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Cu–Zn SUPEROXIDE DISMUTASE INHIBITS LACTATE DEHYDROGENASE RELEASE AND PROTECTS AGAINST CELL DEATH IN MURINE FIBROBLASTS PRETREATED WITH ULTRAVIOLET RADIATION
Cell Biology International 2000, Vol. 24, No. 7, 459–465 doi:10.1006/cbir.2000.0513, available online at http://www.idealibrary.com on
Cu–Zn SUPEROXIDE DISMUTASE INHIBITS LACTATE DEHYDROGENASE RELEASE AND PROTECTS AGAINST CELL DEATH IN MURINE FIBROBLASTS PRETREATED WITH ULTRAVIOLET RADIATION HIROKAZU KIMURA1,2, HISANORI MINAKAMI3, KUNIO OTSUKI2 and AKIRA SHOJI1 1 2
Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma 371-0052, Japan 3 Department of Obstetrics and Gynecology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan Received 17 January 2000, accepted 2 February 2000
The effects of adding Cu–Zn superoxide dismutase (Cu–Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312 nm, 480 mJ/cm2) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1–0.3 Cu–Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu–Zn SOD treatment inhibited this. Heat-denatured Cu–Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu–Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3 exogenous Cu–Zn SOD to the medium did not affect intracellular Cu–Zn SOD activity. These results establish that Cu–Zn SOD added to extracellular medium can protect cells against injury caused by UV-B 2000 Academic Press exposure. K: Cu–Zn SOD; ultraviolet; apoptosis; LDH; fibroblast. A: Cu–Zn SOD, Cu–Zn superoxide dismutase; UV-B, ultraviolet-B; LDH, lactate dehydrogenase; FCS, fetal calf serum; EDTA, ethylene-diaminetetraacetic acid; PBS, phosphate buffered saline.
INTRODUCTION Cells at the surface of the body are often being bombarded by radiation, including ultraviolet (UV) radiation (Doll and Peto, 1981). The latter may be harmful events to cells by causing, for example, DNA damage (Hall et al., 1988), activation of proto-oncogenes (Angel and Karin, 1991), and lysis (Michaelson, 1977). Exposure to excessive UV radiation induces cell death (Martin and Cotter, 1991), and, as shown in murine fibroblasts by Kimura et al. (1999), UV-B irradiation causes apoptosis. Unfortunately, the risk of expo*To whom correspondence should be addressed: Akira Shoji, PhD, Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan. E-mail: [email protected] 1065–6995/00/070459+07 $35.00/0
sure of human beings to UV radiation is gradually increasing with the continuing erosion of the ozone layer of the stratosphere (Crutzen, 1992). Cells that metabolize oxygen will inevitably be exposed to superoxide radicals, and although their effects on specific cellular components are not well understood, they are potentially cytotoxic (Michaelson, 1977; Fridovich, 1995). UV as well as gamma-irradiation can react with water molecules to generate superoxide radicals (Michaelson, 1977; Hall et al., 1988; Fridovich, 1995). Superoxide radicals could therefore be partly responsible for the cellular injury caused by UV radiation. SOD (EC.1.15.1.1) catalytically scavenges this radical and protects against its potential cytotoxity (Michaelson, 1977; Fridovich, 1995). Michaelson (1977) also reported that gamma irradiation causes 2000 Academic Press
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morphologic changes and death of myoblasts, changes resembling those observed with the superoxide radical-generating system, and which were reduced by SOD treatment. Podda et al. (1998) demonstrated that UVirradiation depletes such antioxidants as ubiquinol, ubiquinone, alpha-tocopherol, urate and ascorbate in a model of human skin. Ascorbate inhibited UV-induced lipid peroxidation and LDH release in human keratinocytes, thereby protecting against cell death (Tebbe et al., 1997), which indicates that active oxygen species are probably involved in UV-induced skin injury. Fibroblasts are important components of such connective tissues as skin and vascular walls, and extracellular SOD (EC-SOD) is abundant in them (Marklund, 1984; Ookawara et al., 1998). Hypothetically, EC-SOD could play a role in reducing superoxide-induced injuries of these tissues (Marklund, 1984; Fridovich, 1995; Ookawara et al., 1998). Thus it was considered possible that adding exogenous SOD to culture medium would reduce UV-induced cytotoxity in fibroblasts, but this has not been demonstrated to our knowledge. We have therefore explored the effects of adding exogenous Cu–Zn SOD to culture medium of murine fibroblast cells (L-929) pretreated with UV-B radiation, and found that it acts as a reducer of these oxidative and genotoxic stresses. MATERIALS AND METHODS Cell lines and cell cultures Murine fibroblasts L-929 (NCTC clone 929) were purchased from Dainippon Pharmaceutical Co. Ltd (Osaka, Japan) and grown in Eagle’s minimal essential medium (MEM, Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 10% fetal calf serum (FCS) (Kimura et al., 1999). UV-B exposure and addition of Cu–Zn superoxide dismutase The confluent L-929 cells (3.2–3.4105/cm2), grown in 25 cm2 tissue culture flasks (Corning Co. Ltd, New York, U.S.A.), were obtained. The culture medium was replaced by 5 ml of MEM containing 2% FCS. Before the exposure to UV-B radiation, either Cu–Zn SOD, from bovine erythrocytes, 3400 units/mg protein (Sigma, St. Louis, MO., U.S.A.), or heat-denatured Cu–Zn SOD (121C, 20 min) was added to the medium. The cells were irradiated with UV-B (312 nm, 8 mW/
