Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

A new LC-MS/MS method for the quantification of endogenous and vinyl chloride-induced 7-(2-Oxoethyl)guanine in sprague-dawley rats.

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [(13)C(2)]-7-OEG from the reaction of calf thymus DNA with [(13)C(18)]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10(6) guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days was 104.0 ± 23.0 adducts per 10(6) guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10(6) guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [(13)C(2)]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10(6) guanine in liver. Studies on the persistence of [(13)C(2)]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [(13)C(2)]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.

Formation of hydroxymethyl DNA adducts in rats orally exposed to stable isotope labeled methanol.

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol's well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [¹³CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [¹³CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.

1,3-Butadiene: Biomarkers and application to risk assessment.

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ∼200 times more DEB than humans at exposures of 0.1-1.5ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2-3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ∼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.

Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.

There is a strong need for science-based risk assessment that utilizes known data from diverse sources to arrive at accurate assessments of human health risk. Such assessments will protect the public health without mandating unreasonable regulation. This paper utilizes 30 years of research on three "known human carcinogens": formaldehyde, vinyl chloride (VC), and ethylene oxide (EO), each of which forms DNA adducts identical to endogenous DNA adducts in all individuals. It outlines quantitative data on endogenous adducts, mutagenicity, and relationships between endogenous and exogenous adducts. Formaldehyde has the richest data set, with quantitative data on endogenous and exogenous DNA adducts from the same samples. The review elaborates on how such data can be used to inform the current risk assessment on formaldehyde, including both the biological plausibility and accuracy of projected risks. Finally, it extends the thought process to VC, EO, and additional areas of potential research, pointing out needs, nuances, and potential paths forward to improved understanding that will lead to strong science-based risk assessment.

Development and application of an LC-MS/MS method for the detection of the vinyl chloride-induced DNA adduct N(2),3-ethenoguanine in tissues of adult and weanling rats following exposure to [(13)C(2)]-VC.

In the 1970s, exposure to vinyl chloride (VC) was shown to cause liver angiosarcoma in VC workers. We have developed a new LC-MS/MS method for analyzing the promutagenic DNA adduct N(2),3-ethenoguanine (εG) and have applied this to DNA from tissues of both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days or 1100 ppm VC for 1 day. This assay utilizes neutral thermal hydrolysis and an HPLC cleanup prior to quantitation by LC-MS/MS. The number of endogenous and exogenous εG adducts in DNA from tissues of adult rats exposed to [(13)C(2)]-VC for 5 days was 4.1 ± 2.8 adducts/10(8) guanine of endogenous and 19.0 ± 4.9 adducts/10(8) guanine of exogenous εG in the liver, 8.4 ± 2.8 adducts/10(8) guanine of endogenous and 7.4 ± 0.5 adducts/10(8) guanine of exogenous εG in the lung, and 5.9 ± 3.3 adducts/10(8) guanine of endogenous and 5.7 ± 2.1 adducts/10(8) guanine of exogenous εG in the kidney (n = 4). Additionally, the data from weanling rats demonstrated higher numbers of exogenous εG, with ∼4-fold higher amounts in the liver DNA of weanlings (75.9 ± 17.9 adducts/10(8) guanine) in comparison to adult rats and ∼2-fold higher amounts in the lung (15.8 ± 3.6 adducts/10(8) guanine) and kidney (12.9 ± 0.4 adducts/10(8) guanine) (n = 8). The use of stable isotope labeled VC permitted accurate estimates of the half-life of εG for the first time by comparing [(13)C(2)]-εG in adult rats with identically exposed animals euthanized 2, 4, or 8 weeks later. The half-life of εG was found to be 150 days in the liver and lung and 75 days in the kidney, suggesting little or no active repair of this promutagenic adduct.

Genotoxicity of 1,3-butadiene and its epoxy intermediates.

