Sister Chromatid Exchange Analysis Research Articles

Mutagenic effects of ozone on human cells exposed in vivo and in vitro based on sister chromatid exchange analysis

A sister chromatid exchange (SCE) analysis was performed on circulating lymphocytes collected from young human volunteers exposed to 0.5 ppm ozone for 2 hr, an exposure simulating conditions in severe air pollution episodes. SCE analysis was also performed on diploid human fetal lung cells (WI-38) exposed to 0, 0.25, 0.50, 0.75, and 1.00 ppm ozone for 1 hr in vitro. No increase in SCEs over controls was demonstrated in the lymphocyte chromosomes. However, a clear dose-related increase in SCEs was observed in the WI-38 cells exposed in vitro.

Bioassay Procedure for the Detection of Mutagenic Metabolites in Human Urine With the Use of Sister Chromatid Exchange Analysis<xref ref-type="fn" rid="FN2">2</xref>

A short-term bioassay system for the detection of activated mutagenic metabolites in urine from humans exposed to promutagens was described. Human diploid fibroblasts were grown in medium containing 5--20% urine from smokers, from nonsmokers, and from individuals undergoing cyclophosphamide (Cp) chemotherapy for treatment of cancer. The cells were then subjected to sister chromatid exchange (SCE) analysis. Activated Cp metabolic products in urine specimens produced up to a ten-fold increase in SCE's over preinjection SCE levels for the same individuals. Linear dose-response curves over a urine concentration range from 5 to 20% in culture medium were obtained from cells grown in urine specimens from 7 nonsmokers and 8 cigarette smokers. This test system proved to be sensitive to ambient exposure levels of environmental mutagens and demonstrated that urine from smokers was significantly more mutagenic than was urine from nonsmokers. Replicate experiments showed highly reproducible SCE values for each individual as well as for average SCE values for each group of subjects. The ability of this bioassay system to detect trace mutagenic activity in human urine reproducibly makes it an attractive choice for the monitoring of humans who have been exposed to environmental and/or industrial mutagens.

Studies on sister chromatid exchanges and chromosomal aberrations in tonsillar lymphocytes caused by several factors (author's transl)

Using primarily and continuously cultured lymphocytes from tonsils, various factors causing the changes of occurrence in sister chromatid exchanges (SCE) together with the chromosomal aberrations (CA) were examined. The most optimal culture conditions for the SCE observation were studied in primary tonsillar lymphocytes and determined as follows: The number of cells at initiation was; 2 X 106 cells/ml, the culture medium was RPMI-1640 containing 20% fetal calf serum, 15pg/ml of phytohemagglutinin-P and 1011g/ml of 5-bromodeoxyuridine (BUdR) ; the incubation period was 72 hours at 37-C. During 72 hours, the most of cells performed two rounds of DNA replication and showed the metaphases in which each chromosome had one chromatid unifilarly substituted with BUdR and one bifilarly. These cells were submitted to the fluorescence plus Giemsa technique (FPG) for the SCE observation following the usual procedures for chromosomal analysis (flame-drying method). The mean number of SCE in primary tonsillar lymphocytes from 13 patients showing tonsillar hypertrophy or recurrent tonsillitis without inflammatory signs was 5. 134-0.16 SCEs/cell, while 6.57-0. 15 SCEs/cell in 14 patients with chronic inflammatory tonsils. No significant changes of the SCE scores related to the ages, the grades of hypertrophy and the mitotic indices were observed. In the case of tonsillar lymphocytes infected by Epstein-Barr virus (EBV), the SCE scores showed the similar values to those in the chronic tonsillitis. The treatment of these cells with an antitumor drug (mitomycin C), u chemical mutagen (4-nitroquinoline 1-oxide) and the irradiation with ultraviolet light induced significantly the increases of SCE scores before the chromatid breaks occured. Caffein known as the specific inhibitor for DNA repair enzyme increased the breaks without the SCE changes. Nicotine which seems to be related to generation of lung cancer did not show any effects on the SCE or break changes even at 10-3 M. The infection by several DNA-viruses (adeno type 5 and 12, cowpox) or RNA-viruses (rubella, Sendai and Coxsackie B-5), which induced oral or upper respiratory tract infection did not cause the significant changes of SCE scores. However, it resulted in the increases of the breaks before the viral cytopathogenic effect was appeared. These results seem to indicate that the analysis of SCE and CA in tonsillar lymphocytes may be a useful procedure for the follow-up of the minor action of chemical mutagens and the initial change by viral infection with the other cellular responses.