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cm2) for 60 sec using a UV-B generator (Atto, DT-20MP, Tokyo, Japan) (Kimura et al., 1999). The concentrations of Cu–Zn SOD ranged from 0.0 to 0.3 . Cells were incubated at 37C in an atmosphere of 5% CO2 in air. Measurement of cell viability Some cells were detached from the culture flask wall, suspended in medium at harvest, and collected by centrifugation (1500g, 15 min, 4C). Cells that were attached to the wall of the tissue culture flask were washed twice with PBS, trypsinized in PBS with 0.02 % EDTA, neutralized by FCS, and collected by centrifugation (700g, 10 min, 4C) (Kimura et al., 1999). Cell viability was measured by the trypan blue exclusion test. Cells were counted with Burker-Turk’s cell counter. Analysis of DNA fragmentation Analysis of DNA fragmentation was performed separately using cells attached to the wall of the flask and detached cells that were suspended in the culture medium. DNA extracted and purified by the method of Laird et al. (1991) was electrophoresed on 1.5% agarose gels and stained with ethidium bromide (Wako Pure Chemicals, Co. Ltd, Tokyo, Japan). Assays of culture medium LDH and intracellular Cu–Zn SOD activity Culture medium was centrifuged to remove suspended cells and the supernatants used to determine LDH activity by the conventional method of Sasaki et al. (1992). Preparation of Cu–Zn SOD from L-929 cells was carried out by the method of Nakano et al. (1990). Briefly, cells were suspended in 2 ml of buffered sucrose solution (0.25 in 20 m Tris-HCl pH 7.4 buffer containing 1 m EDTA). The suspended cells were sonicated on ice in 30 consecutive 0.5 s bursts at 0.5 s intervals at power setting of 30 W (Branson, Sonifire). This procedure was repeated once at a 1 min interval. The sonicated samples were centrifuged at 78,000g for 60 min. Aliquots (0.4 ml) of the supernatant were treated with sodium dodecyl sulfate (Wako Pure Chemicals Co, Ltd, Tokyo, Japan) to inactivate Mn SOD activity. Activity of Cu–Zn SOD in the L-929 cells was measured by the chemiluminescence method of Kimura and Nakano (1988; see also Nakano et al., 1990), using Cypridina luciferin analogue (Tokyo Kasei Co. Ltd, Tokyo, Japan). Intensity of chemiluminescence was
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Fig. 1. Microscopic findings in cells treated with UV-B alone and with UV-B plus Cu–Zn SOD. (a): Control cells (not exposed to UV-B); (b): cells incubated with 0.3 Cu–Zn SOD alone for 72 h; (c): cells 72 h after UV-B exposure; (d): cells incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure. Bar indicates 100 m.
measured with a BLR-310 reader (Aloka Co. Ltd, Tokyo, Japan), and Cu–Zn SOD activity was expressed as ng/105 cells (Kimura and Nakano, 1988; Nakano et al., 1990). Statistical analysis Data are presented as meanstandard error and analyzed by the unpaired Student’s t-test. A level of P<0.05 was accepted as statistically significant. RESULTS Light microscopical findings and DNA fragmentation Serial changes were investigated microscopically in cells treated with Cu–Zn SOD alone, UV-B alone, and with combined UV-B and Cu–Zn SOD [Fig. 1(a–d)]. The shape or density of cells incubated with 0.3 Cu–Zn SOD alone [Fig. 1(b)] or 0.3 heat-denatured Cu–Zn SOD (data not shown) for
72 h did not differ from their controls [Fig. 1(a)], nor did they detach from the flask wall. After 72 h of UV-B exposure, cells became rounder in shape and started to detach from the flask wall [Fig. 1(c)]. The shape of the cells treated with UV-B plus 0.3 of Cu–Zn SOD [Fig. 1(d)] was similar to that of the cells treated with UV-B alone. However, detachment of the cells from the flask wall was partially blocked after combined treatment with UV-B and 0.3 of Cu–Zn SOD [cf. Fig. 1 (d and c)]. Since the cells detached from the flask wall and suspended in the medium were considered apoptotic, cells that were detached from, rather than attached to the flask wall after 72 h incubation were examined separately for DNA fragmentation (Fig. 2). No fragmentation was observed in control cells (not exposed to UV-B), cells treated with 0.3 Cu–Zn SOD alone, or 0.3 heatdenatured Cu–Zn SOD alone (Fig. 2, lanes 1 to 3, respectively). After 72 h UV-B exposure, a significant number of cells were detached from the flask wall irrespective of the presence or absence of
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Fig. 2. Fragmentation of DNA in cells treated with UV-B and/or Cu–Zn SOD. M, marker (X174/Hinc II digest ); lane 1, control cells (not exposed to UV-B); lane 2, cells incubated with 0.3 Cu–Zn SOD alone for 72 h; lane 3, cells incubated with 0.3 heat-denatured Cu–Zn SOD for 72 h; lane 4, attached cells to the flask wall 72 h after UV-B exposure; lane 5, attached cells to the flask wall that were incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure; lane 6, detached cells from the flask wall 72 h after UV-B exposure; lane 7, detached cells from the flask wall that were incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure. Ori, origin.
