Evaluation of frequencies of HPRT mutant lymphocytes in butadiene polymer workers in a Southeast Texas facility.

We examined the frequency of mutant lymphocytes (VFs) in workers (n = 30) occupationally exposed to the petrochemical, 1,3-butadiene (BD), using the autoradiographic HPRT mutant lymphocyte assay. Current exposures were determined with organic vapor monitors that had a 12-hr method detection limit (MDL) of 2.5 parts per billion (ppb). HPRT VFs were analyzed with respect to current exposure estimates, age in years, and occupational longevity (OL; defined as years working in the BD industry at this facility). Current exposures were low (mean 93.5 ppb, median 2.5 ppb) with only one individual's estimate (1683.5 ppb) exceeding the Occupational Safety and Health Administration's permissible exposure limit of 1,000 ppb. The majority (>50%) of current exposures were below the MDL. HPRT VFs were not significantly associated with current exposures (n = 29), and they were not significantly associated with age (n = 29). HPRT VFs were, however, significantly associated with OL (n = 29, R(2) = 0.107, P < 0.046). This result suggests that chronic and/or past, high-level exposures might leave a mutagenic signature that is revealed by the HPRT assay, possibly through the retention of mutant, long-term memory T-cells. While it is encouraging that current occupational exposures to BD in this facility do not appear to be increasing the frequency of mutant T-lymphocytes, evidence from workers with a lengthy history in the industry (>or=30 years in this case) indicates that these individuals likely require additional biomonitoring for possible mutagenic effects resulting from chronic, past exposures.

Pharmacological agents for the prevention and treatment of toxic radiation exposure in spaceflight.

BACKGROUND: Astronauts are exposed to toxic ionizing radiation sources, including galactic cosmic radiation and solar particle events (SPE). Exposure to these radiation sources can lead to cataracts, heritable genetic mutations, cancer, acute life-threatening physiological compromise, and death. Current countermeasures focus on spacecraft shielding and creation of heavily shielded safe havens. At issue is the extraordinarily high cost of launching these heavy structures into space and their inability to provide adequate shielding from heavy ions at a feasible shield thickness. Pharmacological enhancement of cellular radiation resistance, an alternative method to limiting radiation toxicity, has received less attention. METHODS: We have conducted an extensive literature review and critical evaluation of the scientific data pertaining to this field of study. Publications for review were identified through a Medline search using relevant terms, including radiotherapeutics, galactic cosmic radiation, radiopharmacology, radioprotectants, radiation countermeasures, solar particles, solar flares, radiation toxicity, and radiotoxicity. RESULTS: We identified 15 agents with significant radiation dose reduction factors, ranging from 1.1 to 2.4, in experimental models. Of these, only amifostine is FDA approved for use in treating radiation toxicity. CONCLUSIONS: Current data do not support the use of radiopreventive agents in the treatment of low-level ionizing radiation exposures. However, pharmacological countermeasures should be instituted for life-threatening, high-level radiation exposures, as occur with SPE. Given the catastrophic effects of SPE, the risk of toxicity from radioprotective agents is warranted. The current data supports treatment with high-dose amifostine (at 910 mg m(-2)) 30 min prior to radiation exposure.

Detoxification of olefinic epoxides and nucleotide excision repair of epoxide-mediated DNA damage: Insights from animal models examining human sensitivity to 1,3-butadiene.

1,3-Butadiene (BD) is a well-documented mutagen and carcinogen in rodents and is currently classified as a probable carcinogen in humans. Studies investigating workers exposed to BD indicate that, in some plants, there may be an increased genetic risk, and that polymorphisms in biotransformation and DNA repair proteins may modulate genetic susceptibility. To investigate the role of genetic polymorphisms in microsomal epoxide hydrolase (mEH) or nucleotide excision repair (NER) in contributing to the mutagenicity of BD, we conducted a series of experiments in which mice lacking mEH or NER activity were exposed to BD by inhalation or to the reactive epoxide metabolites of BD (epoxybutene-EB or diepoxybutane-DEB) by i.p. injection. Genetic susceptibility was measured using the Hprt cloning assay. Both deficient strains of mouse were significantly more sensitive to the mutagenic effects of BD and the injected epoxides. These studies provide support for the critical role that mEH plays in the biotransformation of BD, and the role that NER plays in maintaining genomic integrity following exposure to BD. Additional studies are needed to examine the importance of base excision repair (BER) in maintaining genomic integrity, the differential formation of DNA and protein adducts in deficient strains, and the potential for enhanced sensitivity to BD genotoxicity in mice either lacking or deficient in both biotransformation and DNA repair activity.

Variability in human sensitivity to 1,3-butadiene: influence of polymorphisms in the 5'-flanking region of the microsomal epoxide hydrolase gene (EPHX1).

