In Vitro Mouse Lymphoma Cell (L5178Y Tk+/- -3.7.2.C) Forward Mutation Assay.

The in vitro mouse lymphoma cell assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk+/- -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions and multilocus, chromosomal rearrangements, mitotic recombination and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA; small and large colony mutants. The induction of small colony mutants is associated with chemicals inducing gross chromosomal aberrations, whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development Test Guideline 490 is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.

Dose-Response for Multiple Biomarkers of Exposure and Genotoxic Effect Following Repeated Treatment of Rats with the Alkylating Agents, MMS and MNU.

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

A novel approach for concurrent quantitation of glutathione, glutathione disulfide, and 2-hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide, using liquid chromatography-positive electrospray tandem mass spectrometry.

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.

LC-MS/MS simultaneous quantitation of 2-hydroxyethylated, oxidative, and unmodified DNA nucleosides in DNA isolated from tissues of mice after exposure to ethylene oxide.

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2'-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2'-deoxyadenosine (N1HEdA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), N2,3-etheno-2'-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2'-deoxyguanosine (dG), and 2'-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks' inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.

Characterization of nuclear receptor-mediated murine hepatocarcinogenesis of the herbicide pronamide and its human relevance.

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.

Concurrent evaluation of general, immune, and genetic toxicity endpoints as part of an integrated testing strategy.

Integrated testing strategies involve the assessment of multiple endpoints within a single toxicity study and represent an important approach for reducing animal use and streamlining testing. The present study evaluated the ability to combine general, immune, and genetic toxicity endpoints into a single study. Specifically, this study evaluated the impact of sheep red blood cell (SRBC) immunization, as part of the T-cell dependent antibody response (TDAR) assay, on organ weights, micronuclei (MN) formation (bone marrow and peripheral blood), and the Comet assay response in the liver of female F344/DuCrl rats treated with cyclophosphamide (CP) a known immunosuppressive chemical and genotoxicant. For the TDAR assay, treatment with CP resulted in a dose-dependent decrease in the antibody response with a suppression of greater than 95% at the high dose. Injection with SRBC had no impact on evaluated organ weights, histopathology, hematology, and clinical chemistry parameters. Analysis of MN formation in bone marrow and peripheral blood revealed a dose-dependent increase in response to CP treatment. Injection with SRBC had no impact on the level of MN in control animals and did not alter the dose response of CP. There was a slight increase in liver DNA damage in response to CP as measured by the Comet assay; however, injection with SRBCs did not alter this endpoint. Overall these data provide strong support for the concurrent assessment of general, immune, and genetic toxicology endpoints within a single study as part of an integrated testing strategy approach.

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system.

In vitro mouse lymphoma (L5178Y Tk⁺/⁻-3.7.2C) forward mutation assay.

The in vitro mouse lymphoma assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk (+/-) -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions (intragenic) and multilocus, chromosomal rearrangements, mitotic recombination, and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA: small- and large-colony mutants. The induction of small-colony mutants is associated with chemicals inducing gross chromosomal aberrations whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test.

The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay.

Measurement of deoxyinosine adduct: Can it be a reliable tool to assess oxidative or nitrosative DNA damage?

Adenosine deaminases (ADA) are key enzymes that deaminate adenosine (A) or deoxyadenosine (dA) and produce inosine or deoxyinosine (dI), respectively. While ADA only deaminates free dA, reactive nitrogen species (RNS) or reactive oxygen species (ROS) deaminate adenine base on the DNA and leave dI, which is a pre-mutagenic lesion. Therefore, dI adduct in the genomic DNA has been considered a biomarker of DNA damage caused by RNS or by ROS. In the presented study, genomic DNA was isolated from frozen calf thymus in low or room temperature, with or without an addition of antioxidant. The number of dI in the DNA was measured using liquid chromatography-tandem mass spectrometry. While low temperature (LT) work-up with an addition of antioxidant in reagents helped to prevent artifactual formation of oxidative DNA lesions in the calf thymus DNA (CTD), it also significantly inhibited activities of proteinase, which in turn resulted in significant ADA contamination in the final DNA samples. ADA remained in LT-CTD completely deaminated most dA when the DNA was subjected to enzymatic hydrolysis to single nucleosides. The ADA contamination in the DNA was significantly reduced when DNA was isolated from pre-isolated nuclear fraction rather than from entire tissue homogenates. However, enzymes used for DNA hydrolysis were confirmed to contain significant amounts of ADA. Therefore, these enzymes would increase deamination of dA during DNA hydrolysis. Artifactual dI production by contaminated ADA was dramatically reduced by an addition of EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), which is a potent inhibitor of ADA. However, time- and temperature-dependent dI production from dA in phosphate buffer solution was observed. More importantly, TEMPO, an antioxidant commonly used to prevent DNA oxidation, was found to deaminate dA independent to ADA. Overall, these findings indicate that assay methods measuring dI or other dA DNA adducts in genomic DNA should be carefully validated to minimize artificial errors caused by dA deamination. Recommendations to overcome those technical challenges were discussed in this presentation.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

