Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.     

Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro

The main lesion produced in DNA by UV-C irradiation of spores of Bacillus subtilis is 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP). Using high-performance liquid chromatography-mass spectrometry analysis of the photoproducts, we showed that in wild-type B. subtilis spores (1) UV-C irradiation generates almost exclusively SP with little if any CPD and 6-4PP; (2) the SP generated is approximately 99% of the intrastrand derivative, but approximately 1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV-C irradiation of spores lacking the majority of their alpha/beta-type SASP gave less SP than with wild-type spores and significant levels of CPD and 6-4PP. The binding of an alpha/beta-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6-4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.     

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.     

Naringenin protects HaCaT human keratinocytes against UVB-induced apoptosis and enhances the removal of cyclobutane pyrimidine dimers from the genome

Many naturally occurring agents are believed to protect against UV-induced skin damage. In this study, we have investigated the effects of naringenin (NG), a naturally occurring citrus flavonone, on the removal of UVB-induced cyclobutane pyrimidine dimers (CPD) from the genome and apoptosis in immortalized p53-mutant human keratinocyte HaCaT cells. The colony-forming assay shows that treatment with NG significantly increases long-term cell survival after UVB irradiation. NG treatment also protects the cells from UVB-induced apoptosis, as indicated by the absence of the 180 base pair DNA ladders and the appearance of sub-G1 peak using agarose gel electrophoresis and flow cytometric analysis, respectively. The UVB-induced poly (ADP-ribose) polymerase-1 (PARP-1) cleavage, caspase activation and Bax/Bcl2 ratio were modulated following NG treatment, indicating an antiapoptotic effect of NG in UVB-damaged cells that occurs at least in part via caspase cascade pathway. Moreover, treatment of UVB-irradiated HaCaT cells with NG enhances the removal of CPD from the genome, as observed by both direct quantitation of CPD in genomic DNA and immuno-localization of the damage within the nuclei. The study provides a molecular basis for the action of NG as a promising natural flavonoid in preventing skin aging and carcinogenesis.     

Circadian rhythms of resistance to UV-C and UV-B radiation in Euglena as related to "escape from light" and "resistance to light"

Radiation-induced stress, either from visible or UV light, is strongest at midday. We found that, in the absence of stress or time cues, Euglena gracilis Z was the most resistant to UV-C and UV-B at subjective midday, whether judged from immediate or reproductive survival. The circadian UV-resistance rhythms were free-running in stationary cultures under 1-h light/1-h dark cycles or continuous darkness, indicating that cell-cycle dependent DNA susceptibility to UV was not involved. We moreover examined what was the primary cause of the circadian UV resistance, estimated as the immediate cell survival. The half-maximal lethal dose (LD(50)) of UV-C at subjective midday (the most resistant phase) was 156 J/m(2), which is approximately 3-fold that at subjective midnight. The same was true for UV-B, except the LD(50) was approximately 13-fold that of UV-C. Temperature during UV irradiation had little effect, indicating that survival was not mediated via enzymatic reactions. Non-enzymatic antioxidants were added 5 min before UV irradiation. Dimethylsulfoxide (a hydroxyl radical scavenger) increased survival after UV-B, but had little effect after UV-C; conversely, sodium ascorbate increased survival after UV-C, but not after UV-B. These findings suggest that circadian rhythms of resistance to UVs involve a common mechanism for maximizing non-enzymatic antioxidative capacity at subjective midday, but the specific antioxidants differ.     

