Evaluation of a new procedure for the flow cytometric analysis of in vitro,chemically induced micronuclei in V79 cells

Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical treatment is widely used to analyze cytogenetic damage. The microscopic scoring of micronuclei is a tedious and time-consuming procedure. Therefore, attempts have been made to automate micronuclei scoring by means of image analysis or flow cytometry. A new procedure for the flow cytometric analysis of chemically induced micronuclei in V79 Chinese hamster cells has been established in our laboratory. Debris was separated from micronuclei by means of a new gating procedure using area and width fluorescence of the stained suspension of micronuclei and nuclei. In order to test the sensitivity and specificity of this improved method of flow cytometric analysis, five well-known mutagenic compounds were tested. With the new technique, the frequency of micronuclei measured and analyzed corresponded well with results obtained by conventional microscopy. In addition, a large series of negative compounds, and weak, middle, and strong micronuclei inducers, were tested in order to establish criteria for discrimination between genotoxic and nongenotoxic compounds by flow cytometry. This new procedure for flow cytometric detection of micronuclei represents a quick, reliable, and relatively simple method for in vitro micronucleus testing. Environ. Mol. Mutagen. 32: 387–396, 1998 © 1998 Wiley-Liss, Inc.     

Cytogenetic biomonitoring in traffic police workers: Micronucleus test in peripheral blood lymphocytes

Atmospheric pollution represents a relevant environmental hazard which has been associated with considerable excess mortality, morbidity, and increased rates of respiratory diseases in humans. To date, more than 3,000 environmental chemical compounds have been identified in the ambient atmosphere, including a variety of mutagenic and/or carcinogenic agents, such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and heterocyclic compounds. Positive associations between cytogenetic markers and airborne levels of PAHs have been reported by experimental and human studies. Traffic has been implicated as the major determinant for the concentration of PAHs and, therefore, for the genotoxic activity of urban air. A biomonitoring study has been conducted in 82 Italian traffic police workers exposed to air pollutants and 34 control subjects (matched by age, gender, and smoking habits) not exposed to traffic pollutants. The aim of this study was to assess the cytogenetic effects, such as micronucleus frequency in peripheral blood lymphocytes, and to estimate the association with individual exposure to PAH. Statistical analysis of the frequency of micronuclei in binucleated cells showed higher mean levels in referent subjects (4.03%) than in traffic police officers (3.73%). Smoking showed no effect on the frequency of micronuclei. The study failed to detect any association between micronucleus frequency and individual level of benzo(a)pyrene, considered a marker of exposure to PAHs. These findings indicate that exposure to urban air pollutants does not result in increased levels of micronuclei in peripheral white blood cells. Environ. Mol. Mutagen. 30:396–402, 1997 © 1997 Wiley-Liss, Inc.     

Genotoxic and antigenotoxic effects of Hemidesmus indicus R. Br. root extract in cultured lymphocytes

Aim of the study

The genotoxic and antigenotoxic potential of the ethanolic extract of Hemidesmus indicus roots were evaluated in cultured human lymphocytes using cisplatin as the positive mutagen.

Materials and methods

Cytogenetic damage and cytotoxicity were determined in cells exposed to different doses of the extract, ranging from 2 to 32 μg/ml of culture medium, either alone or together with cisplatin.

Results

There was a significant reduction in cisplatin-induced frequencies of sister chromatid exchanges, chromosome aberrations and micronucleated binucleate cells at the lower concentrations of 4 and 8 μg/ml (P < 0.05). However, the extract by itself reduced the proliferative rate index, mitotic index and cytokinesis-block proliferative index (P < 0.05). Further, a significant increase in the percentage of chromosome aberrations was noticed at the higher concentrations.

Conclusion

Hemidesmus indicus root extract possesses significant genoprotective effect at the lower concentrations although it is cytotoxic and probably genotoxic at higher doses.     

养精种玉汤对KM小鼠骨髓细胞微核率的影响

目的：探讨养精种玉汤对KM小鼠骨髓细胞微核率的影响,对其遗传安全性进行研究。方法：采用不同时间点、不同剂量对KM小鼠进行体内骨髓细胞染毒,观察其致小鼠骨髓细胞染色体完整性受损及染色体分离异常等情况。结果：采用不同时间点取样,其骨髓细胞微核率与溶剂对照相比无明显增加（P〉0．05）,说明在时效关系上,不同时间点给药对小鼠骨髓细胞微核率无明显影响。采用不同剂量进一步染毒,其骨髓细胞微核率分别与正常对照相比也无明显增加（P〉0．05）,说明在量效关系上,无明显诱发小鼠骨髓细胞微核率增加作用。正常对照组和高剂量组嗜多染红细胞与成熟红细胞比值未见明显降低,表明该药对小鼠骨髓细胞也无明显毒性。结论：养精种玉汤药液100g／kg的剂量范围以内,初步认为无明显致小鼠骨髓细胞染色体完整性受损及染色体分离异常。     

