Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Effect of UVC light on growth, incorporation of thymidine, and DNA chain elongation in cells derived from the Indian meal moth and the cabbage looper.

After exposure to 10 or 20 J/m2 UVC light, cells of the UMN-PIE-1181 line, an embryonic cell line derived from the Indian meal moth, Plodia interpunctella, exhibited a rapid and prolonged depression in the rate of incorporation of [3H]thymidine, whereas cells of the TN-368 line, an ovarian cell line derived from Trichoplusia ni, the cabbage looper, showed only a slight drop in incorporation and a rapid recovery after exposure to 10 or 40 J/m2 UVC light. The extent of this depression was not correlated to the amount of cell killing by UVC light in these cell lines or in IAL-PID2 cells. Blockage of fork progression was correlated to the depression in thymidine incorporation. TN-368 cells exhibited little blockage after exposure to 10 or 20 J/m2 UVC light, whereas UMN-PIE-1181 cells exhibited significant blockage at these fluences. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation, or cell killing, indicating that, although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions such as the (6-4) photoproduct may play a role.     

Sensitivity threshold and response characteristics of infrared detection in the beetle Melanophila acuminata (Coleoptera: Buprestidae)

The minimum detection threshold of the infrared sensitive beetle, Melanophila acuminata, was measured with a helium-neon laser that emitted light at a wavelength of 3.39 microm. Extracellular recordings were taken both at the pit organ responsible for detection and at the interganglionic connectives in the thorax of the beetle. At the pit organ, generator and action potentials from single neurons were measured with a sharpened tungsten electrode. At the connectives that linked the fused second meso-/metathoracic and prothoracic ganglia, compound action potentials were measured with a tungsten hook electrode that encircled the connective. The latter recordings confirmed conveyance of infrared information through specific pathways to rostrally-situated sites in the nervous system of the beetle. The 50% probability irradiance threshold at which action potentials were elicited from the receptor and connectives occurred at 17.3 and 14.6 mW/cm(2), respectively. In addition to sensitivity threshold, several other characteristics of the response were quantified including dependence of generator potential latency, generator potential duration, spike frequency, and spike latency on irradiance, dependence of response strength (spike count) on exposure time, and flicker fusion frequency. The ability to detect infrared radiation is rare in nature, and these results provide valuable information necessary to understand this unique sensitivity.     

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S.

The effects of irradiances of 920 and 1200 mW m(-2) (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61 degrees N to 54 degrees S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m(-2) for the lower irradiance and from 4.3 to 25.9 kJ m(-2) for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m(-2) irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose.     

Ultraviolet light exposure triggers nuclear accumulation of p21(WAF1) and accelerated senescence in human normal and nucleotide excision repair-deficient fibroblast strains

Induction of the p21(WAF1) protein (hereafter called p21) following genotoxic stress is known to inhibit proliferating cell nuclear antigen (PCNA)-dependent DNA repair, downregulate apoptosis, and trigger a sustained growth-arrested phenotype called accelerated senescence. Studies with immortalized human and murine cell lines have revealed that exposure to ultraviolet light (UVC; 254 nm) results in the degradation of p21 to facilitate DNA repair and promote cell survival, or may lead to apoptotic cell death. The objective of the present study was to determine whether exposure of non-transformed human fibroblast strains to relatively low fluences of UVC (i.e., fluences typically used in the clonogenic survival assay) might induce sustained nuclear accumulation of p21, leading to accelerated senescence. We have evaluated the responses of normal human fibroblast (NHF) strains and nucleotide excision repair (NER)-deficient fibroblast strains representing xeroderma pigmentosum (XP) complementation groups A and G and Cockayne syndrome (CS) complementation groups A and B. We report that exposure of NHFs to < or =15 J/m(2) of UVC, and NER-deficient fibroblasts to < or =5 J/m(2) of UVC, results in sustained nuclear accumulation of p21 and growth arrest through accelerated senescence. With each fibroblast strain examined, exposure to UVC fluences that resulted in approximately 90% loss of clonogenic potential triggered significant (>60%) accelerated senescence, but only marginal (<5%) apoptosis. We conclude that nuclear accumulation of p21 accompanied by accelerated senescence may be an integral component of the response of human fibroblasts to UVC-induced DNA damage, irrespective of their DNA repair capabilities.     

High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.     

Effect of helium-neon laser radiation on the morphology of experimental allergic contact dermatitis

Clinical and morphological studies of guinea-pig skin in the areas of dermatitis induced by application of dinitrochlorobenzene (DNCB) have shown the decreased skin response to DNCB after treatment with helium-neon laser (wave length 632.8 nm, power density 8-10 mvt /cm2). The ultrastructural signs of cell metabolism activation in the epidermis and derma as well as activation of capillary transport have been discovered in allergic contact dermatitis areas after treatment with helium-neon laser.     

