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Genotoxicity assessment of Aloe vera gel in mice with the comet assay
S104
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1142 Genotoxicity assessment of Aloe vera gel in mice with the comet assay E. Durmaz 1,∗ , O. Kenan Ulutas¸ 1 , R. Güler 1 , I. Gürbüz 2 , I. C¸ok 1
human skin than keratinocytes and epidermal skin equivalents and are therefore recommended to be used for toxicity testing. Keywords: Xenobiotics metabolism; Enzyme activity; Reconstructed human skin model. doi:10.1016/j.toxlet.2011.05.377
1
Faculty of Pharmacy, Department of Toxicology, Gazi University, Ankara, Turkey, 2 Faculty of Pharmacy, Department of Pharmacognosy, Gazi University, Ankara, Turkey
Aloe vera gel contains multiple constituents with potential biological and toxicological activities. Toxic substances like many kinds of anthraquinones in latex part of leaf may be transferred to the gel through a total leaf extract incidentally. Due to the mutagenic and genotoxic properties of these chemicals the contaminated gel may induce serious health problems on public usage. In this study, genotoxic potential of one of the commercial Aloe vera gel products, have been investigated in mice using comet assay. Suggested human dose of the commercial Aloe vera gel adjusted to experimental animals per kilogram basis and given to mice by gastric gavage for 14 days with normal, half and double doses once per day. Balb-c, male and female mice, aged 4 weeks, weighted 30–45 g were used and a control group was accompany the experiment groups (n = 10). Venous bloods of mice were taken at 1st, 7th and 14th days of the experiment. For the half dose groups, statistical significance have been found when the results of 1st and 14th days of experiments compared (p < 0.01). Similar significances have been observed when the results of 1st and 7th days of experiments compared (p < 0.01). In the double doses groups, statistical significance has been found when the results of 1st and 7th (p < 0.01) and 1st and 14th (p < 0.05) days of experiments compared as well. On the other hand significant differences have been observed when double dose groups compared with control groups in 7th day (p < 0.01). This study was supported by Gazi University Scientific Research Fund (Project No. 02/2010-09). doi:10.1016/j.toxlet.2011.05.376
P1143 Investigation of the biotransforming capacity of human reconstructed skin models for genotoxicity testing E.J. Fabian 1,∗ , V. Blatz 1 , C. Jäckh 1 , B. van Ravenzwaay 1 , K. Reisinger 2 , R. Landsiedel 1 1
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany, 2 Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany Purpose: The objective of this comparative study was to investigate xenobiotic metabolizing enzyme activities of keratinocytes, three reconstructed human skin models (epidermal and fullthickness) and native human skin to specify a metabolic active human skin equivalent which is appropriate for toxicity testing. Methods: Enzyme activities were investigated by incubation of model substrates with S9 or microsomal fractions which were obtained from keratinocytes, epidermal or full-thickness in vitro skin models and native human skin. The investigated activities included enzymes such as CYPs and flavin-dependent monooxygenases (FMO) as well as N-acetyltransferases (NATs) and UDP-glucuronyltransferases (UDP-GT). Results: CYP enzyme activities (EROD, PROD and BROD) were below the limit of quantification in all investigated models. FMO, UDP- GT1 and NAT1 activities though were demonstrated in all in vitro skin systems. Enzyme activities in full-thickness skin models were more similar to native
