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.

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.

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.

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.

Frequencies of micronucleated reticulocytes, a dosimeter of DNA double-strand breaks, in infants receiving computed tomography or cardiac catheterization.

The use of computed tomography (CT scans) has increased dramatically in recent decades, raising questions about the long-term safety of CT-emitted x-rays especially in infants who are more sensitive to radiation-induced effects. Cancer risk estimates for CT scans typically are extrapolated from models; therefore, new approaches measuring actual DNA damage are needed for improved estimations. Hence, changes in a dosimeter of DNA double-strand breaks, micronucleated reticulocytes (MN-RETs) measured by flow cytometry, were investigated in mice and infants exposed to CT scans. In male C57BL/6N mice (6-8 weeks-of-age), there was a dose-related increase in MN-RETs in blood samples collected 48h after CT scans delivering targeted exposures of 1-130 cGy x-rays (n=5-10/group, r=0.994, p=0.01), with significant increases occurring at exposure levels as low as 0.83 cGy x-rays compared to control mice (p=0.002). In paired blood specimens from infants with no history of a prior CT scan, there was no difference in MN-RET frequencies found 2h before (mean, 0.10±0.07%) versus 48h after (mean, 0.11±0.05%) a scheduled CT scan/cardiac catheterization. However, in infants having prior CT scan(s), MN-RET frequencies measured at 48h after a scheduled CT scan (mean=0.22±0.12%) were significantly higher than paired baseline values (mean, 0.17±0.07%; p=0.032). Increases in baseline (r=0.722, p<0.001) and 48-h post exposure (r=0.682, p<0.001) levels of MN-RETs in infants with a history of prior CT scans were significantly correlated with the number of previous CT scans. These preliminary findings suggest that prior CT scans increase the cellular responses to subsequent CT exposures. Thus, further investigation is needed to characterize the potential cancer risk from single versus repeated CT scans or cardiac catheterizations in infants.

1,3-Butadiene, CML and the t(9:22) translocation: A reality check.

Epidemiological studies of 1,3-butadiene have suggest that exposures to humans are associated with chronic myeloid leukemia (CML). CML has a well-documented association with ionizing radiation, but reports of associations with chemical exposures have been questioned. Ionizing radiation is capable of inducing the requisite CML-associated t(9:22) translocation (Philadelphia chromosome) in appropriate cells in vitro but, thus far, chemicals have not shown this capacity. We have proposed that 1,3-butadiene metabolites be so tested as a reality check on the epidemiological reports. In order to conduct reliable testing in this regard, it is essential that a positive control for induction be available. We have used ionizing radiation to develop such a control. Results described here demonstrate that this agent does in fact induce pathogenic t(9:22) translocations in a human myeloid cell line in vitro, but does so at low frequencies. Conditions that will be required for studies of 1,3-butadiene are discussed.

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.

The stress response resolution assay. II. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium.

Cellular stress responses consist of a complex network of pathways and linked processes that, when perturbed, are postulated to have roles in the pathogenesis of various human diseases. To assess the impact of environmental insults upon this network, we developed a novel stress response resolution (SRR) assay for investigation of cellular stress resolution outcomes and the effects of environmental agents and conditions thereupon. SRR assay-based criteria identified three distinct groups of surviving cell clones, including those resembling parental cells, those showing Hprt/HPRT mutations, and a third type, "Phenotype-altered" clones, that occurred predominantly in cells pretreated with a chemical mutagen, was heterogeneous in nature, and expressed significant alterations in cell morphology and/or function compared with parental cells. Further evaluation of Phenotype-altered clones found evidence of various alterations that resembled epithelial-to-mesenchymal transition, phenotype switching, checkpoint dysfunction, senescence barrier bypass, and/or epigenetic reprogramming. Phenotype-altered clones were found to occur spontaneously in a cell line with a mutator phenotype, to represent the major surviving clone type in a variation of the SRR assay, and to be tumorigenic in nude mice. Assessment of SRR assay final results showed that pretreatment with a chemical mutagen induced significant changes in cellular stress response prosurvival capacity, in damage avoidance versus damage tolerance stress resolution outcomes, and in the damage burden in the final surviving cell populations. Taken together, these results support the conclusion that use of the SRR assay can provide novel insights into the role of environmental insults in the pathogenesis of cancer and other human diseases.

The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium.

The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the "stress response resolution" (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6-thioguanine). Pilot studies conducted with a spontaneously-immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N-ethyl-N-nitrososurea/ml medium for 1 hr, or two hTERT-immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post-exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle-treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub-cytotoxic pretreatments with chemical mutagens have long-term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors.

