Effect of 5-azacytidine (5-azaC) on the induction of chromatid aberrations (CA) and sister-chromatid exchanges (SCE)

Experiments were carried out using human lymphocytes from a male donor in order to test the action of 5-azaC treatment on the induction of SCE and chromatid aberrations. The 5-azaC was found to increase the frequency of both baseline and MMC-induced SCEs. Using the same 5-azaC treatment conditions it was found that the frequency of X-ray-induced CA did not increase.     

Chromosome aberrations (CA), sister-chromatid exchanges (SCE) and mitogen-induced blastogenesis in cultured peripheral lymphocytes from 48 control individuals sampled 8 times over 2 years

Confidence in the measurement of positive effects determined by monitoring of environmentally or occupationally exposed individuals can be enhanced by a knowledge of the normal variability in these endpoints in the general population. Confounding effects can be determined and study interpretation improved by correlation of this variability with various lifestyle factors such as sex and age of donor, smoking and drinking habits, viral infections, exposure to diagnostic X-rays, etc.

8 blood samples were taken from each of 24 male and 24 female volunteers over a period of 2 years. Questionnaires pertaining to lifestyle were completed at the time of each sampling. Whole blood was cultured and slides prepared for CA or SCE analysis. Separated mononuclear cells were cultured with a range of phytohaemagglutinin concentrations and the maximum level of mitogen-induced blastogenesis was determined by measurement of [³H]thymidine uptake.

There was a significant effect of both year and season of sampling for all 3 endpoints. No significant effects in any of the 3 endpoints were found with respect to sex or age of donor nor any of the other lifestyle factors, although SCE frequency and mitogen-induced blastogenesis were nearly always higher in females than males. These results point to the need for concurrent sampling of controls with exposed populations.     

Induction of chromosomal aberrations, sister-chromatid exchanges and cell-cycle delay in cow peripheral blood lymphocytes treatment with two N-nitroso compounds in vitro

We investigated the effect of NDMA and DNSGU on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs), as well as the influence of the former compound on cell-cycle kinetics in cultured cow peripheral lymphocytes. A clastogenic effect was observed in treated cell cultures at 6 or 12 × 10⁻⁵ M concentrations of NDMA and DNSGU, respectively, but no increase of chromosomal breaks was seen at the lowest dose. NDMA at 6 × 10⁻⁴ M was toxic to cow lymphocytes. NDMA and DNSGU induced statistical increases of SCEs at the test doses (6 or 12 × 10⁻⁶ and 6 or 12 × 10⁻⁵ M, respectively). In addition, treatment with NDMA at a dose of 6 × 10⁻⁵ M revealed significant heterogeneity of the first, second and third metaphases between treated and untreated groups. A reduction of the proliferation index and proliferation delay per cycle was shown too.     

Aging and smoking increase the frequency of sister-chromatid exchanges (SCE) in man

The frequency of SCE was determined in lymphocytes of 88 healthy human subjects, not occupationally exposed to known genotoxic agents, who were uniformly distributed in several classes of age (from 16 to 70 years), including an equal number of smokers and non-smokers, and of males and females. Our results indicate that the frequency of SCE increases linearly with age and that smoking enhances the frequency of SCE independently of age and sex.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

Higher incidence of spontaneous sister-chromatid exchanges (SCEs) and X-ray-induced chromosome aberrations in peripheral blood lymphocytes during pregnancy

In vitro cultures of peripheral blood lymphocytes from human and muntjac (barking deer) females who were at an advanced stage of pregnancy (32-37 weeks pregnant women and 20-24 weeks pregnant muntjacs) showed an enhanced frequency of SCEs and X-ray-induced chromosome aberrations when compared with those of nonpregnant females. Lymphocyte cultures of nonpregnant females to which sex hormones progesterone, oestrogen and human chorionic gonadotropin (HCG) were added together exogenously also showed higher frequency of SCEs. The plausible reason(s) for such high incidence of SCEs during pregnancy is discussed.     

