The effects of 4-thujanol on chromosome aberrations,sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

4-Thujanol, a bicyclic monoterpene alcohol, is present in the essential oils of many medicinal and aromatic plants. It is commonly used as a fragrance and flavouring ingredient in a lot of different products. The potential genotoxic effects of 4-thujanol on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests. The cells were treated with 13, 26 and 52 μg/mL 4-thujanol in the presence and absence of a metabolic activator (S9 mix). 4-Thujanol induced CA (P < 0.001) and MN formation (P < 0.05) at all concentrations (13, 26 and 52 μg/mL) in the presence and absence of the S9 mix without a concentration-dependent manner. However, the treatment of peripheral lymphocytes with 4-thujanol did not produce a statistical difference in the frequency of SCEs when compared with control group. Furthermore, this monoterpene did not significantly decrease the mitotic index (MI), proliferation index (PI), and nuclear division index (NDI). In conclusion, 4-thujanol had a significant clastogenic effect at the tested concentrations (13, 26 and 52 μg/mL) for human PBLs. In addition, no cytotoxic and/or cytostatic effects were observed regardless of the concentrations used. This work presents the first report on genotoxic properties of 4-thujanol.     

Chromosome aberrations and sister chromatid exchanges in cultured human lymphocytes treated with sodium metabisulfite, a food preservative

The aim of this study was to investigate the ability of sodium metabisulfite (SMB) which is used as an antimicrobial substance in food, to induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in human lymphocytes. SMB-induced CAs and SCEs at all concentrations (75, 150 and 300 μg/ml) and treatment periods (24 and 48 h) dose-dependently. However, SMB decreased the replication index (RI) and the mitotic index (MI) at the concentrations of 150 and 300 μg/ml for 24 and 48 h treatment periods. This decrease was dose-dependent as well.     

The genotoxic and anti-genotoxic effects of <Emphasis Type="Italic">Stachys petrokosmos</Emphasis> leaf extract in human lymphocytes using microsomal fractions

The genotoxic and anti-genotoxic effects of Stachys petrokosmos leaf extracts (Sp) were investigated in human lymphocytes. The cells were treated with 1.5, 3.0 and 6.0 μL/mL concentrations of Sp leaf extracts for 24 and 48 h treatment periods in the absence and presence of metabolic activator (S9mix). In the absence of S9mix, Sp alone did not induce chromosome aberrations and formation of micronucleus while inducing the mean sister chromatid exchange at the highest concentration. In addition, Sp decreased the mutagenic effect of mitomycin-c. Sp alone showed a cytotoxic effect determined by a decrease in the proliferation index, mitotic index and nuclear division index. On the other hand a mixture of Sp and mitomycin-c resulted in a higher cytotoxic effect especially for 48 h treatment period. In the presence of S9mix, Sp was not genotoxic and cytotoxic however, it showed an anti-genotoxic effect by decreasing the effects of cyclophosphamide.     

Clastogenic effects of food additive citric acid in human peripheral lymphocytes

Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant.     

Effects of copaene,a tricyclic sesquiterpene,on human lymphocytes cells in vitro

In this study, the cytotoxic, genotoxic/antigenotoxic and antioxidant/oxidant activity of copaene (COP), a plant-derived tricyclic sesquiterpene, on human lymphocyte cultures (n = 5) was investigated. COP was added into culture tubes at various concentrations (0, 10, 25, 50, 100, 200 and 400 mg/L). While the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used for viability and cytotoxic evaluations, the micronucleus (MN) and sister chromatid exchange (SCE) assays were used for genetic evaluations. Moreover, total antioxidant capacity (TAC) and total oxidative status analysis were used for biochemical evaluations. According to LDH and MTT assays COP significantly reduced cell proliferation at high concentrations (200 and 400 mg/L). In addition, there was no significant increase (P < 0.05) in both SCE and MN frequencies of cultures treated with COP as compared to controls. We have also concluded that concentrations of COP of 50 and 100 mg/L increased TAC level when compared to the controls. In conclusion, in this study it has been reported for the first time that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures.     

Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro

N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes. To evaluate its genotoxicity, we examined whether it can induce sister chromatid exchanges (SCEs) in human lymphocytes. We found that N,N-diethylaniline significantly increased the frequency of SCEs both in the absence and presence of S-9 mix. The SCEs from cultures treated by N,N-diethylaniline in the presence of S-9 mix displayed a marked increase which was about 5-fold greater than the control. ANOVA analyses indicated that there is a dose–response relationship between doses of N,N-diethylaniline and the frequency of SCEs, especially in the presence of S-9 mix. The results suggested that N,N-diethylaniline has genotoxicity.     