Modulation of human peripheral blood neutrophil apoptosis by ultraviolet C-range radiation and by gamma-irradiation

It was found that the irradiation with in vitro UVC (254 nm) in the dose range of 6-600 J/m2 accelerates the apoptosis of human peripheral blood neutrophils in a dose-dependent manner, with saturation occurring at UVC doses of 250-300 J/m2. gamma-Irradiation with a dose of 2 Gy accelerates the apoptosis of neutrophils, whereas the irradiation with doses of 10 and 20 Gy suppresses it (by 9 h of cultivation). Lipopolysaccharide (1 microgram/ml) suppresses the UVC-induced apoptosis of neutrophils.     

Using ultraviolet light for improved antifouling performance on ship hull coatings

Abstract

A two-part study was designed to investigate the efficacy of using UVC to prevent biofouling in the context of ship hull coatings. The first study determined the frequency of UVC required for a coating that does not have any additives (epoxy). It was found that 1?min/day was effective at preventing hard fouling but not biofilm development. The second study addressed several variables: coating type (epoxy, copper, fouling release), frequency of UVC (no exposure, continuous exposure, 1min/6h, 1?min/day), and distance from the lamp (25 and 50?mm). Continuous UVC exposure resulted in no biofouling settlement but it did damage the copper coating. Intermittent UVC exposure was effective at preventing biofouling recruitment to both the copper and the fouling release coatings. Variations were observed with regards to the fouling composition, especially biofilms, sedimentary tubeworms and barnacles, suggesting tolerances within the community.     

The action of helium-neon laser radiation on the growth of Mycobacterium tuberculosis

The growth properties of M. tuberculosis subjected to the action of helium-neon laser radiation was studied. Laser radiation was shown to change the quantitative and qualitative composition of mycobacterial population. Disturbances in the viability of mycobacteria appear as a consequence of changes in the morphological structure of mycobacterial cells. The maximum effect of helium-neon laser radiation was achieved after the irradiation of M. tuberculosis culture on days 2-3 after inoculation. These results made it possible to suggest that the effect of helium-neon laser radiation was most pronounced in cells at the stage of mitosis (the logarithmic stage of growth) with the highest degree of metabolism.     

Mechanistic investigation of doxycycline photosensitization by picosecond-pulsed and continuous wave laser irradiation of cells in culture

In order to elucidate the photophysical mechanisms of cellular phototoxicity sensitized by doxycycline, MGH-U1 human bladder carcinoma cells in vitro were treated with 20.7 microM doxycycline and irradiated with either a pulsed (lambda = 355 nm, pulse duration = 24 ps) or a continuous wave (lambda = 351 nm) laser. Cumulative radiant exposure and irradiance were systematically varied in experiments with both lasers. Phototoxicity was assessed by epifluorescence microscopy of unfixed cells using rhodamine 123 labeling of mitochondria. With the continuous wave source, the cumulative radiant exposure required for induction of phototoxic injury was independent of irradiance. With the 24-ps-pulsed source, a significantly lower cumulative radiant exposure was required to induce the phototoxicity when the peak irradiance was 5.8 x 10(7) or 1.3 x 10(8) watts cm-2 compared with when peak irradiance was either lower (6.0 x 10(6) watts cm-2) or higher (7.6 x 10(8) watts cm-2). The measured fluorescence lifetimes of doxycycline in buffered saline solution were longer than the laser pulse duration of 24 ps. The increased efficiency of photosensitization at the optimal peak irradiance in the ps domain appears to result from sequential multiphoton absorption involving higher excited states of the singlet manifold. At the highest irradiance studied, on the other hand, reduced efficiency of photosensitization is attributed to increased photodegradation of doxycycline from higher excited states by processes such as photoionization. A model consistent with these observations is presented along with calculations, based on simple rate equations, that fit the essentials of the proposed model.     

Influence of laser light on mycelial growth of Hebeloma mesophaeum and ectomycorrhizal development on Scots pine

 The effects of exposure to helium-neon (He-Ne) or Argon (Ar) laser light (λ=632.8 nm and 514 nm, respectively) on the growth of Hebeloma mesophaeum mycelium in pure culture were studied. Growth rates were highest after exposure to the He-Ne laser (1×60 s)+Ar laser (2×60 s) and to the He-Ne laser for 3×30. Container-grown Pinus sylvestris (pine) seedlings were inoculated with a water suspension of H. mesophaeum mycelium previously exposed to different kinds of laser light. After 3 months, the percentage of mycorrhizal associations on pine roots was 34.3% higher after He-Ne laser treatment and 47.1% higher after Ar laser treatment than in the controls with untreated fungus. However, seedlings infected with treated fungus were smaller than the control. Overall, laser light stimulated growth of H. mesophaeum mycelia in pure culture and enhanced mycorrhizal development on Scots pine seedlings.     

