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Hydroxybenzene induction of mitotic segregation in Aspergillus nidulans
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The results show that the synthetic fec-12 is a potent direct clastogen in human lymphocytes. This is in accordance with findings with the Salmonella assay and suggests that this compound is a potential colonic carcinogen.
49 Lafi, A., and J.M. Parry, School of Biological Sciences, University College of Swansea, Swansea SA2 8PP (Great Britain) Spindle fidelity and the induction of structural and numerical chromosome aberrations in primary Chinese hamster cells after exposure to cigarette smoke condensate Cigarette smoke condensate (CSC) has been shown to be genotoxic in a variety of assay systems (for review see De Marini, 1983). We have undertaken a comparative study of the ability of CSC to induce aberrations of spindle fidelity and both numerical and structural chromosome aberrations in primary Chinese hamster (Luc 1) cells. Cells treated with CSC (up to 90 /~g/ml) show elevated levels of chromosome structural aberrations, endoreduplication and both hypodiploidy and hyperdiploidy. Structural aberrations and endoreduplication were induced in a linear dose-dependent manner over the concentration range tested. In contrast numerical aberrations showed a biphasic induction curve with a plateau of response at high doses. CSC exposure produced little or no modification of the fidelity of mitotic cell division spindle synthesis or dislocations of chromosomes from the spindle apparatus as measured by the use of the differential staining technique (Parry et al., 1982). Our data suggest that induced aneuploidy observed in Chinese hamster cells exposed to CSC is unlikely to result from chromosome structural damage, nuclear spindle damage or damage to the kinetochore. References De Marini, D.M. (1983) Mutation Res., 114, 59-89. Parry, E.M., N. Danford and J.M. Parry (1982) Mutation Res., 105, 243-252.
50 Crebelli, R., J. Franekic 1, R. Benigni, G. Conti, L. Conti and A. Carere, Istituto Superiore di Sanith, Rome (Italy), and a Faculty of Food and Biotechnology, Zagreb (Yugoslavia) Induction of mitotic segregation by chloromethanes and chloroethanes in Aspergillus nidulans Several chloromethanes and chloroethanes were assayed for their ability to induce mitotic segregation in A. nidulans. All tested chemicals turned out to be active, to various degrees, in a narrow range of concentrations. Active doses were immediately below the concentration arresting conidial germination and nuclear division. Among the halomethanes assayed, CC14 was the most active (LEC 0.04% v/v), increasing up to 10-fold the spontaneous frequency of mitotic segregants. Chloroform and CHzC12 were similarly active (ca. 5-fold increases) at higher concentrations (LEC 0.16 and 0.4%, respectively). Among the haloethanes, 1,2-dichloroethane and 1,1-dichloroethane showed identical cytotoxic and cytostatic properties and were active in the same range of concentrations (0.2-0.3%) but exerted different mutagenic potencies, the former increasing the spontaneous frequencies up to 30-fold and the latter being only borderline active. Mutagenic potencies of tested chemicals were poorly correlated with log P, melting point and MW but showed a significant correlation with the refractive index.
51 Crebelli, R., G. Conti and A. Carere, Istituto Superiore di SanitY, Rome (Italy) Hydroxybenzene induction of mitotic segregation in Aspergillus nidulans Hydroquinone, catechol and phenol, the principal hydroxy metabolites of benzene, were assayed in tests for mitotic segregation induction in Aspergillus nidulans diploid strain 19. Hydroquinone was the most active chemical, increasing up to 10-fold the frequency of mitotic segregants at 1-3 mM. Catechol was similarly active at 10-20
224
mM and phenol was weakly positive at 15 mM. Genetic characterization of induced abnormal colonies segregating colour markers by replating and complementary assays with haploid strain 35 suggests that gross chromosomal aberrations, instead of numerical abnormalities, are the primary genetic damages induced by hydroxybenzenes in A. nidulans. The protecting activity exerted by L-cysteine against equimolar concentrations of hydroquinone s u p p o r t s a free radical m e c h a n i s m for hydroxybenzene genotoxicity in A. nidulans. (Partially supported by the E.E.C. contract No. 530 ENV-I-s.)
52 Danielsen, H., A. Brogger and A. Reith, The Norwegian Radium Hospital and Institute for Cancer Research, Oslo (Norway) Cytogenetic differences between diploid and tetraploid hepatocytes isolated from premalignant lesions The liver is one of the organs most widely used in studies of carcinogenesis, and several models for the identification of premalignant liver lesions have been developed. So far, few cytogenetic studies have been made on such premalignant cells from mammalian tissues. The present study is based on direct preparation and G-banding of chromosomes of premalignant nodules isolated from the livers of mice after treatment with diethylnitrosamine and partial hepatectomy. Our preliminary results show a significant difference between diploid hepatocytes and tetraploid hepatocytes with respect to the distribution of chromosomes after induced mitosis in premalignant lesions. The diploid hepatocytes demonstrated apparently normal karyotypes, with only 1 cell out of 15 showing trisomy (for chromosome 19). Tetraploid or near-tetraploid cells demonstrated on the other hand pentasomy for 1 or 2 autosomes in almost all (15 out of 16) cells, and 62.5% of the tetraploid cells studied had pentasomy for chromosome 19. We have no experimental data so far to explain the preferential gain of pentasomy for chromosome 19. The difference observed between di-
ploid and tetraploid premalignant cells might be taken as an indication of a preferentially expressed genetic a n d / o r kinetic lability, induced by the carcinogen treatment.