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.

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.

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.

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.

Exposure-response of 1,2:3,4-diepoxybutane-specific N-terminal valine adducts in mice and rats after inhalation exposure to 1,3-butadiene.

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. 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 extensive species differences in carcinogenicity. This study presents a comprehensive exposure-response for the formation of the DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in mice and rats. Using nano-ultra high pressure liquid chromatography-tandem-mass spectrometry allowed analysis of pyr-Val in mice and rats exposed to BD as low as 0.1 and 0.5 ppm BD, respectively, and demonstrated significant differences in the amounts and exposure-response of pyr-Val formation. Mice formed 10- to 60-fold more pyr-Val compared to rats at similar exposures. The formation of pyr-Val increased with exposures, and the formation was most efficient with regard to formation per parts per million BD at low exposures. While formation at higher exposures appeared linear in mice, in rats formation saturated at exposures > or = 200 ppm for 10 days. In rats, amounts of pyr-Val were lower after 20 days than after 10 days of exposure, suggesting that the lifespan of rat erythrocytes may be shortened following exposure to BD. This research supports the hypothesis that the lower susceptibility of rats to BD-induced carcinogenesis results from greatly reduced formation of DEB following exposure to BD.

Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene.

Hprt mutant frequency and p53 gene status were assessed in wild-type and p53 heterozygous (p53+/-) mice exposed chronically by inhalation to benzene. Benzene exposures to 100 ppm for 6h on Monday-Friday, 100 ppm for 10h on Monday-Wednesday-Friday, or 200 ppm for 5h on Monday-Wednesday-Friday yielded the same total exposures (concentration x time) of 3000 ppm x h/week. Hprt mutations in splenic T-lymphocytes were significantly increased in all benzene groups, ranging from 3.8- to 8.0-fold greater than control values. Wild-type and p53+/- mice were equally susceptible to benzene mutagenesis. Hprt wild-type and mutant isolates from control and exposed animals were examined for TCR gene rearrangements (as markers of in vivo clonality) and for loss of p53 wild-type or mutant alleles. Moderate clonal amplifications were observed among the Hprt mutant but not Hprt wild-type isolates but was not sufficient to account for the increases in Hprt mutant frequencies. Most isolates, whether Hprt wild-type or mutant, retained both p53 alleles in the benzene-exposed p53+/- animals (54% and 63%, respectively, for the Hprt wild-type and mutants). However, 37% of the Hprt wild-type isolates and 46% of the Hprt mutant isolates lost the p53 mutant allele. Only a small percentage of either type of isolate lost the p53 wild-type allele, and this was always in isolates that that previously lost the p53 mutant allele. Loss of the p53 mutant allele was independent of benzene exposure, Hprt status, or 6-thioguanine selection. These findings contrast with the p53 status of thymic lymphomas that had preferentially lost the wild-type p53 allele in some of these same mice. Possible reasons for loss of the mutant p53 allele in the Hprt mutant and wild-type isolates are discussed.

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)

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.

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.

Molecular dosimetry of 1,2,3,4-diepoxybutane-induced DNA-DNA cross-links in B6C3F1 mice and F344 rats exposed to 1,3-butadiene by inhalation.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen based on epidemiologic studies in occupationally exposed workers and animal studies in laboratory rats and mice. BD is metabolically activated to three epoxides that can react with nucleophilic sites in biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogen due to its high genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links. Our laboratory has developed quantitative high-performance liquid chromatography-muESI(+)-tandem mass spectrometry methods for two DEB-specific DNA-DNA cross-links, 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) and 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD). This report describes molecular dosimetry analysis of these adducts in tissues of B6C3F1 mice and F344 rats exposed to a range of BD concentrations (0-625 ppm). Much higher (4- to 10-fold) levels of DEB-DNA cross-links were observed in mice compared with rats exposed to the same BD concentrations. In both species, bis-N7G-BD levels were 1.5- to 4-fold higher in the liver than in other tissues examined. Interestingly, tissues of female animals exposed to BD contained higher concentrations of bis-N7G-BD adducts than tissues of male animals, which is in accord with previously reported differences in tumor incidence. The molecular dosimetry data presented herein suggest that species and gender differences observed in BD-induced cancer are directly related to differences in the extent of BD metabolism to DEB. Furthermore, a rat model of sensitivity to BD may be more appropriate than a mouse model for assessing human risk associated with BD exposure, because rats and humans seem to be similar with respect to DEB formation.