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FISH for micronuclei analysis in barley cells
Abstracts / Toxicology Letters 164S (2006) S1–S324
methylating genotoxins when MGMT is depleted. p53 and c-Fos knockout mouse fibroblasts are more sensitive to UV light than the corresponding wild-type cells, indicating that in fibroblasts p53 and c-Fos play a protective rather than a pro-apoptotic role, which is due to regulation of DNA repair genes. Human lymphoblastoid cells and malignant glioma cell lines are more sensitive to methylating agents if they are wild-type for p53 compared to p53 mt. Methylation-induced apoptosis in lymphocytes and glioblastoma cells was preceded by DSB formation, p53 stabilization, and death receptor (Fas/CD95/Apo1) upregulation. O6 MeG lesions do not induce immediate early-signaling. This is in contrast to cisplatin which is highly effective in inducing the MAP kinase pathway provoking sustained JNK/p38 kinase activation and death receptor ligand (Fas-L) upregulation. For methyl methanesulfonate, JNK/P38 kinase activation requires DNA-PK and CSB protein that appear to be involved in sensing critical DNA damage. Overall, the data show that non-repaired DNA damage triggers apoptotic cell death in a cell-type and lesion-specific way by activating the death receptor or mitochondrial damage pathway. Although many downstream players may have impact on cellular sensitivity, DNA repair seems to be most important in determining the genotoxin-induced apoptotic response. Work was supported by DFG, Ka724/12-1, Ka724/13-1, 13-2 and SFB432/B7. doi:10.1016/j.toxlet.2006.06.119 112 Effects of dietary flavonoids on detoxyfing cell system Ksenija Durgo 1 , Lidija Vukovi´c 3 , Gordana Rusak 2 , ˇ c1 Maja Osmak 3 , Jasna Franeki´c Coli´ 1 Faculty
of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; 2 Faculty of Natural – Science, University of Zagreb, Zagreb, Croatia; 3 Ruder Boˇskovi´c Institute, Zagreb, Croatia Flavonoids are phytochemicals widely distributed in plants. In this work we have investigated five structurally similar flavonoids for their ability to: (i) to cause antioxidant/prooxidant effect in cells; (ii) to change basal level of total cellular glutathione and glutathione-Stransferases; (iii) to cause cytochrome 1A1 expression and (iv) their influence on antiapoptotic proteins expression (survivin and Bcl-2) and their ability to cause PARP degradation. For this purpose, laryngeal carcinoma cell lines HEp2 and drug resistant cell line CK2 were used.
S57
Previously, we have concluded that cell line CK2 was more resistant to cytotoxic effect of investigated flavonoids than parental, HEp2 cells. Quercetin, luteolin and fisetin have acted as prooxidants in cell lines where cytochrome 1A1 was highly expressed (cell line HEp2). In such conditions, fisetin and luteolin have caused enhanced prooxidative response when cells were treated with free radicals of other origin. In cells where cytochrome 1A1 was slightly expressed, quercetin has caused protein PARP degradation and survivin expression. Luteolin has caused PARP degradation and antiapoptotic proteins expression in cells with low cytochrome 1A1 and glutathione expression. In cells where expression of cytochrome 1A1 is low and glutathione-S-transferases activity and glutathione level high, fisetin has caused PARP degradation and antiapoptotic proteins repression. These results show clear evidence of proapoptotic nature of fisetin. Naringin has shown antioxidant property, it has caused cell membrane stabilisation and higher selectivity. In cells where cytochrome 1A1 was highly expressed, together with total cellular glutathione and glutathione-S-transferases, naringin has caused Bcl-2 repression, and has shown proapoptotic action. Myricetin did not cause prooxidative damage; it has caused expression of phase II enzymes and Bcl-2 expression, pointing its antiapoptotic nature. In conclusion, small differences in chemical structure of flavonoids lead to drastic change of their biological effects. These effects are strongly dependent of cell type as well as test systems used. Extensive studies on structure–function relationship of flavonoids in different test systems could provide rational approach to drug and chemopreventive agent design. doi:10.1016/j.toxlet.2006.06.120 113 FISH for micronuclei analysis in barley cells Jolanta Juchimiuk, Barbara Hering, Jolanta Maluszynska University of Silesia, Poland Chromosomal aberrations (CA) can be detected with simple, classical chromosome staining methods, nevertheless fluorescent in situ hybridization method (FISH) provides new tools for their analysis. The identification of individual chromosomes or chromosome arms using FISH with specific probes is very helpful in the detection and detailed characterization of chromosome rearrangements. Additionally, the possibility of the detection of chromosome or chromosome fragments in interphase
