RESUMO
This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15⯵g/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.
Assuntos
Brometos/normas , Carcinógenos/normas , Contaminação de Medicamentos , Indicadores e Reagentes/normas , Mutagênicos/normas , Animais , Brometos/classificação , Brometos/toxicidade , Carcinógenos/toxicidade , Indústria Farmacêutica , Humanos , Indicadores e Reagentes/toxicidade , Mutagênicos/toxicidade , Medição de RiscoRESUMO
This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.
Assuntos
Testes de Mutagenicidade/métodos , Testes de Mutagenicidade/tendências , Medição de Risco , Animais , Aberrações Cromossômicas , Análise Citogenética , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Seguimentos , Humanos , Mutagênicos/toxicidade , Reprodutibilidade dos Testes , Fuso Acromático/efeitos dos fármacosRESUMO
We have developed a model system for assessing the demethylating potential of external agents. Disruption in the DNA methylation pattern was evaluated at the translational level of the Escherichia coli beta-galactosidase coding gene (lacZ). We have constructed a clonal cell line (A4/4 cells) derived from the adenovirus-transformed human embryonic kidney 293 strain. The A4/4 cells contain the E. coli lacZ gene under the control of the mouse metallothionein 1 promoter which is down-regulated by a natural DNA methylation pattern. Furthermore, the lacZ transcription is also regulated by the E. coli lac operator/repressor system and by mouse metallothionein 1 metal responsiveness offering a wide range in lacZ expression. In this system, the beta-galactosidase activity was only recovered in the presence of a demethylating agent such as 5-azacytidine. The demethylating potential of 5-azacytidine, 5-aza 2'-deoxycytidine and sodium butyrate was rapidly assessed by a flow cytometric method using fluorescein di-beta-D galactopyranoside as a fluorescent probe. A tremendous induction of lacZ expression was triggered by these drugs. Analysis of cell cycles showed little disruptions with 5-azacytidine and sodium butyrate, but an important blockage in the S-phase following 5-aza 2'-deoxycytidine treatment was observed. This approach allows a rapid identification and study of environmental demethylating agents.
Assuntos
Azacitidina/análogos & derivados , Azacitidina/análise , Butiratos/análise , DNA Bacteriano/efeitos dos fármacos , Azacitidina/farmacologia , Sequência de Bases , Butiratos/farmacologia , Ácido Butírico , Ciclo Celular/efeitos dos fármacos , Transformação Celular Viral/fisiologia , Células Cultivadas , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Decitabina , Escherichia coli/enzimologia , Escherichia coli/genética , Citometria de Fluxo , Fluorometria , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/citologia , Óperon Lac/efeitos dos fármacos , Óperon Lac/genética , Metilação , Dados de Sequência Molecular , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/genéticaRESUMO
Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.
Assuntos
Técnicas de Cultura de Células/normas , Dano ao DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfoma/tratamento farmacológico , Testes de Mutagenicidade/normas , Mutagênicos/toxicidade , Padrões de Referência , Animais , Células CHO , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Cariotipagem Espectral , Proteína Supressora de Tumor p53/metabolismoRESUMO
beta-Propiolactone (BPL) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are two direct alkylating agents that induce multiple genetic lesions and tumors in the rodent stomach. We measured the kinetics of the induction of DNA damage by using the single-cell gel electrophoresis assay (SCGE) and the induction of gene mutations by using the MutaMouse model in the glandular stomach mucosa of mice exposed to a single oral administration of BPL or MNNG. The aims were to determine the optimal sampling time and to investigate the cause-effect relationship between DNA damage and gene mutations. The induction of comets, evaluated in individual cells with the tail moment, was analyzed 1, 2, 4, 24, and 72 hr after a single oral administration of 25 mg/kg BPL or 20 mg/kg MNNG. The effects of both compounds were most intense at the earlier sampling times (1-2 hr), tailing off 4 hr after treatment and becoming undetectable at 72 hr. The lacZ mutant frequency (MF) was measured 3, 7, 14, 28, and 50 days after a single oral administration of 150 mg/kg BPL or 100 mg/kg MNNG, and 3 and 14 days after a single administration of 25 mg/kg BPL or 20 mg/kg MNNG. The MF was strongly enhanced at the highest doses and all sampling times, the most marked effects being observed 14 days (11.1-fold) and 28 days (19.0-fold) after BPL and MNNG administration, respectively. At the lowest doses, only a small increase in MF ( approximately 2.5- to 3.5-fold) was found at both sampling times. Primary DNA damage detected with SCGE shortly after treatment (1-2 hr) was rapidly (3 days) transformed into stable gene mutations that remained detectable for 50 days. These results illustrate the ability and complementarity of the SCGE and MutaMouse models to assess the genotoxicity of direct alkylating agents in the mouse gastric mucosa in vivo.
