RESUMO
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.
Assuntos
Etilnitrosoureia , Linfócitos T , Camundongos , Humanos , Animais , Etilnitrosoureia/toxicidade , Mutação , Mutagênese/genética , Mutagênicos/toxicidade , Hipoxantina Fosforribosiltransferase/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Citometria de Fluxo/métodos , Proteínas de Membrana/genética , Testes para Micronúcleos , Mutação , Reticulócitos/ultraestrutura , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
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 â¼15 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.
Assuntos
Acrilonitrila/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/análise , Guanina/análise , Hipoxantina Fosforribosiltransferase/genética , Acrilonitrila/administração & dosagem , Acrilonitrila/metabolismo , Administração Oral , Animais , Carcinógenos/administração & dosagem , Carcinógenos/metabolismo , Células Cultivadas , Adutos de DNA/biossíntese , Relação Dose-Resposta a Droga , Óxido de Etileno/administração & dosagem , Óxido de Etileno/análogos & derivados , Óxido de Etileno/metabolismo , Óxido de Etileno/toxicidade , Feminino , Guanina/análogos & derivados , Guanina/biossíntese , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Masculino , Camundongos , Ratos , Ratos Endogâmicos F344RESUMO
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.
Assuntos
Acrilonitrila/toxicidade , Carcinógenos/toxicidade , Citocromo P-450 CYP2E1/metabolismo , Hipoxantina Fosforribosiltransferase/metabolismo , Acrilonitrila/administração & dosagem , Acrilonitrila/metabolismo , Administração Oral , Animais , Biomarcadores/análise , Carcinógenos/administração & dosagem , Carcinógenos/metabolismo , Citocromo P-450 CYP2E1/análise , Citocromo P-450 CYP2E1/genética , Dano ao DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Hipoxantina Fosforribosiltransferase/análise , Hipoxantina Fosforribosiltransferase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Testes de Mutagenicidade , Mutação , Baço/efeitos dos fármacos , Baço/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
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 HL60â¯cells, a promyelocytic leukemia cell line lacking the Philadelphia chromosome, can produce t(9;22) translocations. In HL60â¯cells exposed for 3â¯h to 0-10⯵M DEB, overlapping dose-response curves suggested a direct relationship between 1,4-bis-(guan-7-yl)-2,3-butanediol crosslink adduct formation (Râ¯=â¯0.977, Pâ¯=â¯0.03) and cytotoxicity (Râ¯=â¯0.961, Pâ¯=â¯0.002). Experiments to define the relationships between cytotoxicity and the induction of micronuclei (MN), a dosimeter of DNA DSBs, showed that 24â¯h exposures of HL60â¯cells to 0-5.0⯵M DEB caused significant positive correlations between the concentration and (i) the degree of cytotoxicity (Râ¯=â¯0.998, pâ¯=â¯0.002) and (ii) the frequency of MN (Râ¯=â¯0.984, pâ¯=â¯0.016) at 48â¯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, HL60â¯cells were exposed for 24â¯h to 0, 1, 2.5, or 5⯵M DEB or to 0, 2.0, 3.5, or 5.0â¯Gy x-rays, or treatments demonstrated to yield 0, 20%, 50%, or 80% cytotoxicity. Treatments between 0 and 3.5â¯Gy x-rays caused significant dose-related increases in both MN (pâ¯<â¯0.001) and t(9;22) translocations (pâ¯=â¯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 HL60â¯cells did not produce the Philadelphia chromosome obligatory for CML.
Assuntos
Adutos de DNA/metabolismo , Compostos de Epóxi/toxicidade , Translocação Genética/efeitos dos fármacos , Butadienos/metabolismo , Adutos de DNA/análise , Compostos de Epóxi/química , Células HL-60 , Humanos , Radiação Ionizante , Translocação Genética/efeitos da radiaçãoRESUMO
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.
Assuntos
Cateterismo Cardíaco/efeitos adversos , Quebras de DNA de Cadeia Dupla , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Reticulócitos/patologia , Tomografia Computadorizada por Raios X/efeitos adversos , Animais , Relação Dose-Resposta à Radiação , Feminino , Citometria de Fluxo , Instabilidade Genômica , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Micronúcleos com Defeito Cromossômico/estatística & dados numéricos , Projetos Piloto , Gravidez , Estudos Prospectivos , Doses de Radiação , Irradiação Corporal TotalRESUMO
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.
