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Okadaic acid genotoxicity investigated by the comet assay
PosterSession lAo Genotoxicity
Posters PtA. Genotoxicity
IP1 A1 lis
A POSITIVE CONTROL USEFULIN THERAT IN VIVO MICRONUCLEUS TEST? A COMPARATIVE STUDY OF DIFFERENT ROUTES OF ADMINISTRATION OF CYCLOPHOSPHAMIDE
M. Aujoulat *1 , A. Forichon 1 , J. Descotes'[. I CHRYSAUS, L'ArbresIe; 2 Laboratoire de Pharmacologie, Toxicologie Medicate et Medecine de l'Environnement, INSERM U 80, Faculte de Medecine, Lyon, France Mice and rats are the rodent species recommended by regulatory guidelines for the in vivo bone marrow micronucleus test (MNT). As ADME information is generally available for the rat, the current trend is to use this species. The objective of this work was to test whether different routes (oral, intravenous, intraperitoneal) routinely used to administer the positive control cyclophosphamide, induced different responses in micronucleated polychromatic erythrocyte (MPCE) frequency or in the polychromatic/normochromatic erythrocyte ratio (PCElNCE). There was no differences in MPCE frequency between the three routes following administration of 100 mglkg of cyclophosphamide to OFA rats, so the I.P. route could be routinely used for the positive control article whatever the route used for the test article. This approach would be especially relevant for MNT evaluation performed as one of the end points in chronic toxicology studies using a parenteral route like continuous infusion. The need for a positive control in the standard regulatory in vivo MNT then becomes questionable when historical data are available in the testing facility.
IP1 A2j
IP1 A31
D. Brault *, D. Renault, V.Thybaud. Rhone-Poulenc RorerS.A.,
Wtrysur Seine, France We used the single cell gel electrophoresis assay (SCGE) to detect organ-specific activity of N-methyl-N' -nitro-nitrosoguanidine (MNNG) and tl-propiolactone (BPL), two highly reactive alkylating agents known to induce genetic damage and tumors in rodent stomach when administered orally. In order to define the optimal sampling time for the detection of DNA strand-breaks, we measured the tail moment of comets induced in the mouse gastric mucosa, 1, 2, 4, 24 and 72 hours after a single oral administration of MNNG (20 mglkg) or BPL (25 mglkg). For both compounds, a marked induction of DNA strand-breaks was observed I and 2 hours after treatment and the effect decreased rapidly 4 hours after teatment. At 2-hour sampling time, the induction of comets was also assessed in liver and bone marrow cells. While more than a lO-fold increase in tail moment was noted for gastric mucosa cells, no changes were measured in the liver and bone marrow cells. These data indicate that the SCGE can be used to predict the organo-specificity of genotoxic carcinogens. (Supported by the E.U. Environment Program).
OKADAICACIDGENOTOXICITY INVESTIGATED BY THE COMETASSAY
V. Fessard *1 , 1. Puech 2 ,1. I CNEVA Fougeres, Laboratoire des Medicaments Yeterinaires. La Haute Marche, BP 203, 35302 Fougeres; 2CNEVA Paris, Unitedes Toxines Microbiennes, 10 rue PierreCurie, 94 704 MaisonsAlfort Cedex, France Okadaic acid (OA) is a marine toxin involved in Diarrhetic Shellfish Poisoning (DSP), a seafood intoxication present on the five continents. This toxin is a SerlThr protein phosphatase (l and 2A) inhibitor. Carcinogenic studies have shown that it is a potent tumor promoter on rodent skin and glandular stomach. Because of a negative Ames test, this compound has been considered as non genotoxic. However, few studies have suggested an effect on DNA. Indeed, we have previously shown DNA adduct formation after OA exposure on fibroblast and keratinocyte cell lines. Similar results were also obtained with hepatocytes and zebrafish embryos. In this study, we have used the Comet assay on Chinese Hamster Ovary cell line to study the effect of OA on DNA strand break formation directly after exposure or when OA exposure is followed by an incubation in fresh medium for repair. A topoisomerase II inhibitor, etoposide (0.5 jLglml), was used all along the study as a positive control. Preliminary results showed that no DNA strand breaks were observed after 1, 3 or 24 h exposure to a OA dose range between 5 and 40 oM. Since DNA effects seen in the Comet assay by adduct forming compounds mainly represent strand break formation during excision repair, we suggest that OA may induce DNA adduct formation concomitantly to inhibition of the repair system. Experiments concerning the effects on DNA breakage when cells are allowed to repair in fresh medium after OA are in progress. They should provide interesting results and will be further discussed.
