The Effect of Up- and Downregulation of MnSOD Enzyme on Oxidative Stress in Human Lens Epithelial Cells

Abstract

purpose. Gene knockouts serve as useful experimental models to investigate the role of antioxidant enzymes in protection against oxidative stress in the lens. In the absence of gene knockout animals for Mn-containing superoxide dismutase (MnSOD), the effect of this enzyme on oxidative stress was investigated in a human lens epithelial cell line (SRA 01/04) in which the enzyme was up- or downregulated by transfection with sense and antisense expression vectors for MnSOD.

methods. Human lens epithelial cells (SRA 01/04) were transfected with plasmids containing sense and antisense human cDNA for MnSOD. The enzyme levels were determined by Western blot analysis and immunocytochemistry. The protective effect of the enzyme against the cytotoxicity of H₂O₂ was evaluated by cell viability, DNA strand breaks, and morphologic changes observed by transmission electron microscopy. In addition, the protective effect of this enzyme against photochemically induced stress and UVB irradiation in cells was assessed by measuring the damage of cellular DNA.

results. The MnSOD level in upregulated cells was three times higher than in downregulated cells, and the enzyme surrounded the nucleus. Cells with elevated enzyme levels were more resistant to the cytotoxic effect of H₂O₂ with greater cell viability. MnSOD-deficient cells showed dramatic mitochondrial damage when exposed to 50 μM H₂O₂ for 1 hour. Similarly, oxidative challenge by H₂O₂, photochemically generated reactive oxygen species, or UVB irradiation produced greater DNA strand breaks in MnSOD-deficient cells than in those in which the enzyme was upregulated.

conclusions. These findings demonstrate the protective effect of MnSOD in antioxidant defense of cultured lens epithelial cells. This approach to modulating the enzyme level in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens.

Cataract remains the leading cause of blindness worldwide, with an estimated 17 million people experiencing profound or total loss of vision due to lens opacification. ¹ The burden of this avoidable cause of vision loss falls primarily on developing countries, where fewer than 20% of patients with cataract are estimated to receive surgery. ² However, if the risk factors for cataract could be identified and strategies for prevention implemented, the number of people blinded by cataract could be diminished. In addition, if a factor could be found that simply delayed the onset of cataract by 10 years, the number of cataract operations needed would decline by 45%. ³  

Oxidative stress ⁴ is one of the risk factors and has been implicated in the pathogenesis of age-related cataract. ^{4 5 6 7 8} Oxidative damage resulting from free radicals and/or H₂O₂ has been considered a major factor in the development of age-onset cataracts. The oxidative modification of a number of cellular constituents, such as proteins, ^{9 10 11 12} cytoskeletal elements, ⁷ membrane sulfhydryls, ⁹ glutathione levels, and alterations in transport systems in the epithelium are thought to result in lens opacification and nuclear cataracts. ¹¹ A group of antioxidant enzymes that are known to serve as a defense system to protect cells against oxidative stress ¹² includes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase. 

SOD is one of the key enzymes that detoxifies the superoxide radical O₂ ⁻ and generates H₂O₂ which in turn is detoxified by GPx ¹³ and catalase. The two major isoforms of SOD are Mn- and CuZn-containing enzymes. CuZnSOD, a cytosolic enzyme, accounts for nearly 90% of total SOD. ¹⁴ The role of CuZnSOD in protecting against oxidative stress can be investigated by using transgenic and gene knockouts of this enzyme. ^{15 16} However, it is difficult to study the animal model with a complete knockout of MnSOD, ^{17 18} because the animals die within 10 days. ^{19 20 .} Useful information on the role of MnSOD in inhibiting oxidative insult can be obtained by comparing heterozygous (partial knockout) and wild-type animals. ^{20 21}  

The mitochondrial enzyme (MnSOD), which is localized in the epithelium, may be involved in protecting the lens against free-radical–induced injury. Moreover, it has been reported that MnSOD is induced by oxidative stress mediators, including radiation, ^{22 23 24} lipopolysaccharides, ²⁵ tumor necrosis factor (TNF)-α, ^{23 24 25 26} and interleukin (IL)-1. ^{24 25 27} Lens epithelium plays an important role in the maintenance of normal physiology, metabolic activity, and homeostasis of the lens. ^{28 29 30 31 32}  

One approach to studying the role of MnSOD in lens epithelium is by up- and downregulation of the enzyme through introduction of sense and antisense cDNA. Accordingly, in the present investigation, an established human lens epithelial (HLE) cell line SRA 01/04 ³³ was transfected with plasmids containing sense and antisense cDNA, and the protective effect of this enzyme against H₂O₂-induced oxidative challenge, photochemically generated oxidative stress, and UVB irradiation was evaluated. 