Current risk assessments of 1,3-butadiene (BD*) are complicated by limited evidence of its carcinogenicity in humans. Hence, there is a critical need to identify early events and factors that account for the heightened sensitivity of mice to BD-induced carcinogenesis and to deter-mine which animal model, mouse or rat, is the more useful surrogate of potency for predicting health effects in BD-exposed humans. HEI sponsored an earlier investigation of mutagenic responses in mice and rats exposed to BD, or to the racemic mixture of 1,2-epoxy-3-butene (BDO) or of 1,2,3,4-diepoxybutane (BDO2; Walker and Meng 2000). In that study, our research team demonstrated (1) that the frequency of mutations in the hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene of splenic T cells from BD-exposed mice and rats could be correlated with the species-related differences in cancer susceptibility; (2) that mutagenic-potency and mutagenic-specificity data from mice and rats exposed to BD or its individual epoxy intermediates could provide useful information about the BD metabolites responsible for mutations in each species; and (3) that our novel approach to measuring the mutagenic potency of a given chemical exposure as the change in Hprt mutant frequencies (Mfs) over time was valuable for estimating species-specific differences in mutagenic responses to BD exposure and for predicting the effect of BD metabolites in each species. To gain additional mode-of-action information that can be used to inform studies of human responses to BD exposure, experiments in the current investigation tested a new set of five hypotheses about species-specific patterns in the mutagenic effects in rodents of exposure to BD and BD metabolites: 1. Repeated BD exposures at low levels that approach the occupational exposure limit for BD workers (set by the U.S. Occupational Safety and Health Administration) are mutagenic in female mice. 2. The differences in mutagenic responses of the Hprt gene to BD in similarly exposed rodents of a given species (reported in various earlier studies) are primarily associated with age-related thymus activity and trafficking of T cells and with sex-related differences in BD metabolism. 3. The mutagenic potency of the stereochemical forms of BD's epoxy intermediates plays a significant role in the species-related mutagenicity of BD. 4. The hydrolysis-detoxification pathway of BD through 1,2-dihydroxy-3-butene (BD-diol) is a major contributor to mutagenicity at high-level BD exposures in mice and rats. 5. Significant and informative species-specific differences in mutation spectra can be identified by examining both large- and small-scale genetic alterations in the Hprt gene of BD-exposed mice and rats. The first four hypotheses were tested by exposing mice and rats to BD, meso-BDO2, or BD-diol and measuring Hprt Mfs as the primary biomarker. For this, we used the T-cell-cloning assay of lymphocytes isolated from the spleens of exposed and control (sham-exposed) mice and rats. The first hypothesis was tested by exposing female B6C3F1 mice (4 to 5 weeks of age) by inhalation for 2 weeks (6 hours/day, 5 days/week) to 0 or 3 ppm BD. Hprt Mfs were measured at the time of peak mutagenic response after exposure for this age of mice. We then compared the resulting data to those from mutagenicity studies with mice of the same age that had been exposed in a similar protocol to higher levels of BD (Walker and Meng 2000). In mice exposed to 3 ppm BD (n = 27), there was a significant 1.6-fold increase over the mean background Hprt Mf in control animals (n = 24, P = 0.004). Calculating the efficiency of Hprt mutant induction, by dividing induced Hprt Mfs by the respective BD exposure levels, demonstrated that the mutagenic potency of 3 ppm BD was twice that of 20 ppm BD and almost 20 times that of 625 or 1250 ppm BD in exposed female mice. Sample-size calculations based on the Hprt Mf data from this experiment demonstrated the feasibility of conducting a future experiment to find out whether induced Mfs at even lower exposure levels (between 0.1 and 1.0 ppm BD) fit the supralinear exposure-response curve found with exposures between 3.0 and 62.5 ppm BD, or whether they deviate from the curve as Mf values approach the background levels found in control animals. The second hypothesis was tested by estimating mutagenic potency for female mice exposed by inhalation for 2 weeks to 0 or 1250 ppm BD at 8 weeks of age and comparing this estimate to that reported for female mice exposed to BD in a similar protocol at 4 to 5 weeks of age (Walker and Meng 2000). For these two age groups, the shapes of the mutant splenic T-cell manifestation curves were different, but the mutagenic burden was statistically the same. These results support our contention that the disparity in responses reported in earlier Hprt-mutation studies of BD-exposed rodents is related more to age-related T-cell kinetics