The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Kiyoshi Sasaki - One of the best experts on this subject based on the ideXlab platform.
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photo catalogue for the classification of foci in the balb c 3t3 Cell Transformation Assay
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Claire B ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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recommended protocol for the balb c 3t3 Cell Transformation Assay
Mutation Research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
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prevalidation study of the balb c 3t3 Cell Transformation Assay for assessment of carcinogenic potential of chemicals
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Jessica Ponti, B. Munaro, E Sabbioni, Kiyoshi SasakiAbstract:Abstract The Cell Transformation Assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 Cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the Assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the Cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC ( http://monographs.iarc.fr ) as group 3 “not classifiable as to its carcinogenicity to human”, were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the Assay itself. During the study it became clear that selecting the most appropriate concentrations for the Transformation Assay is crucial.
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Photo catalogue for the classification of foci in the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, B. Claire ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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Recommended protocol for the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2011Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
Ayako Sakai - One of the best experts on this subject based on the ideXlab platform.
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photo catalogue for the classification of foci in the balb c 3t3 Cell Transformation Assay
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Claire B ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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recommended protocol for the balb c 3t3 Cell Transformation Assay
Mutation Research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
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prevalidation study of the balb c 3t3 Cell Transformation Assay for assessment of carcinogenic potential of chemicals
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Jessica Ponti, B. Munaro, E Sabbioni, Kiyoshi SasakiAbstract:Abstract The Cell Transformation Assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 Cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the Assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the Cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC ( http://monographs.iarc.fr ) as group 3 “not classifiable as to its carcinogenicity to human”, were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the Assay itself. During the study it became clear that selecting the most appropriate concentrations for the Transformation Assay is crucial.
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Photo catalogue for the classification of foci in the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, B. Claire ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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Recommended protocol for the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2011Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
Makoto Umeda - One of the best experts on this subject based on the ideXlab platform.
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A Bhas 42 Cell Transformation Assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity.
Mutation research, 2010Co-Authors: Ayako Sakai, Dai Muramatsu, Kiyoshi Sasaki, Sachiko Kuroda, Kumiko Hayashi, Shoko Arai, Nobuko Endou, Yeon-mi Lim, Shojiro Yamazaki, Makoto UmedaAbstract:The Bhas 42 Cell Transformation Assay is a short-term system using a clone of the BALB/c 3T3 Cells transfected with an oncogenic murine ras gene (v-Ha-ras). The Assay has previously been reported to be capable of detecting the tumor-initiating and tumor-promoting activities of chemical carcinogens according to the different protocols, an initiation Assay and a promotion Assay, respectively. We applied this short-term Assay to 98 chemicals to characterize the Assay and evaluate its performance for the detection of chemical carcinogenicity. When the Assay results were compared with the existing genotoxicity data, the Bhas 42 Cell Transformation Assay could detect a considerable number of Ames-negative and Ames-discordant carcinogens: and the promotion Assay detected most of those Ames-negative and -discordant carcinogens. This fact suggested that the Bhas 42 Cells behaved as initiated Cells in the Transformation Assay. The performance indices were calculated from the Assay results of 52 carcinogens and 37 non-carcinogens. The concordance was 78%, sensitivity 73%, specificity 84%, positive predictivity 86%, negative predictivity 69%, false negative 27% and false positive 16%. Of these values, the concordance, specificity, negative predictivity and false positive were superior and the other performance indices were equivalent to those of conventional genotoxicity tests. From overall results, we concluded that the accuracy of prediction of chemical carcinogenicity would be improved by introducing the Bhas 42 Cell Transformation Assay into the battery of in vitro Assays.
