RESUMO
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
During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.
Assuntos
Toxinas Bacterianas/metabolismo , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/metabolismo , Mutagênicos/efeitos adversos , Exposição Ocupacional/efeitos adversos , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Convulsões/metabolismo , Urânio/efeitos adversos , Guerra do Golfo , Humanos , Militares , Mutação/efeitos dos fármacos , Estados Unidos , VeteranosRESUMO
Fifty Veterans of the first Gulf War in 1991 exposed to depleted uranium (DU) were studied for glycosylphosphatidylinositol-anchor (GPIa) deficient T-cell mutants on three occasions during the years 2009, 2011, and 2013. GPIa deficiency was determined in two ways: cloning assays employing aerolysin selection and cytometry using the FLAER reagent for positive staining of GPIa cell surface proteins. Subsequent molecular analyses of deficient isolates recovered from cloning assays (Nicklas JA et al. [2019]: Environ Mol Mutagen) revealed apparent incomplete selection in some cloning assays, necessitating correction of original data to afford a more realistic estimate of GPIa deficient mutant frequency (MF) values. GPIa deficient variant frequencies (VFs) determined by cytometry were determined in the years 2011 and 2013. A positive but nonsignificant association was observed between MF and VF values determined on the same blood samples during 2013. Exposure to DU had no effect on either GPIa deficient MF or VFs. Environ. Mol. Mutagen. 60:494-504, 2019. © 2019 Wiley Periodicals, Inc.
Assuntos
Glicosilfosfatidilinositóis/deficiência , Mutagênicos/efeitos adversos , Mutação/efeitos dos fármacos , Exposição Ocupacional/efeitos adversos , Convulsões/metabolismo , Linfócitos T/efeitos dos fármacos , Urânio/efeitos adversos , Estudos de Coortes , Glicosilfosfatidilinositóis/metabolismo , Guerra do Golfo , Humanos , Estudos Longitudinais , Militares , VeteranosRESUMO
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
Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI-a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X-linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI-a deficient isolates, real-time analysis for all the 25 autosomal GPI-a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5' region of PIGL (17p12-p22) extending 5' (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000 bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712 bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI-a deficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI-a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously.
Assuntos
Proteínas de Membrana/genética , Mutação , N-Acetilglucosaminiltransferases/genética , Linhagem Celular , Deleção Cromossômica , Metanossulfonato de Etila/toxicidade , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Homozigoto , Humanos , Íntrons , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismoRESUMO
A total of 70 military Veterans have been monitored for HPRT T-cell mutations in five separate studies at 2-year intervals over an 8-year period. Systemic depleted uranium (DU) levels were measured at the time of each study by determining urinary uranium (uU) excretion. Each HPRT study included 30-40 Veterans, several with retained DU-containing shrapnel. Forty-nine Veterans were evaluated in multiple studies, including 14 who were in all five studies. This permitted a characterization of the HPRT mutation assay over time to assess the effects of age, smoking and non-selected cloning efficiencies, as well as the inter- and intra-individual variability across time points. Molecular analyses identified the HPRT mutation and T-cell receptor (TCR) gene rearrangement in 1,377 mutant isolates. An unexpected finding was that in vivo clones of HPRT mutant T-cells were present in some Veterans, and could persist over several years of the study. The calculated HPRT mutant frequencies (MFs) were repeatedly elevated in replicate studies in three outlier Veterans with elevated urinary uranium excretion levels. However, these three outlier Veterans also harbored large and persistent in vivo HPRT mutant T-cell clones, each of which was represented by a single founder mutation. Correction for in vivo clonality allowed calculation of HPRT T-cell mutation frequencies (MutFs). Despite earlier reports of DU associated increases in HPRT MFs in some Veterans, the results presented here demonstrate that HPRT mutations are not increased by systemic DU exposure. Additional battlefield exposures were also evaluated for associations with HPRT mutations and none were found.
Assuntos
Hipoxantina Fosforribosiltransferase/genética , Mutagênicos/toxicidade , Exposição Ocupacional , Urânio/toxicidade , Adulto , Células Cultivadas , Análise Mutacional de DNA , Frequência do Gene , Guerra do Golfo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Militares , Mutação , Urânio/urina , Adulto JovemRESUMO
Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures.
Assuntos
Hipoxantina Fosforribosiltransferase/genética , Mutagênicos/toxicidade , Mutação , Exposição Ocupacional , Urânio/toxicidade , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Frequência do Gene , Guerra do Golfo , Humanos , Hipoxantina Fosforribosiltransferase/química , Estudos Longitudinais , Masculino , Militares , Dados de Sequência MolecularRESUMO
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
A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.