Assuntos
Dano ao DNA , Mucosa Gástrica/efeitos dos fármacos , Óperon Lac , Metilnitronitrosoguanidina/farmacologia , Mutação , Propiolactona/farmacologia , Animais , Eletroforese/métodos , Cinética , Camundongos , Camundongos Transgênicos , Mutagênicos/farmacologiaRESUMO
Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.
Assuntos
Testes de Mutagenicidade , Animais , Animais Geneticamente Modificados , Camundongos , Camundongos Transgênicos , Ratos , Ratos Endogâmicos F344 , Manejo de EspécimesRESUMO
As part of the Germ Cell Collaborative Study, we used the positive-selection Muta Mouse model to evaluate the effects of two direct alkylating agents, ethylnitrosourea (ENU) and methyl methanesulfonate (MMS), on male germ cells. The LacZ mutation frequency in seminiferous tubule cells and epididymis spermatozoa was measured 3, 14, 25 and 50 days after a single intraperitoneal (i.p.) administration of 150 mg/kg ENU and 3 and 14 days after a single i.p. administration of 40 mg/kg MMS. Three and 14 days after ENU treatment, the mutation frequency was slightly but significantly increased in seminiferous tubule cells (3.5- and 3.6-fold, respectively), while it remained unchanged in epididymis spermatozoa. After 25 and 50 days, time-dependent increase in the mutation frequency was observed in seminiferous tubule cells (8.9- and 14.3-fold, respectively) and epididymis spermatozoa (3.4- and 7.9-fold, respectively), confirming the sensitivity of premeiotic cells to the mutagenic activity of ENU. Three and 14 days after MMS administration, the mutation frequency remained unchanged in seminiferous tubule cells and epididymis spermatozoa. The inability of Muta Mouse model to reveal the mutagenic activity of MMS was confirmed in bone marrow cells, 14 days after treatment. These data indicate that the Muta Mouse model can be used to detect the induction of gene mutations but not chromosome damage in germ cells.
Assuntos
Etilnitrosoureia/toxicidade , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Bacteriófago lambda/genética , Células da Medula Óssea , Epididimo/citologia , Óperon Lac , Masculino , Camundongos , Testes de Mutagenicidade , Túbulos Seminíferos/citologiaRESUMO
We used the positive selection Muta Mouse model to detect organ-specific activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL), two highly reactive alkylating agents known to induce genetic damage and tumors in rodent stomach when administered orally. Seven days after a single oral administration of MNNG (100 mg/kg) or BPL (150 mg/kg), the mutation frequency in the Muta Mouse stomach increased significantly by 6.4-fold and 8.8-fold, respectively. A slight (1.8-fold) but significant increase in mutation frequency was also observed in the livers of BPL-treated mice, but not in the livers of MNNG-treated mice or the bone marrow of MNNG- and BPL-treated animals. These data indicate that the Muta Mouse model can be used to predict the gastric specificity of genotoxic carcinogens.
Assuntos
Medula Óssea/efeitos dos fármacos , Fígado/efeitos dos fármacos , Metilnitronitrosoguanidina/toxicidade , Mutagênicos/toxicidade , Propiolactona/toxicidade , Estômago/efeitos dos fármacos , Administração Oral , Animais , Reparo do DNA , Masculino , Metilnitronitrosoguanidina/administração & dosagem , Camundongos , Mutagênicos/administração & dosagem , Propiolactona/administração & dosagemRESUMO
The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.
Assuntos
Testes de Mutagenicidade/métodos , Mutagênicos , Salmonella typhimurium/genética , Plasmídeos , Salmonella typhimurium/efeitos dos fármacos , Sensibilidade e EspecificidadeRESUMO
Because of its rapidness, simplicity and potential for automation, the measurement of micronucleated cells in vivo is not only equivalent to the analysis of chromosome aberrations, but often even preferred within routine genotoxicity testing. In order to evaluate the correlation between the in vitro micronucleus assay (MNT) and the in vitro chromosome aberration test (CA), we collected data from four pharmaceutical companies obtained either in Chinese hamster cell lines (CHO-K5, CHO-K1, V79) or in human peripheral blood lymphocytes. Among the 57 compounds included in this comparison, 45 compounds gave rise to concordant results in both assays (26 compounds negative in both assays; 19 compounds positive in both assays). The high percentage of concordance, i.e. about 79% is very promising and can be even increased to about 88% by omitting the 3 aneugenic compounds and 2 compounds inducing endoreduplicated chromosomes which were found positive only in the in vitro MNT. The results are remarkable in particular considering that most of the compounds evaluated are 'standard' pharmaceutical compounds and thus are at most weak inducers of chromosome damage. Our comparison strongly supports that the in vitro micronucleus test is a suitable alternative to the in vitro chromosome aberration assay. Moreover, the MNT has the potential of not only detecting clastogens but additionally aneuploidy inducing chemicals.
