Micronucleus induction by metronidazole in rat vaginal mucosa

Metronidazole (MTZ) is used for the treatment of many infectious diseases, including vaginal infections. While data indicate that MTZ is mutagenic and induces micronuclei in rodents, there is no information on the genotoxicity of MTZ in epithelial vaginal cells or cervical cells. In the present study, we have instilled MTZ into the vagina of rats and evaluated the micronucleus (MN) frequency in proestrus rat vaginal mucosal cells. The first identified proestrus before treatment was used to establish basal proestrus micronucleated cell (PMNC) frequencies. Rats then were assigned to one of five groups: a negative control, three MTZ treatment groups (30, 50, or 100 mg/kg MTZ), and a positive control treated with 2.5 mg of 5-fluorouracil (5-Fu) per rat. Following treatment for five consecutive days, vaginal cell samples were taken daily until three cycles of estrus were completed. Smears prepared from the samples were evaluated for micronuclei in proestrus cells. No differences were found between the PMNC frequencies of the negative control and the 30 and 50 mg/kg MTZ groups. The group treated with 100 mg/kg MTZ, however, had significantly elevated PMNC frequencies in the first and second proestrus samples, while 5-Fu treatment produced significant increases in PMNC frequency in the second and third proestrus. These results indicate that topical administration of relatively high concentrations of MTZ is genotoxic in rat vaginal mucosa cells.     

Induction of micronucleated cells in the shed skin of salamanders (Ambystoma sp.) treated with colchicine or cyclophosphamide

The micronucleus (MN) assay can be used to detect the genotoxic effects of chemical agents in virtually any cell that divides frequently. Salamanders (Ambystoma sp.) are amphibians that can be easily maintained and bred in the laboratory and spontaneously shed their skin every 2.5-4 days. In this present study, we have evaluated the usefulness of this shed skin for the MN assay. We exposed salamanders to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNCs) in their sheds. Fragments of shed skin were placed on clean slides, fixed, stained, observed with a light microscope, and the number of MNCs was counted. The MNC frequency was increased significantly by all doses of COL and CP tested, administered either as single or repeated exposures. The presence of MNCs in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, are suitable alternative models for detecting genotoxic exposures relevant to aquatic environments.     

Spontaneous and mitomycin C-induced micronuclei in peripheral blood reticulocytes from severely malnourished rats

Severe malnutrition caused by deficiencies in protein, calorie, and micronutrient intake is widely distributed throughout the world and is a particular problem in developing countries. Animal models have been useful for studying the effects of malnutrition under different experimental conditions. In this study, we have evaluated the effect of malnutrition on the frequency of spontaneous and mitomycin C (MMC)-induced micronuclei in the peripheral blood of rats measured using a flow cytometric analysis technique. Neonatal rats were experimentally malnourished during lactation and assayed at weaning (21 days of age). The malnourished rats weighed 49.2% less than well-nourished controls and had lower concentrations of serum protein, triglycerides, and cholesterol. In rats not treated with MMC, the frequency of micronucleated reticulocytes (MN-RETs) was 1.6 times greater in malnourished rats than in well-nourished rats (0.48% +/- 0.16% vs. 0.31% +/- 0.09%). The mean MN-RET frequency measured 32 hr after treatment with single i.p. doses of 0.5, 0.75, or 1.0 mg/kg of MMC was 0.60 +/- 0.10 vs. 0.84 +/- 0.14, 1.21 +/- 0.52 vs. 2.36 +/- 0.47, and 2.50 +/- 0.06 vs. 4.64 +/- 1.14 for well-nourished vs. malnourished rats, respectively. Statistical comparisons indicate significant differences between the two groups of rats at all doses tested. Malnourishment and MMC treatment had no significant effects on the frequencies of RETs or micronucleated normochromatic erythrocytes. The data indicate that protein-calorie malnutrition during lactation is associated with increased frequencies of MN-RETs, which are indicative of chromosome damage. These findings suggest that malnutrition could result in greater susceptibility to environmental damage.     