than to age-specific differences in the metabolism of BD. The third hypothesis was tested by estimating mutagenic potency for female mice and rats (4 to 5 weeks of age) exposed by inhalation to 2 or 4 ppm meso-BDO2 and comparing these estimates to those previously obtained for female mice and rats of the same age and exposed in a similar protocol to (+/-)-BDO2 (Meng et al. 1999b; Walker and Meng 2000). These exposures to stereospecific forms of BDO2 caused equivalent mutagenic effects in each species. This suggests that the small differences in the mutagenic potency of the individual stereoisomers of BDO2 appear to be of less consequence in characterizing the sources of BD-induced mutagenicity than the much larger differences between the mutagenic potencies of BDO2 and the other two BD epoxides (BDO and 1,2-dihydroxy-3,4-epoxybutane [BDO-diol]). The fourth hypothesis was tested in several experiments. First, female and male mice and rats (4 to 5 weeks of age) were exposed by nose only for 6 hours to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure levels that would yield similar plasma concentrations of BD-diol. Second, animals were exposed in inhalation chambers for 4 weeks to 0, 6, 18, or 36 ppm BD-diol to determine the mutagenic potency estimates for these exposure levels and to compare these estimates with those reported for BD-exposed female mice and rats (Walker and Meng 2000) in which similar blood levels of BD-diol had been achieved. Measurements of plasma concentrations of BD-diol (via a gas chromatography and mass spectrometry [GC/MS] method developed for this purpose) showed these results: First, BD-diol accumulated in a sublinear manner during a single 6-hour exposure to more than 200 ppm BD. Second, BD-diol accumulated in a linear manner during single (6-hour) or repeated (4-week) exposure to 6 or 18 ppm BD and in a sublinear manner with increasing levels of BD-diol exposure. Third, exposure of female mice and rats to 18 ppm BD-diol produced plasma concentrations equivalent to those produced by exposure to 200 ppm BD (exposure to 36 ppm BD-diol produced plasma concentrations of about 25% of those produced by exposure to 625 ppm BD). In general, 4-week exposure to 18 or 36 ppm BD-diol was significantly mutagenic in female and male mice and rats. The differences in mutagenic responses between the species and sexes were not remarkable, except that the mutagenic effects were greatest in female mice. The substantial differences in the exposure-related accumulation of BD-diol in plasma after rodents were exposed to more than 200 ppm BD compared with the relatively small differences in the mutagenic responses to direct exposures to 6, 18, or 36 ppm BD-diol in female mice provided evidence that the contribution of BD-diol-derived metabolites to the overall mutagenicity of BD has a narrow range of effect that is confined to relatively high-level BD exposures in mice and rats. This conclusion was supported by the results of parallel analyses of adducts in mice and rats concurrently exposed to BD-diol (Powley et al. 2005b), which showed that the exposure-response curves for the formation of N-(2,3,4-trihydroxybutyl)valine (THB-Val) in hemoglobin, formation of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) in DNA, and induction of Hprt mutations in exposed rodents were remarkably similar in shape (i.e., supralinear). Combined, these data suggest that trihydroxybutyl (THB) adducts are good quantitative indicators of BD-induced mutagenicity and that BD-diol-derived BDO-diol (the major source of the adducts) might be largely responsible for mutagenicity in rodents exposed to BD-diol or to hight levels of BD. The mutagenic-potency studies of meso-BDO2 and BD-diol reported here, combined with our earlier studies of BD, (+/-) BDO, and(+/-)-BDO2 (Walker and Meng 2000), revealed important trends in species-specific mutagenic responses that distinguish the relative degree to which the epoxy intermediates contribute to mutation induction in rodents at selected levels of BD exposures. These data as a whole suggest that , in mice, BDO2 largely causes mutations at exposures less than 62.5 ppm BD and that BD-diol-derived metabolites add to these mutagenic effects at higher BD exposures. In rats, it appears that the BD-diol pathway might account for nearly all the mutagenicity at the hight-level BD exposures where significant increases in Hprt Mfs are found and cancers are induced. Additional exposure-response studies of hemoglobin and DNA adducts specifics to BDO2, BDO-diol, and other reactive intermediates are needed to determine more definitively the relative contribution of each metabolite to the DNA alkylation and mutation patterns induced by BD exposure in mice and rats. For the fifth hypothesis, a multiplex polymerase chain reaction (PCR) procedure for the analysis of genomic DNA mutations in the Hprt gene of mice was developed. (ABSTRACT TRUNCATED)

