The role of Bach1 in ultraviolet A-mediated human heme oxygenase 1 regulation in human skin fibroblasts

Up-regulation of heme oxygenase 1 (HO-1) by ultraviolet A (UVA; 320-380 nm) irradiation of human skin cells protects them against oxidative stress. The role of Nrf2 in up-regulation of HO-1 and other phase II genes is well established. The mechanism underlying Bach1-mediated HO-1 repression is less well understood although cellular localization seems to be crucial. Because prolonged HO-1 overexpression is likely to be detrimental, it is crucial that activation of the gene is transient. We now show that UVA irradiation of cultured human skin fibroblasts enhances accumulation of Bach1 mRNA and protein severalfold. Endogenous Bach1 protein accumulates in the nucleus after 8h and may occupy MARE sites after HO-1 activation thus providing a compensatory mechanism to control HO-1 overexpression. Overexpression of Bach1, together with MafK, represses basal and UVA-mediated HO-1 protein expression, whereas silencing of the Bach1 gene by Bach1-specific siRNAs causes robust enhancement of constitutive HO-1 levels. UVA treatment of cells in which Bach1 has been silenced leads to higher levels of induction of the HO-1 protein. Although Bach1 protein is exported from the nucleus 12h after UVA irradiation, the release of free cellular heme from microsomal heme-containing proteins is immediate rather than delayed. Although heme does promote the export of Bach1 via the Crm1/exportin 1 pathway and is involved in the delayed UVA-mediated export of the protein, it is not clear how this occurs.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture

Ultraviolet A radiation (UVA, 320–400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 μM) or zinc chloride (100 μM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm². In the Comet assay, the tail moment increased by 45% (1J/cm²) to 89% (6J/cm²) in non-supplemented cells (p<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm² (p<0.05). For the highest UVA dose (6J/cm²), Se and Zn were more effective than NAC (p<0.01).     

High levels of zinc ions induce loss of mitochondrial potential and degradation of antiapoptotic Bcl-2 protein in in vitro cultivated human prostate epithelial cells

Prostate epithelial cells contain the highest levels of zinc among all organs and tissues in the human body. Zinc is accumulated primarily in the mitochondria, where it is responsible for inhibition of mitochondrial aconitase activity, thereby increasing citrate production. The present study was designed to clarify the role of zinc for human prostate epithelial cell growth and apoptosis. Apoptosis of in vitro cultivated human prostate epithelial cells exposed to ZnCl(2) was analyzed by determination of phospholipid membrane asymmetry, nuclear fragmentation, DNA strand breaks, changes of mitochondrial potential and cellular pro/antiapoptotic proteins. Zinc induced apoptosis without involvement of p53 by decreasing mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 protein levels in proliferating epithelial cells. Thus, the high local concentrations of zinc ions in the prostatic lumen seem to be necessary to regulate proliferative activities and to enforce epithelial differentiation processes.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

Formation of DNA strand breaks in UV-irradiated human fibroblast preparations

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m2; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m2. Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblast strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m2 indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m2. Moreover, after 25 or 50 J/m2, strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scraping. Permeabilization in situ saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m2, UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.     

Immediate and delayed apoptotic cell death mechanisms: UVA versus UVB and UVC radiation

The mechanism of cell death induced by the different waveband regions of ultraviolet radiation (UVR), i.e., UVA1 (340-400 nm), UVB (290-320 nm) and UVC (200-290 nm) was investigated, using equilethal doses (90% reproductive death) on L5178Y-R murine lymphoma cells. To distinguish between necrosis and apoptosis, the following endpoints were monitored over time using flow cytometry and transmission electron microscopy: percentage of remaining cells, membrane permeabilized cells, dead cells, apoptotic cells, and ultrastructural changes. All waveband regions of UVR were found to cause apoptosis as opposed to necrosis. However, UVA1-induced immediate (0-4 h) apoptosis, while UVB- or UVC-induced delayed apoptosis (<34 h). Moreover, the membrane permeability changes that only result from exposure to UVA1 radiation, especially to red blood cells, suggests that the immediate apoptotic mechanism involves membrane damage. Therefore, the results suggest that there are three death mechanisms available to one cell type: necrosis, immediate apoptosis, and delayed apoptosis (or programmed cell death).     

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.     

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.     

Rufloxacin induced photosensitization in bio-models of increasing complexity.

