RESUMEN
Drinking of arsenic (As) contaminated well water has become a serious threat to the health of many millions in Bangladesh. However, the implications of contamination of agricultural soils from long-term irrigation with As-contaminated groundwater for phyto-accumulation in food crops, and thence dietary exposure to As, and other metals, has not been assessed previously in Bangladesh. Various vegetables were sampled in Samta village in the Jessore district of Bangladesh, and screened for As, Cd, Pb, Cu and Zn by inductively coupled plasma emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). These local food products are the basis of human nutrition in this region and of great relevance to human health. The results revealed that the individual vegetables containing the highest mean As concentrations microg x g(-1)) are snake gourd (0.489), ghotkol (0.446), taro (0.440), green papaya (0.389), elephant foot (0.338) and Bottle ground leaf (0.306), respectively. The As concentration in fleshy vegetable material is low. In general, the data show the potential for some vegetables to accumulate heavy metals with concentrations of Pb greater than Cd. Some vegetables such as bottle ground leaf, ghotkol, taro, eddoe and elephant foot had much higher concentrations of Pb. Other leafy and root vegetables, contained higher concentrations of Zn and Cu. Bioconcentration factors (BCF) values, based on dry weight, were below 1 for all metals. In most cases, BCF values decreased with increasing metal concentrations in the soil. From the heavily As-contaminated village in Samta, BCF values for As in ladies finger, potato, ash gourd, brinjal, green papaya, ghotkol and snake gourd were 0.001, 0.006, 0.006, 0.014, 0.030, 0.034 and 0.038, respectively. Considering the average daily intake of fresh vegetables per person per day is only 130 g, all the vegetables grown at Samta had Pb concentrations that would be a health hazard for human consumption. Although the total As in the vegetables was less than the recommended maximum intake of As, it still provides a significant additional source of As in the diet.
Asunto(s)
Arsénico/análisis , Exposición a Riesgos Ambientales , Contaminación de Alimentos , Metales Pesados/análisis , Verduras/química , Bangladesh , Dieta , Monitoreo del Ambiente , Contaminación de Alimentos/análisis , Humanos , Espectrometría de Masas , Salud Pública , Medición de Riesgo , Abastecimiento de AguaRESUMEN
The concentrations of 13 elements were determined in the muscle, liver, intestine, kidney, and gonads of cultured and wild carp caught at two sites in Lake Kasumigaura, Japan, between September 1994 and September 1995. Despite having a reputation for being heavily polluted, the carp were not heavily burdened with metals. Our results suggest that despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in the same manner and that aquaculture practices are not increasing metal concentrations in these fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The differences in metal concentrations between cultivated and wild carp are negligible and should pose no health problems for consumers of either type of fish.
Asunto(s)
Carpas , Metales Pesados/farmacocinética , Oligoelementos/farmacocinética , Alimentación Animal , Animales , Animales Salvajes , Acuicultura , Dieta , Contaminación de Alimentos , Japón , Metales Pesados/análisis , Medición de Riesgo , Distribución Tisular , Oligoelementos/análisisRESUMEN
The mechanism of arsenic toxicity is believed to be due to the ability of arsenite (As(III)) to bind protein thiols. Glutathione (GSH) is the most abundant cellular thiol, and both GSH and GSH-related enzymes are important antioxidants that play an important role in the detoxification of arsenic and other carcinogens. The effect of arsenic on the activity of a variety of enzymes that use GSH has been determined using purified preparations of glutathione reductase (GR) from yeast and bovine glutathione peroxidase (GPx) and equine glutathione S-transferase (GST). The effect on enzyme activity of increasing concentrations (from 1 microM to 100 mM) of commercial sodium arsenite (As(III)) and sodium arsenate (As(V)) and a prepared arsenic(III)-glutathione complex [As(III)(GS)(3)] and methylarsenous diiodide (CH(3)As(III)) has been examined. GR, GPx, and GST are not sensitive to As(V) (IC(50) > 50 mM), and none of the enzymes are inhibited or activated by physiologically relevant concentrations of As(III), As(III)(GS)(3), or CH(3)As(III), although CH(3)As(III) is the most potent inhibitor (0.3 mM < IC(50) < 1.5 mM). GPx is the most sensitive to arsenic treatment and GST the least. Our results do not implicate a direct interaction of As with the glutathione-related enzymes, GR, GPx, and GST, in the mechanism of arsenic toxicity. CH(3)As(III) is the most effective inhibitor, but it is unclear whether this product of arsenic metabolism is produced at a sufficiently high concentration in critical target tissues to play a major role in either arsenic toxicity or carcinogenesis.
