Olive Tail Moment Research Articles

Comparison of comet assay parameters for estimation of genotoxicity by sum of ranking differences

The genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or single-cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gill cells, and liver cells, were used for assessing DNA damage. Images of randomly selected cells were analyzed with a Leica fluorescence microscope and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length-l, tail intensity-i, and Olive tail moment-m) were analyzed on 1,700 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity, and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm), and gill cells (rgl, rgi, rgm), the SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in the case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (~5 % probability of random ranking).

Effect of short-term storage on quality parameters, DNA integrity, and oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon sperm

The sperm of Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii) was used to evaluate the effects of short-term (liquid) storage on functional parameters (spermatozoa motility and velocity), DNA integrity and oxidative stress indices. Spermatozoa showed >50% motility during 6 days of storage with an average velocity of 133.12±15.4 to 87.9±11.23μm s−1 in both species. No motile spermatozoa were recorded after nine days of storage. Analysis of Russian sturgeon sperm showed no significant differences in DNA damage expressed as percent tail DNA and Olive Tail Moment for first three days of storage. In Siberian sturgeon significant differences in DNA damage were detected after two days of storage. The level of oxidative stress indices (TBARS, CP) and antioxidant activity (SOD) increased significantly with storage time in both species. Results of this study can be utilized for successful reproduction management and cryopreservation protocols of these endangered species.

Effect of dihydrokainate on the capacity of repair of DNA damage and apoptosis induced by doxorubicin

The intention of the current study was to investigate the effect of non-toxic doses of dihydrokainate on the capacity of repair of DNA damage and apoptosis induced by doxorubicin in mouse bone-marrow cells. The scoring of micronuclei and olive tail moment was undertaken in the current study as markers of DNA damage and repair. Apoptosis was analysed by the occurrence of a hypodiploid DNA peak. Oxidative stress markers such as bone-marrow reactive oxygen species, lipid peroxidation, reduced and oxidised glutathione were assessed as a possible mechanism underlying this amelioration. In addition, the influence of dihydrokainate on doxorubicin-induced topoisomerase II inhibition was examined. Dihydrokainate was neither genotoxic nor apoptogenic at doses equivalent to 10 or 20mg/kg/day for 7 days. Pre-treatment of mice with dihydrokainate significantly enhances the repair of doxorubicin-induced DNA damage and reduced doxorubicin-induced apoptosis depending on dose. Doxorubicin induced marked biochemical alterations characteristic of oxidative stress, including increased reactive oxygen species, enhanced lipid peroxidation and reduction in the reduced/oxidised glutathione ratio. Prior administration of dihydrokainate ahead of doxorubicin challenge ameliorated these oxidative stress markers. Importantly, dihydrokainate treatment had no antagonising effect on doxorubicin-induced topoisomerase II inhibition. Conclusively, this study provides for the first time that dihydrokainate enhances the repair of doxorubicin-induced DNA damage, which resides, at least in part, in its ability to modulate the cellular antioxidant levels. Based on our data presented, strategies can be developed to enhance the repair of doxorubicin-induced genomic damage in normal cells using dihydrokainate without diminishing doxorubicin's anti-topoisomerase II activity. Thus, improvement of doxorubicin's therapeutic index may be achieved by using dihydrokainate.

Evaluation of the in vivo genotoxic effects of gamma radiation on the peripheral blood leukocytes of head and neck cancer patients undergoing radiotherapy