Evaluation of the immunosuppression in an experimental model of autoimmunity: suppressor activity of spleen cells from cyclophosphamide-treated rats

Rats immunized with chemically modified rat male accessory glands (MRAG) and injected 3 days later with cyclophosphamide (CY) were unable to develop humoral and cellular immune response to the autoantigen of MRAG. The present report demonstrates that the spleen mononuclear (SpM) cells transference from rats injected with CY 3 days after the antigen to normal male or female syngeneic animals before immunization with MRAG did not suppress the immune response to this antigen, whereas the transference of SpM cells from suppressed animals to animals previously immunized, depressed the delayed type hypersensitivity (DTH) response against MRAG (suppression of the expression) only in male rats. Similar results were obtained by transference of purified T cells. SpM cells did not suppress an established humoral immune response induced in male or female rats. The results suggest that non-adherent cells present in the spleen of male suppressed rats might be one of the responsible mechanisms for suppression of the efferent phase of the cellular autoimmune response to MRAG.     

Effects of vinblastine and colchicine on male rat meiosis in vivo: Disturbances in spindle dynamics causing micronuclei and metaphase arrest

We have studied the effects of vinblastine sulfate (VBL) and colchicine (COL) on male rat in vivo and in vitro meiosis. A novel methodology based on isolating a segment of seminiferous tubules containing meiotically dividing spermatocytes was applied. During meiotic divisions at stage XIV of rat spermatogenesis, both chemicals induced only low frequencies of micronu-clei (MN), 0.8–3.2 MN/1,000 spermatids. Fluorescence in situ hybridization experiments in mice with the mouse centromere-specific y-sat-ellite DNA probe showed that 50.7% of VBL-induced MN and 56.6% of COL-induced MN were centromere positive, indicating that the MN induced by both chemicals contained detached chromosomes. The inhibition of cell proliferation was determined by counting the number of cells arrested at metaphase during the first meiotic (MI) or the second meiotic (MII) division. VBL was found to be a potent inducer of cell death while COL was not. The direct effects of VBL and COL on the meiotic spindles were evaluated using immunohistochemistry with anti-a-tubulin and confocal microscopy. In the control animals a significant difference was observed between the mean length of metaphase spindles of MI and MII Both were dramatically decreased 6 hr after treatment with 2.0 mg/kg of VBL and 0.8 mg/kg of COL, respectively. At 18 hr after COL injection the spindles had about the same length as in the controls. However, the VBL-induced shortening was even more evident at 18 hr for both Ml and Mll. The possible reasons for observed differences between the two chemicals and between meiosis and mitosis are discussed. © 1995 Wiley-Liss, Inc.     

Effect of 5-azacytidine on frequency of polycentric chromosomes induced in cells with micronuclei by 5-bromodeoxyuridine

Tissue Culture Laboratory, Institute of Poliomyelitis and Virus Encephalitis, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. G. Drozdov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 4, pp. 479–480, April, 1988.     

Bleomycin sulfate-induced micronuclei in human,rat, and mouse peripheral blood lymphocytes

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 μg/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37°C in a 5% CO₂ atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species concentration-response curves gave good fits (r² values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]^0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations ≧ 20 μg/ml. The nucleation index was significantly decreased in all species at either 80 or 160 μg/ml. © 1995 Wiley-Liss, Inc.     

兔肾小管基底膜所致Wistar大鼠肾小管间质性肾炎模型的建立及环磷酰胺对其防治作用

选用兔肾小管基底膜（ＴＢＭ）、完全福氏佐剂及百白破疫苗免疫雌性Ｗｉｓｔａｒ大鼠，成功地建立了肾小管间质性肾炎（ＴＩＮ）模型。实验证明在ＴＩＮ发生时，ＴＢＭ上有ＩｇＧ和Ｃ３沉积，肾间质大量单个核细胞浸润，主要是淋巴细胞，部分有肉芽肿形成。随后出现肾小管萎缩和肾间质纤维化。用环磷酰胺可以抑制ＴＩＮ的发生和进展，其作用机制可能是抑制细胞免疫反应     

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.     

