Comparison of baseline sister-chromatid exchanges (SCE), cyclophosphamide-, ethylnitrosourea (ENU)-induced SCE, ENU-induced cell-cycle delay and chromosome aberrations between Peru and laboratory mice

The baseline sister-chromatid exchange (SCE) frequency and sensitivity to the effects of the mutagens cyclophosphamide (CPP) and ethylnitrosourea (ENU) in bone-marrow cells of descendants of wild mice trapped from Rimac valley in Peru (Peru mice) were studied and compared to the same effects in laboratory mice. Baseline SCE of the Peru mice were significantly higher than those of the C57BL/6J and DBA/2 mice. The average SCE/cell of 4 Peru mice was 5.4 (range 3.8-7.6), while the average of SCE/cell of either 4 C57BL or 5 DBA mice was 3.2 (range 3.0-3.4). The variation of SCE/cell among Peru mice studied was statistically significant whereas among C57BL or DBA mice it was not. SCE frequencies of primary cultures derived from the ear tissue of 10 Peru (mean SCE/cell = 8.5) were also significantly higher than those of 6 C57BL mice (mean SCE/cell = 7.4). CPP treatment resulted in a dose-dependent increase of SCE frequencies in bone-marrow cells of all the mice. However, some of Peru mice treated with CPP had significantly higher SCE than the other Peru mice and than all of the C57BL and DBA mice treated with equivalent dose. ENU induced increased SCE frequencies in Peru and C57BL mice. Again some of Peru mice either had significantly higher SCE, greater extent induced cell-cycle delay or chromosome aberrations (CA) than other Peru mice and than of all the C57BL mice treated with equivalent dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of in vivo and in vitro SCE induction

In vivo and in vitrod sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. II. Relationship to baseline SCE frequency

Environmental and genetic factors have been implicated as important sources of individual variation in baseline sister-chromatid exchange (SCE) frequency in humans. The current study was designed to test whether the frequency of baseline SCEs in 58 normal blood donors is associated with previously observed variations in SCE frequencies induced by diepoxybutane (DEB). Because 12 subjects were current cigarette smokers and smoking is known to be an in vivo inducer of baseline SCE frequencies, we specifically tested whether higher baseline SCE frequencies in smokers would be associated with in vitro sensitivity to SCE induction by DEB. Analysis of variance showed that DEB-induced SCE frequencies were significantly associated with baseline SCE frequencies; those who were sensitive to SCE induction by DEB were more likely to have higher baseline SCE frequencies. This effect, however, was independent of in vivo induction of SCE by smoking. Chromosomal sensitivity to the induction of SCE by DEB explained approx. 15-20% of the variation in baseline SCE. This was similar in magnitude to the effect of cigarette smoking. Because increased sensitivity to DEB-induced SCEs is common in normal blood donors (approx. 24%) and is associated with an increase in baseline SCEs, it should be investigated as a source of bias and/or a potential marker of sensitivity to environmental mutagens in future cytogenetic studies.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Comparative in vivo mutagenicity testing by SCE and micronucleus induction in mouse bone marrow

Summary The treatment of mice with repeated injections of BUdR and FUdR allows for the demonstration of differentially stained metaphases from bone marrow after FPG (fluorescence plus Giemsa; Perry and Wolff, 1974) treatment. Thus, it is possible to determine the number of SCE's under in vivo conditions, which appears as a very promising system for mutagenicity testing. We studied the response of this system in comparison to the micronucleus test using six mutagenic agents: triaziquone, cyclophosphamide (CP), dimethylphenyltriazene (PDMT), methylnitronitrosoguandine (MNNG), dimethylnitrosamine (DMNA), and diethylnitrosamine (DENA). With the exception of MNNG and DENA, all these agents induce both, SCE and micronuclei, MNNG and DENA being ineffective in both systems. The most potent SCE-inducing agent was triaziquone, followed by PDMT, CP, and DMNA. The quantitative comparison indicates that SCE are induced at 1/10–1/100 of the concentrations which are required for the detection of micronuclei.     

SCE induction and harlequin staining in mycoplasma-contaminated Chinese hamster cells

Chinese hamster V79 and CHO cells infected with Mycoplasma hyorhinis show elevated sister-chromatid exchange (SCE) levels but normal cell proliferation and levels of chromosomal aberrations when compared with uninfected cells. Harlequin staining patterns differ from those seen with uninfected cells at similar levels of bromodeoxyuridine (BrdUrd), indicating that BrdUrd is rapidly depleted from the medium by the mycoplasmal uridine phosphorylase and therefore becomes unavailable over the two cell cycles necessary for harlequin staining. Continuous treatment with the antibiotic minocycline restores the SCE level and harlequin staining to that seen in uncontaminated cells. The results suggest that mycoplasma infection should be suspected if harlequin staining patterns indicate a sudden decrease in incorporation of BrdUrd in cells grown in normal levels of BrdUrd.     

