RESUMEN
Single-crystal B4.3C boron carbide is investigated through the pressure-dependence and inter-relation of atomic distances, optical properties and Raman-active phonons up to ~70 GPa. The anomalous pressure evolution of the gap width to higher energies is striking. This is obtained from observations of transparency, which most rapidly increases around 55 GPa. Full visible optical transparency is approached at pressures of >60 GPa indicating that the band gap reaches ~3.5 eV; at high pressure, boron carbide is a wide-gap semiconductor. The reason is that the high concentration of structural defects controlling the electronic properties of boron carbide at ambient conditions initially decreases and finally vanishes at high pressures. The structural parameters and Raman-active phonons indicate a pressure-dependent phase transition in single-crystal (nat)B4.3C boron carbide near 40 GPa, likely related to structural changes in the C-B-C chains, while the basic icosahedral structure appears to be less affected.
RESUMEN
A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations.
Asunto(s)
Aberraciones Cromosómicas , Citometría de Flujo/métodos , Índice Mitótico/métodos , Adulto , Benzo(a)pireno/toxicidad , Ciclofosfamida/toxicidad , Etopósido/toxicidad , Humanos , Linfocitos/efectos de los fármacos , Mitomicina/toxicidad , Análisis de RegresiónRESUMEN
Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal alpha-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.
Asunto(s)
Inhibidores Enzimáticos/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Extractos Vegetales/toxicidad , Salacia/química , Animales , Aberraciones Cromosómicas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Inhibidores de Glicósido Hidrolasas , Humanos , Masculino , Medicina Ayurvédica , Ratones , Ratones Endogámicos , Micronúcleos con Defecto Cromosómico/inducido químicamente , Mutágenos/clasificación , Mutágenos/metabolismo , Extractos Vegetales/clasificación , Extractos Vegetales/metabolismo , Plantas Medicinales , Ratas , Ratas Sprague-Dawley , Proteína Ribosómica S9 , Proteínas Ribosómicas/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMEN
Aristolochic acid (AA), the active compound found in Aristolochia extracts, has been used as a traditional medicine. However, products containing AA were withdrawn from the market in the early 1980s because AA was found to be a potent carcinogen. Some genotoxicity studies of AA were conducted after the carcinogenicity of AA was reported. The purpose of this study was to check the ability of simplified, screening tests for genotoxicity to indicate the genotoxic activities of AA. Four commonly used in vitro genotoxicity endpoints were examined. In a bacterial mutation screening test, AA was mutagenic to tester strains TA98 and TA100 with and without rat liver S9. In the L5178Y mouse lymphoma cell gene mutation test, mutagenic activity was observed at > or = 25 microg/ml with or without S9. A concentration-dependent increase in structural chromosome aberrations was observed in CHO cells, with significant increases at 50 microg/ml without S9 and at 25 microg/ml with S9. Significant increases in micronucleated binucleated cells were observed in CHO cells treated with AA at > or = 25 microg/ml with or without S9. These results demonstrated that the genotoxicity of AA would have been easily detected if simple screening versions of in vitro genotoxicity assays had been used during early product development. It is suggested that simplified screening tests such as those used in this study would be a rapid and economical way of obtaining the preliminary genotoxicity profiles of new substances or products as an aid to decision-making for further development.