The present study aimed to evaluate the genotoxic effects of ionizing radiation on non-target cells of Head and Neck Squamous Cell Carcinoma (HNSCC) patients exposed to various cumulative doses of gamma rays during radiotherapy. The ten patients (P1–P10) were treated with cobalt 60 gamma radiation (External Beam Radiotherapy) for a period of five to six weeks with a daily fraction of 2Gy for 5 days each week. The genotoxic effects of radiation (single strand breaks – SSBs) in these patients were analyzed using the alkaline single cell gel electrophoresis (SCGE) technique, with the Olive Tail Moment (OTM) as the critical parameter. A sample of each patient's peripheral blood before starting with radiotherapy (pre-therapy) served as the control, and blood collected at weekly time intervals during the course of the radiotherapy served as treated (10, 20, 30, 40, 50 and 60Gy) samples. In vivo radiosensitivity of these patients, as indicated by SSB's after the cumulative radiation doses at the various times, was assessed using Student's t-test. Significant DNA damage relative to the individual patient's pre-therapy baseline data was observed in all patients. Inter-individual variation of the genotoxic effects was analyzed using two-way ANOVA. The correlation between doses for the means of smoker and non-smoker patients was calculated using the Pearson test. The results of this study may indicate the need to reduce the daily radiotherapy dose further to prevent genotoxic effects on non-target cells, thus improving safety. Furthermore, these results may indicate that the estimation of DNA damage following exposure to a gamma radiation, as measured by the comet assay in whole blood leukocytes, can be used to screen human populations for radiation-induced genetic damage at the molecular level.

Assessment of exposure to organochlorine pesticides and levels of DNA damage in mother–infant pairs of an agrarian community

Exposure to organochlorine pesticides was studied in a group of mother-infant pairs living in a rural area where agriculture is the main economic activity. Fumigation in this zone is performed with airplanes, thus affecting the inhabited areas around them, including schools. Heparinized venous blood of mothers and umbilical cords was used to evaluate the olive tail moment in the comet assay, and micronuclei, chromatin buds, and nucleoplasmic bridges in peripheral blood lymphocytes. Cord blood samples were taken at the moment of birth only from natural and normal parturitions. Determinations of hexachlorobenzene, aldrin, heptachlor epoxide, oxichlordane, t and c-chlordane, cis-nonachlor, mirex, alpha and beta-endosulfan, alpha, beta and gamma hexachlorocyclohexane, and p'p'-DDT, p'p'-DDE were conducted to establish the differential distribution of the toxicants between compartments, i.e., mother and umbilical cord. Significantly higher pesticide levels were found in umbilical cord plasma than in mothers' plasma for almost all compounds tested, except DDE and oxychlordane. Significantly higher olive tail moments were found in umbilical cords than in mothers, whereas micronuclei frequencies were higher in mothers than in umbilical cords. However, neither the levels of micronuclei nor the olive tail moment were correlated with pesticide levels. Given that no other exposure to toxic compounds has been identified in this region, the lack of correlation between genotoxicity biomarkers and pesticide levels may be due to the variability of the exposure and to endogenous processes related to lipid mobility during pregnancy, the metabolism of the compounds, and individual susceptibilities.

Influence of GSM Signals on Human Peripheral Lymphocytes: Study of Genotoxicity

Exposure to radiofrequency (RF) electromagnetic fields (EMF) is continuously increasing worldwide. Yet, conflicting results of a possible genotoxic effect of RF EMF continue to be discussed. In the present study, a possible genotoxic effect of RF EMF (GSM, 1,800 MHz) in human lymphocytes was investigated by a collaboration of six independent institutes (institutes a, b, c, d, e, h). Peripheral blood of 20 healthy, nonsmoking volunteers of two age groups (10 volunteers 16-20 years old and 10 volunteers 50-65 years old) was taken, stimulated and intermittently exposed to three specific absorption rates (SARs) of RF EMF (0.2 W/kg, 2 W/kg, 10 W/kg) and sham for 28 h (institute a). The exposures were performed in a setup with strictly controlled conditions of temperature and dose, and randomly and automatically determined waveguide SARs, which were designed and periodically maintained by ITIS (institute h). Four genotoxicity tests with different end points were conducted (institute a): chromosome aberration test (five types of structural aberrations), micronucleus test, sister chromatid exchange test and the alkaline comet assay (Olive tail moment and % DNA). To demonstrate the validity of the study, positive controls were implemented. The genotoxicity end points were evaluated independently by three laboratories blind to SAR information (institute c = laboratory 1; institute d = laboratory 2; institute e = laboratory 3). Statistical analysis was carried out by institute b. Methods of primary statistical analysis and rules to adjust for multiple testing were specified in a statistical analysis plan based on a data review before unblinding. A linear trend test based on a linear mixed model was used for outcomes of comet assay and exact permutation test for linear trend for all other outcomes. It was ascertained that only outcomes with a significant SAR trend found by at least two of three analyzing laboratories indicated a substantiated suspicion of an exposure effect. On the basis of these specifications, none of the nine end points tested for SAR trend showed a significant and reproducible exposure effect. Highly significant differences between sham exposures and positive controls were detected by each analyzing laboratory, thus validating the study. In conclusion, the results show no evidence of a genotoxic effect induced by RF EMF (GSM, 1,800 MHz).