Production of chromosome aberrations, micronuclei, and sister-chromatid exchanges by 24-keV epithermal neutrons in human G0 lymphocytes

The induction of chromosome aberrations, micronuclei and sister-chromatid exchanges in human G0 lymphocytes by 24-keV epithermal neutrons has been measured. Positive linear dose responses were obtained for the 3 end points, with a tendency to saturation at higher dose for SCE production. In all cases, the responses to 24-keV neutrons were characteristic of high-LET radiations.     

cis-diamminedichloroplatinum(II)-induced sister-chromatid exchanges and chromosome aberration formation in cultured human lymphocytes and their inhibition by sodium thiosulfate

cis-Diamminedichloroplatinum(II) (cis-DDP)-induced sister-chromatid exchanges (SCEs) and chromosome aberration formation were studied in human lymphocytes. The mitotic index decreased abruptly at 2 X 10(-6) M cis-DDP and the frequency of SCEs was dose-related; a marked increase was recorded at 10(-6) M cis-DDP. A dose-dependent effect was also found for chromosome aberration formation at concentrations between 10(-11) and 4 X 10(-6) M. The aberrations observed were primarily chromatid breaks and gaps. We also examined the inhibition of these genotoxicities by treating the cells with sodium thiosulfate (STS). Simultaneous treatment with 10(-4)-10(-3) M STS (100-1000-fold molar ratio to cis-DDP) significantly reduced the frequency of SCEs induced by 10(-6) M cis-DDP. Furthermore, a 3-h delay in treating with STS significantly reduced cis-DDP-induced SCEs, but not chromosome aberration formation.     

Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations of Chinese hamster bone marrow cells

Summary The in vivo SCE formation and the induction of chromosome aberrations in the bone marrow of Chinese hamsters (Cricetulus griseus) were studied after various concentrations of cyclophosphamide, and the sensitivity of the two test methods was compared. The administration of 1.0, 5.0, 13.3, 25.0, and 40.0 mg/kg body weight induced a dose-dependent increase in SCE. The frequency of chromosome aberration, however, was not increased significantly with doses of 1.0 and 5.0 mg/kg body weight. Only with doses of more than 13.3 mg is a significant induction of chromosome aberrations seen. Therefore the SCE test system seems to be 10 times more sensitive than the induction of chromosome aberrations in the same cell type.This work is a part of the M.D. thesis of G. Roszinsky-Köcher, to whom offprint requests should be sent     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks, and the relation to toxicity

Substantial increases in chromosome aberrations were induced in Chinese hamster ovary cells by medium made hyperosmotic with NaCl, KCl, sucrose, sorbitol or dimethyl methylphosphonate. The increases were associated with cytotoxicity but occurred in the range (e.g., 70% survival) commonly included in in vitro tests for 'genotoxicity'. The relation between increased osmotic pressure and chromosome aberrations is compound-dependent, e.g., some compounds may have a direct effect in addition to an effect mediated by osmotic pressure/ionic strength. Also, glycerol at high osmolality was not toxic and did not induce aberrations, probably because rapid equilibration across the cell membrane precluded severe osmotic stress to the cells. Weak increases in DNA single-strand breaks (NaCl and KCl) and double-strand breaks (NaCl) were also detectable, at higher concentrations and more toxic levels than those required to produce aberrations. Slight elevations in sister-chromatid exchange frequencies caused by hyperosmotic medium were found in the presence of toxicity and severe cell cycle delay. Our data on cell growth inhibition suggest that this is the result of increased incorporation of bromodeoxyuridine per cell due to decreased numbers of growing cells, although other mechanisms cannot be ruled out. The observations on chromosome aberrations demonstrate the need for keeping in vitro test conditions in the physiological range, and provide a means for investigation of indirect DNA damage.     

Cytogenetic effects of pulsing electromagnetic field on human lymphocytes in vitro: chromosome aberrations, sister-chromatid exchanges and cell kinetics

Exposure of human lymphocyte cultures to a pulsing electromagnetic field (PEMF; 50 Hz, 1.05 mT) for various durations (24, 48 and 72 h) resulted in a statistically significant suppression of mitotic activity and a higher incidence of chromosomal aberrations. Furthermore, the shorter exposure times (24 and 48 h) did not cause a significant delay in cell turnover (cell proliferation index) or an increase in the baseline frequency of sister-chromatid exchanges (SCE). However, cultures continuously exposed to PEMF for 72 h exhibited significant reduction of the cell proliferation index (CPI) and an elevation of SCE rate. These results suggest that exposure to PEMF may induce a type of DNA lesions that lead to chromosomal aberrations and cell death but not to SCE, except probably at longer exposure times.     