Biological Impact of Shorter Wavelength Ultraviolet Radiation-C†

Life on earth has constantly coped with the impact of solar radiation, especially solar ultraviolet radiation (solar UV). Various biological mechanisms protect us from solar UV. New devices emitting shorter wavelengths UV-C, i.e. <254 nm emitted by conventional UV germicidal lamps, have emerged. These shorter wavelength UV-C emitting devices are useful for various purposes, including microorganism inactivation. However, as solar UV-C does not reach the earth surface, biological impacts of UV-C has been studied using 254 nm germicidal lamps, and those using shorter wavelength UV-C is rarely known. To balance the utility and risk of UV-C, the biological effect of these new UV-C emitting devices must be investigated. In addition, our knowledge of biological impacts of the wavelength-dependent entire UV (100–400 nm) must be enhanced. In this review, we briefly summarize the biological impacts of shorter wavelength UV-C. Mechanisms of UV-C-induced cellular damage and factors affecting the microorganism inactivation efficiency of UV-C have been discussed. In addition, we theoretically estimate the probable photocarcinogenic action spectrum of shorter wavelength UV-C. We propose that increasing the knowledge on UV-C will facilitate the adoption of shorter wavelength UV-C emitting new devices in an optimal and appropriate manner.     

Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.     

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 μmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.     

Organic/inorganic double-layered shells for multiple cytoprotection of individual living cells

The cytoprotection of individual living cells under in vitro and daily-life conditions is a prerequisite for various cell-based applications including cell therapy, cell-based sensors, regenerative medicine, and even the food industry. In this work, we use a cytocompatible two-step process to encapsulate Saccharomyces cerevisiae in a highly uniform nanometric (<100 nm) shell composed of organic poly(norepinephrine) and inorganic silica layers. The resulting cell-in-shell structure acquires multiple resistance against lytic enzyme, desiccation, and UV-C irradiation. In addition to the UV-C filtering effect of the double-layered shell, the biochemical responses of the encapsulated yeast are suggested to contribute to the observed UV-C tolerance. This work offers a chemical tool for cytoprotecting individual living cells under multiple stresses and also for studying biochemical behavior at the cellular level.     

RECOVERY OF VIRAL CAPACITY IN IRRADIATED EXPONENTIALLY GROWING CELLS

Exponentially growing cells of the PtK-2 line (ATCC No. CCL56, from the marsupial Potorous tridactylus) require protein and RNA synthesis in a limited period following UV-radiation damage for optimal recovery as colony formers [Overberg et al. (1988) Mutat. Res. 194, 83-92]. Overall behavior suggests the operation of damage-induced recovery processes. The capacity of confluent cell monolayers for infection with unirradiated herpes simplex virus 1 (HSV-1) is sharply reduced by UV-irradiation. We have followed capacity changes in exponentially growing cells after irradiation and varying amounts of photoreactivation by means of an infectious center assay. These changes closely parallel changes of colony formation. Spontaneous recovery of capacity in the dark occurs over approximately the same time period that the UV sensitivity of colony formation depends on macromolecular synthesis. The effect of photoreactivation is complementary rather than additive to this recovery, suggesting that the dark recovery in this period concerns pyrimidine dimers in cell DNA.     

INHIBITION OF TRANSIENT GENE EXPRESSION IN CHINESE, HAMSTER OVARY CELLS BY TRIPLET-SENSITIZED UV-B IRRADIATION OF TRANSFECTED DNA

The biological effectiveness of thymine-thymine cyclobutane dimers specifically induced by photosensitized ultraviolet-B irradiation was analyzed by host-cell reactivation of triplet-sensitized, UV-B irradiated plasmid pRSV beta gal DNA transfected into normal and repair-deficient Chinese hamster ovary cells. For comparison, pRSV beta gal DNA was also UV-C irradiated and transfected into the same cell lines. Ultraviolet endonuclease-sensitive site induction was determined after UV-C irradiation or acetophenone-sensitized UV-B irradiation of plasmid pRSV beta gal DNA. These data were used to calculate the number of cyclobutane pyrimidine dimers required to inactivate expression of the lacZ reporter gene in each irradiation condition. Transfection with UV-C-irradiated plasmid DNA resulted in a significantly greater reduction of reporter gene expression than did transfection with acetophenone-sensitized UV-B-irradiated pRSV beta gal DNA at equivalent induction of enzyme-sensitive sites. Since only a fraction of the inhibition could be accounted for by noncyclobutane dimer photoproducts, these results suggest that cytosine-containing pyrimidine cyclobutane dimers may be more effective than thymine-thymine dimers in inhibiting transient gene expression as measured in such host-cell reactivation experiments in mammalian cells.     

