Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

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.

Bis-butanediol-mercapturic acid (bis-BDMA) as a urinary biomarker of metabolic activation of butadiene to its ultimate carcinogenic species.

Human carcinogen 1,3-butadiene (BD) undergoes metabolic activation to 3,4-epoxy-1-butene (EB), hydroxymethylvinyl ketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD) and 1,2,3,4-diepoxybutane (DEB). Among these, DEB is by far the most genotoxic metabolite and is considered the ultimate carcinogenic species of BD. We have shown previously that BD-exposed laboratory mice form 8- to 10-fold more DEB-DNA adducts than rats exposed at the same conditions, which may be responsible for the enhanced sensitivity of mice to BD-mediated cancer. In the present study, we have identified 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA) as a novel DEB-specific urinary biomarker. Isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was employed to quantify bis-BDMA and three other BD-mercapturic acids, 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxy-but-3-ene (MHBMA, from EB), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA, from HMVK) and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA, from EBD), in urine of confirmed smokers, occupationally exposed workers and BD-exposed laboratory rats. Bis-BDMA was formed in a dose-dependent manner in urine of rats exposed to 0-200 p.p.m. BD by inhalation, although it was a minor metabolite (1%) as compared with DHBMA (47%) and THBMA (37%). In humans, DHBMA was the most abundant BD-mercapturic acid excreted (93%), followed by THBMA (5%) and MHBMA (2%), whereas no bis-BDMA was detected. These results reveal significant differences in metabolism of BD between rats and humans.

VDJ recombinase-mediated TCR ß locus gene usage and coding joint processing in peripheral T cells during perinatal and pediatric development.

The generation of TCR proteins is the result of V(D)J recombinase-mediated genomic rearrangements at recombination signal sequences (RSS) in human lymphocytes. V(D)J recombinase can also mediate rearrangements at nonimmune or "cryptic" RSS in normal and leukemic human peripheral T cells. We previously demonstrated age- and gender-specific developmental differences in V(D)J coding joint processing at cryptic RSS within the HPRT locus in peripheral T cells from healthy children (Murray et al. 2006. J. Immunol. 177: 5393-5404). In this study, we investigated developmentally specific V(D)J recombinase TCRß immune gene rearrangements and coding joint processing at RSS in peripheral T cells in the same pediatric population. This approach provided a unique opportunity to investigate site-specific V(D)J recombinase rearrangements and coding joint processing at immune and nonimmune genes from the same individual T cell population. We determined the genomic sequence of 244 TCRß coding junctions from 112 (63 male, 49 female) subjects from the late stages of fetal development through 9 y of age. We observed both age- and gender-specific V(D)J recombinase-mediated TCRß gene usage and coding joint processing at immune RSS. To the best of our knowledge, these data represent the first description of age- and gender-specific developmental differences in TCR gene usage and coding joint processing that could directly influence TCR diversity and immune specificity. It will be important for future studies to ascertain the mechanistic etiology of these developmental and gender differences in TCR diversity and specificity, as well as their importance with respect to the age and gender risks for infectious and autoimmune diseases in humans.

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

WR1065 conjugated to thiol-PEG polymers as novel anticancer prodrugs: broad spectrum efficacy, synergism, and drug resistance reversal.

