COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

Comparative Action Spectrum for Ultraviolet Light Killing of Mouse Melanocytes from Different Genetic Coat Color Backgrounds

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony-forming ability after irradiation. The cell lines were wild-type black (melan-a) and the mutants brown (melan-b) and albino (melan-c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200–280 Dm), 82.3% output at 254 nm, TL01 (UVB, 280–320 nm), 64.2% at 310–311 nm, FS20, broadband with peak output at 312 nm and Alisun-S (UVA, 320–400 nm), broadband with peak output at 350–354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild-type melan-a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan-c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan-b was more sensitive than melan-a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6–4 photoproducts) produced in melan-a than in melan-c cells by UVC, UVB and FS20 lamps. Unlike melan-c, melan-a and melan-b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin-like centers. The contribution of pheome-lanin was higher in melan-b than in melan-a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild-type melan-a melanocytes were well melanized and ellipsoidal, whereas those of melan-b melanocytes tended to be spherical. In the albino line (melan-c) the melanocytes contained only early-stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.     

LONG-WAVELENGTH UVA RADIATION INDUCES OXIDATIVE STRESS,CYTOSKELETAL DAMAGE and HEMOLYSIS*

Abstract— We investigated the ability of the different wavelength regions of UV radiation, UVA(320–400 nm), UVB(290–320 nm) and UVC(200–290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (>340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA 1-exposed samples. The decrease in RCN was dependent on dose(0–1625 kj/m²), time(0–78 h postirradiation) and cell density (10⁶-10⁷ cells/mL). Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy(200–700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.     

Natural UV-Screening Mechanisms of Norway Spruce (Picea abies [L.] Karst.) Neediest

Abstract— Ultraviolet-light screening potential of Norway spruce (Picea abies [L.] Karst.) needles was investigated by UV-spectroscopic, microscopic, fluorescence spectroscopic techniques as well as by HPLC, mass spectrometry and NMR spectroscopy. Results showed four potential barriers of UV screening by Norway spruce needles: (1) UV-light screening via reflectance of UV/violet light by epidermis, (2) UV-light screening via reduction of transmission of UV light by special anatomical arrangement of the epidermal cells containing the UV-screening allomelanins as well as by the light-reflecting hyaline hypodermal cells, (3) conversion of UV light by epidermis into photosynthetically active radiation (PAR; blue and red spectral bands) via fluorescence and (4) UV-light screening by absorption of UV light by UV-screening substances contained in the epidermis, whereby the latter was found to be the most important UV-screening mechanism. Staining of needle cross sections with Naturstoffreagenz A showed the localization of bound flavonoids and its derivatives in the cell walls of the outer epidermal cell layer as revealed by confocal laser scanning microscopy. By fluorescence spectroscopy and confocal laser scanning microscopy, the conversion of UVA light into PAR in the epidermis was related to various UV-screening substances contained in the epidermis. The methanol-soluble UV-absorbing substances were found to create novel UV-screening barrier zones: UVC, >200–253 nm; UVC/UVB, >253–300/303 nm; and UVB/UVA, >300–362/368 nm in epidermis as well as in mesophyll (±vascular bundles) tissues, suggesting the protective functions of epidermis for the underlying mesophyll as well as of mesophyll for the underlying vascular bundles. The following sequence of efficiency of UV-screening barrier zones of the methanol-soluble extracts of the needle epidermis and mesophyll (± vascular bundles) for various UV-spectral bands was detected: UVC screening at less than 265 nm > UVC screening at 265–280 nm > UVB screening at 280–320 nm > UVA screening at 280–320 nm, whereby the UV screening at 280–320 nm was suggested as the most relevant barrier against enhanced UVB radiation. A blend of various UV-screening substances occurred in the methanol-soluble fractions of needle epidermis, whereby p-hydroxybenzoic acid 4-O-β-D-glucopyranoside, picein, (+)-catechin, p-hydroxyacetophenone, benzoic acid and astragalin were identified as UVC/UVB-screening substances; picein, (+)-catechin, astringin, p-hydroxyacetophenone and astragalin(s) as UVB-screening substances and astragalin(s) as UVA/B-screening substances. Alkaline hydrolysis of methanol-insoluble epidermal cell wall fractions released p-coumaric acid, ferulic acid and as-tragalin(s) as major UVB-screening substances. Loss of vitality of Norway spruce trees (forest decline disease) led to a significant reduction of UVB (315 nm)-screening ability of methanol-soluble fractions from epidermis, mesophyll (±vascular bundles) and whole needles. The HPLC analysis showed that the loss of vitality is due to a reduction in accumulation of UVB-absorbing substances, mainly picein, (+)-catechin, isorhapontin and astragalin(s) in the epidermis of needles from the second needle year in accordance with the detected loss of UVB-screening ability. It is concluded that the natural UV-screening mechanisms of Norway spruce needles are highly complex but mainly actively mediated by the ability of methanol-soluble UV-absorbing substances to form variable UVB-AJVA-screening barrier zones and passively by the ability of epidermal cell wall-bound UV-screening substances to screen UV light, whereby in the epidermis a conversion of excess UV light into PAR takes place.     