Materials and Methods

Cell Culture

The human lens epithelial cell line SRA 01/04 was established by transformation of primary cultured human lens epithelium with a DNA plasmid containing the large T antigen of simian virus (SV)40. ³³ These cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) with 15% fetal bovine serum (FBS; Gibco) in a 100 × 20-mm culture dish (Falcon, Lincoln Park, NJ) in a 5% CO₂ environment. After the cells were confluent and subcultured, they were used in triplicate in each experiment. 

Construction of Expression Vectors

To construct the human MnSOD expression vectors, ³⁴ the 1.0-kb EcoR1 DNA fragment containing the full-length human MnSOD cDNA, as described previously, was ligated into the EcoR1 site of the expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Expression of the cDNA insert in this vector is driven by the promoter of the human cytomegalovirus immediate early gene. Digestion of the expression vectors with BamH1, which cut the cDNA asymmetrically, allowed determination of the orientation of the inserted cDNA (Fig. 1) . 

Transfection and Selection of Stably Transfected Cells

Established SRA 01/04 cells were transfected with expression vectors containing human cDNA for MnSOD and the neomycin-resistant gene by using a lipofectin system (LipofectAmine 2000 Reagent, LF-2000; Gibco). Cells (n = ∼100,000) were cultured in 12-well plates (Corning Costar, Corning, NY) to 95% confluence on the day of transfection. Two micrograms of cDNA was diluted in 100 μL medium without serum (Opti-MEM I; Gibco); 2 μL of liposome (LF-2000 Reagent; Gibco) was diluted in another 100 μL of serum-free medium (Opti-MEM I) and incubated for 5 minutes at room temperature. The two diluted media were mixed and incubated at room temperature for 20 minutes. Into each well, 200 μL of cDNA and the liposome complex (LF2000 Reagent; Gibco) was pipetted. The cells were incubated at 37°C in a 5% CO₂ atmosphere for 6 hours. After the medium was changed to DMEM containing 15% fetal bovine serum, cells were incubated overnight, and the medium was replaced once again. Cell culture was continued until confluence. To obtain selected transfected cells, the cells were cultured in 200 μg/mL of geneticin (G418; Gibco) in DMEM containing 15% serum for 2 weeks to kill all nontransfected cells. The floating dead cells were washed out, and the culture of live cells continued with change of medium twice a week to obtain stably transfected cells. Two sets of controls were used: one consisting of the original established SRA 01/04 cell line and the other in which the cells were transfected with plasmid alone. These cells were cultured and treated with geneticin in a manner similar to cells transfected with expression vectors. 

Western Blot Analysis

Cultured cells were dissolved in 2% sodium dodecyl sulfate (SDS) buffer. SDS-PAGE (12%) was run for protein separation. The separated proteins were transferred to nitrocellulose membrane (Trans-Blot Transfer Medium; Bio-Rad, Hercules, CA). For blocking, the membrane was incubated in 5% fat-free milk (Blotting Grade Blocker Nonfat Dry Milk; Bio-Rad) overnight at 4°C and was incubated with sheep anti-human MnSOD enzyme antibody (Ab; The Binding Site, Birmingham, UK), which was diluted 1:200, overnight at 4°C. After it was washed with Tris-buffered saline (TBS; Bio-Rad), the membrane was incubated for 1 hour at room temperature with a secondary antibody (anti-sheep goat IgG labeled with biotin; Sigma-Aldrich, St. Louis, MO), which was diluted 1:3000. After a thorough wash with TBS, the membrane was incubated in a conjugated complex of streptavidin and biotinylated alkaline phosphatase (Amplified Alkaline Phosphatase Immun-Blot Assay Kit; Bio-Rad), and color was developed with an alkaline phosphatase conjugate substrate kit (Bio-Rad). A prestained protein marker (Prestained SDS-PAGE Standards, broad range; Bio-Rad) was used to estimate the molecular weight. The staining reaction was analyzed by quantitative densitometry by using a computerized image analysis program (NIH Image, ver.1.61; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). 

