Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes

Aim of the present paper was to assess by using the in vitro micronucleus (MN) test in human lymphocytes the effect of two plant extracts isolated from Bupleurum fruticosum (saponins) on the clastogenicity and cytotoxicity of the anticancer drugs mitomycin C (MMC) and bleomycin (BLM). One saponin showed a dose-dependent MMC-induced mutagenesis inhibition together with co-genotoxic effect on BLM-treated cultures. The remaining saponin did not significantly alter MN induction of both chemotherapeutic agents whereas it enhanced BLM cytotoxicity.     

Nuclear abnormalities of marine fish erythrocytes

The aim of this study was to monitor erythrocyte nuclear abnormalities (NA) including micronuclei (MN) in cultured and wild sea bass Dicentrarchus labrax and wild mullet Mugil spp. Seasonal sampling was performed at seven locations along the eastern coast of the Adriatic Sea. The frequency of NA and MN was positively correlated to temperature (NA: P < 0·05, r = 0·11; MN P < 0·05, r = 0·10), and there was also a positive correlation between NA and MN frequency ( P < 0·001, r = 0·43). The lowest NA and MN values for both fish species were recorded in spring, while the highest were recorded in autumn. Significantly higher frequency of NA was seen in D. labrax compared to Mugil spp., while MN frequency was low in both species and not significantly different. There was no significant difference in NA and MN frequency between cultured and wild D. labrax sampled in the same month, and there was no difference between wild Mugil spp. sampled near or far from fish farms. In view of sampling sites, the highest values were detected in fishes from the Limski Channel, the lowest from the Janjina location.     

Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus

In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.     

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays,and reproductive performance of male mice exposed to cyclophosphamide

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.     

Association between human papillomavirus (HPV) DNA and micronuclei in normal cervical cytology

The aim of the present study was to investigate the association between HPV-DNA and micronucleus (MN) frequency in women with normal cervical cytology. A total of 158 normal cervical smears were analyzed cytologically. The HPV genome was amplified using the GP5+/bioGP6+ consensus primers. HPV-DNA of high-risk types 16, 18, 31, 33, 39, 45 and 59 were also investigated. Of the 158 samples, 20 (12.7%) and 47 (29.7%) were positive for HPV-DNA and MN, respectively. Evidence for MN was found in 11 out of 20 (55%) HPV-DNA positive samples and in 36 out of 138 (26.1%) HPV-DNA negative ones. MN presence was significantly higher in HPV-DNA positive samples (p = 0.016). On the other hand, the absence of MN observed in a considerable number of HPV-DNA negative samples (102) may be of great value in predicting the absence of HPV. The mean age of HPV-DNA positive women (34.2 ± 12.6) was significantly lower than the mean age of HPV-DNA negative women (43.9 ± 13.7) (p = 0.003). Infection by one or multiple HPV types was found in 11 out of 20 (55.0%) and 9 out of 20 (45.0%) samples, respectively. The evaluation of MN using cervical smears collected for cytology tests could, thus, be used as additional information to monitor a population’s exposure to HPV.     

Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake

Lake Paranoá is a tropical reservoir for the City of Brasilia, which became eutrophic due to inadequate sewage treatment associated with intensive population growth. At present, two wastewater treatment plants are capable of processing up to 95% of the domestic sewage, thereby successfully reducing eutrophization. We evaluated both genotoxic and cytotoxic parameters in several fish species (Geophagus brasiliensis, Cichla temensis, Hoplias malabaricus, Astyanax bimaculatus lacustres, Oreochromis niloticus, Cyprinus carpio and Steindachnerina insculpita) by using the micronucleus (MN) test, the comet assay and nuclear abnormality assessment in peripheral erythrocytes. The highest frequencies of MN were found in Cichla temensis and Hoplias malabaricus, which were statistically significant when compared to the other species. However, Steindachnerina insculpita (a detritivorous and lake-floor feeder species) showed the highest index of DNA damage in the comet assay, followed by C. temensis (piscivorous). Nuclear abnormalities, such as binucleated, blebbed, lobed and notched cells, were used as evidence of cytotoxicity. Oreochromis niloticus followed by Hoplias malaricus, ominivorous/detritivotous and piscivorous species, respectively, presented the highest frequency of nuclear abnormalities, especially notched cells, while the herbivorous Astyanax bimaculatus lacustres showed the lowest frequency compared to the other species studied. Thus, for biomonitoring aquatic genotoxins under field conditions, the food web should also be considered.     