S58
Abstracts / Toxicology Letters 164S (2006) S1–S324
nuclei in order to analysis of chromosomal aberrations, is unquestionable advantage of FISH technique. Until now, FISH is not widely applied in plant mutagenesis for detection and precise localization of chromosome aberrations, as DNA probes required for chromosomes of particular plant species are very limited. The application of FISH in chromosomal aberrations analysis is more common in human cytogenetics and carcinogenicity studies in mammals. However, there are plant species, which most of the chromosomes can be distinguished by presence/specific location of available DNA probes and thus make possible detailed CA analysis with FISH. In present study fluorescent in situ hybridization (FISH) using telomere- and centromere-specific DNA and rDNA (5S and 25S rDNA) as probes was used to compare the cytogenetic effects of two chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic acid hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n = 14). The micronucleus test (MN) combined with FISH allowed quantitative analysis of the involvement of chromosome fragments in micronuclei formation and thus enabled explanation the origin of mutagens-induced micronuclei. The analysis of the frequency of micronuclei with signals of investigated DNA probes did not show significant differences between the MH- and MNU-induced micronuclei. Additionally, especially the application of rDNA as probes to FISH enabled more detailed study of the contribution of particular chromosomes/chromosome fragments in micronuclei formation. doi:10.1016/j.toxlet.2006.06.121
unscrupulous traders. Broadly, the common pollutants causing concern to food industry and consumers could be classified as (a) intentional additives/adulterants and (b) non-intentional contaminants. The commonly encountered food adulterants which pose a serious health hazard are: (i) Non-permitted colours in sweets, savoury, crushed ice, hard boiled sugar confectioneries, spices, etc. (ii) Adulterants in edible oils such as Argemone seed oil; butter yellow for colouration of cheaper colourless oil; tricresyl phosphate, an odourless and colourless industrial chemical. (iii) Lathyrus sativus pulse and its flour in Cajanus indicus pulse and dehusked Bengal gram flour. Among the contaminants of concern are: (i) polycyclic aromatic hydrocarbons, a group of ubiquitous environmental pollutant in several foodstuffs including several edible oils. (ii) Toxic metals in various raw and processed foodstuffs. (iii) Pesticide residues especially organochlorine compounds in food grains, vegetables, fruits, milk and edible oils. (iv) Mycotoxins which represent a major group of chemicals that can occur in a variety of plant food especially in tropical countries. Among these compounds, aflatoxin B1 appears to be the most hazardous. Thus, there is need to restrict the presence of various contaminants and adulterants in food within the maximum permissible levels. It is only after ensuring good quality food, by keeping the contaminants as low as achievable, that we shall be able to fulfill the basic requirement of safe food for humans. doi:10.1016/j.toxlet.2006.06.122 115 Food safety nets in Latin America Elizabeth de Souza Nascimento
W8 Food Safety in Relation to Chemical Contaminants in Developing Countries 114 Common adulterants and food safety status in India Mukul Das Food Toxicology Laboratory, Industrial Toxicology Research Centre, Lucknow 226001, India Food is required for the growth and maintenance of living organisms. The chemical constituents of food that are important to safety include a wide spectrum of substances. These chemical substances may exist as naturally occurring components, or as contamination of the environment in which the food is produced and stored, or as additives introduced by man in the course of food manufacture and preparation, or as deliberate adulterants added for the purpose of earning undue profits by
Faculty of Pharmaceutical Sciences/University of S˜ao Paulo, S˜ao Paulo, Brazil Food security exists when all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life. The four pillars of food security are availability, stability of supply, access and utilization. There are many nets involved with attacking hunger, and they are necessary as FAO estimates that 852 million people worldwide are undernourished: 815 million in developing countries, 28 million in the countries in transition and 9 million in the industrialized countries. It is a fact that the percentage of population undernourished in the Latin America and Caribbean decreased from 19 millions in 1967 to 10 million at the turn of the new millennium. So, supply food to hunger people still an issue in this part of the world. But also food safety is of