53 Russo, A., and F. Pacchierotti, Lab. Toxicology, ENEA CRE Casaccia, C.P. 2400, Rome (Italy) Cytogenetic effects of vinblastine on mouse oocytes The effect of vinblastine has been studied in mouse oocytes after in vivo treatment; literature data on mouse bone marrow (Liang and SatyaPrakash (1985) Mutation Res., 155, 61) showed that cell division is arrested at metaphase stage and that the incidence of this effect is dose-dependent. In the present experiment vinblastine was injected i.p. in young superovulated BC3F1 female mice at doses between 0.001 mM (0.9 m g / k g b.w.) and 0.01 mM (9 m g / k g b.w.), following the experimental design recommended in Mailhes et al. (1986) Mutation Res., 167, 139. Nearly all the oocytes collected (184) appeared arrested at the metaphase I stage, irrespective of the dose; in some cases, the presence of both bivalents and univalents with a metaphase II-like morphology has been observed, suggesting that endoreduplicative phenomena may occur. In untreated animals only 1 oocyte at the metaphase I stage has been detected over 371 eggs collected. From literature data, mouse oocytes (Tease and Fisher (1986) Mutation Res., 173, 31) appear to be more sensitive than bone marrow cells (Liang and Satya-Prakash (1985) Mutation Res., 155, 61) to the blocking activity of colchicine; similarly, a concentration 10 times lower than the maximum tested still produced, in our experimental conditions, a 100% arresting effect, while at the same two concentrations the mitotic indices recorded in bone marrow preparations differed by a factor of 2. It remains to detect the minimum effective dose, and, if it exists, the range in which the incidence of arrested cells is dose-dependent. Finally, different intervals between treatment and sacrifice may be considered to detect possible phenomena of recovery, as observed in mitotic
The results show that the synthetic fec-12 is a potent direct clastogen in human lymphocytes. This is in accordance with findings with the Salmonella assay and suggests that this compound is a potential colonic carcinogen.
49 Lafi, A., and J.M. Parry, School of Biological Sciences, University College of Swansea, Swansea SA2 8PP (Great Britain) Spindle fidelity and the induction of structural and numerical chromosome aberrations in primary Chinese hamster cells after exposure to cigarette smoke condensate Cigarette smoke condensate (CSC) has been shown to be genotoxic in a variety of assay systems (for review see De Marini, 1983). We have undertaken a comparative study of the ability of CSC to induce aberrations of spindle fidelity and both numerical and structural chromosome aberrations in primary Chinese hamster (Luc 1) cells. Cells treated with CSC (up to 90 /~g/ml) show elevated levels of chromosome structural aberrations, endoreduplication and both hypodiploidy and hyperdiploidy. Structural aberrations and endoreduplication were induced in a linear dose-dependent manner over the concentration range tested. In contrast numerical aberrations showed a biphasic induction curve with a plateau of response at high doses. CSC exposure produced little or no modification of the fidelity of mitotic cell division spindle synthesis or dislocations of chromosomes from the spindle apparatus as measured by the use of the differential staining technique (Parry et al., 1982). Our data suggest that induced aneuploidy observed in Chinese hamster cells exposed to CSC is unlikely to result from chromosome structural damage, nuclear spindle damage or damage to the kinetochore. References De Marini, D.M. (1983) Mutation Res., 114, 59-89. Parry, E.M., N. Danford and J.M. Parry (1982) Mutation Res., 105, 243-252.
50 Crebelli, R., J. Franekic 1, R. Benigni, G. Conti, L. Conti and A. Carere, Istituto Superiore di Sanith, Rome (Italy), and a Faculty of Food and Biotechnology, Zagreb (Yugoslavia) Induction of mitotic segregation by chloromethanes and chloroethanes in Aspergillus nidulans Several chloromethanes and chloroethanes were assayed for their ability to induce mitotic segregation in A. nidulans. All tested chemicals turned out to be active, to various degrees, in a narrow range of concentrations. Active doses were immediately below the concentration arresting conidial germination and nuclear division. Among the halomethanes assayed, CC14 was the most active (LEC 0.04% v/v), increasing up to 10-fold the spontaneous frequency of mitotic segregants. Chloroform and CHzC12 were similarly active (ca. 5-fold increases) at higher concentrations (LEC 0.16 and 0.4%, respectively). Among the haloethanes, 1,2-dichloroethane and 1,1-dichloroethane showed identical cytotoxic and cytostatic properties and were active in the same range of concentrations (0.2-0.3%) but exerted different mutagenic potencies, the former increasing the spontaneous frequencies up to 30-fold and the latter being only borderline active. Mutagenic potencies of tested chemicals were poorly correlated with log P, melting point and MW but showed a significant correlation with the refractive index.