Assuntos
Aberrações Cromossômicas , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Animais , Células CHO , Células Cultivadas , Cricetinae , Humanos , Linfócitos , Mutagênicos/toxicidade , Reprodutibilidade dos TestesRESUMO
7H-Dibenzo[c,g]carbazole (DBC) is an environmental pollutant that produces DNA adducts and tumors in mouse liver and skin following subcutaneous injection and topical application. The two synthetic derivatives 5,9-dimethyl-DBC (DMDBC) and N7-methyl-DBC (NMDBC) induce tissue-specific lesions. DNA adducts and tumors are observed only in liver following exposure to DMDBC and only in skin following exposure to NMDBC. We used the positive selection MutaMouse model to measure the induction of mutations in the two target organs, 28 days after a single subcutaneous injection or topical application of DBC, DMDBC and NMDBC. In liver, DBC and DMDBC induced 30- to 50-fold increases in mutant frequency (MF), while NMDBC had only a weak effect, regardless of the route of administration. After topical application, DBC and NMDBC produced 3.4- to 7.9-fold increases in MF in skin, while DMDBC had a weak effect. After subcutaneous injection, the three compounds had no or weak effect in skin. This study shows gene mutations arise in the respective target organs in which primary DNA damage and tumors are observed. These results illustrate the relevance of the MutaMouse model for testing organ-specific mutagens.
Assuntos
Carbazóis/toxicidade , Carcinógenos/toxicidade , Fígado/efeitos dos fármacos , Pele/efeitos dos fármacos , Administração Cutânea , Animais , Injeções Subcutâneas , Óperon Lac , Masculino , Camundongos , Testes de Mutagenicidade , MutaçãoRESUMO
Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.
Assuntos
Testes de Mutagenicidade , Mutagênicos/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Interpretação Estatística de Dados , Metanossulfonato de Etila/toxicidade , Etilnitrosoureia/toxicidade , Cooperação Internacional , Laboratórios , Óperon Lac , Masculino , Mesilatos/toxicidade , Metanossulfonato de Metila/toxicidade , Metilnitrosoureia/toxicidade , Camundongos , Camundongos TransgênicosRESUMO
Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose-response data (Gollapudi et al., 2013), here we present analyses of 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) dose-response data and additional approaches for the determination of genetic toxicity point-of-departure (PoD) metrics. We previously described methods to determine the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously named Td), and the benchmark dose (BMD10 ) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non-linear slope-transition-dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making.
Assuntos
Ecotoxicologia/métodos , Etilnitrosoureia/toxicidade , Metilnitrosoureia/toxicidade , Medição de Risco/métodos , Animais , Benchmarking , Bases de Dados Factuais , Relação Dose-Resposta a Droga , Metanossulfonato de Etila/toxicidade , Humanos , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Nível de Efeito Adverso não ObservadoRESUMO
Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL(10) ) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data.
Assuntos
Relação Dose-Resposta a Droga , Modelos Genéticos , Testes de Mutagenicidade/métodos , Animais , Humanos , Mutação , Nível de Efeito Adverso não Observado , Medição de RiscoAssuntos
Linfoma/genética , Testes de Mutagenicidade/métodos , Testes de Mutagenicidade/normas , Adenosina/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Aberrações Cromossômicas , Relação Dose-Resposta a Droga , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Camundongos , Mutação , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Software , Timidina/toxicidade , Fatores de Tempo , Células Tumorais Cultivadas , Zalcitabina/toxicidadeRESUMO
In order to validate the in vivo micronucleus test in mouse splenocytes using the cytokinesis block method, 14 compounds with various mechanisms of action were tested: three direct alkylating agents (mitomycin C, ethylnitrosourea, beta-propiolactone), seven indirect alkylating agents (cyclophosphamide, benzo[a]pyrene, diethylnitrosamine, dimethylnitrosamine, 4-aminophenol, 4-aminobiphenyl, 1,1-dimethylhydrazine), two intercalating agents (acridine orange, ethidium bromide) and two spindle poisons (vincristine, colchicine). Male mice were dosed once with the compound, and spleen samples were taken 2 or 14 days after treatment. A significant increase in the binucleated micronucleated splenocyte rate was observed with all the alkylating and intercalating agents at at least one sampling time. In contrast, no increase in the binucleated micronucleated splenocyte rate was observed with the spindle poisons. In conclusion, under these experimental conditions, this in vivo test seems appropriate for the detection of clastogenic compounds including compounds that cannot be detected in the bone marrow micronucleus test. The limit of this test, as expected, is the lack of detection of aneugenic compounds.