Immunocytochemistry

The cells were cultured on a coverslip that had been placed on the bottom of the culture plate and fixed with methanol which had been cooled to −20°C. After rehydrating with PBS, they were blocked with 10% goat serum and rinsed and washed. The washed cells were incubated with sheep anti-human MnSOD Ab (1:20 dilution; Binding Site). Goat anti sheep Ig-G Ab (1:40 dilution) labeled with FITC (anti-sheep Ig-G FITC conjugate; Sigma-Aldrich) was used as a secondary antibody. The cells were photographed with a fluorescence microscope (VANOX-S; Olympus, Melville, NY). 

Cell Viability Assay

Cell viability was examined in both transfected and control cells under either normal culture conditions (normal medium) or after the cells were subjected to oxidative stress. The cells were cultured in 12-well culture plates to 95% confluence, and trypsinized with 0.025% trypsin-EDTA (Gibco). The living cells in each suspension were stained with trypan blue dye (0.4% in saline; Gibco) and counted with a hemocytometer. Cell viability was expressed by calculating the percentage of nonstained cells to the initial number of cells plated in the culture dish. 

DNA Single Strand Breaks Assay

To assess DNA damage induced by oxidative stress, the single-cell gel electrophoresis ³⁵ technique was used. The cells (transfected and control), with or without oxidative stress, were harvested by scraping and centrifuging and mixed with 0.8% low-melting-temperature agarose at 37°C and cast onto a precleaned frosted microscope slide that had been coated with a thin layer of 0.8% normal melting agarose to promote adhesion of a second layer. The slides were covered with coverslips and kept at 4°C for 5 minutes. After the coverslips were removed, the slides were covered with a third layer of 0.8% normal melting agarose at 37°C, which acted as a protective layer, and then were covered with a coverslip and kept at 4°C for 5 minutes. The coverslips were removed, and cells were incubated in the dark for 1 hour at 4°C with lysing solution (1% N-laurylsarcosine sodium, 2.5 M NaCl, 100 mM EDTA, 10 mM Tris [pH 10.0] and 1% Triton X-100). These sample slides were placed into running buffer for 20 minutes at 4°C in the dark. This buffer had been freshly prepared with 1 mM EDTA and 300 mM NaOH and electrophoresed at 17 V for 20 minutes at 4°C in the dark. The slides were then placed in 0.4 M Tris at pH 7.5 for 5 minutes. After slides were stained with 20 μg/mL ethidium bromide, coverslips were placed on the samples, and the DNA migration in the cells was photographed. The migration (in millimeters) was measured from enlarged photographs (5 × 7 in.). 

Transmission Electron Microscopy

Cells with up- and downregulated enzyme levels along with the control (nontransfected) cells were cultured in 12-well plates and treated with a single bolus of 50 μM H₂O₂ in Hank’s balanced buffered solution (HBBS) for 1 hour. The samples were fixed in 2.5% (vol/vol) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) overnight and postfixed with 1% osmium tetroxide in the same buffer for 1 hour. They were then dehydrated in an ascending series of ethanol and embedded in epoxy resin (PolyBed 812; Polysciences, Warrington, PA). ³⁶ En face sections were cut from embedded samples and stained with lead citrate and uranyl acetate and examined under a transmission electron microscope (model EM 410; Philips, Mahwah, NJ). 

Oxidative Stress

Three types of oxidative stress were used: exposure to H₂O₂, exposure to reactive oxygen species derived by photochemical reaction, or exposure UVB irradiation. For H₂O₂-induced damage, cells were seeded in DMEM containing 15% FBS for 24 hours to allow attachment to the culture plates. After cell attachment was confirmed, the DMEM-containing serum was replaced by serum-free DMEM containing a single bolus of 50 μM H₂O₂. 