Cu–Zn SOD. A small amount of DNA fragmentation was observed in attached cells that were treated with UV-B alone and with UV-B plus Cu–Zn SOD (Fig. 2, lanes 4 and 5, respectively). In contrast, detached cells treated with UV-B alone and with UV-B plus Cu–Zn SOD (Fig. 2, lanes 6 and 7, respectively) showed marked DNA fragmentation. Effects of addition of Cu–Zn SOD to the medium on cell viability Serial changes of cell in the viability of the L-929 cells treated with UV-B alone, UV-B plus various concentrations of Cu–Zn SOD, and UV-B plus heat-denatured Cu–Zn SOD are shown in Figure 3. Some cells were detached from the flask wall after exposure to UV-B. All the cells detached from the flask wall by the treatments, and between 10 and 20% of those attached to the flask wall stained with trypan blue. The percentage of unstained (‘viable’) cells gradually decreased over time following UV-B
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Fig. 3. Effects of addition of Cu–Zn SOD on cell viability. Exposure to UV-B radiation (312 nm, 480 mJ/cm2) was performed at 0 h. However, before the exposure to radiation, amounts of 0.0, 0.05, 0.1, 0.2, 0.3 Cu–Zn SOD, or 0.3 heat-denatured Cu–Zn SOD, were added to the culture medium. Control cells were not exposed to UV-B or incubated with Cu–Zn SOD. The vertical bar indicates the mean1 standard error (SE) of triplicate experiments. A significantly larger number of cells that were incubated with Cu–Zn SOD (P<0.05 for 0.1 Cu–Zn SOD, P<0.01 for 0.2 Cu–Zn SOD and 0.3 Cu–Zn SOD) were alive at 72 h compared with those exposed to UV-B alone. Line 1, control cells (not exposed to UV-B); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
treatment (Fig. 3, line 8). Those not exposed to UV-B remained viable. A significant number of cells were rescued from cell death by the addition of 0.1 to 0.3 Cu–Zn SOD to the culture medium just before exposure to UV-B radiation (Fig. 3, line 3–5), whereas addition of heat-denatured Cu–Zn SOD failed to rescue cells (Fig. 3, line 7). Cu–Zn SOD activity of cells attached to the flask wall in the presence or absence of exogenous Cu–Zn SOD To investigate whether the addition of exogenously supplied enzyme affected total Cu–Zn SOD activity in the cells, intracellular enzyme activity was assayed in cells incubated with or without exogenous Cu–Zn SOD (Fig. 4). After 72 h of UV-B exposure, Cu–Zn SOD activity decreased by approximately 20% in cells that were treated with UV-B alone (Fig. 4, line 8). In contrast, no significant difference
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Fig. 4. Cu–Zn SOD activity in cells attached to flask wall in the presence or absence of SOD. Experimental conditions are the same as those in Figure 3. The vertical bar indicates the meanSE of triplicate experiments. Line 1, control cells (not exposed to UV-B); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
was found between cells that were exposed to UV-B alone and treated with UV-B plus various concentrations of Cu–Zn SOD. Effects of Cu–Zn SOD on LDH activity Serial changes in LDH activity in culture medium in which L-929 cells had been treated with UV-B alone, UV-B plus various concentrations of Cu–Zn SOD, or UV-B plus heat-denatured Cu–Zn SOD were followed (Fig. 5). Although LDH activity increased slightly with time even in the control cells (Fig. 5, line 1), enzyme activity rose markedly in the cells pretreated with UV-B (cf. Fig. 5, line 8). Addition of 0.1 to 0.3 Cu–Zn SOD to the medium significantly inhibited the rise caused by UV-B radiation (Fig. 5, lines 3 -5), whereas heatdenatured 0.3 Cu–Zn SOD had no such effect (Fig. 5, line 7). On its own, 0.3 Cu–Zn SOD did not affect LDH activity of intact cells (Fig. 5, line 2). DISCUSSION The addition of Cu–Zn SOD to culture medium partially inhibited LDH release and protected against cell death in UV-B exposed murine fibroblasts. Cytosolic enzymes tend to be released when the cell membrane is injured (Sasaki et al., 1992)
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Fig. 5. Effects of Cu–Zn SOD on LDH activity in the culture medium. Experimental conditions are the same as those in Figure 3. LDH activity was assayed in the culture medium after centrifugation. The vertical bar indicates the meanSE of triplicate experiments. LDH activity at 0 h was approximately 10 IU/L because the medium contained 2% FCS. The LDH corrected activity was shown in this Figure. LDH activity was significantly decreased in the medium containing 0.1 to 0.3 Cu–Zn SOD at 72 h as compared with that in the medium containing no Cu–Zn SOD (P<0.01). Line 1, control cells (not exposed to UV-B ); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
and, therefore, release of LDH in the present study could be taken as a measure of the impairment of the cell membrane integrity following UVirradiation. Because the activity of Cu–Zn SOD in the cells incubated with Cu–Zn SOD did not differ from that in the cells incubated without Cu–Zn SOD, it is unlikely that Cu–Zn SOD added to the culture medium penetrated the cell membrane. This suggests that exogenous SOD acted only within the medium, perhaps scavenging superoxide radicals that might have been generated by UV-irradiation, thereby reducing impairment of membrane integrity. It has recently been demonstrated that -arginine (a substrate of nitric oxide synthase) increases UV-A cytotoxicity in irradiated human keratinocytes, suggesting that nitric oxide may play a causative role in cell death (Didier et al., 1999), although these authors did not specify whether cell death was due to apoptosis. Exogenous SOD and -thiocitrulline (an inhibitor of nitric oxide synthesis) prevented this cytotoxic effect, suggesting
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that the ratio of nitric oxide to superoxide radicals is important in UV-A-induced cytotoxicity (Didier et al., 1999). Our culture medium contained 0.6 m -arginine, and therefore the possibility that nitric oxide played a role in causing apoptotic cell death cannot be excluded. Exogenous SOD may have altered the ratio of nitric oxide to superoxide radicals, and partially abrogated this effect. However, the precise mechanisms of protection from cell death of exogenous SOD remains to be elucidated. Activities of Cu–Zn SOD and Mn SOD are very low in extracellular fluid, although the former has been identified in the extracellular fluid (Marklund, 1984; Fridovich, 1995). Much of the EC-SOD is circulating, but it is otherwise mainly located in connective tissues and vascular walls in mice (Ookawara et al., 1998). One role of EC-SOD may be related to the control of oxidative stress in cells and tissues adjacent to extracellular fluid (Marklund, 1984; Fridovich, 1995; Oury et al., 1996; Ookawara et al., 1998). Our experimental results seem to support the idea that endogenous EC-SOD reduces oxidative stress occurring in cell membranes adjacent to the extracellular fluid/ medium. The biological role of SOD in the extracellular fluid was extensively studied in late 1970s and early 1980s using reconstructed systems (Michaelson, 1977; Lynch and Fridovich, 1978; Takahashi and Asada, 1983), but results were inconsistent as to whether SOD added to medium could reduce the cytotoxicity of superoxide radicals. The superoxide radicals generated by the xanthine–xanthine oxidase system were able to lyse erythrocyte membranes, which was monitored by leakage of the radioactive sucrose (Lynch and Fridovich, 1978). Either the SOD sealed into the erythrocyte or the SOD in the suspending medium prevented the leakage of radioactive sucrose, which suggested to these authors that the superoxiderelated lysis of the cell membrane could be blocked by exogenous SOD in the medium. Furthermore, exogenous SOD protected gamma-irradiated myoblasts against cell death (Michaelson, 1977). However, exogenous SOD did not prevent the lysis of the erythrocytes induced by exposure to UV radiation (Michaelson, 1977). Some of these conflicting results may be due to differences in experimental conditions. We used UV-B light (312 nm, 8 mW/cm2) for 60 s to irradiate cells, which corresponds to 8 mJ/ sec/cm2 60 sec (=480 mJ/cm2). The amount of solar UV-B reaching the surface of the earth is 0.3 to 0.5 mW/cm2 at sea level, a dose corresponding to 18 to 30 mJ/cm2/min (Madhu and Thomas, 1993).