Increased frequency of sister chromatid exchanges in peripheral lymphocytes of alcoholics and cigarette smokers

Sister chromatid exchange (SCE) is a sensitive indicator of genotoxicity. In this study we investigated the effects of alcohol consumption and cigarette smoking on the frequency of SCE in cultures of peripheral lymphocytes. The rate was higher in alcoholics who smoked (10.89+/-2.46) and in smokers (positive controls) (7.64+/-1.01) than in healthy non-smokers (negative controls) (6.96+/-2.18). Statistical analysis suggested that the increases were related to alcohol consumption and cigarette smoking (p<0.05).     

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

Fenvalerate, a synthetic pyrethroid insecticide, is commonly used in agriculture and other domestic applications due to its high insecticidal activity and low mammalian-, avian- and phyto-toxicities. However, the genotoxic effect of fenvalerate is highly equivocal. In the present study the genotoxic effects of fenvalerate was evaluated using structural chromosome aberration (CA) and sister chromatid exchange (SCE) assays in mice. Out of the three doses (5, 10 and 20 mg/kg) tested, statistically significant increase in CA was found following intra peritoneal (i.p.) treatment of 20 mg/kg of fenvalerate for 24 h (P<0.01) and 48 h (P<0.05) only. Neither the acute doses of 5 and 10 mg/kg, nor the sub-acute dose (5×4 mg/kg) of fenvalerate could induce any significant effect. All the three acute doses induced significant increase in the frequency of SCEs (P<0.01) in the bone marrow cells, which showed a significant dose-response correlation (r=0.9541, P<0.05). With certain reservations to possible impurities, from the present findings technical grade fenvalerate may be considered as a weak clastogen and a potent inducer of SCEs in mice.     

Correlation between the adaptive response and individual sensitivity to monoepoxybutene in in vitro experiments on human lymphocytes

Individual variations in the susceptibility to mutagenic/carcinogenic chemicals depend on the activity of xenobiotic metabolizing enzymes and on DNA- and chromosome-damage repair systems. Monoepoxybutene (MEB) is a genotoxic metabolite of 1,3-butadiene (BD), which has been classified as a probable carcinogen in humans. The purpose of the present study was to investigate by in vitro experiments on human whole blood lymphocytes (WBL), whether an individual sensitivity to MEB correlates with the adaptive response to the tested agent. In the analyzed group, 8.3% of blood donors were relatively sensitive to MEB. The comparison of SCE induction in cultures pretreated and not pretreated with an adaptive dose (AD) of MEB showed, that there was an adaptive response to MEB. The adaptive response in the group of relatively sensitive donors was similar to that of the relatively resistant ones. This result suggests that individual sensitivity to the tested agent and adaptive response depend on different biological mechanisms.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

黄鳝染色体体内SCE检测系统的建立

应用IdU-毛玉米油体内SCE技术,以不同剂量的典型诱变剂MMC和CP对70尾黄鳝的脾、肾、血淋巴细胞进行了体内诱发SCE敏感性测试。结果:三种细胞的染色体SCE自发频率均较低,不同剂量MMC和CP诱发黄鳝三种细胞SCE频率均较对照组显著增加。诱变剂剂量与诱发SCE频率呈线性关系。三种细胞染色体SCE对MMC和CP的敏感性次序为肾>脾>血淋巴细胞。与几种鱼和其它动物比较,黄鳝三种细胞的SCE自发频率均较低,对MMC和CP诱发SCE的敏感性均较高,因此认为黄鳝可作为较理想的体内SCE检测系统。     

In vitro genotoxic effects of ZnO nanomaterials in human peripheral lymphocytes

In this study, possible genotoxic effects of zinc oxide (ZnO) nanoparticles were investigated in cultured human peripheral lymphocytes by using chromosome aberrations and micronucleus assays (MN). For this purpose, the cells were treated with ZnO (1, 2, 5, 10, 15 and 20 μg/mL) for 24 and 48 h. In this research, four types of chromosome aberrations were observed as chromatid and chromosome breaks, fragment and dicentric chromosomes. ZnO induced significant increase of the ratio of chromosomal aberrations as well as percentage of abnormal cells at concentrations of 1, 5, 10 and 20 μg/mL in 24 h treatments. In 48 h treatments, while ZnO nanomaterials induced significant increase of the percentage of abnormal cells only at a concentration of 10 μg/mL, and of chromosome aberration per cell in comparison to the control at concentrations of 5 and 10 μg/mL. On the other hand, this material significantly increased the micronuclei frequency (MN) at concentrations of 10 and 15 μg/mL in comparison to the control. Cytokinesis-block proliferation index was not affected by ZnO treatments. It also decreased the mitotic index in all concentrations at 24 h but not at 48 h. The present results indicate that ZnO nanoparticles are clastogenic, mutagenic and cytotoxic to human lymphocytes in vitro at specific concentrations and time periods.     