Asunto(s)
Ácidos Aristolóquicos/toxicidad , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Animales , Células CHO , Línea Celular Tumoral , Aberraciones Cromosómicas/efectos de los fármacos , Cricetinae , Medios de Cultivo , Histidina/metabolismo , Técnicas In Vitro , Linfoma/genética , Ratones , Pruebas de Micronúcleos , Microsomas Hepáticos/enzimología , Extractos Vegetales/toxicidad , Ratas , Salmonella typhimurium/genética , Fracciones Subcelulares/metabolismoRESUMEN
An improved protocol was developed to detect light-induced clastogenic photoproducts in Chinese hamster ovary (CHO) cells. Dishes (60 mm) containing cells and the test material or vehicle control in 3 mL of phosphate-buffered saline were exposed to light using a SUNTEST CPS solar simulation unit. Importantly, cells were exposed at about 25 cm from the light source, thereby allowing a short exposure time of 2 min. With this exposure the assay was conducted with lids removed during the UV exposure with minimal risk of contamination. After preliminary experiments an exposure of 165.6 mJ/cm(2) UVA: 17.0 mJ/cm(2) UVB was selected for treatments with the different phototoxins. Under these exposure conditions about 10-15% aberrant cells were induced in vehicle control cultures with no or minimal cytotoxicity. The well-known photoclastogens 8-methoxypsoralen (8-MOP) and chlorpromazine (CLZ) were tested. In agreement with published data, 8-MOP and CLZ were clastogenic (lowest observed effect level, LOEL, was 0.0159 microg/mL and 1.03 microg/mL, respectively). In the absence of UV, 8-MOP was clastogenic at a much higher concentration (LOEL 251 microg/mL without UV vs. 0.0159 microg/mL with UV) while CLZ was negative up to a toxic concentration of 35 microg/mL. 7,12-Dimethylbenz[a]anthracene (DMBA), which is photomutagenic in bacteria, was clastogenic at > or =0.005 microg/mL with UV light (without S9) and at > or =2.53 microg/mL with S9 (without UV light). These results demonstrate the utility of the protocol for the detection of photoclastogenicity and expand the characterization of DMBA's photogenotoxic activity.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/toxicidad , Mutágenos/toxicidad , Luz Solar , Animales , Células CHO , Clorpromazina/toxicidad , Aberraciones Cromosómicas , Cricetinae , Metoxaleno/toxicidadRESUMEN
Genotoxicity evaluations have been proposed as regulatory requirements for establishing German MAK values for inhaled fibrous dusts. The objective of this in vitro assay was to assess the potential for para-aramid (p-aramid) respirable-sized, fiber-shaped particulates (RFP) to induce chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. The highest concentration tested in this assay was limited by the physical characteristics of p-aramid RFP. The test substance was suspended in fully supplemented RPMI culture medium with 1% Pluronic F68. All dosing was achieved using a dosing volume of 90% (900 microl/ml), and the vehicle control cultures were treated with 900 microl/ml of fully supplemented RPMI culture medium with 1% Pluronic F68. In the chromosomal aberrations assay, the treatments were either 3 or 19 h without metabolic activation. Cultures were harvested 22 h from the initiation of treatment. Replicated cultures of human whole blood lymphocytes were incubated with p-aramid RFP concentrations of 6.30, 12.6, 25.2, 50.4, 101, 201, and 401 microg/ml. Cultures treated with concentrations to 50.4 microg/ml for 3 h and 6.30, 12.6, 25.2, and 201 microg/ml for 19 h were analyzed for structural and numerical chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The results demonstrated that p-aramid RFP was negative for inducing chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. In addition, we conclude that the utility of these tests for evaluating the genotoxicity of fibrous or particulate materials is questionable.
Asunto(s)
Aberraciones Cromosómicas , Linfocitos/efectos de los fármacos , Mutágenos/toxicidad , Polímeros/toxicidad , Adulto , Células Cultivadas , Humanos , Técnicas In Vitro , Linfocitos/ultraestructura , Microscopía Electrónica de Rastreo , Mitomicina/toxicidad , Pruebas de Mutagenicidad , Inhibidores de la Síntesis del Ácido Nucleico/toxicidadRESUMEN
The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.
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Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Aberraciones Cromosómicas , Daño del ADN , Hepatocitos/efectos de los fármacos , Herbicidas/toxicidad , Mutágenos , Salmonella typhimurium/efectos de los fármacos , Ácido 2,4-Diclorofenoxiacético/toxicidad , Animales , Arachis , Biotransformación , Células CHO , Células Cultivadas , Cricetinae , Reparación del ADN/efectos de los fármacos , Europa (Continente) , Hepatocitos/citología , Hordeum , Masculino , Medicago sativa , Ratones , Microsomas Hepáticos/metabolismo , Pruebas de Mutagenicidad , Poaceae , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Glycine max , Estados UnidosRESUMEN
ARASCO and DHASCO oils are microbially-derived triglycerides rich in arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids, respectively. Both oils were tested for mutagenic activity in three different in vitro mutagenesis assays. All assays were conducted with and without metabolic activation. Neither ARASCO nor DHASCO oil was mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains, nor were the oils mutagenic in the mouse lymphoma TK(+/-) forward mutation assay. The oils showed no clastogenic activity in chromosomal aberration assays performed with Chinese hamster ovary cells. Based on these assays, neither ARASCO nor DHASCO oils appear to have any genotoxic potential.