For photochemical stress, attached cells were incubated in serum-free DMEM containing 15 μM riboflavin in HBBS (overnight, 15 hours), exposed to a 15-W circular daylight fluorescent lamp placed 10 cm above the plate, and irradiated for 30 minutes. ³⁷  

For UVB irradiation, attached cells were exposed to either 0.05 or 0.09 J/cm² UVB. A broad-spectrum lamp, with a wavelength peak of 310 nm (Spectroline, model EB-160C; Spectronics Corp., Westbury, NY) was used as the source of the UVB radiation. The cells were cultured in 12-well plates to 90% confluence and irradiated according to the desired dosage. The UVB dose was monitored and measured by a radiometer and sensor (UVX-31; UVP Inc., San Gabriel, CA). The radiation dose was adjusted by varying the duration of exposure time for each set of experiments. The maximum time of UVB irradiation was less than 2 minutes. 

Statistical Analysis

The comparison of results obtained in cell viability assay and DNA strand breaks were performed by Student’s t-test. P < 0.05 was considered significant. 

Results

Because the major objective was to establish stably transfected cells with up- and downregulated levels of MnSOD, we first compared the level of the enzyme in sense and antisense transfected cells with that in the control (nontransfected cells and cells transfected with plasmid alone). To quantify the expression of MnSOD, transfected and control cells were cultured in dishes to 95% confluence and then were lysed with SDS sample buffer. Equal amounts of protein (25 μg) from each cell extract were separated with electrophoresis and transferred to nitrocellulose membrane. Protein blot analysis with antibody to human MnSOD revealed 25-kDa bands in each of the samples (Fig. 2A) . Density scanning of these immunoblots showed that the expression of MnSOD in upregulated cells was approximately 20% higher than in the two control cells (Fig. 2B) . However, the expression level in antisense transfected cells was approximately 50% lower than the endogenous level of the enzyme in control cells. Thus, the upregulated cells had roughly a 2.5 times higher enzyme level than did antisense transfected cells. 

Experiments were also performed to examine the localization of MnSOD by a histochemical technique in which intact cells were stained with anti-human MnSOD antibody (Binding Site). The immunocytochemistry revealed stronger fluorescence intensity near or surrounding the nucleus (Fig. 3) . Consistent with the immunoblot results (Fig. 2) , the intensity of MnSOD staining in sense transfected cells was clearly greater than in antisense transfected cells (compare 1 and 3 in Fig. 3 ), whereas the intensity of fluorescence in the control cells (SRA 01/04) appeared to be intermediate between up- and downregulated cells. 

Cell Morphology and Viability

In view of the differences noted in MnSOD levels in the up- and downregulated cells, we examined the viability of these cells under both normal culture conditions and oxidative stress. Figure 4 shows phase contrast micrographs of cells at 95% confluence. The cell shape and size of the transfected and nontransfected cells appeared epithelial-cell–like and no differences in morphology was observed with a phase-contrast microscope (Fig. 4) . 

It has been reported that MnSOD can increase the resistance against oxidative stress. ^{16 17 18} We therefore examined the effect of H₂O₂-induced cytotoxicity in the up- and downregulated cells by assessing cell viability after exposure to H₂O₂. Transfected cells were seeded in quadruplicate in culture dishes in DMEM containing 15% serum for 24 hours for attachment. The medium was changed to HBBS containing a bolus of 50 μM H₂O₂, and the cells were cultured further for 12 hours. Parallel cell cultures without H₂O₂ treatment served as the control. To evaluate cell viability, live cells (not stained with trypan blue) were counted and compared with cell numbers in non-H₂O₂–treated control cells. Figure 5 shows that the number of upregulated cells was 95% (5% decrease) of that in non-H₂O₂–treated cells. However, in the cells in which MnSOD was downregulated, H₂O₂ treatment resulted in a cell loss of nearly 30% (P < 0.01), compared with non-H₂O₂ treated cells. Thus, the cells with lower MnSOD levels were less resistant to H₂O₂-induced cytotoxicity. 