The lack of anticancer agents that overcome innate/acquired drug resistance is the single biggest barrier to achieving a durable complete response to cancer therapy. To address this issue, a new drug family was developed for intracellular delivery of the bioactive aminothiol WR1065 by conjugating it to discrete thiol-PEG polymers: 4-star-PEG-S-S-WR1065 (4SP65) delivers four WR1065s/molecule and m-PEG6-S-S-WR1065 (1LP65) delivers one. Infrequently, WR1065 has exhibited anticancer effects when delivered via the FDA-approved cytoprotectant amifostine, which provides one WR1065/molecule extracellularly. The relative anticancer effectiveness of 4SP65, 1LP65, and amifostine was evaluated in a panel of 15 human cancer cell lines derived from seven tissues. Additional experiments assessed the capacity of 4SP65 co-treatments to potentiate the anticancer effectiveness and overcome drug resistance to cisplatin, a chemotherapeutic, or gefitinib, a tyrosine kinase inhibitor (TKI) targeting oncogenic EGFR mutations. The CyQUANT®-NF proliferation assay was used to assess cell viability after 48-h drug treatments, with the National Cancer Institute COMPARE methodology employed to characterize dose-response metrics. In normal human epithelial cells, 4SP65 or 1LP65 enhanced or inhibited cell growth but was not cytotoxic. In cancer cell lines, 4SP65 and 1LP65 induced dose-dependent cytostasis and cytolysis achieving 99% cell death at drug concentrations of 11.2 ± 1.2 µM and 126 ± 15.8 µM, respectively. Amifostine had limited cytostatic effects in 11/14 cancer cell lines and no cytolytic effects. Binary pairs of 4SP65 plus cisplatin or gefitinib increased the efficacy of each partner drug and surmounted resistance to cytolysis by cisplatin and gefitinib in relevant cancer cell lines. 4SP65 and 1LP65 were significantly more effective against TP53-mutant than TP53-wild-type cell lines, consistent with WR1065-mediated reactivation of mutant p53. Thus, 4SP65 and 1LP65 represent a unique prodrug family for innovative applications as broad-spectrum anticancer agents that target p53 and synergize with a chemotherapeutic and an EGFR-TKI to prevent or overcome drug resistance.

Persistence and repair of bifunctional DNA adducts in tissues of laboratory animals exposed to 1,3-butadiene by inhalation.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen. The mechanism of BD-mediated cancer is of significant interest because of the widespread exposure of humans to BD from cigarette smoke and urban air. BD is metabolically activated to 1,2,3,4-diepoxybutane (DEB), which is a highly genotoxic and mutagenic bis-alkylating agent believed to be the ultimate carcinogenic species of BD. We have previously identified several types of DEB-specific DNA adducts, including bis-N7-guanine cross-links (bis-N7-BD), N(6)-adenine-N7-guanine cross-links (N(6)A-N7G-BD), and 1,N(6)-dA exocyclic adducts. These lesions were detected in tissues of laboratory rodents exposed to BD by inhalation ( Goggin et al. (2009) Cancer Res. 69 , 2479 -2486 ). In the present work, persistence and repair of bifunctional DEB-DNA adducts in tissues of mice and rats exposed to BD by inhalation were investigated. The half-lives of the most abundant cross-links, bis-N7G-BD, in mouse liver, kidney, and lungs were 2.3-2.4 days, 4.6-5.7 days, and 4.9 days, respectively. The in vitro half-lives of bis-N7G-BD were 3.5 days (S,S isomer) and 4.0 days (meso isomer) due to their spontaneous depurination. In contrast, tissue concentrations of the minor DEB adducts, N7G-N1A-BD and 1,N(6)-HMHP-dA, remained essentially unchanged during the course of the experiment, with an estimated t(1/2) of 36-42 days. No differences were observed between DEB-DNA adduct levels in BD-treated wild type mice and the corresponding animals deficient in methyl purine glycosylase or the Xpa gene. Our results indicate that DEB-induced N7G-N1A-BD and 1,N(6)-HMHP-dA adducts persist in vivo, potentially contributing to mutations and cancer observed as a result of BD exposure.

Column switching HPLC-ESI(+)-MS/MS methods for quantitative analysis of exocyclic dA adducts in the DNA of laboratory animals exposed to 1,3-butadiene.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen on the basis of epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its carcinogenicity in laboratory rats and mice. BD is metabolically activated to epoxide intermediates that can react with nucleophilic sites of cellular biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to the ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. DEB mutagenesis studies suggest that adducts formed at adenine bases may be critically important, as DEB induces large numbers of A --> T transversion mutations. We have recently identified two regioisomeric exocyclic DEB-dA adducts, 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-gamma-HMHP-dA) and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-alpha-HMHP-dA) ( Seneviratne et al. ( ( 2010 ) Chem. Res. Toxicol. 23 , 118 - 133 ), which were detected in DEB-treated calf thymus DNA and in tissues of BD-exposed laboratory animals. In the present work, we describe a column switching HPLC-ESI(+)-MS/MS methodology for the quantitative analysis of 1,N(6)-HMHP-dA isomers in the DNA of laboratory mice exposed to BD by inhalation. On the basis of their exocyclic structure, which prevents normal Watson-Crick base pairing, these adducts could be responsible for mutations at the A:T base pairs observed following exposure to DEB.