Asunto(s)
Arsenicales/metabolismo , Arsenicales/farmacología , Carcinógenos/toxicidad , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Transferasa/antagonistas & inhibidores , Carcinógenos/metabolismo , Relación Dosis-Respuesta a Droga , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
The molecular mechanism of arsenic toxicity is believed to be due to the ability of arsenite [As(III)] to bind protein thiols. Numerous studies have shown that arsenic is cytotoxic at micromolar concentrations. Micromolar As can also induce chromosomal damage and inhibit DNA repair. The mechanism of arsenic-induced genotoxicity is very important because arsenic is a human carcinogen, but not a mutagen, and there is a need to establish recommendations for safe levels of As in the environment. We have measured the dose-response for arsenic inhibition of several purified human DNA repair enzymes, including DNA polymerase beta, DNA ligase I and DNA ligase III and have found that most enzymes, even those with critical SH groups, are very insensitive to As. Many repair enzymes are activated by millimolar concentrations of As(III) and/or As(V). Only pyruvate dehydrogenase, one of eight purified enzymes examined so far, is inhibited by micromolar arsenic. In contrast to the purified enzymes, treatment of human cells in culture with micromolar arsenic produces a significant dose-dependent decrease in DNA ligase activity in nuclear extracts from the treated cells. However, the ligase activity in extracts from untreated cells is no more sensitive to arsenic than the purified enzymes. Our results show that direct enzyme inhibition is not a common toxic effect of As and that only a few sensitive enzymes are responsible for arsenic-induced cellular toxicity. Thus, arsenic-induced co-mutagenesis and inhibition of DNA repair is probably not the result of direct enzyme inhibition, but may be an indirect effect caused by As-induced changes in cellular redox levels or alterations in signal transduction pathways and consequent changes in gene expression.
Asunto(s)
Arsénico/toxicidad , ADN Ligasas/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Mutágenos/toxicidad , Células Cultivadas , ADN Ligasas/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas/metabolismo , Escherichia coli , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidoresRESUMEN
Certain particulate compounds of hexavalent chromium are well-known occupational and environmental human carcinogens. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction; however, the ultimate trivalent oxidation state of chromium, Cr(III), predominates within the cell. DNA-bound Cr(III) has been previously shown to decrease the fidelity of replication in the M13 phage mutation assay. This study was done to understand how Cr(III), in the presence of physiological concentrations of magnesium, affects the kinetic parameters of steady-state DNA synthesis in vitro across site-specific O6-methylguanine (m6dG) residues by DNA polymerase beta (pol beta). Cr(III) binds to the short oligomer templates in a dose-dependent manner and stimulates the activity of pol beta. Cr(III) stimulates the mutagenic incorporation of dTTP opposite m6dG more than the nonmutagenic incorporation of dCTP, and thereby Cr(III) further decreases the fidelity of DNA synthesis across m6dG by pol beta. In contrast, Cr(III) does not affect the fidelity of DNA synthesis across the normal template base, dG. Both the enhanced activity and the mutagenic lesion bypass in the presence of Cr(III) may be associated with Cr(III)-dependent stimulation of pol beta binding to DNA as reported here. This study shows some of the mechanisms by which mutagenic chromium affects DNA synthesis.
