Nondisjunction rates of mouse chromosomes involved in heterozygous Rb rearrangements measured by chromosome painting of spermatocytes. II. The effects of trivalent combinations and genetic background.

Chromosome specific nondisjunction rates were quantified by dual-colour FISH in spermatocytes II of Robertsonian heterozygous mice with different trivalent combinations or, alternatively, with different genetic backgrounds. We found that such factors do not influence the proneness to nondisjunction of specific chromosomes.

Nondisjunction rates of mouse specific chromosomes involved in heterozygous Rb rearrangements measured by chromosome painting of spermatocytes II. I. The effects of the number of trivalents.

Dual-colour FISH painting with alternative fluorescent chromosome-specific probes allowed us to distinguish chromosomes 1, 4, 6 and 14. The purpose was to check whether nondisjunction rates of specific chromosomes involved in heterozygous Robertsonian fusions are independent of the number of trivalents, or an epistatic effect among Rb chromosomes takes place affecting nondisjunction rates. Probes were used on DAPI-stained metaphases of spermatocytes II of laboratory strains of mice with reconstructed karyotypes heterozygous for one, two, three or four Robertsonian metacentrics in an all-acrocentric background. The existence of such epistatic interactions was not verified.

Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization.

The detection of a possible genotoxic effect of surface water treated with disinfectants for potabilization is the aim of the present work. The Comet assay and the micronucleus test were applied in circulating erythrocytes of Cyprinus carpio. Young specimens (20-30 g) were exposed in experimental basins, built within the potabilization plant of Castiglione del Lago (Perugia, Italy). In this plant the water of the Trasimeno Lake is treated and disinfected for potabilization before it is distributed to the people in the net of drinkable water. A continuous flow of water at a constant rate was supplied to basins; the water was continuously treated at a constant concentration with one of the three tested disinfectants (sodium hypochlorite, peracetic acid and chloride dioxide), one control basin being supplied with untreated water. Three sampling campaigns were performed: October 2000, February 2001 and June 2001. Repeated blood samplings through intracardiac punctures allowed to follow the same fish populations after different exposure times: before introduction of the disinfectant, and 10 or 20 days afterwards. An additional blood sampling was performed 3 h after addition of the disinfectant in other, simultaneously exposed, fish populations. Genotoxic damage was shown in fish exposed to water disinfected with sodium hypochlorite and chloride dioxide. The Comet assay showed an immediate response, i.e. DNA damage that was induced directly in circulating erythrocytes, whereas micronuclei reached their highest frequencies at later sampling times, when a genotoxic damage in stem cells of the cephalic kidney is expressed in circulating erythrocytes. The quality of the untreated surface water seems to be the most important parameter for the long-term DNA damage in circulating erythrocytes.

Cosegregation of Robertsonian metacentric chromosomes in the first meiotic division of multiple heterozygous male mice as revealed by FISH analysis of spermatocyte II metaphases.

Contrasting results (random segregation or cosegregation of isomorphic chromosomes) have been reported up to now on the segregation pattern of Robertsonian metacentric chromosomes of Mus musculus domesticus in multiple heterozygotes, using different approaches (karyotypical analysis of the progeny or of second meiotic metaphases). In the present contribution data are presented based on FISH (Fluorescence In Situ Hybridisation) analysis with telomeric probes, which allowed us to distinguish metacentric chromosomes from pairs of acrocentric chromosomes with their centromeric regions close to each other. Probes were hybridized to DAPI stained metaphases of spermatocytes II of mice heterozygous for two, three or four Robertsonian metacentrics in an all-acrocentric background, the karyotype of which has been reconstructed starting from laboratory strains. Isomorphic chromosomes tend to cosegregate (metacentrics with metacentrics, acrocentrics with acrocentrics); the values found for cosegregation have a clear even if moderate effect on the reproductive isolation caused by underdominant chromosomal rearrangements.

Genotoxicity of surface water treated with different disinfectants using in situ plant tests.