The carcinogenic effects of 1,3-butadiene (BD), a mutagenic chemical widely used in the manufacture of synthetic rubber, are likely initiated through its epoxide metabolites. In humans, these epoxides are detoxified predominantly by hydrolysis, a reaction mediated by the microsomal epoxide hydrolase (mEH; EPHX1) enzyme. It appears reasonable to hypothesize that BD-exposed individuals possessing lower mEH detoxification capacity may have elevated risk of adverse health effects. The interindividual levels of mEH enzymatic activity vary considerably, and polymorphisms in the mEH gene may contribute to this variability. In addition to the well-studied coding region polymorphisms encoding Tyr113His and His139Arg substitutions, seven other polymorphic sites in the 5'-flanking region of the mEH gene have been reported. These polymorphisms appear to differentially affect mEH gene transcriptional activities. The 5'-flanking region polymorphisms exist in two linkages, the -200 linkage (-200C/T, -259C/T, -290T/G) and the -600 linkage (-362A/G, -613T/C, -699T/C), whereas the -399T/C polymorphism exists as an independent site. Because these polymorphisms may affect total mEH enzymatic activity, we hypothesized that they influence the mutagenic response associated with occupational exposure to BD. We genotyped the 5'-region of the mEH gene in 49 non-smoking workers from two styrene-butadiene rubber facilities in southeast Texas and evaluated the linkage patterns against results obtained from an autoradiographic HPRT mutant lymphocyte assay, used as a biomarker of genotoxic effect. In the study population, 67% were exposed to low BD levels, <150 parts per billion, and 33% were exposed to >150 ppb. We used the observed HPRT mutant (variant) frequency (VF) in the studied population and a 4-way first-order interaction statistical model to estimate parameters that describe the influence of exposure, genotypes and the interaction between the two on the HPRT VF in the target population. The background (baseline) VF, defined as the VF (x 10(-6)) +/- S.E.M. at low levels of BD exposure (<150 ppb) where all the genotypes under study are homozygous wild-type, was estimated to be 4.02 +/- 1.32. Exposure to >150 ppb of BD alone resulted in an estimated increase in VF of 3.42 +/- 2.47 above the baseline level. Inheritance of the variant ATT allele in the -600 linkages resulted in an estimated increase in VF of 3.39 +/- 1.67 above the baseline level. When the interaction between BD exposure and the ATT allele in the -600 linkage group was considered, a statistically significant positive interaction was observed, with an estimated increase in the VF of 10.89 +/- 2.16 (95% CI = 6.56-15.20; p = 0.0027) above baseline. These new data confirm and extend our previous findings that sensitivity to the genotoxic effects of BD is inversely correlated with predicted mEH activity.

A model of sensitivity: 1,3-butadiene increases mutant frequencies and genomic damage in mice lacking a functional microsomal epoxide hydrolase gene.

The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO(2)]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO(2) (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P < 0.05) 12.4-fold increase in MF over controls and a significant 5.4-fold increase in MF over exposed WT mice. Additionally, KO mice exposed to BDO(2) exhibited a significant 4.5-fold increase in MF over controls and a significant 1.7-fold increase in MF over exposed WT mice. We also compared genomic damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO(2). KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5-12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO(2) exhibited significantly more DNA damage than all other groups (2.3-27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r(2) = 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO(2). This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD.

Biomarkers in Czech workers exposed to 1,3-butadiene: a transitional epidemiologic study.

