Sister-chromatid exchanges induced by mitomycin C after acute or chronic exposure of human lymphocytes to a low dose of X-rays

Human peripheral blood lymphocytes from 10 male donors were exposed to mitomycin C with and without prior irradiation with 0.01 Gy X-rays. Acute or chronic irradiation of lymphocytes in G1 resulted in a decrease in the subsequent level of mitomycin C-induced sister-chromatid exchange aberrations. The effect was small (approximately 10%) with evidence of some variability between donors. By contrast no adaptive response was observed if the cells were treated in G0.     

Sister-chromatid exchanges induced by vinyl esters and respective carboxylic acids in cultured human lymphocytes.

Vinyl acetate--an efficient inducer of sister-chromatid exchanges (SCEs)--is known to be hydrolyzed in mammalian cells into acetic acid and acetaldehyde, the latter being the likely metabolite responsible for the SCE induction. As similar hydrolysis to acetaldehyde and to a carboxylic acid is also expected for other vinyl esters, five such compounds--vinyl formate, vinyl chloroformate, vinyl propionate, vinyl crotonate and vinyl-2-ethylhexanoate--and five carboxylic acids--formic acid, acetic acid, propionic acid, crotonic acid and 2-ethylhexanoic acid--were tested for their ability to induce SCEs in cultured (72 h) human lymphocytes with a 48-h treatment, starting at 24 h after culture initiation. Vinyl formate, vinyl propionate and vinyl crotonate induced a clear dose-dependent increase in the number of SCEs/cell at concentrations of 0.125-0.5 mM and vinyl chloroformate at 0.063-1 mM, i.e., at roughly the same concentration range as vinyl acetate and acetaldehyde. Vinyl-2-ethylhexanoate required slightly higher concentrations (0.25-4 mM) for SCE induction. All of the carboxylic acids tested also elevated SCEs, but only slightly. Formic acid and crotonic acid produced some SCE increase at a concentration of 10 mM, acetic acid at 5 and 10 mM and propionic acid at 2.5 mM. 2-Ethylhexanoic acid induced SCEs at a lower concentration range (0.63-2.5 mM) than the other acids. The positive concentrations of the first three carboxylic acids lowered the pH of the culture medium immediately after the treatment by 0.5-1.0 pH unit (lowest observed pH 6.53). The pH differences from the control cultures became smaller in measurements done 24 h and 48 h after the beginning of treatment. Propionic acid and 2-ethylhexanoic acid affected medium pH only slightly (maximum drop 0.2 pH units) at the concentrations that induced SCEs. The results lend support to the idea that the efficient SCE induction observed with the vinyl esters results from the formation of acetaldehyde, with carboxylic acids--with the possible exception of 2-ethylhexanoic acid--playing no significant role. The slight SCE induction obtained with the carboxylic acids cannot be explained by lowered pH alone.     

Sister-chromatid exchanges in human lymphocytes exposed in vitro to therapeutic ultrasound

Human lymphocytes were exposed in vitro to therapeutic levels of ultrasound (1 W/cm2, CW, 0.87 MHz, durations of 80 and 160 sec). There were no significant differences in sister-chromatid exchange frequencies between controls and ultrasound-exposed cells. Exposure of lymphocytes to the positive control (mitomycin C) resulted in a significant increase in sister-chromatid exchanges. The data do not verify a report by Stella et al. (Mutation Res., 138 (1984) 75-85) that such exposures result in increased frequencies of SCEs.     

Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure

Somatic intrachromosomal recombination can result in inversions and deletions in DNA, which are important mutations in cancer. The pKZ1 chromosomal inversion assay is a sensitive assay for studying the effects of DNA damaging agents using chromosomal inversion as a mutation end-point. We have previously demonstrated that the chromosomal inversion response in pKZ1 spleen after single low doses of X-radiation exposure does not follow the linear no-threshold dose–response model. Here, we optimised a chromosomal inversion screening method to study the effect of low dose X-radiation exposure in pKZ1 prostatic tissue. In the present study, a significant induction in inversions was observed after ultra-low doses of 0.005–0.01 mGy or after a high dose of 1000 mGy, whereas a reduction in inversions to below the sham-treated frequency was observed between 1 and 10 mGy exposure. This is the first report of a reduction to below endogenous frequency for any mutation end-point in prostate. In addition, the doses of radiation studied were at least three orders of magnitude lower than have been reported in other mutation assays in prostate in vivo or in vitro. In sham-treated pKZ1 controls and in pKZ1 mice treated with low doses of 1–10 mGy the number of inversions/gland cross-section rarely exceeded three. Up to 4 and 7 inversions were observed in individual prostatic gland cross-sections after doses ≤0.02 mGy and after 1000 mGy, respectively. The number of inversions identified in individual cross-sections of prostatic glands of untreated mice and all treated mice other than the 1000 mGy treatment group followed a Poisson distribution. The dose–response curves and fold changes observed after all radiation doses studied were similar in spleen and prostate. These results suggest that the pKZ1 assay is measuring a fundamental response to DNA damage after low dose X-radiation exposure which is independent of tissue type.     

Sister-chromatid exchanges in lymphocytes of workers exposed to trichloroethylene

To detect mutagenic effects of trichloroethylene (TCE) on humans, sister-chromatid exchanges (SCEs) were analyzed in lymphocytes of 22 workers occupationally exposed to TCE and 22 matched controls. Although urinalysis in the workers revealed their obvious exposure to TCE, no increase in SCE frequencies was found in lymphocytes of the workers. SCE analysis in lymphocytes could not detect mutagenic effects by occupational exposure to TCE on humans.     

Sister-chromatid exchanges in human lymphocytes after a non-S-phase incubation period to allow excision DNA repair-in vitro exposure to N-acetoxy-2-acetylaminofluorene and ethylene oxide

Sister-chromatid exchanges (SCE) were analyzed in human peripheral blood lymphocytes at the baseline level, after induction of DNA damage by N-acetoxy-2-acetylaminofluorene (NA-AAF) and ethylene oxide (EO), and after a subsequent 18-h DNA-repair incubation period. There was a significant difference between the baseline SCE frequencies as compared to those after 1 h of NA-AAF or EO treatment. There was no significant difference between the SCE frequencies after 1 h of NA-AAF treatment and those after 18 h of DNA-repair incubation, suggesting that only a low level of NA-AAF damage to DNA had been removed. However, there was a significant difference between the SCE frequencies after 1 h of EO treatment and those after 18 h of DNA-repair incubation, indicating that a significant level of EO induced DNA lesions had been repaired. Thus, it seems likely that the EO induced DNA damage is more easily recognized, and hence more rapidly repaired than the NA-AAF induced damage. The reason for this may be the different chemical nature of the DNA lesions induced, which, in turn, leads to different kinetics of DNA repair.     

DNA polymerase delta mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Sister-chromatid exchanges in human lymphocytes induced by dimethoate, omethoate, deltamethrin, benomyl and their mixture.

Dimethoate and omethoate, two common organophosphorus insecticides, induced a dose-related increase in the frequency of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (P of the regression lines less than 0.01). Two other common pesticides, the pyrethroid insecticide deltamethrin and the systemic fungicide benomyl, induced a modest increase in SCEs which bordered on statistical significance (P = 0.053 and 0.055, respectively). Mixtures of the four pesticides at total concentrations of 41.5 and 83 micrograms/ml (composed of 43% dimethoate, 43% omethoate, 12% deltamethrin and 1.2% benomyl) induced a dose-dependent increase in SCEs (P less than 0.01). The effects of these mixtures of pesticides were variable using lymphocytes from different individuals, although these differences did not attain statistical significance. Moreover, low concentrations of the four pesticides that did not increase SCEs significantly when tested alone, were positive for SCE induction when tested as a mixture. The experiments show that sub-threshold doses of pesticides may increase SCEs when present in a mixture.     

Chromosome-type aberrations induced in chromosome 9 after treatment of human peripheral blood lymphocytes with mitomycin C at the G(0) phase.