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Abstract 4365: A Bhas 42 Cell Transformation Assay sensitive to Ames-negative and Ames-discordant carcinogens: Its performance for the prediction of chemical carcinogenicity
Carcinogenesis, 2010Co-Authors: Ayako Sakai, Dai Muramatsu, Kiyoshi Sasaki, Sachiko Kuroda, Kumiko Hayashi, Shoko Arai, Nobuko Endou, Yeon-mi Lim, Shojiro Yamazaki, Makoto UmedaAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: A Cell Transformation Assay (CTA) can detect the carcinogenicity of chemicals, but is classified into a category different from a genotoxicity Assay which has been used for the prediction of chemical carcinogenicity. CTA detects non-genotoxic carcinogens as well as genotoxic ones. Most of non-genotoxic carcinogens are considered to be tumor-promoters and many of them have been detected as promoters in a two-stage BALB/c 3T3 CTA, which mimics a in vivo two-stage carcinogenesis test. The Bhas 42 Cells were established from the BALB/c 3T3 Cells through transfection of v-Ha-ras gene and regarded as initiated Cells in the two-stage carcinogenesis theory. Using the Bhas 42 Cells, a short-term CTA was developed. Bhas 42 CTA is superior to conventional CTAs in cost and labor performance. It consists of an initiation Assay and a promotion Assay to detect initiating activity and promoting activity, respectively. We applied this short-term CTA to 92 chemicals to characterize the Assay and evaluate its performance for the prediction of chemical carcinogenicity. Methods: In the initiation Assay, the Bhas 42 Cells were seeded at a density of 2000 Cells/mL onto 6-well microplates (Day 0), treated with test chemicals from Day 1, when the Cells were sparse, to Day 4, and thereafter cultured in normal medium until Day 21. In the promotion Assay, the Cells were seeded at a density of 7000 Cells/mL, treated with chemicals from Day 4, when the Cells were sub-confluent, to Day 14, and maintained in normal medium until Day 21. The cultures were fixed and stained with Giemsa's solution. The transformed foci were judged on the basis of morphological characteristics. A statistical analysis for the number of transformed foci per well was performed by the multiple comparison using the Dunnett method. Results and Discussion: Our results of Bhas 42 CTA were compared with existing genotoxicity data. Bhas 42 CTA could detect Ames-negative and Ames-discordant chemicals, and the promotion Assay detected most of them, confirming that the Bhas 42 Cells act as initiated Cells in the CTA. The concordance, sensitivity, specificity, positive predictivity, negative predictivity, false positive and false negative were calculated from the Assay results for known carcinogens and non-carcinogens. Among these performance characteristics, the specificity and positive predictivity were high and exceeded 80%. The other performance characteristics were equivalent to those of genotoxicity tests. From these results, we considered that the accuracy of prediction for chemical carcinogenicity would be improved by introducing Bhas 42 CTA into the battery of in vitro Assays. Acknowledgement: This work was supported by the New Energy and Industrial Technology Development Organization/the Ministry of Economy, Trade and Industry of Japan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4365.
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detection of initiating as well as promoting activity of chemicals by a novel Cell Transformation Assay using v ha ras transfected balb c 3t3 Cells bhas 42 Cells
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2005Co-Authors: Shin Asada, Kiyoshi Sasaki, Noriho Tanaka, Ken Takeda, Makoto Hayashi, Makoto UmedaAbstract:Cell Transformation Assay using BALB/c 3T3 Cells, C3H10T1/2 Cells and others, can simulate the two-stage carcinogenesis utilized for formation of transformed foci. A sensitive Cell Transformation Assay for tumor initiators as well as promoters has been developed using a v-Ha-ras-transfected BALB/c 3T3 Cell line, Bhas 42; these Cells are regarded as initiated in the two-stage paradigm of carcinogenesis. To distinguish between initiation and promotion, the initiation Assay involves a 2-day treatment of low-density Cells, obtained one day after plating, with a test chemical, and the promotion Assay involves treatment of near-confluent Cells with a test chemical for a period of 12 days (Day 3-14). When Bhas 42 Cells were treated with tumor initiators, N-methyl-N'-nitro-N-nitrosoguanidine and 3-methylcholanthrene, transformed foci were induced in the initiation Assay but not in the promotion Assay. In contrast, tumor promoters, 12-O-tetradecanoylphorbol-13-acetate, lithocholic acid and okadaic acid, gave negative responses in the initiation Assay but positive responses in the promotion Assay. The results were reproducible with various treatment protocols. Sixteen polycyclic aromatic hydrocarbons were examined using both Assays. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene induced focus formation only in the initiation Assay. Increase of focus formation was observed in the promotion Assay with benzo[e]pyrene, benzo[ghi]perylene, 1-nitropyrene and pyrene. Benz[a]anthracene, benz[b]anthracene, chrysene and perylene showed positive responses in both initiation and promotion Assays. Results of initiation and promotion Assays of acenaphthylene, anthracene, coronene, 9,10-diphenylanthracene, naphthalene and phenanthrene were negative or equivocal. The present Bhas Assays for the detection of either/both initiating and promoting activities of chemicals are sensitive and of high performance compared with other Cell Transformation Assays.