Mutagenicity of stereochemical configurations of 1,3-butadiene epoxy metabolites in human cells.

The mutagenic and carcinogenic effects of 1,3-butadiene (BD*) are related to its bioactivation to several DNA-reactive metabolites, including 1,2-epoxy-3-butene (BDO), 1,2,3,4-diepoxybutane (BDO2), and 1,2-dihydroxy-3,4-epoxybutane (BDO-diol). Accumulated evidence indicates that stereochemical configurations of BD metabolites may play a role in the mutagenic and carcinogenic action of BD. The objective of this study was to evaluate the cytotoxicity and mutagenicity of each stereoisomer of major BD metabolites in human cells. For this purpose, nine stereochemical forms of BDO, BDO2, and BDO-diol were synthesized. TK6 cells, a human lymphoblastoid cell line, were exposed to each stereoisomer. Cytotoxicity was measured by comparing cloning efficiencies (CEs) in chemical-exposed cells versus those in control cells. Based on the results of cytotoxicity tests, TK6 cells were exposed to 0; 2, 4, or 6 pM of each form of BDO2, or to 0, 200, 400, or 600 pMof each form of BDO for 24 hours to determine the mutagenic efficiencies. The exposure concentrations for BDO-diol ranged from 5 to 1000 pM. The mutagenicity was measured by determining, in a lymphocyte cloning assay, the mutant frequencies (Mfs) in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) genes. HPRT mutants collected from cells exposed to the three forms of BDO2 were analyzed by polymerase chain reaction (PCR) to characterize large genetic alterations. All three stereoisomers of BDO2 [(2R,3R)-BDO2, (2S,3S)-BDO2, and meso-BDO2] caused increased HPRT and TK Mfs compared with the concurrent control samples, with P values ranged from 0.05 to 0.001. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of BDO2. Molecular analysis ofHPRTmutants revealed similar distributions of deletion mutations caused by the three isomers of BDO2. There were also no statistical differences in mutagenic efficiencies between the two isomers of BDO [(2R)-BDO and (2S)-BDO] in TK6 cells. These results were consistent with the in vivo finding that there was little difference in the mutagenic efficiencies of (+)-BDO2 versus meso-BDO2 in rodents. Thus, in terms of mutagenic potency, there was no evidence that stereochemical configurations of BDO and BDO2 play a significant role in the mutagenicity and carcinogenicity of BD. The most significant results of this study were the marked differences in cytotoxicity and mutagenicity among the four stereoisomers of BDO-diol [(2R,3R)-BDO-diol, (2R,3S)-BDO-diol, (2S,3R)-BDO-diol, and (2S,3S)-BDO-diol]. (2R,3S)-BDO-diol was at least 30-fold more cytotoxic and mutagenic than the other three forms of BDO-diol. This was consistent with the finding that 75% of the adduct N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) originated from (2R,3S)-BDO-diol in the lungs of mice exposed to BD. The mutagenic potency of (2R,3S)-BDO-diol was much closer to that of BDO2 than previously demonstrated in experiments in which stereochemistry was not considered. The current study demonstrated that the mutagenic potency of (2R,3S)-BDO-diol was only 5-to-l0-fold less than the average equimolar effect of BDO2 stereoisomers in the HPRT and TK genes, and was 10-to-20-fold greater than the average equimolar effect of BDO stereoisomers in the HPRT and TKgenes. Previous DNA and hemoglobin adduct data demonstrated that BDO-diol is the dominant BD metabolite available to react with macromolecules in vivo after BD exposure (Pérez et al. 1997; Swenberg et al. 2001). Thus, the differences in BD carcinogenesis among rodent species may be significantly accounted for by the stereochemistry-dependent distributions of BDO-diol metabolites and BDO-diol-DNA adducts, and by the mutagenic efficiencies of BDO-diol in mice and rats.

Human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra- and inter-subject variation.