Asunto(s)
Cromo/farmacología , ADN Polimerasa beta/efectos de los fármacos , ADN Polimerasa beta/metabolismo , Sitios de Unión/efectos de los fármacos , Cromo/metabolismo , ADN/metabolismo , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/metabolismo , Desoxicitidina Monofosfato/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/metabolismo , Activación Enzimática/efectos de los fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Cinética , Moldes Genéticos , Nucleótidos de Timina/metabolismoRESUMEN
Several pathways of DNA repair are essential for maintaining genomic integrity in mammalian cells. Mismatch repair is the final line of defense against polymerase errors during normal cellular replication. Base excision repair removes endogenous DNA damage resulting from normal cellular metabolism. Nucleotide excision repair removes bulky, transcription blocking, lesions resulting from endogenous and environmental insults to the DNA. The role of DNA repair in mammalian development is not well understood. Nevertheless, clues to the essential nature of these processes are evident in the human DNA repair syndromes, in the nature of the interactions between DNA repair and other proteins, and in the phenotypes of genetically engineered, knockout mice lacking functional repair genes. Questions remain: what is the relative importance of endogenous vs. environmental DNA damage and is repair itself critical for normal development or are transcription-repair interactions more crucial?
Asunto(s)
Reparación del ADN , Desarrollo Embrionario y Fetal , Animales , División Celular , Humanos , Ratones , Ratones Noqueados , Transducción de Señal , Transcripción GenéticaRESUMEN
The transgenic cell lines G12 and G10, each with a bacterial gpt gene stably integrated at a single but different position in the Chinese hamster genome, were evaluated for deletion of the gpt transgene following exposures to several clastogens. More than 150 independently cloned G12 and G10 6-thioguanine-resistant mutants have been characterized by polymerase chain reaction (PCR) amplification and Southern blots in this study. Despite differences in the integration sites for the gpt gene in the G12 and G10 cells, PCR amplification of the gpt gene from both cell lines can be performed using the same single set of primers. By PCR deletion screening, about 20% of recovered spontaneous 6-thioguanine resistant (6TG) gpt G12 mutants had deleted the transgene, whereas the deletion mutant frequency was increased to about 50% of the X-ray- and bleomycin-induced G12 mutants. In contrast, both spontaneous and induced deletion frequencies are considerably higher for the G10 cell line. Among spontaneous G10 mutants, up to 50% have deleted the gpt transgene, whereas almost all of the X-ray- and bleomycin-induced G10 mutants have lost the integrated gene sequence. These results are discussed in the context of the transgene integration sites and the influences of the surrounding genome that may render certain genetic regions prone to deletion.
Asunto(s)
Alanina Transaminasa/genética , Eliminación de Gen , Transgenes/genética , Animales , Secuencia de Bases , Southern Blotting , Línea Celular , Cricetinae , Cricetulus , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase beta (pol beta) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol beta. Our results show that m6dG is a strong but not absolute block to replication by pol beta. pol beta exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol beta preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol beta extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo-KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol beta has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG. This suggests that pol beta, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG. Our results also suggest that replication across m6dG by pol beta may contribute to m6dG-induced G --> A transition mutations.
Asunto(s)
ADN Polimerasa I/metabolismo , Reparación del ADN , Guanosina/análogos & derivados , Secuencia de Bases , Replicación del ADN , Guanosina/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Moldes GenéticosRESUMEN