P1144 Metafer image analysis platform: A useful tool for chromosome damage assessment in genetic toxicology F. Finot ∗ , N. Bichet, A. Essahli, F. Paillard Covance Laboratory SAS, Porcheville, France Assessment of potential genotoxicity for drug candidates is mandatory to enter Phase I. Chromosomal damage is estimated either by the analysis of chromosomes (chromosome aberration test) or by micronuclei visualization (chromosome fragments or whole chromosomes). We perform the chromosome aberration test on human lymphocytes whereas micronuclei induction is evaluated using different kind of cells depending on the study type: mouse lymphoma L5178Y cells in Discovery, human lymphocytes for regulatory purpose. In Covance Laboratory SAS (Porcheville, France), the Metafer platform was fully validated (scientific and software validation) for chromosomal aberration assay to automatically score the mitotic index and to allow a manual seizure for chromosome aberrations through a user-interface. It was also validated for the scoring of micronuclei in mononucleated L5178Y cells, after DAPI staining and for visualization of the cytoplasm by phase contrast (in discovery studies). This image analysis system improves statistical analysis power by increasing the number of cells counted leading to a better objectivity compared with manual scoring. For regulatory purpose, the decision was made to perform a new micronucleus test validation on Metafer platform using human lymphocytes with cytochalasine B to automatically and manually quantify micronuclei in binucleated cells after Giemsa and DAPI staining and to compare the results obtained. doi:10.1016/j.toxlet.2011.05.378
P1145 Assessment of ␥H2AX as an endpoint for DNA damage: A potential novel in vitro genotoxicity assay C. Garcia Canton ∗ , K. Luettich, C. Meredith Group Research and Development, British American Tobacco, Southampton, UK H2AX is a histone that is rapidly phosphorylated to become ␥H2AX after exposure to DNA-damaging agents that cause doublestrand DNA breaks (DSBs). ␥H2AX can be detected and quantified, giving a correlation with the number of DSBs. This feature makes the ␥H2AX assay a potential complement to the current genotoxicity battery of in vitro assays. Using a panel of genotoxins and non-genotoxins, we are assessing the sensitivity and specificity of ␥H2AX as an endpoint for DNA damage in the human bronchial epithelial cell line BEAS-2B. Here we report our initial findings using the genotoxins ethyl methanesulfonate (EMS) and etoposide (ETO) and the non-genotoxin, dimethyl formamide (DMF). To determine suitable concentrations for assessment of genotoxic effects (maximum 50% reduction in cell viability), compounds were tested over a range of concentrations for 1, 3 and 24 h. Cytotoxicity was assessed with the Cell-TiterGlo® kit. DSBs were identified
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1142 Genotoxicity assessment of Aloe vera gel in mice with the comet assay E. Durmaz 1,∗ , O. Kenan Ulutas¸ 1 , R. Güler 1 , I. Gürbüz 2 , I. C¸ok 1
human skin than keratinocytes and epidermal skin equivalents and are therefore recommended to be used for toxicity testing. Keywords: Xenobiotics metabolism; Enzyme activity; Reconstructed human skin model. doi:10.1016/j.toxlet.2011.05.377
1
Faculty of Pharmacy, Department of Toxicology, Gazi University, Ankara, Turkey, 2 Faculty of Pharmacy, Department of Pharmacognosy, Gazi University, Ankara, Turkey
Aloe vera gel contains multiple constituents with potential biological and toxicological activities. Toxic substances like many kinds of anthraquinones in latex part of leaf may be transferred to the gel through a total leaf extract incidentally. Due to the mutagenic and genotoxic properties of these chemicals the contaminated gel may induce serious health problems on public usage. In this study, genotoxic potential of one of the commercial Aloe vera gel products, have been investigated in mice using comet assay. Suggested human dose of the commercial Aloe vera gel adjusted to experimental animals per kilogram basis and given to mice by gastric gavage for 14 days with normal, half and double doses once per day. Balb-c, male and female mice, aged 4 weeks, weighted 30–45 g were used and a control group was accompany the experiment groups (n = 10). Venous bloods of mice were taken at 1st, 7th and 14th days of the experiment. For the half dose groups, statistical significance have been found when the results of 1st and 14th days of experiments compared (p < 0.01). Similar significances have been observed when the results of 1st and 7th days of experiments compared (p < 0.01). In the double doses groups, statistical significance has been found when the results of 1st and 7th (p < 0.01) and 1st and 14th (p < 0.05) days of experiments compared as well. On the other hand significant differences have been observed when double dose groups compared with control groups in 7th day (p < 0.01). This study was supported by Gazi University Scientific Research Fund (Project No. 02/2010-09). doi:10.1016/j.toxlet.2011.05.376