ISOLATION OF V79 FIBROBLAST CELL LINES CONTAINING ELEVATED METALLOTHIONEIN LEVELS THAT HAVE INCREASED RESISTANCE TO THE CYTOTOXIC EFFECTS OF ULTRAVIOLET-A RADIATION

Abstract Isolated clones of V79 Chinese hamster lung fibroblasts, selected for resistance against cadmium toxicity, were exposed to monochromatic 365 nm ultraviolet-A (UVA, 320 nm to visible light) radiation and examined for cell survival. All three of the Cd-resistant V79 clones (V79Cd) tested exhibited significant increases in survival after irradiation compared with control cultures similar to the increased survival observed in Zn acetate-induced V79 cells. Dose-modifying factors calculated for these survival experiments were all approximately 1.5. When characterized for steady-state levels of metallothionein (MT) mRNA and associated Cd-binding activity, all of the Cd-resistant V79Cd clones demonstrated elevated constitutive levels of both, implicating MT as the mechanism responsible for the observed cellular resistance to Cd and also to 365 nm UVA radiation. However, whereas levels of intracellular MT protein correlated with differences in survival against Cd, MT intracellular levels did not correlate well with protection against 365 nm UVA. Increased cell survival after exposure to 365 nm UVA radiation mediated by MT appeared to reach a threshold level and MT only provided a limited degree of protection. Since UVA radiation is known to cause cell death mediated through the intracellular generation of reactive oxygen species (ROS), these results suggest that the role of MT in ameliorating cellular photooxidative damage produced by UVA is by reducing intracellular ROS.     

Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose-dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 +/- 8, 36 +/- 10, 57 +/- 12 and 80 +/- 10, and total percent dermal cells positive for CPD ranged from 1 +/- 1, 7 +/- 3, 16 +/- 3 and 20 +/- 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.     

Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens

The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (10(8) cells per mL) were incubated with hypericin (0-40 μM) for 30 min and followed by light irradiation of 600-800 nm at 5-30 J cm(-2). Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.     

Human BCL-2 Expression Delays Ultraviolet-Induced Apoptosis in Marsupial Cells

We have introduced the human bcl-2 gene under the control of the human metallothionein MTII_A promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl-2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV-induced apoptosis. Overexpression of Bcl-2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl-2 did not impose a definitive block for cell death. The elimination of cyclobutane py-rimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl-2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl-2 in the cell. Therefore, although the human Bcl-2 protein can delay the onset of UV-induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl-2 action, lead the cell to a state committed to die.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.     

UVB dose-toxicity thresholds and steady-state DNA-photoproduct levels during chronic irradiation of inbred Xenopus laevis tadpoles

Environmental stressors that severely impact some species more than others can alter ecosystems and threaten biodiversity. Genotoxic stress, such as solar UV-B irradiance, may induce levels of DNA damage at rates that exceed repair capacities in some species but remain below repair capacities in other species. Repair rates would seem to establish toxicity thresholds. We used inbred Xenopus laevis tadpoles in the laboratory to test the hypothesis that balances between rates of induction of cyclobutane pyrimidine dimers (CPDs; the major UV-B photoproduct in DNA) and rates of CPD removal (repair) can determine UV-B toxicity thresholds. As rates of chronic UV-B irradiance were progressively increased by decreased shielding of lamps, survival decreased sharply over a relatively narrow range of dose rates. Apparent toxicity thresholds were associated with large increases in steady-state CPD levels. Induction at twice the measured removal (repair) rate produced sustained high CPDs and 100% mortality. Induction at one-half the removal rate resulted in negligible CPD levels and low mortality. Increased intensity of visible radiation available to drive CPD photoreactivation, mimicking interspecies variation in DNA repair capacity, reduced steady-state CPD levels and increased survival at UV-B dose rates that were previously toxic, resulting in increased thresholds of apparent toxicity. We suggest that threshold effects due to DNA repair should generally be considered in assessments of effects of genotoxic agents on species-specific population decreases and human health risks.