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Detection of initiating as well as promoting activity of chemicals by a novel Cell Transformation Assay using v-Ha-ras-transfected BALB/c 3T3 Cells (Bhas 42 Cells).
Mutation research, 2005Co-Authors: Shin Asada, Kiyoshi Sasaki, Noriho Tanaka, Ken Takeda, Makoto Hayashi, Makoto UmedaAbstract:Cell Transformation Assay using BALB/c 3T3 Cells, C3H10T1/2 Cells and others, can simulate the two-stage carcinogenesis utilized for formation of transformed foci. A sensitive Cell Transformation Assay for tumor initiators as well as promoters has been developed using a v-Ha-ras-transfected BALB/c 3T3 Cell line, Bhas 42; these Cells are regarded as initiated in the two-stage paradigm of carcinogenesis. To distinguish between initiation and promotion, the initiation Assay involves a 2-day treatment of low-density Cells, obtained one day after plating, with a test chemical, and the promotion Assay involves treatment of near-confluent Cells with a test chemical for a period of 12 days (Day 3-14). When Bhas 42 Cells were treated with tumor initiators, N-methyl-N'-nitro-N-nitrosoguanidine and 3-methylcholanthrene, transformed foci were induced in the initiation Assay but not in the promotion Assay. In contrast, tumor promoters, 12-O-tetradecanoylphorbol-13-acetate, lithocholic acid and okadaic acid, gave negative responses in the initiation Assay but positive responses in the promotion Assay. The results were reproducible with various treatment protocols. Sixteen polycyclic aromatic hydrocarbons were examined using both Assays. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene induced focus formation only in the initiation Assay. Increase of focus formation was observed in the promotion Assay with benzo[e]pyrene, benzo[ghi]perylene, 1-nitropyrene and pyrene. Benz[a]anthracene, benz[b]anthracene, chrysene and perylene showed positive responses in both initiation and promotion Assays. Results of initiation and promotion Assays of acenaphthylene, anthracene, coronene, 9,10-diphenylanthracene, naphthalene and phenanthrene were negative or equivocal. The present Bhas Assays for the detection of either/both initiating and promoting activities of chemicals are sensitive and of high performance compared with other Cell Transformation Assays.
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Cell Transformation Assay using balb c 3t3 Cells or bhas 42 Cells for the efficient detection of tumour promoters
Atla-alternatives To Laboratory Animals, 2004Co-Authors: Makoto UmedaAbstract:In this paper, we report three interlaboratory validation studies with special interest in detecting tumour promoters. The former two validation studies were on the improved two-stage in vitro Transformation Assay using Balb/c 3T3 Cells. The third one was on the Bhas 42 Cell Assay. From the results, we could confirm that, on the whole, the improved two-stage Transformation Assay was sensitive, reproducible and reliable, while the Bhas 42 Cell Assay was a very sensitive and reproducible method for detecting tumour-promoting potential. However, there was some variation in the sensitivity among the laboratories. In order to cope with this sensitivity variation, further refinement is necessary, which will increase the applicability of the methods.
Noriho Tanaka - One of the best experts on this subject based on the ideXlab platform.
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recommended protocol for the balb c 3t3 Cell Transformation Assay
Mutation Research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
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photo catalogue for the classification of foci in the balb c 3t3 Cell Transformation Assay
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Claire B ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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prevalidation study of the balb c 3t3 Cell Transformation Assay for assessment of carcinogenic potential of chemicals
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2012Co-Authors: Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, Jessica Ponti, B. Munaro, E Sabbioni, Kiyoshi SasakiAbstract:Abstract The Cell Transformation Assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 Cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the Assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the Cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC ( http://monographs.iarc.fr ) as group 3 “not classifiable as to its carcinogenicity to human”, were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the Assay itself. During the study it became clear that selecting the most appropriate concentrations for the Transformation Assay is crucial.