Effect of annatto on micronuclei induction by direct and indirect mutagens in HepG2 cells

Annatto (AN), a natural food colorant rich in carotenoids, has been reported as being an effective antioxidant, but little is known about its potential chemopreventive properties. In this study, we evaluated the ability of AN to protect human hepatoma cells (HepG2) from micronucleus (MN) induction against three different mutagens: benzo(a)pyrene (B(a)P), doxorubicin (DXR), and methyl methanesulfonate (MMS). In an attempt to clarify the possible mechanism of antimutagenicity of AN, three protocols of treatment were applied (pretreatment; simultaneous treatment, and post‐treatment with AN following treatment with the mutagens). Also, cells exposed only to AN were assayed for cytotoxicity and mutagenicity. A dosage up to 10 μg/ml of AN was devoid of mutagenic activity. Protective effects were seen on micronuclei induced by B(a)P and DXR using pre and simultaneous treatment, but AN had no significant effect on MN induction by MMS in any of the protocols. Our results also show that exposure of cells to concentrations of AN higher than 10 μg/ml decreased cell viability. Taken together, our findings indicate that AN presents antimutagenic activity in vitro, but its protective effect is dependent on the mutagen and on type of treatment suggesting its potential use as a chemopreventive agent. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

The spread and persistence of influenza viruses in normal and cyclophosphamide-treated mice

The persistence and extrapulmonary spread of three strains of influenza virus, the mouse neuro-adapted A/NWS virus, the wild-type strain A/Victoria/75, and a recombinant virus RIT4050, bearing surface antigens derived from A/ Victoria/75, were studied in both normal and cyclophosphamide-treated CBA mice following either intranasal or intracerebral inoculation. All three viruses showed increased lethality in mice in the presence of cyclophosphamide but exhibited distinctive patterns of replication and spread. The recombinant virus RIT4050 showed a reduced ability to replicate, persist, and spread in CBA mice compared to either A/NWS or A/Victoria/7 5 viruses, and in general, the A/NWS virus persisted to a greater extent than the A/Victoria/75 virus in both normal and treated mice. However, in the presence of cyclophosphamide, no extrapulmonary spread of A/NWS virus was observed. The reasons for the differences are discussed.     

雌激素对大鼠阴道平滑肌细胞RhoA/Rho激酶表达的影响

目的 研究去势及雌激素替代对大鼠阴道平滑肌细胞RhoA/Rho激酶表达的影响.方法 30只10周龄雌性SD大鼠随机均分成:对照4和6周组(C4,C6)、去势4和6周组(OE4,OE6)和去势+雌二醇(E2)干预4和6周组(E4,E6).E4和E6组分别于去势后每周皮下注射E2(250 μg/kg)4和6周,OE4和OE6组注射等量0.9％氯化钠注射液,4和6周时测定血清E2水平及用免疫组化和Western blot检测阴道平滑肌细胞Rock1、Rock2的表达.结果 OF4和OE6组血清E2水平显著低于C4和C6(P＜0.01),OF4和OE6组阴道平滑肌细胞Rock1表达显著高于C4和C6(0.261 ±0.039 vs 0.207 ±0.032;0.270 ±0.037 vs 0.208 ±0.032,P＜0.05),OE4和OE6组阴道平滑肌细胞Rock2表达显著高于C4和C6(0.377 ±0.035 vs 0.294 ±0.033;0.388 ±0.038 vs 0.300 ±0.034,P＜0.05).结论 低雌激素水平上调RhoA/Rho激酶在大鼠阴道平滑肌中的表达,E2替代治疗可抑制RhoA/Rho激酶在去势大鼠阴道平滑肌中的高表达,雌激素可能通过调控RhoA/Rho激酶表达影响阴道平滑肌功能.     