methylating genotoxins when MGMT is depleted. p53 and c-Fos knockout mouse fibroblasts are more sensitive to UV light than the corresponding wild-type cells, indicating that in fibroblasts p53 and c-Fos play a protective rather than a pro-apoptotic role, which is due to regulation of DNA repair genes. Human lymphoblastoid cells and malignant glioma cell lines are more sensitive to methylating agents if they are wild-type for p53 compared to p53 mt. Methylation-induced apoptosis in lymphocytes and glioblastoma cells was preceded by DSB formation, p53 stabilization, and death receptor (Fas/CD95/Apo1) upregulation. O6 MeG lesions do not induce immediate early-signaling. This is in contrast to cisplatin which is highly effective in inducing the MAP kinase pathway provoking sustained JNK/p38 kinase activation and death receptor ligand (Fas-L) upregulation. For methyl methanesulfonate, JNK/P38 kinase activation requires DNA-PK and CSB protein that appear to be involved in sensing critical DNA damage. Overall, the data show that non-repaired DNA damage triggers apoptotic cell death in a cell-type and lesion-specific way by activating the death receptor or mitochondrial damage pathway. Although many downstream players may have impact on cellular sensitivity, DNA repair seems to be most important in determining the genotoxin-induced apoptotic response. Work was supported by DFG, Ka724/12-1, Ka724/13-1, 13-2 and SFB432/B7. doi:10.1016/j.toxlet.2006.06.119 112 Effects of dietary flavonoids on detoxyfing cell system Ksenija Durgo 1 , Lidija Vukovi´c 3 , Gordana Rusak 2 , ˇ c1 Maja Osmak 3 , Jasna Franeki´c Coli´ 1 Faculty
of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; 2 Faculty of Natural – Science, University of Zagreb, Zagreb, Croatia; 3 Ruder Boˇskovi´c Institute, Zagreb, Croatia Flavonoids are phytochemicals widely distributed in plants. In this work we have investigated five structurally similar flavonoids for their ability to: (i) to cause antioxidant/prooxidant effect in cells; (ii) to change basal level of total cellular glutathione and glutathione-Stransferases; (iii) to cause cytochrome 1A1 expression and (iv) their influence on antiapoptotic proteins expression (survivin and Bcl-2) and their ability to cause PARP degradation. For this purpose, laryngeal carcinoma cell lines HEp2 and drug resistant cell line CK2 were used.
S57
Previously, we have concluded that cell line CK2 was more resistant to cytotoxic effect of investigated flavonoids than parental, HEp2 cells. Quercetin, luteolin and fisetin have acted as prooxidants in cell lines where cytochrome 1A1 was highly expressed (cell line HEp2). In such conditions, fisetin and luteolin have caused enhanced prooxidative response when cells were treated with free radicals of other origin. In cells where cytochrome 1A1 was slightly expressed, quercetin has caused protein PARP degradation and survivin expression. Luteolin has caused PARP degradation and antiapoptotic proteins expression in cells with low cytochrome 1A1 and glutathione expression. In cells where expression of cytochrome 1A1 is low and glutathione-S-transferases activity and glutathione level high, fisetin has caused PARP degradation and antiapoptotic proteins repression. These results show clear evidence of proapoptotic nature of fisetin. Naringin has shown antioxidant property, it has caused cell membrane stabilisation and higher selectivity. In cells where cytochrome 1A1 was highly expressed, together with total cellular glutathione and glutathione-S-transferases, naringin has caused Bcl-2 repression, and has shown proapoptotic action. Myricetin did not cause prooxidative damage; it has caused expression of phase II enzymes and Bcl-2 expression, pointing its antiapoptotic nature. In conclusion, small differences in chemical structure of flavonoids lead to drastic change of their biological effects. These effects are strongly dependent of cell type as well as test systems used. Extensive studies on structure–function relationship of flavonoids in different test systems could provide rational approach to drug and chemopreventive agent design. doi:10.1016/j.toxlet.2006.06.120 113 FISH for micronuclei analysis in barley cells Jolanta Juchimiuk, Barbara Hering, Jolanta Maluszynska University of Silesia, Poland Chromosomal aberrations (CA) can be detected with simple, classical chromosome staining methods, nevertheless fluorescent in situ hybridization method (FISH) provides new tools for their analysis. The identification of individual chromosomes or chromosome arms using FISH with specific probes is very helpful in the detection and detailed characterization of chromosome rearrangements. Additionally, the possibility of the detection of chromosome or chromosome fragments in interphase
