Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0 mg/L, induced a 1.9–3.9-fold increase in MN frequency and a 1.5–1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P < 0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15–80% decrease in mitotic indices (MI), but at the lowest concentration (0.005 mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

Insights into the mechanisms of sister chromatid exchange formation

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies.     

Environmental monitoring for genotoxicity: in vivo sister chromatid exchange in the house mouse (Mus musculus)

Sister chromatid exchange (SCE) values were determined in bone marrow cells isolated from mouse (Mus musculus) femurs after injections of 5-bromo-2'-deoxyuridine (BrdU) and 5-fluorodeoxyuridine (FrdU). Male mice of C3H/J, C57BL/6J, and DBA/2 strains maintained in the laboratory gave mean SCE values of 3.42 +/- 0.07, 3.62 +/- 0.08, and 3.97 +/- 0.13, respectively. Males obtained from natural populations of southwestern Ontario had a higher mean SCE value (6.02 +/- 0.16), as did inbred males maintained in outdoor enclosures for at least 3 weeks (5.07 +/- 0.22). Wild mice housed in the laboratory for 9 months or longer had SCE values similar to laboratory bred mice (3.46 +/- 0.05). The SCE values in wild-caught mice were inversely proportional (r = -0.49) to the distance between the sites where these animals were collected and the nearest major industrial center. Based on these results, SCE analysis in mice is proposed as a possible first-line monitoring procedure for the detection of general changes in environmental genotoxicity.     

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.     

Human sister chromatid exchange caused by methylazoxymethanol acetate

The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [3H]-thymidine incorporation in PHA-stimulated short-term lymphocyte cultures versus MAM AC contraction. The lower concentration was less than the TD50 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.     

Statistical and image analysis of sister chromatid exchange in maize

The present study reports the use of the fluorescence plus Giemsa (FPG) technique, image analysis and statistical methods to assess the sister chromatid exchanges (SCEs) frequency in maize. Roots derived from germinated maize seeds were treated with BrdU solution and fixed. The slides were prepared by enzymatic cellular dissociation, air-drying technique, stained with Hoechst 33258 fluorochrome, and incubated in salt solution. The chromosomes were irradiated with ultraviolet light and stained with Giemsa solution. The FPG technique associated with digital analysis system was used to measure the length of 597 BrdU-incorporated maize chromosomes and to identify 0.5243 SCE per chromosome. A range from 0 to 4 SCE events were classified and the chi-square test (chi2=1.586, P=0.662) showed a good fit to the hypothesis that the SCEs are independent and random events that follow Poisson distribution. The SCE frequencies in long and short chromosome arms corresponded to a mean value of 0.876 SCE microm(-1). Considering that the maize line used in this study contains 5.78 picogram (pg) DNA (2C value) in interphasic G0/G1 nuclei or 11.56 pg DNA (4C value) in metaphase, and that the DNA mean value corresponds to 0.578 pg/metaphasic chromosome, the analysis suggests an occurrence of approximately 0.9 SCE/pg DNA.     

Elevated rates of sister chromatid exchange at chromosome ends

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.     

Spontaneous and mutagen-induced sister chromatid exchange in motor neurone disease

Spontaneous and mutagen-induced sister-chromatid exchange frequencies have been studied in the peripheral blood lymphocytes of 6 patients with motor neurone disease. Their values were compared with those obtained in age- and sex-matched healthy controls. No significant differences were observed between the 2 groups. These results do not support the hypothesis of a defect in the repair of DNA damage as the primary abnormality in the development of the disease.     

Aging and sister chromatid exchange : II. The effect of the in vitro passage level of human fetal lung fibroblasts on baseline and mutagen-induced sister chromatid exchange frequencies

The frequencies of baseline and mutagen-induced sister chromatid exchanges (SCE) were examined in human fetal lung fibroblasts (IMR-90, WI38) as a function of in vitro serial passage (in vitro aging). Although baseline SCE levels remained relatively constant throughout the in vitro lifespan of these cell cultures, a significant decline was observed at middle and late passage in the levels of SCE induced by mitomycin-C, ethyl methane-sulfonate and N-acetoxy-2-acetylaminofluorene. These findings indicate that cellular aging results in an altered response to certain types of induced DNA damage.     