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Photo catalogue for the classification of foci in the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2012Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Noriho Tanaka, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Susan Salovaara, B. Claire ThomasAbstract:Abstract This catalogue is a display of focus photos representative of the BALB/c 3T3 Cell Transformation Assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the Assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the Assay results.
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Recommended protocol for the BALB/c 3T3 Cell Transformation Assay
Mutation research, 2011Co-Authors: Kiyoshi Sasaki, Dai Muramatsu, Kumiko Hayashi, Ayako Sakai, Albrecht Poth, Susanne Bohnenberger, Thorsten Kunkelmann, Pascal Phrakonkham, Susan Salovaara, Noriho TanakaAbstract:The present protocol has been developed for the BALB/c 3T3 Cell Transformation Assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes Cell culture maintenance, the dose-range finding (DRF) experiment and the Transformation Assay, including cytotoxicity and morphological Transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.
Robert J. Isfort - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the Transforming Potential of Growth and Differentiation Factors in Syrian Hamster Embryo Cells: Reversible and Irreversible Transformation.
In vitro & molecular toxicology, 1999Co-Authors: Robert J. Isfort, Gary A. KerckaertAbstract:The mitogenic growth and differentiation factor (GDFs) oncostatin M (OM), epidermal growth factor (EGF), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor AA (PDGF AA), PDGF AB, and PDGF BB and the anti-mitogenic GDF, transforming growth factor beta one (TGF-beta1), were tested in the 7-day continuous exposure and 24-h transient exposure Syrian hamster embryo (SHE) Cell Transformation Assay to determine their reversible and irreversible transforming potential. OM was negative while EGF, FGF-4, and PDGF AA were positive for statistically significant morphological Transformation (MT) in the 7-day exposure SHE Cell Transformation Assay. PDGF AB and PDGF BB (but not EGF, FGF-4, and PDGF AA) were positive for statistically significant MT in the 24-h transient exposure SHE Cell Transformation Assays. TGF-beta1 was not only negative for the induction of MT in the 7-day and 24-h exposure SHE Cell Transformation Assays, but suppressed the spontaneous background Transformation response. Investigation of the Transformation suppression potential of TGF-beta1 demonstrated that TGF-beta1 was able to irreversibly suppress the Transformation potential of a variety of transforming agents including growth factors, Ames Assay positive carcinogens, and Ames Assay negative carcinogens. PDGF AA and PDGF BB were investigated to better understand the reversible and irreversible Transformation response. Differences in the receptors activated, the proteins phosphorylated by the receptors, and immediate early gene expressed were observed in SHE Cells treated with either PDGF AA or PDGF BB. Importantly, SHE Cells treated with TGF-beta1 and PDGF BB, two GDFs, which modulate SHE Cell Transformation irreversibly, altered DNA methylation; PDGF AA did not demonstrate this effect. Together these data demonstrate that the SHE Cell Transformation Assay can be utilized to evaluate the Transformation potential and mechanism of activation of GDFs.
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Assessing the Predictiveness of the Syrian Hamster Embryo Cell Transformation Assay for Determining the Rodent Carcinogenic Potential of Single Ring Aromatic/Nitroaromatic Amine Compounds
Toxicological sciences : an official journal of the Society of Toxicology, 1998Co-Authors: Gary A. Kerckaert, Robert A. Leboeuf, Robert J. IsfortAbstract:Abstract The pH 6.7 Syrian hamster embryo (SHE) Cell Transformation Assay was used to test the morphological Transformation potential of 5 rodent carcinogenic single ring aromatic/nitroaromatic amine compounds: 2-amino-4-nitrotoluene, 2,4-diaminotoluene, 2,4-dinitrotoluene, o -anisidine hydrochloride, and o -toluidine; and 5 noncarcinogenic single ring aromatic/nitroaromatic amine compounds: 2,6-diaminotoluene, 2,4-dimethoxyaniline hydrochloride, 4-nitro- o -phenylenediamine, p -phenylenediamine dihydrochloride, and HC Blue No. 2. All 5 rodent carcinogens produced significant morphological Transformation in a dose-responsive manner. None of the 5 noncarcinogens yielded significant Transformation at any of the doses tested. Therefore, the concordance between the pH 6.7 SHE Cell Transformation Assay and rodent carcinogenicity for these 10 single ring aromatic/nitroaromatic amine compounds is 100%. In contrast, the concordance between the standard SHE Cell Transformation Assay and rodent carcinogenicity for 13 single ring aromatic/nitroaromatic amine compounds was 62%. For 5 aromatic/nitroaromatic amine compounds which were tested in both standard and pH 6.7 SHE Cell Transformation Assays (i.e., a subset of the above two databases), the concordance between the standard SHE Cell Transformation Assay and the rodent bioAssay was 40%, while the concordance between the pH 6.7 SHE Cell Transformation Assay and the rodent bioAssay was 100%. This relatively high concordance between the pH 6.7 SHE Cell Transformation Assay and rodent bioAssay results demonstrates the utility of the pH 6.7 SHE Cell Transformation Assay for predicting the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds.