The divalent nickel ion (Ni2+) is one of several metal ions that can substitute for Mg2+ in the activation of DNA polymerases in vitro, but usually with very low efficiency. We have purified and partially characterized a Ni(2+)-binding protein (p40) from HeLa cell extracts that can specifically enhance the polymerase activity of DNA polymerase alpha (pol alpha) and other DNA polymerases in response to Ni2+. This protein, with a molecular mass of 40 kDa, is a single stranded DNA binding protein that binds to a M13 DNA template-primer with an optimum stoichiometry of approximately 90 equiv of protein per equiv of DNA template and enhances the affinity of pol alpha for the primer-template. In the presence of Ni2+, p40 exhibits an increased affinity for DNA. The p40 increased by 3- to 6-fold the rates at which pol alpha and the Klenow fragment of Escherichia coli DNA polymerase I (KF) replicate different DNA templates in response to Ni2+. The low processivity of Ni(2+)-activated pol on primed M13 ssDNA was also enhanced by the presence of p40. The rates of Ni(2+)-dependent replication by inherently more processive enzymes, DNA polymerase delta and T4 DNA polymerase, were not significantly increased by p40 when M13 ssDNA was used as a template; however, p40 did increase the activity of T4 polymerase on an activated calf thymus DNA template. The protein did not stimulate Mg(2+)-activated DNA replication.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Células HeLa/química , Níquel/farmacología , ADN Polimerasa II/metabolismo , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Magnesio/farmacología , Peso Molecular , Níquel/metabolismo , Moldes Genéticos , Timidina Monofosfato/metabolismoRESUMEN
Chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis. Chromium(VI) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable Cr(III) species. Chromium(III) forms tight complexes with biological ligands, such as DNA and proteins, which are slow to exchange. In vitro, CrCl3.6H2O primarily interacts with DNA to form outer shell charge complexes with the DNA phosphates. However, at micromolar concentrations, the Cr(III) binds to a low number of saturable tight binding sites on single-stranded M13 DNA. Additional chromium interacts in a nonspecific manner with the DNA and can form intermolecular DNA cross-links. Although high concentrations of Cr(III) inhibit DNA replication, micromolar concentrations of Cr(III) can substitute for Mg2+, weakly activate the Klenow fragment of E. coli DNA polymerase I (Pol I-KF), and act as an enhancer of nucleotide incorporation. Alterations in enzyme kinetics induced by Cr(III) increase DNA polymerase processivity and the rate of polymerase bypass of DNA lesions. This results in an increased rate of spontaneous mutagenesis during DNA replication both in vitro and in vivo. Our results indicate that chromium(III) may contribute to chromate-induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis.
Asunto(s)
Cromo/toxicidad , Replicación del ADN/efectos de los fármacos , ADN Polimerasa I/efectos de los fármacos , ADN Polimerasa I/metabolismo , ADN de Cadena Simple/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Mutagenicidad , Nucleótidos/metabolismo , Moldes GenéticosRESUMEN
Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium.
Asunto(s)
Carcinógenos/toxicidad , Metales/toxicidad , Mutágenos/toxicidad , Animales , Animales Modificados Genéticamente , Línea Celular , Cromatos/toxicidad , Cricetinae , Cricetulus , Mercurio/toxicidad , Níquel/toxicidad , Solubilidad , Vanadio/toxicidadRESUMEN
An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Células HeLa/efectos de los fármacos , Níquel/farmacología , División Celular/efectos de los fármacos , Escherichia coli/metabolismo , Células HeLa/metabolismo , Humanos , Níquel/metabolismo , Unión Proteica , Estimulación QuímicaRESUMEN
The effects of the carcinogenic metal nickel on DNA polymerase alpha (pol alpha) activity and fidelity have been analyzed. In the absence of Mg2+, the presence of Ni2+ ions at concentrations below 0.25 mM gave rise to a dose-dependent activation of pol alpha as monitored by [3H]dTMP incorporation into an activated DNA template. The apparent Km for Ni(2+)-dependent pol alpha incorporation of dTTP was estimated to be 25 microM, which was about 10 times higher than the Km for Mg2+ (2.3 microM). Above 0.25 mM, Ni2+ caused a dose-dependent inhibition of pol alpha activity and the Ki was calculated to be 1.5 mM. Scatchard analyses showed that Ni2+ binds to affinity-purified pol alpha and associated proteins at two tight binding sites with a Kd of approximately 50 microM and at eight weak binding sites with a Kd of approximately 4 mM. In the presence of 2 mM Mg2+, the addition of Ni2+ to the reactions caused an inhibition of polymerase activity. The inhibition patterns tended to switch from competitive to mixed-type to noncompetitive as a function of Ni2+ concentration. Lastly, Ni2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP.