WR1065 mitigates AZT-ddI-induced mutagenesis and inhibits viral replication.

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.

Toxicity, DNA binding, and cell proliferation in male F344 rats following short-term gavage exposures to trans-2-hexenal.

Hexenal is a genotoxic compound to which humans are exposed daily through the consumption of foods and beverages. The present studies were conducted to examine the relationships between the dose-responses of trans-2-hexenal-induced toxicity, DNA adduct formation, and cell proliferation. Male F344 rats were exposed by gavage to single doses of up to 500 mg/kg and killed 1, 2, or 4 days after dosing or were exposed to repeat doses of up to 100 mg/kg once daily for 5 days or 5 days per week for 4 weeks and killed 1 day after the end of the dosing period. Histologically, the primary observations were necroulcerative lesions, inflammation, and hyperplasia in the forestomach and inflammation in the glandular stomach. Hexenal-derived DNA adduct formation and cell proliferation were induced in the forestomach at doses of hexenal that also induced gastric toxicity; DNA adducts were not observed in the glandular stomach. These findings suggest that the toxicity of hexenal was limited to the site of contact (stomach) and that the observed DNA adduct formation and cell proliferation occurred in the setting of severe tissue damage.

The importance of 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone in 3-butene-1,2-diol associated mutagenicity.

1,2:3,4-Diepoxybutane is hypothesized to be the main intermediate involved in mutagenicity following exposure to low levels of 1,3-butadiene (BD) in mice, while metabolites of 3-butene-1,2-diol (BD-diol) are thought to become involved in both rats and mice at higher exposures. BD-diol is biotransformed to hydroxymethylvinyl ketone (HMVK), a potentially mutagenic metabolite, and 3,4-epoxy-1,2-butanediol (EB-diol), a known mutagen. To determine the relative importance of HMVK and EB-diol in BD-diol associated mutagenesis, we have examined the dosimetry of a HMVK derived DNA adduct, as well as EB-diol derived DNA and hemoglobin adducts, in rodents exposed to BD-diol. We previously demonstrated similarities in the shapes of the dose-response curves for EB-diol derived DNA adducts, hemoglobin adducts, and Hprt mutant frequencies in BD-diol exposed rodents, indicating that EB-diol was involved in the mutagenic response associated with BD-diol exposure. To examine the role of HMVK in BD-diol mutagenicity, a method to quantify the alpha-regioisomer of HMVK derived 1,N(2)-propanodeoxyguanosine (alpha-HMVK-dGuo) was developed. The method involved enzymatic hydrolysis of DNA, HPLC purification, and adduct measurement by liquid chromatography - tandem mass spectrometry. Intra- and inter-experimental variabilities were determined to be 2.3-18.2 and 4.1%, respectively. The limit of detection was approximately 5 fmol of analyte standard injected onto the column or 5 fmol/200 microg DNA. The method was used to analyze liver DNA from control female F344 rats and female F344 rats exposed to 36 ppm BD-diol. In addition, liver samples from female Sprague-Dawley rats exposed to 1000 ppm BD were analyzed. alpha-HMVK-dGuo was not detected in any of the samples analyzed. Several possible explanations exist for the negative results including the possibility that alpha-HMVK-dGuo may be a minor adduct or may be efficiently repaired. Alternatively, HMVK itself may be readily detoxified by glutathione (GSH) conjugation. While experiments must be conducted to understand the exact mechanism(s), these results, in addition to published EB-diol derived adduct dosimetry and existing HMVK derived mercapturic acid data, suggest that EB-diol is primarily responsible for BD-diol induced mutagenicity in rodents.

Evaluation of effects from repeated inhalation exposure of F344 rats to high concentrations of propylene.

