Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Ways of apoptosis development in human lymphocytes, induced by UV-irradiation

The level of DNA damage and cytochrome c content in human lymphocytes in the dynamics of apoptosis induced by UV light (240?C390 nm) at doses of 151, 1510 and 3020 J/m² is studied. DNA fragmentation is revealed in 20 h after UV irradiation of lymphocytes at doses mentioned above. It is shown that DNA damages (single strand breaks) appear immediately after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² (comets of C1 type) and reach their maximum 6 h after cell modification (comets of C2 and C3 types). It is concluded that p53-dependent and receptor caspase pathways are involved in apoptosis development in the human lymphocytes, modified after UV irradiation.     

Fragmentation of DNA lymphocytes the human in dynamics of development apoptosis, induced influence of UV-radiation and reactive oxygen species

It is established that UV-light (240-390 nm) in doses of 151, 1510 and 3020 J/m2 and reactive oxygen species and singlet oxygen induce DNA fragmentation lymphocytes cells of the human 20 h after influence. Using a method of DNA-comets it is revealed that DNA damages (single strand breaks) are found out right after UV-irradiations of lymphocytes in doses of 1510 and 3020 J/m2 and additions hydrogen peroxide in concentration of 10-6 mol/l (a comet of type C1) and reach a maximum through 6 h after influence on of cells UV-light and ROS (comets of types C2 and C3). Assumption about the leading part of a p53-dependent way in realization apoptosis human lymphocytes in the conditions of influence of UV-light and reactive oxygen species is put forward.     

Apoptosis and necrosis induced by ultraviolet radiation in the presence of autologous plasma

This paper deals with the effect of ultraviolet (UV) light (240–390 nm) at doses of 151–3020 J/m²* on the character of the death of donors’ blood lymphocytic cells (with the use of markers of apoptotic and necrotic cell death) and on the level of the CD95 receptor—a marker of apoptotic predisposition. UV irradiation has been shown to increase the expression of CD95 receptors, which is due mainly to synthesis de novo. It is revealed that over the course of the 24-h incubation of photomodified lymphocytes (at irradiation doses of 151 and 755 J/m²) without autologous blood plasma, cell death occurs by receptor-mediated apoptosis. Action of high irradiation doses (1510 and 3020 J/m²) leads to the massive necrotic death of immunocytes. The use of autologous blood plasma during the incubation of photomodified lymphocytes allows a reduction in the amount of both apoptotic and necrotic cells.     

Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

Green light effects on biological systems: a new biophysical phenomenon

This paper reports a new phenomenon connected with the influence of green light (GL) on biological systems. Our experiments have revealed an antioxidant effect of GL on cells subjected to lethal doses of UV at the cellular level and a protective effect of GL on DNA denatured by UV, coupled with a structural modification of DNA macromolecules under GL irradiation, at the molecular level. Mouse melanocyte cultures are subjected to UV irradiations with L₅₀ fluxes of 16.0 J m^− 2 s^− 1. GL is obtained from a strontium aluminate pigment, which emits GL under UV activation. Cells grown in GL, prior to UV irradiation, present a clear surprising protective effect with surviving values close to the controls. A GL antioxidant effect is suggested to be mediated through GL influence on cellular water cluster dynamics. To test this hypothesis, reactive oxygen species (ROS) are determined in cell cultures. The results revealed a decrease of cellular ROS generation in the UV-irradiated samples protected by a previous 24 h of GL irradiation. At the DNA level, the same type of GL protection against UV damage is recorded by gel electrophoresis and by UV spectroscopy of the irradiated DNA molecules. Two physical methods, impedance spectroscopy and chronoamperometry, have revealed at the level of GL-irradiated DNA molecules spectral modifications that correlate with the UV spectroscopy results. The interaction between the chargeless photons and the field of water molecules from the cellular compartments is discussed in relation with the new field of macroscopic quantum coherence phenomena.     

Ultraviolet B and A irradiation induces fibromodulin expression in human fibroblasts in vitro

Ultraviolet (UV) radiation affects the extracellular matrix (ECM) of the human skin. The small leucine-rich repeat protein fibromodulin interacts with type I and II collagen fibrils, thereby affecting ECM assembly. The aim of this study was to evaluate whether short wave UV (UVB) or long wave UV (UVA) irradiation influences fibromodulin expression. Exponentially growing human fibroblasts (IMR-90 cells) were exposed to increasing doses of UVB (2.5–60 mJ/cm²) or UVA (0.5–10 J/cm²). After UV irradiation fibromodulin, p21 and GADD45 levels were evaluated as well as cell viability, reactive oxygen species formation (ROS) and DNA damage. We found that fibromodulin expression: (i) increased after UVB and UVA irradiation; (ii) was 10-fold higher after UVA (10 J/cm²) versus 5-fold with UVB (10 mJ/cm²); (iii) correlated with reactive oxygen species formation, particularly after UVA; and (iv) was linked to the DNA damage binding protein (DDB1) translocation in the nucleus, particularly after UVB. These results further suggest that the UV-induced fibromodulin increase could counteract the UV-induced connective tissue damage, promoting the assembly of new collagen fibrils.     

