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).     

Sister chromatid exchange induction near the baseline with low doses of the alkylating agent CCNU

Sister chromatid exchange (SCE) and cell-cycle kinetics were examined at near-baseline levels in human peripheral lymphocytes exposed to low doses of the potent SCE inducer 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), in vitro. A preliminary SCE dose-response curve was determined with a broad range of doses of CCNU using a single donor. SCE induction was approximately linear over the entire dose range from 5 to 200 microM CCNU. Cell cycle kinetics were retarded in a dose-dependent manner. A second dose-response curve from the same donor was constructed using several doses of CCNU between 0.5 and 10 microM to evaluate linearity and uniformity of SCE response near baseline levels. SCE induction was approximately linear between 1.0 and 10.0 microM CCNU. Finally, SCE and cell-cycle kinetics were examined in 12 donors at doses of 1.0, 5.0, and 10.0 microM CCNU to evaluate the reproducibility of near-baseline SCE induction over a range of subjects. Cell-cycle kinetics were retarded at all three doses with a highly significant increase in SCE frequencies at 5.0 and 10.0 microM CCNU. These data suggest that increases in SCE less than twice background can be reliable indicators of genotoxic exposure.     

Sister chromatid exchange in vivo in mice: I. The influence of increasing doses of bromodeoxyuridine

Bromodeoxyuridine (BrdUrd) pellets weighing either 20, 25, 30, 35, 40, 45, or 53 mg were implanted subcutaneously in strain DBA/2J male mice. Femoral bone marrow cells were analyzed for sister chromatid exchange (SCE) and chromosome aberration frequencies, mitotic indices, and cellular replication kinetics. Small but statistically significant BrdUrd dose-dependent increases of SCEs were observed, whereas chromosome aberrations were induced at low frequencies and occurred independently of BrdUrd dose. The mitotic index remained relatively constant for all doses. A slight inhibition of cellular replication, as manifested by a reduction in third division cells at the 40- and 45-mg doses, was observed. The use of an intense fluorescent light source in the fluorescence-plus-Giemsa technique provided consistently good sister chromatid differentiation at pellet doses substantially lower than those previously used by other investigators.     

Inhibitors of poly(ADP-ribose) polymerase enhance DNA strand breaks,excision repair,and sister chromatid exchanges induced by alkylating agents

Benzamide and 3-aminobenzamide, inhibitors of poly(ADP-ribose) polymerase, synergistically enhanced the frequencies of unscheduled DNA synthesis and sister chromatid exchanges in Chinese hamster ovary (CHO) cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These inhibitors also increased methyl methanesulfonate (MMS)- or MNNG-induced DNA strand breaks and significantly retarded the rejoining of strand breaks in CHO and HeLa S₃ cells. These results suggest that poly(ADP-ribose) is important in the repair of DNA damage after exposure to alkylating agents and exercises a regulatory role in stabilizing chromatin structure whenever strand breaks occur in DNA.     

Replication index in cultured human lymphocytes: methods for statistical analysis and possible role in genetic toxicology

The replication index (RI) is an index of cytokinetics in cultured cells. This paper presents statistical methods for the analysis of RI data. It also proposes formulas for the calculation of RI and its standard error. The proposed statistical analysis may be applied only in cases when replicate cultures are used. If duplicate or repeated cultures are used, the interculture variation may provide a more suitable error term for RI. Determining the RI in human blood cultures may be useful in genotoxicity testing, because inhibition of DNA synthesis will result in a decrease of the RI. Monte Carlo methods are used to show that the sensitivity of the proposed method is roughly equivalent to that of a chi 2 test. Power studies indicate that for the study sample, no less than 200 cells should be analyzed when assessing the RI.     

Cytogenetic effects of shale-derived oils in murine bone marrow

The cytogenetic effects of exposure of mice to shale-derived oils by either skin painting or intraperitoneal injection were examined. Skin painting with 40 mg crude oil every other day for five weeks had no effect on the frequency of chromosomal aberrations observed in the bone marrow. Three daily intraperitoneal injections of 0.5–2.0 ml/kg per day of a crude shale oil from an aboveground retort induced a dose-related increase in the frequency of aberrations observed. A hydrotreated sample of this oil produced a similar pattern of aberration induction, but at lower frequencies. Crude shale oil from a modified in situ retort induced the highest frequency of chromosomal aberrations at the 0.5 ml/kg dose; lower frequencies were induced at the 1.0 ml and 2.0 ml/kg doses. Of the three shale oils tested, only the crude oil from the aboveground retort induced increased frequencies of sister chromatid exchanges and only at doses that also induced structural aberrations. These studies indicate that structural aberration analysis is a more sensitive test than sister chromatid exchange analysis for the type of DNA damage induced by shale-derived oils in murine bone marrow cells.     