Chronic exposure to propylene does not result in any increased incidence of tumors, yet does increase N7-hydroxypropylguanine (N7-HPGua) adducts in tissue DNA. To investigate any potential for genotoxicity (mutagenicity or clastogenicity), male F344 rats were exposed via inhalation to up to 10,000 ppm propylene for 1, 3, or 20 days (6 h/day, 5 days/week). The endpoints examined included gene (Hprt, splenocytes) and chromosomal (bone marrow micronucleus [MN]) mutations, hemoglobin (hydroxypropylvaline, HPVal) adducts in systemic blood, and DNA adducts (N7-HPGua) in several tissues. Similarly exposed female and male F344 rats, implanted with bromodeoxyuridine (BrdU) minipumps, were evaluated for nasal effects (irritation via histopathology and cell proliferation via BrdU). Internal dose measures provided clear evidence for propylene exposure, with HPVal increased for all exposures; N7-HPGua was increased in all tissues from rats exposed for more than 1 day (except lymphocytes). Saturation of propylene conversion to propylene oxide was apparent from the adduct dose-response curves. There were no biologically significant genotoxic effects demonstrated at any exposure level, with no increase in Hprt mutant frequency or in bone marrow MN formation. In addition, no histopathological changes were noted in rodent nasal tissues nor any induction of cell proliferation in nasal tissues. These results demonstrate that repeated exposure of rats to high concentrations of propylene (< or = 10,000 ppm) does not produce evidence of local nasal cavity toxicity or evidence of systemic genotoxicity to hematopoietic tissue, despite the formation of N7-HPGua adducts. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic.

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene.

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene.

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.

N-terminal globin adducts as biomarkers for formation of butadiene derived epoxides.

The aim of this review is to summarize our recent data on butadiene (BD) derived hemoglobin adducts as biomarkers for the internal formation of the individual epoxides formed by butadiene (BD). It is well known that BD is oxidized by cytochrome P450s to several epoxides that form DNA and protein adducts. 1,2-Epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol) form N-(2-hydroxy-3-butenyl)-valine (HB-Val), N,N-(2,3-dihydroxy-1,4-butadiyl)-valine (pyr-Val) and N-(2,3,4-trihydroxybutyl)-valine (THB-Val) adducts, respectively. The analysis of HB-Val and THB-Val by the modified Edman degradation and GC-MS/MS has generated valuable insights into BD metabolism across species. In addition, a recently established method for the analysis of pyr-Val has been proven to be suitable for detection of pyr-Val in rodents exposed to BD as low as 1 ppm. These technologies have been applied to study a wide range of exposures to BD, EB, DEB, and 3-butene-1,2-diol as a precursor of EB-diol in male and female mice and rats. Altogether the data have shown that BD metabolism is species and concentration dependent, consistent with metabolism and carcinogenesis data. Mice form much more HB-Val and pyr-Val than rats, especially at low exposures. After 10 days of inhalation exposure to 3 ppm BD, mice formed 12.5-fold more pyr-Val than rats. In contrast, the amounts of THB-Val were similar in mice and rats exposed to 3 or 62.5 ppm BD. Furthermore, it appears that the formation of THB-Val is supralinear in mice and rats due to saturation of metabolic activation pathways. Gender differences in metabolism are less well established. One study with male and female rats exposed to 1000 ppm BD for 90 days demonstrated a 1.6-, 3.5- and 2.0-fold gender difference in formation of HB-Val, pyr-Val and THB-Val, respectively, with females being more efficient in epoxide formation. The analyses of BD derived protein adducts correlate well with the observed species and gender differences in BD-carcinogenesis and suggest that DEB may indeed be the most important metabolite.

Pyrimido[1,2-a]-purin-10(3H)-one, M1G, is less prone to artifact than base oxidation.

Pyrimido[1,2-a]-purin-10(3H)-one (M1G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M1G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M1G adducts measured in either control or positive control tissue samples. The number of M1G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M1G/10(8) nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M1G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M1G is a reliable way to assess oxidative DNA damage because the number of M1G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M1G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports.

Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.

Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.

Analysis of diepoxide-specific cyclic N-terminal globin adducts in mice and rats after inhalation exposure to 1,3-butadiene.

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane-specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)-valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers.

Molecular dosimetry and repair of N(2),3-ethenoguanine in rats exposed to vinyl chloride.