The formation of DNA single-strand breaks and alkali-labile sites in human blood lymphocytes exposed to 365-nm UVA radiation

The potency of UVA radiation, representing 90% of solar UV light reaching the earth׳s surface, to induce human skin cancer is the subject of continuing controversy. This study was undertaken to investigate the role of reactive oxygen species in DNA damage produced by the exposure of human cells to UVA radiation. This knowledge is important for better understanding of UV-induced carcinogenesis. We measured DNA single-strand breaks and alkali-labile sites in human lymphocytes exposed ex vivo to various doses of 365-nm UV photons compared to X-rays and hydrogen peroxide using the comet assay. We demonstrated that the UVA-induced DNA damage increased in a linear dose-dependent manner. The rate of DNA single-strand breaks and alkali-labile sites after exposure to 1 J/cm² was similar to the rate induced by exposure to 1 Gy of X-rays or 25 μM hydrogen peroxide. The presence of either the hydroxyl radical scavenger dimethyl sulfoxide or the singlet oxygen quencher sodium azide resulted in a significant reduction in the UVA-induced DNA damage, suggesting a role for these reactive oxygen species in mediating UVA-induced DNA single-strand breaks and alkali-labile sites. We also showed that chromatin relaxation due to hypertonic conditions resulted in increased damage in both untreated and UVA-treated cells. The effect was the most significant in the presence of 0.5 M Na⁺, implying a role for histone H1. Our data suggest that the majority of DNA single-strand breaks and alkali-labile sites after exposure of human lymphocytes to UVA are produced by reactive oxygen species (the hydroxyl radical and singlet oxygen) and that the state of chromatin may substantially contribute to the outcome of such exposures.     

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m² there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m² the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m² did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.     

Complex repair kinetics of DNA strand breaks induced by γ-rays or UV radiation in Ehrlich ascites tumour cells

Fluorometric analysis of DNA unwinding (FADU) – a sensitive technique for the detection of strand breaks in DNA – has been modified and used for the detailed investigation of repair kinetics of DNA-strand breaks arising under different conditions in Ehrlich ascites tumour (EAT) cells irradiated by γ-rays or ultraviolet (UV) radiation. The repair kinetics of DNA-strand breaks induced in EAT cells by γ-radiation was measured at radiation doses of 8, 20 and 50 Gy. We found complex repair curves in all cases, probably reflecting the combined processes of break rejoining and break generation during repair. In order to affect the above-mentioned processes, we have used different conditions of repair and different types of radiation. Lowering of the temperature of incubation and treating the cells by 5-fluoro-2′-deoxyuridine (FUdR) lead to complex changes of the repair curve with a reduced ``wave' pattern. In order to change the type of damage to DNA, we used UV radiation (254 nm, 10 and 20 J/m²). Detailed studies of the repair kinetics showed that the repair curve for 10 J/m² had a second maximum within 70 min after irradiation. Received: 17 May 1995 / Accepted in revised form: 15 March 1996     

Role of mitochondria in ultraviolet‐induced oxidative stress

The biological effects of ultraviolet radiation (UV), such as DNA damage, mutagenesis, cellular aging, and carcinogenesis, are in part mediated by reactive oxygen species (ROS). The major intracellular ROS intermediate is hydrogen peroxide, which is synthesized from superoxide anion (^•O₂⁻) and further metabolized into the highly reactive hydroxyl radical. In this study, we examined the involvement of mitochondria in the UV‐induced H₂O₂ accumulation in a keratinocyte cell line HaCaT. Respiratory chain blockers (cyanide‐p‐trifluoromethoxy‐phenylhydrazone and oligomycin) and the complex II inhibitor (theonyltrifluoroacetone) prevented H₂O₂ accumulation after UV. Antimycin A that inhibits electron flow from mitochondrial complex III to complex IV increased the UV‐induced H₂O₂ synthesis. The same effect was seen after incubation with rotenone, which blocks electron flow from NADH‐reductase (complex I) to ubiquinone. UV irradiation did not affect mitochondrial transmembrane potential (ΔΨ_m). These data indicate that UV‐induced ROS are produced at complex III via complex II (succinate‐Q‐reductase). J. Cell. Biochem. 80:216–222, 2000. © 2000 Wiley‐Liss, Inc.     

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Extreme ultraviolet radiation-sensitivity of respiratory adaptation in Saccharomyces cerevisiae cells during transition.

The respiratory adaptation process in both wild-type and UV-sensitive strains of $\text{Saccharomyces}\text{cerevisiae}$ was sensitive to small doses of UV-radiation (10 and 0.7 J/m², respectively). These doses of irradiation were ineffective in arresting induced synthesis of acid phosphatase and catalase. Exposure of the irradiated cells to visible light (370 – 800 nm) could completely restitute the impaired respiratory adaptation process. UV irradiation at these doses affected DNA and RNA synthesis in maturing mitochondria in both the yeast strains. The UV-induced block could however be eliminated by exposure of the cells to visible light. These results suggest that the lesion in the UV-induced block in the respiratory adaptation may be in the DNA of promitochondria.     