Effects of age, sex and genes on sister chromatid exchange

Sister chromatid exchange (SCE) was studied in cultured lymphocytes from a limited series of 21 like-sexed twin pairs; 11 monozygotic (MZ) and 10 dizygotic (DZ) pairs. The 18 subjects, who were between 57 and 61 years old, had an SCE mean value () of 8.0 whereas the 24 subjects between 33 and 39 years of age had a mean of 6.8. The difference was statistically significant ( P <0.001). The effect of age appeared to be present in both sexes. No significant difference was found between females (%7.3) and males (%7.5), nor between smokers (%7.3) and non-smokers (%7.4). Drug users had a slightly higher mean (%7.9) than non-users (= 7.0) ( P < 0.05). This trend was found in each age group. The within-pair variance was slightly higher in DZ than in MZ pairs. The difference was not significant. We conclude that genetic factors are probably not a major source of subject variation in SCE mean value.     

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.     

Clastogenic effect of fenfluramine in mice bone marrow cells in vivo

Fenfluramine, an amphetamine derivative used in the treatment of obesity, has been evaluated in vivo in the bone marrow cells of Swiss albino mice using two cytogenetic endpoints for assessing its genotoxic and clastogenic potentials. Concentrations of 0.75, 1.5, 3.0, and 5.0 mg/kg b.w. were administered orally for the study of sister chromatid exchange frequencies and chromosome aberrations (CA). SCE frequencies showed a positive dose response; 1.5 mg/kg being the minimum effective concentration. Fen caused a prolongation of cell cycle at all concentrations. Except for the minimum therapeutic dose (0.75 mg), all other doses (1.5, 3.0, and 5.0 mg) showed a significant increase in the percentage of damaged cells over that of the vehicle control. The degree of clastogenicity was directly proportional to the dosage used and inversely related with the duration of treatment. A gradual reduction of the clastogenic potential was observed after 12 and 24 hr of exposure, indicating that the maximum effect occurs at the middle or late synthetic phase of the cell cycle. This study, probably the first detailed screening of the drug for its genotoxicity, shows that Fen is moderately clastogenic and a DNA damaging agent in vivo.     

烟草和酒精诱导人淋巴细胞SCE和微核的研究

目的　研究烟草和酒精共同作用 ,观察其对人体是否存在有害的叠加效应。方法　采用人全血培养淋巴细胞染色体SCE和微核在酒精和烟草酒精提取物作用下的改变。结果　酒精组与对照组相比 ,SCE频率增高 (P <0 0 5 ) ,各烟草处理组SCE高于酒精组 ,与对照组相比更高 ,存在显著差异 (P <0 0 1)。微核在酒精组与对照组相比 ,显著提高。结果　酒精与烟草之间存在叠加效应。     

The longevity of chemically induced sister chromatid exchanges in chinese hamster ovary cells

The persistence of the lesions leading to sister chromatid exchanges (SCEs) following acute exposure of Chinese hamster ovary (CHO) cells to direct-acting chemical mutagens was measured. The results revealed that these lesions (and consequently SCEs) are rapidly eliminated from cells. SCE levels fell to near control values by the third or fourth day (six and eight cell cycles, respectively) following exposure of CHO cells to quinacrine mustard (QM) and mitomycin C (MMC). In contrast, cells exposed to methyl methanesulfonate (MMS) showed a small but significant increase in SCE level over control up to and including the fifth day following exposure (approximately ten cell cycles), suggesting that the behavior of these lesions in cells is influenced, at least in part, by the mechanism by which a specific agent interacts with DNA. The possibility that the decline in SCE level was due to the loss of cell populations with high numbers of exchanges was eliminated by the assessment of cloning efficiencies of treated and control cultures.     

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.     

Evaluation of the genotoxicity of Fusarium mycotoxin moniliformin in human peripheral blood lymphocytes

Mycotoxins are fungal secondary metabolites that can be found in contaminated food and feed. There is some evidence to suggest that certain mycotoxins may be mutagenic. Here, we investigate the genotoxicity of the mycotoxin moniliformin (MON) (3‐hydroxycyclobut‐3‐ene‐1,2‐dione) in human peripheral blood lymphocytes using chromosomal aberration (CA), sister‐chromatid exchange (SCE), and micronucleus (MN) analysis. Lymphocyte cultures were treated for 48 h with six different concentrations of MON between 2.5 and 25 μM. CA, SCE, and MN frequencies were significantly increased in a dose‐dependent manner compared with the negative control. The mitotic, replication, and cytokinesis‐block proliferation indices were not affected by treatment with MON. The results provide evidence to demonstrate that MON can exert cytogenetic effects in human cells in culture. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Activity of nitro-polynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation

Nitro-polynuclear aromatic hydrocarbons are found in diesel particulates. These compounds are potent mutagens in the Ames test. To determine whether nitro-polynuclear aromatic hydrocarbons are active in a mammalian cell assay, 1-nitropyrene, 1,8-dinitropyrene, 2-nitrofluorene, and 4-nitrobiphenyl were incubated with cultures of Chinese hamster ovary cells. The frequency of sister chromatid exchange (SCE) was measured in the presence and absence of rat liver S-9 mix. The addition of S-9 mix resulted in a large increase in the SCEs induced by all four compounds.     