Asunto(s)
ADN Polimerasa II/metabolismo , Replicación del ADN , Níquel/metabolismo , Citidina Trifosfato/metabolismo , ADN Polimerasa II/efectos de los fármacos , Células HeLa , Humanos , Magnesio/metabolismo , Níquel/farmacologíaRESUMEN
Propylene oxide (PO) is a widely used industrial reagent which is mutagenic and carcinogenic. We have recently shown that a variety of aliphatic epoxides, including propylene oxide, can react with DNA to form hydroxyalkyl adducts at N-3 of cytosine which rapidly undergo hydrolytic deamination to produce uracil adducts. These 3-hydroxyalkyl uracil adducts are stable in DNA and are postulated to be an important class of potentially mutagenic lesions. Mutagenesis at cytosine residues due to PO modification of single-stranded M13mp2/C141 DNA was studied by transfection of modified DNA into SOS and non-SOS induced E. coli host cells. Mutations of the proline (CCC) codon at C141 which result in reversion of the lacZ phenotype (blue plaques) were scored. It was found that PO treatment of single-stranded DNA results in dose-dependent mutagenesis that is highly SOS dependent. The spectrum of base-substitution mutations found at this site differed when PO-modified DNA was transfected into E. coli with different DNA repair backgrounds. These results indicate that propylene oxide induced DNA adducts at template cytosine residues are mutagenic in E. coli and that this mutagenesis is greatly increased by SOS processing. They also show that these lesions may be repaired by one or more mechanisms.
Asunto(s)
Citosina/metabolismo , ADN Glicosilasas , Compuestos Epoxi/toxicidad , Proteínas de Escherichia coli , Mutagénesis , Mutágenos/metabolismo , Mutación Puntual , Respuesta SOS en Genética , Bacteriófago M13/genética , Distribución de Chi-Cuadrado , Daño del ADN , Reparación del ADN , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/metabolismo , Compuestos Epoxi/metabolismo , Escherichia coli/genética , Operón Lac , Mutágenos/toxicidad , N-Glicosil Hidrolasas/metabolismo , Moldes Genéticos , Uracil-ADN GlicosidasaRESUMEN
Several gpt+ transgenic cell lines were derived from hprt V79 cells to study mutagenesis mechanisms in mammalian cells. The G12 cell line was previously shown to be hypermutable by X-rays and UV at the gpt locus compared to the endogenous hprt gene of the parental V79 cells (Klein and Rossman, 1990), and is now shown to be highly mutable by the clastogenic anti-tumor agent bleomycin sulfate. A second transgenic cell line G10, which has a different gpt insertion site, was studied in comparison with G12. Both G12 and G10 cell lines carry the stable gpt locus at a single integration site in the Chinese hamster genome, and neither spontaneously deletes the integrated gpt sequence at a high frequency. Although spontaneous mutation to 6-thioguanine resistance in G10 cells is 3-4 times higher than in G12 cells, the cell lines differ to a much greater extent when mutated by clastogens. In comparison to G12 cells, the gpt locus in G10 cells is up to 13 times more sensitive to bleomycin mutagenesis and 5 times more responsive to X-ray mutagenesis. In contrast, there is much less difference in UV-induced mutagenesis and no differences in mutagenesis induced by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The dose-dependent decrease in survival of the transgenic cells is the same for all mutagens tested, and does not differ from that of V79 cells. Neither transgenic cell line is generally hypermutable, since mutagenesis at an endogenous gene, Na+K+/ATPase, is similar to that of the parental V79 cell line. Although both cell lines can be induced to delete the transgene following clastogen exposure, deletions are not the only recovered mutations, and the cell lines can also be used to study mutations within the PCR recoverable gpt gene. The utility of these transgenic cells to investigate genome position effects related to mammalian mutagenesis mechanisms is discussed.
Asunto(s)
Línea Celular , Hipoxantina Fosforribosiltransferasa/genética , Mutagénesis Sitio-Dirigida , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Bleomicina/toxicidad , Línea Celular/efectos de los fármacos , Línea Celular/efectos de la radiación , Cricetinae , Cricetulus , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Tolerancia a Medicamentos , Eliminación de Gen , Metilnitronitrosoguanidina/toxicidad , Datos de Secuencia Molecular , Mutágenos/toxicidad , Mutación Puntual , Reacción en Cadena de la Polimerasa , Tolerancia a Radiación , ATPasa Intercambiadora de Sodio-Potasio/genética , Rayos UltravioletaRESUMEN
Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.