ACTIVATION OF NF-KB IN HUMAN SKIN FIBROBLASTS BY THE OXIDATIVE STRESS GENERATED BY UVA RADIATION

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)-KB by oxidant stress generated via the UVA (320–380nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF-KB that bound to its recognition sequence in DNA. The UVA radiation-dependent activation of NF-KB in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation-dependent activation of NF-KB appeared to be correlated with membrane damage, and activation could be prevented by a-tocopherol and butylated hydroxytol-uene, agents that inhibited UVA radiation-dependent peroxidation of cell membrane lipids. The activation of NF-KB by the DNA damaging agents UVC (200–290nm) and UVB (290–320nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation-dependent activation of NF-KB over all wavelength ranges examined.     

Photodynamic therapy resistant human colon carcinoma HT29 cells show cross-resistance to UVA but not UVC light

The isolation of photodynamic therapy (PDT)-resistant HT29 human colon adenocarcinoma cells has been reported previously. These PDT-resistant variants show increased expression of the Hsp27 and BNip3 proteins and a decreased expression of mutant p53 protein compared with parental HT29 cells. Because mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. In the present report, we examined the colony survival of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells to ultraviolet light (UV) treatment. The HT29 PDT-resistant variants showed cross-resistance to long-wavelength UVA (320-400 nm) but not to short-wavelength UVC (200-280 nm) light. Cell sensitivity to UVA or UVC was then correlated with Hsp27, BNip3 and mutant p53 protein levels in the PDT-resistant variants as well as in several clonal variants of HT29 cells that express different levels of Hsp27, BNip3 and mutant p53. We show that increased expression of Hsp27 and BNip3 and decreased expression of mutant p53 correlated with increased resistance to UVA. In contrast, increased expression of Hsp27 and BNip3 correlated with increased sensitivity to UVC, whereas increased expression of mutant p53 showed no significant correlation with sensitivity to UVC. These results suggest that the PDT-resistant HT29 cell variants are differentially sensitized to UVA compared with UVC due, in part at least, through the altered expression levels of BNip3, Hsp27 and mutant p53.     

Simultaneous Irradiation with Different Wavelengths of Ultraviolet Light has Synergistic Bactericidal Effect on Vibrio parahaemolyticus

Ultraviolet (UV) irradiation is an increasingly used method of water disinfection. UV rays can be classified by wavelength into UVA (320–400 nm), UVB (280‐320 nm), and UVC (<280 nm). We previously developed UVA sterilization equipment with a UVA light‐emitting diode (LED). The aim of this study was to establish a new water disinfection procedure using the combined irradiation of the UVA‐LED and another UV wavelength. An oxidative DNA product, 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG), increased after irradiation by UVA‐LED alone, and the level of cyclobutane pyrimidine dimers (CPDs) was increased by UVC alone in Vibrio parahaemolyticus. Although sequential irradiation of UVA‐LED and UVC‐induced additional bactericidal effects, simultaneous irradiation with UVA‐LED and UVC‐induced bactericidal synergistic effects. The 8‐OHdG and CPDs production showed no differences between sequential and simultaneous irradiation. Interestingly, the recovery of CPDs was delayed by simultaneous irradiation. The synergistic effect was absent in SOS response‐deficient mutants, such as the recA and lexA strains. Because recA‐ and lexA‐mediated SOS responses have crucial roles in a DNA repair pathway, the synergistic bactericidal effect produced by the simultaneous irradiation could depend on the suppression of the CPDs repair. The simultaneous irradiation of UVA‐LED and UVC is a candidate new procedure for effective water disinfection.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