Effect of BrdUrd and low doses of gamma radiation on sister chromatid exchange,chromosome breaks,and mitotic delay in mouse bone marrow cells in vivo

We have measured, in mouse bone marrow cells in vivo, the ability of low doses of gamma radiation to induce sister chromatid exchanges (SCEs) in unifilarly 5-bromodeoxyuridine (BrdUrd)-substituted DNA. Also examined was the effect of BrdUrd incorporation on SCE induction by radiation, the comparative frequency of SCE and chromosome breaks induced by gamma radiation, the ability of ionizing radiation to interfere with normal cellular proliferation, and the relationship between proliferative inhibition and SCE and chromosome break frequency. A direct relationship between the number of SCEs and gamma radiation dose was observed, an effect which was dependent on BrdUrd incorporation. The frequency of SCE was 80 times higher than that of chromosome aberrations in cells with BrdUrd-substituted DNA, and there was no difference between the frequency of SCE in cells with or without chromosome breaks. The mitotic delay increased with the time between irradiation and harvesting. There was no relationship between the extent of mitotic delay and the number of SCEs.     

Modulation of streptonigrin's clastogenic effects in CHO cells by the metal-chelating agent 1,10-phenanthroline.

The effect of the metal-chelating agent 1,10-phenanthroline (PNT) on the clastogenesis induced by streptonigrin (SN) in CHO cells was investigated. When CHO cells were exposed to SN, chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner (P < 0.05). When PNT was present in the culture medium, the production of CAs by SN was strongly inhibited (inhibition range = 54.9-80.8%). Similarly, the induction of SCEs by SN was significantly decreased by the addition of PNT to CHO cultures (P < 0.05), although the effect was minor. This finding suggests that intracellular transition metals are implicated in the clastogenesis by SN, and that the Fenton reaction (Fe(2+) + H2O2 --> OH* + OH(-) + Fe(3+)) may be responsible for the production of CAs by this compound. Moreover, the fact that PNT did not completely inhibit the induction of SCEs by SN suggests that this phenomenon might be attributable to a different mechanism, in which transition metals and free radicals play a minor role.     

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.     

Differential induction of sister chromatid exchanges by indirect-acting mutagen-carcinogens at early and late stages of embryonic development

To examine the development of drug-metabolizing enzyme systems in the early chick embryo, the procarcinogens, aflatoxin B₁ (AF-B₁) and 2-acetylamino-fluorene (2-AAF), and the direct-acting carcinogen, ethyl methanesulfonate (EMS) (positive control), were given to embryos; the sister-chromatid-exchange (SCE) technique was used as an indicator of conversion to active mutagenic metabolities. Chick embryos at two stages of incubation (3-day and 6-day) were exposed to the same graded series of dosages of the compounds for a period of 22 hours. All three mutagens increased the frequency of SCE above the control rate of 1.8 SCEs/cell. While a dose-dependent increase in SCE was obtained for both procarcinogens at each age, the mean SCE frequency was significantly higher in the 6-day embryos for each dosage given. In contrast, the direct-acting mutagen, EMS, gave a reduced level of SCEs at the older age. These results suggest that the ability of early chick embryos to activate promutagens to forms capable of inducing SCE increases as development advances from three to six days of incubation (DI). In the 6-day embryo, the metabolic conversion is enhanced, resulting in a significant increase in the mutagenicity of the test chemicals.     

The key radiologic and cytomorphologic features of oncocytic and oncocytoid lesions of the salivary gland

Oncocytic and oncocytoid lesions represent a distinct subset of salivary gland lesions. True oncocytic lesions of the salivary gland are entirely composed of oncocytes. These are characterized by the presence of abundant eosinophilic granules due to the presence of abundant cytoplasmic mitochondria. Oncocytic lesions of the salivary gland include oncocytosis, oncocytoma, and oncocytic carcinoma. In addition to the true oncocytic lesion, there exists another group of salivary gland lesions, which demonstrate cells with abundant and occasionally granular cytoplasm. These are often termed as “oncocytoid” lesions. The recently proposed Milan System for reporting salivary gland cytology clearly states that fine‐needle aspiration specimens representing oncocytic/oncocytoid lesions of salivary gland cannot effectively distinguish between a nonneoplastic lesion, benign and malignant neoplasms. Therefore, most lesions lacking classic cytomorphologic features will be classified under the umbrella diagnostic term of “Salivary Gland Neoplasm of Uncertain Malignant Potential” (SUMP). In this review, we discuss and illustrate key clinicopathologic and radiologic features that can help the practicing cytopathologist narrow down the differential and provide the best management based diagnosis.