Deregulation of cyclin-dependent kinase 2 activity correlates with UVC-induced apoptosis in Chinese hamster ovary cells

Progression through the cell cycle relies on the activities of cyclin-dependent kinases (Cdk), which in turn are modulated by inhibitory proteins such as p21(waf1/cip1) that are induced when genomic damage occurs. In this study, we show that exposure of normal mammalian cells, such as NIH3T3 fibroblasts, to UVC (25 J/m2, at 254 nm) induces the expression of p21 without causing significant apoptosis, whereas similar treatment of Chinese hamster ovary (CHO-K1) cells with UVC causes apoptosis without inducing p21. The absence of p21 in UV-irradiated CHO-K1 cells is accompanied by the deregulation of Cdk2 activity. The elevation of Cdk2 activity correlates with the increase of UV-induced apoptosis, which can be suppressed by small-molecule Cdk2 inhibitors such as roscovitine and pyrrolidine dithiocarbamate. The results of this study suggest that the deregulation of Cdk2 activity may be critical to UV-induced apoptosis in CHO-K1 cells.     

Effects of exposure of different skin areas to low-power laser light

The effect of helium-neon laser light of extremely low power of 0.2 mW/cm2 and wavelength 632.8 nm on the immune status of mice bearing solid tumors was studied. The evaluation of the status of tumor-bearing animals was provided by taking into account the number of immune cells, cytokine concentration (tumor necrosis factor, interleukin 2, production of nitric oxide, expression of heat shock proteins (Hsp70 and Hsp90), and activity of natural killers. The model of a solid tumor was formed by subcutaneous injection of Ehrlich carcinoma cells, and average life span of tumor-bearing mice achieved about 55 days. Different areas of the skin of tumor-bearing mice were subjected either to a single (1 min, dose 0.012 J/cm3) or repeated exposure to laser light (1 min, 48-h intervals, 30 days). Two different areas were irradiated: the thymus projection area or a hind limb with solid tumors. The results showed that chronic exposure of tumor-bearing mice in the thymus projection area, and especially, hind limb, reduced the resistance, which manifested itself in the acceleration of tumor growth and a tendency of mouse life span to decrease. On the contrary, a single exposure stimulated the antitumor immunity for several days after the exposure. The results show the expediency of further investigation of the immunomodulative effects of low-power laser light and the necessity of monitoring the immune system during laser therapy.     

The relationship of decreased serum thyrotropin and increased colonic temperature in rats exposed to microwaves

Although decreased serum thyrotropin (TSH) concentration has been found to be part of the endocrine response pattern in rats exposed to microwaves and other stimuli, the response of individual endocrine organs was not activated simultaneously by a given irradiance. Therefore, analytical evaluation of the function of endocrine organs individually as well as collectively is required to characterize the extent of biological involvement in microwave exposure. We have studied the changes in TSH concentration in unanesthetized rats exposed to 2.45 GHz amplitude modulated (120 Hz) microwaves in the far field for 2 and 4 h, between 0 and 55 mW/cm2, and from 1 to 10 times to demonstrate any possible cumulation, acclimation, or sensitization process. Ether inhalation was administered to test the responsiveness of TSH in groups of rats that failed to respond to microwave exposure by lowering TSH concentration. In addition, groups of rats were sampled 24 h after microwave exposure to test the persistency of the microwave effect on serum TSH concentration. Results showed that TSH concentration decreased in rats after microwave exposure. Influence of microwave exposure on serum TSH concentration was independent of the number of exposures indicating absence of cumulation, acclimation, or sensitization. The microwave effect on serum TSH could be dependent on duration of exposure. Decreased TSH concentration was usually accompanied by increased colonic temperature. For 4-h exposure, the lowest irradiance was 20 mW/cm2 or a 0.3 degree C increase in colonic temperature independent of the number of exposures. For 2-h exposure, the lowest irradiance was 30 mW/cm2 or a 1.1 degree C increase in colonic temperature regardless of the number of exposures. All the rats exposed at 10 mW/cm2 for 2 h had a lower TSH concentration than those of sham-exposed rats. Occasionally, significant reduction in TSH concentration could not be found in rats exposed to 20 or 25 mW/cm2 for 2 h. None of the rats exposed at an irradiance lower than 10 mW/cm2 had any change in TSH concentration. Failure of change in TSH concentration in response to microwave exposure was not a reflection of a deficiency since these rats responded to ether inhalation by lowering their TSH concentration. The effect of microwave exposure on TSH concentration was not persistent after exposure. The relation between TSH concentration and colonic temperature was curvilinear (exponential). From these results, two mechanisms and their implications for man were discussed.(ABSTRACT TRUNCATED AT 400 WORDS)     

