The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform
Siegfried Knasmüller - One of the best experts on this subject based on the ideXlab platform.
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single cell gel electrophoresis assays with human derived hepatoma hep g2 cells
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 1999Co-Authors: Christoph Helma, Siegfried KnasmüllerAbstract:Abstract The purpose of the present study was the development of a protocol for detecting chemically-induced DNA damage, using the alkaline single-cell gel electrophoresis (SCGE) assay with human-derived, metabolically competent hepatoma (Hep G2) cells. Previous studies indicated that Hep G2 cells have retained the activities of certain phase I and phase II enzymes and reflect the metabolism of genotoxins in mammals better than other in vitro models which require addition of exogenous activation mixtures. The optimal trypsin concentration for the removal of the cells from the plates were found to be 0.1%. Dimethylsulfoxide, at concentrations up to 2%, was an appropriate solvent for water-insoluble compounds. To determine the optimal exposure periods for mutagen treatment, the time kinetics of comet formation was investigated with genotoxic chemicals representing various classes of Promutagens namely benzo[a]pyrene (B[a]P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and N-nitrosodimethylamine (NDMA) and with N-nitrosomethylurea (NMU). All compounds caused a statistically significant induction in DNA damage. With the Promutagens, comet formation increased gradually as a function of the exposure duration, and reached maximum values between 20–24 h. With NMU, comet induction maximized already after a short exposure (1 h) and remained at a constant level for up to 24 h. Based on these results, the Hep G2/SCGE assay appears to be a suitable approach for investigating DNA damaging potential of chemicals. Further experiments with IQ and B[a]P showed that the assays are highly reproducible. Comparisons of the present results with those from earlier experiments in which other endpoints (induction of sister chromatid exchanges, micronuclei and chromosomal aberrations) were measured in Hep G2 cells, indicated that the sensitivity of the SCGE assays is more or less identical. Since the SCGE assay is less time consuming than other genotoxicity assays we anticipate that it might be a suitable approach to investigate DNA damaging effects of chemicals in the human-derived, metabolically competent cell line.
John E Casida - One of the best experts on this subject based on the ideXlab platform.
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1 3 dichloropropene epoxides intermediates in bioactivation of the Promutagen 1 3 dichloropropene
Chemical Research in Toxicology, 1998Co-Authors: Manfred Schneider, Gary B Quistad, John E CasidaAbstract:1,3-Dichloropropene (1,3-D), a major soil fumigant nematicide, is genotoxic in many types of assays, leading to its classification as possibly carcinogenic in humans. This study tests in three steps the hypothesis that 1,3-D is a Promutagen activated by epoxidation and further reaction of the 1,3-D-epoxides. Stereospecific epoxidation of 1,3-D (examined as the cis/trans mixture and as individual isomers) to the corresponding cis- and trans-1,3-D-epoxides is demonstrated here for the first time, both in vitro in a mouse liver microsome−NADPH system and in vivo in the liver of ip-treated mice, using GC/MS for product identification and quantitation. The cis epoxide is observed in higher yield than the trans epoxide, both in vitro and in vivo, and the cis isomer also reacts slower than the trans isomer with GSH alone or catalyzed by GSH S-transferase. cis- and trans-1,3-D-Epoxides are stable in acetone or chloroform but degrade completely in Me2SO exclusively to 2-chloroacrolein (30 min at 40 °C). Epoxide de...
Christoph Helma - One of the best experts on this subject based on the ideXlab platform.
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single cell gel electrophoresis assays with human derived hepatoma hep g2 cells
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 1999Co-Authors: Christoph Helma, Siegfried KnasmüllerAbstract:Abstract The purpose of the present study was the development of a protocol for detecting chemically-induced DNA damage, using the alkaline single-cell gel electrophoresis (SCGE) assay with human-derived, metabolically competent hepatoma (Hep G2) cells. Previous studies indicated that Hep G2 cells have retained the activities of certain phase I and phase II enzymes and reflect the metabolism of genotoxins in mammals better than other in vitro models which require addition of exogenous activation mixtures. The optimal trypsin concentration for the removal of the cells from the plates were found to be 0.1%. Dimethylsulfoxide, at concentrations up to 2%, was an appropriate solvent for water-insoluble compounds. To determine the optimal exposure periods for mutagen treatment, the time kinetics of comet formation was investigated with genotoxic chemicals representing various classes of Promutagens namely benzo[a]pyrene (B[a]P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and N-nitrosodimethylamine (NDMA) and with N-nitrosomethylurea (NMU). All compounds caused a statistically significant induction in DNA damage. With the Promutagens, comet formation increased gradually as a function of the exposure duration, and reached maximum values between 20–24 h. With NMU, comet induction maximized already after a short exposure (1 h) and remained at a constant level for up to 24 h. Based on these results, the Hep G2/SCGE assay appears to be a suitable approach for investigating DNA damaging potential of chemicals. Further experiments with IQ and B[a]P showed that the assays are highly reproducible. Comparisons of the present results with those from earlier experiments in which other endpoints (induction of sister chromatid exchanges, micronuclei and chromosomal aberrations) were measured in Hep G2 cells, indicated that the sensitivity of the SCGE assays is more or less identical. Since the SCGE assay is less time consuming than other genotoxicity assays we anticipate that it might be a suitable approach to investigate DNA damaging effects of chemicals in the human-derived, metabolically competent cell line.
Manfred Schneider - One of the best experts on this subject based on the ideXlab platform.
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1 3 dichloropropene epoxides intermediates in bioactivation of the Promutagen 1 3 dichloropropene
Chemical Research in Toxicology, 1998Co-Authors: Manfred Schneider, Gary B Quistad, John E CasidaAbstract:1,3-Dichloropropene (1,3-D), a major soil fumigant nematicide, is genotoxic in many types of assays, leading to its classification as possibly carcinogenic in humans. This study tests in three steps the hypothesis that 1,3-D is a Promutagen activated by epoxidation and further reaction of the 1,3-D-epoxides. Stereospecific epoxidation of 1,3-D (examined as the cis/trans mixture and as individual isomers) to the corresponding cis- and trans-1,3-D-epoxides is demonstrated here for the first time, both in vitro in a mouse liver microsome−NADPH system and in vivo in the liver of ip-treated mice, using GC/MS for product identification and quantitation. The cis epoxide is observed in higher yield than the trans epoxide, both in vitro and in vivo, and the cis isomer also reacts slower than the trans isomer with GSH alone or catalyzed by GSH S-transferase. cis- and trans-1,3-D-Epoxides are stable in acetone or chloroform but degrade completely in Me2SO exclusively to 2-chloroacrolein (30 min at 40 °C). Epoxide de...
Augustinus Bader - One of the best experts on this subject based on the ideXlab platform.
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comparison of primary human hepatocytes and hepatoma cell line hepg2 with regard to their biotransformation properties
Drug Metabolism and Disposition, 2003Co-Authors: Stefan Wilkening, Frank Stahl, Augustinus BaderAbstract:Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of Promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food Promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three Promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these Promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes P450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.