51 Crebelli, R., G. Conti and A. Carere, Istituto Superiore di SanitY, Rome (Italy) Hydroxybenzene induction of mitotic segregation in Aspergillus nidulans Hydroquinone, catechol and phenol, the principal hydroxy metabolites of benzene, were assayed in tests for mitotic segregation induction in Aspergillus nidulans diploid strain 19. Hydroquinone was the most active chemical, increasing up to 10-fold the frequency of mitotic segregants at 1-3 mM. Catechol was similarly active at 10-20
224
mM and phenol was weakly positive at 15 mM. Genetic characterization of induced abnormal colonies segregating colour markers by replating and complementary assays with haploid strain 35 suggests that gross chromosomal aberrations, instead of numerical abnormalities, are the primary genetic damages induced by hydroxybenzenes in A. nidulans. The protecting activity exerted by L-cysteine against equimolar concentrations of hydroquinone s u p p o r t s a free radical m e c h a n i s m for hydroxybenzene genotoxicity in A. nidulans. (Partially supported by the E.E.C. contract No. 530 ENV-I-s.)
52 Danielsen, H., A. Brogger and A. Reith, The Norwegian Radium Hospital and Institute for Cancer Research, Oslo (Norway) Cytogenetic differences between diploid and tetraploid hepatocytes isolated from premalignant lesions The liver is one of the organs most widely used in studies of carcinogenesis, and several models for the identification of premalignant liver lesions have been developed. So far, few cytogenetic studies have been made on such premalignant cells from mammalian tissues. The present study is based on direct preparation and G-banding of chromosomes of premalignant nodules isolated from the livers of mice after treatment with diethylnitrosamine and partial hepatectomy. Our preliminary results show a significant difference between diploid hepatocytes and tetraploid hepatocytes with respect to the distribution of chromosomes after induced mitosis in premalignant lesions. The diploid hepatocytes demonstrated apparently normal karyotypes, with only 1 cell out of 15 showing trisomy (for chromosome 19). Tetraploid or near-tetraploid cells demonstrated on the other hand pentasomy for 1 or 2 autosomes in almost all (15 out of 16) cells, and 62.5% of the tetraploid cells studied had pentasomy for chromosome 19. We have no experimental data so far to explain the preferential gain of pentasomy for chromosome 19. The difference observed between di-
ploid and tetraploid premalignant cells might be taken as an indication of a preferentially expressed genetic a n d / o r kinetic lability, induced by the carcinogen treatment.
53 Russo, A., and F. Pacchierotti, Lab. Toxicology, ENEA CRE Casaccia, C.P. 2400, Rome (Italy) Cytogenetic effects of vinblastine on mouse oocytes The effect of vinblastine has been studied in mouse oocytes after in vivo treatment; literature data on mouse bone marrow (Liang and SatyaPrakash (1985) Mutation Res., 155, 61) showed that cell division is arrested at metaphase stage and that the incidence of this effect is dose-dependent. In the present experiment vinblastine was injected i.p. in young superovulated BC3F1 female mice at doses between 0.001 mM (0.9 m g / k g b.w.) and 0.01 mM (9 m g / k g b.w.), following the experimental design recommended in Mailhes et al. (1986) Mutation Res., 167, 139. Nearly all the oocytes collected (184) appeared arrested at the metaphase I stage, irrespective of the dose; in some cases, the presence of both bivalents and univalents with a metaphase II-like morphology has been observed, suggesting that endoreduplicative phenomena may occur. In untreated animals only 1 oocyte at the metaphase I stage has been detected over 371 eggs collected. From literature data, mouse oocytes (Tease and Fisher (1986) Mutation Res., 173, 31) appear to be more sensitive than bone marrow cells (Liang and Satya-Prakash (1985) Mutation Res., 155, 61) to the blocking activity of colchicine; similarly, a concentration 10 times lower than the maximum tested still produced, in our experimental conditions, a 100% arresting effect, while at the same two concentrations the mitotic indices recorded in bone marrow preparations differed by a factor of 2. It remains to detect the minimum effective dose, and, if it exists, the range in which the incidence of arrested cells is dose-dependent. Finally, different intervals between treatment and sacrifice may be considered to detect possible phenomena of recovery, as observed in mitotic