In vitro DEMONSTRATION OF A FUNDAMENTAL DIFFERENCE IN THE PROLIFERATION OF MURINE and HUMAN BONE MARROW and LYMPHOCYTES FOLLOWING ULTRAVIOLET IRRADIATION: RELEVANCE TO BONE MARROW TRANSPLANTATION

Exposure of rodent allogeneic donor marrow and splenocyte grafts to ultraviolet radiation (UVR) has been shown to permit durable engraftment at doses that abolish graft-versus-host disease (GVHD) and graft rejection. We have compared both murine and human alloreactive and mitogen-induced lymphoid responses and bone marrow proliferation in mixed lymphocyte culture (MLC), phy-tohemagglutinin (PHA)-induced proliferation and colony-forming unit-granulocyte/macrophage (CFU-GM) assays using germicidal UVC (200–290nm), broadband and narrowband UVB (290–320nm) and UVA (320^100 nm) sources. Our data show a wavelength and dose-dependent reduction in lymphoid proliferation in the mouse with CFU-GM survival of50–75% of control at doses required to abolish allogeneic lymphocyte responses for all lamps. In contrast, human lymphocyte responses are more resistant to UVC with CFU-GM proliferation reduced to zero when allostimulation is abolished. Mito-gen-induced lymphoid responses show a similar wavelength-dependent sensitivity. Abolition of response in MLC using UV-irradiated stimulator cells was less sensitive than proliferation with UV-irradiated responder cells at all wavelengths in both species. With all sources, murine CFU-GM proliferation is less susceptible to UVR than human marrow at doses required to abolish lymphoid responses.     

Bystander Me45 Melanoma Cells Increase Damaging Effect in UVC‐irradiated Cells

The aim of our study was to investigate the possible mechanism(s) of the bystander effect induced by UVC light in malignant melanoma Me45 cells that were co‐incubated with irradiated cells of the same line. We have found that the UVC band effectively generated apoptosis, premature senescence, single and double DNA strand breaks and reduced clonogenic survival of bystander cells. However, in the feedback response, the bystander cells intensified damage in directly irradiated cells, especially seen at the level of apoptosis and survival of clonogenic cells. Pretreatment of bystander cells with inhibitor of inducible nitric oxide synthase blocks this signaling. It seems that the mediators of this phenomenon produced and secreted by neighboring cells are superoxide, nitric oxide and TGF‐β. The reverse deleterious effect caused by cells not exposed to UVC in directly exposed cells is opposed to the protective/rescue effect exerted by the bystander cells in the case of ionizing radiation known in the literature. Whether this opposite adverse effect is a feature of only Me45 melanoma cells or whether it is a general phenomenon occurring between cells of other types exposed to ultraviolet radiation requires further research.     

The Monodelphis melanoma model: initial report on large ultraviolet A exposures of suckling young

The objective of this study was to determine whether exposure of early suckling young of the opossum Monodelphis domestica to ultraviolet A (UVA) radiation (320-400 nm) can lead to the development of melanocytic lesions similar to those induced after exposure to ultraviolet B (UVB) radiation (280-320 nm) to total doses as low as 380 J/m2. A total of 576 sucklings received nine exposures of 0.6, 2.6 or 15.5 kJ/m2 per dose (total doses approximately 6, 23 and 140 kJ/m2, respectively) from a Blak Ray lamp source with a narrow range emission at 365 nm. A further 280 sucklings were exposed in the same way to doses of 2.6 kJ/m2 per dose (total approximately 23 kJ/m2) broad-band UVA with visible wavelengths from a Dermalight lamp. Frequency of litter loss following all of the UVA-exposure protocols was similar to that within the same stocks in the colony at large. Only one of the 856 UVA-exposed individuals possessed a melanocytic lesion at the 5 month assessment point. No radiation-induced lesions of any type were evident on the skin of the other animals exposed as sucklings. The affected male was from a group of 70 individuals exposed to the highest total dose (140 kJ/m2) from the Blak Ray light source. The melanocytic hyperplasia was provisionally identified as a potential melanoma but it slowly regressed as the animal aged. We conclude that in the opossum suckling exposure system, the potency of UVA for melanoma induction is extremely low compared with that of UVB. Possible explanations, amenable to further investigations, are given for the low UVA sensitivity of the suckling model compared to the adult exposure model of Ley (Ley, R. D. [1997] Cancer Res. 57, 3682-3684).     

