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Effects of variation in superoxide dismutases (SOD) on oxidative stress and apoptosis in lens epithelium
Experimental Eye Research 79 (2004) 859–868 www.elsevier.com/locate/yexer
Effects of variation in superoxide dismutases (SOD) on oxidative stress and apoptosis in lens epithelium V.N. Reddya,*, E. Kasaharaa, M. Hiraokab, L.-R. Lina, Y.-S. Hoc a
The Departments of Ophthalmology and Visual Sciences, University of Michigan, Kellogg Eye Center, 1000 Wall St., Ann Arbor, MI 48105, USA b Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA c Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA Received 2 March 2004; accepted in revised form 14 April 2004 Available online 10 July 2004
Abstract Among the critical antioxidant enzymes that protect the cells against oxidative stress are superoxide dismutases: CuZnSOD (Sod1) and MnSOD (Sod2). The latter is also implicated in apoptosis. To determine the importance of these enzymes in protection against reactive oxygen species (ROS) in the lens, we analysed DNA strand breaks in lens epithelium from transgenic and knockout (Sod1) mice following exposure to H2O2 in organ culture. Since Sod2 knockouts do not survive, comparison was made of lenses of partially-deficient (heterozygote) for Sod2 and the wild-type controls which have twice the enzyme level. Antioxidant potential of Sod2 was also studied in human lens epithelial cells (SRA01/04) in which the enzyme was up- and down-regulated by transfection with plasmids containing sense and antisense human cDNA for MnSOD. DNA strand breaks in the epithelium of Sod1 knockouts and Sod2 heterozygotes were much greater than in the corresponding wild-type or in transgenic mice over-expressing the enzymes when the lenses were exposed to H2O2. The functional role of Sod2 in apoptosis was examined in cultured human lens epithelial cells. Cells with higher enzyme levels were more resistant to the cytotoxic effects of H2O2, O2 2 and UV –B radiation. Furthermore, Sod2-deficient cells showed dramatic mitochondrial damage, cytochrome C leakage, caspase 3 activation and increased apoptotic cell death when they were challenged with O2 2 . Thus, mitochondrial enzyme (Sod2) deficiency plays an important role in the initiation of apoptosis in the lens epithelium. q 2004 Elsevier Ltd. All rights reserved. Keywords: CuZnSOD; MnSOD; reactive oxygen species; apoptosis; DNA strand breaks; transgenic mouse models; human lens epithelium; gene regulation; oxidative stress; mitochondria; cataract
1. Introduction Oxidative damage resulting from reactive oxygen species (ROS) due to light catalysed reactions in the transparent ocular media, aqueous humor and lens, has been thought to be a major factor in the development of age-onset cataracts (Giblin et al., 1982; Reddy and Giblin, 1984; Spector, 1984). It is well established that both visible and UV radiation to which the lens is constantly exposed give rise to ROS that includes O2 2 , singlet oxygen, and hydroxyl radicals, (Varma et al., 1979; Zigler et al., 1985; Pitts et al., 1986). These are capable of causing oxidative modification of cellular * Corresponding author. Dr V.N. Reddy, The Departments of Ophthalmology and Visual Sciences, University of Michigan, Kellogg Eye Center, Room 2341, 1000 Wall St., Ann Arbor, MI 48105, USA. Tel..: þ 1-734 763 7246; fax: þ 1-734 615 0542. E-mail address: [email protected] (V.N. Reddy). 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.04.005
macromolecules such as lens crystallins, (Buckingham and Pirie, 1972; Van Heyningen, 1973; Goosey et al., 1980), glutathione (Reddy et al., 1980; Reddy, 1990), cytoskeletal elements (Padgaonkar et al., 1999), and membrane thiols thus leading to lens opacification and cataract formation. In addition to the detoxification of ROS involving direct reaction with cellular constituents, the lens is equipped with a number of antioxidant enzymes (Bhuyan and Bhuyan, 1978; Reddy et al., 1980; Giblin et al., 1985; Spector et al., 1993). One of the key enzymes that detoxify the reactive O2 2 anion through dismutation to form H2O2 is superoxide dismutase (SOD) (Bhuyan and Bhuyan, 1978; Reddy et al., 1980; Carlsson et al., 1995). The peroxide formed is detoxified by the enzymes of the glutathione redox cycle and catalase which have been extensively studied (Spector and Garner, 1981; Giblin et al., 1982; Giblin et al., 1984, Reddy and Giblin, 1984; Giblin et al., 1990). However, the role of superoxide dismutases in antioxidant defense of
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the lens has not been well delineated. There are three isoforms of this enzyme that include the cytosolic-CuZnSOD (Sod1), mitochondrial superoxide dismutase MnSOD (Sod2) and the extracellular secreted enzyme which is anchored to proteoglycans and cell surface (Carlsson et al., 1995). However, the major isoforms are Sod1 which accounts for nearly 90% while the mitochondrial enzyme, Sod2, is approximately 10%. While all three isoforms are capable of converting O2 2 to H2O2, their individual contribution to defend against oxidation-induced cellular damage is not known. One approach to studying the role of the two major isoforms of SOD in the lens epithelium is to vary the cellular level of these enzymes in transgenic animals in which a single enzyme is either over-expressed or made deficient through gene knockout (Ho et al., 1998a,b; Lebovitz et al., 1996; Li et al., 1995; Carlsson et al., 1995; Reddy et al., 1997, 2001; Spector et al., 2001). Alternatively, the enzyme level in cultured epithelial cells may be varied by up- and down-regulation by transfection with sense and antisense expression vectors for the specific enzyme (Matsui et al., 2003). We have recently shown that oxidant mediated DNA strand breaks in lens cultures of HLE cells correlated with the level of Sod2 (Matsui et al., 2003). In the present study, we have used transgenic and gene knockouts for Sod1 to demonstrate that oxidative damage in lens epithelium in situ resulting from H2O2 exposure of organ cultured lens correlated with the enzyme levels in this animal model. Furthermore, Sod1 knockout animals developed delayed-onset cataracts at approximately 12 months of age compared to lenses of wild-type controls, suggesting a critical role of this enzyme in protection of the lens against oxidation-induced injury. Since Sod2 knockout is lethal and animals do not survive for more than a few postnatal days (Li et al., 1995; Lebovitz et al., 1996), a comparison of heterozygote, transgenic mice over-expressing the enzyme and wild-type revealed that Sod2 also plays a protective role against oxidative damage and that the protective effect is related to the enzyme level in these animals. There are a number of studies in which Sod2 has been implicated in apoptosis (Karbowski et al., 1999; Liu and Keefe, 2000; Hengartner, 2000; McCarthy, 2003; Parone et al., 2003). Towards this end, we examined the functional role of Sod2 in apoptosis in HLE cells in which the enzyme was up- and down-regulated. The results demonstrate that cells with higher levels of the enzyme are resistant to the cytotoxic effects of H2O2, O2 2 and UV –B radiation (Matsui et al., 2003) and the enzyme-deficient cells show increased apoptotic cell death when they are challenged with O2 2.