Induction of sister-chromatid exchanges (SCE), polyploidy, and micronuclei by plant flavonoids in human lymphocyte cultures. A comparative study of 19 flavonoids

Nineteen naturally occurring flavonoids were studied with regard to their SCE-inducing potency and their capability of inducing polyploidy and micronuclei in human lymphocyte cultures. The cells were treated for a period of 48 h. The flavone C-glycosides, vitexin and orientin, exhibited a moderate SCE-inducing activity, whereas the other compounds displayed only weak effects or were inactive. Polyploidy was induced by procyanidins consisting of 3 or 4 flavanol units and to a lesser extent by flavone, flavonol, and anthocyanidin aglycones. The aglycones as well as the C-glycosides and the O-glycosides, spiraeoside and luteolin-7-glucoside, were more or less active in inducing micronuclei in the lymphocytes. The flavonol O-glycosides, rutin and hyperoside, and the monomeric and dimeric flavanols failed to produce any genotoxic effects. The results are discussed with respect to a possible structure-activity relationship.     

Chromosome aberrations and sister-chromatid exchanges (SCEs) in peripheral blood lymphocytes of patients suffering from poliomyelitis

The frequencies of sister-chromatid exchanges (SCEs) and various chromosome aberrations were studied in blood lymphocyte cultures of individuals suffering from polio virus infection. The frequency of SCEs was found to be within the normal range in polio patients whereas the frequency of chromatid breaks, gaps and other chromosome aberrations showed a significant (p less than 0.001) increase when compared with that of controls. It indicates that the mechanism(s) responsible for polio virus-induced chromosomal damage may not be related to or affect the molecular process(es) that functions in SCE formation.     

Baseline frequency of sister-chromatid exchanges (SCE) in newborn lymphocytes and its relationship to in vivo aging in humans

Heparinised cord blood from newborns and peripheral venous blood from three other age groups of individuals (1-75 years) have been cultured in vitro to obtain baseline frequencies of SCE and to see if the frequency of baseline SCE in vitro varies as a function of aging in vivo. The results demonstrate an age-dependent variation in the frequency of SCEs. Although the SCE frequency was lowest (5.10/cell) in 1-5-year-old infants, a significantly higher (P less than 0.001) frequency (8.97/cell) was observed in the cord blood of newborns. In old age, the level of SCE also increased. The plausible reason(s) for such observations is discussed.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Induction of sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse peripheral blood B lymphocytes exposed to mutagenic carcinogens in vivo

To determine the sensitivity of the mouse peripheral blood lymphocyte (PBL) culture system, male B6C3f1 mice were injected i.p. with either 2-acetylaminofluorene (AAF) (20, 40, 80, 160 mg/kg), benzo[a]pyrene (BP) 25, 75, 150, 300 mg/kg), dichlorvos (DCV) (5, 15, 25, 35 mg/kg), ethyl methanesulfonate (EMS) (10, 30, 90, 180, 270 mg/kg), or N-nitrosomorpholine (NM) (37.5, 75, 150, 300 mg/kg) dissolved in either RPMI 1640 (DCV, EMS, NM) or sunflower oil (AAF, BP). 24 h later blood was removed by cardiac puncture, and the lymphocytes were cultured in the presence of lipopolysaccharide for analysis of SCE in B lymphocytes. All 4 mutagenic carcinogens (AAF, BP, EMS, NM) induced significant dose-related increases in SCE frequency. DCV, a potent neurotoxicant, caused no change in the baseline SCE frequency. At the highest concentration of each chemical examined, AAF caused a 1.6-fold increase, EMS a 1.8-fold increase, NM a 3.0-fold increase, and BP a 3.1-fold increase in SCE frequency compared to concurrent controls. A comparison of these results for PBLs with those reported in the literature for bone marrow cells indicates that PBLs offer a good quantitative and qualitative estimate of the SCE-inducing potential for these 5 compounds in bone marrow cells.