Asunto(s)
Ácido Araquidónico/toxicidad , Ácidos Docosahexaenoicos/toxicidad , Mutágenos/toxicidad , Animales , Ácido Araquidónico/análisis , Células CHO , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas , Cromosomas/efectos de los fármacos , Cricetinae , ADN/efectos de los fármacos , Ácidos Docosahexaenoicos/análisis , Leucemia L5178/enzimología , Leucemia L5178/genética , Leucemia L5178/patología , Ratones , Pruebas de Mutagenicidad , Aceites/química , Aceites/toxicidad , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Células Tumorales CultivadasRESUMEN
Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity.
Asunto(s)
Pruebas de Mutagenicidad , Rayos Ultravioleta , Animales , Línea Celular , Relación Dosis-Respuesta en la Radiación , Guías como Asunto , FotoquímicaRESUMEN
The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/toxicidad , Pruebas de Micronúcleos/métodos , Mutágenos/toxicidad , Animales , Células de la Médula Ósea/efectos de los fármacos , Carcinógenos/toxicidad , Eritrocitos/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , RatasRESUMEN
The genotoxic and cytotoxic potential of mainstream cigarette smoke condensate (CSC) from a new cigarette that primarily heats tobacco (TOB-HT) was compared with that of CSC from a Kentucky reference low "tar" cigarette (1R4F) representative of the current US cigarette market, and Kentucky Reference 1R5F, representative of ultra-low "tar" cigarettes on the US market. TOB-HT was evaluated at concentrations which induced concentration-dependent positive responses with 1R4F and 1R5F in an in vitro toxicology test battery which included sister chromatid exchange, chromosome aberration, and neutral red cytotoxicity assays in CHO cells, and the Ames bacterial mutagenicity assay. CSC from 1R4F and 1R5F was positive in the Ames assay with Salmonella typhimurium strains TA98, TA100, TA1538 and TA1537, and negative with TA1535, while CSC from TOB-HT was negative in all five strains. CSC from 1R4F and 1R5F cigarettes was positive in sister chromatid exchange (SCE), chromosome aberration (CA) and neutral red cytotoxicity assays, while CSC from the TOB-HT cigarette yielded negative results in all the above endpoints. These data indicate that in these assays the genotoxic and cytotoxic potential of CSC from the new cigarette that primarily heats tobacco is significantly less than CSC from Kentucky reference 1R4F and 1R5F cigarettes, which are representative of cigarettes currently sold in the US.
Asunto(s)
Nicotiana/química , Plantas Tóxicas , Fumar/tendencias , Industria del Tabaco/tendencias , Contaminación por Humo de Tabaco/efectos adversos , Animales , Células CHO , Ciclo Celular , Aberraciones Cromosómicas , Cricetinae , Relación Dosis-Respuesta a Droga , Pruebas de Mutagenicidad , Rojo Neutro , Estándares de Referencia , Intercambio de Cromátides Hermanas , Estados Unidos , United States Federal Trade Commission/normasRESUMEN
Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER/NO ([H2N(CH2)3NH(CH2)4N[N(O)NO-](CH2)3 NH3+]), which generate NO spontaneously with half-lives at 37 degrees C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/-), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the Ames Salmonella plate assay. In the experiments reported here, Salmonella typhimurium strain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mM (3.5 x control) and greater, while SPER/NO was mutagenic at 0.5 mM (2.7 x control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mM, inducing a mutant frequency per survivor that was 2.5 x control, while SPER/NO was mutagenic at 0.5 mM with a mutant frequency of 2.5 x control. When the CHO cells were given 10 repetitive 20 mM DEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 x control. In the L5178Y mouse lymphoma cell TK+/- assay, DEA/NO doubled the mutation rate at 1.82 mM, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mM. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mM that was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mM. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.
