MATERIALS and METHODS

Chemicals

DDC, 6-hydroxypurine (hypoxanthine), ferricytochrome c (type III), and buttermilk xanthine oxidase were obtained from Sigma, (St Louis, MO). Recombinant human TNF- and TGF-₁ were purchased from Genzyme (Cambridge, MA). 2',7'-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR).

Cell preparations

The human keratinocytes utilized in this study were HaCaT cells (^{Boukamp et al. 1988}), transformed human keratinocytes provided by Professor N.E. Fusenig. HaCaT cells were cultured in Dulbecco's modified Eagle's medium (Nikken Biomedical Laboratory, Kyoto, Japan), supplemented with 5% fetal bovine serum (ICN Biomedicals, Costa Mesa, CA), a 2% mixture of penicillin (100 IU per ml) and streptomycin (100 g per ml), and 0.2 mg per ml L-glutamine (Gibco-BRL, New York, NY). Cells were seeded with 1 10⁶ cells in each culture dish (6 cm in diameter) and incubated at 37°C in 5% CO₂.

UV irradiation source

The UVB source was a bank of seven fluorescent sunlamps (FL.20SE.30, Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 275–375 nm peaking at 305 nm, emitting mainly in the UVB range but also small amounts of UVA and UVC. The irradiance was 0.3 mW per cm² at a distance of 35 cm measured with a radiometer (UVR-305/365D(II), Toshiba Medical Supply).

Treatment with DDC, TNF-, or TGF-₁

After washing HaCaT cells three times with phosphate-buffered saline (PBS), the cells were treated with each reagent. HaCaT cells were incubated with 1 mM DDC dissolved in PBS (pH 7.4) for 1.5 h, 1 ng per ml of TNF- for 24 h, or 10 ng per ml of TGF-₁ for 24 h.

UVB irradiation

After the treatment with 1 mM DDC, 1 ng per ml of TNF-, or 10 ng per ml of TGF-₁, HaCaT cells were washed three times with PBS. Then, 2 ml of PBS was added to each culture dish. Thereafter, each culture dish of cells was exposed to a single dose of UVB irradiation (10, 20, or 30 mJ per cm²).

Assay of LDH activity

LDH leakage from damaged HaCaT cells in the supernatant was measured 8 and 24 h after each dose of UVB irradiation. The LDH activity was spectrophotometrically determined by measuring the reduced nicotinamide adenine dinucleotide (NADH) disappearance rate at 340 nm as a main wavelength during the LDH-catalyzed conversion of pyruvate to lactate according to the Wróblewski-La Due method (^{Wróblewski & John 1955}). The LDH activity is expressed as U per l at 37°C. Incubation with either 1 mM DDC, 1 ng per ml of TNF-, or 10 ng per ml of TGF-₁ for an adequate time did not significantly affect LDH release.

Cell viability

Cell viability of HaCaT cells treated with 1 mM DDC, 1 ng per ml of TNF-, or 10 ng per ml of TGF-₁ was evaluated by the trypan blue dye exclusion assay 8 and 24 h after each dose of UVB irradiation. We collected both the floating cells in the supernatant and the remaining cells adhering to the dish together using 0.02% ethylenediamine tetraacetic acid (EDTA) and 0.25% trypsin 8 and 24 h after UVB irradiation. Then, both the dead and alive cells were counted using the trypan blue dye exclusion assay. Incubation with 1 mM DDC, 1 ng per ml of TNF-, or 10 ng per ml of TGF-₁ for an adequate time did not significantly affect cell viability.

Measurement of intracellular peroxides by flow cytometry

Intracellular peroxide levels were assessed using an oxidation-sensitive fluorescent probe DCFH-DA. In the presence of a variety of intracellular peroxides, DCFH is oxidized to a highly fluorescent compound, 2',7'-dichlorofluorescein (^{Bass et al. 1983}). HaCaT cells, both untreated and treated with 1 mM DDC for 1.5 h, were exposed to a single dose of 10 mJ per cm² UVB; both untreated and treated cells were collected using 0.02% EDTA and 0.25% trypsin 8 h after UVB irradiation. Then, HaCaT cells were incubated with 5 M DCFH-DA. The cellular fluorescence intensity, which was directly proportional to levels of intracellular peroxides after 30 min DCFH-DA oxidation, was measured using FACScan (Becton Dickinson, San Jose, CA). For each analysis, 10,000 events were recorded. For image analysis, cells were analyzed for fluorescence intensity using a lysis cell analysis system.

