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In vitro and in vivo studies of the toxic and mutagenic effects of neoprene
408
Klebsiella pneumoniae in the fluctuation test but not for Salmonella typhimurium TA100 in the Ames test without metabolic activation. The mutagenic activity of the following substances was investigated: acetamide, chloroacetamide, dichloroacetamide, trichloroacetamide, N-chlorodichloroacetamide, N-chlorotrichloroacetamide, bromoacetamide, dichloroacetonitrile, acetaldoxime, dichloroacetohydroxamoylchloride, trichlorohydroxamoylchloride, dichloroacetic acid and trichloroacetic acid. With the fluctuation test mutagenic activity was found in Klebsiella pneumoniae for chloroacetamide, dichloroacetamide, trichloroacetamide, dichloroacetonitrile, acetaldoxime and probably bromoacetamide. With the Ames test mutagenic activity in the Salmonella typhimurium strains TA100 and TA98 was found for trichloroacetamide and dichloroacetonitrile and in strain TA100 with acetaldoxime. On the basis of the obtained results from the mutagenicity and analytical chemistry investigations a revised reaction scheme could be made. The mutagenic activity of the mixture of reaction products caused by the chlorination of cyanoethanoic acid was caused by dichloroacetonitrile.
was increased 7-fold and that of 30 #g/plate 4-fold by the liver microsomal enzymes ($9), but only in the presence of NADP. Induction of the liver enzymes with Aroclor 1254 did not further activate quercetin mutagenicity. Quercetin is known to undergo oxidative degradation in aqueous solution. This spontaneous degradation can be measured spectrophotometrically. When SOD, $100 or $9 with or without NADP was added, no changes in the spectrum of quercetin were observed. The enhancement of quercetin mutagenicity by these components can be explained as prevention of degradation. The additional activation of quercetin mutagenicity by $9 in the presence of NADP, however, imphes that quercetin must undergo metabolic transformation.
In vitro and in vivo studies of the toxic and mutagenic effects of neoprene
Venegas, W., and I. Chouroulinkov, Genetic Toxicology Laboratory, University of Concepci6n, Concepci6n (Chile) and Institut de Recherches Scientifiques sur le Cancer, CNRS, 84802 Villejuif (France)
Metabolic activation of quercetin mutagenicity
Vrijsen, R., A. Broos and A. Boey6, Department of Microbiology and Hygiene, Vrije Universiteit Brussel, B-1090 Brussels (Belgium) The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test (MacGregor, 1984). Using test strain TA98, quercetin is weakly mutagenic in the absence, but strongly mutagenic in the presence of a liver metabolic activation system. The requirements of the metabolic activation system were examined. The addition of rat liver cytosol (S100) with or without NADP enhanced the mutagenic activity of 15 /xg quercetin/plate by 200% and that of 30/~g/plate by 100%. We confirmed that the cytosol enzyme superoxide dismutase (SOD) was responsible for the enhancement of the mutagenic activity of quercetin by the S100 preparation (Ochiai et al., 1984). The mutagenic activity of 15/~g quercetin/plate
Inhalation of neoprene, a mix of volatile organic solvents incorporated into a type of glue and sniffed by some infants and youngsters of many countries, became a worrying health problem in the beginning of the 1970s in Chile. In order to study the genotoxic effects of this solvent mix, in vitro and in vivo experiments were carried out. Cell cultures of the cell line V79 were treated with the volatile solvents of the glue neoprene during variable periods of time under sterile conditions. Cells were then treated for the study of sisterchromatid exchanges (SCE). For in vivo study, 20 specimens of Cricetus griseus were used in inhalation experiments for 35 days with a daily average time of 3.5 h of inhalation. Hamsters were killed at regular time intervals and tissue samples were prepared for the study of SCE frequency and of damage in different areas of the respiratory tract by histological analysis. The results show a significant increase in SCE frequency in relation to the time of exposure in both in vitro and in vivo
409 experiments. Histological analysis revealed that animals under inhalation conditions showed not only inflammatory lesions of the trachea and larynx, but lung hypertrophy as well.
DNA damages by hydroquinone and duroquinone Walles, S.A.S., Department of Toxicology, National Institute of Occupational Health, Solna (Sweden) Hydroquinone (HQ) and duroquinone (tetramethyl-benzoquinone, DQ) have earlier been shown to induce single-strand breaks (SSB) in liver D N A of rats. Primary cultures of hepatocytes were used in order to make mechanistic studies of the DNA-damaging effects of HQ and DQ. The hepatocytes were incubated with the chemicals in Krebs-Henseleit buffer in rotating flasks for 1 h at 37 o C. 1 million cells/ml were used. SSB were measured by the DNA-unwinding technique and the alkaline elution technique (AE). With AE different types of elution curves were found. D N A from hepatocytes treated with HQ showed straight elution curves like those from cells exposed to y-radiation. The D N A damage induced by 7-radiation is known to be caused by O H radicals. The elution curve for DQ-treated cells was initially steep and then slowed with time, which indicates double-strand breaks in DNA. HQ gave linear dose curves. SSB induced by increasing doses of DQ reached a plateau value. The same phenomenon has been reported for TPA, and indicates an influence of the superoxide anion. For both substances the viability remained unchanged, while the SSB levels increased. AE was used to evaluate if HQ and DQ induced alkali-labile sites (ALS). The D N A was eluted at pH 12.1 and at pH 12.8. The elution rates for D N A from cells treated with HQ and DQ were higher at p H 12.8 than at pH 12.1. This indicates ALS. The results suggest that H Q mostly attacks D N A by means of OH radicals and that the active species of DQ is the superoxide anion.