SCYTONEMIN, A CYANOBACTERIAL SHEATH PIGMENT, PROTECTS AGAINST UVC RADIATION: IMPLICATIONS FOR EARLY PHOTOSYNTHETIC LIFE

During the Precambrian, ultraviolet (UV) radiation reaching the Earth's surface, including UVC wavelengths (190–280 nm), was considerably higher than present because of the lack of absorbing gases (e.g. O₂ and O₃) in the atmosphere. High UV flux would have been damaging to photosynthetic organisms exposed to solar radiation. Nevertheless, fossil evidence indicates that cyanobacteria-like ancestors may have evolved as early as 3.5 × 10⁹ yr ago, and were common in shallow marine habitats by 2.5 × 10⁹ years ago. Scytonemin, a cyanobacterial extracellular sheath pigment, strongly absorbs UVC radiation. Exposure to high-irradiance conditions caused cells to synthesize scytonemin and resulted in decreased UVC inhibition of photosynthetic carbon uptake. It was further demonstrated that scytonemin alone was sufficient for substantial protection against UVC damage. This represents the first experimental demonstration of biological protection against UVC radiation in cyanobacteria. These results suggest that scytonemin may have evolved during the Precambrian and allowed colonization of exposed, shallow-water and terrestrial habitats by cyanobacteria or their oxygenic ancestors.     

Effect of low-intensity laser radiation (632.8 nm) on immune cells isolated from mice

The effect of in vitro exposure to low-power laser light with a power density of 0.2 mW/cm2 and a wavelength of 632.8 nm induced by helium-neon laser on the functional activity of macrophages and splenic lymphocytes was studied. If the exposure period did not exceed 60 sec, the stimulation in interleukin-2 (IL-2) and nitric oxide (NO) production, as well as an increase in the activity of natural killer cells were observed. The increase of irradiation dose by prolongation of the exposure duration up to 180 s induced a significant decrease in NO production and natural killer cell activity, but IL-2 production was not different from the control level. A remarkable decrease in interferon-gamma (IFN-gamma) production was observed following laser light exposure of cells for 60 or 180 s, whereas under lower doses (exposure for 5 or 30 sec) IFN-gamma production increased. Irradiation of isolated macrophages induced a significant stimulation of cellular tumor necrosis factor-alpha (TNF- alpha) production at all dboes used, and, what is more important, an enhancement in both TNF-a phaand interleukin-6 (IL-6) production was revealed as early as after a 5-s exposure. In this case, more prolonged exposure periods, 60 and 180 s, either did not induce changes in IL-6 production (in macrophages) or decreased IL-6 production (in lymphocytes). Thus, upon in vitro exposure of cells to extremely low-power laser light, a basic tendency was observed: short-term irradiation predominantly induced stimulation in secretory activity of cells, whereas prolongation of exposure mainly induced immunosuppression. The only exception to the rule was a change in interleukin-3 (IL-3) production, which decreased after short-time exposure, but, on the opposite, increased when the cells were exposed for 180 s. In addition, a high sensitivity to extremely low-power laser light was supported by expression of the inducible heat shock protein, Hsp70, the effect being observed at all doses used, including the exposure for 5 s. At the same time, expression of another heat shock protein, Hsp90, was somewhat reduced after irradiation of cells with laser light.     

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

Demonstration of elevated type I and type III procollagen mRNA levels in cutaneous wounds treated with helium-neon laser. Proposed mechanism for enhanced wound healing

To assess laser modulation of wound healing, full-thickness cutaneous wounds were produced in the backs of pigs, and subjected to treatment with helium-neon laser. For comparison, some wounds were treated with non-laser energy source (a tungsten light) or left untreated as controls. Type I and type III procollagen mRNA levels were determined in the wounds by molecular hybridization with cDNA probes. The results indicated that type I and type III mRNA levels were markedly increased at days 17 and 28 of the healing in wounds treated with He-Ne laser, when compared to control or tungsten light-treated wounds. The results suggest that helium-neon laser stimulates wound healing by enhancing procollagen gene expression. These observations may have relevance to previous clinical studies suggesting that helium-neon laser stimulates wound healing.