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Thus, the dose of the UV-B used in this study corresponds to 16–27 min sunbathing at sea level, if we neglect biological effects of UV-B in the calculation. However, it must be remembered that the biological effects of UV-B depend on wavelength. The dose of the UV-B used here may have actually corresponded to several hours of sunbathing at sea level (Madhu and Thomas, 1993). We recently reported that UV-B radiation induces the expression of Fas (CD 95) and Fas ligand in L-929 cells, which induced the cells to undergo apoptosis (Kimura et al., 1999). A marked DNA fragmentation was observed in the cells detached from the flask wall 72 h after the UV-B exposure, irrespective of the presence or absence of exogenous Cu–Zn SOD in the culture medium in this study (Fig. 2). Thus, cell death in the present study was due to apoptosis induced by UV-B radiation, which could be partially inhibited by the presence of Cu–Zn SOD in the medium. However, it is unlikely that zinc ions, well-known to inhibit apoptosis (Thompson, 1995) and contained within Cu–Zn SOD, inhibited cell death in the present study because heat-denatured Cu–Zn SOD was ineffective. Cu–Zn SOD activity in fibroblasts decreased by 20% after UV-B exposure in the present study, consistent with result of previous study in keratinocytes (Punnonen et al., 1991). In another study, Cu–Zn SOD activity in human keratinocytes increased transiently in response to UV-B irradiation, and then gradually declined (Sasaki et al., 1997). Thus, activity of intracellular Cu–Zn SOD changes in response to genotoxic stress caused by UV radiation. The decreased Cu–Zn SOD activity seen in the present study may be associated with an increased oxidative stress. However, we could not determine whether a decrease in intracellular Cu–Zn SOD activity was responsible for apoptotic cell death in the present study. In conclusion, our results indicated that SODs present in the extracellular fluid can play a role in reducing oxidative and genotoxic stresses in L-929 cells that may occur after UV exposure, which is consistent with the results by other investigators in various other cells (Michaelson, 1977; Fridovich, 1995; Oury et al., 1996; Didier et al., 1999). ACKNOWLEDGEMENTS We thank Dr Atsushi Tachibana (Department of Pediatrics, Gunma University, School of Medicine) and Mr Yasuyuki Kobayashi (Gunma Prefectural
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Cardiovascular Center) for their skilful assistance. We thank CBI’s Manuscript Presentation Service for help in the presentation of this article. REFERENCES A P, K M, 1991. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129–157. C PJ, 1992. Ultraviolet on the increase. Nature 356: 104–105. D C, E-P N, BA JC, C J, R MJ, 1999. L-arginine increases UVA cytotoxity in irradiated human keratinocyte 2000 cell line. FASEB J 13 (13): 1817– 1824. D R, P R, 1981. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66: 1191–1308. F I, 1995. Superoxide radical and superoxide dismutase. Annu Rev Biochem 64: 97–112. H EJ, A M, B J, B C, C SB, F M, G C, H T, M J, O N, 1988. Basic Radiobiology. Am J Clin Oncl 11: 220–252. K H, N M, 1988. Highly sensitive and reliable chemi-luminescence method for the assay of superoxide dismutase in human erythrocytes. FEBS Lett 239 (9): 347–350. K H, M H, S I, O K, S A, 1999. Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation. J Biochem 126 (2): 340–346. L PW, Z A, L K, R MA, J R, B R, 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293. L RE, F I, 1978. Effects of superoxide on the erythrocyte membrane. J Biol Chem 253 (6): 1838–1845. M AP, T BF, 1993. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sun-protective agents. In: Thomas BF, Arthur ZE, Klaus W, Irwin MFK, Frank A, eds. Dermatology in General Medicine (4th edition). New York, McGraw-Hill Inc. 1689–1717. M SL, 1984. Extracellular superoxide dismutase and other superoxide dismutase isozymes in tissues from nine mammalian species. Biochem J 222: 649–655.