2. Materials and methods Experimental Animals: all animals used in this study were treated in strict accordance with institutional
guidelines and with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.1. CuZnSOD (Sod1) transgenic and knockouts Generation and characterization of Sod1 knockout mice have been described previously (Ho et al., 1998a). Briefly, exon 5 of the mouse Sod1 gene in the knockout mice was replaced with a neomycin resistance cassette. This modification completely abolishes expression of the Sod1 gene. Homozygous Sod1 knockout mice are phenotypically normal, except female mice are not very fertile. To generate a large population of experimental animals, breeding between two heterozygous knockout mice (on a 129SV/ C57BL/6 genetic background) was initially performed to generate mice with three genotypes in the Sod1 allele (wildtype mice and heterozygous and homozygous knockout mice). Breeding between wild-type mice was then performed to generate the wild-type control mice used in the study. Homozygous Sod1 knockout mice were generated from breeding between male homozygous knockout mice and female heterozygous knockout mice. Only 50% of the progeny from the latter breeding were homozygous Sod1 knockout mice, which were identified by the lack of Sod1 activity in blood samples collected from the mouse tails using a native polyacrylamide gel (Beauchamp and Fridovich, 1971). For cataract evaluation, five Sod1 knockout animals and five age-matched controls (wild-type) were observed for a period of 1 year. Lens opacities were evaluated subjectively after 2 months of age at monthly intervals with a slit lamp without anesthesia to avoid cold cataract development. This subjective eye examination was preceded by mydriasis with 1% tropicamide hydrochloride. At 12 months of age when there was a distinct impression of lens opacification in Sod1deficient animals, both knockout and controls were sacrificed with CO2 asphyxiation and the lenses were carefully removed and photographed under dark field microscope. 2.2. Genetically altered MnSOD (Sod2) mice with deficiency or over-expression of the enzyme Initially, a breeding pair of heterozygous Sod2 knockout mice (stock# 002973, strain B6.129S7-Sod2 tm1Leb) was obtained from The Jackson Laboratory, Bar Harbor, ME. The offspring from this breeding pair will have about 50% heterozygote, 25% wild-type and 25% homozygote. However, the homozygote ( –/ – ) died within 2 – 3 days after birth and therefore could not be used as an experimental animal model. Genotyping protocol ‘Human HPRT Version 2’ from The Jackson Laboratory was used for genotyping of the offspring. This is a PCR (polymerase chain reaction) protocol to detect the human HPRT (hypoxanthine guanine phosphoribosyl transferase) gene. If the PCR products are positive for both HPRT and Sod2, the mouse is
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a heterozygote; if negative for HPRT and positive for Sod2, the animal is wild type. The homozygous knockout mice are expected to be positive for HPRT but will be lacking Sod2. The primers designed for HPRT were, forward: TGT TCT CCT CTT CCT CAT CTC C, and reverse: ACC CTT TCC AAA TCC TCA GC. Product size of PCR was 240 bp. The primers for mouse Sod2 were, forward: CCG CGT TCT GAG GAG AGC AG, and reverse: CCT TGG CCA GAG CCT CGT GG. Product size of PCR was 380 bp. The Sod2 transgenic mice were first generated by microinjecting fertilized mouse eggs isolated from mating of (C57BL/6 X C3H) (abbreviated B6C3 F1) male and female mice with the human MnSOD expression vector as described previously (Ho et al., 1998b). Experimental animals were generated by breeding of hemizygous Sod2 transgenic mice with wild-type B6C3 F1 mice. PCR was used for genotyping of the offspring. The primers for human Sod2 were (forward: CCC TGG AAC CTC ACA TCA AC and reverse: GCC GTC AGC TTC TCC TTA AA, PCR product was 301 bp); they only amplify the human Sod2 transgene to yield a fragment of 301 bp but not the endogenous mouse Sod2 gene. Protein blot study showed that the transgenic mice had approximately 158% MnSOD levels in heart mitochondria compared to wild-type animals. Subsequently, lenses of hemizygous transgenic mice and wild-type littermates were used to study the effect of Sod2 on protection against oxidant-induced DNA strand breaks and apoptosis. 2.3. Cell culture Human lens epithelial cells (HLE, SRA 01/04) (Ibaraki et al., 1998) were cultured at 378C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 15% fetal bovine serum (FBS; Gibco) in a 100 £ 20 mm culture dish (Falcon; Lincoln Park, NJ, USA) in a 5% CO2 environment. These cells were transfected with expression vectors for sense- and antisense human cDNA for Sod2 as described previously (Matsui et al., 2003). Two sets of controls were used: one consisting of the original established cell line (SRA 01/04) and the other in which the cells were transfected with vector alone.
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For H2O2-induced oxidative damage, cultured cells were seeded in DMEM containing 15% FBS for 24 h to allow attachment to the culture dish. After cell attachment, DMEM containing serum was replaced by serum-free medium containing a single bolus of 50 mM H2O2 and DNA strand breaks determined following oxidative challenge for 30 min. To study the oxidative stress induced by photochemically generated O2 2 , cultured cells with varying levels of Sod2 were seeded in DMEM containing 15% FBS for 24 h. After attachment to the culture dish, the medium was replaced with serum-free medium containing 15 mM riboflavin (Spector et al., 1993; Matsui et al., 2003) for 15 hr and exposed to a 15-W circular daylight fluorescent lamp placed 10 cm above the plate and irradiated for 1 hr. The cells were then re-incubated at 378C for 4 hr and assayed for apoptosis. 2.5. Analysis of (Sod2) protein expression Trypsinized cultured cells were washed with phosphate buffered saline (PBS) and centrifuged at 1000g for 5 min. Collected cells were dissolved in 0·5% triton buffer containing proteinase inhibitor (Roche Diagnostics, Mannheim, Germany) and incubated for 10 min on ice. After incubation, each sample was centrifuged at 10 000g for 10 min at 48C to remove the debris. Supernatants were assayed for protein and all samples adjusted to the same protein concentration, then mixed with lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) and 2-mercaptoethanol according to Invitrogen protocols. The samples were subjected to SDS/PAGE followed by Western-blot analysis using protein expression assay (Western Breeze; Invitrogen). Briefly, electrophoresed proteins were transferred from gel onto nitrocellulose membrane (Trans-Blot transfer medium; Bio-Rad, Hercules, CA) and incubated with primary antibody against human MnSOD (Anti MnSOD; Product #SOD-111, Stressgen, Victoria, BC, Canada) overnight at 48C and then incubated for 30 min at room temperature with alkaline phosphatase-conjugated secondary antibody. The staining reaction was quantified by densitometry using computerized image analysis program (NIH Image, Ver. 1·63). 2.6. Analysis of apoptosis
2.4. DNA strand breaks in lens epithelium in situ and in cell cultures Individual lenses removed from animals with varying levels of Sod1 and Sod2 were cultured at 378 for 30 min in a 2·0 ml Hank’s Solution containing 25 mM H2O2. Following incubation, the lens epithelial cells were harvested from the capsule. They were then embedded in agarose gel and processed for single cell gel electrophoresis (Singh et al., 1988.). The extent of DNA strand breaks was estimated by measuring DNA migration from enlarged photographs (Reddy et al., 1997; Matsui et al., 2003).
The cells were cultured on a cover slip and fixed with 1% methanol-free formaldehyde and further post-fixed with precooled ethanol acetic acid solution (ethanol:acetic acid ¼ 2:1), and subjected to terminal deoxynucleotidyltransferase dUTP nickend labeling (TUNEL) staining using ApopTag Apoptosis detection system (Intergen, Purchase, NY). To confirm the evidence of apoptosis, the cells were also immunocytochemically stained with antibody to cleaved caspase-3, one of the ‘executioner’ factors of apoptosis (Hengartner, 2000; Zhivotovsky, 2003), and analysed by fluorescent microscopy. The cultured cells on
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5% albumin. To further confirm and quantify apoptosis, Annexin V staining, which detects translocation of the membrane phospholipid phosphatidylserine from the inner face of the plasma membrane to the cell surface after initiating apoptosis (Fadok et al., 1992), was performed and analysed by flow cytometry. Cells (5 £ 105) were gently trypsinized and collected by centrifugation at 1000g for 5 min and subjected to Annexin V-FITC apoptosis detection kit (Bio Vision; Mountain View, CA, USA). The cells were analysed by flow cytometry (FACS, BD Biosciences, San Jose, CA, USA); Ex ¼ 558 nm; Em ¼ 530 nm using FITC signal detector [FL1]). 2.7. Analysis of cytochrome C leakage from mitochondria
Fig. 1. The effect of H2O2 on DNA strand breaks in lenses of normal, transgenic and CuZn SOD (Sod1) knockout mice. Isolated lenses were exposed to 25 mM peroxide (single bolus) for 30 min. DNA migration was measured in 50 cells in each group. Data are means ^ SD [p , 0·01 for trans vs. KO; p , 0·05 for WT vs. trans; p . 0·05 for KO vs. WT]; trans, transgenic; WT, wild-type; KO, knockout.
