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P XIV A.5 Metabolic activation of promutagenic carcinogens with human hepatoma (Hep G2) cells
SI24
S-XIV: New mutagenicity tests and their evaluation
Ip XIV A.21
Salmonella spiral assay: Manually countlnt: and data analysis
Lone Lillemarlc, Mona-Lise Binderup, Birgitte Plesing Moller, Vivian Jorgensen. National Food Agency of Denmark, Murkhe} Bygade /9, DK-
2860 Seborg, Denmark A method for manually counting of colonies in the Spiral Salmonella Assay by the use of a template has been established, and a model and a spreadsheet for data analysis have been set up. Upon analysis, each spiral plate yields a dose-response curve consisting of 6 points with a concentrallon range of 13-fold. In the literature the order of application of bacteria. test substance and S9-mix have been discussed as well as the possibility of pouring bacteria andlor S9-mix onto the plate with top agar after spiral plating of the test substance. For evaluation of the method four different concentrations of two known mutagens 2-nitrofluorene (2-NF) and 2-aminoanthracene (2-AA) dissolved in dimethyl sulfoxide (OM SO) were tested. To establish the optimum test conditions the application order of bacteria, test substance and S9-mix (only 2-AA) were varied. Also the application of bacteria and S9-mix were performed with two different techniques: spiral plating and top agar. As it appeared that the relatively high volume of DMSO plated in the first part of the spiral was toxic for the bacteria. the test was repeated with the more volatile solvents diethyl ether and ethanol. The results will be given and discussed. Keyword(s): Spiral Salmonella Assay; Ames test; Volatile solvents
Ip XIV A.31
Deteetlon ofl:enomle DNA damage (ndueed by genotoxins In mammalian eell lines by a ehemllumlneseenee mleroplate DNA repair assay
R.-Y. u', C. Provot l,l.-P'laeg1, C. Bozzato", B. Salles 1. IS.F.R./.. Berganton, 33127 Saint Jean d'J1Jac, France; 2IPBS. UPR 9062 CNRS. 205 route de Narbonne. 3/077 Toulouse. France To easily screen genotoxic compounds, we developed a Damaged DNA Detection assay (3D assay). The 3D-assay is an adaptation of the repair assay initially reported by Wood et al, (I) which takes advantage of an adsoption step of plasmid DNA in sensitized microplate wells. After DNA damaging treatments, digoxigenylated-modified nucleotides were incorporated during the repair synthesis step and their presence was detected by an ELISA-like reaction with chemiluminescence detection (2). Here, we show that this assay allows the analysis of damaged genonuc DNA Isolated from mammalian cells following treatment with chemicals. thus making this method interesting for the following applications: (i) detection of DNA damage, (ii) determination of the repair rate and (iii) evaluation of protective agents. (i) DNA alkylation provoked by EMS and MNNG or oxidative damage induced by H101 were detected. Moreover, genotoxicity of pro-drugs in metabolically competent human cell line expressing cytochromes P450s (3) was tested. Oi) After a post-treatment incubation step in drug free medium, kinetics of DNA repair were determined in different cells line by analysing the decrease amount of DNA damage. (iii) Following H101 treatment the protective effects of different antioxidants (NAC, silymarin ...) was shown. (I) Wood, R. D., Robins, P. & Lindahl, T. (1988) Cell. 53, 97-106. (2) B. Salles, C. Provot et al. (1995) Anal. Biochem., 132,37-42. (3) Crespi (1991) Chern. Res. Toxicol 4, 566-572.