Although the DNA adducts of vinyl chloride (VC) have been well characterized, previous studies have used single concentrations of VC that are well above contemporary human exposures. This study examined the exposure response to VC in male Sprague Dawley rats with respect to the molecular dose of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonG). Adult rats were exposed by inhalation to 0, 10, 100, or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week). Weanling rats were similarly exposed for 5 days. The amount of N(2),3-epsilonG in hepatocyte (HEP) and nonparenchymal cell (NPC) fractions obtained from the liver was measured with a sensitive immunoaffinity/gas chromatography/high-resolution mass spectrometry assay. Endogenous N(2),3-epsilonG was present in HEPs and NPCs from all unexposed rats. The exposure response to VC in each group and cell population was supralinear, with a linear increase from 0 to 100 ppm, and a plateau between 100 and 1100 ppm. There was no statistically significant difference in N(2),3-epsilonG concentrations between HEPs and NPCs in any adult exposure group, which suggests that factors other than adduct concentrations contribute to the particular susceptibility of NPCs to VC-induced carcinogenesis. The accumulation of N(2),3-epsilonG with respect to time was nearly linear in rats exposed to 600 ppm VC for 1, 2, 4, or 8 weeks (4 h/day, 5 days/week), and no repair of N(2),3-epsilonG was detected in rats exposed to VC for 4 weeks and allowed to recover for 1 week. N(2),3-epsilonG concentrations in HEPs from weanling rats were 2-3-fold greater than those in adult rats exposed for the same time. Higher adduct concentrations in young rats may contribute to their greater susceptibility to VC-induced hepatic angiosarcoma as well as their particular susceptibility to hepatocellular carcinoma. The molecular dosimetry of N(2),3-epsilonG in liver appears to be a sensitive and informative biomarker of genotoxic effect after exposure to VC. N(2),3-epsilonG was the predominant etheno adduct measured in vivo after exposure to VC, and the saturable nature of VC metabolism was reflected in its molecular dose. The relationships between endogenous N(2),3-epsilonG and that formed by low exposures to VC were demonstrated. Conclusions drawn from these exposures may be more relevant for risk assessment purposes than those drawn from high exposures where activation, detoxication, and repair pathways may be saturated or otherwise perturbed. These data are well suited for consideration in future risk assessments of VC that incorporate nontumor mode of action data.

Analysis of DNA adducts in rats exposed to pentachlorophenol.

Pentachlorophenol (PCP) is a widely used biocide that has been reported to be hepatocarcinogenic in mice. Its effects in rats are equivocal, but the liver clearly is not a target organ for carcinogenesis. The carcinogenic effects of PCP in mice may relate to reactive oxygen species generated during metabolism. PCP is known to increase the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine (ohdG), in the liver of exposed mice. To investigate whether the generation of oxidative DNA damage and direct DNA adducts may explain the species difference in carcinogenicity, we have analyzed ohdG in hepatic DNA from PCP-exposed rats. Rats were exposed acutely to PCP for 1 or 5 days. Tissues also were obtained from a 27 week interim sacrifice of the 2 year National Toxicology Program carcinogenesis bioassay. We used HPLC with electrochemical array detection for ohdG analysis. Single or 5 day exposure to PCP (up to 120 or 60 mg/kg/day, respectively) did not increase ohdG. Dietary exposure to 1000 p.p.m. PCP (equivalent to 60 mg/kg/day) for 27 weeks induced a 2-fold increase in ohdG (1.8 versus 0.91x10(-6) in controls). In parallel, formation of direct DNA adducts was analyzed by 32P-post-labeling following nuclease P1 adduct enrichment. We detected two major DNA adducts with relative adduct labeling of 0.78x10(7) adducts per total nucleotides. One of these adducts was found to co-migrate with the adduct induced by the metabolite, tetrachloro-1,4-benzoquinone. We observed differences in DNA adduct formation between acute and chronic studies, with acute studies not inducing any detectable amount of DNA adducts. These results indicated that chronic, but not acute exposure to PCP increased ohdG and direct adducts in hepatic DNA. As the same exposure conditions that enhanced ohdG did not produce liver cancer in rats, the generation of reactive oxygen species, oxidative DNA damage and direct DNA adducts is not sufficient for the induction of hepatocarcinogenesis by PCP in the rat.

Methods for measuring DNA adducts and abasic sites II: methods for measurement of DNA adducts.

This unit contains protocols for analyzing DNA adducts separated from the DNA backbone. HPLC is used to quantify total guanine or ribo- or deoxynucleotides as well as methods for analyzing specific adducts. These methods include HPLC with electrochemical detection, immunoaffininty chromatography to enrich for specific adducts, and gas and liquid chromatography in combination with HPLC and mass spectrometry.