We previously described flow cytometry-based methods for scoring the incidence of micronucleated reticulocytes (MN-RET) and PIG-A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71-positive reticulocytes prior to MN-RET scoring, and procedures for storing frozen blood for later PIG-A analysis. Technical replicate variability in MN-RET and MUT RET frequencies based on blood specimens from 14 subjects, intra-subject variability based on serial blood draws from 6 subjects, and inter-subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter-subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra-subject and technical replicate variability. The relatively large degree of inter-subject variation is apparent from mean and standard deviation values for MN-RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter-subject variation were investigated, and neither factor affected MN-RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN-RET and MUT RET are automation-compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter-individual variability reported herein.

Antiretroviral activity of the aminothiol WR1065 against Human Immunodeficiency virus (HIV-1) in vitro and Simian Immunodeficiency virus (SIV) ex vivo.

BACKGROUND: WR1065 is the free-thiol metabolite of the cytoprotective aminothiol amifostine, which is used clinically at very high doses to protect patients against toxicity induced by radiation and chemotherapy. In an earlier study we briefly reported that the aminothiol WR1065 also inhibits HIV-1 replication in phytohemagglutinin (PHA)-stimulated human T-cell blasts (TCBs) infected in culture for 2 hr before WR1065 exposure. In this study we expanded the original observations to define the dose-response curve for that inhibition, and address the question of additive effects for the combination of WR1065 plus Zidovudine (AZT). Here we also explored the effect of WR1065 on SIV by examining TCBs taken from macaques with well-established infections several months with SIV. RESULTS: TCBs from healthy human donors were infected for 2 hr with HIV-1, and viral replication (p24) was measured after 72 hr of incubation with or without WR1065, AZT, or both drugs. HIV-1 replication, in HIV-1-infected human TCBs, was inhibited by 50% at 13 microM WR1065, a dose at which 80% of the cells were viable. Cell cycle parameters were the same or equivalent at 0, 9.5 and 18.7 microM WR1065, showing no drug-related toxicity. Combination of AZT with WR1065 showed that AZT retained antiretroviral potency in the presence of WR1065. Cultured CD8+ T cell-depleted PHA-stimulated TCBs from Macaca mulatta monkeys chronically infected with SIV were incubated 17 days with WR1065, and viral replication (p27) and cell viability were determined. Complete inhibition (100%) of SIV replication (p27) was observed when TCBs from 3 monkeys were incubated for 17 days with 18.7 microM WR1065. A lower dose, 9.5 microM WR1065, completely inhibited SIV replication in 2 of the 3 monkeys, but cells from the third macaque, with the highest viral titer, only responded at the high WR1065 dose. CONCLUSION: The study demonstrates that WR1065 and the parent drug amifostine, the FDA-approved drug Ethyol, have antiretroviral activity. WR1065 was active against both an acute infection of HIV-1 and a chronic infection of SIV. The data suggest that the non-toxic drug amifostine may be a useful antiretroviral agent given either alone or in combination with other drugs as adjuvant therapy.

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)

1,3-Butadiene metabolite 1,2,3,4 diepoxybutane induces DNA adducts and micronuclei but not t(9;22) translocations in human cells.

Epidemiological studies of 1,3-butadiene (BD) exposures have reported a possible association with chronic myelogenous leukemia (CML), which is defined by the presence of the t(9;22) translocation (Philadelphia chromosome) creating an oncogenic BCR-ABL fusion gene. Butadiene diepoxide (DEB), the most mutagenic of three epoxides resulting from BD, forms DNA-DNA crosslink adducts that can lead to DNA double-strand breaks (DSBs). Thus, a study was designed to determine if (±)-DEB exposure of HL60 cells, a promyelocytic leukemia cell line lacking the Philadelphia chromosome, can produce t(9;22) translocations. In HL60 cells exposed for 3 h to 0-10 µM DEB, overlapping dose-response curves suggested a direct relationship between 1,4-bis-(guan-7-yl)-2,3-butanediol crosslink adduct formation (R = 0.977, P = 0.03) and cytotoxicity (R = 0.961, P = 0.002). Experiments to define the relationships between cytotoxicity and the induction of micronuclei (MN), a dosimeter of DNA DSBs, showed that 24 h exposures of HL60 cells to 0-5.0 µM DEB caused significant positive correlations between the concentration and (i) the degree of cytotoxicity (R = 0.998, p = 0.002) and (ii) the frequency of MN (R = 0.984, p = 0.016) at 48 h post exposure. To determine the relative induction of MN and t(9;22) translocations following exposures to DEB, or x-rays as a positive control for formation of t(9;22) translocations, HL60 cells were exposed for 24 h to 0, 1, 2.5, or 5 µM DEB or to 0, 2.0, 3.5, or 5.0 Gy x-rays, or treatments demonstrated to yield 0, 20%, 50%, or 80% cytotoxicity. Treatments between 0 and 3.5 Gy x-rays caused significant dose-related increases in both MN (p < 0.001) and t(9;22) translocations (p = 0.01), whereas DEB exposures causing similar cytotoxicity levels did not increase translocations over background. These data indicate that, while DEB induces DNA DSBs required for formation of MN and translocations, acute DEB exposures of HL60 cells did not produce the Philadelphia chromosome obligatory for CML.