IP1 A41 USEOF SINGLECELL GEL ELECTROPHORESIS ASSAYFORTHE DETECTION OF TlSSUE-5PECIFIC DNA DAMAGE IN MOUSEGASTRICMUCOSAAFTER ACUTE ORAL ADMINISTRATION OF N-METHYL-N'-NITRO-NITROSOGUANIDINE AND I/-PROPIOLACTONE
37
DNA DAMAGE EVALUATED BY THE ALKALINE COMET ASSAYIN LYMPHOCYTES OF HUMANS ANAESTHETIZED WITH ISOFLURANE
S. Sardas *, 1. Karabiyik, N. Aygiin, A.E. Karakaya. Department of Toxicology, Gazi University, Faculty of Pharmacy and Department
of Anaesthesia, Turkish State RailwaysHospital, Ankara, Turkey In the present paper, we report data on the possible DNA damage, induced in vivo by isofturane using the alkaline single cell gel electrophoresis technique (SCGE-comet assay) in patients before/after anaesthesia and in control group. 20 patients, aged 22--66 year, were anaesthetized for elective abdominal surgery with isofturane in oxygen for 120--162 min (mean: 133.2 min). Venous blood samples were obtained from the patients before the induction of anaesthesia, during the 60 th and 120 min of anaesthesia and on the first, third and fifth following days of anaesthesia. SCGE was examined in 100 cells from each specimen graded as undamaged, stretched and comet imaged. The number of SCGE per cell was almost the same in control and in patients before anaesthesia. However, significant differences were observed in undamaged, stretched and comet imaged cells of patients during 60 th, 120 th min of anaesthesia and on the following first day. DNA damage in cells started to return to normal rates after third day of anaesthesia and were almost identical with the rates of control group five days later. We conclude that examination of comet assay in peripheral blood lymphocytes of patients during anaesthesia with isofturane in oxygen revealed indication of genotoxic effect before DNA repair begins.
Posters PtA. Genotoxicity
IP1 A1 lis
A POSITIVE CONTROL USEFULIN THERAT IN VIVO MICRONUCLEUS TEST? A COMPARATIVE STUDY OF DIFFERENT ROUTES OF ADMINISTRATION OF CYCLOPHOSPHAMIDE
M. Aujoulat *1 , A. Forichon 1 , J. Descotes'[. I CHRYSAUS, L'ArbresIe; 2 Laboratoire de Pharmacologie, Toxicologie Medicate et Medecine de l'Environnement, INSERM U 80, Faculte de Medecine, Lyon, France Mice and rats are the rodent species recommended by regulatory guidelines for the in vivo bone marrow micronucleus test (MNT). As ADME information is generally available for the rat, the current trend is to use this species. The objective of this work was to test whether different routes (oral, intravenous, intraperitoneal) routinely used to administer the positive control cyclophosphamide, induced different responses in micronucleated polychromatic erythrocyte (MPCE) frequency or in the polychromatic/normochromatic erythrocyte ratio (PCElNCE). There was no differences in MPCE frequency between the three routes following administration of 100 mglkg of cyclophosphamide to OFA rats, so the I.P. route could be routinely used for the positive control article whatever the route used for the test article. This approach would be especially relevant for MNT evaluation performed as one of the end points in chronic toxicology studies using a parenteral route like continuous infusion. The need for a positive control in the standard regulatory in vivo MNT then becomes questionable when historical data are available in the testing facility.
IP1 A2j
IP1 A31
D. Brault *, D. Renault, V.Thybaud. Rhone-Poulenc RorerS.A.,
Wtrysur Seine, France We used the single cell gel electrophoresis assay (SCGE) to detect organ-specific activity of N-methyl-N' -nitro-nitrosoguanidine (MNNG) and tl-propiolactone (BPL), two highly reactive alkylating agents known to induce genetic damage and tumors in rodent stomach when administered orally. In order to define the optimal sampling time for the detection of DNA strand-breaks, we measured the tail moment of comets induced in the mouse gastric mucosa, 1, 2, 4, 24 and 72 hours after a single oral administration of MNNG (20 mglkg) or BPL (25 mglkg). For both compounds, a marked induction of DNA strand-breaks was observed I and 2 hours after treatment and the effect decreased rapidly 4 hours after teatment. At 2-hour sampling time, the induction of comets was also assessed in liver and bone marrow cells. While more than a lO-fold increase in tail moment was noted for gastric mucosa cells, no changes were measured in the liver and bone marrow cells. These data indicate that the SCGE can be used to predict the organo-specificity of genotoxic carcinogens. (Supported by the E.U. Environment Program).