小麦愈伤组织细胞的姐妹染色单体交换

用BrdU标记,改良的FPG法染色,建立了一种植物愈伤组织细胞姐妹染色单体分染的方法。并对培养基的不同附加成分对SCE的影响进行了研究。所有培养基上愈伤组织细胞的SCE率都显著高于正常根尖分生组织细胞。6-BA,AgNO_3,高浓度的2,4-D,蔗糖均可诱发SCE。     

Influence of duration of exposure to styrene oxide on sister chromatid exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro

Styrene-7,8-oxide, an intermediate of styrene, is a known alkylating mutagen. The present study was carried out to investigate the influence of duration of exposure to styrene-7,8-oxide (styrene oxide) on induction of sister chromatid exchanges (SCEs) and inhibition of cell-cycle kinetics using cultured human blood lymphocytes in vitro. Phytohemagglutinin-stimulated whole-blood lymphocyte cultures obtained from heparinized whole blood from healthy donors were exposed to 100 μM styrene oxide for 22, 36, 48 and 72 h. A reduction of SCEs induction with increase in duration of exposure to styrene oxide was observed, i.e. a clear significant inverse relationship between exposure time and frequencies of SCEs induction due to styrene oxide was obtained. Styrene oxide induces significant elevations in unscheduled DNA synthesis DNA repair as well as S-phase synthesis in human blood lymphocytes in vitro, depending on the duration of exposure. The decrease in the induction of SCEs due to styrene oxide with increasing duration of its exposure may be principally due to an increased DNA repair and partly due to an increasing metabolic transformation to styrene glycol with increasing duration of its exposure as well as to some extent due to cell death at the maximum period of exposure, i.e. 72 h. Although the proliferations of lymphocytes exposed to 100 μM styrene oxide were significantly inhibited at different durations of exposure, no linear relationship between the replication index and the duration of exposure was noticed (r=0.47, p>0.05). Similarly, there was no relationship between replication index and SCE frequency (r=−0.36, p>0.05), suggesting that these two parameters may reflect two different endpoints for the cytogenotoxic effects of styrene oxide.     

Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (Mus spretus) exposed to cadmium, lead and zinc

As a consequence of human activities, large amounts of cadmium, lead and zinc are released in the environment, often simultaneously. The aim of this study was to investigate under experimental conditions the DNA damage induced in Algerian mice (Mus spretus) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) separately, or in selected combinations. Three cytogenetic end points were considered: the frequencies of micronucleated cells (MN) and sister chromatid exchange (SCE) in the bone marrow and the frequency of sperm abnormalities. Mice were treated by intraperitoneal (i.p.) injections with 5 or 10 doses of aqueous solutions of cadmium acetate, lead acetate and zinc acetate in concentrations corresponding to 1/10 of the LD50, respectively, 21.5, 0.46 and 1.5 mg/kg bw. The control groups were injected in the same way with distilled water.With only one exception (Cd + Zn group treated with 5 doses), the results show a significant increase of MN in all groups for both treatments (5 and 10 doses). Similarly, the results concerning the SCE revealed a statistically significant increase in all treated animals, with the exception of the Zn group treated with 5 doses. The number of sperm abnormalities was significantly higher in animals treated with 5 doses, except in the group Pb + Zn. In animals treated with 10 doses the number of sperm abnormalities was always statistically higher compared with controls.This study indicates that cadmium, lead and zinc can induce MN, SCEs and sperm abnormalities in Algerian mice and that the clastogenic potential is dependent on the time of exposure and the interaction between the three elements, confirming the environmental damage that may result from the simultaneous action of several metals. Most relevant is the toxic potential for Zn, related with the dose, which may compromise its protective effect against other metal contaminations, such as cadmium.     

