Sister Chromatid Exchanges In Cells Research Articles

Potentiation by caffeine of cytogenetic damage induced by steroidal derivatives in human lymphocytes in vitro

We studied the effects of three newly synthesized steroidal derivatives of nitrogen mustards, alone or in combination with caffeine, on sister chromatid exchange (SCE) frequencies and on human lymphocyte proliferation kinetics. The agents have as alkylator functionalities either P-N,N-bis(2-chloroethyl)aminophenyl-buturate (CHL) or P-N,N-bis(2-chloroethyl)aminophenyl-acetate (PHE), esterified with a modified steroidal nucleus. An enhancement of SCE frequency was seen with compounds which contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17-position of the D-ring. The exocyclic insertion of an –NHCO– group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) gave a compound showing increased SCE frequency. Enhanced cytogenetic damage was observed when lymphocytes were exposed in vitro to caffeine. The compounds, alone or in combination with caffeine, caused a concentration-dependent increase in SCE frequencies and cell division delays, and caffeine was found to act synergistically with the steroidal alkylators.

Evaluation of genotoxic and oxidative effects in workers exposed to jet propulsion fuel

Jet fuel is a common occupational exposure risk among military and civilian populations. The purpose of this study was to evaluate genotoxic and oxidative effects in workers occupational exposure to jet propulsion fuel (JP-8). In this study, sister-chromatid exchange (SCE), high frequency of SCE cells (HFCs), and micronuclei (MN) were determined for 43 workers exposed to JP-8 and 38 control subjects. We measured the antioxidant enzyme activities including that of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). The levels of thiobarbituric acid-reactive substances (TBARS) were also studied. Urinary 1- and 2-naphthol excretion was used as a biomarker of occupational exposure to JP-8. The results obtained from cytogenetic analysis show a statistically significant increase in frequency of SCE in the exposed workers when compared to controls (P<0.05). Interestingly, the mean value of the frequency (%o) of MN and HFCs for workers and controls did not show any statistical differences (P>0.05). Oxidative stress parameters were not statistically different between exposed and control groups except for TBARS levels. Urinary 1-and 2-naphthol levels of exposed workers were found to be significantly higher than those of control subjects. Occupational exposure to JP-8 resulted in no significant genotoxic and oxidative effects, while smoking is the principal confounding factor for the some parameters. To understand the genotoxic and oxidative effects of JP-8 exposure, further studies should be planned to find out whether human populations may be at increased risk for cancer because of the exposures related to occupation and lifestyle.

White blood cell sister chromatid exchange among a sample of Thai subjects exposed to lead: Lead-induced genotoxicity

Epidemiological and experimental studies indicated that inorganic lead compounds are associated with increased risks of tumorigenesis. This study was aimed at determination for sister chromatid exchange (SCE), a marker for genotoxicity with correlation to lead blood levels in Thai exposed subjects. A total of 32 police (all males) were included into this study. The average (mean ± SD) blood lead in these police was 2.1 ± 1 µmol L−1. The average SCE among the police with high blood lead was significant higher than those with low metal exposure. Data suggest that cytogenetic response to Pb is significantly different between the subjects with high versus low lead exposure. High exposure to lead seems related to high chromosome aberrations.

White blood cell sister chromatid exchange among a sample of Thai subjects exposed to toluene, an observation

There is a particular concern with toluene because some research has indicated that toluene exposure could result in chronic toxicity including mutagenesis and carcinogenesis. This study aimed to determine the rate of sister chromatid exchanges (SCE), a marker for genotoxicity, and its correlation to the classical urine biomarker for toluene exposure, urine hippuric acid, among a sample of Thai exposed subjects. A total of 26 police (all males) were included in this study. The average (mean +/- SD) urine hippuric acid level in these police was 0.8 +/- 0.4 mg/g creatinine. The average (mean +/- SD) SCE level in these police was 4.5 +/- 1.0/cell. The average SCE among the police with high urine hippuric acid levels was non-significantly higher than the average SCE level of those without (P = 0.41). This implies that the cytogenetic response to toluene was not different between the subjects with and without high toluene exposure. High exposure to toluene seems not to be related to high SCE.