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M SJ, C TG, 1991. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis. Int J Radiat Biol 59: 1001–1016. M AM, 1977. Toxicity of superoxide radical anions. In: Michaelson AM, McCord JM, Fridovich I, eds. Superoxide and Superoxide Dismutase. New York, Academic Press. 245–255. N M, K H, H M, K M, K M, T K, Y T, 1990. A highly sensitive method for determinating both Mn- and Cu-Zn superoxide dismutase activities. Anal Biochem 187: 277–280. O T, I N, M O, K T, O-I S, N C, S Y, O H, 1998. Tissue distribution of immunoreactive mouse extracellular superoxide dismutase. Am J Physiol 275: 840–847. O TD, D BJ, C JD, 1996. Extracellular superoxide dismutase: A regulatory of nitric oxide bioavailability. Lab Invest 75: 617–636. P M, T MG, W C, Y LJ, P L, 1998. UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med 24 (1): 55–65. P K, J CT, P A, A M, 1991. Effects of in vitro UVA irradiation and PUVA treatment on membrane fatty acids and activities of antioxidant enzymes in human keratinocytes. J invest Dermatol 96 (2): 255–259. S H, A H, H T, 1997. Effects of single exposure to UVB radiation on the activities and protein levels of copper-zinc and manganese superoxide dismutase in cultured human keratinocytes. Photochem Photobiol 6 (4): 707–713. S T, K K, S-K K, O T, 1992. Detergent cytoxity: Simplified assay of cytolysis by measuring LDH activity. Toxicol in vitro 6: 451–457. T M, A K, 1983. Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids. Arch Biochem Biophys 226: 558–566. T B, W S, G CC, E J, K V, O CE, 1997. L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1 alpha and IL-6 cultured human keratinocytes in vitro. J Invest Dermatol 108 (3): 302–306. T CB, 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456–1462.
Cu–Zn SUPEROXIDE DISMUTASE INHIBITS LACTATE DEHYDROGENASE RELEASE AND PROTECTS AGAINST CELL DEATH IN MURINE FIBROBLASTS PRETREATED WITH ULTRAVIOLET RADIATION HIROKAZU KIMURA1,2, HISANORI MINAKAMI3, KUNIO OTSUKI2 and AKIRA SHOJI1 1 2
Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma 371-0052, Japan 3 Department of Obstetrics and Gynecology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan Received 17 January 2000, accepted 2 February 2000
The effects of adding Cu–Zn superoxide dismutase (Cu–Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312 nm, 480 mJ/cm2) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1–0.3 Cu–Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu–Zn SOD treatment inhibited this. Heat-denatured Cu–Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu–Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3 exogenous Cu–Zn SOD to the medium did not affect intracellular Cu–Zn SOD activity. These results establish that Cu–Zn SOD added to extracellular medium can protect cells against injury caused by UV-B 2000 Academic Press exposure. K: Cu–Zn SOD; ultraviolet; apoptosis; LDH; fibroblast. A: Cu–Zn SOD, Cu–Zn superoxide dismutase; UV-B, ultraviolet-B; LDH, lactate dehydrogenase; FCS, fetal calf serum; EDTA, ethylene-diaminetetraacetic acid; PBS, phosphate buffered saline.
INTRODUCTION Cells at the surface of the body are often being bombarded by radiation, including ultraviolet (UV) radiation (Doll and Peto, 1981). The latter may be harmful events to cells by causing, for example, DNA damage (Hall et al., 1988), activation of proto-oncogenes (Angel and Karin, 1991), and lysis (Michaelson, 1977). Exposure to excessive UV radiation induces cell death (Martin and Cotter, 1991), and, as shown in murine fibroblasts by Kimura et al. (1999), UV-B irradiation causes apoptosis. Unfortunately, the risk of expo*To whom correspondence should be addressed: Akira Shoji, PhD, Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan. E-mail: [email protected] 1065–6995/00/070459+07 $35.00/0
sure of human beings to UV radiation is gradually increasing with the continuing erosion of the ozone layer of the stratosphere (Crutzen, 1992). Cells that metabolize oxygen will inevitably be exposed to superoxide radicals, and although their effects on specific cellular components are not well understood, they are potentially cytotoxic (Michaelson, 1977; Fridovich, 1995). UV as well as gamma-irradiation can react with water molecules to generate superoxide radicals (Michaelson, 1977; Hall et al., 1988; Fridovich, 1995). Superoxide radicals could therefore be partly responsible for the cellular injury caused by UV radiation. SOD (EC.1.15.1.1) catalytically scavenges this radical and protects against its potential cytotoxity (Michaelson, 1977; Fridovich, 1995). Michaelson (1977) also reported that gamma irradiation causes 2000 Academic Press
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morphologic changes and death of myoblasts, changes resembling those observed with the superoxide radical-generating system, and which were reduced by SOD treatment. Podda et al. (1998) demonstrated that UVirradiation depletes such antioxidants as ubiquinol, ubiquinone, alpha-tocopherol, urate and ascorbate in a model of human skin. Ascorbate inhibited UV-induced lipid peroxidation and LDH release in human keratinocytes, thereby protecting against cell death (Tebbe et al., 1997), which indicates that active oxygen species are probably involved in UV-induced skin injury. Fibroblasts are important components of such connective tissues as skin and vascular walls, and extracellular SOD (EC-SOD) is abundant in them (Marklund, 1984; Ookawara et al., 1998). Hypothetically, EC-SOD could play a role in reducing superoxide-induced injuries of these tissues (Marklund, 1984; Fridovich, 1995; Ookawara et al., 1998). Thus it was considered possible that adding exogenous SOD to culture medium would reduce UV-induced cytotoxity in fibroblasts, but this has not been demonstrated to our knowledge. We have therefore explored the effects of adding exogenous Cu–Zn SOD to culture medium of murine fibroblast cells (L-929) pretreated with UV-B