Chromosomal aberrations and SCEs as biomarkers of cancer risk

Previous studies have suggested that the frequency of chromosomal aberrations (CAs), but not of sister chromatid exchanges (SCEs), predicts cancer risk. We have further examined this relationship in European cohorts comprising altogether almost 22,000 subjects, in the framework of a European collaborative project (CancerRiskBiomarkers). The present paper gives an overview of some of the results of the project, especially as regards CAs and SCEs. The results confirm that a high level of CAs is associated with an increased risk of cancer and indicate that this association does not depend on the time between CA analysis and cancer detection, i.e., is obviously not explained by undetected cancer. The present evidence indicates that both chromatid-type and chromosome-type CAs predict cancer, even though some data suggest that chromosome-type CAs may have a more pronounced predictive value than chromatid-type CAs. CA frequency appears to predict cancers at various sites, although there seems to be a particular association with gastrointestinal cancers. SCE frequency does not appear to have cancer predictive value, at least partly due to uncontrollable technical variation. A number of genetic polymorphisms of xenobiotic metabolism, DNA repair, and folate metabolism affect the level of CAs and might collectively contribute to the cancer predictivity of CAs. Other factors that may influence the association between CAs and cancer include, e.g., exposure to genotoxic carcinogens and internal generation of genotoxic species. Although the association between CA level and cancer is seen at the group level, an association probably also exists for the individual, although it is not known if an individual approach could be feasible. However, group level evidence should be enough to support the use of CA analysis as a tool in screening programs and prevention policies in occupational and environmental health.     

The mechanism of sister chromatid cohesion

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 μg/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Monitoring of occupational exposure to epichlorohydrin by genetic effects and hemoglobin adducts

The present work is focused on the determination of in vivo doses and studies of genetic effects in workers exposed to epichlorohydrin (ECH). The studied endpoints were hemoglobin (Hb) adducts, frequencies of hprt mutants, micronuclei in cytochalasin B blocked binucleated lymphocytes, sister chromatid exchanges (SCE) and high frequency cells (HFC). Blood samples were collected from office clerks and ECH exposed factory workers at an industrial plant in Germany. The workers were exposed to 0.11–0.23 ppm ECH in the air 45 h per week and to 0.2–2.6 ppm for 3 h per week. Some Swedish non-exposed subjects were also used for Hb adduct measurements. The genetic data, HFC and SCE, showed a significant difference between exposed and unexposed donors. In contrast to earlier studies on SCE, no impact of smoking was observed. Effects on micronuclei were on the borderline of significance, whereas there was no effect for HPRT mutants. The average Hb adduct level was higher in exposed than in non-exposed donors, although the difference was only significant when the exposed group was compared to Swedish controls. Smoking gave significantly increased adduct levels. The absence of significant correlations between individual data for Hb adducts and genetic effects, may be explained by the different periods of time covered by the responses in these endpoints. Whereas Hb adducts reflect the exposure during up to 4 months (i.e. the life span of human erythrocytes), the SCE, and particularly the HFC, seem to accumulate for years in a long-lived fraction of T-lymphocytes without DNA repair. Thus, the adduct data does not reflect the exposure backwards in time unless it can be shown that exposure conditions have remained unchanged. The origin of the background adduct levels in non-smoking control persons is at present not known.     

Protective effects of cimetidine on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes

The radioprotective effects of cimetidine, which has been used clinically as an antagonist of H 2 receptor, on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes (PBL) prepared from healthy donors were studied. Cells were treated with cimetidine before or after X-irradiation, and then cytokinesis-blocked micronucleus assay and flow cytometry for measurement of phosphatidylserine externalization were utilized to evaluate the radiation-induced cytogenetic damage and apoptosis. The protective effect of pre-irradiation treatment of cimetidine on radiation-induced micronuclei was dependent on the concentration. The maximum protection rates of cimetidine (1 mM) on frequencies of micronuclei were 38.8 and 30.2% for cells treated before and after X-irradiation (5 Gy), respectively. Protective effects of pre- and post-irradiation treatment with cimetidine on radiation-induced early apoptosis and decreased activity of caspase-3 were observed. A study of electron paramagnetic resonance-spin trapping with 5,5'-dimethyl-1- N -oxide revealed that the rate constant of cimetidine with radiation-induced OH radicals is about 4.5 ×10 9 l/mol/s. Cimetidine did not significantly increase the intracellular concentration of glutathione. These results suggest that cimetidine suppresses radiation-induced micronuclei and apoptosis via OH radical scavenging and an intracellular antioxidation mechanism. Cimetidine appears to be a useful candidate for the future development of post-irradiation radioprotectors.