a cover slip were fixed as previously described and washed with PBS. They were blocked with a 5% serum in PBS for 1 hr at 258C, and then incubated with primary cleaved caspase-3 antibody (Cell Signaling Technology; Beverly, MA) diluted in PBS containing 5% albumin overnight at 48C. The antibody detects activated caspase-3 which is a cleaved form of procaspase-3. The cells were then incubated for 1 hr at room temperature with TRITC-conjugated (tetramethylrhodamine isomer R) secondary antibody (Dako, Glostrup, Denmark) diluted with PBS containing
The cells were homogenized in hypotonic lysis buffer (20 mM Hepes-KOH pH 7·5, 10 mM KCl, 1·5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 600g for 5 min at 48C to remove the debris. The supernatant was then centrifuged at 10 000g for 30 min at 48C. The pellet was used for mitochondrial fraction, and the supernatant was further centrifuged at 100 000g for 30 min at 48C, and the resulting pellet was used for cytosol fraction. These mitochondrial and cytosolic fractions were analysed for cytochrome C by western-blot analysis (anti cytochrome C antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3. Results 3.1. Mice with deficiency and over-expression of Sod1 In order to examine the protective role of each of the major superoxide dismutase enzymes in the lens against ROS, we first studied the animal model in which Sod1 was overexpressed or made deficient by its knockout. It has been
Fig. 2. Cataract formation in Sod1 knockout mice (representative photograph). Five knockout and five WT controls were followed for 12 months of age and killed by CO2 asphyxiation. The isolated lenses were photographed under dark field microscope. Dark field photographs showed lamellar cataracts with higher nuclear density in all knockouts while lenses of wild-type controls were clear.
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Fig. 3. Genotyping of mice with altered Sod2. Genomic DNA was isolated from tail samples and PCR was performed using primers as described in Section 2. Lanes 1 and 2 from W.T. showing absence of HPRT (240 bp) and positive for Sod2 (380 bp); lanes 3 and 4 from heterozygote showing presence of both HPRT and Sod2; lanes 5 and 6 from knockouts showing presence of HPRT but absence of Sod2.
previously reported that the knockout of Sod1 does not affect the other antioxidant enzymes including Sod2 (Ho et al., 1998a) so that the individual contribution of this enzyme can be assessed in knockout animals. When lenses of transgenic and knockouts were exposed to 25 mM H2O2 in situ for 30 min, DNA strand breaks in lens epithelium of knockout mice were approximately 20% higher than in wild-type controls. While the differences between knockout and wild type were marginal ðp , 0:05Þ the difference between knockout and transgenic were highly significant ðp , 0·01Þ; the DNA damage in the knockouts was twice as great compared to the lenses of transgenic animals over expressing the enzyme (Fig. 1). Thus, the lenses with higher level of Sod1 in transgenic animals exerted a greater protection against H2O2-induced DNA damage compared to normal (wild-type) or gene knockout mouse lenses. In view of greater oxidative damage in Sod1 deficient mice, it was considered possible that these animals might show lens changes in vivo. To examine such a phenotype, lens changes in knockout and wild type control animals (five knockouts and five age-matched controls) were followed by slit lamp examination for a period of 12 months. Because of deep anesthesia required for slit lamp photographs, lens opacities were subjectively evaluated in conscious animals with a slit lamp following mydriasis with tropicamide.
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This subjective evaluation showed no lens opacities in both groups of animals for the first 10 months when there was an impression of cataract formation in the knockouts. To document the changes in lens opacification, all animals in both groups were sacrificed at 12 months of age and isolated lenses were photographed under dark field microscope. Fig. 2 shows a representative dark field photograph of lens from an animal lacking Sod1 and a control animal. Opacities were observed in all 5 Sod1 knockouts with higher nuclear density and lamellar type of cataracts. In contrast, the lenses from control animals remained clear. This is the first phenotype observed in the Sod1 knockout animal model. The late onset lens opacity observed in these animals is similar to mice lacking glutathione peroxidase-1 (GPX-1). However, at present, it is not known if cataract formation in Sod1 knockout model develops gradually as in GPX1 knockouts (Reddy et al., 2002) or abruptly at 1 year of age and needs to be further investigated. 3.2. Mice with deficiency and over-expression of Sod2 As seen in Fig. 3, genotyping with PCR as described in Section 2 showed that HPRT gene was present in both heterozygote and homozygous (pups) but absent in wildtype. On the other hand, Sod2, while present in heterozygote and wild-type, was absent in homozygous pups. Therefore, the protective effect of this enzyme against oxidative injury was examined by comparing H2O2-induced DNA strand breaks in epithelium of lenses removed from heterozygote (half knockouts) with wild-type controls and transgenic mice in which the enzyme was over-expressed by the introduction of human cDNA into fertilized mouse oocytes, as described under Methods (Ho et al., 1998b). The enzyme level in the three groups of mice is shown in Fig. 4. The heart mitochondria from heterozygote had 44% Sod2 level compared to the wild-type controls and the expression of the enzyme in the transgenic mouse was 158% of that in
Fig. 4. Sod2 expression in the heart—mitochondria of heterozygote (half knockout),wild-type control and hemizygous mice over-expressing the enzyme. The enzyme level in heart-mitochondria of transgenic animals was nearly 3·6 £ that of heterozygote.
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the wild-type. As shown in Fig. 5, when the lenses from the three genotypes for Sod2 were exposed to 25 mM H2O2, DNA damage was highest in the epithelium of lenses from heterozygote while the lenses from transgenic mice showed the least DNA damage (p , 0·01 values), clearly demonstrating the protective effect of Sod2 on H2O2-induced DNA strand breaks. Thus, the mitochondrial enzyme (Sod2) also protects the lens epithelium against oxidation-induced injury. Furthermore, the protective effect is correlated with the cellular enzyme level. 3.3. Sod2 deficiency and apoptosis
Fig. 5. Effect of H2O2 on DNA strand breaks in lens epithelium from heterozygote, wild-type controls and transgenic mice over-expressing Sod2. Isolated lenses were exposed to 25 mM peroxide (single bolus) for 30 min. DNA migration was measured in 50 cells isolated from epithelium in each group. Data are means ^ SD (p-value between heterozygote and transgenic was p , 0·01).
There are a number of studies in which Sod2 has been implicated in cell death as a result of a decrease in the mitochondrial enzyme (Li et al., 1995; Lebovitz et al., 1996; Fujimura et al., 1999). Although young mutant mice (5 – 18 days old) lacking Sod2 have been observed to display widely ranging pathologies including cardiomyopathy and neurodegeneration (Lebovitz et al., 1996), ocular manifestation of this deficiency is not known. Also, it is not known if partial deficiency of the enzyme leads to apoptosis resulting from the accumulation of ROS. To get an insight
Fig. 6. Regulation of Sod2 in human lens epithelial cells. Western blot analysis of SDS-PAGE from cell cultures. (A) Cells were transfected with (lane S) sense MnSOD, (lane AS) anti-sense MnSOD cDNA and lane VEC, plasmid alone. Lane 01/04: non-transfected cell line. Extracts containing 25 mg of protein from each experiment were used for separation on SDS-PAGE. Transblots were immuno-stained with anti 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) using a computerized image analysis program (NIH image, ver. 1·61) with permission. Matsui et al., Invest Ophthal Vis Sci 44:3467–3475, 2003.
