Reduction of sister chromatid exchange frequency with time after mutagen exposure in chinese hamster ovary cells in the presence of 3-aminobenzamide

3-Aminobenzamide (3AB) is a potent inhibitor of poly(ADP-ribose) synthesis and has been reported to inhibit DNA repair. Inhibition of poly(ADP-ribose) synthesis by 3AB results in a potentiation of the cytotoxic effects of alkylating agents and synergistically increases the frequency of sister chromatid exchanges (SCEs) induced in Chinese hamster ovary cells by methyl methanesulfonate (MMS). Experiments were carried out to determine if this synergism was related to an inhibition of the repair or the removal of SCE-forming lesions by 3AB. Cells were treated with MMS or ethyl nitrosourea (ENU) and either held in a nonproliferative state or maintained in exponential growth for up to 48 hr before SCE frequencies were measured. During this time, the SCE frequency decreased greatly. 3AB did not affect this decrease in either cycling or noncycling cells. Furthermore, 3AB appears to exert its effect only in cycling cells when bromodeoxyuridine is present. Therefore, the synergism between 3AB and alkylating agents in the induction of SCEs is unrelated to effects of 3AB on the repair or removal of SCE-forming lesions.     

Tissue-specific induction of sister chromatid exchanges by ethyl carbamate in mice

Sister chromatid exchange (SCE) techniques were used to analyze the genetic effects of ethyl carbamate (urethane) in cultured mouse-bone marrow cells, and in several different mouse tissues in vivo. Ethyl carbamate concentrations up to 5.0 mg/ml were ineffective in causing a significant elevation of SCE in vitro. After in vivo drug administration, bone marrow, liver and spermatogonial cells all revealed significant dose-related increases in SCE. Baseline and relative incremental levels of SCE were somatic vs germ tissue-specific. Regenerating liver cells exhibited significantly greater absolute SCE values than all other tissues examined. Marrow cells revealed intermediate values, while germ cells were the least sensitive in SCE responsiveness. Spermatogonia required a fourfold higher dose, over that effective in somatic tissues, to promote an approximate doubling of the baseline SCE level. In vivo SCE analysis affords sensitive risk assessments for different tissues. Thus, this approach should be generally useful for studying compounds with questionable mutagenic potential, and/or those exerting target organ specificities of related biological activity (eg, toxicity, carcinogenesis).     

Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals

The National Toxicology Program has undertaken a study to assess the ability of four genetic toxicology assays to predict the carcinogenicity of chemicals in 2-year rodent studies [Tennant et al.: Science 236:933-941, 1987]. Two of the assays, used for evaluating in vitro cytogenetic damage, were the SCE and chromosome aberration assays in Chinese hamster ovary cells. The results and data for 15 of the chemicals tested in these two assays are presented here. Each chemical was tested with and without exogenous metabolic activation. The chemicals tested were bisphenol A, 2-chloroethanol, C.I. acid orange 10, C.I. disperse yellow 3, C.I. solvent yellow 14, cytembena, D&C red 9, 1,2-dibromoethane, FD&C yellow 6, malaoxon, D,L-menthol, phenol, sulfisoxazole, titanium dioxide, and tris(2-ethylhexyl)phosphate. In vitro cytogenetic results from the other chemicals presented by Tennant et al. (Science 236:933-941, 1987) have been published by Galloway et al. (Environmental and Molecular Mutagenesis 10(Suppl 10): 1-175, 1987), Gulati et al. (Environmental and Molecular Mutagenesis 13:133-193, 1989), and Love-day et al. (Environmental Mutagenesis 13:60-94).     

Factors underlying variation in spontaneous and clastogen-induced sister chromatid exchanges and chromosome breakage frequencies.

The latent "factors" influencing spontaneous and clastogen-induced genetic damage, measured by rates of sister chromatid exchange (SCE) and chromosome breakage (CB), were investigated in a small sample of 20 unrelated, healthy individuals. The covariation of spontaneous and clastogen-induced (bleomycin [BLM], streptonigrin [SN], mitomycin-C [MMC], 4-nitroquinoline-1-oxide [4NQO]) SCEs and CBs was analyzed by maximum-likelihood factor analysis. A single-factor model resulted in large standardized regression coefficients of measured variables on the factor for spontaneous and BLM- and SN-induced SCE frequencies, and a modest regression coefficient for MMC-induced SCEs. A two-factor model, after varimax rotation, yielded one factor strongly associated with spontaneous and BLM- and SN-induced SCE frequencies, and a second factor associated with spontaneous and BLM- and SN-induced CBs. A bootstrap analysis of this data set indicated the statistical significance of one regression coefficient (i.e., P less than or equal to 0.05) and borderline significance (0.07 less than or equal to P less than or equal to 0.11) of three other regression coefficients on the first factor, to be interpreted as an effector of SCE frequencies. However, for the second factor, none of the bootstrapped regression coefficients was significant (P greater than 0.22). Due to the modest sample utilized in this study, the validity of this model should be further explored using additional, larger data sets.     

Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo

Diethylstilbestrol diphosphate (DES-dp) clastogenesis was examined in the bone marrow of C57B1/6 male and female mice. Significant and sex-related dose effects were observed for the induction of chromatid-type chromosomal aberrations and for the inhibition of cellular proliferation. Females were more sensitive to the effects of DES-dp than males when assessed for either induced chromosomal aberrations or proliferative inhibition. Contrary to other published results, we did not observe either an increase in sister chromatid exchanges or an increased incidence of aneuploidy. Ovariectomy reduced the ability of DES-dp to inhibit cellular proliferation and decreased the high degree of variability between animals at high doses of DES-dp. The results of our studies show that DES is a clastogenic agent in vivo which may relate to its carcinogenicity.     

Induction by inorganic metal salts of sister chromatid exchanges and chromosome aberrations in human and syrian hamster cell strains

Sister chromatid exchange (SCE) and chromosome aberration induction were determined for several inorganic metal salts. Arsenic, nickel, and beryllium salts at concentrations effective in causing transformation of Syrian hamster cells (HEC) induced SCE and chromosome aberrations of HEC and human lymphocytes, whereas sodium tungstate, a non-transforming chemical, neither induced SCE nor chromosome aberrations. Normal human and hamster cells exhibited equal sensitivity to SCE induction; nontoxic concentrations of sodium arsenite, beryllium sulfate, and nickel sulfate caused an increase of 8–10 SCE/cell over control values. Sodium arsenite, a trivalent arsenic, and sodium arsenate, a pentavalent arsenic, produced increases in SCE but the former was effective at lower concentrations. Both arsenic salts were less efficient in inducing SCE in human whole blood than in purified lymphocyte cultures. Sodium arsenite, sodium arsenate, nickel sulfate, and beryllium sulfate also caused damage consisting primarily of chromatid type of aberrations. In HEC, with doses most effective in SCE induction, all four metals produced aberrations in 16–21% of cells. In human lymphocytes, 34 and 30% of the cells had chromosome damage after sodium arsenite and sodium arsenate, respectively, whereas beryllium sulfate or nickel sulfate caused damage in about 10% of the cells. The induction of SCE and chromosomal aberrations by metals reemphasizes the sensitivity of cytological assays and their importance for detecting genetic damage caused by carcinogens.     

Induction of sister chromatid exchanges in human diploid fibroblasts by mutagens with and without rat liver microsomal activation

The frequency of sister chromatid exchange (SCE) in WI-38 cells was estimated by the 5-bromodeoxyuridine (BrdUrd)-dye technique after one hour's exposure to cyclophosphamide (CY), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), and maleic hydrazide (MH) with and without the addition of rat liver microsomal suspension (S-9) fraction with co-factors (S-9 mix). CY at concentrations from 1 × 10^?5M to 1 × 10^?3M with S-9 mix increased the number of SCEs per cell in a dose-dependent manner. Without S-9 mix, CY at concentrations below 1 × 10^?3M failed to produce more SCEs than the controls. MNNG at 1 × 10^?8M and 4NQO at 1 × 10^?7M without S-9 produced significant increases in SCEs per cell. Addition of S-9 during treatment slightly decreased the effects of MNNG and 4NQO in the formation of SCEs. MH was tested at pH 6.4 and pH 7.6. At pH 7.6, MH at 1 × 10^?3M without S-9 mix inhibited cell multiplication, but did not cause a significant increase of SCEs per cell. There were no interactions between MH (2 × 10^?4M) and S-9 mix nor between MH and the pH levels tested. These results indicate that in the presence of metabolic activation, SCE formation in human diploid fibroblasts in vitro may be used as a potential assay for mutagenicity.     

Sister chromatid exchange response of human diploid fibroblasts and chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene

In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in all cell lines. The response of CHO cells and three human diploid fibroblast strains (IMR-90, WI-38, S-3299) to benzo(a)pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-responses to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.     