We also examined the effect of H₂O₂-induced oxidative stress on mitochondria in sense and antisense transfected cells by electron microscopy. The cells were exposed to a single bolus of 50 μM H₂O₂ in HBBS medium for 60 minutes. After fixation and embedding, en face sections were examined with a transmission electron microscope, as described in the Materials and Methods section. As shown in Figure 6 , MnSOD-deficient cells showed dramatic mitochondrial damage and loss after exposure to H₂O₂. Although almost all mitochondrial matrix and the inner membrane in antisense transfected cells were damaged or lost, the integrity of the mitochondria in sense transfected cells was better preserved. The morphology of these cells was similar and comparable to that of the nontransfected controls (Fig. 6B) . These results suggest that elevated levels of MnSOD protect mitochondria against oxidative damage resulting from treatment with H₂O₂. 

Effect of Oxidative Stress on DNA Damage

The effect of H₂O₂-induced stress on DNA, as assessed by single-strand breaks in cells with different levels of MnSOD, is shown in Figure 7 . One hundred thousand sense and antisense transfected cells along with control cells were seeded in 12-well plates and cultured to 95% confluence. After they were washed with PBS, the cells were exposed to a single bolus of 50 μM H₂O₂ in 2 mL HBBS medium for 30 minutes. Catalase (60 units, Sigma-Aldrich) was then added to deplete the residual H₂O₂ in the buffer, and the cells were subjected to electrophoresis, as described in the Materials and Methods section. The data in Figure 7 show that DNA damage in MnSOD-deficient cells was more than 2.5 times greater than in upregulated cells (Fig 7 , right; P < 0.01), whereas that in both control cell groups was intermediate. Also, the extent of DNA damage in the two sets of control cells was indistinguishable. 

Because H₂O₂ is a substrate for GPx-1 and catalase, the oxidative damage resulting from peroxide may not directly involve MnSOD. In addition, H₂O₂ is known to give rise to OH radicals through the Fenton reaction. ³⁸ Therefore, we assessed the protective effect of MnSOD against DNA damage induced by other oxidants known to generate free radicals, such as photochemically generated O₂ ⁻ and UVB radiation. In view of the observations that the maximum difference in DNA damage induced by H₂O₂ was noted between up- and downregulated cells only these cells, were further subjected to photochemical insult or UV-B radiation in the following series of experiments. 

Irradiation of riboflavin with visible light generates reactive oxygen radicals including O₂ ⁻ which is dismutated by SOD. The results in Figure 8 show that DNA strand breaks were much greater in MnSOD-deficient cells than in cells in which the enzyme levels were upregulated. DNA migration in downregulated cells was 3.5 times greater that that in upregulated cells. 

Similarly, a higher level of MnSOD exerted a significant protective effect against UVB-induced DNA damage (Fig. 9) . This was evident particularly when the cells were exposed to a lower dose (0.05 J/cm²) of UVB. At a higher level of irradiation (0.09 J/cm²), the protective effect of MnSOD on DNA damage, although observable, appeared less significant. This may suggest that a higher irradiation dose may overwhelm the protective effect of MnSOD on DNA damage. 

Discussion

In the present study, we succeeded in transfecting an established HLE cell line with sense and antisense expression vectors for MnSOD and obtained HLE cells with different expression levels of MnSOD. Our results also demonstrate that cells with elevated MnSOD levels were more resistant to the cytotoxic effect of H₂O₂. In addition, the cells with elevated levels of MnSOD protected against DNA strand breaks induced by H₂O₂. Furthermore, the mitochondrial integrity of the cells exposed to H₂O₂ was better preserved in cells transfected with sense cDNA for MnSOD than in cells in which the enzyme was downregulated (Fig. 7) . 

The defense against the harmful effects of free radicals is achieved by endogenous antioxidant compounds and enzymes present in the lens. Other investigators have also used gene transfer of MnSOD to study the function of this enzyme, ^{17 22 39} and it has been reported that MnSOD is important in affording resistance to paraquat-mediated cytotoxicity, ¹⁶ X-irradiation, ^{22 23 24} and the cytotoxic effects of TNF-α ^{23 24 25 26} or IL-1. ^{24 25 27} At present, the mechanism by which MnSOD protects against H₂O₂-induced stress is unclear, because this enzyme detoxifies superoxide radicals rather than H₂O₂ directly. It is known, however, that H₂O₂ can serve as a substrate for the iron-mediated (Fe²⁺) Fenton reaction ³⁸ and can generate O₂ ⁻. ⁴⁶ Therefore, MnSOD may protect the cells by removing the O₂ ⁻ generated from the increased concentration of H₂O₂. Alternatively, O₂ ⁻ can reduce Fe³⁺ to form Fe²⁺, which can then participate in the Fenton reaction. Such an event suggests that MnSOD also functions in blocking the generation of the hydroxyl radical by preventing the regeneration of Fe²⁺ from Fe³⁺. 