P1143 Investigation of the biotransforming capacity of human reconstructed skin models for genotoxicity testing E.J. Fabian 1,∗ , V. Blatz 1 , C. Jäckh 1 , B. van Ravenzwaay 1 , K. Reisinger 2 , R. Landsiedel 1 1
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany, 2 Henkel AG & Co. KGaA, 40191, Düsseldorf, Germany Purpose: The objective of this comparative study was to investigate xenobiotic metabolizing enzyme activities of keratinocytes, three reconstructed human skin models (epidermal and fullthickness) and native human skin to specify a metabolic active human skin equivalent which is appropriate for toxicity testing. Methods: Enzyme activities were investigated by incubation of model substrates with S9 or microsomal fractions which were obtained from keratinocytes, epidermal or full-thickness in vitro skin models and native human skin. The investigated activities included enzymes such as CYPs and flavin-dependent monooxygenases (FMO) as well as N-acetyltransferases (NATs) and UDP-glucuronyltransferases (UDP-GT). Results: CYP enzyme activities (EROD, PROD and BROD) were below the limit of quantification in all investigated models. FMO, UDP- GT1 and NAT1 activities though were demonstrated in all in vitro skin systems. Enzyme activities in full-thickness skin models were more similar to native
P1144 Metafer image analysis platform: A useful tool for chromosome damage assessment in genetic toxicology F. Finot ∗ , N. Bichet, A. Essahli, F. Paillard Covance Laboratory SAS, Porcheville, France Assessment of potential genotoxicity for drug candidates is mandatory to enter Phase I. Chromosomal damage is estimated either by the analysis of chromosomes (chromosome aberration test) or by micronuclei visualization (chromosome fragments or whole chromosomes). We perform the chromosome aberration test on human lymphocytes whereas micronuclei induction is evaluated using different kind of cells depending on the study type: mouse lymphoma L5178Y cells in Discovery, human lymphocytes for regulatory purpose. In Covance Laboratory SAS (Porcheville, France), the Metafer platform was fully validated (scientific and software validation) for chromosomal aberration assay to automatically score the mitotic index and to allow a manual seizure for chromosome aberrations through a user-interface. It was also validated for the scoring of micronuclei in mononucleated L5178Y cells, after DAPI staining and for visualization of the cytoplasm by phase contrast (in discovery studies). This image analysis system improves statistical analysis power by increasing the number of cells counted leading to a better objectivity compared with manual scoring. For regulatory purpose, the decision was made to perform a new micronucleus test validation on Metafer platform using human lymphocytes with cytochalasine B to automatically and manually quantify micronuclei in binucleated cells after Giemsa and DAPI staining and to compare the results obtained. doi:10.1016/j.toxlet.2011.05.378
P1145 Assessment of ␥H2AX as an endpoint for DNA damage: A potential novel in vitro genotoxicity assay C. Garcia Canton ∗ , K. Luettich, C. Meredith Group Research and Development, British American Tobacco, Southampton, UK H2AX is a histone that is rapidly phosphorylated to become ␥H2AX after exposure to DNA-damaging agents that cause doublestrand DNA breaks (DSBs). ␥H2AX can be detected and quantified, giving a correlation with the number of DSBs. This feature makes the ␥H2AX assay a potential complement to the current genotoxicity battery of in vitro assays. Using a panel of genotoxins and non-genotoxins, we are assessing the sensitivity and specificity of ␥H2AX as an endpoint for DNA damage in the human bronchial epithelial cell line BEAS-2B. Here we report our initial findings using the genotoxins ethyl methanesulfonate (EMS) and etoposide (ETO) and the non-genotoxin, dimethyl formamide (DMF). To determine suitable concentrations for assessment of genotoxic effects (maximum 50% reduction in cell viability), compounds were tested over a range of concentrations for 1, 3 and 24 h. Cytotoxicity was assessed with the Cell-TiterGlo® kit. DSBs were identified