ENDOGENOUS GLUTATHIONE PROTECTS HUMAN SKIN FIBROBLASTS AGAINST THE CYTOTOXIC ACTION OF UVB, UVA AND NEAR-VISIBLE RADIATIONS

Both the UVB (290-320 nm) and UVA (320-380 nm) regions of sunlight damage human skin cells but, particularly at the longer wavelengths, information is scant concerning the mechanism(s) of damage induction and the roles of cellular defense mechanisms. Following extensive glutathione depletion of cultured human skin fibroblasts, the cells become strongly sensitized to the cytotoxic action of near-visible (405 nm), UVA (334 nm, 365 nm) and UVB (313 nm) but not UVC (254 nm) radiations. In the critical UVB region, the magnitude of the protection afforded by endogenous glutathione approaches that of the protection provided by excision repair. The results suggest that a significant fraction of even UVB damage can be mediated by free radical attack and that a major role of glutathione in human skin cells is to protect them from the cytotoxic action of sunlight.     

INDUCTION OF phr GENE EXPRESSION BY PYRIMIDINE DIMERS IN Escherichia coli

The photoreactivating enzyme (PRE) is concerned with mainly two kinds of light wavelength. The PRE splits UVC (254 nm)-induced pyrimidine dimer by absorbing UVA (320–380 nm) or visible light in its chromophore. The present paper demonstrates that the phr gene expression was efficiently induced in an excision defective strain (uvrA∼) after irradiation by UVC and UVB (290-320 nm), but not by UVA and visible light. In addition, the induced activity was significantly depressed by irradiation with UVA and visible light. Therefore we conclude that the phr gene expression can be induced by pyrimidine dimers.     

INHIBITION OF DNA REPAIR SYNTHESIS BY SUNLIGHT

Abstract— DNA repair synthesis as determined by thymidine incorporation in the presence of hydroxyurea reached a much lower maximum level after solar compared with UVC exposure in five human melanoma cell lines, in HeLa cells, and in two human fibroblast strains. This finding was confirmed by determination of unscheduled DNA synthesis where both the number of labelled nuclei and grain count per nucleus were lower in sun-exposed cells. In a cloned human melanoma line (MM253cl), glass-filtered sunlight inhibited UVC repair synthesis, and solar UVB alone induced a higher level of repair synthesis than either complete sun or solar UVA plus solar UVB. The fluence response of filtered sunlight for inhibition of UVB (sunlamp) and UVC showed that most inhibition was obtained at low fluences (5-10 min), further exposure giving a plateau at 40% of the original level. Ultraviolet C and sunlight inactivated adenovirus 5 giving F ₀ values for virus survival 40-fold higher than for cell survival. Replication of either UVC- or solar-irradiated virus was not affected by prior irradiation of cells with glass-filtered sunlight. Stathmokinetic analysis of cell cycle progression by DNA flow cytometry showed that UVC and sunlamp UVB retarded cell movement from the G1 and S phases whereas equitoxic sunlight and glass-filtered sunlight (nontoxic) had no effect. These results indicate that solar UVA at low environmental fluences partially inhibits UVB repair synthesis in a range of human cell types but does not affect the replication of a UVB- or UVC-damaged virus when applied to the genome alone or to the host cell.     

Effect of cyclobutane pyrimidine dimers on apoptosis induced by different wavelengths of UV.

Ultraviolet radiation within three different wavelength ranges, UVA (340-400 nm), UVB (290-320 nm) or UVC (200-290 nm), was shown to induce apoptosis in OCP13 cells, derived from the medaka fish. Morphological changes such as cell shrinkage and a decrease in the number of nucleoli appeared 4 h after UVA, UVB or UVC irradiation, although with different relative efficiencies. Doses required to induce apoptosis with similar efficiencies were about 2500-fold higher for UVA and 10-fold higher for UVB than for UVC. The following phenomena occurred after UVA irradiation but not after UVB or UVC irradiation. (1) Ultraviolet-A-induced cell detachment occurred with or without cycloheximide pretreatment. (2) Cells attached to plastic showed morphological changes such as rounding up of nuclei without a change in the cell distribution. (3) Morphological changes after UVA irradiation could not be evaded by photorepair treatment. (4) Morphological changes did not occur in cells attached to glass coverslips but only those in plastic dishes. (5) Apoptosis occurred without detectable increase of caspase-3-like activity. (6) Morphological changes were inhibited by N-acetylcysteine, a scavenger of active oxygen species. These results suggest the existence of two different pathways leading to apoptosis, one for long- (UVA) and the other for short- (UVB or UVC) wavelength radiation.     