Use of sister chromatid exchange analysis as a bioassay for detecting mutagenic metabolites in urine

Use of sister chromatid exchange analysis as a bioassay for detecting mutagenic metabolites in urine

Analysis of sister chromatid exchange and chromosome replication kinetics using Brd-U-dye techniques.

The basis of BrdU-dye techniques for detecting DNA synthesis in cytological preparations is reviewed. General procedural modifications are described for applying this approach for the study of chromosome replication kinetics and sister chromatid exchange formation. Implications of sister chromatid exchange analysis for detecting mutagen-carcinogens and for differentiating human chromosome fragility diseases predisposing to neoplasia are discussed.

New staining method for the detection of sister-chromatid exchanges in BrdU-labelled chromosomes.

A direct Giemsa technique is described for the selective staining of Brd-U-substituted chromatin. It can advantageously be used for sister-chromatid exchange analysis and for the detailed cytological study of the mouse kinetochores. Presumptive inversions within the centromeric region of chromosomes 4 and 18 in the C57Black/6J mouse are described.

Bromodeoxyuridine tablet methodology for in vivo

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.

Sister chromatid exchange induced by promutagens/carcinogens in Chinese hamster cells cultured in diffusion chambers in mice.

SummaryA system for in vivo analysis of sister chromatid exchange (SCE) was described. It involved culturing Chinese hamster V-79 cells in diffusion chambers (DC) in mice. Treatment of the hosts with repeated injections of 5-bromodeoxyuridine permitted the demonstration of differentially stained metaphases in the V-79 target cells after using the fluorescence plus Giemsa technique. Thus, it was possible to determine the number of SCE's under in vivo conditions. Induction of SCE in V-79 cells in vivo were studied following treatment with seven known promutagens/carcinogens: Cyclophosphamide, 1-(pyridyl-3)-3,3-dimethyltriazene, dimethylnitrosamine, diethylnitrosamine, benzo(α)-pyrene, 7,12-dimethylbenz(α)anthracene, 3-methylcholanthrene and three nonmutagens/noncarcinogens: perylene, pyrene, and anthracene. With the exception of DEN, all the promutagens/carcinogens caused a significant increase in the frequency of SCE. DEN was effective at the highest dose studied only after pretreatment with Aroclor 1254, ...

1022 PHOTOTHERAPY DOES NOT INCREASE THE SISTER CHROMATID EXCHANGE FREQUENCY IN PREMATURE INFANTS

Conventional cytogenetic techniques have failed to detect chromosome breakage in infants due to phototherapy. Sister chromatid exchange (SCE) analysis now permits a more sensitive examination of the impact of bilirubin plus light on human chromosomes. In vitro exposure of adult lymphocytes to 20 mg/dl bilirubin, 2 × 105 J/m2 white fluorescent light, or to both resulted in 16.6, 17.8, or 17.0 SCE/cell, respectively; levels not significantly greater than the control of 17.2 SCE/cell (54-61 cells per value, all cultures 1% DMSO). In vivo response was examined in a double-blind study of 9 premature infants, 5 undergoing phototherapy (35 ± 8.4h) and 4 controls matched for wt. (1531 ± 375g), age (30-35 wks. gestation) and serum bilirubin (7.5 ± 1.7 mg/dl). The pre- and post-treatment SCE frequencies in the treated group (8.2 - 11.7 and 7.1 - 10.7/cell, approx. 20 cells examined per infant) did not differ significantly either between themselves or from parallel measurements on controls (9.0 - 11.7 and 7.7 - 11.6/cell), and chromosomal breakage was negligible in all cases. Our results differ from those of a differently designed study (Lancet 2: 1084, 1977) which observed increased SCE frequency due to phototherapy. Interindividual variations in SCE in our study were significant, and infant SCE frequencies were significantly lower than those typical for untreated adult cells (15.5 ± 0.2/cell). Our present data provide evidence that phototherapy does not demonstrably alter SCE frequency in premature infants.

Chapter 25 Bromodeoxyuridine Differential Chromatid Staining Technique: A New Approach to Examining Sister Chromatid Exchange and Cell Replication Kinetics

The chapter discusses the methodology for Bromodeoxyuridine (BrdU) differential staining, both in vitro as well as in vivo, and an examination of the many applications of this technique. The chapter focuses on the BrdU labeling of mammalian chromosomes in vivo. They involve multiple subcutaneous injections, multiple intraperitoneal injections, or continuous intravenous infusion of BrdU. Examination of the concentrations of BrdU used in vitro for differential chromatid staining reveals extreme variation, even for the same cellular system. The chapter discusses the two most important areas of the applications of BrdU differential staining techniques are the utilization of sister chromatid exchanges (SCEs) as an indication of DNA damage and repair and the employment of BISACK for examining cellular replication. SCE analysis potentially detects an agent, which may not be mutagenic for bacterial systems and yet be mutagenic, and perhaps even carcinogenic, for human cells. SCE analyses have been employed to examine various human genetic disorders. The system for analyzing cell replication kinetics (BISACK), permit new insight into the nature of replicating cell populations both in vivo and in vitro.