S58
Abstracts / Toxicology Letters 164S (2006) S1–S324
nuclei in order to analysis of chromosomal aberrations, is unquestionable advantage of FISH technique. Until now, FISH is not widely applied in plant mutagenesis for detection and precise localization of chromosome aberrations, as DNA probes required for chromosomes of particular plant species are very limited. The application of FISH in chromosomal aberrations analysis is more common in human cytogenetics and carcinogenicity studies in mammals. However, there are plant species, which most of the chromosomes can be distinguished by presence/specific location of available DNA probes and thus make possible detailed CA analysis with FISH. In present study fluorescent in situ hybridization (FISH) using telomere- and centromere-specific DNA and rDNA (5S and 25S rDNA) as probes was used to compare the cytogenetic effects of two chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic acid hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n = 14). The micronucleus test (MN) combined with FISH allowed quantitative analysis of the involvement of chromosome fragments in micronuclei formation and thus enabled explanation the origin of mutagens-induced micronuclei. The analysis of the frequency of micronuclei with signals of investigated DNA probes did not show significant differences between the MH- and MNU-induced micronuclei. Additionally, especially the application of rDNA as probes to FISH enabled more detailed study of the contribution of particular chromosomes/chromosome fragments in micronuclei formation. doi:10.1016/j.toxlet.2006.06.121
unscrupulous traders. Broadly, the common pollutants causing concern to food industry and consumers could be classified as (a) intentional additives/adulterants and (b) non-intentional contaminants. The commonly encountered food adulterants which pose a serious health hazard are: (i) Non-permitted colours in sweets, savoury, crushed ice, hard boiled sugar confectioneries, spices, etc. (ii) Adulterants in edible oils such as Argemone seed oil; butter yellow for colouration of cheaper colourless oil; tricresyl phosphate, an odourless and colourless industrial chemical. (iii) Lathyrus sativus pulse and its flour in Cajanus indicus pulse and dehusked Bengal gram flour. Among the contaminants of concern are: (i) polycyclic aromatic hydrocarbons, a group of ubiquitous environmental pollutant in several foodstuffs including several edible oils. (ii) Toxic metals in various raw and processed foodstuffs. (iii) Pesticide residues especially organochlorine compounds in food grains, vegetables, fruits, milk and edible oils. (iv) Mycotoxins which represent a major group of chemicals that can occur in a variety of plant food especially in tropical countries. Among these compounds, aflatoxin B1 appears to be the most hazardous. Thus, there is need to restrict the presence of various contaminants and adulterants in food within the maximum permissible levels. It is only after ensuring good quality food, by keeping the contaminants as low as achievable, that we shall be able to fulfill the basic requirement of safe food for humans. doi:10.1016/j.toxlet.2006.06.122 115 Food safety nets in Latin America Elizabeth de Souza Nascimento
W8 Food Safety in Relation to Chemical Contaminants in Developing Countries 114 Common adulterants and food safety status in India Mukul Das Food Toxicology Laboratory, Industrial Toxicology Research Centre, Lucknow 226001, India Food is required for the growth and maintenance of living organisms. The chemical constituents of food that are important to safety include a wide spectrum of substances. These chemical substances may exist as naturally occurring components, or as contamination of the environment in which the food is produced and stored, or as additives introduced by man in the course of food manufacture and preparation, or as deliberate adulterants added for the purpose of earning undue profits by
Faculty of Pharmaceutical Sciences/University of S˜ao Paulo, S˜ao Paulo, Brazil Food security exists when all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life. The four pillars of food security are availability, stability of supply, access and utilization. There are many nets involved with attacking hunger, and they are necessary as FAO estimates that 852 million people worldwide are undernourished: 815 million in developing countries, 28 million in the countries in transition and 9 million in the industrialized countries. It is a fact that the percentage of population undernourished in the Latin America and Caribbean decreased from 19 millions in 1967 to 10 million at the turn of the new millennium. So, supply food to hunger people still an issue in this part of the world. But also food safety is of