RESUMO
To assess the effects of acrylonitrile (AN) exposure on reproduction, Sprague-Dawley rats (25/sex/group) were exposed to vapor atmospheres of AN via whole-body inhalation at concentrations of 0, 5, 15, 45 (two offspring generations) and 90 ppm (one offspring generation), 6 h daily, 1 litter/generation, through F2 weanlings on postnatal day 28. After approximately 3 weeks of direct exposure following weaning, exposure of the F1 animals at 90 ppm was terminated due to excessive systemic toxicity in the males. There were no exposure-related mortalities in adult animals, no functional effects on reproduction or effects on reproductive organs, and no evidence of cumulative toxicity or of enhanced toxicity in pregnant and lactating dams or in developing animals. Adult systemic toxicity was limited to body weight and/or food consumption deficits in both sexes and generations (greater in males) at 45 and 90 ppm and increased liver weights in the 90 ppm F0 males and females and 45 ppm F1 males. Neonatal toxicity was expressed by F1 offspring weight decrements at 90 ppm. Clinical signs of local irritation during and immediately following exposure were observed at 90 ppm. Microscopic lesions of the rostral nasal epithelium, representing local site-of-contact irritation, were observed in some animals at 5 to 45 ppm. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity over two generations and neonatal toxicity of AN administered to rats via whole-body inhalation was 45 ppm. The NOAEL for reproduction was 90 ppm for the first generation. The NOAEL for parental systemic toxicity was 15 ppm.
Assuntos
Acrilonitrila/toxicidade , Reprodução/efeitos dos fármacos , Acrilonitrila/administração & dosagem , Administração por Inalação , Animais , Câmaras de Exposição Atmosférica , Peso Corporal/efeitos dos fármacos , Colinesterases/sangue , Determinação de Ponto Final , Ciclo Estral/efeitos dos fármacos , Feminino , Crescimento/efeitos dos fármacos , Masculino , Mucosa Nasal/patologia , Tamanho do Órgão/efeitos dos fármacos , Ovário/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Maturidade Sexual , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
Meiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues. If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop. We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex. Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins. Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large "bubble"; repair of this substrate was dependent on Rad1p. Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination.
Assuntos
DNA/química , Heterozigoto , Meiose , Conformação de Ácido Nucleico , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Pareamento Incorreto de Bases , Southern Blotting , Reparo do DNA , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Modelos Genéticos , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , Ácidos Nucleicos Heteroduplexes/química , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
Exonuclease I was originally identified as a 5' --> 3' deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs). We report here that exo1 mutants of S. cerevisiae exhibit subtle but complex defects in meiosis. Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold. Exo1p functions in the same pathway as Msh5p for intergenic recombination. The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation. These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes. In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.
Assuntos
Exodesoxirribonucleases/genética , Proteínas Fúngicas/genética , Meiose , Não Disjunção Genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/ultraestrutura , DNA Fúngico/genética , Exodesoxirribonucleases/fisiologia , Proteínas Fúngicas/fisiologia , Deleção de Genes , Análise HeteroduplexRESUMO
Tandem repeats of the pentanucleotide 5'-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y. -H. Wang and J. D. Griffith, Proc. Natl. Acad. Sci. USA 93:8863-8867, 1996). To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene of Saccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)(12) tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)(48) tract repressed recombination (cold spot activity). In addition, a "pure" tract of (CCGAT)(12) activated both transcription and meiotic recombination. We suggest that the cold spot activity of the (CCGNN)(48) tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol. Cell. Biol. 15:1679-1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes, Genetics 145:661-670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55-66, 1995; L. Xu and N. Kleckner, EMBO J. 16:5115-5128, 1995).
Assuntos
Nucleossomos/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequências de Repetição em Tandem , Ativação Transcricional , Oxirredutases do Álcool , Aminoidrolases , Sequência de Bases , Cruzamentos Genéticos , Proteínas Fúngicas/genética , Conversão Gênica , Regulação Fúngica da Expressão Gênica , Meiose , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Insercional , Pirofosfatases , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae.
Assuntos
Adenosina Trifosfatases , Pareamento Incorreto de Bases , DNA Ligases/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Meiose/fisiologia , Dímeros de Pirimidina/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , DNA Ligases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endonucleases/genética , Endonucleases/fisiologia , Células Eucarióticas/citologia , Células Eucarióticas/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Humanos , Meiose/genética , Modelos Genéticos , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent).
Assuntos
Proteínas de Arabidopsis , Produtos do Gene vpr/fisiologia , Meiose/fisiologia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Actinas/metabolismo , Oxirredutases do Álcool , Aminoidrolases , Diploide , Proteínas Fúngicas/metabolismo , Haploidia , Repetições Minissatélites , Modelos Biológicos , Modelos Genéticos , Plasmídeos , Pirofosfatases , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismoRESUMO
alpha-Methylstyrene (AMS) is a chemical intermediate used in the synthesis of specialty polymers and copolymers. Inhalation studies of AMS were conducted because of the lack of toxicity data and the structural similarity of AMS to styrene, a toxic and potentially carcinogenic chemical. Male and female B6C3F1 mice were exposed to 0, 600, 800, or 1000 ppm AMS 6 h/day, 5 days/week, for 12 days. After 1 exposure, 21% (5/24) of female mice were found dead in the 1000-ppm group, 56% (10/18) in the 800-ppm group, and 6% (1/18) in the 600-ppm concentration group. After 12 exposures, relative liver weights were significantly increased and relative spleen weights were significantly decreased in both male and female mice at all concentrations. No microscopic treatment-related lesions were observed. A decrease in hepatic glutathione (GSH) was associated with AMS exposure for 1 and 5 days. Male and female F344 rats were exposed to 0, 600 or 1000 ppm AMS for 12 days. No mortality or sedation occurred in AMS-exposed rats. Relative liver weights were significantly increased in both males and females after 12 exposures to 600 or 1000 ppm. An increased hyaline droplet accumulation was detected in male rats in both concentration groups; no significant microscopic lesions were observed in other tissues examined. Exposure of male and female F344 rats and male NBR rats to 0, 125, 250 or 500 ppm AMS, 6 h/day for 9 days resulted in increased accumulation of hyaline droplets in the renal tubules of male F344 rats in the 250 and 500 ppm concentration groups. Although AMS and styrene are structurally very similar, AMS was considerably less toxic for mice and more toxic for male rats than styrene.