Melatonin enhances DNA repair capacity possibly by affecting genes involved in DNA damage responsive pathways

BackgroundMelatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis.ResultsIn the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”.ConclusionsThese findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.

Emodin regulating excision repair cross-complementation group 1 through fibroblast growth factor receptor 2 signaling

To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC₅₀) and reversal index (IC₅₀ in experimental group/IC₅₀ in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. Compared with the IC₅₀ of 120.78 μmol/L in HepG2/OXA cells, the IC₅₀ decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/OXA/T cells, in which FGFR2 was silenced by FGFR2 shRNA. In the examined HepG2 cells, the FGFR2, p-ERK1/2 and ERCC1 expression levels demonstrated increasing trends in the OXA group and OXA + FGF7 group. Compared with the OXA group and OXA + FGF7 group, the FGFR2, p-ERK1/2, and ERCC1 expression levels were significantly lower in the OXA + emodin group, and these differences were statistically significant. In the HepG2/OXA/T cell line that was transfected with FGFR2 shRNA, the FGFR2, p-ERK1/2 and ERCC1 expression levels were significantly inhibited, but there were no significant differences in these expression levels among the OXA, OXA + FGF7 and OXA + emodin groups. Emodin markedly reversed OXA resistance by enhancing OXA DNA damage in HepG2/OXA cells, and the molecular mechanism was related to the inhibitory effect on ERCC1 expression being mediated by the FGFR2/ERK1/2 signaling pathway.

DNA damage induced by oridonin involves cell cycle arrest at G2/M phase in human MCF-7 cells

Aim of the studyTo study the mechanisms of inhibition of cell growth and induction of DNA damage in oridonin-treated MCF-7 human breast cancer cells.Material and methodsThe cytotoxicity of oridonin-treated MCF-7 cells was measured by MTT assay. Cell cycle phase distribution was analyzed by flow cytometry. P-ATM, P-CHK2, γH2AX and P-P53 protein expressions were detected by Western blot analysis. The expression of r-h2ax and P-ATM was also detected by immunofluorescence staining. The degree of cellular damage of oridonin-induced MCF-7 human breast cancer cells was confirmed by the comet assay analysis of DNA fragmentation.ResultsOridonin inhibited cell growth in a time- and dose-dependent manner. The IC50 values at 48 and 72 hours were 78.3 and 31.62 µmol/l, respectively. Oridonin induced G2/M phase arrest in MCF-7 cells. MCF-7 cells treated with oridonin showed significant DNA damage as shown by an increase in olive tail moment (OTM). The protein expression levels of P-ATM, P-CHK2, γH2AX and P-P53 were increased significantly in a dose-dependent manner.ConclusionsDNA damage provokes p53-mediated G2/M cell cycle arrest in oridonin-induced MCF-7 cells through the mechanism of CHK2 activation by activated ATM protein kinase, which is induced by oridonin.