SPECTRAL DEPENDENCE OF UV-INDUCED IMMEDIATE AND DELAYED APOPTOSIS: THE ROLE OF MEMBRANE AND DNA DAMAGE

Abstract— The phototoxicity of each waveband region of UV radiation (UVR), i.e., UVA (32CM100 nm), UVB (290–320 nm) and UVC (200–290 nm), was correlated with an apoptotic mechanism using equilethal doses (10% survival) on murine lymphoma L5178Y-R cells. Apoptosis was qualitatively monitored for DNA "ladder" formation (multiples of 200 base pair units) using agarose gel electrophoresis, while the percentages of apoptotic and membrane-permeabilized cells were quantified over a postexposure time course using flow cytometry. The UVA1 radiation (340–400 nm) induced both an immediate (<4 h) and a delayed (>20 h) apoptotic mechanism, while UVB or UVC radiation induced only the delayed mechanism. The role of membrane damage was examined using a lipophilic free-radical scavenger, vitamin E. Immediate apoptosis and membrane permeability increased in a UVA1 dose-dependent manner, both of which were reduced by vitamin E. However, vitamin E had little effect on UVR-induced delayed apoptosis. In contrast, the DNA damaging agents 2,4- and 2,6-diaminotoluene exclusively induced delayed apoptosis. Thus, immediate apoptosis can be initiated by UVA 1-induced membrane damage, while delayed apoptosis can be initiated by DNA damage. Moreover, the results suggest that immediate and delayed apoptosis are two independent mechanisms that exist beyond the realm of photobiology.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: SEDIMENTATION STUDIES WITH THE USE OF 5''-BROMODEOXYURIDINE PHOTOLYSIS

Abstract Relative to their L5178Y-S counterparts, L5178Y-R cells have an impaired capacity to form patches in DNA after exposure to UVC radiation. The photolysis of 5'-bromodeoxyuridine (BrdUrd) incorporated into DNA was used to estimate the number of 'repair patches'formed in response to a 254 nm UV (UVC) exposure. L5178Y-S cells, typical of rodent cell lines, formed a small number of patches in exposed DNA (1-2 patches per 1 times 10⁸ dalton during a 6 h recovery after an exposure of 20 J/m²). In contrast, DNA extracted from L5178Y-R cells exposed to UVC and subsequently incubated with BrdUrd for 6 h showed no evidence of BrdUrd incorporation indicating no capacity to form sites of repair (fewer than 0.5 sites of BrdUrd incorporation per 1 times 10⁸ dalton). Moreover, in L5178Y-R cells high fluences of UVC caused an extensive DNA degradation. Such degradation was not observed in L5178Y-S cells during the 24-h post-exposure period. These results are consistent with the notion that L5178Y-R cells have a reduced capacity to repair DNA damage induced by UVC radiation.     

DIFFERENTIAL SENSITIVITY TO INACTIVATION OF NUR AND NUR+ STRAINS OF ESCHERICHIA COLI AT SIX SELECTED WAVELENGTHS IN THE UVA,UVB AND UVC RANGES*

The radiation response of stationary-phase cells of Escherichia coli strains RT4 (nur⁺) and RT2 (nur) was measured at 6 selected wavelengths between 254 and 405 ran. The relative response of the nur⁺. and nur strains was almost the same at 254 and 290 nm. However, the differential sensitivity of the RT4 and RT2 strains (ratio of the initial F₃₇ values of the nur⁺ to the nur strains) was 2.7 at 313 nm, 3.2 at 334 nm, 3.1 at 365 nm, and 2.3 at 405 nm. Thus, the fluence enhancing effect of the nur genotype extends over the wavelength range of approximately 300 to 420 nm. The substantial effect of nur at 313 nm strongly suggests that the increased sensitivity of the nur strain is the consequence of a repair deficiency that reduces the efficiency of mending DNA lesions produced by UVA (320–400 nm) and UVB (290–320 nm), but not UVC (200–290 nm) radiation.