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into Sod2-associated apoptosis, we modulated the enzyme level in lens epithelial cell cultures with expression vectors for Sod2 as described previously (Matsui et al., 2003). Fig. 6 shows Sod2 levels in HLE cells transfected with sense and antisense expression vectors of Sod2 cDNA compared with controls. When challenged with O2 2 , cells deficient in Sod2 (down regulated enzyme) showed greater number of apoptotic cells compared to controls or enzyme-rich cells (upregulated) as observed by TUNEL assay (Fig. 8). While the mechanism of apoptosis is complex and involves a cascade of reactions (Hengartner, 2000; Rich et al., 2000; Parone et al., 2003), one of the key steps leading to apoptosis is the leakage of cytochrome C from the mitochondria and activation of caspase 3 (Hengartner, 2000). In view of our earlier observation that oxidative challenge with H2O2 leads to mitochondrial membrane damage in Sod2-deficient lens epithelial cells (Matsui et al., 2003), we measured the appearance of cytochrome C in the cytoplasm of these cells following exposure to O2 2 anions. Cytochrome C in the cytosol and mitochondrial fractions were assayed by western blotting. While cytochrome C levels in mitochondrial fractions from the four cell extracts were indistinguishable (data not shown) its leakage into
Fig. 7. Effect of oxidative stress (O2 2 ) on cytochrome C leakage from mitochondria of human lens epithelial cells in which Sod2 was up- and down-regulated. Following exposure of the cells to O2 2 as described under Methods, they were dissolved in hypotonic buffer and cytoplasmic and mitochondrial fractions were separated and analysed for cytochrome C by Western blot analysis using anti-cytochrome C antibody. The bar graph on top is the scan of the immunoblots of cytosol fractions. The leakage of cytochrome C from antisense transfected cells (Sod2 deficient) is twice as great as from sense transfected cells or controls (SRA01/04, transfected with vector alone).
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Fig. 8. Effect of oxidative stress (O2 2 ) on apoptosis as determined by TUNEL assay in human lens epithelial cells with up- and down-regulation of Sod2. Cells cultured on cover slips, exposed to O2 2 and fixed as described in Section 2 were subjected to terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining using ApopTag apoptosis detection kit, and analysed with fluorescence microscopy. It may be noted that there are many more TUNEL positive cells in which the enzyme is downregulated (anti-sense) compared to controls or those in which Sod2 is upregulated (sense).
the cell cytoplasm was greater from Sod2 deficient cells compared with those in which enzyme was upregulated or from non transfected or cells tranfected with vector alone (Fig. 7); the bar graph illustrates the relative scanned density of cytochrome C present in the cytosol fractions from the four cell extracts; the leakage of cytochrome C into cell cytoplasm is twice as large from Sod2-deficient cells compared with controls and those in which the enzyme level was up-regulated. TUNEL staining of sense- and anti-sense transfected HLE cells compared with nontransfected and those transfected with vector alone are shown in Fig. 8. It may be noted that there are very few tunel positive cells in the two controls and sense transfected cells whereas there is a marked increase in tunel positive staining in antisense transfected cells which are deficient in Sod2. Thus the cells with lower level of Sod2 undergo greater apoptosis. Since cytochrome C is known to activate caspase-3 enzyme, which is thought to initiate apoptosis, experiments were carried out to determine the activation of this enzyme in cells with up- and down-regulated Sod2 following exposure to O2 2 . The data in Fig. 9 demonstrate increased caspase-3 activity in Sod2-deficient cells compared to controls and up-regulated cells and are consistent with the enhanced leakage of cytochrome C; they also correlate with the level of Sod2 in the respective cell cultures.
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To further confirm and quantify the effect of ROS on apoptosis in cultured cells with Sod2-deficiency, the cells exposed to O2 2 were stained with annexin V-FITC and analysed by flow cytometry (Fig. 10). Fluorescence intensity, a measure of apoptosis, is greater in cells in which the enzyme was down-regulated compared to non-transfected controls or cells in which the enzyme was up-regulated. Increase in fluorescence intensity of the cells is indicated by the shift of the curve to the right. Based on the log scale for X-axis it is estimated that fluorescence intensity of antisense transfected cells is approximately 1·4 times that of upregulated cells. The value for non transfected controls is intermediate to the up- and down regulated cells. Thus, Sod2 plays a critical role in oxidationinduced apoptosis.
4. Discussion
Fig. 9. Effect of oxidative stress (O2 2 ) on the activation of caspase-3 in human lens epithelial cells with up- and down-regulation of Sod2. Cells cultured on cover slips, exposed to O2 2 and fixed as described in Section 2 were reacted first with antibody to cleaved caspase-3. They were then incubated with TRITC-conjugated secondary antibody and analysed by fluorescence microscopy. It may be noted that there are greater number of fluorescent cells in Sod2-deficient cells (anti-sense) compared to controls or in cells with up-regulated enzyme (sense).
Fig. 10. Effect of oxidative stress on apoptosis of cultured lens epithelial cells with up- and down-regulation of Sod2; Annexin V staining was performed in these cells after exposure to photochemically generated 5 oxidative stress (O2 2 ). Cells (5 £ 10 ) were incubated with riboflavin for 15 hr and then irradiated with a 15-W circular daylight fluorescent lamp for 1 hr. After reincubation of 4 hr at 378C, cells were collected and subjected to Annexin V-FITC staining. The Intensity of Annexin VFITC signal in cells was analysed by flow cytometry (FACS, Ex ¼ 448 nm; Em ¼ 530 nm) using FITC signal detector. Annexin V-FITC signal in Sod2-deficient cells was more intense (red line) than in control cells (black line), or cells in which the enzyme was up regulated (green line). Increase in fluorescence intensity is reflected by the shift in curves to the right (see text).
This study describes the effect of variation of Sod1 and Sod2 both in vivo and in cultured lens epithelial cells and their response to oxidative stress in lens epithelium. A major objective of this investigation was to assess the relative role of the two enzymes by making use of transgenic animals in which a specific enzyme was over-expressed by gene manipulation or made deficient through gene knockout. While both isoenzymes serve to protect the cells against O2 2 induced injury, it is expected that their function may differ since they are localized in different compartments of the cell. In contrast to Sod1, which is present in the cytoplasm, Sod2 is sequestered in mitochondria so that oxidative injury may involve apoptosis. The studies with transgenic mice over-expressing Sod1, or its knockout model clearly demonstrate that this enzyme plays a critical role in protection against H2O2-induced DNA damage to lens epithelium. Lens epithelium from animals lacking Sod1 show greater DNA strand breaks and thus more sensitive to oxidative challenge as compared to lenses from wild-type control mice. Furthermore, up-regulation of the enzyme in transgenic animals results in a greater protection against H2O2-induced DNA damage. Thus, the ability of the lens epithelium to protect itself against oxidation-induced damage appears to be directly correlated with Sod1 levels (Fig. 1). Another significant finding in this study is that near absence or lack of the enzyme in knockout animal results in lens opacities approximately at 12 months of age (Fig. 2). This is the first report of cataract development in Sod1 knockout mice and point to the critical role of this enzyme in defense against oxidative stress. Previous studies (Ho et al., 1998a) have shown that mice null to Sod1 are phenotypically normal except that the female mice are less fertile. Therefore, cataract development in these animals suggests that lens may be more sensitive to oxidation-induced damage compared to other tissues. Since animals null to Sod1 do not appear to affect other antioxidant enzymes,