Validation of DNA Damage Progression with Days after Single Exposure of Sublethal Dosage of Electron Beam Radiation

Article history: The aim of this study was to asses the DNA damage progression with days after single exposure of 6Gy electron beam radiation (EBR). Molecular DNA damage in lymphocytes of mice was assessed using alkaline comet assay and in bone marrow cells by micronucleus assay. In comet assay %DNA in tail, Comet length, Olive Tail Moment (OTM) served as a measure of DNA damage and in micronucleus test, frequency of micronucleus formation in bone marrow cells was measured to evaluate the DNA damage. The experiment was performed by taking 24 healthy Swiss albino mice with body weight 30+5g. The animals were grouped into four. Group1 served as control, Group 2, 3 and 4 were irradiated by 6Gy EBR. Animals of Group 2, 3 and 4 were sacrificed on 5 th , 10 th and 15 th day post irradiation respectively. The comet assay procedure was carried out for all these groups by using lymphocytes separated from EDTA treated blood. The micronucleus test was performed in bone marrow cells (PCE-Poly chromatic erythrocytes, NCE-normochromatic erythrocytes). The slides prepared for this were scored for the measure of DNA damage. The results showed all comet parameter were significantly (P<0.05) affected by prolonging the post irradiation days from zero (control) to 5, 10 and 15. There was an alteration found in the PCE/PCE+NCE ratio in irradiated mice. A linear increase in the micronucleus formation was observed in post irradiated days. These results conclude a progression in DNA damage with days, post irradiation.

Biodegradation of cypermethrin by Bacillus sp. in soil microcosm and in-vitro toxicity evaluation on human cell line

Soil microcosms were set up with cypermethrin (100 mg/kg of dry soil) to assess the biodegradation by Bacillus sp. strain ISTDS2. Soil samples from the microcosms were collected at regular intervals during the biodegradation of cypermethrin and metabolites were extracted with hexane/Acetone (1:1v/v). GC–MS analysis of the soil extract showed formation of degraded compounds like cyclopropane, carboxylic acid, hydroxyacetonitrile and benzene ethanamine. The toxic effects of degraded product were studied on a human hepatocarcinoma cell line HuH7 before and after bacterial treatment. Methyltetrazolium (MTT) assay for cytotoxicity and alkaline comet assay for genotoxicity were carried for toxicity evaluation. The bacterium reduced toxicity as shown by a 105-fold increase in LC50 value, and a 3-fold reduction in Olive Tail Moment after 30 days of treatment. Biodegradation ability of the strain without toxic by-products reveals its potential for further study as a biological agent for the remediation of soil contaminated with cypermethrin.

Antioxidant defense system responses and DNA damage of earthworms exposed to Perfluorooctane sulfonate (PFOS)

The use of earthworms as a sublethal endpoint has significantly contributed to the ecological risk assessment of contaminated soils. Few studies have focused on the potential toxicity of PFOS to earthworms in the soil. In this work, artificial soils were tested, and contact filter paper studies were used. The results showed that earthworm growth was generally inhibited. The antioxidant activities of the enzymes superoxide dismutase, peroxidase, catalase and glutathione peroxidase were initially activated and then inhibited. Reduced glutathione content was observed, and malondialdehyde content was elevated over the duration of the exposure. These results suggested that PFOS induced oxidative stress in earthworms. In addition, the values of olive tail moment, tail DNA% and tail length using SCGE showed similar frequency distributions and increased with increases in the PFOS concentration. These results suggest that all concentrations of PFOS cause DNA damage.

Using the stable HSPA1A promoter-driven luciferase reporter HepG2 cells to assess the overall toxicity of coke oven emissions

Using the stable HSPA1A (HSP70-1) promoter-driven luciferase reporter HepG2 cells (HepG2/HSPA1A cells) to assess the overall toxicity of coke oven emissions. The stable HepG2/HSPA1A cells were treated with different concentrations of coke oven emissions (COEs) collected from the top, side, and bottom of a coke oven battery for 24 h. After the treatments, luciferase activity, cell viability, malondialdehyde (MDA) concentration, Olive tail moment, and micronuclei frequency were determined, respectively. The bottom COEs induced significant increases (P < 0.01) in relative luciferase activity up to 1.4 times the control level at 0.15 µg/L. The low dose of side COEs (0.02 µg/L) led to a significant increase (P < 0.01) in relative luciferase activity that progressively increased to 2.1 times the control level at 65.4 µg/L. The top COEs produced a strong dose-dependent induction of relative luciferase activity up to over 5 times the control level at the highest concentration tested (202 µg/L). In HepG2/HSPA1A cells treated with the bottom COEs, relative luciferase activity was positively correlated with MDA concentration (r = 0.404, P < 0.05). For the three COEs samples, positive correlations were observed between relative luciferase activity and Olive tail moment and micronuclei frequency. The relative luciferase activity in HepG2/HSPA1A cells can sensitively reflect the overall toxicity of COEs. The stable HepG2/HSPA1A cells can be used for rapid screening of the overall toxicity of complex air pollutants in the workplace.