Interaction between benzoylperoxide and TPA in the V79 metabolic cooperation assay W~rng&rd, L. 1, S. Duddy 2 and G. Kass 2, 1 Institute of Environmental Medicine and 2 Department of Toxicology, Karolinska Instituter, S-104 01 Stockholm (Sweden) An in vitro assay measuring inhibition of metabolic cooperation between fibroblasts of Chinese hamster cells (V79) was used to study the effect of benzoylperoxide (BP) on metabolic cooperation. The principle of the assay is to cultivate 6thioguanine (6-TG)-sensitive ( H G - P R T - ) and 6TG-resistant ( H G - P R T +) cells in 6-TG-containing medium. The endpoint is to measure the recovery of H G - P R T - colonies in the presence and absence of a test agent. BP was tested alone and together with increasing doses of the tumour promoter TPA. The doses of BP were 3 and 5 /~M. The dose range of TPA was 0.1-1.0 nM. BP alone did not enhance the recovery of mutant cells. TPA alone increased the recovery as expected. When BP was added together with TPA we observed a potentiation of the response demonstrated as a shift in the dose-response curve for TPA. Preliminary data with redox-cycling quinones have also demonstrated an enhancement of the TPA-induced inhibition of intercellular communication. These results suggest that oxidative stress is important but not sufficient for the inhibition of intercellular communication. Inhibition of intercellular communication has been associated with the activation of protein kinase C. Studies on the effect on protein kinase C after coexposure with BP and TPA are in progress.
Genotoxicity of formamidine sulfinate, a metabolite of thiourea, in V79 cells Ziegler-Skylakakis, K., and U. Andrae, GSF-Institut ftir Toxikologie, D-8042 N e u h e r b e r g (F.R.G.) Thiourea (TU) is a carcinogen which causes thyroid tumors by a thyroid-specific non-geno-
Klebsiella pneumoniae in the fluctuation test but not for Salmonella typhimurium TA100 in the Ames test without metabolic activation. The mutagenic activity of the following substances was investigated: acetamide, chloroacetamide, dichloroacetamide, trichloroacetamide, N-chlorodichloroacetamide, N-chlorotrichloroacetamide, bromoacetamide, dichloroacetonitrile, acetaldoxime, dichloroacetohydroxamoylchloride, trichlorohydroxamoylchloride, dichloroacetic acid and trichloroacetic acid. With the fluctuation test mutagenic activity was found in Klebsiella pneumoniae for chloroacetamide, dichloroacetamide, trichloroacetamide, dichloroacetonitrile, acetaldoxime and probably bromoacetamide. With the Ames test mutagenic activity in the Salmonella typhimurium strains TA100 and TA98 was found for trichloroacetamide and dichloroacetonitrile and in strain TA100 with acetaldoxime. On the basis of the obtained results from the mutagenicity and analytical chemistry investigations a revised reaction scheme could be made. The mutagenic activity of the mixture of reaction products caused by the chlorination of cyanoethanoic acid was caused by dichloroacetonitrile.
was increased 7-fold and that of 30 #g/plate 4-fold by the liver microsomal enzymes ($9), but only in the presence of NADP. Induction of the liver enzymes with Aroclor 1254 did not further activate quercetin mutagenicity. Quercetin is known to undergo oxidative degradation in aqueous solution. This spontaneous degradation can be measured spectrophotometrically. When SOD, $100 or $9 with or without NADP was added, no changes in the spectrum of quercetin were observed. The enhancement of quercetin mutagenicity by these components can be explained as prevention of degradation. The additional activation of quercetin mutagenicity by $9 in the presence of NADP, however, imphes that quercetin must undergo metabolic transformation.
