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1.
Int J Oncol ; 14(5): 833-43, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10200332

RESUMO

Fumonisin B1 stimulates apoptosis in a variety of cell types and tissues. We examined the role of sphingolipid changes in fumonisin B1-stimulated apoptosis. Sphinganine accumulated rapidly, sphingosine levels remained unchanged, and ceramides decreased during fumonisin B1 exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to fumonisin B1, sphinganine, or N-acetylsphingosine. Co-exposure to N-acetylsphingosine or beta-chloroalanine, which blocks sphinganine accumulation, partially protected cells from fumonisin B1-induced apoptosis. These results illustrate three sphingolipid-dependent mechanisms for inducing apoptosis: accumulation of excess ceramide, accumulation of excess sphinganine, and depletion of ceramide or complex sphingolipids derived from ceramide.


Assuntos
Apoptose , Ácidos Carboxílicos/farmacologia , Ceramidas/metabolismo , Fumonisinas , Queratinócitos/efeitos dos fármacos , Esfingosina/análogos & derivados , Teratogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ensaio de Unidades Formadoras de Colônias , Fragmentação do DNA/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Esfingolipídeos/farmacologia , Esfingosina/metabolismo , Esfingosina/farmacologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia
2.
Environ Mol Mutagen ; 11(2): 215-23, 1988.
Artigo em Inglês | MEDLINE | ID: mdl-3345738

RESUMO

To determine the positive and negative classification error rates associated with the HTA in our laboratory, F1 sons of TEM-exposed CD-1 male mice were evaluated by the sequential fertility method with subsequent cytogenetic analysis. Males who sired three litters of size 10 or less when mated to primiparous females from either the B6C3F1 or the BCF1 strain were classified as partial steriles. When meiotic chromosome analyses revealed the presence of at least two cells containing multivalent figures, males were classified as translocation heterozygotes. When the fertility evaluation and the cytogenetic analysis were compared, normal fertility was observed on 5 of 83 (6.02%) translocation-bearing F1 males mated to B6C3F1 tester females and on 3 of 83 (3.61%) F1 males mated to BCF1 tester females. Thus, the false-negative error rates were 6.02% and 3.61% with these two tester strains. Multivalent figures were not observed in the meiotic chromosomes of 410 F1 males. Of these, 12 (2.93%) had reduced fertility when mated to the B6C3F1 tester strain as did 7 (1.71%) mated to the BCF1 strain. Thus, the false-positive error rates with these two tester strains were 2.93% for the B6C3F1 strain and 1.71% for the BCF1 strain. Our results indicate that non-zero error rates, both false-positive and false-negative, are associated with the sequential mating method HTA. In addition, the magnitude of these error rates was influenced not only by the tester female strain but also by the genotype of the F1 male.


Assuntos
Infertilidade Masculina/induzido quimicamente , Translocação Genética/efeitos dos fármacos , Trietilenomelamina/toxicidade , Animais , Feminino , Fertilidade/efeitos dos fármacos , Infertilidade Masculina/genética , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Masculino , Meiose/efeitos dos fármacos , Camundongos , Mitose/efeitos dos fármacos , Valor Preditivo dos Testes
3.
Environ Mol Mutagen ; 14(2): 107-14, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2767057

RESUMO

We have developed methods in our laboratory whereby the effects of toxicant exposure on cell proliferation can be evaluated flow cytometrically. We sought to relate the flow cytometric analyses to other biological response measurements. Thus, we exposed P3 cells to increasing concentrations of bromodeoxyuridine (BRdU) and measured sister-chromatid exchange (SCE) frequency, average generation time (AGT), and relative cloning ability. Each of these is well documented (see introduction) to respond to BRdU exposure in a concentration-dependent manner. In this study, SCE frequency remained constant between the concentrations of 2.5 and 10 microM of BRdU. However, a small, but significant, increase in SCE frequency was observed between the concentrations of 10 microM and 50 microM BRdU. A significant increase in AGT was noted in 50 microM BRdU-exposed cells. Relative cloning efficiency decreased in a concentration-dependent manner when cells were cultured for 24, 48, or 72 hours with BRdU. When cell proliferation was assessed by flow cytometric analysis in cells exposed to 0, 10, or 50 microM BRdU, a statistically significant delay in the cell-cycle was observed in BRdU-exposed cells. These results may be interpreted to mean that inhibition of cell proliferation is detected by this type of analysis at toxicant concentrations that induce other biological endpoints. The inclusion of flow cytometric analysis in a test battery to evaluate toxicant effects is warranted.