OKADAICACIDGENOTOXICITY INVESTIGATED BY THE COMETASSAY
V. Fessard *1 , 1. Puech 2 ,1. I CNEVA Fougeres, Laboratoire des Medicaments Yeterinaires. La Haute Marche, BP 203, 35302 Fougeres; 2CNEVA Paris, Unitedes Toxines Microbiennes, 10 rue PierreCurie, 94 704 MaisonsAlfort Cedex, France Okadaic acid (OA) is a marine toxin involved in Diarrhetic Shellfish Poisoning (DSP), a seafood intoxication present on the five continents. This toxin is a SerlThr protein phosphatase (l and 2A) inhibitor. Carcinogenic studies have shown that it is a potent tumor promoter on rodent skin and glandular stomach. Because of a negative Ames test, this compound has been considered as non genotoxic. However, few studies have suggested an effect on DNA. Indeed, we have previously shown DNA adduct formation after OA exposure on fibroblast and keratinocyte cell lines. Similar results were also obtained with hepatocytes and zebrafish embryos. In this study, we have used the Comet assay on Chinese Hamster Ovary cell line to study the effect of OA on DNA strand break formation directly after exposure or when OA exposure is followed by an incubation in fresh medium for repair. A topoisomerase II inhibitor, etoposide (0.5 jLglml), was used all along the study as a positive control. Preliminary results showed that no DNA strand breaks were observed after 1, 3 or 24 h exposure to a OA dose range between 5 and 40 oM. Since DNA effects seen in the Comet assay by adduct forming compounds mainly represent strand break formation during excision repair, we suggest that OA may induce DNA adduct formation concomitantly to inhibition of the repair system. Experiments concerning the effects on DNA breakage when cells are allowed to repair in fresh medium after OA are in progress. They should provide interesting results and will be further discussed.
IP1 A41 USEOF SINGLECELL GEL ELECTROPHORESIS ASSAYFORTHE DETECTION OF TlSSUE-5PECIFIC DNA DAMAGE IN MOUSEGASTRICMUCOSAAFTER ACUTE ORAL ADMINISTRATION OF N-METHYL-N'-NITRO-NITROSOGUANIDINE AND I/-PROPIOLACTONE
37
DNA DAMAGE EVALUATED BY THE ALKALINE COMET ASSAYIN LYMPHOCYTES OF HUMANS ANAESTHETIZED WITH ISOFLURANE
S. Sardas *, 1. Karabiyik, N. Aygiin, A.E. Karakaya. Department of Toxicology, Gazi University, Faculty of Pharmacy and Department
of Anaesthesia, Turkish State RailwaysHospital, Ankara, Turkey In the present paper, we report data on the possible DNA damage, induced in vivo by isofturane using the alkaline single cell gel electrophoresis technique (SCGE-comet assay) in patients before/after anaesthesia and in control group. 20 patients, aged 22--66 year, were anaesthetized for elective abdominal surgery with isofturane in oxygen for 120--162 min (mean: 133.2 min). Venous blood samples were obtained from the patients before the induction of anaesthesia, during the 60 th and 120 min of anaesthesia and on the first, third and fifth following days of anaesthesia. SCGE was examined in 100 cells from each specimen graded as undamaged, stretched and comet imaged. The number of SCGE per cell was almost the same in control and in patients before anaesthesia. However, significant differences were observed in undamaged, stretched and comet imaged cells of patients during 60 th, 120 th min of anaesthesia and on the following first day. DNA damage in cells started to return to normal rates after third day of anaesthesia and were almost identical with the rates of control group five days later. We conclude that examination of comet assay in peripheral blood lymphocytes of patients during anaesthesia with isofturane in oxygen revealed indication of genotoxic effect before DNA repair begins.