Sister chromatid exchange (SCE) for the first time in Casertana pig breed

Lymphocyte cell cultures from 30 Casertana pigs (13 males and 17 females), reared in southern Italy, underwent the sister chromatid exchange (SCE) test. The Casertana pig is an endangered native breed from the region of Campania, raised chiefly half-wild. In the 1500 cells we studied, the mean SCE was 6.32+/-2.92 and SCE frequency did not follow a Poisson distribution. A higher mean value of SCE cell(-1) was found in the older group (SCE cell(-1)=6.68+/-2.95) compared with the younger (SCE cell(-1)=5.94+/-2.84), the difference being statistically significant (P<0.01). To our knowledge, this is the first investigation in a representative sample of Italian pig breed using the SCE test. Furthermore, this is the first report where the differences found in the mean SCE values were related to age in domestic species.

Biomarkers of environmental contaminants in field population of green mussel (Perna viridis) from Karnataka–Kerala coast (South West coast of India)

The green mussel Perna viridis was sampled from relatively clean and contaminated sites along the Kartanata-Kerala coast (south west coast of India) to study the tissue concentration of trace metals and biological responses to stress (biomarkers) such as sister chromatid exchange (SCE), chromosomal aberration, micronucleus (MN) test, hemic neoplasia (HN), Chromotest (Ames test) and comet assay. In general, mean tissue concentrations of toxic trace metals collected from 25 sampling sites were found to be below the World Health Organisation (WHO) permissible concentration given for seafood. The digestive gland extract of mussels from all 25 sampling sites showed negative reaction for mutagenic activity (Ames test) in the absence of metabolic activation. Very low levels of chromosomal aberration, SCE, MN, HN and comet cells were observed in mussels collected from both the urban associated and relatively clean sites. This study seems to indicate that that the coastal waters of Karnataka and Kerala are minimally contaminated with genotoxic and carcinogenic chemicals.

Role of mismatch repair in the induction of chromosomal aberrations and sister chromatid exchanges in cells treated with different chemotherapeutic agents.

The mismatch repair (MMR) system plays a major role in mediating the cytotoxicity and clastogenicity of agents generating O(6)-methylguanine in DNA. Loss of MMR has also been associated with tumor cell resistance to the cytotoxic effects of 6-thioguanine and cisplatin and with hypersensitivity to N-(2-chloroethyl)- N'-cyclohexyl- N-nitrosourea (CCNU). The aim of the present investigation was to elucidate the role played by the MMR system in the generation of chromosomal damage in cells exposed to 6-thioguanine, cisplatin or CCNU. The MMR-proficient cell lines TK6 and HCT116/3-6, and their MMR-deficient counterparts MT1 and HCT116, were treated with 6-thioguanine, cisplatin or CCNU, and analyzed for cell growth inhibition and chromosomal damage. As a control, similar experiments were performed with the methylating agent temozolomide. Cytotoxicity, chromosomal aberrations and sister chromatid exchanges induced by 6-thioguanine and temozolomide were significantly reduced in the MMR-deficient cell lines with respect to their MMR-proficient counterparts. In contrast, although conferring some protection against cytotoxicity, the loss of MMR did not affect cytogenetic damage induced by cisplatin. CCNU produced comparable levels of cytotoxicity, chromosomal aberrations and sister chromatid exchanges in both MMR-proficient and MMR-deficient cell lines. The MMR system is involved in the generation of chromosomal damage in cells exposed to 6-thioguanine. The system does not play a relevant role in the generation of chromosomal damage in cells treated with CDDP and does not confer protection against the clastogenic effects of CCNU, at least in the cell lines investigated.

SCE formation after exposure of CHO cells prelabelled with BrdU or biotin-dUTP to various DNA-damaging agents.