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Computerized image analysis of morphologically transformed and nontransformed syrian hamster embryo (SHE) Cell colonies : application to objective SHE Cell Transformation Assay scoring
Carcinogenesis, 1997Co-Authors: Gregg M. Ridder, Gary A. Kerckaert, Robert A. Leboeuf, Sharon B. Stuard, David B. Cody, Robert J. IsfortAbstract:We have developed an automated image analysis system that provides comparable classification of morphologically transformed SHE Cell colonies to the current visual classification method used in the in vitro SHE Cell Transformation Assay. Visual classification of morphologic Transformation in this Assay has been shown to accurately predict the carcinogenic potential of chemical, biological and physical agents. The image analysis system is quantitative, based on measuring features of colony color, texture and growth patterns. A linear combination of feature measurements produces a classification process that agrees with visual assessment 93% of the time. All identifiable sources of error are explored and the method is found to be robust in analyzing nearly 500 colonies from a variety of studies performed over a one year period. The high degree of correlation between the visual classification and the objective measurements of the image analysis system validates the reproducibility of the visual scoring process and serves as a basis for automation of the Assay.
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Induction of micronuclei in Syrian hamster embryo Cells: comparison to results in the SHE Cell Transformation Assay for National Toxicology Program test chemicals.
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 1997Co-Authors: David P. Gibson, Gary A. Kerckaert, Robert A. Leboeuf, Robert J. Isfort, Roger Brauninger, Hussain S Shaffi, Marilyn J. AardemaAbstract:Sixteen chemicals currently being tested in National Toxicology Program (NTP) carcinogenicity studies were evaluated in the Syrian hamster embryo (SHE) Cell in vitro micronucleus Assay. Results from these studies were compared to the results from the SHE Cell Transformation Assay for the same chemicals The overall concordance between induction of micronuclei and Transformation of SHE Cells was 56%, which is far lower that the 93% concordance between these two tests reported previously by Fritzenschaf et al. (1993; Mutation Res. 319, 47-53). The difference between our results appears to be due to differences in the types of chemicals in the two studies. Overall, there is good agreement between the SHE Cell micronucleus and Transformation Assays for mutagenic chemicals, but, as our study highlights, the SHE Cell Transformation Assay has the added utility of detecting nonmutagenic carcinogens. The utility of a multi-endpoint assessment in SHE Cells for carcinogen screening is discussed.
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The low pH Syrian hamster embryo (SHE) Cell Transformation Assay: A revitalized role in carcinogen prediction
Mutation research, 1996Co-Authors: Marilyn J. Aardema, Robert J. Isfort, E. D. Thompson, Robert A. LeboeufAbstract:Abstract A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236, 933–941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964–971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160–179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291–295 and Ashby (1994) 308, 113–114; Madle (1993) Mutation Res., 300, 73–76 and Madle (1994) 308, 111–112; Zeiger (1994) Mutation Res., 304, 309–314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) Cell Transformation Assay alone and in combination with the Salmonella mutation Assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE Cell Transformation Assay and an extensive review of the databases which support our conclusion that the SHE Cell Transformation Assay provides an improved prediction of rodent bioAssay results relative to other in vitro genotoxicity test batteries.