Assuntos
Bromodesoxiuridina/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Citometria de Fluxo , Troca de Cromátide Irmã/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Teratoma/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco
4.
Environ Mol Mutagen ; 18(2): 139-49, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1879406

RESUMO

In order to determine the relationships among the reduction in relative cloning efficiency (RCE), sister-chromatid exchange (SCE) formation, and interference with progression through the cell-cycle, human teratocarcinoma-derived (P3) cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate. The relationship between SCE and toxicity was quantified, the progression through the cell-cycle was evaluated with flow cytometric methods, and the effects of these chemicals on cell growth and average generation time (AGT) were determined. A strong correlation existed between RCE and SCE (r = -0.978, p less than .001) which was accompanied by an inhibition of growth as evidenced by a significant (p less than .0001) negative linear effect of concentration on the relative cell count from 24 to 72 hours after exposure and by a concentration-dependent increase (p less than .0001) in the AGT. Delays in the transit through S-phase were evident 4 hours after exposure to toxic concentrations of either carcinogen and by 8 to 12 hours post-exposure at the lower concentrations. Increases in the percentage of nuclei in G2 + M, indicative of G2 arrest, occurred from 12 to 24 hours after exposure. One interpretation of these results is that those effects of EMS and MMS exposure which result in S-phase delay and G2 arrest may be those elements common to the induction of SCE and cellular toxicity.


Assuntos
Ciclo Celular/efeitos dos fármacos , Metanossulfonato de Etila/toxicidade , Metanossulfonato de Metila/toxicidade , Troca de Cromátide Irmã , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Técnicas In Vitro , Teratoma , Fatores de Tempo , Células Tumorais Cultivadas
5.
Environ Mol Mutagen ; 27(1): 10-8, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8625943

RESUMO

AHH-1 tk +/- cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (GO/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of subdiploid peak, consistent with cells undergoing apoptosis.


Assuntos
Amsacrina/toxicidade , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Substâncias Intercalantes/toxicidade , Mutagênicos/toxicidade , Inibidores da Topoisomerase II , Linhagem Celular Transformada , Cromossomos Humanos/efeitos dos fármacos , Meios de Cultura Livres de Soro , Dano ao DNA , Citometria de Fluxo , Fase G2/efeitos dos fármacos , Humanos , Timidina Quinase/genética
6.
Environ Mol Mutagen ; 15(1): 10-8, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2298197

RESUMO

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.


Assuntos
Interleucina-2/farmacologia , Linfócitos/efeitos dos fármacos , Mercaptoetanol/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Interpretação Estatística de Dados , Interações Medicamentosas , Proteínas de Choque Térmico/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Técnicas In Vitro , Masculino , Índice Mitótico/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fosforilação , Fito-Hemaglutininas/farmacologia , Ratos , Ratos Endogâmicos F344 , Troca de Cromátide Irmã/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos
7.
Mutat Res ; 142(4): 193-8, 1985 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-3982428

RESUMO

We recently identified and confirmed 8 induced mutations in the N2 and N3 progeny of ethyl methanesulfonate (EMS) treated C57BL/6J mice. Each of these mutations altered specific enzyme activities. These separate mutant sublines have been maintained through several generations as heterozygous mutant carriers. The percent decrease of the specific enzyme activity from normal in each subline was calculated for each generation. Additionally, the percentage of breeders within each mutant subline producing abnormal progeny and the fraction of such breeders' total progeny possessing abnormal activity were determined. The aberrant activity values observed in progeny of a confirmed mutant carrier were all lower than normal. 4 of the mutant sublines had decreases in enzyme activities which were constant across the generations analyzed. 3 of the mutant sublines had decreases in activities which were consistent over early generations but changed significantly in later generations. Another subline with decreased enzyme activity was lost. For 7 of the sublines, the number of progeny having altered activity and the number of breeders producing mutant progeny approximated that expected for single gene inheritance. In the remaining subline, a change in the decrease in enzyme activity probably accounts for the deviation from expected inheritance. Although the phenotypes for these quantitative traits are considered to be quasi-continuous, the data indicate that the mutations are probably of major genes.