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including Sod2, one is tempted to ascribe the protective effect of Sod1 against H2O2-induced DNA strand breaks to this cytosolic enzyme. The delayed onset cataracts observed in Sod1 knockout mice is similar to cataracts observed in GPX-1 knockout mice (Reddy et al., 2001), which also grow and reproduce normally (Ho et al., 1997; Spector et al., 2001). At present, it is unclear whether the mechanisms leading to cataract formation in the two animal models are similar or distinct and remain to be investigated further. In GPX-1 knockout mice, cataract development was gradual and appear to involve changes in nuclear fiber membranes. In any case, both GPX-1 and Sod1-deficient mice serve as useful animal models for age-onset cataracts (Spector et al., 1996; Reddy et al., 1997; Ho et al., 1998c; Reddy et al., 2001; Spector et al., 2001). The investigations on the antioxidant role of Sod2 involve genetically altered (Sod2) mice with deficiency or over-expression of the enzyme as well as up- and downregulation of the enzyme in cultured HLE cells. Unlike the Sod1 model, it was not possible to use intact lenses from homozygous mice since these animals did not survive beyond a few days after birth. Although there are previous reports (Li et al., 1995; Lebovitz et al., 1996) in which Sod2 knockout mice survived from 5 to 18 days after birth, in our hands Sod2-deficient mice did not live beyond 3 days after birth. However, using heterozygote (half-knockout) containing 50% of the enzyme compared to wild-type controls, we have demonstrated that lenses from these animals are much more sensitive to oxidative insult compared to wildtype controls. Furthermore, the protective effect of Sod2 on DNA damage to epithelium from in situ lenses of mice in which the enzyme was genetically elevated by the introduction of human Sod2 cDNA was significantly enhanced compared to lenses from heterozygote ðp ,0·01Þ: The enzyme level in transgenic mice was nearly three times higher than in heterozygote (relative levels, 158 to 44%—Fig. 4). Thus, the ability of Sod2 to protect against oxidative damage is also directly related to the enzyme levels in the respective genotypes. These findings are consistent with our previous studies (Matsui et al., 2003) which showed that cells with elevated enzyme levels were more resistant to the cytotoxic effect of H2O2, photochemically generated O2 2 or UV-B irradiation. While the results of this study demonstrate the critical role of Sod2 (as well as Sod1) in protection against oxidation-induced DNA damage in lens epithelial cells, they do not reveal the specific effect of Sod2 deficiency in the mitochondria. Since mutant mice lacking Sod2 do not survive, it is difficult to separate its effect on oxidative insult from other forms of SOD. The direct role of Sod2 in antioxidant defense of mitochondria was therefore studied by varying the level of Sod2 in cultured HLE cells. Mitochondria are especially sensitive to oxidative damage and it has been reported that oxidative damage can cause mitochondrial membrane damage (Zhang et al., 1990,
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Kabrowski et al., 1999) and cell death (Zhang et al., 1990; Karbowski et al., 1999; Liu and Keefe, 2000). Consistent with such reports, our results have shown that cells deficient in Sod2 display mitochondrial membrane damage (Matsui et al., 2003) and undergo apoptosis (Figs. 8 –10). In the model system we have employed in which Sod2 was up- and downregulated by sense-and antisense expression vectors, oxidative challenge with photochemically generated O2 2 results in greater apoptotic cell death. Additonally, caspase 3 activation (Fig. 9) and apoptosis correlate with cytochrome C leakage from the mitochondria into the cytoplasm. Although the mechanism by which cytochrome C leaks from mitochondria is complex, in the present study, its leakage into the cell cytoplasm may be due to membrane damage, as seen by the swelling of mitochondria (Matsui et al., 2003). Thus, it appears that in lens epithelium as in many other cell types, Sod2 deficiency plays a critical role in apoptosis. Finally, present approach to modulate the Sod2 enzyme level in cultured cells may provide a model system to explore the mechanism of Sod2-associated apoptosis in a wide variety of mammalian cells.
Acknowledgements Supported in part by National Institutes of Health grants EY-00484 (V.N.R.), Core center grant EY07003, and HL56421 (Y.-S.H.). This investigation is part of US Cooperative Cataract Research Group (CCRG) Program.
References Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276– 287. Bhuyan, K.C., Bhuyan, D.K., 1978. Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage. Biochim. Biophys. Acta 542, 28– 38. Buckingham, R.H., Pirie, A., 1972. The effect of light on lens proteins in vitro. Exp. Eye Res. 14, 297–299. Carlsson, L.M., Jonsson, J., Edlund, T., Maklund, S.L., 1995. Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia. Proc. Nat. Acad. Sci. USA 92, 6264–6268. Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., Henson, P.M., 1992. Exposure of phophatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216. Fujimura, M., Morita-Fujimura, Y., Kawase, M., Copin, J.C., Calagui, B., Epstein, C.J., Chan, P.H., 1999. Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent fragmentation after permanent focalcerebral ischemia in mice. J. Neurosci. 19, 3414–3422. Giblin, F.J., Reddan, J.R., Schrimscher, L., Dziedzic, D.C., Reddy, V.N., 1990. The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 by cultured rabbit lens epithelial cells. Exp. Eye. Res. 50, 795 –804.
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Giblin, F.J., McCready, J.P., Reddan, J.R., Dziedzic, D.C., Reddy, V.N., 1985. Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle. Exp. Eye. Res. 40, 827– 840. Giblin, F.J., McCready, J.P., Reddy, V.N., 1982. The role of glutathione metabolism in the detoxification of H2O2 in rabbit lens. Invest. Ophthalmol. Vis. Sci. 22, 330–335. Goosey, J.D., Zigler Jr, J.S., Kinoshita, J.H., 1980. Cross linking of lens crystallins in a photodynamic system a process mediated by singlet oxygen. Science 208, 1278–1280. Hengartner, M.O., 2000. The biochemistry of apoptosis (insight review). Nature 407, 776 –779. Ho, Y.-S., Gargano, M., Cao, J., Bronson, R.T., Heimler, I., Hutz, R.J., 1998a. Reduced fertility in female mice lacking copper–zinc superoxide dismutase. J. Biol. Chem. 273, 7765–7769. Ho, Y.S., Vincent, R., Dey, M.S., Slot, J.W., Crapo, J.D., 1998b. Transgenic models for the study of lung antioxidant defense: enhanced manganese-containing superoxide dismutase activity gives partial protection to B6C3 hybrid mice exposed to hyperoxia. Am. J. Respir. Cell Mol. Biol. 18, 538 –547. Ho, Y.-S., Magnenat, J.L., Gargano, M., Cao, J., 1998c. The nature of antioxidant defense mechanisms: a lesson from transgenic studies. Environ. Health Prospect. 106, 1219–1228. Ho, Y.S., Magnenat, J.L., Bronson, R.T., Cao, J., Gargano, M., Sugawara, M., Funk, C.D., 1997. Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia. J. Biol. Chem. 272, 16644–16651. Ibaraki, N., Chen, S.C., Lin, L.R., Okamoto, H., Pipas, J.M., Reddy, V.N., 1998. Human lens epithelial cell line. Exp. Eye Res. 67, 577–585. Karbowski, M., Kurono, C., Wozniak, M., Ostrowski, M., Teranishi, M., Soji, T., Wakabayashi, T., 1999. Free radical-induced megamitochondria formation and apoptosis. Free Rad. Biol. Med. 26, 396 –409. Lebovitz, R.M., Zhang, H., Vogel, H., Cartwright Jr, J., Dionne, L., Lu, N., Huang, S., Matzuk, M.M., 1996. Neurodegeneration, myocardial injury and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc. Nat. Acad. Sci. USA 93, 9782–9787. Li, Y., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C., Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H., 1995. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nature Genetics 11, 376 –381. Liu, L., Keefe, D.L., 2000. Cytoplasm mediates both development and oxidation-induced apoptotic cell death in mouse zygotes. Biol. Repro. 62, 1828–1834. Matsui, M., Lin, L.-R., Ho, Y.-S., Reddy, V.N., 2003. The effect of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells. Invest.Ophthalmol. Vis. Sci. 44, 3467–3475. McCarthy, J.V., 2003. In: Cotter, T.G., (Ed.), Apoptosis and Development in Programmed Cell Death, vol. 39. Portland Press, UK, pp. 11–24. Padgaonkar, V., Lin, L.-R., Leverenz, V.R., Rinke, A., Reddy, V.N., Giblin, F.J., 1999. Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus. Exp. Eye Res. 68, 493–504. Parone, P., Priault, M., James, D., Nothwehr, S.F., Martinou, J.C., 2003. Apoptosis: bombarding the mitochondria. In: Cotter, T.G., (Ed.), Programmed Cell Death, vol. 39. Portland, London, UK, pp. 41–51.
