DNA Damage Recovery Research Articles

Vitrification in Open and Closed Carriers at Different Cell Stages: Assessment of Embryo Survival, Development, DNA Integrity and Stability during Vapor Phase Storage for Transport

BackgroundHigh cooling rates with vitrification can be achieved through the use of carriers that allow cryopreservation in fluid volumes < one μl. Open carriers allow direct contact of embryos with liquid nitrogen (LN2) whereas closed carrier systems sequester the embryo within a sealed system during immersion in LN2. The use of closed systems may be preferable to reduce the possibility of cross-contamination. In the present study, we compare open and closed carriers for vitrification of embryos. We also examine their ability to retain embryo viability during vapor phase transport.MethodsFrozen one-cell mouse embryos were thawed and randomly allocated to treatment groups. Embryos were cultured and vitrified at the 8-cell (CL) or at the blastocyst (BL) stage. The cryoloop, an open carrier was tested against two closed systems, the Cryotip and the HSV straw. Carriers were tested for their ability to maintain embryo viability when held in the vapor phase of a dry shipper for a period of 96 hours. Outcome parameters monitored were embryo survival, recovery, subsequent development and signs of DNA damage.ResultsA total of 561 embryos were vitrified. The only parameter significantly affected by the type of carrier was the percentage of embryos recovered after warming. Vitrification of both CL and BL stage embryos in the Cryotip resulted in significantly lower recovery rates (P < 0.001). The subsequent developmental parameters were unaffected by either the carrier or the cell stage. Vapor phase storage for 96 hours under "transport conditions" did not appear to adversely affect the viability after warming. Quantitative analysis for DNA damage showed that <5% of cells were TUNEL positive. Interestingly, the overall percent of cells exhibiting DNA damage was lower after CL stage vitrification (P < 0.001).ConclusionThis study is one of the first to examine DNA integrity after vitrification on different carriers and at different cell stages. It also provides insight on relative safety of short term vapor storage of vitrified embryos during transport. Within the limits of this study we could not detect an adverse effect of vapor storage on blastomere DNA or other measured outcome parameters.

Polo‐like kinase 1 phosphorylation of G2 and S‐phase‐expressed 1 protein is essential for p53 inactivation during G2 checkpoint recovery

In response to G2 DNA damage, the p53 pathway is activated to lead to cell-cycle arrest, but how p53 is eliminated during the subsequent recovery process is poorly understood. It has been established that Polo-like kinase 1 (Plk1) controls G2 DNA-damage recovery. However, whether Plk1 activity contributes to p53 inactivation during this process is unknown. In this study, we show that G2 and S-phase-expressed 1 (GTSE1) protein, a negative regulator of p53, is required for G2 checkpoint recovery and that Plk1 phosphorylation of GTSE1 at Ser 435 promotes its nuclear localization, and thus shuttles p53 out of the nucleus to lead to its degradation during the recovery.

Polo-like kinase 1 is essential to DNA damage recovery

Radiation treatment and chemotherapy can both cause DNA damage to tumor cells. Tumor cells have specific self-protecting mechanisms to escape death. In this study, we explored the effects of polo-like kinase 1 on the DNA damage recovery system in lymphoma cell lines. DNA damage caused inhibition of polo-like kinase 1 activity and induced cell cycle arrest at the G2/M phase. Once the DNA damage was repaired, polo-like kinase 1 activity was restored and cell cycle arrest was removed. Unexpectedly, polo-like kinase 1 depletion hampered the cell cycle progress of cells recovered from DNA damage, and induced apoptosis.

DYNAMICS OF MICRONUCLEI IN MOUSE SKIN FIBROBLASTS AFTER GAMMA IRRADIATION

Micronuclei (MN) were assessed in dermal fibroblasts from C3H HeJ and C57 Bl6 mice (6-10 mo of age) irradiated in vivo as a potential method of biodosimetry. Radiation-induced MN [per 1,000 binucleated (BN) cells], assessed in fibroblasts obtained 1 d post-irradiation, increased in a dose-dependent manner in the range of 1 - 10 Gy per single dose. Analysis at 1 wk post irradiation showed some attenuation of MN response in C3H HeJ male mice, suggesting partial recovery of DNA damage. This was not observed in C57 Bl6 mice. Monomicronucleated cells predominated in unirradiated fibroblasts, whereas in irradiated fibroblasts multimicronucleated cells predominated at dose levels above about 5 Gy (more than 1,000 MN per 1,000 BN cells). Modeling of the data indicate that assaying total MN in dermal fibroblasts from samples of irradiated skin taken up to 1 wk after irradiation can provide biodosimetric information (with an accuracy of 1-2 Gy) for doses up to at least 6 Gy with lesser accuracy at higher doses. Percentage of multimicronucleated cells may be useful as a substitute of total MN at the higher doses, but total percentage of micronucleated cells is of limited value as a substitute.