Nanosecond electric pulses induce DNA breaks in cisplatin-sensitive and -resistant human ovarian cancer cells

Human ovarian cancer cells COC1 and COC1/DDP (cisplatin-resistant subline) were exposed to 6kV/cm nanosecond electric pulses (nsEP) with a pulse length of 8, 16 or 24ns. The potential in a subcellular unit was calculated using a multilayer dielectric spherical model, and area under the voltage–time curves (AUC) integrated with a lower limit of 0.2V. Cell viability was determined, and double-stand and total DNA breaks detected with the neutral and alkaline comet assays. nsEP evoked a higher voltage and AUC in nucleoplasm, and the levels in COC1 cells was just above those in COC1/DDP cells. Comets only appeared in the alkaline assay demonstrating single-stand DNA break. Fewer DNA break (16.51% vs. 35.13% at 24ns, p=0.0150) and more survival (22.42% vs. 13.19% at 24ns, p=0.0015) occurred in COC1/DDP cells despite an equal electric energy and almost equal cell sizes. 24-ns EP led to higher rates of cell-death and comet. The comet rate correlated with cell-death fraction in either cell line (r=0.5701, p=0.0135; r=0.5110, p=0.0302). There was no a correlation between the tail length, tail moment or Olive tail moment and cell-death rate. The data showed that response of chemosensitive cells differed from that of chemoresistant cells and DNA damage contributed to percent of cell death.

Comet Assay Determine DNA Damage Baseline Level of Mollusca Hemolymph Cells in Poyang Lake and Surrounding Waters

Mollusc species were sampled in different sites based on pollution level in Poyang lake and its surrounding waters during withered water period (from Oct to Dec) and plentiful water period (from May to June) from 2007 to 2008. Single-cell gel electrophoresis (SCGE) was used to analyze their haemolymph cells Arbitrary Unit(AU) and Olive tailmoment (OTM) value of DNA damage in molluca. The results showed that the mean DNA damage value Jin river&gt;Nanxin&gt;Xiu stream in withered water period; and in Plentiful water period, Hukou&gt;Qinglan lake&gt;Xiyuan. Two mollusc species Corbicula fluminea and Bellamya aeruginosa have higher DNA damage value than other mussel in all sites. Comparing the DNA damage value of Bellamya aeruginosa collected in twenty sites in 2008, the highest DNA damage value was observed in Dacha lake and lowest in Xiyuan. Meanwhile, the highest DNA damage value of Unio douglasiae was recorded Chucha river from ten sites, and lowest in Xiyuan river.

Amelioration of radiation-induced damages in mice by carotenoid meso-zeaxanthin

Purpose: The present study was undertaken to evaluate the radioprotective effect of meso-zeaxanthin, a xanthophyll carotenoid with profound antioxidant activity.Materials and methods: Swiss albino mice were treated with different doses of meso-zeaxanthin (50 and 250 mg/kg body weight, orally) five days before irradiation and sacrificed at different time points. The protective effects of meso-zeaxanthin on mortality, haematological parameters, bone marrow cellularity and gastrointestinal system of irradiated mice were studied. The anti-genotoxic action of meso-zeaxanthin was studied by measuring micronuclei formation, chromosomal aberrations and DNA damage (comet assay).Results: Meso-zeaxanthin administration significantly increased the lifespan of irradiated mice and reduced myelosuppression as evident from increases in white blood cell counts, bone marrow cellularity and the number of maturing monocytes when compared to the myelosuppression in radiation control animals. Meso-zeaxanthin significantly elevated the radiation-induced reduction in the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione in both liver and intestinal mucosa. The carotenoid-treated animals showed a profound reduction in genotoxic activity which was apparent in decreases in micronuclei formation and chromosomal aberrations. Irradiation also induced damage to cellular DNA as was obvious from increases in comet parameters like tail DNA%, tail moment, tail length and Olive tail moment in the radiation control group. These parameters were decreased by meso-zeaxanthin treatment.Conclusion: Results indicated a radioprotective potential of meso-zeaxanthin.