Pitts, D.G., Commerce, L.L., Jose, J.G., 1986. Optical radiation and cataracts. In: Waxler, M., Hitchins, V.M. (Eds.), Optical Radiation and Visual Health, CRC Press, Boca Raton, FL, pp. 5–42. Reddy, V.N., Giblin, F.J., Lin, L.R., et al., 2001. Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice. Invest. Ophthal. Vis. Sci. 42, 3247–3255. Reddy, V.N., Lin, L.R., Ho, Y.S., Magnenat, J.L., Ibaraki, N., Giblin, F.J., Dang, L., 1997. Peroxide-induced damage in lenses of transgenic mice with deficient and elevated levels of glutathione peroxidase. Ophthalmologica 211, 192 –200. Reddy, V.N., 1990. Glutathione and its function in the lens: an overview. Exp. Eye Res. 50, 771–778. Reddy, V.N., Giblin, F.J., 1984. In: Nugent, J., Whelan, J. (Eds.), Metabolism and Function of Glutathione in the Lens (Ciba Foundation Symposium in: Human Cataract Formation), Pitman, London, pp. 65–87. Reddy, V.N., Giblin, F.J., Matsuda, H., 1980. In: Srivastawa, S.K., (Ed.), Defense system of the lens against oxidative damage in Second International Symposium on Red Blood Cell and Lens, Elsevier/North Holland, Amsterdam, pp. 139 –154. Rich, T., Allen, R.L., Wyllie, A.H., 2000. Defying death after DNA damage. Nature 407, 777–783.(insight Review). Singh, N.P., McCoy, M.L., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low level DNA damage in individual cells. Exp. Cell Res. 175, 184– 191. Spector, A., Kuszak, J.R., Wanchao, M.A., Wang, R.-R., 2001. The effect of aging on glutathione peroxidase-1 knockout mice: resistance of the lens to oxidative stress. Exp. Eye Res. 72, 533–545. Spector, A., Yang, Y., Ho, Y.-S., Magnenat, J.L., Wang, R.R., Ma, W., Li, W.C., 1996. Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress. Exp. Eye Res. 62, 521 –540. Spector, A., Wang, G.-M., Wang, R.-R., 1993. Photochemically-induced cataracts in rat lenses can be prevented by AL-3823A, a glutathione peroxidase mimic. Proc. Nat. Acad. Sci. USA 90, 7485–7489. Spector, A., 1984. The search for a solution to senile cataracts. Proctor Lecture. Invest. Ophthalmol. Vis. Sci. 25, 130 –146. Spector, A., Garner, W.H., 1981. Hydrogen peroxide and human cataract Exp. Eye Res. 33, 673–681. Van Heyningen, R., 1973. Photooxidation of lens proteins by sunlight in the presence of fluorescent derivatives of kynurenine isolated from human lens. Exp. Eye Res. 17, 137–141. Varma, S.D., Kumar, S., Richards, R.D., 1979. Light-induced damage to the ocular lens cation pump: prevention by vitamin C. Proc. Nat. Acad. Sci. USA 76, 3504–3506. Zhang, Y., Marcillat, O., Giulivi, C., Davies, K.J., 1990. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J. Biol. Chem. 265, 16330– 16336. Zhivotovsky, B., 2003. Caspases: the enzymes of death. In: Cotter, T.G., (Ed.), Programmed Cell Death, vol. 39. Portland, London, UK, pp. 25 –40. Zigler Jr, J.S., Jernigan, H.M., Garland, D., Reddy, V.N., 1985. The effect of ‘oxygen radicals’ generated in the medium on lenses in organ culture: inhibition of damage by chelated iron. Arch. Biochem. Biophys. 241, 163 –172.
Effects of variation in superoxide dismutases (SOD) on oxidative stress and apoptosis in lens epithelium V.N. Reddya,*, E. Kasaharaa, M. Hiraokab, L.-R. Lina, Y.-S. Hoc a
The Departments of Ophthalmology and Visual Sciences, University of Michigan, Kellogg Eye Center, 1000 Wall St., Ann Arbor, MI 48105, USA b Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA c Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA Received 2 March 2004; accepted in revised form 14 April 2004 Available online 10 July 2004
Abstract Among the critical antioxidant enzymes that protect the cells against oxidative stress are superoxide dismutases: CuZnSOD (Sod1) and MnSOD (Sod2). The latter is also implicated in apoptosis. To determine the importance of these enzymes in protection against reactive oxygen species (ROS) in the lens, we analysed DNA strand breaks in lens epithelium from transgenic and knockout (Sod1) mice following exposure to H2O2 in organ culture. Since Sod2 knockouts do not survive, comparison was made of lenses of partially-deficient (heterozygote) for Sod2 and the wild-type controls which have twice the enzyme level. Antioxidant potential of Sod2 was also studied in human lens epithelial cells (SRA01/04) in which the enzyme was up- and down-regulated by transfection with plasmids containing sense and antisense human cDNA for MnSOD. DNA strand breaks in the epithelium of Sod1 knockouts and Sod2 heterozygotes were much greater than in the corresponding wild-type or in transgenic mice over-expressing the enzymes when the lenses were exposed to H2O2. The functional role of Sod2 in apoptosis was examined in cultured human lens epithelial cells. Cells with higher enzyme levels were more resistant to the cytotoxic effects of H2O2, O2 2 and UV –B radiation. Furthermore, Sod2-deficient cells showed dramatic mitochondrial damage, cytochrome C leakage, caspase 3 activation and increased apoptotic cell death when they were challenged with O2 2 . Thus, mitochondrial enzyme (Sod2) deficiency plays an important role in the initiation of apoptosis in the lens epithelium. q 2004 Elsevier Ltd. All rights reserved. Keywords: CuZnSOD; MnSOD; reactive oxygen species; apoptosis; DNA strand breaks; transgenic mouse models; human lens epithelium; gene regulation; oxidative stress; mitochondria; cataract
1. Introduction Oxidative damage resulting from reactive oxygen species (ROS) due to light catalysed reactions in the transparent ocular media, aqueous humor and lens, has been thought to be a major factor in the development of age-onset cataracts (Giblin et al., 1982; Reddy and Giblin, 1984; Spector, 1984). It is well established that both visible and UV radiation to which the lens is constantly exposed give rise to ROS that includes O2 2 , singlet oxygen, and hydroxyl radicals, (Varma et al., 1979; Zigler et al., 1985; Pitts et al., 1986). These are capable of causing oxidative modification of cellular * Corresponding author. Dr V.N. Reddy, The Departments of Ophthalmology and Visual Sciences, University of Michigan, Kellogg Eye Center, Room 2341, 1000 Wall St., Ann Arbor, MI 48105, USA. Tel..: þ 1-734 763 7246; fax: þ 1-734 615 0542. E-mail address: [email protected] (V.N. Reddy). 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.04.005
macromolecules such as lens crystallins, (Buckingham and Pirie, 1972; Van Heyningen, 1973; Goosey et al., 1980), glutathione (Reddy et al., 1980; Reddy, 1990), cytoskeletal elements (Padgaonkar et al., 1999), and membrane thiols thus leading to lens opacification and cataract formation. In addition to the detoxification of ROS involving direct reaction with cellular constituents, the lens is equipped with a number of antioxidant enzymes (Bhuyan and Bhuyan, 1978; Reddy et al., 1980; Giblin et al., 1985; Spector et al., 1993). One of the key enzymes that detoxify the reactive O2 2 anion through dismutation to form H2O2 is superoxide dismutase (SOD) (Bhuyan and Bhuyan, 1978; Reddy et al., 1980; Carlsson et al., 1995). The peroxide formed is detoxified by the enzymes of the glutathione redox cycle and catalase which have been extensively studied (Spector and Garner, 1981; Giblin et al., 1982; Giblin et al., 1984, Reddy and Giblin, 1984; Giblin et al., 1990). However, the role of superoxide dismutases in antioxidant defense of
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the lens has not been well delineated. There are three isoforms of this enzyme that include the cytosolic-CuZnSOD (Sod1), mitochondrial superoxide dismutase MnSOD (Sod2) and the extracellular secreted enzyme which is anchored to proteoglycans and cell surface (Carlsson et al., 1995). However, the major isoforms are Sod1 which accounts for nearly 90% while the mitochondrial enzyme, Sod2, is approximately 10%. While all three isoforms are capable of converting O2 2 to H2O2, their individual contribution to defend against oxidation-induced cellular damage is not known. One approach to studying the role of the two major isoforms of SOD in the lens epithelium is to vary the cellular level of these enzymes in transgenic animals in which a single enzyme is either over-expressed or made deficient through gene knockout (Ho et al., 1998a,b; Lebovitz et al., 1996; Li et al., 1995; Carlsson et al., 1995; Reddy et al., 1997, 2001; Spector et al., 2001). Alternatively, the enzyme level in cultured epithelial cells may be varied by up- and down-regulation by transfection with sense and antisense expression vectors for the specific enzyme (Matsui et al., 2003). We have recently shown that oxidant mediated DNA strand breaks in lens cultures of HLE cells correlated with the level of Sod2 (Matsui et al., 2003). In the present study, we have used transgenic and gene knockouts for Sod1 to demonstrate that oxidative damage in lens epithelium in situ resulting from H2O2 exposure of organ cultured lens correlated with the enzyme levels in this animal model. Furthermore, Sod1 knockout animals developed delayed-onset cataracts at approximately 12 months of age compared to lenses of wild-type controls, suggesting a critical role of this enzyme in protection of the lens against oxidation-induced injury. Since Sod2 knockout is lethal and animals do not survive for more than a few postnatal days (Li et al., 1995; Lebovitz et al., 1996), a comparison of heterozygote, transgenic mice over-expressing the enzyme and wild-type revealed that Sod2 also plays a protective role against oxidative damage and that the protective effect is related to the enzyme level in these animals. There are a number of studies in which Sod2 has been implicated in apoptosis (Karbowski et al., 1999; Liu and Keefe, 2000; Hengartner, 2000; McCarthy, 2003; Parone et al., 2003). Towards this end, we examined the functional role of Sod2 in apoptosis in HLE cells in which the enzyme was up- and down-regulated. The results demonstrate that cells with higher levels of the enzyme are resistant to the cytotoxic effects of H2O2, O2 2 and UV –B radiation (Matsui et al., 2003) and the enzyme-deficient cells show increased apoptotic cell death when they are challenged with O2 2.