Mitochondria are especially sensitive to oxidative damage, and it has been reported that mitochondrial damage induced by oxidants can cause release of calcium, ⁴⁰ protein oxidation, ⁴¹ loss of electron transport capacity, ⁴² and mitochondrial DNA damage. ⁴³ Oxidative stress can cause damage to mitochondrial function by decreasing mitochondria-derived products, which can result in damage to cell function and cause cell death. ^{44 45} In the present study, mitochondria were more intact after exposure to H₂O₂ in MnSOD-upregulated cells than in downregulated cells. This suggests that MnSOD may have a protective effect on mitochondria against H₂O₂-induced stress. Because MnSOD is located near and surrounding the nucleus (Fig. 3) , it may protect nuclear DNA against H₂O₂-induced stress, indirectly. To the extent that lens epithelium contains mitochondria, in contrast to the cortex, MnSOD may play an important role in lens homeostasis. 

The experiments involving photochemical stress and UVB irradiation were undertaken to examine the protective role of MnSOD against oxidative damage resulting directly from O₂ ⁻ radicals, which are scavenged by this enzyme. Although photochemical reaction and UVB irradiation are known to give rise to a number of reactive oxygen species, the effect of MnSOD against oxidative damage by photochemical insult and UVB irradiation must involve the scavenging of O₂ ⁻ radicals. 

Taken together, the present results clearly demonstrate that MnSOD exerts a protective effect against oxidative damage induced by H₂O₂, the photochemical reaction, and UVB irradiation. The present approach of modulating the enzyme in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens. 

 Supported by National Eye Institute Grants EY00484 (VNR); Vision Core Grant EY07003; National Institute of Environmental Health Sciences Grant P30-ES006639 (Y-SH); an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, University of Michigan; and an unrestricted grant from Shojin Research Associates, Studio City, California (VNR).

 Submitted for publication August 15, 2002; revised December 4 and December 18, 2002; accepted January 7, 2003.

 Disclosure: H. Matsui, None; L.-R. Lin, None; Y.-S. Ho, None; V.N. Reddy, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

(A) Representation of sense and antisense expression vectors of human MnSOD. A 1-kb EcoR1 DNA fragment containing full-length human MnSOD cDNA was isolated and ligated into the EcoR1 site of the expression vector. The cells were transfected with plasmids containing sense and antisense cDNA for MnSOD. Sense orientation is shown in the top line and antisense orientation in the bottom line. (B) The orientation of the inserted cDNA was determined by digestion of the expression vectors with BamH1. The fragment size of sense and antisense cDNA after digestion with BamH1 is shown by the DNA gel. The sense expression vector is marked by the release of a 603-bp BamH1 fragment (lane 2) and antisense expression vector by the release of a 434-bp fragment (lane 3); 1-kb DNA ladder (lane 1).

Figure 2.

 

Western blot analysis of SDS-PAGE from cell cultures. (A) Cells were transfected with (lane S) sense MnSOD, (lane AS) antisense MnSOD cDNA (lane V) and plasmid alone. Lane 01/04: nontransfected cell line. Extracts containing 25 μg of protein from each experiment were used for separation on SDS-PAGE. Transblots were immunostained with anti-human MnSOD antibody. (B) The levels of MnSOD expressed in the four cell extracts were quantified by relative integrated density scanning of the immunoblots shown in (A).

Figure 3.

 

Immunofluorescence staining of transfected and nontransfected SRA 01/04 cells. (1) Transfected with sense cDNA, (2) nontransfected control, (3) transfected with antisense cDNA, as detected by anti-human MnSOD antibody. The cells were cultured on coverslipped culture slides and fixed with precooled (−20°C) methanol. After rehydration with PBS and blocking with 10% goat serum, they were incubated with anti-human MnSOD antibody and viewed under a fluorescence microscope.

Figure 4.