Nuclear anomalies in the buccal cells of calcite factory workers

The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and 'broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.     

Toxicity and genotoxicity in Astyanax bimaculatus (Characidae) induced by microcystins from a bloom of Microcystis spp

Studies of genotoxicity in fish caused by cyanobacterial microcystins can be useful both in determining the sensitivity of native species, as well as comparing exposure routes. The genotoxicity caused by the microcystins LR and LA from a bloom collected in a eutrophic lake, was revealed in the fish Astyanaxbimaculatus, a native species from South America. LC50 (72 h) was determined as 242.81 μg L (-1) and LD50 (72 h) as 49.19 μg kg (-1) bw. There was a significant increase of DNA damage in peripheral erythrocytes, following intraperitoneal injection (ip) with tested concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw, as well as through body exposure to a concentration of 103.72 μg L (-1) . Micronucleus (MN) induction was observed after ip injections of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw for 72 h, as well as following body exposure for 72 at 103.72 μg L (-1) . Thus, both exposure routes resulted in MN induction and DNA damage. Apoptosis-necrosis testing was carried out only by ip injection with concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg- 1 bw. Exposure to microcystins at lower concentrations induced more apoptosis than necrosis in peripheral erythrocytes, whereas exposure at higher concentrations gave rise to both conditions. Thus, Astyanax bimaculatus can be considered as a species sensitive to the genotoxic effects caused by microcystins.     

Evolution of programmed DNA rearrangements in a scrambled gene

Gene unscrambling in spirotrichous ciliates involves massive genome-wide DNA deletion and rearrangement events during development. During each sexual cycle, the somatic nucleus (macronucleus) regenerates from the germ line nucleus (micronucleus). Development of the polyploid somatic genome requires programmed DNA deletion of micronuclear-limited intragenic noncoding sequences and permutation and amplification of the protein-coding regions. Recent studies suggest that, despite novel insertions of endogenous transposon or foreign DNA into the germ line genome, ciliates possess a whole-genome surveillance system that guides the recapitulation of a functional somatic genome. This renders the germ line genome an extremely dynamic structure over evolutionary time. Here we describe the germ line and somatic architectures of the gene encoding alpha-telomere-binding protein in three early-diverging species (Holosticha sp., Uroleptus sp., and Paraurostyla weissei) and trace the natural history of DNA rearrangements in this gene in six species, including three previously studied oxytrichids. Comparisons of homologous coding regions between earlier and later diverging species provide evidence for fusion of scrambled germ line fragments as small as 24 bp during evolution, as well as simultaneous fragmentation and scrambling of the germ line locus and shifting of the boundaries between coding and noncoding DNA, leading to distinct gene architectures in each species. We infer an evolutionary recombination pathway that passes through identified intermediate species and gives rise to the observed patterns in all known species, capitalizing on their unique DNA rearrangement machinery and germ line flexibility.     

Complex germline architecture: two genes intertwined on two loci

The germline micronuclear genome of some ciliated protists can be scrambled, with coding segments disordered relative to the expressed macronuclear genome. Here, we report a surprisingly complex pair of genes that assemble from interwoven segments on two germline loci in the ciliate Uroleptus. This baroque organization requires two scrambled genes to be disentangled from each other from two clusters in the genome, one containing segments 1-2-4-5-6-8-11-13-15-16 and the other 7-9-3-10-12-14, with pieces 1-5 comprising the first gene and 6-16 the second gene. Both genes remain linked in the somatic genome on a 1.5-kb "nanochromosome." This study is the first to reveal that two genes can become scrambled during evolution with their coding segments intertwined. These twin scrambled genes underscore the beauty and exceptions of protist genome architecture, pointing to the critical need for evolutionary biologists to survey protist genomes broadly.