2. Materials and methods Experimental Animals: all animals used in this study were treated in strict accordance with institutional
guidelines and with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.1. CuZnSOD (Sod1) transgenic and knockouts Generation and characterization of Sod1 knockout mice have been described previously (Ho et al., 1998a). Briefly, exon 5 of the mouse Sod1 gene in the knockout mice was replaced with a neomycin resistance cassette. This modification completely abolishes expression of the Sod1 gene. Homozygous Sod1 knockout mice are phenotypically normal, except female mice are not very fertile. To generate a large population of experimental animals, breeding between two heterozygous knockout mice (on a 129SV/ C57BL/6 genetic background) was initially performed to generate mice with three genotypes in the Sod1 allele (wildtype mice and heterozygous and homozygous knockout mice). Breeding between wild-type mice was then performed to generate the wild-type control mice used in the study. Homozygous Sod1 knockout mice were generated from breeding between male homozygous knockout mice and female heterozygous knockout mice. Only 50% of the progeny from the latter breeding were homozygous Sod1 knockout mice, which were identified by the lack of Sod1 activity in blood samples collected from the mouse tails using a native polyacrylamide gel (Beauchamp and Fridovich, 1971). For cataract evaluation, five Sod1 knockout animals and five age-matched controls (wild-type) were observed for a period of 1 year. Lens opacities were evaluated subjectively after 2 months of age at monthly intervals with a slit lamp without anesthesia to avoid cold cataract development. This subjective eye examination was preceded by mydriasis with 1% tropicamide hydrochloride. At 12 months of age when there was a distinct impression of lens opacification in Sod1deficient animals, both knockout and controls were sacrificed with CO2 asphyxiation and the lenses were carefully removed and photographed under dark field microscope. 2.2. Genetically altered MnSOD (Sod2) mice with deficiency or over-expression of the enzyme Initially, a breeding pair of heterozygous Sod2 knockout mice (stock# 002973, strain B6.129S7-Sod2 tm1Leb) was obtained from The Jackson Laboratory, Bar Harbor, ME. The offspring from this breeding pair will have about 50% heterozygote, 25% wild-type and 25% homozygote. However, the homozygote ( –/ – ) died within 2 – 3 days after birth and therefore could not be used as an experimental animal model. Genotyping protocol ‘Human HPRT Version 2’ from The Jackson Laboratory was used for genotyping of the offspring. This is a PCR (polymerase chain reaction) protocol to detect the human HPRT (hypoxanthine guanine phosphoribosyl transferase) gene. If the PCR products are positive for both HPRT and Sod2, the mouse is
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a heterozygote; if negative for HPRT and positive for Sod2, the animal is wild type. The homozygous knockout mice are expected to be positive for HPRT but will be lacking Sod2. The primers designed for HPRT were, forward: TGT TCT CCT CTT CCT CAT CTC C, and reverse: ACC CTT TCC AAA TCC TCA GC. Product size of PCR was 240 bp. The primers for mouse Sod2 were, forward: CCG CGT TCT GAG GAG AGC AG, and reverse: CCT TGG CCA GAG CCT CGT GG. Product size of PCR was 380 bp. The Sod2 transgenic mice were first generated by microinjecting fertilized mouse eggs isolated from mating of (C57BL/6 X C3H) (abbreviated B6C3 F1) male and female mice with the human MnSOD expression vector as described previously (Ho et al., 1998b). Experimental animals were generated by breeding of hemizygous Sod2 transgenic mice with wild-type B6C3 F1 mice. PCR was used for genotyping of the offspring. The primers for human Sod2 were (forward: CCC TGG AAC CTC ACA TCA AC and reverse: GCC GTC AGC TTC TCC TTA AA, PCR product was 301 bp); they only amplify the human Sod2 transgene to yield a fragment of 301 bp but not the endogenous mouse Sod2 gene. Protein blot study showed that the transgenic mice had approximately 158% MnSOD levels in heart mitochondria compared to wild-type animals. Subsequently, lenses of hemizygous transgenic mice and wild-type littermates were used to study the effect of Sod2 on protection against oxidant-induced DNA strand breaks and apoptosis. 2.3. Cell culture Human lens epithelial cells (HLE, SRA 01/04) (Ibaraki et al., 1998) were cultured at 378C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 15% fetal bovine serum (FBS; Gibco) in a 100 £ 20 mm culture dish (Falcon; Lincoln Park, NJ, USA) in a 5% CO2 environment. These cells were transfected with expression vectors for sense- and antisense human cDNA for Sod2 as described previously (Matsui et al., 2003). Two sets of controls were used: one consisting of the original established cell line (SRA 01/04) and the other in which the cells were transfected with vector alone.