Inhibition of PARP activity by PJ‐34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment.

DNA protective properties of vanillin against γ-radiation under different conditions: Possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 ( in vitro) upon exposure to 50 Gy of γ-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes ( ex vivo) to γ-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of γ-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, ‘tail moment’ and ‘Olive tail moment’. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice ( in vivo) to γ-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS + radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against γ-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.

Solar UV radiation modulates daily production and DNA damage of marine bacterioplankton from a productive upwelling zone (36°S), Chile

In upwelling ecosystems, such as the Humboldt Current system (HCS) off Concepción, the effects of solar radiation on bacterioplankton incorporation rates have been related to previous light acclimation and responses to irradiance. In this paper, we study the daily effect of Photosynthetic Active Radiation (PAR, 400–700 nm) and ultraviolet radiation UVR (280–400 nm) on bacterial secondary production (BSP). We also considered the DNA damage–repair response to solar radiation stress by the induction of cyclobutane pyrimidine dimers (CPDs). Experiments were conducted with natural bacterioplankton assemblages (0.2–0.7 μm) collected off Concepción (36°S), during the austral Spring, October–November, 2004. Surface (0.5 m) and subsurface (80 m) bacterioplankton samples were exposed to different solar radiation treatments for 5–20 h. BSP was estimated by 14C-leucine and 3H-thymidine incorporation at several time intervals, whereas CPDs accumulation was assessed using immunoassay techniques. During high irradiance periods, BSP was mainly affected by PAR in both surface and subsurface assemblages and, to a lesser, but significant (Tukey < 0.05) extent, by UV-A (320–400 nm) and UV-B (280–320 nm) radiation. Maximum inhibition of BSP in surface waters was 78%; growth rates ( μ) and bacterial growth efficiency (BGE) were also low (78% and 66% respectively). Subsurface water assemblages, on the contrary, showed a ∼ 25 fold enhancement of BSP, μ, and BGE. Both types of assemblages had a rapid CPDs accumulation (maximum 60 CPDs Mb − 1 ) during high irradiance periods. Recovery of BSP inhibition and DNA damage in surface bacteria was total after sunset and after the night incubation period, resembling pre-exposure levels. Despite subsurface BSP enhancement during day–night exposure, residual DNA damage was detected at the end of the experiment (20 CPDs Mb − 1 ) suggesting a chronic DNA damage. Our results represent the worst case scenario (i.e., assemblages receiving surface irradiances as may occur in this upwelling zone) and indicate that surface and subsurface bacterial assemblages in the HCS are both highly sensitive to solar irradiance. However, they showed different responses, with surface bacteria having more effective photorepair mechanisms, and sustaining higher BSP than subsurface assemblages.

In Vitro Mitigation of Arsenic Toxicity by Tea Polyphenols in Human Lymphocytes

The groundwater arsenicals have brought dreadful misery for the people residing in the endemic regions of West Bengal, India. Arsenic-related anomalies include arsenicosis, hyperkera-tosis, gastric complications, liver fibrosis, peripheral neuropathy, and cancer. Some of these diseases have been frequently associated with overproduction of reactive oxygen species that cause DNA damage and improper functioning of body's antioxidant defense mechanism. Natural polyphenols present in tea serve as excellent antioxidants. In the present study, an attempt has been made to elucidate the role of representative polyphenols and extracts of green and black tea in modulating sodium arsenite (As III)-induced DNA damage in normal human lymphocytes. Comet assay was used to detect the DNA damage. Arsenic-induced oxidative stress was measured with generation of reactive oxygen species, lipid peroxidation, and activity of some antioxidant enzymes. Expression of some repair enzymes such as poly(ADP-ribose) polymerase and DNA polymerase beta was measured to assess the effect of tea on DNA repair. Tea afforded efficient reduction of As III-induced DNA damage in human lymphocytes. Tea also quenched the excessive production of reactive oxygen species by arsenic, reduced the elevated levels of lipid peroxidation, and increased the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Furthermore, tea enhanced recovery of DNA damage, which was indicative of repair as confirmed by unscheduled DNA synthesis and pronounced expression of DNA repair enzyme poly(ADP-ribose) polymerase. It is speculated that the antioxidant potential and repair-inducing capacity of tea might help in combating the severe genotoxic effects induced by arsenic in the human population.

Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress

Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(−/−) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of γ-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of γ-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.

Role of DNA Repair Systems Inhibition in Carcinogenicity of Metal Compounds

Several metals have been shown to be carcinogenic to humans and/or experimental animals. The mechanisms of their carcinogenicity are not completely understood, even if DNA repair inhibition seems to be the most probable. Current evidence suggests that DNA repair systems are very sensitive target for nickel (II), cadmium (II), cobalt (II) and arsenic (III) leading to a diminished removal of endogenous and exogenously induced DNA damage, which increase the risk of tumor formation. Metal compounds inhibit DNA repair probably by oxidative damage and direct interactions of metal-ions with reparative enzyme. Metals carcinogenic potential depends from their solubility and oxidation state. Oxidative DNA damage by metal compounds is due to ROS generation by Fenton-type reactions and inactivation of anti-oxidative enzymes. DNA repair inhibition seems to depend on the ability of metal ions to compete with magnesium ions or to displace zinc ions in zinc finger structures of DNA repair enzymes with consequent their inactivation. The DNA repair inhibition has been shown for low non-cytotoxic metal concentrations. DNA repair systems inhibited by metal compounds include essentially base-excision repair (BER) including Formamidopyrimidinglycosilase (Fpg)-oxidized base excision repair and nucleotide excision repair (NER). Several studies on metal inhibition activity use prevalently the comet test, a rapid and sensitive technique, to evaluate direct and oxidative DNA damage and its repair in human cells. Comet test consents to study the influence of metals on the different steps involved in DNA repair, evaluating the recovery of DNA damage induced by exposure to different genotoxic agents in absence or presence of metal compounds. This technique and its modifications can clarify the mechanisms of inhibitory processes of enzymes involved in carcinogenesis and could be a useful tool in the development of new anticancer drugs. Keywords: metals, dna repair inhibition, cancer, comet assay, oxidative damage

Sensitivity of Laminariales zoospores from Helgoland (North Sea) to ultraviolet and photosynthetically active radiation: implications for depth distribution and seasonal reproduction

ABSTRACTDepth distribution of kelp species in Helgoland (North Sea) is characterized by occurrence of Laminaria digitata in the upper sublittoral, whereas L. saccharina and L. hyperborea dominate the mid and lower sublittoral region. Laminaria digitata is fertile in summer whereas both other species are fertile in autumn/winter. To determine the light sensitivity of the propagules, zoospores of L. digitata, L. saccharina and L. hyperborea were exposed in the laboratory to different exposure times of photosynthetically active radiation (PAR; 400–700 nm), PAR + UVA radiation (UVAR; 320–400 nm) and PAR + UVAR + UVB radiation (UVBR; 280–320 nm). Optimum quantum yield of PSII and DNA damage were measured after exposure. Subsequently, recovery of photosynthetic efficiency and DNA damage repair, as well as germination rate were measured after 2 and 3 d cultivation in dim white light. Photosynthetic efficiency of all species was photoinhibited already at 20 µmol photons m−2 s−1 PAR, whereas UV radiation (UVR) had a significant additional effect on photoinhibition. Recovery of the PSII function was observed in all species but not in spores exposed to irradiation longer than 4 h of PAR + UVA + UVB and 8 h of PAR + UVA. The amount of UVB‐induced DNA damage measured as cyclobutane–pyrimidine dimers (CPDs) increased with exposure time and highest damage was detected in the spores of lower subtidal L. hyperborea relative to the other two species. Significant removal of CPDs indicating repair of DNA damage was observed in all species after 2 d in low white light especially in the spores of upper subtidal L. digitata. Therefore, efficient DNA damage repair and recovery of PSII damage contributed to the germination success but not in spores exposed to 16 h of UVBR. UV absorption of zoospore suspension in L. digitata is based both on the absorption by the zoospores itself as well as by exudates in the medium. In contrast, the absorption of the zoospore suspension in L. saccharina and L. hyperborea is based predominantly on the absorption by the exudates in the medium. This study indicates that UVR sensitivity of zoospores is related to the seasonal zoospore production as well as the vertical distribution pattern of the large sporophytes.