Formation of sister chromatid exchanges (SCE) is a mechanism of repair or bypass of DNA damage during S phase. Although SCE have been studied for a long time, the types of DNA lesions involved and the role of 5-bromodeoxyuridine (BrdU) in SCE formation are a matter of debate. We have developed a novel method of differential labelling of sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) and could show that a substantial proportion of radiation-induced SCE arise via damage to BrdU-moieties. The present investigations were performed to examine the role of BrdU in the formation of SCE by the endonucleases AluI and DNase I, as well as the alkylating agent mitomycin C (MMC). CHO cells unifilarily prelabelled with biotin-dUTP or BrdU were treated and the frequencies of SCE analysed in the first post-treatment mitoses. AluI induced similar frequencies of SCE in cells prelabelled with BrdU or biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP than BrdU, but the difference was not significant. The possible mechanisms responsible for the enhanced SCE frequency following DNase I treatment of cells prelabelled with BrdU are discussed.

Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue® mouse and rat fibroblasts

We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue® mouse (BBM1) and Big Blue® rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H 2O 2. Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H 2O 2. GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 μM DEB plus 0, 2 or 20 units of GSH-Px with and without 2×10 8 species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.

Sister chromatid exchanges and ion release in patients wearing fracture fixation devices.

The quantification of sister chromatid exchange (SCE) during mitosis is a useful index for evaluating genotoxic effects in subjects occupationally or incidentally exposed to potentially toxic substances. The authors investigated the hypothesis that ions released by corrosion from prosthetic components of fracture fixation devices are associated with change in SCE incidence. In the present study, ten patients with implants were examined, and fifteen subjects with no implants were used as controls. SCE and high frequency cell (HFC) numbers were evaluated in circulating lymphocytes. In addition, nickel (Ni) and chromium (Cr) ion values in the serum were measured because, after iron, these metals are major components of stainless steel. A significant increase in SCE numbers was observed in patients compared to the control population (4.9 +/- 1.3 vs. 3.5 +/- 1.4). Ni concentration was 1.71 +/- 1.49 ng/mL in patients and 0.72 +/- 0.52 ng/mL in control subjects; Cr concentration was, respectively, 1.01 +/- 0.77 ng/mL and 0.19 +/- 0. 27 ng/mL. The increase of serum Cr and Ni was statistically significant. No correlation was found between the increased Cr concentrations and SCE number while Cr ion levels were found to be significantly correlated to HFC. An inverse correlation between Ni level and SCE numbers was observed. Our findings suggest that Cr release by stainless steel implants could have a genotoxic effect; thus it would be useful to carefully monitor implanted subjects with regard to serum ion dosage, SCE analysis, and HFC evaluation. In any case, it would be appropriate to remove the implant when fracture fixation is reached.

Cytogenetic biomonitoring of workers exposed to bitumen fumes

Bitumen samples and fumes consist essentially of polycyclic hydrocarbons (PAH) and their derivatives, some of which are known to be carcinogenic or co-carcinogenic in animals. The level of total PAH is low when compared with coal-tar products. There is very limited data on possible health risk from exposure to bitumen fumes in workers. In this study, sister-chromatid exchange (SCE), micronuclei (MN) and high frequency of SCE cells (HFCs) were determined for 28 workers exposed to bitumen fumes and 28 control subjects. Urinary 1-hydroxypyrene (1-OHP) excretion was used as a biomarker of occupational exposure to PAH. The mean value of 1-OHP excretion of workers was 0.78±0.46 μmol/mol creatinine and for controls 0.52±0.44 μmol/mol creatinine ( p<0.05). The mean values of SCE per cell and the frequency (‰) of MN in peripheral lymphocytes from the workers and controls were 5.13±0.64, 4.71±0.67, and 2.25±0.42, 1.79±0.32 respectively ( p<0.05, p<0.0001). The mean value of HFCs for workers and controls were 7.85±2.3 and 7.05±3.16, respectively ( p>0.05). Our data reveal that bitumen fumes during road paving operations are absorbed by workers and that bitumen fume exposure is able to significantly induce cytogenetic damage in peripheral lymphocytes of workers after controlling some possible confounding factors, such as age, sex and smoking habits.

Optimization of gene-disruption methods for reverse genetics in the nematode C. elegants

Exposure in vitro of cultivated peripheral leukocytes to trimethylpsoralen and UV light (365nm) under conditions that are known to produce DNA cross-links caused a greater increase in the number of sister chromatid exchanges in cells from two patients with dyskeratosis congenita (DKC) than in normal cells. Absence of this enhancing phenomenon in an unaffected sibling of one patient as well as its presence to a partial degree in an obligate carrier suggest that the observation may be etiologically significant. The data suggest that DKC is associated with a heritable defect in the repair of DNA cross-links.

Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes

The effect of P[ N, N-bis(2-chloroethyl)amino]phenylacetate esters of 3β- hydroxy- methyl-17α- aza- d - homo-5α- androstan-17- one (compound 3) and 3β-hydroxy-17α-aza- d-homo-5α-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[ N, N-bis(2-chloroethyl)phenylacetate esters of 3β-hydroxy-17α-aza- d-homo-5α-androstan-17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.

DNA-protein cross-links and sister chromatid exchange frequencies in lymphocytes and hydroxyethyl mercapturic acid in urine of ethylene oxide-exposed hospital workers.

The lymphocytes of 25 hospital workers exposed to ethylene oxide and of a standardized control group were investigated for DNA damage (measured by alkaline filter elution) and sister chromatid exchange (SCE) frequencies. Additionally, the excretion of hydroxyethyl mercapturic acid (HEMA) in the 24-h urine of ten workers and ten control persons was determined. The peak levels of ethylene oxide in air during the first 8 min after opening of the sterilization unit were measured. Peak levels of ethylene oxide in the air of up to 417 ppm after opening of the sterilization unit were detected. In the alkaline filter elution assay we found significantly reduced elution rates in the exposed workers, indicating DNA-protein cross-links. The reduction of the elution rates through HVLP filters correlated significantly with the exposure classification (low, medium, high) (r = -0.45, P < 0.05) and the ethylene oxide peak level after opening of the sterilization unit (r = -0.42, P < 0.05). The SCE frequencies in the standardized control group were significantly elevated. With respect to (n = 78) historic control SCE values of our institute, the SCE values of the disinfectors were not significantly elevated (6.54 vs 6.27). The ethylene oxide-exposed workers did not have a greater percentage of high-frequency SCE cells. The mean HEMA concentration in the urine of the exposed workers was significantly elevated, but there were wide variations in HEMA concentrations and no correlation to ethylene oxide exposure. We conclude that the alkaline filter elution assay may be a sensitive parameter for ethylene oxide-exposed workers.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aroyl-1-nitrosohydrazinecarboxamides: synthesis, in vivo and in vitro antitumor activity

4-Benzoyl-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamide ( I), 1-(2-chloroethyl)-1-nitroso-4-phenylacetylhydrazinecarboxamide ( II), 1-(2-chloroethyl)-4-(2-hydroxybenzoyl)-1-nitrosohydrazinecarboxamide ( III), 4-(4-aminobenzoyl)-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamide ( IV), 4-(4-chlorobenzoyl)-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamide ( V), all novel class nitrosoureas, were synthesized. All five compounds showed a dose-dependent activity against leukemias L1210, P388 and Ehrlich ascites tumor (EAT). Compound I was superior, yielding T/C% values of 400%, resulting in many cures, reaching 100% in EAT mice. The effect of the above substances on the incorporation of radioactive precursors into DNA, RNA and proteins of EAT cells was investigated. It was found that treatment of cells with 50 μg/ml of the compounds causes significant inhibition (approximately 70%) of the incorporation of 3H-thymidine into DNA. Finally, the effect of these compounds on sister chromatid exchange (SCE) values and on cell kinetics in cultured human lymphocytes was studied. Compound I was found to be the most effective in causing markedly increased SCE values and cell division delays.

Some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchange (SCE) in Hordeum vulgare

A study of some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchanges (SCE) in Hordeum vulgare is reported. After we studied the influence of 5-fluorodeoxyuridine (FdU) and growth temperature on SCE in barley cells, and the effect of FdU, growth temperature, the growth time of plant cells in 5-bromo-2-deoxyuridine (BrdU) solution on SCD, we found an experimental condition under which the frequency of SCE is lower, but the percentage of SCD is higher. Our data show that ascorbic acid, mitomycin C, adriamycin, and maleic hydrazide induce SCEs in cells of Hordeum vulgare by means of free radicals. This can be shown from the two observations: (1) sulfhydryl compounds such as cysteine and glutathione can completely or partially inhibit the SCEs induced by ascorbic acid, mitomycin C, adriamycin and maleic hydrazide; (2) the amounts of free radicals in root tips correlate with the frequencies of SCE in root tip cells.