Quinoline-induced chromosome aberrations and sister chromatid exchanges in rat liver

Induction of chromosome aberrations and sister chromatid exchanges (SCEs) was studied in hepatocytes of F344 rats exposed in vivo to the hepatocarcinogen quinoline (Q). Hepatocytes were isolated 4–48 hr after a single dose of 200 mg/kg body weight or 24 hr after 28 repeated doses (once a day) of 25–200 mg/kg body weight/day by gastric intubation, and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 hr. A single dose of Q induced chromosome aberrations in up to 22% of metaphase cells, and SCEs with a frequency of up to 1.27 per chromosome 12 hr after the dose, while the control values were 1% and 0.63 per chromosome, respectively. Treatment with 28 repeated doses of Q induced significant chromosome aberrations and SCEs dose-dependently. Cytogenetic damage induced in the liver by repeated doses of Q was greater than induced by a single dose. Furthermore, Q induced replicative DNA synthesis in the liver, but failed to induce micronucleus formation in the bone marrow. The noncarcinogen 8-hydroxyquinoline was also examined and found to be essentially non-genotoxic to rat liver. These results show that Q is a genotoxic carcinogen to rat liver and the present method of in vivo cytogenetic assay should be useful for evaluating the genotoxicity of hepatocarcinogens. Environ. Mol. Mutagen. 30:459–467, 1997 © 1997 Wiley-Liss, Inc.     

Failure to demonstrate an effect of in vivo diagnostic ultrasound on sister chromatid exchange frequency in amniotic fluid cells

Antepartum use of diagnostic ultrasound has markedly reduced radiation exposure of the fetus. Previous investigations have documented the safety of ultrasound, but concern persists regarding its long-term effects. As new methods become available to study possible subtle effects of ultrasound, it is important to reevaluate this technique continually because of its universal use in obstetrics and elsewhere. We report results of in vivo studies of effect of diagnostic ultrasound on the sister chromatid exchange (SCE) frequency in amniotic fluid cells. SCE is a cytogenetic phenomenon believed to be a sensitive indicator of environmental perturbations and chromosome stability. In amniotic fluid cells from six pregnancies without ultrasound exposure and in 34 pregnancies that received varying amount of ultrasound immediately before amniocentesis, there was no difference in SCE frequency in exposed versus nonexposed cells. These data, which appear to confirm again the safety of ultrasound, are reassuring to both patients and clinicians.     

Simultaneous analysis of sister chromatid exchanges and cell cycle delay

Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 5, pp. 589–591, May, 1988.     

Effects of acute and chronic administration of mitomycin C on the induction of sister chromatid exchanges in vivo

Frequencies of sister chromatid exchanges (SCE) were analyzed in bone marrow cells of mice injected with mitomycin C (MMC) both before and during infusion with bromodeoxyuridine (BrdU). Administration of MMC at 1, 6.5, and 13 hours after the onset of BrdU infusion resulted in the induction of approximately 45 SCE/cell, independent of time of administration. When MMC was injected 26 hours prior to BrdU infusion, only baseline levels of SCE were noted. The effects of multiple doses of MMC (chronic administration) were examined in mice treated with 1–5 mg/kg on a weekly or bimonthly basis. SCE analysis was performed one week after the final injection. At all doses and with all treatment regimes, SCE frequencies did not differ from control levels. The results indicate that most or all MMC-induced DNA damage that results in SCE formation is removed in a single cell cycle after its administration.     

Time course of frequency of sister chromatid exchanges after exposure to thiophosphamide in vivo

Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 85–87, January, 1989.     

Effect of deoxycytidine on frequency of sister chromatid exchanges in human lymphocytes detected with 5-bromodeoxyuridine

The frequency of sister chromatid exchanges (SCE) during abolition of the inhibitory action of 5-bromodeoxyuridine (BdU, 0.05 mM) on DNA synthesis by means of deoxycytidine (dC, 0.1 and 0.01 mM) was investigated in cultures of lymphocytes from the blood of three normal individuals. The frequency of SCE was significantly increased in all three experiments in the presence of dC in a concentration of 0.1 mM. Parallel with the increase in the frequency of SCE, dC also increased the yield of second-division metaphases. Elevation of the SCE level under the influence of dC was connected with its normalizing action on DNA replication.Laboratory of General Cytogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 744–745, June, 1978.