Assuntos
Butadienos/toxicidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Cromossomo Filadélfia/efeitos dos fármacos , Translocação Genética/efeitos dos fármacos , Translocação Genética/genética , Linhagem Celular Tumoral , Células HL-60 , Humanos , Células K562 , Células Mieloides/efeitos dos fármacosRESUMO
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.
Assuntos
Butadienos/metabolismo , Carcinógenos/metabolismo , Animais , Biomarcadores/metabolismo , Biomarcadores/urina , Butadienos/administração & dosagem , Butadienos/química , Butadienos/urina , Carcinógenos/administração & dosagem , Carcinógenos/química , Cromatografia Líquida de Alta Pressão , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Inalação , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Redes e Vias Metabólicas , Exposição Ocupacional , Ratos , Fumar , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Células Epiteliais/efeitos dos fármacos , Mutagênicos/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Células Epiteliais/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etilnitrosoureia/farmacologia , Etilnitrosoureia/toxicidade , Nucleotídeos de Guanina/farmacologia , Nucleotídeos de Guanina/toxicidade , Humanos , Hipoxantina Fosforribosiltransferase/genética , Lamivudina/farmacologia , Lamivudina/toxicidade , Camundongos , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Fenótipo , Tionucleotídeos/farmacologia , Tionucleotídeos/toxicidade , Testes de Toxicidade , Zidovudina/farmacologia , Zidovudina/toxicidadeRESUMO
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.
Assuntos
Células Epiteliais/efeitos dos fármacos , Mutagênicos/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Testes de Toxicidade , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Calibragem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Exposição Ambiental , Poluentes Ambientais/farmacologia , Poluentes Ambientais/toxicidade , Células Epiteliais/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etilnitrosoureia/farmacologia , Etilnitrosoureia/toxicidade , Nucleotídeos de Guanina/farmacologia , Nucleotídeos de Guanina/toxicidade , Humanos , Lamivudina/farmacologia , Lamivudina/toxicidade , Camundongos , Mutagênicos/farmacologia , Tionucleotídeos/farmacologia , Tionucleotídeos/toxicidade , Zidovudina/farmacologia , Zidovudina/toxicidadeRESUMO
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.
Assuntos
Rearranjo Gênico do Linfócito T/imunologia , Loci Gênicos/imunologia , Região de Junção de Imunoglobulinas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , VDJ Recombinases/fisiologia , Criança , Estudos de Coortes , Feminino , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Humanos , Recém-Nascido , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/imunologiaRESUMO
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.
Assuntos
Poluentes Atmosféricos/toxicidade , Butadienos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Reparo do DNA , Compostos de Epóxi/metabolismo , Poluentes Atmosféricos/metabolismo , Animais , Butadienos/metabolismo , Carcinógenos/metabolismo , DNA/metabolismo , DNA Glicosilases/genética , Deleção de Genes , Humanos , Camundongos , Ratos , Ratos Endogâmicos F344 , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
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 â¼200 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 â¼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.
Assuntos
Biomarcadores/metabolismo , Butadienos/toxicidade , Animais , Adutos de DNA , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/genética , Masculino , Camundongos , Mutação , Ratos , Medição de RiscoRESUMO
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.
Assuntos
Butadienos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Compostos de Epóxi/toxicidade , Linfócitos/efeitos dos fármacos , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade , Butadienos/administração & dosagem , Butadienos/química , Butadienos/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Humanos , Linfócitos/citologia , Mutagênese/genética , Testes de Mutagenicidade/métodos , Mutagênicos/administração & dosagem , Mutagênicos/química , Mutagênicos/metabolismo , Exposição Ocupacional/efeitos adversos , EstereoisomerismoRESUMO
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.
Assuntos
Butadienos/química , Carcinógenos/química , Cromatografia Líquida de Alta Pressão/métodos , Adutos de DNA/análise , Desoxiadenosinas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenina/metabolismo , Animais , Butadienos/toxicidade , Carcinógenos/toxicidade , DNA/metabolismo , Desoxiadenosinas/química , Compostos de Epóxi/química , Compostos de Epóxi/toxicidade , Inalação , Camundongos , RatosRESUMO
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.
