Micronucleus test in erythrocytes of Barbus plebejus (Teleostei, Pisces) from two natural environments: A bioassay for the in situ detection of mutagens in freshwater

Erythrocyte micronucleus frequencies in wild fish from two riverine environments and in fish reproduced and reared under controlled conditions (control group) were compared, with the aim to evaluate the suitability of the MN test for the in situ detection of mutagens in freshwaters. Fish were caught in different months in two rivers of central ItaIy which have different pollution levels. As indicator species, the barbel (Barbus plebejus) was chosen because of its ecological significance. Blood samplings wen performed on wild fish immediately after capture and repeated at different time intervals on the same individuals, which were maintained in controlled conditions after capture. A total of 10,000 erythrocytes per specimen were scored. No significant differences in micronucleus frequencies were observed between the control group and fish from the unpolluted river (Mignone). A significantly higher frequency of micronuclei was observed in fish caught in the polluted river (Tibet), in comparison to both the controls and the Mignone river fish. No significant seasonal differences were observed. Barbels examined 50 and 100 days after capture presented a remarkable decrease in micronucleus frequency in comparison with the frequency observed in barbels at capture. The micronucleus test in fish erythrocytes was shown to be a sensitive bioassay for detecting mutagenic pollution in fresh water environments

Effect of cytochalasin B on the induction of chromosome missegregation by colchicine at low concentrations in human lymphocytes

The aim of the present work was to investigate the possible interference of cytochalasin B (cyt B) with low concentration treatment with colchicine in the induction of chromosome/chromatid loss and micronuclei in human lymphocytes mitotically activated in vitro. Thus, cells from a single female donor were treated with colchicine (10 or 25 nM, from 24 h after PHA addition to fixation at 66 h) either in the presence or absence of cyt B. Single lagging chromosomes/chromatids were scored in bipolar ana-telophases and greater damage (disrupted and c-anaphases) was scored in cells at anaphase. Micronuclei were scored in the first 4000 nuclei observed in both cyt B-treated (in mononucleate and binucleate cells) and untreated cultures. With the same criterion, FISH analysis was performed on 2000 nuclei where chromosome 7 and 11 centromeric DNA probes were used in pairs. Our results showed that: (i) the frequency of laggards and of micronuclei increased with colchicine concentration but in the presence of cyt B there was a lower frequency of both (with a mean reduction of ~ 49%); (ii) FISH analysis showed a colchicine concentration-dependent increase in nuclei with three spots for chromosome 7; (iii) a colchicine concentration-dependent increase in tetraploid cells was observed. This increase was particularly remarkable (5-fold) in cells grown in the presence of cyt B compared with cyt B-untreated cells. The observed 'cyt B effects' can be explained if it is assumed that in cytokinesis-blocked cells there is a shorter distance between the poles. As a consequence: (i) laggards would be engulfed in the nearest daughter nucleus with a consequent lower induction of micronuclei; (ii) segregating sister chromatids in heavily impaired anaphases would not travel a sufficient distance to give rise to two daughter nuclei, leading to an increased frequency of polyploid nuclei

Direct and indirect non-disjunction in the origin of trisomy in cultured human lymphocytes

The aim of the present work was to investigate the processes involved in the origin of trisomic karyotypes, i.e. co-migration of sister chromatids (mitotic non-disjunction, MND) and recovery of micronuclei (MN) originating from lagging chromosomes/chromatids at anaphase (mitotic indirect non-disjunction, MIND), and to evaluate their relative contribution to aneuploidy in human lymphocytes mitotically activated in vitro. Therefore, phytohaemagglutinin-stimulated human lymphocytes from one donor were treated with 10 and 25 nM colchicine and analysed through two cell cycles by means of both molecular (FISH with centromeric DNA probes specific for chromosomes 7 and 11) and classical cytogenetic techniques. The following events were analysed: (i) chromosome/chromatid Loss (a MN-generating event) in M-1 bipolar ana-telophases; (ii) MN recovery in M2+ prophases; (iii) non-disjunction and loss of chromosomes 7 and 11 by FISH analysis in cytochalasin B-induced binucleate cells; (iv) spontaneous frequency of trisomic cells by chromosome counting and FISH analysis in M-1 c-metaphases; (v) induced frequency of trisomic cells by chromosome counting and FISH analysis in M-2 c-metaphases. Our results indicate that MND plays a major role compared with MIND in the origin of trisomic karyotypes, being similar to 4- to 5-fold higher in colchicine-treated cells. Moreover, remarkable reductions in the observed frequencies of trisomic cells were recorded in comparison with the expected ones, with an observed/ expected frequency ratio of trisomic M-2 c-metaphases ranging between 1/3 and 1/6