Rufloxacin belongs to the class of fluoroquinolones that act mainly as specific inhibitors of bacterial Topoisomerase II. These drugs are widely known to be involved in various diseases ranging from cutaneous reactions to aging. The type II photosensitizing activity of Rufloxacin has been already demonstrated on calf thymus DNA and free nucleosides. The aim of this study is to examine in control untreated and UVA irradiated human fibroblasts the modifications on DNA status induced by Rufloxacin added in the culture medium. This allows to investigate the photosensitizing activity of Rufloxacin in a more complex cell model. Fibroblasts, either in the presence or in the absence of Rufloxacin, were exposed to UVA irradiation for different times. An experimental protocol was followed in order to evaluate the amount of single-strand breaks (SSB) and double-strand breaks (DSB) DNA fragmentation by comet assay, and plasmid photocleavage. The presence of oxidized bases was also evaluated using the 8-OH-dGuo test. The comet assay test was also employed to assess cellular repair capacity. The intracellular drug concentration was verified by HPLC-MS. The results confirming the role of Rufloxacin as photosensitizer were: (i) a time-dependent increase in DNA fragmentation when fibroblasts were irradiated in the presence of Rufloxacin; (ii) the efficiency of the cellular repair machinery to be exhaustive after 2 h (whereas no correlation between irradiation time and DNA damage repair was observed with a higher level of DNA fragmentation after shorter irradiation times); (iii) the increased number of cells exhibiting high DNA fragmentation, seen as comets with long tails, was not accompanied by a similar large extent of oxidised DNA base formation, as measured by 8-OH-dGuo analysis; (iv) the double helix SSB, formed in plasmid photosensitization, agreed with the comet assay results, pointing out a good correlation among the cell system and the simpler models used.     

Modulation of exogenous and endogenous levels of thioredoxin in human skin fibroblasts prevents DNA damaging effect of ultraviolet A radiation

Thioredoxin (Trx) plays important biological roles both intra- and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320--380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm(-2) compared to 4.94 au per Joules.cm(-2) for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.     

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.     

Single strand breakage and repair in eukaryotic DNA as assayed by S1 nuclease.

A sensitive new approach for measuring the repair of single strand breaks in DNA induced by low doses of gamma irradiation was tested in cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and in normal cells of early and late passages. The assay is based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation is shown to follow the relation, in F = -(1/Mn - const) - tbeta where F is the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. Mn is the number-average molecular weight of DNA between single strand breaks. beta less than 1 is an empirically determined constant. The results suggest an increase in the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

Chemical radiosensitization by misonidazole: production and repair of DNA single-strand breaks in Yoshida ascites tumor cells.

Formation of strand-breaks in DNA and its repair in Yoshida ascites tumor cells exposed to gamma radiation (100-400 Gy) in presence and absence of misonidazole (10 mM) were studied. The methodology involved pre-labelling of cellular DNA by 3H-thymidine during cell proliferation in rats, irradiation of cells in vitro and analysing sedimentation profile of DNA by ultracentrifugation in alkaline sucrose density gradients. Irradiation under euoxic conditions resulted in formation of about 1.5 times greater number of strand breaks as compared to those formed during irradiation under hypoxic conditions. Misonidazole (10 mM) by its presence along with the cells during irradiation under hypoxic conditions caused a 3-fold increase in the number of single strand breaks, but under euoxic conditions of irradiation the presence of misonidazole did not enhance the strand break formation. Incubation of cells irradiated in absence of misonidazole for 1 hr in tissue culture medium at 37 degrees C resulted in repair of substantial fraction of the strand breaks while there was no repair of the DNA strand breaks in cells irradiated in the presence of the chemical.     

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.     

Method for detecting DNA strand breaks in mammalian cells using the Deinococcus radiodurans PprA protein

In a previous study, we identified the novel protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans. In this study, we focussed on the ability of PprA protein to recognize and bind to double-stranded DNA carrying strand breaks, and attempted to visualize radiation-induced DNA strand breaks in mammalian cultured cells by employing PprA protein using an immunofluorescence technique. Increased PprA protein binding to CHO-K1 nuclei immediately following irradiation suggests the protein is binding to DNA strand breaks. By altering the cell permeabilization conditions, PprA protein binding to CHO-K1 mitochondria, which is probably resulted from DNA strand break immediately following irradiation, was also detected. The method developed and detailed in this study will be useful in evaluating DNA damage responses in cultured cells, and could also be applicable to genotoxic tests in the environmental and pharmaceutical fields.     

Interaction between radiation and drug damage in mammalian cells. V. DNA damage and repair induced in LZ cells by adriamycin and/or radiation

A multi-drug-resistant cell line selected in increasing concentrations of Adriamycin and designated LZ (J. A. Belli, Radiat. Res. 119, 88-100, 1989) is shown to exhibit a survival response characterized by radiation sensitivity and Adriamycin resistance. To determine if this response is due to alterations in either the initial levels of damage induced or the repair of DNA damage, LZ cells and the parental V79 cells were exposed to either radiation or Adriamycin and the damage and repair were measured with alkaline or nondenaturing filter elution. After exposure to radiation, induction and repair of both single-strand and double-strand breaks were equivalent. LZ cells exposed to 100 micrograms/ml Adriamycin for 1 h contained no measurable damage while the same treatment induced breaks and crosslinks in V79 cells. Pretreatment of LZ cells for 1 h with Adriamycin before irradiation did not alter either the initial levels of induced damage or the repair of strand breakage. These results suggest that (1) mechanisms other than differential induction and repair of strand breaks are responsible for the increased radiation sensitivity in LZ, and (2) the lack of Adriamycin-induced DNA damage in LZ is at least partially responsible for the increased cell survival after treatment.     

The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis

Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.