277 Cytogenetic effects of cytostaties on human tumor cells in culture The induction of chromosome damage and of micronuclei by vincristine, azaserine, and methotrexate was examined on breast tumor cell lines MDA MB 231 and 435. In addition, the same endpoints were analyzed in the newly established cell line ER MC 116 (derived from a carcinoma of the cervix uteri) after treatment with methylCCNU (Semustin). In all cases a more or less pronounced increase of the frequency of breakage and interchange aberrations -- even several weeks after treatment - - could be detected which was in good correlation with the increase of the rate of micronuclei. In experiments with vincristine, in addition, a corresponding increase was found of spontaneous PCC formation. Long-term treatment with semustin resulted in some resistance of the tumor cells to the chromosome-damaging action of this cytostatic which neither could be recognized with the other drugs nor be documented by the finding of cells exhibiting cytogenetic equivalents gene amplification. In general, a clear cytogenetic instability of tumor cells was found which varied according to the type and activity of the applied clastogens.
6 Dresp, J., and M. Bauchinger, Institut fiir Strahlenbiologie, GSF, D-8042 Neuherberg (F.R.G.) The analysis of the clastogenic effect of environmental chemicals in human lymphocytes by means of premature chromosome condensation In 1984 Pantelias and Maillie recommended the use of premature chromosome condensation (PCC) to study radiation-induced cytogenetic damage. In subsequent papers they reported on the advantages of this method: (1) Detection of chromosome damage as early as 2 h after venipuncture in G O or G 1 lymphocytes, respectively. (2) Easier and faster scoring of the induced damage. In 1985 Pantelias and Wolff proposed the use of the PCC method also for mutagenicity testing of chemical clastogens. In previous studies we found different aberration types after in vivo and in vitro exposure of
human lymphocytes to formaldehyde (FA). In the present study the PCC technique was applied to analyse the clastogenic effect of 0.125-0.5 mM FA in interphase chromatin of unstimulated lymphocytes in vitro. PCC has been achieved by fusion of lymphocytes with synchronized CHO cells. FA treatment of 1 h induced a dose-dependent increase in PCC chromosome fragments of G O lymphocytes. Advantages and disadvantages of the PCC technique as compared to conventional aberration scoring for the detection of chemical clastogens are discussed.
7 Emerit, I., CNRS, Institut Biomrdical, Universit6 Paris VI, 15, rue de l'Ecole de Mrdecine, F-75006 Paris (France) Formation of the DNA-damaging material during oxidative stress The survival of aerobic organisms in an oxygen environment involves a complicated interplay between the physiological generation of very reactive chemical species called 'free radicals' and the ability of the organism to control their level in the tissues. Disease states, xenobiotics and other environmental stress can overwhelm defense mechanisms and cause cytotoxicity. Oxygen-derived free radicals, such as 0 2 and O H , induce DNA damage and may therefore play a role in mutagenesis and carcinogenesis. However the action of these free radicals on DNA cannot always be 'direct', in particular when they are generated extracellularly. Because of their high reactivity, they cannot reach the target nucleus, and we have to assume the formation of secondary chemical species that can. These reactions may occur with non-DNA targets. Initiation of the chain reaction of lipid peroxidation in cellular membranes may result in metabolites derived from arachidonic acid, which may themselves be clastogenie agents or induce DNA damage again via the generation of free radicals after having reached the target. DNA-damaging material described as chromosome breakage or clastogenic factors (CF) may represent such intermediates. The CF are low
278 molecular weight substances (under 10 000 dalton) and have been observed as an indirect effect of irradiation, in chronic inflammatory conditions and in the hereditary diseases ataxia telangiectasia and Bloom's syndrome, known for their high risk of cancer and leukemia. They may be produced in vitro by photochemical or enzymatic generation of 02-. Superoxide dismutase prevents their formation and their DNA-damaging effect. Since 0 2 is implicated in CF action and at the same time initiates more CF by lipid peroxidation of membranes, this results in an auto-sustaining or even self-amplificating process. CF may be the link between chronic inflammation and cancer. An example herefore are New Zealand Black mice, that combine a chronic inflammatory condition similar to human systemic lupus erythematosus with a high risk of malignant lymphoma. They have increased chromosome damage in bone marrow, and a CF can be isolated from their plasma. Resident macrophages from the peritoneal cavity produce spontaneously 0 2 , as measured by the cytochrome c assay. Clastogenie material can be isolated from the supernatant of these macrophages after 1 h. This is prevented by superoxide dismutase. Since clastogenic material is produced in lymphocyte cultures exposed to xanthin-xanthine oxidase reaction, it is probable that CF formation plays also a role in the phenomenon of postischemic reperfusion injury. In general, CF may be important as intermediates in oxygen pathology, transporting oxygen free radical-induced damage to distant sites.
Favor, J., A. Neuh~iuser-Klaus and U.H. Ehling, Institut ffir S~ugetiergenetik, GSF, D-8042 Neuherberg (F.R.G.) E N U and radiation-induced reverse mutations recovered in mice The frequency of recessive specific locus mutations and dominant cataract mutations was determined in offspring derived from treated male D B A / 2 mice subsequently mated to untreated tester-stock females. Males were treated with either 160 m g / k g E N U or 3 + 3 Gy (24 h fractionation
interval) X-irradiation. Since both D B A / 2 and tester-stock mice are homozygous non-agouti, brown, dilute, the experimental protocol allows for the first time the determination of the induced reverse mutation rate at the a, b and d loci. In the E N U treatment group, one reverse mutation was recovered at each of the a and b loci in 14342 offspring examined, while in 15931 offspring of the irradiation treatment group one reverse mutation was recovered at each of the a and d loci. Comparing these results to the extensive spontaneous mutation rate data (Mutation Res., 11 (1971) 89-96) indicates the observed reverse mutation rate at the a and d loci following paternal mutagenic treatment to be 16-17 times higher than the spontaneous rate. Spontaneous reverse mutations were only observed at the a and d loci. A molecular characterization of the d allele has indicated the mutant allele to be the result of an insertion of an ecotropic-specific retroviral genome in the vicinity of the dilute locus and the instability of the d allele to be due to the mobility of the endogenous proviral sequence. The nonagouti allele is similarly unstable and may be an interesting candidate for characterization at the molecular level. In contrast to the a and d alleles, the b allele was not observed to revert spontaneously and was not expected to revert following paternal mutagenie treatment. It has recently been shown that the brown mutation is associated with a novel endonuclease restriction site within the brown locus (I. Jackson, personal communication). The observed reverse mutation following E N U treatment may have resulted from an induced base substitution. (Research supported in part by contract No. BI6-156-D of the Commission of the European Communities.)
9 Fertig, G., and H.G. Miltenburger, Institut fgr Zoologie, Technische Hochschule Darmstadt, D6100 Darmstadt (F.R.G.) Using flow cytometry in cytotoxicity research and mutagenicity testing Flow cytometric measuring (Flow Cytometer