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.

Transplacentally exposed human and monkey newborn infants show similar evidence of nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity.

Effective reduction in maternal-fetal human immunodeficiency virus-1 (HIV-1) transmission has been achieved by administration of nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy, and although most exposed children are clinically normal at birth, mitochondrial dysfunction has been reported. To examine mitochondrial integrity on a molecular level, we evaluated mitochondrial morphology by electron microscopy (EM) and mitochondrial DNA (mtDNA) quantity in umbilical cords and cord blood from NRTI-exposed and unexposed human and monkey newborns. Human subjects included infants born to HIV-1-infected mothers who received Combivir (Zidovudine [AZT] plus Lamivudine [3TC]) (n = 9) or AZT plus Didanosine [ddI] (n = 2) during pregnancy, and infants born to HIV-1-uninfected mothers (n = 7). NRTI-exposed Erythrocebus patas monkey dams (n = 3 per treatment group) were given human-equivalent dosing regimens containing 3TC, AZT/3TC, AZT/ddI, or Stavudine (d4T)/3TC during gestation. Four infants born to unexposed patas dams served as controls. Mitochondria in umbilical cord endothelial cells from NRTI-exposed monkey and human infants showed substantial abnormal pathology by EM, the extent of which was quantified from coded photomicrographs and shown to be different (P < 0.05) from the unexposed monkey and human newborns. Significant (P < 0.05) mtDNA depletion was found in umbilical cords from both human and monkey NRTI-exposed infants and in human, but not in monkey, cord blood leukocytes. For umbilical cords, an increase in mitochondrial morphological damage correlated with reduction in mtDNA quantity in fetal monkeys (r = 0.94). The treatment-induced mitochondrial compromise in infant monkeys ranked as follows: d4T/3TC > AZT/ddI > AZT/3TC > 3TC. The study demonstrates that transplacental NRTI exposures induce similar mitochondrial damage in cord blood and umbilical cords taken from retroviral-uninfected monkey infants and from human infants born to HIV-1-infected women.

Relative mutagenic potencies of several nucleoside analogs, alone or in drug pairs, at the HPRT and TK loci of human TK6 lymphoblastoid cells.

Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combinations in human TK6 B-lymphoblastoid cells. Three-day treatments with 0, 33, 100, or 300 microM ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach significance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that exposure to 100 microM d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of mutagenicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to combined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 microM ddI-3TC or 100 microM d4T-3TC, and in the TK gene of cells exposed to 100 or 300 microM ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667-126671; Torres SM et al. [2007]: Environ Mol Mutagen) indicate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT-3TC congruent with AZT-3TC-ABC (abacavir) > AZT >/=ddI > d4T-3TC > 3TC > d4T >/= ABC. These collective data suggest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk.

Mutagenicity of zidovudine, lamivudine, and abacavir following in vitro exposure of human lymphoblastoid cells or in utero exposure of CD-1 mice to single agents or drug combinations.

Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12-18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 microM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 microM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 microM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children.

Transplacental carcinogenicity of 3'-azido-3'-deoxythymidine in B6C3F1 mice and F344 rats.