Assuntos
Alelos , Enzimas/genética , Metanossulfonato de Etila/toxicidade , Genes , Mutação , Animais , Encéfalo/enzimologia , Feminino , Frutose-Bifosfato Aldolase/genética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fenótipo
8.
Mutat Res ; 310(1): 45-54, 1994 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-7523883

RESUMO

Cells from the human lymphoblastoid cell line, AHH-1, were exposed to two direct-acting mutagens, ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), and to three carcinogens that require metabolic activation to an electrophile, benzo[a]pyrene (B(a)P), 6-aminochrysene (6-AC), and 6-nitrochrysene (6-NC); mutation induction at the HPRT locus was quantified by resistance to 6-thioguanine (6-TGr). Exposure of AHH-1 cells to either EMS or ENU resulted in a concentration-dependent increase in mutant frequency at the HPRT locus. When AHH-1 cells were exposed to B(a)P, the increase in mutant frequency at the HPRT locus was marginally significant linearly and significant quadratically. The 32P-postlabeling assay revealed the formation of DNA adducts derived from (+/-)anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide which may account for the increase in 6-TGr clones. Although DNA adducts could be detected by the 32P-postlabeling assay in both 6-NC- and 6-AC-treated AHH-1 cells, exposure to 6-AC or 6-NC did not result in a concentration-dependent increase in mutant frequency at the HPRT locus. Our results are consistent with the results of previous studies which indicate that EMS and ENU are effective inducers of 6-TGr clones as is B9(a)P when activated to an electrophile. In 6-NC- and 6-AC-exposed cells, low levels of N-hydroxy-6-aminochrysene-derived adducts were detected in only 6-NC-exposed cells. No 6-aminochrysene-1,2-dihydrodiol-derived adducts were detected following 6-NC or 6-AC exposure. Minimal metabolic activation of 6-NC or 6-AC by AHH-1 cells may account for the lack of a positive mutagenic response for either 6-AC or 6-NC.


Assuntos
Hipoxantina Fosforribosiltransferase/genética , Mutagênicos/toxicidade , Alquilantes/toxicidade , Benzo(a)pireno/toxicidade , Biotransformação , Células Cultivadas , Crisenos/toxicidade , Dano ao DNA , Metanossulfonato de Etila/toxicidade , Etilnitrosoureia/toxicidade , Humanos , Mutagênese
9.
Mutat Res ; 474(1-2): 129-37, 2001 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11239970

RESUMO

Coumestrol, a phytoestrogen found in high levels in alfalfa and red clover, is of concern since endocrine disorders have been observed in farm animals exposed to high levels of phytoestrogens. Previous studies found that coumestrol was an effective inducer of DNA strand breaks, micronuclei, and mutations in the Hypoxanthine phosphoribosyl transferase (HPRT) gene of Chinese hamster ovary cells. In the experiments presented here, we extended the previous studies to examine the effect of coumestrol exposure on AHH-1 TK(+/-) human lymphoblastoid cells. Micronuclei were induced with the highest frequency occurring at day 2 after exposure. Flow cytometric analysis of annexin V-FITC-7-aminoactinomycin D stained cells indicated that the primary pathway of cell death was by apoptosis. Mutations were induced in the Thymidine Kinase (TK) gene and were due primarily to the induction of clones with the slow-growth phenotype. Subsequent molecular analysis revealed the loss of exon 4 in the coumestrol-induced clones, indicative of loss-of heterozygosity and consistent with a proposed inhibition of topoisomerase-II activity as a mechanism of action for coumestrol. Taken together, these results suggest that coumestrol exhibits both mutagenic and clastogenic properties in cultured human lymphoblastoid cells.