Cu,Zn-SOD and Mn-SOD assays

After treatment with DDC (0.01, 0.1, or 1 mM), TNF- (0.1, 0.5, or 1 ng per ml) or TGF-₁ (1, 5, 10 ng per ml), cells were collected using 0.02% EDTA and 0.25% trypsin, ruptured by sonication for 30 s using a W-220 sonicator (Heat System-Ultrasonic, NY) at full power, and then centrifuged at 18,000 rpm for 60 min. The supernatant was kept on ice and used for the SOD activity assay and protein determination. SOD activity in HaCaT cells was determined according to the reduction of ferricytochrome c method of McCord and Fridovich (^{McCord & Fridovich 1969}). A 0.1 ml volume of each supernatant was added to the xanthine-xanthine oxidase O₂^– generating system, which consisted of the SOD assay mixture [8.2 mg per ml of ferricytochrome c, 2 mM hypoxanthine, and 50 mM disodium EDTA, 125 mM phosphate buffer (pH 7.8)] in a total volume of 1.8 ml, and 0.1 ml of 0.12 U xanthine oxidase per ml. SOD activity was measured at 25°C. In this system, the formation of O₂^– is determined by ferricytochrome c (type III) reduction, and the absorbance was measured using a spectrophotometer (U-3200: Hitachi, Tokyo, Japan) at 550 nm. One unit was defined as the amount of SOD sufficient to inhibit the rate of reduction of ferricytochrome c by 50%, and specific activity was expressed as U per mg protein. To determine Mn-SOD activity, KCN (2 mM) was added to the mixture to inhibit Cu,Zn-SOD activity (^{Tyler 1974}). Protein concentration was determined with a BCA assay kit (Pierce).

Statistical analysis

Statistical significance was assessed by Student's t test. Mean differences were considered significant at p <0.05.

RESULTS

Effects of DDC, TNF-, or TGF-₁ on total, Cu,Zn-SOD, and Mn-SOD activities in HaCaT cells

Total SOD activities were reduced to 72% (p <0.05) and 48% (p <0.01) of the control level after 1.5 h incubation with 0.1 and 1 mM DDC, respectively. Cu,Zn-SOD activities were reduced to 50% (p <0.01) and 0.3% (p <0.01) of the control level after incubation with 0.1 and 1 mM DDC, respectively, whereas Mn-SOD activities in HaCaT cells were not affected by incubation with DDC (Figure 1a). Total SOD activities were increased to 117% (p <0.05) and 147% (p <0.01) of the control level after 24 h incubation with 0.5 and 1 ng per ml of TNF-, respectively. Mn-SOD activities were increased to 141% (p <0.01) and 196% (p <0.01) of the control level after incubation with 0.5 and 1 ng per ml of TNF-, respectively, whereas Cu,Zn-SOD activities in HaCaT cells were not affected by incubation with TNF- (Figure 1b). Mn-SOD activities in HaCaT cells were decreased to 78% (p <0.05) of the control level after 24 h incubation with 10 ng per ml of TGF-₁, whereas total and Cu,Zn-SOD activities were not affected by incubation with TGF-₁ (Figure 1c).

Figure 1.

Effects of DDC, TNF-, or TGF-₁ on total, Cu,Zn-, and Mn-SOD activities in HaCaT cells. (a) Total, Cu,Zn-SOD, and Mn-SOD activities in HaCaT cells treated with DDC (0.01, 0.1, 1 mM). Total, Cu,Zn-SOD, and Mn-SOD activities are expressed as a percentage of the control value. Control values for total, Cu,Zn-SOD and Mn-SOD activities were 8.0 U per mg protein, 4.2 U per mg protein, and 3.9 U per mg protein, respectively. Data are expressed as mean one standard deviation from the results of six separate experiments. *p <0.05 versus control; **p <0.01. (b) Total, Cu,Zn-SOD, and Mn-SOD activities in HaCaT cells treated with TNF- (0.1, 0.5, 1 ng per ml). Total, Cu,Zn-SOD, and Mn-SOD activities are expressed as a percentage of the control value. Control values for total, Cu,Zn-SOD and Mn-SOD activities were 7.6 U per mg protein, 4.0 U per mg protein, and 3.6 U per mg protein, respectively. Data are expressed as mean one standard deviation from the results of six separate experiments. *p <0.05 versus control; **p <0.01. (c) Total, Cu,Zn-SOD, and Mn-SOD activities in HaCaT cells treated with TGF-₁ (1, 5, 10 ng per ml). Total, Cu,Zn-SOD, and Mn-SOD activities are expressed as a percentage of the control value. Control values for total, Cu,Zn-SOD, and Mn-SOD activities were 8.1 U per mg protein, 4.3 U per mg protein, and 3.9 U per mg protein, respectively. Data are expressed as mean one standard deviation from the results of six separate experiments. *p <0.05 versus control; **p <0.01.