Assuntos
Impressões Digitais de DNA/métodos , Técnicas Genéticas , Repetições de Microssatélites , Primers do DNA , Genótipo , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real-time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.
Assuntos
Degradação Necrótica do DNA , Impressões Digitais de DNA/métodos , Primers do DNA , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specific modification of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modification strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the efficiency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fluorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.
Assuntos
Genômica , Hipoxantina Fosforribosiltransferase/genética , Linfócitos/enzimologia , Mutação , Sequência de Bases , Células Cultivadas , Meios de Cultura , Primers do DNA , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6-plex assays (with and without gender determination) have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender the other without) have been developed for use in four/five color real-time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (approximately 2 h), much less expensive than STR analysis, and uses a real-time PCR instrument which is found in most forensic and molecular biology labs.
Assuntos
Impressões Digitais de DNA/métodos , Polimorfismo de Nucleotídeo Único , Sequências de Repetição em Tandem , Sondas de DNA , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Masculino , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.
Assuntos
Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Cabelo/química , Primers do DNA , Cabelo/crescimento & desenvolvimento , Humanos , Reação em Cadeia da Polimerase , Sequências de Repetição em TandemRESUMO
Folic acid deficiency (FA-) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA- diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p=0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA- diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA- wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA- mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA-) had no effect on cytotoxicity, but FA- diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA- diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.
Assuntos
DNA Glicosilases/deficiência , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Animais , Antineoplásicos Alquilantes/toxicidade , Ensaio de Unidades Formadoras de Colônias , DNA Glicosilases/genética , Deficiência de Ácido Fólico/patologia , Hipoxantina Fosforribosiltransferase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Baço/efeitos dos fármacos , Baço/patologiaRESUMO
Results of a recent molecular epidemiological study of 1,3-butadiene (BD) exposed Czech workers, conducted to compare female to male responses, have confirmed and extended the findings of a previously reported males only study (HEI Research Report 116, 2003). The initial study found that urine concentrations of the metabolites 1,2-dihydroxy-4-(acetyl) butane (M1) and 1-dihydroxy-2-(N-acetylcysteinyl)-3-butene (M2) and blood concentrations of the hemoglobin adducts N-[2-hydroxy-3-butenyl] valine (HB-Val) and N-[2,3,4-trihydroxy-butyl] valine (THB-Val) constitute excellent biomarkers of exposure, both being highly correlated with BD exposure levels, and that GST genotypes modulate at least one metabolic pathway, but that irreversible genotoxic effects such as chromosome aberrations and HPRT gene mutations are neither associated with BD exposure levels nor with worker genotypes (GST [glutathione-S-transferase]-M1, GSTT1, CYP2E1 (5' promoter), CYP2E1 (intron 6), EH [epoxide hydrolase] 113, EH139, ADH [alcohol dehydrogenase]2 and ADH3). The no observed adverse effect level (NOAEL) for chromosome aberrations and HPRT mutations was 1.794 mg/m(3) (0.812 ppm)--the mean exposure level for the highest exposed worker group in this initial study. The second Czech study, reported here, initiated in 2003, included 26 female control workers, 23 female BD exposed workers, 25 male control workers and 30 male BD exposed workers (some repeats from the first study). Multiple external exposure measurements (10 full 8-h shift measures by personal monitoring per worker) over a 4-month period before biological sample collections showed that BD workplace levels were lower than in the first study. Mean 8-h TWA exposure levels were 0.008 mg/m(3) (0.0035 ppm) and 0.397 mg/m(3) (0.180 ppm) for female controls and exposed, respectively, but with individual single 8-h TWA values up to 9.793 mg/m(3) (4.45 ppm) in the exposed group. Mean male 8-h TWA exposure levels were 0.007 mg/m(3) (0.0032 ppm) and 0.808 mg/m(3) (0.370 ppm) for controls and exposed, respectively; however, the individual single 8-h TWA values up to 12.583 mg/m(3) (5.72 ppm) in the exposed group. While the urine metabolite concentrations for both M1 and M2 were elevated in exposed compared to control females, the differences were not significant, possibly due to the relatively low BD exposure levels. For males, with greater BD exposures, the concentrations of both metabolites were significantly elevated in urine from exposed compared to control workers. As in the first study, urine metabolite excretion patterns in both sexes revealed conjugation to be the minor detoxification pathway (yielding the M2 metabolite) but