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 × 10 −2 M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 μg per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.

Effect of ultrasound on human leucocytes sister chromatid exchange analysis

The effect of ultrasound on human leucocytes was examined using the BUDR-Giemsa procedure. Cells were cultured in Minimum Essential Medium (Eagle) with Spinner Salts for 72 hr after irradiation. Sister chromatid exchange (SCE) rate was evaluated in cells irradiated in G 0. No effect on SCE could be demonstrated at any of the ultrasound intensities used (15.3–36 W/cm 2).

Sister Chromatid Exchange and Chromosome Aberration Analysis With the Use of Several Carcinogens and Noncarcinogens: Brief Communication

Several chemical carcinogens and noncarcinogens were tested for their ability to induce sister chromatid exchanges (SCE) and structural chromosome aberrations in cultured V79-4 Chinese hamster cells. All of the direct-acting carcinogens induced a large increase in SCE frequency. Two chemicals, which are mutagenic in microorganisms but whose carcinogenicity is poorly documented, also increased the frequency of SCE. Carcinogenic polycyclic hydrocarbons caused an increased incidence of SCE only when a metabolizing feeder layer was used, whereas no increase was observed with noncarcinogenic polycyclic hydrocarbons. The other noncarcinogens also did not influence the SCE frequency. Although some chemicals increased the frequency of structural chromosome aberrations, no correlation was found between the frequencies of SCE and aberrations.

A simplified technique for in vivo analysis of sister-chromatid exchanges using 5-bromodeoxyuridine tablets.

A small tablet of 5-bromodeoxyuridine (BrdU), implanted subcutaneously in a mouse, provides sustained release of base analog sufficient to effect substitution of DNA throughout an entire replication period. As illustrated by studies of mouse bone-marrow and spleen cells in the presence or absence of cyclophosphamide, this depot method of BrdU administration greatly simplifies in vivo analysis of sister-chromatid-exchange formation.

Cytogenic studies of opiate addicts: standard, G banded and sister chromatid exchange analyses

Cytogenic studies of opiate addicts: standard, G banded and sister chromatid exchange analyses

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine.

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 mug/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.

Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence.

A number of applications of the detection of deoxyribonucleic acid synthesis by fluorescence microscopy are illustrated. These include (a) the analysis of sister chromatid exchanges and sister chromatid segregation at mitosis, (b) the location of chromosome regions containing deoxyribonucleic acid with an asymmetric distribution of thymine residues between polynucleotide chains and (c) the detection of late replicating regions in metaphase chromosomes. The suppression of 33258 Hoechst fluorescence by 5-bromodeoxyuridine incorporated biosynthetically into interphase nuclei is demonstrated both in fixed cytologic preparations and in unfixed cultured cells. Many of the cytologic observations described might form a basis for future biochemical studies.

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac.

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.

Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C.

Sister chromatid exchanges in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with the bisbenzimidazole dye 33258 Hoechst. These exchanges are much more frequent than chromosome or chromatid breaks and appear to be partly but not entirely due to 5-bromodeoxyuridine incorporation. Sister chromatid exchanges are extremely sensitive indicators of chromosome damage produced by DNA cross-linking agents such as mitomycin C. Significant increases in the sister chromatid exchange frequency occur with 3 ng/ml of mitomycin C; higher concentrations of mitomycin C induce further sister chromatid exchanges. Comparatively few gross chromosomal aberrations are seen in cells exhibiting as many as one hundred or more sister chromatid exchanges. Most of the damage caused by mitomycin C to chromosomal DNA is apparently repaired without detectable changes in chromosome morphology. Analysis of sister chromatid exchanges may permit more sensitive detection of damage to DNA caused by other agents than has previously been possible by classical cytological techniques.

Localization of sister chromatid exchanges in human chromosomes.

The bromodeoxyuridine; sensitivity of 33258 Hoechst fluorescence allows microfluorometric analysis of sister chromatid exchanges in human metaphase chromosomes. The frequency of sister chromatid exchanges among chromosomes correlates with chromosome length. Exchanges appear to occur predominantly in interband regions, as defined by quinacrine fluorescence, or very near band-interband junctions. A few regions are involved unusually frequently.