Dose-response and operational thresholds/NOAELs for in vitro mutagenic effects from DNA-reactive mutagens, MMS and MNU.

The dose-response relationships for in vitro mutagenicity induced by methylmethanesulfonate (MMS) or methylnitrosourea (MNU) in L5178Y mouse lymphoma (ML) cells were examined. DNA adducts (N7-methylguanine, N7MeG and O(6)-methylguanine, O(6)MeG) were quantified as biomarkers of exposure. Both endpoints were assessed using 5replicates/dose (4-h treatment) with MMS or MNU (0.0069-50muM), or vehicle (1% DMSO). Mutant frequency (MF) (thymidine kinase (TK) locus) was determined using the soft agar cloning methodology and a 2-day expression period; in addition, microwell and Sequester-Express-Select (SES) methods were used for MMS. Isolated DNA was acid-hydrolyzed, and adducts quantified by LC/ESI-MS/MS, using authentic and internal standards. MF dose-responses were analyzed using several statistical approaches, all of which confirmed that a threshold dose-response model provided the best fit. NOAELs for MF were 10muM MMS and 0.69muM MNU, based on ANOVA and Dunnett's test (p<0.05). N7MeG adducts were present in all cell samples, including solvent-control cells, and were increased over control levels in cells treated with >/=10muM MMS or 3.45muM MNU. O(6)MeG levels were only quantifiable at >/=10muM MNU; O(6)MeG was not quantifiable in control or MMS-treated cells at current detection limits. Thus, (1) cells treated with

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.

Quantitation of methylated hemoglobin adducts in a signature peptide from rat blood by liquid chromatography/negative electrospray ionization tandem mass spectrometry.

Hemoglobin adducts are often used as biomarkers for exposure to reactive chemicals in toxicology studies. Therefore, fast, sensitive, accurate, and reproducible methods for quantifying these protein adducts are key to evaluate test material dosimetry. A methodology has been developed for the quantitation of methylated hemoglobin adducts isolated from rats exposed to the model alkylating agent: methyl methane sulfonate (MMS). After 4 days of MMS exposure by oral gavage, hemoglobin was isolated from rat blood and digested with trypsin. The tryptic digestion solution was used for the adducted hemoglobin signature peptide quantitation via liquid chromatography/negative tandem mass spectrometry (LC/ESI-MS/MS). The limit of quantitation (LOQ) for the methylated hemoglobin beta chain N-terminal signature peptide (MeVHLTDAEK) was 1.95 ng/mL (5.9 pmol/mg globin). The calibration curves were linear over a concentration range of 1.95 to 625 ng/mL, with a correlation coefficient R2 >0.998, accuracy of 85.8 to 119.3%, and precision of 0.9 to 19.4%.

Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry.

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.

Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive method has been developed for the direct quantitation of the methyl phosphotriester DNA adduct of thymidyl(3'-5')thymidine (dTp(Me)dT) from enzymatic DNA hydrolysates prepared from cultured cells treated with low levels of N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS), by rapid and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). The lower limit of quantitation was 0.1 ng/mL (6.4 adducts per 10(8) nucleotides). Linearity of the calibration curve was greater than 0.999 from 0.1 to 50 ng/mL. Intra-day precision for three levels of quality control samples ranged from 4.27 to 15.62%. Interday precision ranged from 2.46 to 11.95%. Using this method, the levels of dTp(Me)dT in DNA enzymatic hydrolysates obtained from a series of incubations of mouse lymphoma cells with low doses of MNU (50 microM) were quantified.

Simultaneous quantitation of 7-methyl- and O6-methylguanine adducts in DNA by liquid chromatography-positive electrospray tandem mass spectrometry.