Assuntos
Testes para Micronúcleos/métodos , Alquilantes/toxicidade , Animais , Substâncias Intercalantes/toxicidade , Masculino , Camundongos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos/estatística & dados numéricos , Mutagênicos/toxicidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fuso Acromático/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Fatores de TempoRESUMO
Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a large cell population, calcium flux was measured in MRC-5 cell line expressing the B2 receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin. This study confirmed that only the B2 receptor is present on the plasma membrane of MRC-5 cells. Bradykinin binding to the B2 receptor was not modified by a B1 agonist (Des-Arg9-bradykinin) or by a B1 antagonist (Des-Arg9-[Leu8]-bradykinin) but was inhibited by a B2 agonist ([Hyp3]-bradykinin) and a B2 antagonist (HOE 140). The source of free calcium was also studied in comparison with ionomycin. The intensity of the calcium peak after binding of bradykinin is independent of the concentration of extracellular calcium. Preincubation with diltiazem or TMB-8 did not modify calcium flux, indicating that transduction of the signal after bradykinin binding in this cell line is independent of voltage-dependent channels and does not require mobilization of intracellular calcium blocked by TMB-8. In conclusion, scanning cytometry can be used to study ligand-receptor binding and to obtain results rapidly from multiple cells. Recording of individual cell variations and kinetics enables identification of active agonists or antagonists and consequently the selection of new compounds.
Assuntos
Bradicinina/metabolismo , Cálcio/metabolismo , Microscopia Confocal/métodos , Receptores da Bradicinina/metabolismo , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Diltiazem/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Humanos , Ionomicina/farmacologia , Pulmão , Receptores da Bradicinina/efeitos dos fármacos , Transdução de SinaisRESUMO
The purpose of this work was to investigate the impact of cell proliferation on liver mutagenesis. The genotoxic hepatocarcinogen 5, 9-dimethyldibenzo[c,g]carbazole (DMDBC) was administered to lacZ transgenic MutaTMMice at a non-hepatotoxic dose of 10 mg/kg, which induces only a slight increase in the liver lacZ mutant frequency (MF). To determine if cell proliferation stimuli enhanced DMDBC mutagenicity, MF was analyzed in mice first receiving DMDBC 10 mg/kg, then approximately 2 weeks later, either carbon tetrachloride (CCl4, a cytotoxic agent inducing regenerative cell proliferation) or phenobarbital (PB, a mitogenic agent inducing direct hyperplasia). In preliminary studies, the extent of cell proliferation induced by CCl4, PB and DMDBC was determined in non-transgenic CD2F1 mice by means of 5-bromodeoxyuridine labeling. The labeling index was significantly increased after CCl4 and PB, while no change was detected with DMDBC. MF was then determined in MutaTMMice 28 days after initial DMDBC treatment. No increase in MF was detected in mice receiving CCl4 or PB alone. A 2- to 3-fold increase in MF was detected in mice treated with 10 mg/kg DMDBC alone. In contrast, MF was markedly increased in mice receiving DMDBC followed by proliferative treatment (15-fold with CCl4 and 25-fold with PB). These results demonstrate that expression of DMDBC-induced mutations in mouse liver largely depends on the induction of cell proliferation (by a cytotoxic or mitogenic stimulus) and illustrate that MutaTMMouse is a valuable tool to investigate the early events of liver carcinogenesis.
Assuntos
Carbazóis/farmacologia , Óperon Lac , Fígado/citologia , Mitógenos/farmacologia , Animais , Tetracloreto de Carbono/farmacologia , Divisão Celular/efeitos dos fármacos , Análise Mutacional de DNA , Frequência do Gene , Fígado/anatomia & histologia , Fígado/química , Fígado/efeitos dos fármacos , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Masculino , Camundongos , Camundongos Transgênicos , Testes de Mutagenicidade , Mutação , Tamanho do Órgão/efeitos dos fármacos , Fenobarbital/farmacologiaRESUMO
5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.
Assuntos
Carbazóis/toxicidade , Carcinógenos Ambientais/toxicidade , Adutos de DNA/metabolismo , Dano ao DNA , Fígado/efeitos dos fármacos , Animais , Reparo do DNA , Replicação do DNA , Cinética , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Reprodutibilidade dos TestesRESUMO
Transgenic mouse mutation assays, such as MutaMouse (lacZ, CD2F1) and Big Blue (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both MutaMouse and Big Blue should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics assays in bone marrow or peripheral blood.