To determine the fate of chromosome aberrations induced primarily by clastogenic chemicals, aberrations of chromosome 9 in cultured human peripheral blood lymphocytes were analyzed after exposure to mitomycin C (MMC) at G(0) phase. Chromosome 9 painting by fluorescence in situ hybridization revealed that the translocation of 9p or 9q to another chromosome and the centric fragment representing the entire length of 9p were characteristically generated from chromatid-type aberrations involving the centromeric region of chromosome 9. These changes were not observed at 48 h after culture initiation, but persistently appeared at later stages (72-120 h postinitiation). Induction of centric fragments of 9p and micronuclei without the alpha satellite DNA of chromosome 9 suggested that most of the breaks were induced near the alpha satellite DNA locus on 9q. Modified patterns of chromosome 9 aberrations were also observed, being related to the copy number of the short or long arm of the chromosome. Such unbalanced karyotypes could remain in the lymphocyte genome over further cell divisions for at least 120 h after culture initiation, indicating that these aberrant cells can survive and that they could pose a health risk.     

Sister-chromatid exchanges in human lymphocytes treated with 4-chloromethylbiphenyl and benzyl chloride

At non-inhibitory concentrations, 4CMB and BC both induced small, but dose-related increases in SCE. The dose-response curves gave a highly significant correlation for 4CMB (r=0.996, P<0.001) and a significant correlation for BC (r=0.757, P<0.05) which reflects the relative activities of these substances in bacterial mutation tests.     

Sister-chromatid exchanges in association with occupational exposure to ethylene oxide

Sister-chromatid exchange (SCE) frequencies in employees potentially exposed to ethylene oxide (ETO) were compared with those in unexposed control groups. Three worksites where the previous environmental control of ETO was known to have differed were chosen. Within these worksites, subjects were categorized into high potential exposed, low potential exposed and control groups. An additional community control group was obtained. Blood samples for chromosome studies of peripheral lymphocytes were drawn at several time points over a period of 24 months. The effects on SCE of age, sex, smoking habits and reader variation were considered. Worksites I, II and III, respectively, represented increasing levels of exposure. At Worksite III large differences among groups persisted over 24 months. At Worksite II, the SCEs in the high potential exposed workers were higher than those in the other groups. At no time was the low potential exposed group at Worksite II statistically significantly higher in mean SCE than the worksite controls. No consistent differences among groups were noted in Worksite I.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

Sister-chromatid exchanges induced in rabbit lymphocytes by 2-aminofluorene and 2-acetylaminofluorene after in vitro and in vivo metabolic activation

Because short in vitro treatments of Chinese hamster cells with 2-aminofluorene and 2-acetylaminofluorene did not increase SCEs even in the presence of a metabolic activation system, experiments were carried out with rabbits to see if in vivo activation occurred. Rabbits injected with 2-AF could activate the compound and a transient dose-dependent increase in SCEs was found in peripheral lymphocytes cultured at various times after the injection. With 2-AAF, however, the response was more variable: some rabbits showed an increase immediately, but one showed an increase only after a subsequent injection. This indicated that among rabbits differences exist in their ability to detoxify 2-AAF. Because rabbits could activate the compounds, in vitro experiments were carried out to see if their lymphocytes responded differently from Chinese hamster cells and to see if the metabolic changes brought about by PHA stimulation affect the ability of the cells to activate the chemicals. The addition of PHA and the consequent metabolic stimulation do affect the induction of enzymes involved in the activation of 2-AF and 2-AAF. Benzo[a]pyrene, in contrast, can be activated by rabbit lymphocytes independently of PHA stimulation.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Sister-chromatid exchanges in lymphocytes of smokers in an experimental study

The frequency of sister-chromatid exchanges (SCEs) was studied in peripheral blood lymphocytes of 26 young male smokers and 10 non-smokers who had recently entered military service. The levels of SCEs were examined in 4 consecutive blood samples taken after short experimental periods of smoking only low-tar (LT) or medium-tar (MT) cigarettes. The incidence of SCEs was significantly higher in the the group of smokers than in the group of non-smokers. The SCE levels of the smokers were found to be associated with the personal smoking history; the observed increase in the SCE frequency correlated with the years of smoking measured as cumulative pack years. The difference in type of cigarette did not influence the SCE frequencies.