Partial breast irradiation with interstitial 60CO brachytherapy results in frequent grade 3 or 4 toxicity. evidence based on a 12-year follow-up of 70 patients

Purpose To investigate the radiation-induced toxicity and cosmesis of brachytherapy (BT) alone in early stage breast cancer. Methods and materials A total of 70 women diagnosed with Stage I or II breast carcinoma participated in a BT study at the Municipal Oncoradiological Center, Uzsoki Hospital, Budapest, Hungary, between November 1987 and June 1992. They had undergone breast-conserving surgery with an unknown surgical margin. The postoperative tumor bed irradiation was performed with interstitial 60Co sources with an active length of 4 cm, with 10-mm center-to-center spacing arranged in a single plane. The median number of inserted sources was 5 (range, 2–8), with a linear activity of 133–137 MBq/cm at the beginning of the study. The 50 Gy delivered dose at 5 mm from the surface of the 60Co sources was administered during 10–22 h to the virtual postoperative lumpectomy cavity (i.e., plane). For radiobiologic considerations, the clinical target volume (CTV) was calculated retrospectively with a 10-mm safety margin, resulting in a 72-cm 3 median CTV (range, 36–108 cm 3) irradiated with a reference dose of 28 Gy. In the assessment of the skin and subcutaneous toxicity, the RTOG late radiation morbidity scoring system was applied. The radiosensitivity of the cultured fibroblasts was determined by clonogenic assay to check whether individual radiosensitivity played a role in the development and course of radiation-induced side-effects. Results The median follow-up was 12 years (range, 10–15 years). The population of the final study (34 cases) comprised all survivors with tumor-free breasts (27 cases) and patients with breasts erroneously ablated/excised for misinterpreted radiation-induced sequelae (7 patients). A total of 97% of the cohort (33/34) had grade ≥2, and 59% (20/34) had grade ≥3 radiation-induced toxicity. By the end of the follow-up, 85% of the patients experienced Grade ≥2 telangiectasis and 41% had Grade 3 telangiectasis. Eighty-eight percent had fibrosis of some form, and 35% had grade ≥3 fibrosis. Forty-one percent of the cohort displayed fat necrosis, which was always accompanied by Grade ≥3 fibrosis or telangiectasis. The cosmetic results were poor in 50% (17/34) of the patients. The radiosensitivity of the fibroblasts was increased in only 2/24 patients (8% of the investigated cases, in agreement with data published for the general population). Comparisons of our fibrosis prevalence data with those of others allowed an estimate of 0.47 h −1 for the rate of recovery of DNA damage in the fibroblasts. Conclusions Interstitial 60Co BT of the breast tumor bed alone with a limited CTV (median, 72 cm 3) and a total dose of 28 Gy is associated with a high rate (59%) of grade ≥3 radiation-induced toxicity and a high rate (50%) of poor cosmetic outcome at the end of a median follow-up of 12 years. A relatively high BT dose rate (1.3–2.8 Gy/h) applied during a short overall treatment time (10–22 h) and a possible geographic miss (close to skin implantation) might have contributed to the development of these sequelae.

RAD6B overexpression confers chemoresistance: RAD6 expression during cell cycle and its redistribution to chromatin during DNA damage-induced response.