Full figure and legend (38K)

Increased extracellular leakage of LDH from damaged HaCaT cells treated with DDC following UVB irradiation

UVB-induced cytotoxicity was evaluated by measurement of the extracellular leakage of LDH, a high molecular cytosolic enzyme, and LDH release from damaged keratinocytes increased in a UVB-dose-dependent fashion. The LDH activity released in the supernatant from HaCaT cells treated with 1 mM DDC significantly increased 8 h after 20 and 30 mJ per cm² UVB irradiation (p <0.01) and also 24 h after 10, 20, and 30 mJ per cm² UVB irradiation (p <0.01) compared with untreated groups. Incubation with 1 mM DDC for 8 or 24 h did not significantly affect LDH release(Figure 2a). On the other hand, no increase of LDH release in the supernatant from HaCaT cells treated with either 1 ng per ml of TNF- or 10 ng per ml of TGF-₁ was observed 8 and 24 h after 10, 20, and 30 mJ per cm² UVB irradiation compared with untreated groups (Figure 2b,c).

Figure 2.

Increased LDH leakage from HaCaT cells treated with DDC, but not TNF- or TGF-₁ following UVB irradiation. (a) LDH activities released in the supernatant from HaCaT cells treated with DDC (1 mM) 8 and 24 h after a single exposure to UVB at a dose of 10, 20, or 30 mJ per cm^2. LDH activities are expressed as a percentage of the control value, 31 U per l. Data are expressed as mean one standard deviation from the results of six separate experiments. *p <0.01 versus control. (b) LDH activities released in the supernatant from HaCaT cells treated with TNF- (1 ng per ml) 8 and 24 h after a single exposure to UVB at a dose of 10, 20, or 30 mJ per cm^2. LDH activities are expressed as a percentage of the control value, 28 U per l. Data are expressed as mean one standard deviation from the results of six separate experiments. (c) LDH activities released in the supernatant from HaCaT cells treated with TGF-₁ (10 ng per ml) 8 and 24 h after a single exposure to UVB at a dose of 10, 20, or 30 mJ per cm^2. LDH activities are expressed as a percentage of the control value, 34 U per l. Data are expressed as mean one standard deviation from the results of six separate experiments.

Full figure and legend (25K)

Decreased cell viability of HaCaT cells treated with DDC following UVB irradiation

Cell viability following UVB irradiation was evaluated by the trypan blue dye exclusion assay. Viability of HaCaT cells treated with 1 mM DDC significantly decreased 8 h after 20 and 30 mJ per cm² UVB irradiation (p <0.05) and also 24 h after 10, 20, and 30 mJ per cm² UVB irradiation (p <0.01) compared with untreated groups. Incubation with 1 mM DDC for 8 or 24 h did not significantly affect cell viability (Figure 3). On the other hand, no decrease in viability of HaCaT cells treated with either 1 ng per ml of TNF- or 10 ng per ml of TGF-₁ was observed 8 and 24 h after 10, 20, and 30 mJ per cm² UVB irradiation compared with untreated groups (data not shown).

Figure 3.

Decreased cell viability of HaCaT cells treated with DDC following UVB irradiation. Cell viability of HaCaT cells treated with DDC (1 mM) 8 and 24 h after a single exposure to UVB at a dose of 10, 20, or 30 mJ per cm^2. Data are expressed as mean one standard deviation from the results of six separate experiments. *p <0.05 versus control; **p <0.01.

Full figure and legend (9K)

Flow cytometric analysis of intracellular peroxides produced in HaCaT cells treated with DDC following UVB irradiation

The production of intracellular peroxides in HaCaT cells untreated or treated with 1 mM DDC following 10 mJ per cm² UVB irradiation was examined by fluorescence-activated cell sorting scan analysis using a peroxide-sensitive dye, DCFH-DA. No increase of intracellular peroxides was observed in the untreated cells 8 h following UVB irradiation (Figure 4a), whereas the levels of intracellular peroxides in the cells treated with DDC significantly increased 8 h following UVB irradiation (Figure 4b).