Asunto(s)
Mutágenos/toxicidad , Óxido Nítrico/toxicidad , Profármacos/toxicidad , Animales , Células CHO , Cricetinae , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Pruebas de Mutagenicidad , Salmonella typhimurium/genética , Intercambio de Cromátides Hermanas , Células Tumorales CultivadasRESUMEN
Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.
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Aberraciones Cromosómicas , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Animales , Células CHO , Cricetinae , Japón , Estándares de Referencia , Reino Unido , Estados UnidosRESUMEN
Sodium dichloroacetate (DCA) is a drug with potential for treating patients with stroke and head injury. Conflicting evidence has been published on the mutagenic potential of DCA. A series of genetic tests for mutagenicity and clastogenicity was carried out on pharmaceutical grade DCA. Four types of mutagenicity test were included, with and without metabolic activation where appropriate. These studies included: (i) Salmonella and Escherichia coli mutation (Ames) test, (ii) thymidine kinase locus forward mutation in L5178Y mouse lymphoma cells, (iii) tests for chromosomal aberrations in Chinese hamster ovary cells, and (iv) and in vivo rat bone marrow erythroid micronucleus test. In each study, there was no evidence of mutagenic activity attributable to DCA. It is possible that the present test material, of pharmaceutical grade, has fewer impurities than materials studied in previous reports. These data extend, and in some cases contradict, previous published reports on DCA.
Asunto(s)
Ácido Dicloroacético/toxicidad , Mutágenos/toxicidad , Animales , Células CHO/efectos de los fármacos , Cricetinae , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Ratones , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genéticaRESUMEN
BACKGROUND: The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. STUDY DESIGN AND METHODS: Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. RESULTS: Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6). CONCLUSION: Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.
Asunto(s)
Azul de Metileno/farmacología , Mutágenos , Mutación , Timidina Quinasa/genética , Animales , Muerte Celular/efectos de los fármacos , Linfoma/enzimología , Linfoma/patología , RatonesRESUMEN
Perfluoro-n-decanoic acid (PFDA) is a perfluorinated fatty acid that produces hepatomegaly and increased peroxisomal beta-oxidation when administered to rodents. Chlorotrifluoroethylene (CTFE) trimer acid and CTFE tetramer acid are metabolites of the six- and eight-carbon oligomers of CTFE, respectively. They are structurally related to PFDA, and CTFE tetramer acid has caused toxic effects in rodents that are similar to those observed following PFDA administration. Because of the correlation between peroxisome proliferation and hepatocarcinogenesis, CTFE trimer acid, CTFE tetramer acid, and PFDA were evaluated in in vitro and in vivo/in vitro bioassays to assess their potential genotoxic activity. The assays conducted were the Ames Salmonella/microsomal mutagenicity assay, the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus Chinese hamster ovary gene mutation assay, the sister chromatid exchange (SCE) assay, chromosomal aberration assay, and an in vivo/in vitro unscheduled DNA synthesis (UDS) and S-phase DNA synthesis assay. All test articles were negative in the Ames assay, the HGPRT assay, and the SCE assay. In the chromosomal aberration assay CTFE trimer acid and CTFE tetramer acid were negative in cultures with and without S9 metabolic activation. PFDA was also negative in the absence of metabolic activation, but chromosomal aberrations were observed when PFDA was incubated in the presence of S9 fraction. All test articles were negative for inducing UDS but all induced S-phase replicative DNA synthesis 16 hr after administration of the test article to the test animals; only CTFE tetramer acid and PFDA induced S-phase synthesis 48 hr after dosing: the usual timepoint examined for this response.