The HR6A and HR6B genes, homologs of the yeast RAD6 gene, encode ubiquitin conjugating enzymes that are required for postreplication repair (PRR) of DNA and damage-induced mutagenesis. We show here that consistent with its role as a PRR protein, HR6 protein (referred as RAD6) expression is cell cycle regulated, with maximal levels expressed in late S/G2 phases of the cell cycle. Exposure of MCF10A cells to adriamycin (ADR) causes enhancement in the levels of RAD6B mRNA and protein. Inclusion of actinomycin D abolishes both basal and ADR-induced RAD6B transcription indicating that ADR-induced effects on RAD6B transcription result from an increase in transcriptional activity rather than from regulation of RAD6B mRNA stability. The increase in RAD6 protein expression observed in ADR-treated cells is dependent upon transcription and de novo protein synthesis, as addition of actinomycin D and cycloheximide eliminated the induction effects. Using in vivo crosslinking experiments, we demonstrate that only a small proportion of RAD6 is associated with chromatin in untreated MCF10A cells. However, treatment with ADR or cisplatin is accompanied by a significant increase and redistribution of RAD6 to DNA, and RAD6, RAD18, PCNA, phosphohistone H3, as well as p53 proteins are all found in the DNA fractions. These findings suggest that although RAD6 protein is present in the nucleus, its recruitment to the chromatin appears to be modulated by DNA damage. Whereas MCF10A cells engineered to overexpress ectopic RAD6B are significantly more resistant to ADR and cisplatin as compared to empty vector-transfected cells, MCF10A cells stably transfected with antisense RAD6B display hypersensitivity to these damage-inducing drugs. Analysis of PRR capacities in cisplatin-treated MCF10A cells stably transfected with empty vector, RAD6B or antisense RAD6B showed that whereas RAD6B-overexpressing and vector control MCF10A cells possessed the ability to convert newly synthesized DNA to higher molecular weight species, MCF10A cells depleted of RAD6B are PRR-compromised. Although no human diseases have been linked to mutations in the PRR pathway genes, these data suggest that RAD6 may play an essential role in DNA damage tolerance and recovery via modulation of PRR, and that imbalances in the levels of RAD6 could lead to changes in drug sensitivity and damage-induced mutagenesis.

Prevention and repair of DNA damage by selected phytochemicals as measured by single cell gel electrophoresis.

We assessed the ability of some natural products--namely, curcumin, resveratrol, indole-3-carbinol, and ellagic acid--to modify the DNA damaging ability of the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster lung fibroblast cells (CH V-79). MNNG produced DNA single strand breaks in a dose- and time-dependent manner, as observed by increase in the tail moments of the comet, when the cells were subjected to alkaline single cell gel electrophoresis. When the cells were treated in the presence of each of the natural compounds, the DNA damage caused by MNNG was considerably reduced. This effect was found to be dose related. Preincubation of cells with each of these compounds individually afforded significant protection to DNA against damage caused by subsequent treatment with MNNG, indicating a true chemopreventive role of these substances. The most remarkable aspect of the present study was that all four compounds helped in the recovery of DNA damage by accelerating DNA repair efficiency in the damaged cells. This was further substantiated by the observation on unscheduled DNA synthesis. Our results suggest that these agents are chemopreventive by virtue of their ability to protect DNA as well as to induce DNA repair.

Three-phase reactions in honeycombs

Hypericum olympicum L. (Hypericaceae) flowering aerial parts has been utilized in Turkish folk medicine as a remedy against inflamed skin problems.This study was designed to state the effect of H. olympicum on dermatological problems. For this purpose effect of the plant extract on the DNA strand break and matrix metalloproteinase (MMP)–9 activity of human dermal fibroblast (HDFs) cells irradiated with UVB as well as antioxidant activity potential were studied.The methanolic extract of Hypericum olympicum (HOM) was prepared by maceration at room temperature. DNA damage and increased MMP–9 activity in HDFs were induced by UVB irradiation. The cell viability was measured by water–soluble tetrazolium salt (WST)–1 assay. The effects on DNA strand break was investigated by single gel electrophoresis (commonly known as Comet assay), while MMP–9 activity was observed by gelatin zymography assay. In vitro antioxidant tests were performed to indicate the effect on reactive oxygen species (ROS). The major metabolites were identified and their concentrations were measured by high performance thin layer chromatography (HPTLC).HOM was found to recover DNA damage dose–dependently. The enzymatic activity of MMP–9 was inhibited almost 100% by the treatment of 1.5 mg/mL of the extract. It also enhanced cell proliferation in those cells, and also it was shown to possess antioxidant activity. The major metabolites of HOM were identified as chlorogenic acid and quercetin glycosides (rutin, hyperoside, isoquercitrin).Experimental studies have proven the traditional use of Hypericum olympicum in inflamed skin problems acting by inhibition of the inflammatory pathway and recovery of DNA damage induced experimentally.

