Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.     

Genotoxicity of drinking water disinfection by-products (bromoform and chloroform) by using both Allium anaphase-telophase and comet tests

Genotoxic effects of bromoform and chloroform, disinfection by-products of the chlorination of drinking water, were examined by using mitotic index (MI), mitotic phase, chromosome aberrations (CAs) and comet assay on root meristematic cells of Allium cepa. Different concentrations of bromoform (25, 50, 75 and 100 μg/mL) and chloroform (25, 50, 100 and 200 μg/mL) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 μg/mL) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance were employed and p < 0.05 was accepted as significant value. Exposure of both chemicals (except 25 μg/mL applications of bromoform) significantly decreased MI. Bromoform and chloroform (except 25 μg/mL applications) increased total CAs in Allium anaphase-telophase test. A significant increase in DNA damage was also observed at all concentrations of both bromoform and chloroform examined by comet assay. The damages were higher than that of positive control especially at 75–100 μg/mL for bromoform and 100–200 μg/mL for chloroform.     

Zinc deficiency or excess within the physiological range increases genome instability and cytotoxicity, respectively, in human oral keratinocyte cells

Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO₄) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO₄ and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

Effects of long-term serial cell passaging on cell spreading,migration, and cell-surface ultrastructures of cultured vascular endothelial cells

The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

In vitro detection of indirect-acting genotoxins in the comet assay using Hep G2 cells

The induction of DNA damage by four known promutagens (cyclophosphamide (CP), benzo(a)pyrene (BP), dimethylbenz(a)anthracene and 2-acetylaminofluorene (2AAF) was investigated on Hep G2 using the alkaline single cell electroporesis (SCGE) test, most often referred as the "comet assay". After a 3-day incubation, lysed cells embedded in agarose were electrophoresed under alkaline conditions, dyed with a SYBRgold fluorogen and analysed by the Komet software. Among the comet parameters provided by the image analysis program, statistical analysis did not identify any in particular that could best represent the DNA damages. All promutagens, when compared with the control, caused a statistically significant increase in DNA migration as determined by different parameters such as Olive tail moment, tail extent moment, tail/head or tail length. The data demonstrated the ability and the sensitivity of the comet assay when performed on Hep G2 in the detection of DNA damage induced by promutagens, and its suitability in mutagenicity testing in in vitro short-term assays.     

Modification of the alkaline comet assay with human mesenchymal stem cells

MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.     

DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR

The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage.     

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 λunits/g liver at 37°C for 20 λmin) for isolation of intact cells for subsequent comet assay. We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation ( -85°C for 72 λh). To minimize this damage, minced liver (1.0 λg) and kidney (0.5 λg) tissues were added to 20 λml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO ( -85°C for 72 λh and 3 weeks), and to a slightly lesser extent with glycerol (72 λh), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150 λunits/g liver and 300 λunits/g kidney for 10 λmin produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at -85°C for 72 λh. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.     

Protective effect of (−)-epigallocatechin-3-gallate on capsaicin-induced DNA damage and oxidative stress in human erythrocyes and leucocytes in vitro

The aim of this study is to show that protective effects of the main catechin (−)-epigallocatechin-3-gallate (EGCG) against capsaicin (CAP) induced oxidative stress and DNA damage in human blood in vitro. Superoxide dismutase, catalase, glutathione peroxidase and malondialdehyde (MDA) level were studied in erythrocytes and leucocytes with increased concentrations of CAP. DNA damage in leucocytes was measured by the comet assay. Human blood cells have been administered with doses between 0 and 200 μM of CAP and/or EGCG (20 μM) for an hour at 37 °C. Treatment with CAP alone has increased the levels of MDA and decreased antioxidant enzymes in human blood cells. A significant increase in tail DNA%, mean tail length and tail moment indicating DNA damage has been observed at the highest dose of CAP treatment when compared to controls. Treatment of cells with CAP plus EGCG prevented CAP-induced changes in antioxidant enzyme activities and MDA level and mean tail lenght indicating DNA damage. A significant increase in mean tail lenght was observed at high doses of CAP. These data suggest that EGCG can prevent toxicity to human erythrocytes and leucocytes caused by CAP, only at low doses.     

Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH > 13)?

The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt–detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH > 13 [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184–191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205–214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt–detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH > 13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3 M NaOH, 0.02 M Trizma and 1 mM EDTA, pH > 13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium,cobalt, and nickel

The alkaline comet assay is able to identify in individual cells DNA strand breaks associated with different processes. Topoisomerase inhibitors, some of which are used as chemotherapeutic agents, stabilise topoisomerase-DNA cleavable complexes by stimulating DNA strand cleavage and inhibiting religation. This can result in the activation of stress-associated signalling pathways, inducing cell cycle arrest and activation of the biochemical cascade of apoptosis. The aim of our study was to assess the ability of the comet assay to detect stabilisation of cleavable complexes and induction of apoptosis by two topoisomerase II inhibitors, etoposide and ellipticine, and two topoisomerase I inhibitors, camptothecin and topotecan. The study was carried out on Chinese hamster ovary (CHO) cells, DC3F cells and DC3F/C-10, its camptothecin-resistant counterpart. The comet assay was able to identify stabilised cleavable complexes through the presence of DNA strand breaks after 1h treatment that disappeared within 24h after drug removal. Kinetics studies allowed to discriminate between these early DNA damages and DNA fragmentation related to apoptosis characterised by reappearance of DNA strand breaks 48h after treatment.     

Protection against genotoxic damages following whole body gamma radiation exposure in mice by lipoic acid

Alpha-lipoic acid (LA) protected plasmid pBR 322 DNA, under in vitro conditions from gamma radiation induced strand breaks as evidenced by the prevention of the loss of supercoiled covalently closed circular form upon irradiation. It also protected the membrane lipids of liver homogenates from the oxidative damages. Whole body exposure of mice to gamma-radiation resulted in damage to cellular DNA in various tissues and administration of LA prior to the radiation exposure prevented the radiation induced DNA damage as assessed by alkaline comet assay. Administration of LA to mice prior to the radiation exposure also prevented induction of chromosomal damages in bone marrow cells and formation of micronuclei in blood reticulocytes. Thus taken together, LA a normal cellular constituent could be used as a radioprotector against whole body radiation exposure scenarios.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

BACKGROUND: Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV(+) cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). METHODS: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. RESULTS: AV(+) cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV(+) propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. CONCLUSIONS: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.     

HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2 h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10 μM BPDE and p < 0.05 at 20 μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10 μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0454-7) contains supplementary material, which is available to authorized users.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Modulation of DNA damage by pentoxifylline and alpha-tocopherol in skin fibroblasts exposed to Gamma rays

Previous in vivo studies showed the combination pentoxifylline (PTX) and alpha-tocopherol was highly efficient in reducing late radiation-induced skin damage. The present work aimed at investigating the molecular and cellular mechanisms involved in the effects of this combination. Primary cultures of confluent dermal fibroblasts were gamma-irradiated in the presence of PTX and trolox (Tx), the water-soluble analogue of alpha-tocopherol. Drugs were added either before or after radiation exposure and were maintained over time. Their antioxidant capacity and their effect on radiation-induced ROS production was assessed together with cell viability and clonogenicity. DNA damage formation was assessed by the alkaline comet assay and by the micronucleus (MN) test. Cell cycle distribution was also determined. The combination of PTX/ Tx was shown to reduce both immediate and late ROS formation observed in cells after irradiation. Surprisingly, decrease in DNA strand breaks measured by the comet assay was observed any time drugs were added. In addition, the micronucleus test revealed that for cells irradiated with 10 Gy, a late significant increase in MN formation occurred. The combination of PTX/Tx was shown to be antioxidant and to decrease radiation-induced ROS production. The observed effects on DNA damage at any time the drugs were added suggest that PTX/Tx could interfere with the DNA repair process.