Disinfection of surface drinking water, in particular water chlorination, results in many by-products with potential genotoxic and/or carcinogenic activity. In the present study, we evaluated the genotoxicity of surface water after treatment with different disinfectants by means of in situ plant genotoxicity assays (micronucleus and chromosomal aberration tests) which can detect both clastogenic and aneugenic effects. The study was carried out at a pilot plant using lake water after sedimentation and filtration. This water supplied four stainless steel basins: three basins were disinfected with sodium hypochlorite, chlorine dioxide, and peracetic acid and the fourth basin containing untreated lake water was used as a control. Plants were exposed in situ in the basins. The study was carried out using water collected in different seasons over a period of about 1 year in order to assess the treatments in different physical and chemical lake water conditions. The micronucleus test in root cells of Vicia faba (Vicia faba/MCN test) revealed genotoxicity in many samples of disinfected water. The micronucleus test in Tradescantia pollen cells and the chromosome aberration test in root cells of Allium cepa showed genotoxic effects only in some disinfected samples, but also revealed genotoxicity in raw water. The results of the study indicated that the Vicia faba/MCN test was the most sensitive plant assay for disinfected water and that peracetic acid disinfection produced similar or lower genotoxicity than sodium hypochlorite or chlorine dioxide treatment.

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.

Aneuploidy in metaphases II of spermatocytes of wild house mice from a hybrid zone between a Robertsonian population (CD: 2n = 22) and a population with the standard karyotype (2n = 40).

Meiotic metaphases II from archival slides were studied of male house mice caught in a hybrid zone between a population monomorphic for nine centric fusions (2n = 22) and a population with the standard karyotype (2n = 40), near Rome. The frequency of aneuploidy increases, up to 50%, with increasing number of heterozygous centric fusions (1-4).

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.

Mitotic indirect non-disjunction in phytohemagglutinin stimulated human lymphocytes.

In a previous publication we demonstrated that in cells of Vicia faba micronuclei derived from whole lagging chromosomes or chromatids may perform DNA synthesis and mitotic condensation in synchrony with main nuclei and be regained by main nuclei at the next mitosis, giving rise to trisomic cells together with diploids. This process was called 'mitotic indirect non-disjunction' (MIND). In the present work the occurrence of MIND was studied in human lymphocytes cultivated in vitro. Human lymphocytes were treated with low colcemid concentrations until fixation; BrUdR was supplied together with colcemid to distinguish the number of mitoses performed by the cells (M1, M2 and M3 cells). The frequencies of M1 ana-telophases with single lagging chromosomes/chromatids and of M2+ prophases with single micronuclei in synchronous motitic condensation with main nuclei were evaluated. On this basis the expected frequencies of both monosomic and trisomic M2 cells were calculated, according to the hypothesis of MIND. Their observed frequencies were very close to those expected. These results support the hypothesis of the occurrence of MIND in human lymphocytes.

Chromatin bridges between sister chromatids induced in late G2 mitosis in CHO cells by trimethylpsoralen + UVA.

A hypothesis is proposed that sister chromatid pairing is due, at least in part, to the pairing between DNA strands belonging to each of the two sister chromatids (conservative pairing). To test this hypothesis interstrand DNA cross-links were induced in late G2-mitosis in CHO cells in order to bind covalently paired DNA strands eventually coming from both sister chromatids and detect the consequent chromatin bridges between sister chromatids (SCCBs). Therefore cells were treated with trimethylpsoralen (TMP) + UVA (365 or 405 nm). Chromatin bridges in ana-telophase were induced by an UVA irradiation at 365 nm, which gives rise to both monoadducts and cross-links, but not by a 405-nm irradiation, which gives rise only to monoadducts. An analysis of colchicine-induced c-anaphases demonstrated that such chromatin bridges were really SCCBs and that terminal regions of chromosomes were particularly involved. The evolution of SCCBs was studied to rule out that they were masked isochromatid exchanges. So TMP + UVA-treated cells were induced to polyploidize with colchicine and labeled with 5-bromodeoxyuridine. Cells treated with TMP + UVA in G2-mitosis appeared as M1 tetraploid c-metaphases; in such cell populations there was not an increase in isodicentric chromosomes, which are derived from isochromatid exchanges. The present data, as a whole, support the hypothesis that a "conservative pairing" between DNA strands of sister chromatids can be present in mitosis.

The establishment of underdominant chromosomal rearrangements in multi-deme systems with local extinction and colonization.