Assuntos
Butadienos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Compostos de Epóxi/metabolismo , Pirrolidinas/metabolismo , Valina/análogos & derivados , Valina/metabolismo , Animais , Butadienos/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Cromatografia Líquida de Alta Pressão , Adutos de DNA/análise , Compostos de Epóxi/análise , Feminino , Exposição por Inalação , Masculino , Camundongos , Camundongos Endogâmicos , Pirrolidinas/análise , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Valina/análise , Valina/químicaRESUMO
To delineate temporal changes in the integrity and function of mitochondria/cardiomyocytes in hearts from mice exposed in utero to commonly used nucleoside analogs (NRTIs), CD-1 mice were exposed in utero to 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during days 12-18 of gestation and hearts from female mouse offspring were examined at 13 and 26 weeks postpartum. Alterations in cardiac mitochondrial DNA (mtDNA) content, oxidative phosphorylation (OXPHOS) enzyme activities, mtDNA mutations, and echocardiography of NRTI-exposed mice were assessed and compared with findings in vehicle-exposed control mice. A hybrid capture-chemiluminescence assay showed significant twofold increases in mtDNA levels in hearts from AZT- and AZT/3TC-exposed mice at 13 and 26 weeks postpartum, consistent with near doubling in mitochondrial numbers over time compared with vehicle-exposed mice. Echocardiographic measurements at 13 and 26 weeks postpartum indicated progressive thinning of the left ventricular posterior wall in NRTI-exposed mice, relative to controls, with differences becoming statistically significant by 26 weeks. Overall, progressive functional changes occurred in mouse mitochondria and cardiac tissue several months after in utero NRTI exposures; AZT and 3TC acted in concert to cause additive cardiotoxic effects of AZT/3TC compared with either drug alone.
Assuntos
Fármacos Anti-HIV/toxicidade , Coração/efeitos dos fármacos , Lamivudina/toxicidade , Miocárdio/patologia , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Zidovudina/toxicidade , Animais , DNA Mitocondrial/análise , DNA Mitocondrial/efeitos dos fármacos , Interações Medicamentosas , Quimioterapia Combinada , Ecocardiografia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Coração/crescimento & desenvolvimento , Coração/fisiopatologia , Medições Luminescentes/métodos , Exposição Materna , Troca Materno-Fetal , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/ultraestrutura , Fosforilação Oxidativa , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Fatores de TempoRESUMO
The current study was designed to delineate temporal changes in cardiomyocytes and mitochondria at the light and electron microscopic levels in hearts of mice exposed transplacentally to commonly used nucleoside analogs (NRTIs). Pregnant CD-1 mice were given 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during the last 7 days of gestation, and hearts from female mouse pups were examined at 13 and 26 weeks postpartum for histopathological or ultrastructural changes in cross-sections of both the ventricles and the interventricular septum. Using light microscopy and special staining techniques, transplacental exposure to AZT, 3TC, or AZT/3TC was shown to induce significant histopathological changes in myofibrils; these changes were more widespread at 13 weeks than at 26 weeks postpartum. While most light microscopic lesions resolved, some became more severe between 13 and 26 weeks postpartum. Transplacental NRTI exposure also resulted in progressive drug-specific changes in the number and ultrastructural integrity of cardiac mitochondria. These light and electron microscopic findings show that a subset of changes in cardiac mitochondria and myofibrils persisted and progressed months after transplacental exposure of an animal model to NRTIs, with combined AZT/3TC exposure yielding additive effects compared with either drug alone.
Assuntos
Fármacos Anti-HIV/toxicidade , Coração/efeitos dos fármacos , Lamivudina/toxicidade , Miocárdio/patologia , Inibidores da Transcriptase Reversa/toxicidade , Zidovudina/toxicidade , Animais , DNA Mitocondrial/biossíntese , DNA Mitocondrial/genética , Interações Medicamentosas , Ecocardiografia , Feminino , Feto/patologia , Coração/crescimento & desenvolvimento , Masculino , Troca Materno-Fetal , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/ultraestrutura , Mutação/efeitos dos fármacos , Miocárdio/ultraestrutura , Tamanho do Órgão/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Caracteres SexuaisRESUMO
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.