Distribution of the frequency of sister chromatid exchange among Leningrad inhabitants

Peculiarities of frequency variations in sister chromatid exchanges (SCE) were studied in a group of healthy Leningrad citizens who are not engaged in health-risk industries. No relations were found between the SCE frequency and sex, age and smoking habit (10 cigarettes per day as much). The statistical processing of the data obtained was made taking into account the errors in individual measurements of the SCE frequency. Repeated measurements revealed systematic and statistically significant variations in the rate of SCE.     

Further analysis of the replication bypass model for sister chromatid exchange

Summary The replication bypass model for sister chromatid exchange (SCE) proposed by Shafer is examined in detail. When applied through two cell cycles, the model predicts that potentially observable SCEs induced during one S phase will then be cancelled and rendered undetectable during the subsequent S phase. This aspect of replication bypass is inconsistent with the observation of twin SCEs in tetraploid cells. Furthermore, the model cannot account for the efficient induction of SCEs by non-cross-linking chemical agents.     

Gene mutation and sister chromatid exchange in Chinese hamster cells

The mutation in hypoxanthine phosphoribosyl transferase gene and the induction of sister chromatid exchange (SCE) were comparatively studied treating Chinese hamster ovary cells with the mutagens ethylmethanesulphonate. N-methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C and X-ray. All the agents exerted strong mutagenic effects and showed a dose-dependent relationship for the induction of SCEs.     

Unequal sister chromatid exchange in the rDNA array of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA--a minimum of 20 rDNA genes in tandem--and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells.     

Mitomycin C-induced sister chromatid exchange in X chromosomes of Bovidae

Sister chromatid exchanges (SCEs) in early- and late-replicating X chromosomes of seven female cattle (Bos taurus L.) and five female river buffalo (Bubalus bubalis L.) were studied in untreated lymphocytes and lymphocytes treated with mitomycin C (MMC). In the experiment, 577 SCEs on X chromosomes of MMC-untreated cells and 825 SCEs on X chromosomes of MMC-treated cells from both species were observed. No significant differences between the number of SCEs in early- and late-replicating X chromosomes were found even when singular species and subjects were considered.     

A family study of spontaneous sister chromatid exchange frequency.

The frequency of spontaneous sister chromatid exchanges (SCEs) was determined in PHA-stimulated peripheral lymphocytes of 52 individuals, comprising 12 complete 2-generation pedigrees. Neither intraindividual variation between replicate cultures established from the same blood sample nor variation among samples from the same individual initiated at different times was significant. However, familial factors affecting mean SCE frequencies were indicated by detection of significant differences among, but not within, families. Although sample sizes were small, a genetic contribution to the SCE frequency was suggested by the observed pattern of familial correlations.     

Aging and sister chromatid exchange : I. The effect of aging on mitomycin-C induced sister chromatid exchange frequencies in mouse and rat bone marrow cells in vivo

Utilizing the differential staining of chromatids containing BUdR, it was demonstrated that frequencies of mitomycin-C induced sister chromatid exchanges decline with age. The concomitant increase in chromosomal aberrations suggest an altered response to DNA damage with aging.     

Unequal mitotic sister chromatid exchange and different length of Y chromosomes

Summary There is wide variation in the length of the Y chromosome. In the same individual the length varies continuously and is normally distributed. We describe a boy with borderline mental retardation, gross and fine motor coordination difficulty, muscle rigidity, ptosis, clinodactyly, and a Y chromosome of different lengths in two separate cell populations. The most probable explanation of the cytogenetic finding is a mitotic unequal sister chromatid exchange of the Y chromosome.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

Dynamics of the frequencies of sister chromatid exchange and chromosome aberrations in vivo

2.4 and 6 mg/kg thiophosphamide (T) was administered intravenously to New Zealand rabbits. A decrease in sister chromatid exchange (SCE) and chromosome aberration (CA) rate began immediately after the mutagenic action of T was over. The expected SCE rate was more than the investigated one. The difference between expected and investigated SCE rate increased with the dose of T. A calculation of SCE was based on the amount of the administered T, the rate of T removal and cell sensitivity to T. The death of cells with high number of SCE resulted in a fast decrease in SCE rate in the first 4 days. Reparative processes and cell proliferation in lymphocyte tissue resulted in a slow decrease in SCE rate after the 4th day. A number of nuclear cells in the blood was the smallest on the 4 th day, at the same time relative increase in CA rate was observed. The time of sampling and the dose of the substance tested should be taken into account for a more accurate estimation of mutagenic activity of some chemicals in in vivo cytogenetic tests.