The fate of an underdominant chromosomal mutant was investigated in multideme models with high rates of local extinction and colonization. Four models with different patterns of colonization (number of colonists and place of origin of colonists) were studied by performing a large number of computer simulations with the Monte Carlo method for several sets of values of the following parameters: coefficient of selection against the heterozygote, extinction rate of each deme, deme size, and number of demes. The probability of the newly arisen rearrangement being established in the multi-deme system depends strongly on the pattern of colonization, other things being equal. In the three models in which there is absent or scarce mixing of gene pools of different demes when a new deme is founded, the fixation probability of the new chromosomal rearrangement is rather close to that calculated by R. Lande (1979, Evolution 33, 234-251; 1985, Heredity 54, 323-332), which is equal to the corresponding probability in a single deme divided by the number of demes. In the model with extensive mixing of gene pools of demes, the corresponding probability is lower (considerably in some cases). Furthermore, in the models where the fixation probability is higher, the analysis of the time of the process leads to the conclusion that in systems consisting of a large number of demes, overlapping of several different processes of fixation of chromosomal rearrangements occurs.

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells.

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes "lost" as micronuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.

Induction of micronuclei in Vicia faba root tips treated with heavy metals (cadmium and chromium) in the presence of NTA.

Soluble metal compounds (CdCl2, K2Cr2O7) significantly increase the frequency of micronucleated cells in Vicia faba root tips with a clear dose-effect relationship. The addition of NTA (nitrilotriacetic acid) does not enhance the induction of micronucleated cells. Conversely, insoluble metal compounds (CdCO3, PbCRO4) significantly enhance the frequency of micronucleated cells only in the presence of NTA. Since some genotoxic metals are diffused in the environment and are often sequestered as insoluble precipitates in water sediments and sludges, the introduction of NTA is likely to increase the risk of environmental pollution because of its ability to solubilize and make those metals reactive.

A comparison between short-term evolution of micronuclei induced by X-rays and colchicine in root tips of Vicia faba.

The short-term evolution of micronuclei derived from acentric fragments and whole chromosomes was studied in root tips of Vicia faba. Micronuclei were induced by X-rays (30 cGy and 120 cGy) and colchicine (10(-5) M and 3 X 10(-4) M). Frequencies of chromosome breakage or loss of micronuclei in interphase and mitotic cells were studied. The DNA content of micronuclei in interphase cells was also measured. Micronuclei derived from whole chromosome showed a higher probability to survive and to undergo mitotic condensation in synchrony with main nuclei than micronuclei derived from an acentric fragment. PCC (Premature Chromosome Condensation) was not observed for both types of micronuclei in Vicia faba, in contrast to the ones reported in mammalian cells in culture.

Reduction of neutral gene flow due to the partial sterility of heterozygotes for a linked chromosome mutation.

The effect of linkage between a chromosome mutation producing partially sterile heterozygotes and a neutral locus in reducing the gene flow at the neutral locus is studied using a two-population deterministic model. Chromosome mutations are more efficient in reducing gene flow with low migration rates than with high ones. The interaction between high values of partial heterozygote sterility and low recombination rates can produce, in the low migration pattern, a drastic reduction of gene flow. Nevertheless, since only chromosome mutations with low values of partial heterozygote sterility are likely to be involved in chromosomal speciation, a significant reduction of gene flow will probably occur only for a very limited part of the genome. Therefore, a single chromosome mutation is unlikely to play a primary role in speciation.

Micronucleus induction by low doses of X-rays in Vicia faba root tips.

Studies on the micronucleus test in Vicia faba root tips (VM test) were carried out in order to estimate the effects at low doses of X-rays (1, 2, 4, 8 and 12 R). The control value of micronucleus frequency is about 0.44/1000 cells. The dose where the micronucleus frequency is twice that of the control was estimated at 1.384 R. There was a linear kinetic dose response for the low-dose range studied here.

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse.

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.

Effects of actinomycin D and puromycin on colchicine-induced endoreduplication in Chinese hamster cells in vitro.

Experiments were carried out to study the events through which the process of endoreduplication occurs after treatment with colchicine in Chinese hamster cells cultivated in vitro, and to analyse the differences between the processes leading to normal mitosis and those leading to endoreduplication. Chinese hamster cells (line C-125) were treated with colchicine (10(-4) M) for 1 h, in order to induce endoreduplication. Before and after colchicine treatment the cultures were treated at various intervals with actinomycin D and puromycin. For preparation of mitosis specific RNA synthesis is required, and also synthesis of a specific protein, which is not required for endoreduplication. Moreover this suggests not only that the cells that endoreduplicate do not need new RNA and protein synthesis, but that inhibition of their synthesis apparently favours the processes of endoreduplication.