A methodology has been developed and validated for the simultaneous quantitation of O6-methyl- and 7-methylguanine in DNA isolated from in vitro exposure to the model alkylating agents: N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS). After exposure, DNA was isolated and directly hydrolyzed under acid conditions to hydrolytes containing DNA bases (modified and unmodified). The hydrolytes were used for direct O6- and 7-methylguanine quantitation using a rapid and selective liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS). The lower limits of quantitation for O6-methyl- and 7-methylguanine were 75.8 and 151.5 fmol, respectively. Linearity of the calibration curve was greater than 0.999 from 75.8 to 151,600.0 fmol for O6-methylguanine and 0.999 from 151.5 to 303,200.0 fmol for 7-methylguanine. The intra-day assay precision relative standard deviation (R.S.D.) values for O6-methylguanine for quality control (QC) samples were < or =9.2% with accuracy values ranging from 90.8 to 118%, and for 7-methylguanine the R.S.D. values for QC samples were < or =11%, with accuracy values ranging from 92.9 to 119%. The inter-day assay precision (R.S.D.) values for O6-methylguanine QC samples were < or =7.9% with accuracy values ranging from 94.5 to 116%, and for 7-methylguanine QC samples were < or =7.1% with accuracy values ranging from 95.2 to 110.2%. This method was used for simultaneous determination of the levels of 7-methyl- and O6-methylguanine in DNA acidic hydrolytes present in a series of incubations from salmon testis DNA treated with either MNU or MMS.

Quantitation of DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative electrospray tandem mass spectrometry.

A rapid and selective method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the direct quantitation of a methyl phosphotriester DNA adduct, thymidyl (3'-5') thymidine [dTp(Me)dT] from enzymatic hydrolysates of DNA (either in vitro DNA or in cell culture) treated with MNU (N-methyl-N-nitrosourea) or MMS (methyl methane sulfonate). The lower limit of quantitation was 2 ng/mL. Linearity of the calibration curve was greater than 0.999 from 2 to 1000 ng/mL. Intraday precision for four levels of quality controls ranged from 2.8 to 20.1%, and interday precision ranged from 2.9 to 5.6%. This method was used to quantify the levels of dTp(Me)dT in enzymatic hydrolysates of DNA obtained from a series of incubations of salmon testis DNA or mouse lymphoma cells with either MNU or MMS.

Mode of mutagenic action for the biocide Bioban CS-1246 in mouse lymphoma cells and implications for its in vivo mutagenic potential.

The biocidal agent, BIOBAN CS-1246 (7-ethyl bicyclooxazolidine, CAS# 7747-35-5, CS-1246) induced a concentration-dependent mutagenic response in mouse lymphoma (L5178Y TK+/-) cells both with and without the addition of S9 metabolic activation. Previous data indicating the ability of CS-1246 to hydrolyze in aqueous media to generate formaldehyde (FA), led us to investigate the potential role of FA in the CS-1246-induced mutagenic response in the mouse lymphoma assay (MLA). To accomplish this, the MLA on CS-1246 was repeated in the presence of a metabolizing system (formaldehyde dehydrogenase/NAD+), which was shown to successfully inhibit the mutagenic response of formaldehyde in this assay system. Significantly, the observed mutagenicity of CS-1246 was completely abrogated when the cultures were supplemented with formaldehyde dehydrogenase/NAD+, suggesting that the positive MLA response was attributable to the generation of FA in situ. These results demonstrate that in vitro mutagenicity of CS-1246 in the MLA is most likely associated with FA. Negative results from two in vivo assays for genotoxicity were consistent with the known activity of FA in these assays. In the mouse bone marrow micronucleus (MNT), there were no significant increases in micronucleated polychromatic erythrocytes (with evaluation of 2000/animal), after treatment with 0.5, 1, and 2 g/kg/day CS-1246 (6/dose group) for 2 consecutive days and sacrifice 24 h later. Furthermore, in the unscheduled DNA synthesis (UDS) study, male F344 rats (5 /dose group), given a single oral gavage (0, 1, and 2 g/kg) and evaluated at two time points (2-4 and 14-15 h post dosing), did not elicit an UDS response, indicating a lack of DNA reactivity in vivo. Based on the negative in vivo findings, it can be inferred that the FA detoxification mechanisms that exist in intact organisms prevent the likelihood of generating FA at levels capable of causing genotoxicity following exposure to CS-1246 at low, environmentally relevant concentrations. The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.