秦皮水煎剂的遗传毒性研究

目的:研究秦皮水煎剂的遗传毒性.方法:采用小鼠淋巴瘤细胞试验(MLA)和小鼠骨髓微核试验(MNT).MLA试验中,秦皮水煎剂(含生药)1.71,3.42,6.83,13.65 g·L~(-1) 4个质量浓度组,超纯水阴性对照组及甲基甲烷磺酸酯、环磷酰胺阳性对照组,分别在非代谢活化(-S9)和代谢活化(+S9)条件下与L5178Y细胞作用3 h,表达2 d,制备基因突变频率平板并培养12～13 d,计数含大、小突变细胞集落的孔数,计算每组总突变率和小集落突变百分率;MNT试验中,3个剂量组(含生药)7.14,14.28,28.55 g·kg~(-1),氯化钠注射液阴性对照组和环磷酰胺阳性对照组,每组10只ICR小鼠,雌雄各半,间隔24 h实施2次灌胃给药,制作骨髓涂片,镜检每只小鼠2 000个嗜多染红细胞中含微核的嗜多染红细胞数,计算每组动物嗜多染红细胞微核率.结果:MLA试验,-S9条件下各浓度组诱发的总突变率呈现浓度依赖性增加,13.65,6.83 g·L~(-1)组与阴性对照组比较有统计学意义(P<0.01),小集落突变百分率随浓度增加而升高,+S9条件下的各浓度组总突变率和小集落突变百分率均与阴性对照组相近;MNT试验,各剂量组无明显骨髓抑制作用,诱发的小鼠骨髓嗜多染红细胞微核率与阴性对照组比较末见明显增加.结论:秦皮水煎剂在体外非代谢活化条件下可诱发L5178Y细胞tk~(+/-)位点突变并导致染色体损伤,提示其可能存在直接诱变物;秦皮水煎剂对小鼠骨髓细胞染色体无明显损伤作用,经体内、外代谢活化后均未显示遗传毒作用.     

1,2-二氯乙烷致小鼠血淋巴细胞遗传毒性研究

目的研究1,2-二氯乙烷(1,2-DCE)对小鼠血淋巴细胞DNA和骨髓细胞染色体的损伤作用,探讨1,2-二氯乙烷的遗传毒性。方法采用单细胞凝胶电泳和微核试验方法,分别检测1,2-DCE不同染毒剂量(50,100,200,400 mg/kg)小鼠外周血淋巴细胞DNA和骨髓细胞染色体损伤情况。结果除50 mg/kg剂量组外,小鼠血淋巴细胞的彗星细胞率及尾长、骨髓细胞微核率随1,2-DCE染毒剂量的增加而增加(P<0.01)。其中,400 mg/kg剂量组彗星细胞率、平均尾长、微核率分别为45.5%,(37.24±3.17)μm,12.0‰,显著高于阴性对照组和50 mg/kg剂量组(P<0.01)。染毒剂量与彗星细胞率、平均尾长、微核率之间存在着剂量-反应关系(R彗星率=0.980 2,R彗尾长=0.976 6,R微核率=0.975 1,P<0.01)。结论1,2-DCE可导致小鼠血淋巴细胞DNA损伤和骨髓细胞染色体异常。表明1,2-DCE具有细胞遗传毒性。     

Assessment of the Pig-a,micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate

The utility and sensitivity of the newly developed flow cytometric Pig-a gene mutation assay have become a great concern recently. In this study, we have examined the feasibility of integrating the Pig-a assay as well as micronucleus and Comet endpoints into acute and subchronic general toxicology studies. Male Sprague–Dawley rats were treated for 3 or 28 consecutive days by oral gavage with procarbazine hydrochloride (PCZ) or ethyl carbamate (EC) up to the maximum tolerated dose. The induction of CD59-negative reticulocytes and erythrocytes, micronucleated reticulocytes in peripheral blood, micronucleated polychromatic erythrocytes in bone marrow, and Comet responses in peripheral blood, liver, kidney, and lung were evaluated at one, two, or more timepoints. Both PCZ and EC produced positive responses at most analyzed timepoints in all tissue types, both with the 3-day and 28-day treatment regimens. Furthermore, comparison of the magnitude of the genotoxicity responses indicated that the micronucleus and Comet endpoints generally produced greater responses with the higher dose, short-term treatments in the 3-day study, while the Pig-a assay responded better to the cumulative effects of the lower dose, but repeated subchronic dosing in the 28-day study. Collectively, these results indicate that integration of several in vivo genotoxicity endpoints into a single routine toxicology study is feasible and that the Pig-a assay may be particularly suitable for integration into subchronic dose studies based on its ability to accumulate the mutations that result from repeated treatments. This characteristic may be especially important for assaying lower doses of relatively weak genotoxicants. Environ. Mol. Mutagen. 60:56–71, 2019. © 2018 Wiley Periodicals, Inc.     

Assessment of Pig-a,Micronucleus, and Comet Assay Endpoints in Tg.RasH2 Mice Carcinogenicity Study of Aristolochic Acid I

A newly developed in vivo Pig-a gene mutation assay displays great potential for integration into genotoxicity tests. To obtain more evidence for application of the Pig-a assay, we integrated this assay, micronucleus test in peripheral blood (MN-pb test) and bone marrow (MN-bm test), as well as a Comet assay into a transgenic RasH2 mice carcinogenicity study. Fourteen male RasH2 mice and five wild-type (WT) mice were treated with a strong mutagen aristolochic acid I at a dose of 5 mg/kg/day for 4 consecutive weeks. Mice recovered in 5 weeks. Peripheral bloods were collected for Pig-a assay, MN-pb test, and Comet assay at several time points, while bone marrow and target organs were harvested for the MN-bm test and pathological diagnosis after mice were euthanized. Finally, 13 of the 14 RasH2 mice developed squamous cell carcinomas in the forestomach, while there were no carcinomas in the WT mice. Pig-a mutant frequencies (MFs) consecutively increased throughout the study to a maximum value of approximately 63-fold more than background. These frequencies were relative to the incidence, size, and malignant degree of tumors. Micronucleated reticulocytes increased from Day 1 to Day 49, before returning to background levels. No positive responses were observed in either the MN-bm test or the Comet assay. Results suggested that, when compared with the other two tests, the Pig-a assay persistently contributed to sustaining MFs, enhanced detection sensitivity due to the accumulation of Pig-a mutations, and demonstrated better predictability for tumorigenicity. Environ. Mol. Mutagen. 61:266–275, 2020. © 2019 Wiley Periodicals, Inc.