Micronucleus frequency in spleen lymphocytes from severely malnourished rats during lactation

The purpose of this ex vivo study was to determine if severe malnutrition increases the frequency of micronuclei in spleen lymphocytes of experimentally malnourished rats during lactation. Micronucleus frequencies were analyzed in binucleate cells produced by the cytokinesis block method. The overall micronucleus frequency was significantly higher in binucleate cells from malnourished rats (21.3%‰) as compared to that observed in control rats (11.5%‰). The number of binucleate cells with more than one micronucleus was also higher in malnourished rats than in controls (3.1%‰ vs. 1.2%‰). These results indicate that severe malnutrition produces cellular damage in vivo, as was evidenced by the increased micronucleus frequency in rat spleen lymphocytes in vitro. This damage may produce negative effects for the further development of the organism, since the spleen is an important lymphopoietic organ in rodents. © 1995 Wiley-Liss, Inc.     

Induction of micronuclei and sister chromatid exchanges by polycyclic and N-heterocyclic aromatic hydrocarbons in cultured human lymphocytes

Many natural environments are contaminated with carcinogenic polycyclic aromatic hydrocarbons (PAHs) and N-heterocyclic aromatic hydrocarbons (NHAs) as complex mixtures of coal tar, petroleum, and shale oil. These potentially hazardous substances are prevalent at many former tar production and coal gasification sites. Three polycyclic [benzo-(a)pyrene (BaP), benz(a)anthracene (BAA), and 7, 12-dimethylbenz(a)anthracene (DMBA)] and two N-heterocyclic [7H-dibenzo(c, g)carbazole (DBC), and dibenz(a, j)acridine (DBA)] aromatic hydrocarbons were analyzed for cytotoxic and genotoxic effects on human lymphocytes. All of these polyaromatic compounds are normally present in the environment, except for DMBA. Lymphocytes from healthy donors were isolated from whole blood. The 5-ring polycyclic aromatic BaP consistently induced micronuclei in a linear dose-dependent manner with doses from 0.1–10.0 ug/ml, whereas the 4-ring compounds (BAA and DMBA) had no effect on the induction of micronuclei above controls except at 5 and 10 ug/ml. Of the two N-heterocyclic compounds, DBC produced a significant increase in micronuclei in lymphocytes, but the dose response tended to plateau above 0.1 ug/ml. DBA showed an effect on the frequency of micronuclei above controls only at high doses of 5 and 10 ug/ml. The average background frequency of micronuclei for 7 lymphocyte donors averaged 3.1 per 1, 000 stimulated cells, whereas the average frequency of micronuclei at 10 ug/ml BaP was 36.8 per 1,000 stimulated cells. The lowest effective dose in 2 donors for BaP occurred at 0.1 ug/ml. At a challenge dose of 1 ug/ml (4 uM) of BaP, considerable variation in micronuclei induction between 7 individuals was observed, ranging from 2–6-fold increases above spontaneous frequency. Over a dose range of 1–10.0 ug/ml (4–40 uM), BaP also induced sister chromatid exchanges (SCEs) in lymphocytes, whereas BAA had no effect above controls. Parallel studies of both cytogenetic endpoints showed that the micronucleus assay is a more sensitive indicator of BaP exposure at equivalent doses. Mitotic and replication indices of BaP-exposed lymphocytes showed that cell proliferation is only moderately inhibited even at the highest dose; this shows that bulky DNA-adducts are generally compatible with cell survival. The cytogenetic data are consistent, firstoff, with reports that individuals in the population vary widely with respect to the inducibility of the CYP1A1 gene, which is known to be involved in polycyclic aromatic hydrocarbon metabolism, in particular, in BaP. Secondly, the data support the fact that polyaromatic compounds differ with regard to micronucleus induction within the same sample(s) of human lymphocytes, indicating selective metabolism of polyaromatic compounds that may reflect carcinogen sensitivity of the individual. Thirdly, it would appear that the micronucleus induction in human lymphocytes by PAHs is an overall-sensitive endpoint for measuring PAH exposure. Lastly, this is the first report of the use of the micronucleus assay to assess a series of PAHs and NHAs for their ability to induce genetic damage in human lymphocytes. © 1995 Wiley-Liss, Inc.