The prophylactic use of zidovudine (3'-azido-3'-deoxythymidine, AZT) during pregnancy greatly reduces transmission of HIV-1 from infected mothers to their infants; however, the affinity of host cell DNA polymerases for AZT also allows for its incorporation into host cell DNA, predisposing to cancer development. To expand upon previous transplacental carcinogenesis assays performed in CD-1 mice, the transplacental carcinogenicity of AZT was evaluated in a second mouse strain and a second rodent species. Date-mated female mice and rats were gavaged daily with 0, 80, 240, or 480 mg AZT/kg bw during the last 7 days of gestation. At 2 years postpartum, male and female B6C3F1 mouse and F344 rat offspring (n = 44-46 of each sex and species/treatment group) were necropsied for gross and microscopic tissue examinations. Under the conditions of these two-year studies, there was clear evidence of carcinogenic activity based upon significant dose-related trends and increases in the incidences of hemangiosarcoma in male mice and mononuclear cell leukemia in female rats. There was some evidence of carcinogenic activity in the livers of male mice based upon a positive trend and an increased incidence of hepatic carcinoma in the high-dose AZT group. The incidence of gliomas in female rats exceeded the historical background rates for gliomas in F344 rats. P53 overexpression was detected in some AZT-treated mouse neoplasms. These and other cancer-related findings confirm and extend those of previous transplacental carcinogenicity studies of AZT in mice, support the need for long-term follow-up of nucleoside reverse transcriptase inhibitor (NRTI)-exposed children, and indicate the necessity for effective protective strategies against NRTI-induced side effects.

Mitochondrial toxicity in hearts of CD-1 mice following perinatal exposure to AZT, 3TC, or AZT/3TC in combination.

Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), markedly reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To provide animal data modeling the NRTI-induced heart damage reported in human infants, we treated pregnant CD-1 mice throughout gestation and treated their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg body weight (bw)/day AZT, 75 mg/kg bw/day 3TC, 125/62.5 mg/kg bw/day AZT/3TC, or the vehicle control. Half the pups were euthanized on PND 28; the remainder received no further dosing, and were euthanized at week 10. Heart tissue was collected, total DNA was extracted, and mtDNA copy number relative to nuclear DNA copy number, mtDNA damage, and mtDNA mutation assays were performed using PCR-based methods. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC, but not in 10-week-old mice, suggesting that the damage resolved after treatment ceased. Interestingly, 10-week-old females treated with AZT/3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT/3TC, but not 3TC; no increases in mutations were seen in either gender at 4 weeks of age. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and that female mice are more sensitive than males to AZT/3TC-induced mtDNA damage.

Plasma and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism as indicators of DNA damage in cord blood mononuclear cells from infants receiving prepartum NRTIs.

Several systemic and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism and AZT incorporation into nuclear DNA were measured in cord blood from uninfected infants born to HIV-1-infected mothers receiving prepartum therapies based on AZT or AZT in combination with 2',3'-dideoxy-3'-thiacytidine (3TC). In addition, the relationships among these pharmacological end points, levels of AZT-DNA incorporation, and the previously reported mutagenic responses in these infants were evaluated. AZT- and 3TC-specific radioimmunoassays (RIAs), or HPLC coupled with AZT-RIA, were used to measure plasma levels of AZT and the AZT-glucuronide, and cellular levels of AZT, phosphorylated AZT, and DNA incorporation of AZT or 3TC in cord blood mononuclear cells from treated infants compared with unexposed controls born to HIV-uninfected mothers. Fewer infants had detectable AZT-DNA incorporation levels in the group exposed to AZT (71%; n = 7) compared with those receiving AZT-3TC (100%; n = 21), and the mean AZT-DNA incorporation for AZT-exposed infants (14.6 +/- 6.3 AZT/10(6) nucleotides) was significantly lower than that in AZT-3TC exposed infants (51.6 +/- 10.2 AZT/10(6) nucleotides; P = 0.028). Low levels of 3TC-DNA incorporation found in a few AZT-3TC-exposed newborns correlated with AZT-DNA incorporation values in the same samples. Among the metabolites studied, there were positive correlations between levels of AZT-diphosphate and AZT-triphosphate, and AZT-triphosphate and AZT-DNA incorporation, in nucleoside analog-exposed infants. Levels of AZT-DNA incorporation, however, did not correlate well with the reported frequencies of somatic mutations in the same population of nucleoside analog-treated children. While these data support the continued use of AZT-based therapies during pregnancy, infants receiving prepartum AZT should be monitored long-term for adverse health effects.