radiation, and found that it acts as a reducer of these oxidative and genotoxic stresses. MATERIALS AND METHODS Cell lines and cell cultures Murine fibroblasts L-929 (NCTC clone 929) were purchased from Dainippon Pharmaceutical Co. Ltd (Osaka, Japan) and grown in Eagle’s minimal essential medium (MEM, Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 10% fetal calf serum (FCS) (Kimura et al., 1999). UV-B exposure and addition of Cu–Zn superoxide dismutase The confluent L-929 cells (3.2–3.4105/cm2), grown in 25 cm2 tissue culture flasks (Corning Co. Ltd, New York, U.S.A.), were obtained. The culture medium was replaced by 5 ml of MEM containing 2% FCS. Before the exposure to UV-B radiation, either Cu–Zn SOD, from bovine erythrocytes, 3400 units/mg protein (Sigma, St. Louis, MO., U.S.A.), or heat-denatured Cu–Zn SOD (121C, 20 min) was added to the medium. The cells were irradiated with UV-B (312 nm, 8 mW/
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cm2) for 60 sec using a UV-B generator (Atto, DT-20MP, Tokyo, Japan) (Kimura et al., 1999). The concentrations of Cu–Zn SOD ranged from 0.0 to 0.3 . Cells were incubated at 37C in an atmosphere of 5% CO2 in air. Measurement of cell viability Some cells were detached from the culture flask wall, suspended in medium at harvest, and collected by centrifugation (1500g, 15 min, 4C). Cells that were attached to the wall of the tissue culture flask were washed twice with PBS, trypsinized in PBS with 0.02 % EDTA, neutralized by FCS, and collected by centrifugation (700g, 10 min, 4C) (Kimura et al., 1999). Cell viability was measured by the trypan blue exclusion test. Cells were counted with Burker-Turk’s cell counter. Analysis of DNA fragmentation Analysis of DNA fragmentation was performed separately using cells attached to the wall of the flask and detached cells that were suspended in the culture medium. DNA extracted and purified by the method of Laird et al. (1991) was electrophoresed on 1.5% agarose gels and stained with ethidium bromide (Wako Pure Chemicals, Co. Ltd, Tokyo, Japan). Assays of culture medium LDH and intracellular Cu–Zn SOD activity Culture medium was centrifuged to remove suspended cells and the supernatants used to determine LDH activity by the conventional method of Sasaki et al. (1992). Preparation of Cu–Zn SOD from L-929 cells was carried out by the method of Nakano et al. (1990). Briefly, cells were suspended in 2 ml of buffered sucrose solution (0.25 in 20 m Tris-HCl pH 7.4 buffer containing 1 m EDTA). The suspended cells were sonicated on ice in 30 consecutive 0.5 s bursts at 0.5 s intervals at power setting of 30 W (Branson, Sonifire). This procedure was repeated once at a 1 min interval. The sonicated samples were centrifuged at 78,000g for 60 min. Aliquots (0.4 ml) of the supernatant were treated with sodium dodecyl sulfate (Wako Pure Chemicals Co, Ltd, Tokyo, Japan) to inactivate Mn SOD activity. Activity of Cu–Zn SOD in the L-929 cells was measured by the chemiluminescence method of Kimura and Nakano (1988; see also Nakano et al., 1990), using Cypridina luciferin analogue (Tokyo Kasei Co. Ltd, Tokyo, Japan). Intensity of chemiluminescence was
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Fig. 1. Microscopic findings in cells treated with UV-B alone and with UV-B plus Cu–Zn SOD. (a): Control cells (not exposed to UV-B); (b): cells incubated with 0.3 Cu–Zn SOD alone for 72 h; (c): cells 72 h after UV-B exposure; (d): cells incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure. Bar indicates 100 m.
measured with a BLR-310 reader (Aloka Co. Ltd, Tokyo, Japan), and Cu–Zn SOD activity was expressed as ng/105 cells (Kimura and Nakano, 1988; Nakano et al., 1990). Statistical analysis Data are presented as meanstandard error and analyzed by the unpaired Student’s t-test. A level of P<0.05 was accepted as statistically significant. RESULTS Light microscopical findings and DNA fragmentation Serial changes were investigated microscopically in cells treated with Cu–Zn SOD alone, UV-B alone, and with combined UV-B and Cu–Zn SOD [Fig. 1(a–d)]. The shape or density of cells incubated with 0.3 Cu–Zn SOD alone [Fig. 1(b)] or 0.3 heat-denatured Cu–Zn SOD (data not shown) for
72 h did not differ from their controls [Fig. 1(a)], nor did they detach from the flask wall. After 72 h of UV-B exposure, cells became rounder in shape and started to detach from the flask wall [Fig. 1(c)]. The shape of the cells treated with UV-B plus 0.3 of Cu–Zn SOD [Fig. 1(d)] was similar to that of the cells treated with UV-B alone. However, detachment of the cells from the flask wall was partially blocked after combined treatment with UV-B and 0.3 of Cu–Zn SOD [cf. Fig. 1 (d and c)]. Since the cells detached from the flask wall and suspended in the medium were considered apoptotic, cells that were detached from, rather than attached to the flask wall after 72 h incubation were examined separately for DNA fragmentation (Fig. 2). No fragmentation was observed in control cells (not exposed to UV-B), cells treated with 0.3 Cu–Zn SOD alone, or 0.3 heatdenatured Cu–Zn SOD alone (Fig. 2, lanes 1 to 3, respectively). After 72 h UV-B exposure, a significant number of cells were detached from the flask wall irrespective of the presence or absence of
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Fig. 2. Fragmentation of DNA in cells treated with UV-B and/or Cu–Zn SOD. M, marker (X174/Hinc II digest ); lane 1, control cells (not exposed to UV-B); lane 2, cells incubated with 0.3 Cu–Zn SOD alone for 72 h; lane 3, cells incubated with 0.3 heat-denatured Cu–Zn SOD for 72 h; lane 4, attached cells to the flask wall 72 h after UV-B exposure; lane 5, attached cells to the flask wall that were incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure; lane 6, detached cells from the flask wall 72 h after UV-B exposure; lane 7, detached cells from the flask wall that were incubated with 0.3 Cu–Zn SOD for 72 h after UV-B exposure. Ori, origin.