Figure 4.

Increased production of intracellular peroxides in HaCaT cells treated with DDC following UVB irradiation. Flow cytometric analysis of intracelular peroxides in HaCaT cells untreated (A) or treated (B) with 1 mM DDC. Both untreated and treated cells were irradiated with 10 mJ per cm² UVB (black area) or were unirradiated (white area). Relative peroxide concentrations in the cells were quantitated by flow cytometry using a peroxide-sensitive dye, DCFH-DA, 8 h following UVB irradiation.

Full figure and legend (14K)

DISCUSSION

The UVB-induced ROS are generally thought to cause oxidative stress and subsequent photodamage to cellular membrane lipids, proteins, and DNA (^{Breimer 1991;Tyrrell 1995}) in human skin, which leads to skin cancer, photoaging (^{Ananthaswamy & Pierceall 1990;Miyachi 1995}), and many acute or chronic inflammatory skin disorders (^{Black 1987}). Because the surface of the skin is always in contact with oxygen and is one of the major targets for UV light, skin requires efficient mechanisms to protect itself from oxidative stress. Antioxidant enzymes such as SOD in the human epidermis are crucial as the outermost barrier against ROS. With regard to the role of SOD against UVB-induced oxidative stress in the epidermis, it has been reported that endogenous SOD levels are decreased after acute UVB irradiation due to scavenging ROS (^{Hashimoto et al. 1991;Punnonen et al. 1991}). Furthermore, we have recently demonstrated that Cu,Zn-SOD and Mn-SOD change differently following UVB irradiation in the human keratinocyte cell line HaCaT (^{Sasaki et al. 1997}), suggesting that endogenous Cu,Zn-SOD and Mn-SOD may play different defensive roles against UVB-induced keratinocyte injury.

In this study, we demonstrated that Cu,Zn-SOD activities in HaCaT cells treated with 1 mM DDC were markedly decreased, whereas Mn-SOD activities were not. Several reports have shown that DDC inactivates Cu,Zn-SOD activities by chelating copper ion, an active center of the enzyme, without affecting other antioxidant enzymes such as Mn-SOD, catalase, and glutathione peroxidase in vivo and in vitro (^{Heikila et al. 1976;Hiraishi et al. 1994}). We confirmed that 1 mM DDC does not affect either Mn-SOD (Figure 1), catalase, or glutathione peroxidase activities (data not shown) in HaCaT cells. We used a very low concentration of DDC (1 mM) in this study, because DDC itself possesses strong cytotoxicity.

To evaluate the defensive role of Cu,Zn-SOD against UVB-induced injury of HaCaT cells, we measured LDH release in the supernatant from keratinocytes treated or untreated with DDC 8 and 24 h after UVB irradiation. Extracellular LDH release in the supernatant is well known to be associated with the extent of cell membrane damage in culture cells (^{Gaboriau et al. 1993;Tebbe et al. 1997}). The LDH release in the culture medium from HaCaT cells treated with 1 mM DDC significantly increased compared with untreated controls 8 and 24 h following UVB irradiation. The viability of HaCaT cells treated with 1 mM DDC significantly decreased compared with untreated controls 8 and 24 h following UVB irradiation. These results suggest that Cu,Zn-SOD may play a role as an early phase defense mechanism against UVB-induced cytotoxicity in HaCaT cells.

Next, we demonstrated that Mn-SOD activities but not Cu,Zn-SOD activities in HaCaT cells were markedly enhanced after the treatment with 1 ng per ml of TNF-. In a previous study we have shown that 1 ng per ml of TNF- markedly enhances Mn-SOD activities in HaCaT cells (^{Sasaki et al. 1997}). In this study we demonstrated that the treatment with 10 ng per ml of TGF-₁ significantly inhibits Mn-SOD activities in HaCaT cells, whereas Cu,Zn-SOD activities are not affected by TGF-₁. TGF-₁, which plays a central role in negative regulation of cell growth, has been reported to suppress antioxidative enzyme gene expression, mainly Mn-SOD and glutathione-S-transferase, in rat hepatocytes (^{Kayanoki et al. 1994}). We here report that TGF-₁ suppresses Mn-SOD activities in human keratinocytes.