 

Phase-contrast photomicrographs of confluent cultures of transfected and nontransfected cells. SRA 01/04 cells transfected with plasmids containing expression vectors of sense and antisense cDNA for MnSOD were cultured in DMEM with 15% fetal bovine serum. The confluent monolayer of cells was observed by phase-contrast microscope at the end of a 4-day culture. (A) Nontransfected cells and cells transfected with sense (B) or antisense (C) cDNA. Morphology of all cells was similar.

Figure 5.

 

Viability of SRA 01/04 cells transfected with sense (□) and antisense MnSOD (▪) when the cells were cultured in the presence of 50 μM H₂O_2. The cells were exposed to a single bolus of 50 μM H₂O₂ and cultured for 12 hours. Cells not treated with H₂O₂ served as the control. The number of living cells in stressed and nonstressed cultures was determined in trypsinized suspensions by trypan blue staining. Cell viability was calculated from the percentage of nonstaining cells to the initial number of cells plated in the culture dish. Error bars, SD (P < 0.01). The viability of nonstressed cells was not significantly different.

Figure 6.

 

TEM micrographs showing the effect of H₂O₂ on mitochondria of transfected cells and nontransfected control cells. (A) Cells transfected with sense cDNA for MnSOD, (B) nontransfected cells, and (C) cells transfected with antisense cDNA for MnSOD. The cells were exposed to 50 μM H₂O₂ for 60 minutes (single bolus) and fixed and embedded in epoxy resin directly in the culture dish. Ultra thin en fas sections were cut and examined by TEM. The number and integrity of mitochondria (arrow) in sense transfected cells was well preserved (A), whereas almost the entire mitochondrial matrix and inner membrane was damaged or lost in antisense transfected cells (C) Also, there was a ballooning of the mitochondrial structure. In the nontranfected control cells (B), the damage to mitochondria appeared considerably less than MnSOD-deficient cells. Arrows: mitochondria. Scale bar, 1 μm.

Figure 7.

 

The effect of the MnSOD level on DNA strand breaks induced by H₂O₂ in SRA 01/04 cells transfected with sense and antisense cDNA for MnSOD. The cells were cultured for 30 minutes in the presence of 50 μM H₂O₂ (single bolus). DNA strand breaks were determined by single-cell gel electrophoresis. Left: representative micrograph of ethidium bromide–stained cells: (1) cells transfected with sense cDNA, (2) nontransfected cells, (3) cells transfected with plasmid only, and (4) cells transfected with antisense cDNA for MnSOD. Right: DNA migration in the corresponding transfected and control cells was quantified by measuring the length of the comet in at least 50 cells in each experiment (P < 0.01). Error bars: SD.

Figure 8.

 

The effect of MnSOD level on DNA strand breaks induced by photochemical reaction. SRA 01/04 cells transfected with sense and antisense cDNA for MnSOD. The cells were cultured in 15 μM riboflavin in HBBS overnight (15 hours). A 15-W circular daylight fluorescent lamp irradiated the plate for 30 minutes. DNA strand breaks were determined by single-cell gel electrophoresis. (A) Representative micrographs of ethidium bromide–stained cells. Cells transfected with sense (top) and antisense (bottom) cDNA for MnSOD. (B) DNA migration in the two cell lines was quantified by measuring the length of the comet in at least 50 cells in each experiment. (□) Sense and (▪) antisense transfected cells (P < 0.01). Error bars: SD.

Figure 9.

 

The effect of MnSOD level on DNA strand breaks induced by UVB in SRA 01/04 cells transfected with sense and antisense cDNA for MnSOD. The cells were cultured in serum-free DMEM and exposed to 0.05 or 0.09 J/cm² UVB. DNA strand breaks were determined by single-cell gel electrophoresis. (A) Representative micrographs of ethidium bromide–strained cells transfected with sense (top) and antisense (bottom) cDNA for MnSOD. (B) The DNA migration in the two cell lines was quantified by measuring the length of the comet in at least 50 cells in each experiment. (□) Sense and (▪) antisense transfected cells. Significant differences were observed between sense and antisense transfected cells only at 0.05 J/cm² (P < 0.01). Error bars: SD.

The authors thank Pamela C. Sieving, MA, MS, for conducting literature searches for this work.