In vitro and in vivo studies of the toxic and mutagenic effects of neoprene
Venegas, W., and I. Chouroulinkov, Genetic Toxicology Laboratory, University of Concepci6n, Concepci6n (Chile) and Institut de Recherches Scientifiques sur le Cancer, CNRS, 84802 Villejuif (France)
Metabolic activation of quercetin mutagenicity
Vrijsen, R., A. Broos and A. Boey6, Department of Microbiology and Hygiene, Vrije Universiteit Brussel, B-1090 Brussels (Belgium) The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test (MacGregor, 1984). Using test strain TA98, quercetin is weakly mutagenic in the absence, but strongly mutagenic in the presence of a liver metabolic activation system. The requirements of the metabolic activation system were examined. The addition of rat liver cytosol (S100) with or without NADP enhanced the mutagenic activity of 15 /xg quercetin/plate by 200% and that of 30/~g/plate by 100%. We confirmed that the cytosol enzyme superoxide dismutase (SOD) was responsible for the enhancement of the mutagenic activity of quercetin by the S100 preparation (Ochiai et al., 1984). The mutagenic activity of 15/~g quercetin/plate
Inhalation of neoprene, a mix of volatile organic solvents incorporated into a type of glue and sniffed by some infants and youngsters of many countries, became a worrying health problem in the beginning of the 1970s in Chile. In order to study the genotoxic effects of this solvent mix, in vitro and in vivo experiments were carried out. Cell cultures of the cell line V79 were treated with the volatile solvents of the glue neoprene during variable periods of time under sterile conditions. Cells were then treated for the study of sisterchromatid exchanges (SCE). For in vivo study, 20 specimens of Cricetus griseus were used in inhalation experiments for 35 days with a daily average time of 3.5 h of inhalation. Hamsters were killed at regular time intervals and tissue samples were prepared for the study of SCE frequency and of damage in different areas of the respiratory tract by histological analysis. The results show a significant increase in SCE frequency in relation to the time of exposure in both in vitro and in vivo
409 experiments. Histological analysis revealed that animals under inhalation conditions showed not only inflammatory lesions of the trachea and larynx, but lung hypertrophy as well.
DNA damages by hydroquinone and duroquinone Walles, S.A.S., Department of Toxicology, National Institute of Occupational Health, Solna (Sweden) Hydroquinone (HQ) and duroquinone (tetramethyl-benzoquinone, DQ) have earlier been shown to induce single-strand breaks (SSB) in liver D N A of rats. Primary cultures of hepatocytes were used in order to make mechanistic studies of the DNA-damaging effects of HQ and DQ. The hepatocytes were incubated with the chemicals in Krebs-Henseleit buffer in rotating flasks for 1 h at 37 o C. 1 million cells/ml were used. SSB were measured by the DNA-unwinding technique and the alkaline elution technique (AE). With AE different types of elution curves were found. D N A from hepatocytes treated with HQ showed straight elution curves like those from cells exposed to y-radiation. The D N A damage induced by 7-radiation is known to be caused by O H radicals. The elution curve for DQ-treated cells was initially steep and then slowed with time, which indicates double-strand breaks in DNA. HQ gave linear dose curves. SSB induced by increasing doses of DQ reached a plateau value. The same phenomenon has been reported for TPA, and indicates an influence of the superoxide anion. For both substances the viability remained unchanged, while the SSB levels increased. AE was used to evaluate if HQ and DQ induced alkali-labile sites (ALS). The D N A was eluted at pH 12.1 and at pH 12.8. The elution rates for D N A from cells treated with HQ and DQ were higher at p H 12.8 than at pH 12.1. This indicates ALS. The results suggest that H Q mostly attacks D N A by means of OH radicals and that the active species of DQ is the superoxide anion.
Interaction between benzoylperoxide and TPA in the V79 metabolic cooperation assay W~rng&rd, L. 1, S. Duddy 2 and G. Kass 2, 1 Institute of Environmental Medicine and 2 Department of Toxicology, Karolinska Instituter, S-104 01 Stockholm (Sweden) An in vitro assay measuring inhibition of metabolic cooperation between fibroblasts of Chinese hamster cells (V79) was used to study the effect of benzoylperoxide (BP) on metabolic cooperation. The principle of the assay is to cultivate 6thioguanine (6-TG)-sensitive ( H G - P R T - ) and 6TG-resistant ( H G - P R T +) cells in 6-TG-containing medium. The endpoint is to measure the recovery of H G - P R T - colonies in the presence and absence of a test agent. BP was tested alone and together with increasing doses of the tumour promoter TPA. The doses of BP were 3 and 5 /~M. The dose range of TPA was 0.1-1.0 nM. BP alone did not enhance the recovery of mutant cells. TPA alone increased the recovery as expected. When BP was added together with TPA we observed a potentiation of the response demonstrated as a shift in the dose-response curve for TPA. Preliminary data with redox-cycling quinones have also demonstrated an enhancement of the TPA-induced inhibition of intercellular communication. These results suggest that oxidative stress is important but not sufficient for the inhibition of intercellular communication. Inhibition of intercellular communication has been associated with the activation of protein kinase C. Studies on the effect on protein kinase C after coexposure with BP and TPA are in progress.
Genotoxicity of formamidine sulfinate, a metabolite of thiourea, in V79 cells Ziegler-Skylakakis, K., and U. Andrae, GSF-Institut ftir Toxikologie, D-8042 N e u h e r b e r g (F.R.G.) Thiourea (TU) is a carcinogen which causes thyroid tumors by a thyroid-specific non-geno-