Evaluation of oxidative damage and inhibition of DNA repair in an in vitro study of nickel exposure

It has been suggested that Nickel is involved in oxidative damage and inhibition of DNA repair. We studied the effects of NiSO 4 on oxidative stress and DNA repair in Jurkat cells to elucidate its mechanism of action. Cells were treated with H 2O 2 and ROS generation (by flow cytometry), and oxidative DNA damage (as tail moment by Fpg-enzyme comet test), were evaluated immediately and after 4 and 24 h of DNA damage recovery occurred in presence or absence of NiSO 4 (0.017 and 0.17 μ m) to clarify possible interactions of Ni with DNA repair processes. Moreover, cells were exposed to the same doses of NiSO 4 for 4 and 24 hours to evaluate its direct oxidative effect. The results of the comet test showed high tail moment immediately after oxidative burst with a decreasing after 4 h of DNA recovery, and a slight increase after 24 h of recovery. The decreases were more limited for cells treated with NiSO 4 0.17 μ m indicating an inhibition of oxidative DNA damage repair by this substance. An induction of ROS was observed after 4 h of incubation with higher dose of NiSO 4. Cells treated with H 2O 2 showed the highest level of ROS after 4 h of recovery in presence of NiSO 4 0.17 μ m that remained at elevated levels also after 24 h of recovery suggesting a synergistic action of Ni with H 2O 2 in the reduction of cellular anti-oxidative defence activities.

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis.

Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein kinase phosphatase (AtMKP1). Growth of the mkp1 mutant under standard conditions is indistinguishable from wild type, indicating a stress-specific function of AtMKP1. MAP kinase phosphatases (MKPs), the potent inactivators of MAP kinases, are considered important regulators of MAP kinase signaling. Although biochemical data from mammalian cell cultures suggests an involvement of MKPs in cellular stress responses, there is no in vivo genetic support for this view in any multicellular organism. The genetic and biochemical data presented here imply a central role for a MAP kinase cascade in genotoxic stress signaling in plants and indicate AtMKP1 to be a crucial regulator of the MAP kinase activity in vivo, determining the outcome of the cellular reaction and the level of genotoxic resistance.

Uptake, cellular distribution and DNA damage produced by mercuric chloride in a human fetal hepatic cell line

A human hepatic cell line (WRL-68 cells) was employed to investigate the uptake of the toxic heavy metal mercury. Hg accumulation in WRL-68 cells is a time and concentration dependent process. A rapid initial phase of uptake was followed by a second slower phase. The transport does not require energy and at low HgCl 2 concentrations (<50 μM) Hg transport occurs by temperature-insensitive processes. Subcellular distribution of Hg was: 48% in mitochondria, 38% in nucleus and only 8% in cytosolic fraction and 7% in microsomes. Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eucaryotic cells to low concentrations of Hg. Our results showed that Hg induced DNA single-strand breaks or alkali labile sites using the single-cell gel electrophoresis assay (Comet assay). The percentage of damaged nucleus and the average length of DNA migration increased as metal concentration and time exposure increased. Lipid peroxidation, determined as malondialdehyde production in the presence of thiobarbituric acid, followed the same tendency, increased as HgCl 2 concentration and time of exposure increased. DNA damage recovery took 8 h after partial metal removed with PBS–EGTA.

Poly(ADP-ribosylation) and apoptosis.

Poly(ADP-ribosylation) is a post-translational modification playing a relevant role in DNA damage recovery, DNA replication and viral integration. Several reports also suggest a modulation of this process during cell death by apoptosis. The aim of this review is to discuss the possible involvement of poly(ADP-ribosylation) during apoptosis, by dealing with general considerations on apoptosis, and further examining the correlation between NAD consumption and cell death, the regulation of poly(ADP-ribose) metabolism in apoptotic cells, the effect of poly(ADP-ribose) polymerase inhibition on cell death occurrence and the use of enzyme cleavage as a marker of apoptosis. Finally, the future prospects of the research in this area will be addressed.

Establishment of poly(ADP-ribose) polymerase-deficient mouse embryonic stem cell lines

Many studies revealed that poly(ADP-ribose) polymerase (Pare) is involved in DNA-damage recovery, cell-death induction and maintenance of genomic stability. We generated mouse Parp+/- embryonic stem (ES) cell lines by disrupting one allele of Parp exon 1 with a neomycin-resistance gene-cassette and subsequently produced Parp-/- ES cells by disrupting the remaining allele with a puromycin-resistance genecassette. Parp activity was decreased to half in Parp+/- ES cell clones and lost in Parp-/- ES cell clones. Growth rates of Parp+/- and Parp-/- ES cell clones were similar to that of parental J1 ES cells, indicating that Parp is not required for ES cell proliferation. These Parp-/- ES cells will be useful to study biological role of poly(ADP-ribosyl)ation reaction.