Cu–Zn SOD. A small amount of DNA fragmentation was observed in attached cells that were treated with UV-B alone and with UV-B plus Cu–Zn SOD (Fig. 2, lanes 4 and 5, respectively). In contrast, detached cells treated with UV-B alone and with UV-B plus Cu–Zn SOD (Fig. 2, lanes 6 and 7, respectively) showed marked DNA fragmentation. Effects of addition of Cu–Zn SOD to the medium on cell viability Serial changes of cell in the viability of the L-929 cells treated with UV-B alone, UV-B plus various concentrations of Cu–Zn SOD, and UV-B plus heat-denatured Cu–Zn SOD are shown in Figure 3. Some cells were detached from the flask wall after exposure to UV-B. All the cells detached from the flask wall by the treatments, and between 10 and 20% of those attached to the flask wall stained with trypan blue. The percentage of unstained (‘viable’) cells gradually decreased over time following UV-B
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Fig. 3. Effects of addition of Cu–Zn SOD on cell viability. Exposure to UV-B radiation (312 nm, 480 mJ/cm2) was performed at 0 h. However, before the exposure to radiation, amounts of 0.0, 0.05, 0.1, 0.2, 0.3 Cu–Zn SOD, or 0.3 heat-denatured Cu–Zn SOD, were added to the culture medium. Control cells were not exposed to UV-B or incubated with Cu–Zn SOD. The vertical bar indicates the mean1 standard error (SE) of triplicate experiments. A significantly larger number of cells that were incubated with Cu–Zn SOD (P<0.05 for 0.1 Cu–Zn SOD, P<0.01 for 0.2 Cu–Zn SOD and 0.3 Cu–Zn SOD) were alive at 72 h compared with those exposed to UV-B alone. Line 1, control cells (not exposed to UV-B); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
treatment (Fig. 3, line 8). Those not exposed to UV-B remained viable. A significant number of cells were rescued from cell death by the addition of 0.1 to 0.3 Cu–Zn SOD to the culture medium just before exposure to UV-B radiation (Fig. 3, line 3–5), whereas addition of heat-denatured Cu–Zn SOD failed to rescue cells (Fig. 3, line 7). Cu–Zn SOD activity of cells attached to the flask wall in the presence or absence of exogenous Cu–Zn SOD To investigate whether the addition of exogenously supplied enzyme affected total Cu–Zn SOD activity in the cells, intracellular enzyme activity was assayed in cells incubated with or without exogenous Cu–Zn SOD (Fig. 4). After 72 h of UV-B exposure, Cu–Zn SOD activity decreased by approximately 20% in cells that were treated with UV-B alone (Fig. 4, line 8). In contrast, no significant difference
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Fig. 4. Cu–Zn SOD activity in cells attached to flask wall in the presence or absence of SOD. Experimental conditions are the same as those in Figure 3. The vertical bar indicates the meanSE of triplicate experiments. Line 1, control cells (not exposed to UV-B); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
was found between cells that were exposed to UV-B alone and treated with UV-B plus various concentrations of Cu–Zn SOD. Effects of Cu–Zn SOD on LDH activity Serial changes in LDH activity in culture medium in which L-929 cells had been treated with UV-B alone, UV-B plus various concentrations of Cu–Zn SOD, or UV-B plus heat-denatured Cu–Zn SOD were followed (Fig. 5). Although LDH activity increased slightly with time even in the control cells (Fig. 5, line 1), enzyme activity rose markedly in the cells pretreated with UV-B (cf. Fig. 5, line 8). Addition of 0.1 to 0.3 Cu–Zn SOD to the medium significantly inhibited the rise caused by UV-B radiation (Fig. 5, lines 3 -5), whereas heatdenatured 0.3 Cu–Zn SOD had no such effect (Fig. 5, line 7). On its own, 0.3 Cu–Zn SOD did not affect LDH activity of intact cells (Fig. 5, line 2). DISCUSSION The addition of Cu–Zn SOD to culture medium partially inhibited LDH release and protected against cell death in UV-B exposed murine fibroblasts. Cytosolic enzymes tend to be released when the cell membrane is injured (Sasaki et al., 1992)
0
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Fig. 5. Effects of Cu–Zn SOD on LDH activity in the culture medium. Experimental conditions are the same as those in Figure 3. LDH activity was assayed in the culture medium after centrifugation. The vertical bar indicates the meanSE of triplicate experiments. LDH activity at 0 h was approximately 10 IU/L because the medium contained 2% FCS. The LDH corrected activity was shown in this Figure. LDH activity was significantly decreased in the medium containing 0.1 to 0.3 Cu–Zn SOD at 72 h as compared with that in the medium containing no Cu–Zn SOD (P<0.01). Line 1, control cells (not exposed to UV-B ); line 2, control plus 0.3 Cu–Zn SOD; lines 3 to 6, UV-B plus 0.3, 0.2, 0.1, and 0.05 Cu–Zn SOD, respectively; line 7, UV-B plus heat-denatured 0.3 Cu–Zn SOD; line 8, UV-B alone.