Caffeine inhibition of postreplication repair of UV-damaged DNA in mouse epidermal cells

The effect of caffeine (0.25–1.5 mM) on UV-irradiated (5 and 10 J/m²) primary cultures of mouse epidermal cells (EPD) and an in vitro transformed cell line (PDV) was studied at the cellular and molecular levels. A synergistic reduction in cell survival induced by caffeine with UV-irradiation was found in the PDV cells at 10 J/m² but not at 5 J/m². When conversion of low molecular weight newly-synthesized DNA to high molecular weight DNA was studied in both cell types, caffeine at 1.5 mM had no effect on this conversion in unirradiated cultures. At 5 J/m², caffeine had a transitory inhibitory effect on this conversion. However, at 10 J/m² caffeine had a strong permanent inhibitory effect on this conversion at doses higher than 0.5 mM in PDV cells and higher than 0.25 mM in EPD cells. This apparent inhibition of elongation by caffeine in irradiated cells could not be accounted for by an effect on the rate of DNA synthesis. In PDV cells there was a direct correlation in terms of effective caffeine dose level between synergistic reduction in cell survival after UV and the effect on DNA elongation. Irradiated EPD cells were more sensitive to the inhibitory effect of caffeine on DNA elongation.     

Structural and functional changes in complement protein C4 under UV irradiation

The influence of ultraviolet (UV) light on the structural and functional states of the complement factor C4 was investigated using hemolytic and acid-base titration, PAG electrophoresis, and IR and UV spectrophotometry. UV doses of 75.5 and 755 J/m² initiated C4 activation through changes in the globule structure (increased number of aromatic amino acids and ionogenic groups at the surface). The maximal dose of 2265 J/m² has a destructive effect and decreases its C4 activity in the cascade of hemolytic reactions of the complement system.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

Ultraviolet light-induced mutation of diploid human lymphoblasts

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited ³H-TdR uptake into DNA. A shoulder of about 6 J/m² and a D₀ of 6 J/m² was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m²; above 6 J/m², complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m² caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m²), moderately toxic fluences (8.8 J/m²) and toxic fluences (17.6 J/m²). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.     

Developmental changes in the sensitivity of Volvox to ultraviolet light

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m²) and subjected to variable lenght periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m²) is a nonlinear increase in cytotoxicity and is disproportionanately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.     

Ultraviolet radiation response to the physical properties of the reconstituted nucleohistone

The effect of UV irradiation on the reconstituted nucleohistone have been studied with reference to its (nucleohistone) changes in physical properties, after irradiation at different UV doses. The rate of fall of specific viscosity ratio of the reconstituted nucleohistone as a function of UV dose decreased gradually with the increasing histone to DNA weight ratio (r). This effect, was not observed when the histones remained dissociated from DNA, in high ionic strength (1.5 M NaCl). Histone-DNA complex (r=0.97) irradiated up to a dose of 3.6×10⁴ J/m² had a stable melting temperature unlike free DNA where UV irradiation lowered the melting temperature and the heterogeneous melting profiles were observed. Rate of formaldehyde reaction, with DNA recovered from the irradiated complex, was slower than that with native DNA treated at the same dose. All this suggested that the effect of UV in the DNA of the nucleohistone was less, compared to that in free DNA.     

In vivo and in vitro activation of macrophages with a cyanine photosensitizing dye,platonin

A cyanine photosensitizing dye, platonin, is a potent macrophage-activating agent. Four days after the administration to mice of small amounts of platonin (20–40 ng/mouse), peritoneal macrophages exhibited greatly enhanced Fc-receptor-mediated phagocytic and superoxide-generating capacities. Much higher doses (more than 3000 ng/mouse) did not have this effect. Photodynamic experiments for macrophage activation were performed by exposing mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) to white fluorenscent light (3 J m^–2s^–1) in media containing various low concentrations of platonin. A short exposure to white fluorescent light (5 s, 15 J m^–2) of peritoneal cells in a medium containing 3 ng platonin/ml produced a maximal level of phagocytic capacity of macrophages. Although platonin absorbs light poorly at wavelengths longer than 630 nm, the region of the spectrum in which the tissues are transparent allows reasonable penetration of light. Thus, we designed experiments in which peritoneal cells were exposed to a red fluorescent light (0.5 J m^–2s^–1). In a medium containing 10 ng platonin/ml with 15 J m^–2 red light, a markedly enhanced ingestion activity of macrophages was observed. Photodynamic treatment of peritoneal macrophages alone did not activate macrophages. Thus, participation of nonadherent cells is required for photodynamic activation of macrophages, implying that a macrophage-activating factor is generated within the nonadherent cells and transmitted to macrophages.