Ultraviolet-light exposure induces a heritable sensitivity to the induction of SCE by mitomycin-C

The dose-response relationship for mitomycin-C (MMC)-induced sister-chromatid exchange (SCE) has been determined in the progeny of Chinese hamster lung fibroblasts (V79) exposed to 5.0 J/m2 ultraviolet light-C (UVC, 254 nm) and in the progeny of non-UVC-irradiated controls. Progeny of UVC-irradiated cultures exhibited sensitivity to MMC-induced SCE at doses of MMC that were not detectably lethal. This sensitivity was manifest as an increase in SCE per cell in a large proportion of the cells derived from UVC-exposed cultures and thus appears not to result from the expression of a rare event such as mutation.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

Liver tumor potency indicators for technical toxaphene and congeners simulating weathered toxaphene

Technical toxaphene (TT) is a liver tumor promoter in B6C3F1 mice but not in F344 rats. To further evaluate dose-response relationships for weathered toxaphene, B6C3F1 mouse hepatocytes were treated with TT alone, five selected persistent congeners (p-26, p-50, p-62, Hx-Sed, and Hp-Sed), or two selected congener mixtures (simulating weathered toxaphene) and dose-response relationships were characterized for cytotoxicity and gap junction intercellular communication (GJIC) inhibition. Phenobarbital was included as a positive control for mouse liver tumor promotion and GJIC inhibition and dose ranges were calibrated to define benchmark dose concentrations. Each treatment group exhibited significant cytotoxicity and GJIC inhibition for at least one sex (M/F) after 3 and/or 24 h of treatment. Maximum GJIC inhibition was observed at certain noncytotoxic concentrations with sex-specific differences in relative potency estimated as the effective concentration at 20% inhibition (EC20); however, no significant EC20 differences were observed between the treatment groups. Analysis of mixture interactions at the EC20 showed that GJIC inhibition of the two weathered toxaphene mixtures was significantly less than additive compared to that for the component congeners. These findings suggest that the persistent toxaphene congener mixtures tested are not more tumorigenic than the parent insecticide mixture.     

Different effects of 1-beta-D-arabinofuranosylcytosine and aphidicolin in S-phase cells--chromosome aberrations, cell-cycle delay and cytotoxicity

Some effects of a 2-h exposure to either aphidicolin (APC) or cytosine arabinoside (ara-C) on S-phase cells of the cell line JU56 have been measured. At a concentration of 1.5 X 10(-5) M of either drug, incorporation of tritiated thymidine into log-phase cultured was reduced by 97-99%. A 2-h exposure to either drug at the same concentration induced chromosome aberrations in cells in S when they subsequently reached mitosis. However, exposure to ara-C induced small numbers of aberrations per damaged cells, and most cells were undamaged. Exposure to APC induced gross chromosomal damage (pulverized chromosomes) in damaged cells. More cells were delayed, and for longer, after exposure to APC than after exposure to ara-C. The results of clonal assays were consistent with the assumption that chromosome aberrations are the proximal cause of reproductive cell death. In the case of ara-C, the results of this and a previous study are consistent with the assumption that cell death and chromosome aberrations are correlated with incorporation of ara-C into DNA in S-phase cells, but that these biological effects manifest themselves only with doses when inhibition of semi-conservative DNA synthesis is greater than 97%.     

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.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.     

SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either satin (GBI, GBII) at 1.4 x 10⁻³ M or soman (GD) at 1.1 and 2.2 x 10^t-3 M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 x 10⁻³ M and cyclophosphamide (CP) at 10⁻⁴ M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.     

Inhibition of photosynthesis by the organochlorine pesticide toxaphene

Susceptibility to toxaphene, an extensively used organochlorine pesticide, was evident in five cereal types and was widespread in oat (Avena spp.) and barley (Hordeum spp.). In these cereals most of the varieties tested were susceptible, this being assessed biochemically from inhibition of the Hill reaction in chloroplasts from treated plants. Studies with the susceptible Avena sativa variety Blyth showed two sites of inhibition ofphotosynthetic electron flow. The first site was on the oxidizing side of photosystem 2, and the second in the intermediate electron transport chain between the two photosystems. As a consequence of the latter, cyclic photophosphorylation was inhibited and toxaphene may additionally have some uncoupler activity.     

Subpopulation analysis of drug-induced cell-cycle delay in human tumor cells using 90 degrees light scatter

A mitotic cell subset has been identified with nuclear light scatter. Colcemid-treated T-47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90 degrees light scatter. A discrete low-scattering cell cluster could be distinguished from the G2 cell subset on two-dimensional contour plots of 90 degrees light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake-off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell-cycle analysis of DNA fluorescence. Cell sorting confirmed that the low-scattering cell cluster comprised predominantly metaphase and anaphase cells. Identification of mitotic cells with this one-step technique enables rapid analysis of drug-induced cell-cycle delay in cell populations with different rates of cell-cycle traverse. Hence, vincristine-induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug-induced cell-cycle delay and supplements the information obtainable by conventional cell-cycle analysis.