Asunto(s)
Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/efectos de los fármacos , Níquel/farmacología , Cartilla de ADN/metabolismo , Replicación del ADN/fisiología , Exonucleasas/efectos de los fármacos , Exonucleasas/fisiología , Técnicas In Vitro , Iones , Magnesio/farmacología , Níquel/toxicidad , Inhibidores de la Síntesis del Ácido Nucleico , Nucleótidos/metabolismo , Moldes GenéticosRESUMEN
A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI- (am26), and carry the lacZ delta M15 marker for alpha-complementation in beta-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-alpha, the lactose operon is repressed (off). Furthermore, supF suppression of lacIam26 results in a lactose repressor that has an uninducible, lacIS genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF- mutations in pUB3 prevent suppression of lacIam26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is approximately 0.7 and approximately 1.0 x 10(-6) without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Elements; IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for -SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively). The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.
Asunto(s)
Análisis Mutacional de ADN/métodos , Escherichia coli/genética , Genes Supresores , Mutagénesis Insercional , Plásmidos , Secuencia de Bases , Elementos Transponibles de ADN , Genes Bacterianos , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN de Transferencia/genética , Eliminación de Secuencia , Transformación GenéticaRESUMEN
Cancer epidemiology has identified several metal compounds as human carcinogens. Recent evidence suggests that carcinogenic metals induce genotoxicity in a multiplicity of ways, either alone or by enhancing the effects of other agents. This review summarizes current information on the genotoxicity of arsenic, chromium, nickel, beryllium and cadmium compounds and their possible roles in carcinogenesis. Each of these metals is distinct in its primary modes of action; yet there are several mechanisms induced by more than one metal, including: the induction of cellular immunity and oxidative stress, the inhibition of DNA metabolism and repair and the formation of DNA- and/or protein-crosslinks.
Asunto(s)
Carcinógenos/toxicidad , Metales/toxicidad , Animales , Disponibilidad Biológica , Carcinógenos/metabolismo , Carcinógenos/farmacocinética , Daño del ADN , Reparación del ADN , Exposición a Riesgos Ambientales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metales/metabolismo , Metales/farmacocinética , Neoplasias/inducido químicamente , Neoplasias/epidemiología , Enfermedades Profesionales/inducido químicamente , Enfermedades Profesionales/epidemiologíaRESUMEN
Carcinogenic chromium [Cr(VI)] compounds are reduced intracellularly to DNA- and protein-reactive chromium(III) species. However, the role of Cr(III) ions in chromium-induced genotoxicity remains unclear. We have investigated the effects of chromium(III) binding on DNA replication and polymerase processivity in vitro. Chromium ions bind slowly and in a dose-dependent manner to DNA. Micromolar concentrations of free chromium inhibit DNA replication, but if the unbound chromium is removed by gel filtration, the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template is increased greater than 6-fold relative to the control. This increase is paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. These effects are optimum when very low concentrations of chromium ions are bound to the DNA [3-4 Cr(III) ions per 1000 nucleotide phosphates]. Increased concentrations of chromium lead to the production of DNA-DNA cross-links and inhibition of polymerase activity. These results suggest that low levels of DNA-bound chromium(III) ions may contribute to chromium mutagenesis and carcinogenesis by altering the kinetics and fidelity of DNA replication.
Asunto(s)
Cloruros , Compuestos de Cromo , Cromo/farmacología , Replicación del ADN , ADN de Cadena Simple/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Sitios de Unión , Cromo/metabolismo , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/metabolismo , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , Cinética , Mutagénesis , Desnaturalización de Ácido Nucleico , Moldes GenéticosRESUMEN
DNA--protein complexes isolated from CHO cells treated with at least 10-30 microM potassium chromate exhibited an alteration in the degradation of the DNA by restriction enzymes compared to DNA--protein complexes isolated from untreated cells. This alteration in restriction enzyme digestion of DNA--protein complexes induced by chromate was shown to depend upon the binding of trivalent chromium to the DNA and upon the protein associated with the DNA, since both EDTA pretreatment and protease K reversed the inhibition of restriction enzyme degradation of the DNA. The inhibition of restriction enzyme degradation of DNA--protein complexes may be utilized as an indirect way to detect DNA--protein complexing induced by chromate and perhaps other agents.