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Clorofluorocarburos , Ácidos Decanoicos/toxicidad , Fluorocarburos/toxicidad , Hidrocarburos Halogenados/toxicidad , Mutágenos/toxicidad , Animales , Células CHO , Aberraciones Cromosómicas , Cricetinae , ADN/biosíntesis , Hipoxantina Fosforribosiltransferasa/genética , Pruebas de Mutagenicidad , Fase S , Salmonella typhimurium/efectos de los fármacos , Intercambio de Cromátides HermanasRESUMEN
The clastogenic potential of a recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) therapeutic protein formulation was assessed in human peripheral blood lymphocyte (HPBL) cultures. Final formulation, rather than pure protein, was evaluated to simulate exposure conditions encountered in man. Because the formulation excipients (citric acid, sodium phosphate, mannitol, human serum albumin and polyethylene glycol) were found to alter cell-cycle kinetics and interfere with S-9 metabolic activation in vitro, a novel testing sequence and protocol were used to distinguish between rhGM-CSF and excipient effects and to guard against false negative results. Initial trials were performed to establish the volume of formulation alone (with and without rhGM-CSF) that would not cause severe cell-cycle delay, interfere with S-9 metabolic activation or inhibit positive control responses. This maximum tolerated volume of formulation was then incorporated in all dosed and control groups of each respective assay phase to give the same extent of cell-cycle delay. In the main assay, 24-hr HPBL cultures were exposed to dose levels of 0, 50, 100, 200 and 350 mug rhGM-CSF/ml for 24 hr in -S-9 phases and 0, 200, 350, 500 and 650 mug/ml for 2 hr in +S-9 phases, and harvested 24 hr later. No significant increases in the percentage of cells with structural chromosomal aberrations were found in either test phase. These results show that rhGM-CSF is not clastogenic in HPBL at greater than 17,000 times human exposure levels, and demonstrate that valid cytogenetic assay data with formulations containing cytotoxic excipient can be obtained in vitro.
RESUMEN
Previous studies of chromosome stability in the nevoid basal cell carcinoma syndrome have yielded inconsistent results and suffered from small sample sizes and less than optimal controls. We investigated chromosome fragility and sister chromatid exchange in 20 affected individuals from five multiplex pedigrees, and 15 first- or second-degree unaffected relatives. The percentage of case and control cells showing breaks or rearrangements was compared using a test of proportions. A similar procedure was used to compare site-specific sister chromatid exchanges in baseline cultures from affected persons and controls. No significant differences were noted for either chromosome fragility or sister chromatid exchange between the two groups. These results suggest that cancer susceptibility in the nevoid basal cell carcinoma syndrome is not caused by or manifested as chromosome instability.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Carcinoma Basocelular/genética , Intercambio de Cromátides Hermanas , 4-Nitroquinolina-1-Óxido/farmacología , Adulto , Células Cultivadas , Fragilidad Cromosómica , Femenino , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Masculino , Metilnitronitrosoguanidina/farmacología , Persona de Mediana Edad , Mitomicina , Mitomicinas/farmacologíaRESUMEN
Repair of mutagen-induced lesions that result in sister-chromatid exchanges was evaluated in 10 normal individuals. The mutagens used were mitomycin C (MMC), 4-nitroquinoline 1-oxide (4NQO), and N-methyl-N'nitro-N-nitrosoguanidine (MNNG). Cultures of whole blood, freshly purified lymphocytes, or purified lymphocytes cryopreserved for 6 months were analyzed after the mutagen treatments. All 3 mutagens induced reparable damage as evaluated by comparison of sister-chromatid exchanges between cultures that were given time to repair induced damage before 5-bromo-2'-deoxyuridine (BrdUrd) was added to the culture medium with those where BrdUrd was added immediately after the administration of the mutagens (MMC or 4NQO) or at culture initiation (MNNG). Repair of mutagen-induced DNA damage was detected in all 3 culture types; thus cryopreservation did not appear to alter the capacity of lymphocytes to repair mutagen-induced lesions. Quantitative differences in apparent repair capabilities were observed among individuals. Variability also existed among the different culture types within an individual, suggesting that caution should be exercised in interpreting these apparent differences.
Asunto(s)
Reparación del ADN , Linfocitos/fisiología , Intercambio de Cromátides Hermanas , 4-Nitroquinolina-1-Óxido/farmacología , Femenino , Congelación , Humanos , Técnicas In Vitro , Masculino , Metilnitronitrosoguanidina/farmacología , Mitomicina , Mitomicinas/farmacología , Preservación Biológica , FumarRESUMEN
Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (Time 1) and 6 months (Time 6) of cryopreservation and then challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreservation of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.