Genotoxic effects of complex marine sediment extracts on V79 chinese hamster lung fibroblasts

A mammalian in vitro system was used to evaluate the genotoxic potential of two complex environmental samples. Sister chromatid exchanges (SCEs) were measured in Chinese hamster V79 lung fibroblast cells, following exposure to whole extracts of sediments collected from a highly contaminated harbor — Black Rock Harbor, Connecticut (BRH) — and from a reference site — central Long Island Sound (CLIS). Characterization of BRH sediment by analysis of prepared chemical extracts revealed high concentrations of polyaromatic hydrocarbons, anthraquinones, carbazoles, and several inorganic substances that are genotoxic and capable of inducing SCEs. The CLIS sediment, although cleaner than BRH sediment, does contain low levels of similar contaminants. For instance, BRH sediment contains 3.16 μg/g of the polynuclear aromatic hydrocarbon benzo[a]pyrene (BP), whereas CLIS sediment contains only 0.807 μg/g BP. Sediments were extracted with organic solvents and redissolved in dimethyl sulfoxide (DMSO) for culture exposure. Following exposure, extract-treated cells were evaluated for increased frequencies of SCEs. All assays were performed with and without exogenous S9 activation. Positive responses were observed only in the presence of exogenous S9 metabolism. Results indicated a concentration-dependent increase in SCEs in cells exposed to whole BRH sediment extracts and a smaller but significant increase in SCEs in cells exposed to whole CLIS sediment extracts. Four times as much CLIS material (0.13 g extracted sediment [dry wt.]/ml of exposure media) was needed to induce a significant doubling in SCEs, compared to BRH material, in which only 0.03 g extracted sediment (dry wt.)/ml of exposure media was needed. These findings imply that this short-term in vitro bioassay can be used to evaluate the genotoxic potential of complex environmental samples such as contaminated marine sediments.

Cytogenetic analyses of mice exposed to dichloromethane

Chromosome damage was studied in female B6C3F1 mice exposed to dichloromethane (DCM) by subcutaneous or inhalation treatments. No increase in the frequency of either sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) in bone marrow cells was observed after a single subcutaneous injection of 2,500 or 5,000 mg/kg DCM. Inhalation exposure to DCM for 10 days at concentrations of 4,000 or 8,000 ppm resulted in significant increases in frequencies of SCEs in lung cells and peripheral blood lymphocytes, CAs in lung and bone marrow cells, and micronuclei (MN) in peripheral blood erythrocytes. Lung cell CAs and blood erythrocyte MN reached frequencies of approximately two times control levels. Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but significant increases in lung cell SCEs and peripheral blood erythrocyte MN. These findings suggest that genotoxicity may play a role in the carcinogenicity of DCM in the lungs of B6C3F1 female mice.

Sister-chromatid exchanges in human bronchial epithelial cells

One method of assessing the genotoxic effects of exposure to environmental agents is by determining the frequency of sister chromatid exchanges in exposed cells. In the present study, a procedure for observing sister chromatid exchanges in adult human bronchial epithelial cells exposed to a carcinogen has been developed. Using this procedure, the frequencies of sister chromatid exchanges in untreated cells from two individuals were found to be 5.1 ± 1.8 and 6.5 ± 1.4 per metaphase (mean ± SD). Cells from the first individual exposed to 7,12-dimethylbenz[ a]anthracene at 0.5, 1 and 2 μg/ml had 7.8 ± 2.2, 13 ± 3.1, 18.7 ± 3.7 sister chromatid exchanges per metaphase, respectively. This method is likely to be particularly useful for observing sister chromatid exchanges in cells that have a relatively slow growth rate in the presence of bromodeoxyuridine and for which preparation of a large number of metaphase chromosomes is difficult. In addition, the procedure provides a means of assessing genotoxic effects in the lung by examining the direct effects of pollutants on the chromosomes of the target cells for human lung cancer.

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 μM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 μM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.