Assuntos
Estirenos/toxicidade , Administração por Inalação , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Baço/efeitos dos fármacos , Baço/metabolismo , Testes de Toxicidade , VolatilizaçãoRESUMO
Meiotic recombination in yeast is associated with heteroduplex formation. Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected. Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation. Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast. In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene. These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients).
Assuntos
Reparo do DNA/genética , Meiose/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Oxirredutases do Álcool , Alelos , Aminoidrolases , Troca Genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas Fúngicas/genética , Conversão Gênica , Genes Fúngicos , Marcadores Genéticos , Genótipo , Mutação , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Pirofosfatases , Fatores de Transcrição/genéticaRESUMO
A number of enzymes recognize and repair DNA lesions. The DNA-mismatch repair system corrects base-base mismatches and small loops, whereas the nucleotide-excision repair system removes pyrimidine dimers and other helix-distorting lesions. DNA molecules with mismatches or loops can arise as a consequence of heteroduplex formation during meiotic recombination. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, and failure to repair the mismatch results in post-meiotic segregation (PMS). The ratio of gene-conversion to PMS events reflects the efficiency of DNA repair. By examining the PMS patterns in yeast strains heterozygous for a mutant allele with a 26-base-pair insertion, we find that the repair of 26-base loops involves Msh2 (a DNA-mismatch repair protein) and Rad1 (a protein required for nucleotide-excision repair).
Assuntos
Reparo do DNA/fisiologia , DNA Fúngico/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Proteínas Fúngicas/fisiologia , Ácidos Nucleicos Heteroduplexes , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Oxirredutases do Álcool , Aminoidrolases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas Fúngicas/genética , Meiose , Proteína 2 Homóloga a MutS , Mutação , Conformação de Ácido Nucleico , Pirofosfatases , Recombinação Genética , Fatores de Transcrição/genéticaRESUMO
The HIS4-BIK1 interval on chromosome III of Saccharomyces cerevisiae contains a hotspot for meiotic recombination. Previous reports demonstrated that the initiating lesion is a double-stranded break which is subsequently processed in an asymmetric manner. Data presented here show that the efficiency of initiation of meiotic recombination is unaffected by the deletion of flanking ARS elements, and that the distribution of recombinants is not altered in strains heterozygous for these deletions. These results suggest that the initiation of recombination is not affected by the time of replication of the hotspot at HIS4. The data also indicate that altering the direction of replication-fork movement through the HIS4 region does not affect meiotic recombination.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Meiose/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Fatores de Transcrição/genética , Oxirredutases do Álcool , Aminoidrolases , Southern Blotting , Cromossomos/genética , Clonagem Molecular , DNA Fúngico/genética , Plasmídeos , Regiões Promotoras Genéticas , Pirofosfatases , Deleção de SequênciaRESUMO
The purpose of this study was to evaluate the in vivo effects of an acute exposure to low levels of ozone on rat pulmonary alveolar macrophages (PAM). Fisher 344 rats exposed to 0.0, 0.12, 0.8, or 1.5 ppm O3 for 6 h were killed immediately after and 3, 18, 42, or 66 h after ozone exposure and their lungs were lavaged. Compared to sham-exposed (control) rats, exposure to 0.12 ppm O3 had no measurable effect on the total number, labeling index (LI), mitotic index (MI), or morphology of rat alveolar macrophages. The number of neutrophils was significantly (p less than or equal to 0.001) greater than in controls at 3, 18, and 42 h after exposure to 1.5 ppm O3 and 42 h after exposure to 0.8 ppm O3. The number of PAM was approximately twice that of controls 42 and 66 h after exposure to 0.8 and 1.5 ppm O3. There was a significant (p less than or equal to 0.001) increase in PAM MI 42 and 66 h after exposure to 1.5 ppm O3 and 42 h after 0.8 ppm O3. The increase in the number of PAM in mitosis was preceded by an increase in PAM LI. The PAM LI was significantly (p less than or equal to 0.001) greater than controls 18 and 42 h after exposure but returned to near normal levels by 66 h after exposure. There was a transient decrease in the mean nuclear/cytoplasmic ratio of PAM from rats exposed to 1.5 ppm O3 18 and 42 h after exposure due to an increase in the mean PAM cytoplasmic area. Comparison of the PAM population doubling time (Dt) and cell cycle time (Ct) suggest that PAM proliferation played a significant role in the observed increase in PAM following exposure to 0.8 and 1.5 ppm O3. These results highlight the dynamic response of PAM to an acute exposure to ozone and suggest that the proliferative response of pulmonary alveolar macrophages may be a useful indicator of pulmonary damage following inhalation of an irritant oxidant.
Assuntos
Macrófagos/efeitos dos fármacos , Ozônio/farmacologia , Alvéolos Pulmonares/citologia , Animais , Divisão Celular , DNA/biossíntese , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.