Down-regulation of Polη expression leads to increased DNA damage, apoptosis and enhanced S phase arrest in L-02 cells exposed to hydroquinone

DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.

Genotoxic Response of Unionid Mussel Hemolymph to Hydrogen Peroxide and Polycyclic Aromatic Hydrocarbons

The single cell gel electrophoresis or comet assay is widely used to detect DNA damage in isolated cells following exposure to genotoxic compounds. This assay, although commonly used with marine bivalve tissue and circulatory fluid, has received little use or demonstration in freshwater mussels of the order Unionida. Because such a large proportion (>70%) of this faunal group is globally imperiled and is being adversely impacted by environmental contaminants, including many genotoxicants, the aim of this study was to assess the applicability of the comet assay in unionid mussel hemolymph sampled non-lethally with a reference genotoxicant, hydrogen peroxide (H2O2) and polycyclic aromatic hydrocarbons (PAHs), a class of common environmental pollutants of genotoxic action. DNA damage was evaluated in samples of hemolymph from Elliptio complanata in both in vitro and in vivo exposures and quantified using the endpoints % tail DNA, or the percentage of DNA in the comet tail and OTM or olive tail moment, the product of the fraction of DNA in the tail and tail length. Hemocytes were isolated and the comet assay was performed on control, 160 µM H2O2, and PAH treated cells. From the in vitro exposures, 160 µM H2O2, as well as the 50 and 100 µg/L total PAH treatments yielded statistically significant (p < 0.05) levels of DNA damage, with the H2O2 yielding an average of 39.7 % tail DNA and 13.3 OTM and the two PAH treatments yielding 40.7 % and 38.6 % tail DNA, and 12.4 and 11.0 OTM, respectively. An in vivo PAH exposure with adult E. complanata did not detect a similar genotoxic response to that detected with in vitro exposure, indicating that additional research and evaluation may be necessary before implementing the widespread use of a non-lethal, unionid mussel hemolymph based genotoxicity screening tool for environmental biomonitoring.

Genotoxicity evaluation of 4-carboxyl- 2,6-dinitrophenylazohydroxynaphthalenes in mice

A short-term in vivo genotoxicity evaluation of 4-carboxyl-2,6-dinitrophenylazohydronaphthalenes (AZ-01 to AZ-04) has been carried out in mice. Aqueous colloidal solutions of the dyes were administered to mice on each day for 5 successive days using gastric gavages. Two end point assessments of the genotoxicity potentials of the dyes were assessed using comet assay and chromosomal aberration studies using the mice bone marrow cells. The dyes were well tolerated at the doses investigated, as there were no deaths or any adverse pharmacotoxic events. Dose-dependent DNA damage (in terms of percentage of tail DNA and Olive tail moment) occurred with AZ-01 and AZ-02, although the effects were significant only with the highest doses. AZ-03 gave similar patterns with those of AZ-01 and AZ-02, while replacement with butanone in AZ-04 altered the observed pattern. Minimal chromosomal damages were obtained for the four dyes, with AZ-01 and AZ-02 giving nonsignificant damages, while the highest dose of AZ-03 produced significant aberrations in terms of breaks. Some minor isochromatid breaks and gaps were also noticed in the dye-treated mice. Mitotic indices in all cases were not significantly different from concomitantly administered vehicle control showing lack of cytotoxicity of the monoazo dyes at these doses. The monoazo dyes show the potential of being utilized as colorants, pending further required tests.

Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×104M−1) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2′,7′-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V−/7-AAD+ stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor–DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and consequent mitochondrial dysfunction, oxidative DNA damage, and chromosomal breakage, which eventually triggers necrosis in human PBMN cells.