Assuntos
Cumestrol/toxicidade , Linfócitos/efeitos dos fármacos , Sequência de Bases , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais , Primers do DNA , Estudos de Avaliação como Assunto , Humanos , Perda de Heterozigosidade , Linfócitos/citologia , Linfócitos/enzimologia , Testes para Micronúcleos , Mutação , Timidina Quinase/genética
10.
Mutat Res ; 329(1): 79-96, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7770078

RESUMO

One role of programmed cell death (apoptosis) is the removal of cells with DNA damage from the population. Certain cells, however, are able to suppress the signals for apoptotic cell death and maintain viability. This suggests that the susceptibility of a cell to either undergo apoptosis or escape from the apoptotic death pathways may be an important factor in chemical mutagenesis. In order to provide insight into the role of apoptosis in the recovery of chemically induced mutants, AHH-1 cells were exposed to the chromosomal mutagen, m-amsa, and the percentage of cells undergoing apoptosis or necrosis quantified by flow cytometry. Logistic regression analysis revealed that the primary manner of cell death was by apoptosis. Two specific-locus mutations assays, the tk and the hprt, were utilized as markers for cells with DNA damage and that retained clonogenicity under conditions known to induce apoptosis. Analysis of variance indicated that the concentration-dependent increase in the mutant fraction at the tk locus was significant and the result of the recovery of clones with the slow-growth phenotype. Because this phenotype is thought to reflect chromosomal mutations, these results are consistent with the survival and clonogenicity of damaged cells. This suggests that the ability to recover mutant cells may be influenced by the suppression of or an escape from the apoptotic death pathways.


Assuntos
Amsacrina/toxicidade , Apoptose/efeitos dos fármacos , Análise de Variância , Apoptose/genética , Apoptose/fisiologia , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada , Separação Celular , Sobrevivência Celular , Dano ao DNA , Citometria de Fluxo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Necrose , Proteínas Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Supressão Genética/efeitos dos fármacos
11.
Mutat Res ; 121(3-4): 273-80, 1983 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6312303

RESUMO

We conducted dominant lethal studies with chemical mutagens IMS and TEM to investigate the influence which different treated male stocks might have upon individual female response. In both studies, treated males from a CD-1 random bred stock and each of two F1 hybrid stocks (C57BL/6N X C3H; C57BL/6J X BALB/c) were mated to untreated females from the two F1 hybrid stocks. We found that variation in response due to the treatment effect was modulated by the specific male stock/female stock combination involved. The male-dependent variation observed prevented any meaningful evaluation of relative female stock sensitivity but it was obvious that factors other than differences in repair capacity of female germ cells contributed to the response differences observed.


Assuntos
Mesilatos/toxicidade , Trietilenomelamina/toxicidade , Animais , Feminino , Morte Fetal/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos , Testes de Mutagenicidade , Gravidez
12.
Mutat Res ; 357(1-2): 143-65, 1996 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-8876690

RESUMO

The chromosomal mutagen, bleomycin, is also noted for its toxic properties, although the mechanism of cell death is not fully understood. In order to determine if cell death occurred by apoptosis or necrosis, AHH-1 tk+/- cells were exposed to bleomycin and the percentage of viable, apoptotic and necrotic cells quantified by flow cytometry. Logistic regression analysis indicated that the primary manner of cell death was through the apoptosis pathways, that apoptosis was delayed, and that apoptosis was accompanied by an arrest in the G2 phase of the cell cycle. Once apoptosis was established as a mechanism for cell death, the efficiency with which these pathways removed damaged cells from the population was evaluated with the use of specific-locus mutation assays (tk and hprt) as indicators of cells with DNA damage that maintained viability and clonogenicity. Linear regression analysis detected a significant, concentration-dependent increase in the numbers of TFTr clones with the slow-growth phenotype. This suggests that a proportion of cells with bleomycin-induced DNA damage did not undergo cell death by apoptosis and that apoptosis, a mechanism for the destruction of damaged cells, is not fully efficient in the AHH-1 tk +/- cell line.


Assuntos
Apoptose/efeitos dos fármacos , Bleomicina/toxicidade , Mutagênicos/toxicidade , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Mutagênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Timidina Quinase/metabolismo
13.
Mutat Res ; 306(1): 19-34, 1994 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-7512200