and, therefore, release of LDH in the present study could be taken as a measure of the impairment of the cell membrane integrity following UVirradiation. Because the activity of Cu–Zn SOD in the cells incubated with Cu–Zn SOD did not differ from that in the cells incubated without Cu–Zn SOD, it is unlikely that Cu–Zn SOD added to the culture medium penetrated the cell membrane. This suggests that exogenous SOD acted only within the medium, perhaps scavenging superoxide radicals that might have been generated by UV-irradiation, thereby reducing impairment of membrane integrity. It has recently been demonstrated that -arginine (a substrate of nitric oxide synthase) increases UV-A cytotoxicity in irradiated human keratinocytes, suggesting that nitric oxide may play a causative role in cell death (Didier et al., 1999), although these authors did not specify whether cell death was due to apoptosis. Exogenous SOD and -thiocitrulline (an inhibitor of nitric oxide synthesis) prevented this cytotoxic effect, suggesting
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that the ratio of nitric oxide to superoxide radicals is important in UV-A-induced cytotoxicity (Didier et al., 1999). Our culture medium contained 0.6 m -arginine, and therefore the possibility that nitric oxide played a role in causing apoptotic cell death cannot be excluded. Exogenous SOD may have altered the ratio of nitric oxide to superoxide radicals, and partially abrogated this effect. However, the precise mechanisms of protection from cell death of exogenous SOD remains to be elucidated. Activities of Cu–Zn SOD and Mn SOD are very low in extracellular fluid, although the former has been identified in the extracellular fluid (Marklund, 1984; Fridovich, 1995). Much of the EC-SOD is circulating, but it is otherwise mainly located in connective tissues and vascular walls in mice (Ookawara et al., 1998). One role of EC-SOD may be related to the control of oxidative stress in cells and tissues adjacent to extracellular fluid (Marklund, 1984; Fridovich, 1995; Oury et al., 1996; Ookawara et al., 1998). Our experimental results seem to support the idea that endogenous EC-SOD reduces oxidative stress occurring in cell membranes adjacent to the extracellular fluid/ medium. The biological role of SOD in the extracellular fluid was extensively studied in late 1970s and early 1980s using reconstructed systems (Michaelson, 1977; Lynch and Fridovich, 1978; Takahashi and Asada, 1983), but results were inconsistent as to whether SOD added to medium could reduce the cytotoxicity of superoxide radicals. The superoxide radicals generated by the xanthine–xanthine oxidase system were able to lyse erythrocyte membranes, which was monitored by leakage of the radioactive sucrose (Lynch and Fridovich, 1978). Either the SOD sealed into the erythrocyte or the SOD in the suspending medium prevented the leakage of radioactive sucrose, which suggested to these authors that the superoxiderelated lysis of the cell membrane could be blocked by exogenous SOD in the medium. Furthermore, exogenous SOD protected gamma-irradiated myoblasts against cell death (Michaelson, 1977). However, exogenous SOD did not prevent the lysis of the erythrocytes induced by exposure to UV radiation (Michaelson, 1977). Some of these conflicting results may be due to differences in experimental conditions. We used UV-B light (312 nm, 8 mW/cm2) for 60 s to irradiate cells, which corresponds to 8 mJ/ sec/cm2 60 sec (=480 mJ/cm2). The amount of solar UV-B reaching the surface of the earth is 0.3 to 0.5 mW/cm2 at sea level, a dose corresponding to 18 to 30 mJ/cm2/min (Madhu and Thomas, 1993).
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Thus, the dose of the UV-B used in this study corresponds to 16–27 min sunbathing at sea level, if we neglect biological effects of UV-B in the calculation. However, it must be remembered that the biological effects of UV-B depend on wavelength. The dose of the UV-B used here may have actually corresponded to several hours of sunbathing at sea level (Madhu and Thomas, 1993). We recently reported that UV-B radiation induces the expression of Fas (CD 95) and Fas ligand in L-929 cells, which induced the cells to undergo apoptosis (Kimura et al., 1999). A marked DNA fragmentation was observed in the cells detached from the flask wall 72 h after the UV-B exposure, irrespective of the presence or absence of exogenous Cu–Zn SOD in the culture medium in this study (Fig. 2). Thus, cell death in the present study was due to apoptosis induced by UV-B radiation, which could be partially inhibited by the presence of Cu–Zn SOD in the medium. However, it is unlikely that zinc ions, well-known to inhibit apoptosis (Thompson, 1995) and contained within Cu–Zn SOD, inhibited cell death in the present study because heat-denatured Cu–Zn SOD was ineffective. Cu–Zn SOD activity in fibroblasts decreased by 20% after UV-B exposure in the present study, consistent with result of previous study in keratinocytes (Punnonen et al., 1991). In another study, Cu–Zn SOD activity in human keratinocytes increased transiently in response to UV-B irradiation, and then gradually declined (Sasaki et al., 1997). Thus, activity of intracellular Cu–Zn SOD changes in response to genotoxic stress caused by UV radiation. The decreased Cu–Zn SOD activity seen in the present study may be associated with an increased oxidative stress. However, we could not determine whether a decrease in intracellular Cu–Zn SOD activity was responsible for apoptotic cell death in the present study. In conclusion, our results indicated that SODs present in the extracellular fluid can play a role in reducing oxidative and genotoxic stresses in L-929 cells that may occur after UV exposure, which is consistent with the results by other investigators in various other cells (Michaelson, 1977; Fridovich, 1995; Oury et al., 1996; Didier et al., 1999). ACKNOWLEDGEMENTS We thank Dr Atsushi Tachibana (Department of Pediatrics, Gunma University, School of Medicine) and Mr Yasuyuki Kobayashi (Gunma Prefectural
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Cardiovascular Center) for their skilful assistance. We thank CBI’s Manuscript Presentation Service for help in the presentation of this article. REFERENCES A P, K M, 1991. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129–157. C PJ, 1992. Ultraviolet on the increase. Nature 356: 104–105. D C, E-P N, BA JC, C J, R MJ, 1999. L-arginine increases UVA cytotoxity in irradiated human keratinocyte 2000 cell line. FASEB J 13 (13): 1817– 1824. D R, P R, 1981. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66: 1191–1308. F I, 1995. Superoxide radical and superoxide dismutase. Annu Rev Biochem 64: 97–112. H EJ, A M, B J, B C, C SB, F M, G C, H T, M J, O N, 1988. Basic Radiobiology. Am J Clin Oncl 11: 220–252. K H, N M, 1988. Highly sensitive and reliable chemi-luminescence method for the assay of superoxide dismutase in human erythrocytes. FEBS Lett 239 (9): 347–350. K H, M H, S I, O K, S A, 1999. Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation. J Biochem 126 (2): 340–346. L PW, Z A, L K, R MA, J R, B R, 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293. L RE, F I, 1978. Effects of superoxide on the erythrocyte membrane. J Biol Chem 253 (6): 1838–1845. M AP, T BF, 1993. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sun-protective agents. In: Thomas BF, Arthur ZE, Klaus W, Irwin MFK, Frank A, eds. Dermatology in General Medicine (4th edition). New York, McGraw-Hill Inc. 1689–1717. M SL, 1984. Extracellular superoxide dismutase and other superoxide dismutase isozymes in tissues from nine mammalian species. Biochem J 222: 649–655.
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