Micronuclei induced in erythrocytes of Cyprinus carpio (teleostei, pisces) by X-rays and colchicine.

In the present work the induction of micronuclei in erythrocytes of Cyprinus carpio treated with X-rays and colchicine is studied for the evaluation of mutagenic effects of both clastogenic and mitoclastic (spindle poisoning) agents in this system. Three different experiments were performed treating groups of laboratory-reproduced animals with (1) single doses of X-rays (0.1, 0.5 and 2Gy); (2) a single i.p. injection of colchicine at the concentrations: 1.6x10(-2), 8x10(-2), 0.4 and 2mg/kg b.w. so as to mimic an acute exposure to the agent and (3) six repeated i.p. injections of the first three concentrations of colchicine, over a period of 18 days, so as to mimic a chronic exposure. Repeated blood samplings were performed by cardiac puncture over a period of about 2 months after the treatment and micronucleus frequencies were determined at multiple times on the same individuals after mutagen exposure. A dose-dependent increase in the micronucleus frequency was observed in irradiated fish and a peak value detected at 21 days. Slight increases of micronucleus frequencies were also observed in both colchicine experiments only for the highest concentrations at the earliest sampling time. Higher concentrations of colchicine clearly showed a lethal effect. According to the present data the micronucleus frequency induced by the highest colchicine dose is comparable to that observed after 0.1Gy of X-ray irradiation.

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 M(2+) 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 approximately 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.

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 approximately 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.

Mutagenicity (micronucleus test in Vicia faba root tips), polycyclic aromatic hydrocarbons and heavy metal content of sediments collected in Tiber river and its tributaries within the urban area of Rome.

Sediments collected in Tiber river and in its main tributary water courses within the urban area of Rome were tested for mutagenicity by means of Vicia faba root tips micronucleus (MN) test. Representative samples were scored for micronucleus generating events (chromosome/chromatid loss and fragments) too. Sediments were assayed for content of the thirteen most important chemicals of polycyclic aromatic hydrocarbon (PAH) group and for some heavy metal ions (Cd, Cr, Cu, Ni, Pb, Zn). Samples were collected in four tributary rivers (Prima Porta, Acqua Traversa, Aniene and Magliana) just before their confluence with Tiber river and at different stations along the Tiber river itself upstream and downstream the sites of confluence of the sampled tributaries. All samples were collected in July 1992. An alarming level of mutagenicity was reached in most of the tested stations, with an effect comparable to an X-rays exposure up to 0.4 Gy. Chemical analysis showed that the total amount of identified PAHs ranged from 4.5 to 625.2 ng/g of dry matrix in the different stations and the total amount of heavy metals ranged from 130 to 570 ppm. Tiber mutagenicity is likely to be mainly due to local factors such as the confluence of a small polluted tributary rather than to large scale effect due to an upstream-downstream relationship.

Heavy metal content and mutagenic activity, evaluated by Vicia faba micronucleus test, of Tiber river sediments.

Tiber river sediment samples, collected in October 1995, were tested for mutagenicity by the micronucleus test in Vicia faba root tips. Four stations were studied within the urban area of Rome: (1) Castel Giubileo, at the entry of the urban area; (2) Ponte Tor di Quinto, immediately after the confluence of the tributary river Aniene; (3) Ponte Sublicio, in the middle of the city; and (4) Ponte della Magliana, immediately outside Rome, 20 km from the sea. Since no significant increase in micronucleus frequency was observed in any of the tested stations compared to control (while in previous campaigns mutagenic activity was observed in some of the same stations), it can be assumed an interesting recovery from mutagenic pollution in the 2 last years. The samples were analysed for pH value, nitrogen, organic matter and carbonate content, and the concentration of some potentially mutagenic heavy metal ions (Zn, Cd, Ni, V, Cu) was assessed. In all samples, a concentration of heavy metals higher than unpolluted areas was observed. However, the alkaline pH measured should keep them as non-bioavailable elements.

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 Italy which have different pollution levels. As indicator species, the barbel (Barbus plebejus) was chosen because of its ecological significance. Blood samplings were 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 (Tiber), 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.