A multiinstitutional, transitional epidemiologic study was conducted with a worker population in the Czech Republic to evaluate the utility of a continuum of non-disease biological responses as biomarkers of exposure to 1,3-butadiene (BD)* in an industrial setting. The study site included two BD facilities in the Czech Republic. Institutions that collaborated in the study were the University of Vermont (Burlington, Vermont, USA); the Laboratory of Genetic Ecotoxicology (Prague, the Czech Republic); Shell International Chemicals, BV (Amsterdam, The Netherlands); the University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, USA); University of Texas Medical Branch at Galveston (Galveston, Texas, USA); Leiden University (Leiden, The Netherlands); and the Health and Safety Laboratory (Sheffield, United Kingdom). Male volunteer workers (83) participated in the study: 24 were engaged in BD monomer production, 34 in polymerization activities, and 25 plant administrative workers served as unexposed control subjects. The BD concentrations experienced by each exposed worker were measured by personal monitor on approximately ten separate occasions for 8-hour workshifts over a 60-day exposure assessment period before biological samples were collected. Coexposures to styrene, benzene, and toluene were also measured. The administrative control workers were considered to be a homogeneous, unexposed group for whom a series of 28 random BD measurements were taken during the exposure assessment period. Questionnaires were administered in Czech to all participants. At the end of the exposure assessment period, blood and urine samples were collected at the plant; samples were. fractionated, cryopreserved, and kept frozen in Prague until they were shipped to the appropriate laboratories for specific biomarker analysis. The following biomarkers were analyzed: * polymorphisms in genes involved in BD metabolism (Prague and Burlington); * urinary concentrations of 1-hydroxy-2-(N-acetylcysteinyl)-3-butene and 2-hydroxy-1-(N-acetylcysteinyl)-3-butene (M2 [refers to an isomeric mixture of both forms]) (Amsterdam); * urinary concentrations of 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (M1) (Amsterdam); * concentrations of the hemoglobin (Hb) adducts N-(1-[hydroxymethyl]-2-propenyl)valine and N-(2-hydroxy-3-butenyl)valine (HBVal [refers to an isomeric mixture of both forms]) (Amsterdam); * concentrations of the Hb adduct N-(2,3,4-trihydroxybutyl)valine (THBVal) (Chapel Hill); * T cell mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene (autoradiographic assay in Galveston with slide review in Burlington; cloning assay in Leiden with mutational spectra determined in Burlington); and * chromosomal aberrations by the conventional method and by fluorescence in situ hybridization [FISH]), and cytogenetic changes (sister chromatid exchanges [SCEs] (Prague). All assay analysts were blinded to worker and sample identity and remained so until all work in that laboratory had been completed and reported. Assay results were sent to the Biometry Facility in Burlington for statistical analyses. Analysis of questionnaire data revealed that the three exposure groups were balanced with respect to age and years of residence in the district, but the control group had significantly more education than the other two groups and included fewer smokers. Group average BD exposures were 0.023 mg/m3 (0.010 ppm) for the control group, 0.642 mg/m3 (0.290 ppm) for the monomer group, and 1.794 mg/m3 (0.812 ppm) for the polymer group; exposure levels showed considerable variability between and within individuals. Styrene exposures were significantly higher in the polymer group than in the other two groups. We found no statistically significant differences in the distributions of metabolic genotypes over the three exposure groups; genotype frequencies were consistent with those previously reported for this ethnic and national population. Although some specific genotypes were associated with quantitative differences in urinary metabolite concentrations or Hb adduct dose-response characteristics, none indicated a heightened susceptibility to BD. Concentrations of both the M2 and M1 urinary metabolites and both the HBVal and THBVal Hb adducts were significantly correlated with group and individual mean BD exposure levels; the Hb adducts were more strongly correlated than the urinary metabolites. By contrast, no significant relations were observed between BD exposures and HPRT gene mutations (whether determined by the auto-radiographic or the cloning method) or any of the cytogenetic biomarkers (whether determined by the conventional method or FISH analysis). Neither the mutational nor the cytogenetic responses showed any association with genotypes. The molecular spectrum of HPRT mutations in BD-exposed workers showed a high frequency of deletions; but the same result was found in the unexposed control subjects, which suggests that these were not due to BD exposure. This lack of association between BD exposures and genetic effects persisted even when control subjects were excluded from the analyses or when we conducted regression analyses of individual workers exposed to different levels of BD.

Variability in human sensitivity to 1,3-butadiene: Influence of the allelic variants of the microsomal epoxide hydrolase gene.

The carcinogenic effects of 1,3-butadiene (BD), a chemical widely used in the rubber industry, are thought to be due to its epoxide metabolites. In humans, these epoxides are detoxified predominantly by hydrolysis, a reaction mediated by the microsomal epoxide hydrolase (mEH) enzyme. The mEH gene is polymorphic and the most common mEH coding-region variants detected in human populations are the two amino acid polymorphisms Tyr113His and His139Arg. Polymorphic amino acid substitutions at residues 113 and 139 in the human mEH protein can associate in four distinct combinations: Tyr113/His139, Tyr113/Arg139, His113/His139, and His113/Arg139. In vitro studies have shown that each of these genotypes has a unique mEH protein level that can affect net mEH enzymatic activity. In the current study, we examined the relationships among the genotypes involving these two polymorphisms and the mutagenic responses associated with occupational exposure to BD. We studied 49 nonsmoking workers from two styrene-butadiene rubber facilities in southeast Texas using the autoradiographic HPRT mutant lymphocyte assay as a biomarker of genotoxic effect. We genotyped the study participants simultaneously for both polymorphisms, using a multiplex PCR assay developed in our laboratory, and the subjects were assigned to a specific group based on the predicted mEH activity associated with their genotypes (low, intermediate, and high). In the study population, 67% were exposed to low BD levels of <150 ppb (measured by personal badge dosimeters) and 33% were exposed to >150 ppb (mean 2,244 ppb). In the BD low-exposure group, the mEH genotypes had no significant effect on the HPRT variant (mutant) frequency (Vf). In the high-exposure group (BD > 150 ppb), individuals with genotypes associated with low mEH activity had a significant (P < 0.05) 3-fold increase in HPRT Vf (Vf +/- SEM = 13.95 +/- 2.15 x 10(-6)) compared to high-activity individuals (4.41 +/- 1.19 x 10(-6)), and a 2-fold increase in Vf compared to intermediate-activity individuals (6.44 +/- 2.09 x 10(-6)). Our results indicate that mEH genotypes may play a significant role in human sensitivity to the genotoxic effects of exposure to BD.