Ip XIV A.41
Evaluation of the restriction site mutallon assay usInl: murine P53 Intron sequenees
Gareth 1.S. Jenkins, James M. Parry. Centre Jor molecular genetics and toxicology. University of Wales Swansea. Singleto" Parle, Swansea. UK
The restriction site mutation assay, which detects DNA mutations in restriction enzyme sites in target regions, was employed to study induced in vivo mutagenesis. The agents used in this study were: N-ethyl-N-nitrosourea, 2-acetylaminotluorene and 1,2-dimethylhydrazine. Mutations were analYsed in four regions of the murine pS3 gene: exon 4, exon 5, intron 6 and intron 8. The results demonstrated that the two intron regions possessed approximately tenfold more mutations compared with the exon regions. This data was supported by mutation frequency calculations, made possible by the inclusion of an internal standard molecule in the RSM assay, which also showed the higher mutability of the intron regions. Intron regions were sufficiently sensitive such that a number of spontaneous mutations were detected. This is the first time that we have detected spontaneous in vivo mutations in the RSM assay. The mechanism of this increased mutation in intron sequences remains unclear, but may be due to differential DNA repair or due to selection differences between the coding exon regions and the noncoding intron regions. Keyword(s): restriction site mutation assay; intran; exon; mutation
Ip XIV A.51
Metabolle aellvatlon of promutagenle carelnogens witb human hepatoma (Hep G2) eells
F. Darroudil,l, S. Knasrniillef, R. Sanyal", C.M. Meijers", A.T. Natarajan l ,2 . J MGC,
Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Leiden, The Netherlands; 2J.A. Cohen Institute. IRS. Leiden; The Netherlands; j Institute of Tumor Biology and cancer Research, Uniuersity of Vienna, Vienna, Austria We have shown that Hep 02 cells can be used as metabolic activation system as well as target for DNA damage. We have isolated S9-fraction from Hep G2 cells and mvestigated its ability to activate promutagenic carcinogens using Chinese hamster ovary (CHO) cells (in vitro) and Ames Salmonella assay. DIfferent cytogenetic assays, i.e. sister-chromatid exchanges (SCEs), micronuclei (MN) 10 binucleated cells as well as aneuploidy (using MN in combinatio~ ,:"ith a pancentromeric probe and tluorescene in situ hybridization), CytotOXICIty andlor gene mutanoa (at HPRT locus) were used as biological end-points. The mutagenic potential of different types of heterocyclic aromatic amines cooked food mutagens such as IQ, MeIQ. PHIP and Trp-P-l, as well ~ hexamethylphosphoramide (HMPA) and safrole were studied. Usmg a banery consisting of different test systems i.e., human Hep G2, CHO cells as well as Ames test, positive results were found with all chemicals used in this study. This is the first report concerning the mutagenic potential of HMPA and safrole in in vitro eukaryotic test systems as well as in Ames Salmonella assay. The present data indicate that Hep G2 cells are more efficient in activating different classes of indirectly-acting promutagens than the one derived from rat liver S9-fraction. Keyword(s): Bioactivation of mutagens; Hep G2 cell system
Ip XIV A.61
Mutagenle action of myeotoxtns at moleeular level
Marcela Aranda , Manuel Ellahueile 1. IDep. Cs. Biologicas, Universidad de Santiago de Chile. Casilla 40 Correo 33. Santiago. Chile; 1 Laboratorio de Genetica Toxicologica, Fac. Cs. Qcas. y Farm., Uniuersidad de Chile, Casilla 123 Santiago I. Chile 1
It has become evident that many human cancers are associated with changes in p53 gene. The inactivation of its tumor-suppressor activity is an almost universal step in the development of human cancers. Cells without working pS3 arc more susceptible to the mutagenic effects of DNA-damaging agents. In the past few years, researchers have found that some carcinogens leave characteristic fingerprints in p53. Atlatoxin BI, causes a serine substitution at a particular spot on the pS3 protein, some carcinogens in cigarette smoke and oxy radicals also cause a charaeteristic replacement. Such mutational
S-XIV: New mutagenicity tests and their evaluation
Ip XIV A.21
Salmonella spiral assay: Manually countlnt: and data analysis
Lone Lillemarlc, Mona-Lise Binderup, Birgitte Plesing Moller, Vivian Jorgensen. National Food Agency of Denmark, Murkhe} Bygade /9, DK-
2860 Seborg, Denmark A method for manually counting of colonies in the Spiral Salmonella Assay by the use of a template has been established, and a model and a spreadsheet for data analysis have been set up. Upon analysis, each spiral plate yields a dose-response curve consisting of 6 points with a concentrallon range of 13-fold. In the literature the order of application of bacteria. test substance and S9-mix have been discussed as well as the possibility of pouring bacteria andlor S9-mix onto the plate with top agar after spiral plating of the test substance. For evaluation of the method four different concentrations of two known mutagens 2-nitrofluorene (2-NF) and 2-aminoanthracene (2-AA) dissolved in dimethyl sulfoxide (OM SO) were tested. To establish the optimum test conditions the application order of bacteria, test substance and S9-mix (only 2-AA) were varied. Also the application of bacteria and S9-mix were performed with two different techniques: spiral plating and top agar. As it appeared that the relatively high volume of DMSO plated in the first part of the spiral was toxic for the bacteria. the test was repeated with the more volatile solvents diethyl ether and ethanol. The results will be given and discussed. Keyword(s): Spiral Salmonella Assay; Ames test; Volatile solvents
Ip XIV A.31
Deteetlon ofl:enomle DNA damage (ndueed by genotoxins In mammalian eell lines by a ehemllumlneseenee mleroplate DNA repair assay
R.-Y. u', C. Provot l,l.-P'laeg1, C. Bozzato", B. Salles 1. IS.F.R./.. Berganton, 33127 Saint Jean d'J1Jac, France; 2IPBS. UPR 9062 CNRS. 205 route de Narbonne. 3/077 Toulouse. France To easily screen genotoxic compounds, we developed a Damaged DNA Detection assay (3D assay). The 3D-assay is an adaptation of the repair assay initially reported by Wood et al, (I) which takes advantage of an adsoption step of plasmid DNA in sensitized microplate wells. After DNA damaging treatments, digoxigenylated-modified nucleotides were incorporated during the repair synthesis step and their presence was detected by an ELISA-like reaction with chemiluminescence detection (2). Here, we show that this assay allows the analysis of damaged genonuc DNA Isolated from mammalian cells following treatment with chemicals. thus making this method interesting for the following applications: (i) detection of DNA damage, (ii) determination of the repair rate and (iii) evaluation of protective agents. (i) DNA alkylation provoked by EMS and MNNG or oxidative damage induced by H101 were detected. Moreover, genotoxicity of pro-drugs in metabolically competent human cell line expressing cytochromes P450s (3) was tested. Oi) After a post-treatment incubation step in drug free medium, kinetics of DNA repair were determined in different cells line by analysing the decrease amount of DNA damage. (iii) Following H101 treatment the protective effects of different antioxidants (NAC, silymarin ...) was shown. (I) Wood, R. D., Robins, P. & Lindahl, T. (1988) Cell. 53, 97-106. (2) B. Salles, C. Provot et al. (1995) Anal. Biochem., 132,37-42. (3) Crespi (1991) Chern. Res. Toxicol 4, 566-572.