RESUMO

In order to determine the pathway for cell death in alkylating agent-exposed human lymphoblastoid cells, AHH-1 cells were exposed to either ethyl methanesulfonate (EMS) or ethyl nitrosourea (ENU) and the effect on relative cell growth and plating efficiency quantified. Flow cytometric (FCM) assays were utilized to quantify cell viability and to determine if cell death occurred through necrosis or apoptosis. As expected, exposure to the simple ethylating agents resulted in concentration-dependent decreases in plating efficiencies at each time interval after exposure (Days 0, 2, 3 and 7). EMS exposure did not significantly affect the relative cell growth, in contrast to ENU exposure, which inhibited cell growth. The FCM viability assay, based on light scatter characteristics, revealed that exposure to either alkylating agent resulted in a significant reduction in the percentage of viable cells. The results of the FCM dye-exclusion assays revealed that while necrosis occurred in EMS- and ENU-exposed cells, the primary manner of cell death was apoptosis. AHH-1 cells were stained with propidium iodide and fluorescein diacetate, the population of cells sorted electronically and the cell type (necrotic, apoptotic or viable) confirmed morphologically. Our results clearly indicate that exposure to EMS or ENU results in the movement of AHH-1 cells into the pathway for apoptosis and cell death.


Assuntos
Apoptose/efeitos dos fármacos , Metanossulfonato de Etila/toxicidade , Etilnitrosoureia/toxicidade , Mutagênicos/toxicidade , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Humanos , Linfócitos/efeitos dos fármacos
14.
Mutat Res ; 356(2): 129-34, 1996 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-8841477

RESUMO

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.


Assuntos
Genes p53/genética , Genes p53/fisiologia , Mutação Puntual , Apoptose , Células Cultivadas , Éxons , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA
15.
Mutat Res ; 405(1): 41-56, 1998 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-9729267

RESUMO

The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.


Assuntos
Apoptose/efeitos dos fármacos , Genes p53/genética , Genisteína/toxicidade , Mutação/genética , Carcinógenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais/efeitos dos fármacos , Citometria de Fluxo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/farmacologia , Tioguanina/farmacologia , Trifluridina/farmacologia
16.
Mutat Res ; 549(1-2): 43-64, 2004 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-15120962

RESUMO

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Perfilação da Expressão Gênica , Mutagênicos/toxicidade , Sequência de Bases , Células Clonais , Adutos de DNA , Primers do DNA , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
18.
J Hered ; 69(5): 295-8, 1978.
Artigo em Inglês | MEDLINE | ID: mdl-744871

RESUMO

Is abnormal regulation of phaeomelanin synthesis, which results in yellow coat color, inextricably linked to the development of obesity in viable yellow (Avy) house mice? To answer this question, black male mice of genotypes Avy/A eso/e+ and A/ae eso/e+ were produced from (DFT/Wf X C3H-1 vyfB/HeWf X SO/LeWf matings. The sombre (eso) mutation prevents phaeomelanin synthesis; therefore, all mice carrying this dominant allele are black regardless of the alleles present at the agouti locus. These black males were weighed weekly to 18 weeks of age. Simultaneously the agouti locus genotype of each male was determined by test matings to a/a e+/e+ females. The results of these test matings indicated that the two genotypes could be classified rather accurately by the intensity of pigmentation of the belly hair. Black Avy mice tended to have more dilute belly pigmentation that black A/ae mice. These gross observations were confirmed by microscopic examination of several hair types. Black Avy males reached a mean body weight of 44.2 +/- 0.6 g at 18 weeks, while black A males had a mean weight of 30.7 +/- 0.4 g at the same age. It is concluded that excessive weight gain resulting in obesity is induced by the Avy gene even when phaeomelanin synthesis is completely inhibited.


Assuntos
Melaninas/biossíntese , Mutação , Obesidade/genética , Animais , Cruzamentos Genéticos , Genes , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Camundongos Obesos , Fenótipo , Pigmentação
19.
Cell Biol Toxicol ; 4(3): 281-94, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3224305

RESUMO

To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.


Assuntos
Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Testes de Mutagenicidade , Troca de Cromátide Irmã/efeitos dos fármacos , Teratoma , Linhagem Celular , Células Clonais/efeitos dos fármacos , Metanossulfonato de Etila/farmacologia , Humanos , Metanossulfonato de Metila/farmacologia
20.
Cell Biol Toxicol ; 8(1): 75-87, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1591624

RESUMO

Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.


Assuntos
Bromodesoxiuridina/farmacologia , Divisão Celular/efeitos dos fármacos , Metanossulfonato de Etila/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Células Tumorais Cultivadas
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