both M1 and M2 concentration values were lower in males in this second study compared to their concentrations in the first, reflecting the lower external exposures of males in this second study compared to the first. Of note, females showed lower concentrations of both M1 and M2 metabolites in the urine per unit of BD exposure than did males while exhibiting the same M1/(M1+M2) ratio, reflecting the same relative utilization of the hydrolytic (producing M1) and the conjugation (producing M2) detoxification pathways as males. Assays for the N,N-(2,3-dihydroxy-1,4-butadyl) valine (pyr-Val) hemoglobin (Hb) adduct, which is specific for the highly genotoxic 1,2,3,4-diepoxybutane (DEB) metabolite of BD, have been conducted on blood samples from all participants in this second Czech study. Any adduct that may have been present was below the limits of quantitation (LOQ) for this assay for all samples, indicating that production of this important BD metabolite in humans is below levels produced in both mice and rats exposed to as little as 1.0 ppm BD by inhalation (J.A. Swenberg, M.G. Bird, R.J. Lewis, Future directions in butadiene risk assessment, Chem. Biol. Int. (2006), this issue). Results of assays for the HB-Val and THB-Val hemoglobin adducts are pending. HPRT mutations, determined by cloning assays, and multiple measures of chromosome level changes (sister-chromatid exchanges [SCE], aberrations determined by conventional methods and FISH) again showed no associations with BD exposures, confirming the findings of the initial study that these irreversible genotoxic changes do not arise in humans occupationally exposed to low levels of BD. Except for lower production of both urine metabolites in females, no female-male differences in response to BD exposures were detected in this study. As in the initial study, there were no significant genotype associations with the irreversible genotoxic endpoints. However, as in the first, differences in the metabolic detoxification of BD as reflected in relative amounts of the M1 and M2 urinary metabolites were associated with genotypes, this time both GST and EH.
Assuntos
Butadienos/administração & dosagem , Butadienos/efeitos adversos , Indústria Química , Exposição Ocupacional/estatística & dados numéricos , Caracteres Sexuais , Acetilcisteína/análogos & derivados , Acetilcisteína/urina , Adulto , Benzeno/análise , Aberrações Cromossômicas/efeitos dos fármacos , República Tcheca/epidemiologia , Feminino , Genótipo , Hemoglobinas/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Masculino , Epidemiologia Molecular , Mutação/genética , Exposição Ocupacional/efeitos adversos , Troca de Cromátide Irmã/efeitos dos fármacos , Troca de Cromátide Irmã/genética , Estireno/análise , Tolueno/análise , Recursos HumanosRESUMO
A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.
Assuntos
Elementos Alu/genética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Processos de Determinação Sexual , Animais , Cromossomos Humanos Y , Primers do DNA , Sondas de DNA , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
Entrez Gene lists four HPRT1 gene pseudogenes (HPRTP1, HPRTP2, HPRTP3, and HPRTP4) mapping to chromosomes 3, 5, 11q, and 11q, respectively, as originally reported by Patel et al. in 1984 (Patel PI, et al. 1984 Somat Cell Mol Genet 10:483-493). However, the Entrez Gene reports for three of the four pseudogenes (HPRTP1, HPRTP3, and HPRTP4) are currently empty. A BLAST search of both GenBank (Homo sapiens) and the human genome found the chromosome 5 associated HPRTP2 sequence and a single chromosome 11q sequence (HPRTP3 or HPRTP4?). This chromosome 11 sequence had a unique 7.2 kb insert, which may explain why it originally appeared to be two separate pseudogenes. No evidence of a chromosome 3 associated sequence was found; however, a sequence highly homologous to HPRT1 was located on chromosome 4. All of these sequences are intronless processed pseudogenes. Lastly, a sequence highly homologous to HPRT1 exon 8 was found on chromosome 10. This homologous sequence was exactly exon 8 of a gene designated PRTFDC1, for phosphoribosyl transferase domain containing 1. This gene with unknown function is almost completely homologous to HPRT1 in exon structure (except for a 21 bp (seven amino acid) insertion in exon 1) and 68% homologous in amino acid sequence.
Assuntos
Expressão Gênica , Hipoxantina Fosforribosiltransferase/genética , Pseudogenes , Animais , Pareamento Incorreto de Bases , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Cricetinae , DNA/metabolismo , Primers do DNA/química , DNA Complementar/metabolismo , Éxons , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Homologia de Sequência do Ácido Nucleico , Linfócitos T/metabolismoRESUMO
The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).
Assuntos
Elementos Alu , Impressões Digitais de DNA/métodos , DNA/análise , Reação em Cadeia da Polimerase/métodos , Animais , Benzotiazóis , Primers do DNA , Sondas de DNA , Diaminas , Feminino , Corantes Fluorescentes , Humanos , Masculino , Compostos Orgânicos , Quinolinas , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Sequências de Repetição em TandemRESUMO
Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long-distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3-60 days), then decreases and holds fairly steady to 2 years of age.