Ip XIV A.41
Evaluation of the restriction site mutallon assay usInl: murine P53 Intron sequenees
Gareth 1.S. Jenkins, James M. Parry. Centre Jor molecular genetics and toxicology. University of Wales Swansea. Singleto" Parle, Swansea. UK
The restriction site mutation assay, which detects DNA mutations in restriction enzyme sites in target regions, was employed to study induced in vivo mutagenesis. The agents used in this study were: N-ethyl-N-nitrosourea, 2-acetylaminotluorene and 1,2-dimethylhydrazine. Mutations were analYsed in four regions of the murine pS3 gene: exon 4, exon 5, intron 6 and intron 8. The results demonstrated that the two intron regions possessed approximately tenfold more mutations compared with the exon regions. This data was supported by mutation frequency calculations, made possible by the inclusion of an internal standard molecule in the RSM assay, which also showed the higher mutability of the intron regions. Intron regions were sufficiently sensitive such that a number of spontaneous mutations were detected. This is the first time that we have detected spontaneous in vivo mutations in the RSM assay. The mechanism of this increased mutation in intron sequences remains unclear, but may be due to differential DNA repair or due to selection differences between the coding exon regions and the noncoding intron regions. Keyword(s): restriction site mutation assay; intran; exon; mutation
Ip XIV A.51
Metabolle aellvatlon of promutagenle carelnogens witb human hepatoma (Hep G2) eells
F. Darroudil,l, S. Knasrniillef, R. Sanyal", C.M. Meijers", A.T. Natarajan l ,2 . J MGC,
Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Leiden, The Netherlands; 2J.A. Cohen Institute. IRS. Leiden; The Netherlands; j Institute of Tumor Biology and cancer Research, Uniuersity of Vienna, Vienna, Austria We have shown that Hep 02 cells can be used as metabolic activation system as well as target for DNA damage. We have isolated S9-fraction from Hep G2 cells and mvestigated its ability to activate promutagenic carcinogens using Chinese hamster ovary (CHO) cells (in vitro) and Ames Salmonella assay. DIfferent cytogenetic assays, i.e. sister-chromatid exchanges (SCEs), micronuclei (MN) 10 binucleated cells as well as aneuploidy (using MN in combinatio~ ,:"ith a pancentromeric probe and tluorescene in situ hybridization), CytotOXICIty andlor gene mutanoa (at HPRT locus) were used as biological end-points. The mutagenic potential of different types of heterocyclic aromatic amines cooked food mutagens such as IQ, MeIQ. PHIP and Trp-P-l, as well ~ hexamethylphosphoramide (HMPA) and safrole were studied. Usmg a banery consisting of different test systems i.e., human Hep G2, CHO cells as well as Ames test, positive results were found with all chemicals used in this study. This is the first report concerning the mutagenic potential of HMPA and safrole in in vitro eukaryotic test systems as well as in Ames Salmonella assay. The present data indicate that Hep G2 cells are more efficient in activating different classes of indirectly-acting promutagens than the one derived from rat liver S9-fraction. Keyword(s): Bioactivation of mutagens; Hep G2 cell system
Ip XIV A.61
Mutagenle action of myeotoxtns at moleeular level
Marcela Aranda , Manuel Ellahueile 1. IDep. Cs. Biologicas, Universidad de Santiago de Chile. Casilla 40 Correo 33. Santiago. Chile; 1 Laboratorio de Genetica Toxicologica, Fac. Cs. Qcas. y Farm., Uniuersidad de Chile, Casilla 123 Santiago I. Chile 1
It has become evident that many human cancers are associated with changes in p53 gene. The inactivation of its tumor-suppressor activity is an almost universal step in the development of human cancers. Cells without working pS3 arc more susceptible to the mutagenic effects of DNA-damaging agents. In the past few years, researchers have found that some carcinogens leave characteristic fingerprints in p53. Atlatoxin BI, causes a serine substitution at a particular spot on the pS3 protein, some carcinogens in cigarette smoke and oxy radicals also cause a charaeteristic replacement. Such mutational