To evaluate the defensive role of Mn-SOD against UVB-induced injury of HaCaT cells, we measured LDH release in the supernatant from damaged keratinocytes treated or untreated with 1 ng per ml of TNF- after UVB irradiation. The treatment with 1 ng per ml of TNF- had no effect on the LDH release in the culture medium from HaCaT cells 8 and 24 h following UVB irradiation. We also examined the effect of TGF-₁. The treatment with 10 ng per ml of TGF-₁ had no effect on the LDH release in the culture medium from HaCaT cells 8 and 24 h following UVB irradiation. Moreover, we also examined the viability of HaCaT cells treated with TNF- or TGF-₁ after UVB irradiation. The treatment with either 1 ng per ml of TNF- or 10 ng per ml of TGF-₁ had no significant effect on the viability of HaCaT cells 8 and 24 h following UVB irradiation compared with untreated groups (data not shown). These results seem to indicate that Mn-SOD does not participate in an early phase defense mechanism against UVB-induced cytotoxicity. Although there have been no reports concerning the role of Mn-SOD against UVB-induced oxidative stress in the epidermis, Mn-SOD may be related to a chronic phase defense mechanism against UVB-induced cutaneous disorders.

To confirm a protective role of Cu,Zn-SOD against UVB-induced early phase cytotoxicity in keratinocytes, we examined the production of intracellular peroxides following UVB irradiation using flow cytometry. Flow cytometric analysis of peroxides 8 h after UVB irradiation showed that the production of intracellular peroxides was increased in the HaCaT cells treated with 1 mM DDC compared with nonirradiated controls. On the other hand, there were no significant differences between untreated HaCaT cells after UVB irradiation and nonirradiated controls. The results of flow cytometric analysis 24 h after UVB irradiation were similar to those 8 h after irradiation (data not shown). It is generally accepted that UV-induced ROS causes lipid peroxidation of plasma membrane, which is well known to be associated with the extent of cellular damage in vitro (^{Girotti 1990}; Morliére et al. 1990). The induction of lipid peroxidation following UVB or UVA exposure in human keratinocytes or fibroblasts has been documented previously (^{Iizawa et al. 1994;Morliére et al. 1995}). Our results suggest that Cu,Zn-SOD may play an early phase protective role against UVB-induced lipid peroxidation.

In this study, increased cytotoxicity following UVB irradiation was observed in the HaCaT cells whose Cu,Zn-SOD levels were inactivated by DDC, as determined by LDH leakage in the supernatant and cell viability 8 and 24 h after UVB irradiation and also by flow cytometric analysis of intracellular peroxides 8 h after UVB irradiation. Treatment with 1 mM DDC had no effect on either LDH leakage, cell viability (Figure 2a, Figure 3), or intracellular peroxide production (data not shown). On the other hand, no significant differences were observed in HaCaT cells whose Mn-SOD was enhanced or inhibited with cytokines. Our results suggest that Cu,Zn-SOD plays a primary protective role against UVB-induced acute phase keratinocyte injury, presumably due to more widespread and predominant distribution of endogenous Cu,Zn-SOD than Mn-SOD in cells.

It remains unclear whether Mn-SOD does not actually participate in an acute phase defense mechanism against UVB-induced injury of HaCaT cells, although Mn-SOD is decreased following UVB irradiation. It was difficult to evaluate the possible protective role of Mn-SOD against acute phase UVB-induced keratinocyte injury in this study, because there is no reagent that perfectly inhibits Mn-SOD. In fact, we demonstrated that TGF-₁ at a concentration of 10 ng per ml, which is known to suppress Mn-SOD in rat hepatocytes, suppressed only 22% of Mn-SOD activities, whereas DDC at a concentration of 1 mM perfectly inactivated Cu,Zn-SOD activities in HaCaT cells. We have also examined the protective role of Cu,Zn-SOD or Mn-SOD against UVA-induced cytotoxicity in HaCaT cells. We measured LDH release from DDC or TGF-₁ treated cells following 1, 5, or 10 J per cm² UVA irradiation. The results showed that LDH release from HaCaT cells treated with TGF-₁ whose Mn-SOD activity was suppressed significantly increased after UVA irradiation compared with control groups. On the other hand, no significant increase of LDH leakage was observed in the supernatant of the cells whose Cu,Zn-SOD activity was inactivated by DDC after UVA irradiation (data not shown). These results suggest that Mn-SOD may take part in the protection against UVA-induced injury of the human keratinocyte. Further experiments using other methods are required to clarify these points.

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Acknowledgments

We are grateful to Dr. K. Suzuki (Department of Biochemistry, University of Osaka, Osaka, Japan) for useful discussions and help with the flow cytometric analysis.