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For H2O2-induced oxidative damage, cultured cells were seeded in DMEM containing 15% FBS for 24 h to allow attachment to the culture dish. After cell attachment, DMEM containing serum was replaced by serum-free medium containing a single bolus of 50 mM H2O2 and DNA strand breaks determined following oxidative challenge for 30 min. To study the oxidative stress induced by photochemically generated O2 2 , cultured cells with varying levels of Sod2 were seeded in DMEM containing 15% FBS for 24 h. After attachment to the culture dish, the medium was replaced with serum-free medium containing 15 mM riboflavin (Spector et al., 1993; Matsui et al., 2003) for 15 hr and exposed to a 15-W circular daylight fluorescent lamp placed 10 cm above the plate and irradiated for 1 hr. The cells were then re-incubated at 378C for 4 hr and assayed for apoptosis. 2.5. Analysis of (Sod2) protein expression Trypsinized cultured cells were washed with phosphate buffered saline (PBS) and centrifuged at 1000g for 5 min. Collected cells were dissolved in 0·5% triton buffer containing proteinase inhibitor (Roche Diagnostics, Mannheim, Germany) and incubated for 10 min on ice. After incubation, each sample was centrifuged at 10 000g for 10 min at 48C to remove the debris. Supernatants were assayed for protein and all samples adjusted to the same protein concentration, then mixed with lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) and 2-mercaptoethanol according to Invitrogen protocols. The samples were subjected to SDS/PAGE followed by Western-blot analysis using protein expression assay (Western Breeze; Invitrogen). Briefly, electrophoresed proteins were transferred from gel onto nitrocellulose membrane (Trans-Blot transfer medium; Bio-Rad, Hercules, CA) and incubated with primary antibody against human MnSOD (Anti MnSOD; Product #SOD-111, Stressgen, Victoria, BC, Canada) overnight at 48C and then incubated for 30 min at room temperature with alkaline phosphatase-conjugated secondary antibody. The staining reaction was quantified by densitometry using computerized image analysis program (NIH Image, Ver. 1·63). 2.6. Analysis of apoptosis
2.4. DNA strand breaks in lens epithelium in situ and in cell cultures Individual lenses removed from animals with varying levels of Sod1 and Sod2 were cultured at 378 for 30 min in a 2·0 ml Hank’s Solution containing 25 mM H2O2. Following incubation, the lens epithelial cells were harvested from the capsule. They were then embedded in agarose gel and processed for single cell gel electrophoresis (Singh et al., 1988.). The extent of DNA strand breaks was estimated by measuring DNA migration from enlarged photographs (Reddy et al., 1997; Matsui et al., 2003).
The cells were cultured on a cover slip and fixed with 1% methanol-free formaldehyde and further post-fixed with precooled ethanol acetic acid solution (ethanol:acetic acid ¼ 2:1), and subjected to terminal deoxynucleotidyltransferase dUTP nickend labeling (TUNEL) staining using ApopTag Apoptosis detection system (Intergen, Purchase, NY). To confirm the evidence of apoptosis, the cells were also immunocytochemically stained with antibody to cleaved caspase-3, one of the ‘executioner’ factors of apoptosis (Hengartner, 2000; Zhivotovsky, 2003), and analysed by fluorescent microscopy. The cultured cells on
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5% albumin. To further confirm and quantify apoptosis, Annexin V staining, which detects translocation of the membrane phospholipid phosphatidylserine from the inner face of the plasma membrane to the cell surface after initiating apoptosis (Fadok et al., 1992), was performed and analysed by flow cytometry. Cells (5 £ 105) were gently trypsinized and collected by centrifugation at 1000g for 5 min and subjected to Annexin V-FITC apoptosis detection kit (Bio Vision; Mountain View, CA, USA). The cells were analysed by flow cytometry (FACS, BD Biosciences, San Jose, CA, USA); Ex ¼ 558 nm; Em ¼ 530 nm using FITC signal detector [FL1]). 2.7. Analysis of cytochrome C leakage from mitochondria
Fig. 1. The effect of H2O2 on DNA strand breaks in lenses of normal, transgenic and CuZn SOD (Sod1) knockout mice. Isolated lenses were exposed to 25 mM peroxide (single bolus) for 30 min. DNA migration was measured in 50 cells in each group. Data are means ^ SD [p , 0·01 for trans vs. KO; p , 0·05 for WT vs. trans; p . 0·05 for KO vs. WT]; trans, transgenic; WT, wild-type; KO, knockout.
a cover slip were fixed as previously described and washed with PBS. They were blocked with a 5% serum in PBS for 1 hr at 258C, and then incubated with primary cleaved caspase-3 antibody (Cell Signaling Technology; Beverly, MA) diluted in PBS containing 5% albumin overnight at 48C. The antibody detects activated caspase-3 which is a cleaved form of procaspase-3. The cells were then incubated for 1 hr at room temperature with TRITC-conjugated (tetramethylrhodamine isomer R) secondary antibody (Dako, Glostrup, Denmark) diluted with PBS containing
The cells were homogenized in hypotonic lysis buffer (20 mM Hepes-KOH pH 7·5, 10 mM KCl, 1·5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 600g for 5 min at 48C to remove the debris. The supernatant was then centrifuged at 10 000g for 30 min at 48C. The pellet was used for mitochondrial fraction, and the supernatant was further centrifuged at 100 000g for 30 min at 48C, and the resulting pellet was used for cytosol fraction. These mitochondrial and cytosolic fractions were analysed for cytochrome C by western-blot analysis (anti cytochrome C antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3. Results 3.1. Mice with deficiency and over-expression of Sod1 In order to examine the protective role of each of the major superoxide dismutase enzymes in the lens against ROS, we first studied the animal model in which Sod1 was overexpressed or made deficient by its knockout. It has been
Fig. 2. Cataract formation in Sod1 knockout mice (representative photograph). Five knockout and five WT controls were followed for 12 months of age and killed by CO2 asphyxiation. The isolated lenses were photographed under dark field microscope. Dark field photographs showed lamellar cataracts with higher nuclear density in all knockouts while lenses of wild-type controls were clear.
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Fig. 3. Genotyping of mice with altered Sod2. Genomic DNA was isolated from tail samples and PCR was performed using primers as described in Section 2. Lanes 1 and 2 from W.T. showing absence of HPRT (240 bp) and positive for Sod2 (380 bp); lanes 3 and 4 from heterozygote showing presence of both HPRT and Sod2; lanes 5 and 6 from knockouts showing presence of HPRT but absence of Sod2.
previously reported that the knockout of Sod1 does not affect the other antioxidant enzymes including Sod2 (Ho et al., 1998a) so that the individual contribution of this enzyme can be assessed in knockout animals. When lenses of transgenic and knockouts were exposed to 25 mM H2O2 in situ for 30 min, DNA strand breaks in lens epithelium of knockout mice were approximately 20% higher than in wild-type controls. While the differences between knockout and wild type were marginal ðp , 0:05Þ the difference between knockout and transgenic were highly significant ðp , 0·01Þ; the DNA damage in the knockouts was twice as great compared to the lenses of transgenic animals over expressing the enzyme (Fig. 1). Thus, the lenses with higher level of Sod1 in transgenic animals exerted a greater protection against H2O2-induced DNA damage compared to normal (wild-type) or gene knockout mouse lenses. In view of greater oxidative damage in Sod1 deficient mice, it was considered possible that these animals might show lens changes in vivo. To examine such a phenotype, lens changes in knockout and wild type control animals (five knockouts and five age-matched controls) were followed by slit lamp examination for a period of 12 months. Because of deep anesthesia required for slit lamp photographs, lens opacities were subjectively evaluated in conscious animals with a slit lamp following mydriasis with tropicamide.
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This subjective evaluation showed no lens opacities in both groups of animals for the first 10 months when there was an impression of cataract formation in the knockouts. To document the changes in lens opacification, all animals in both groups were sacrificed at 12 months of age and isolated lenses were photographed under dark field microscope. Fig. 2 shows a representative dark field photograph of lens from an animal lacking Sod1 and a control animal. Opacities were observed in all 5 Sod1 knockouts with higher nuclear density and lamellar type of cataracts. In contrast, the lenses from control animals remained clear. This is the first phenotype observed in the Sod1 knockout animal model. The late onset lens opacity observed in these animals is similar to mice lacking glutathione peroxidase-1 (GPX-1). However, at present, it is not known if cataract formation in Sod1 knockout model develops gradually as in GPX1 knockouts (Reddy et al., 2002) or abruptly at 1 year of age and needs to be further investigated. 3.2. Mice with deficiency and over-expression of Sod2 As seen in Fig. 3, genotyping with PCR as described in Section 2 showed that HPRT gene was present in both heterozygote and homozygous (pups) but absent in wildtype. On the other hand, Sod2, while present in heterozygote and wild-type, was absent in homozygous pups. Therefore, the protective effect of this enzyme against oxidative injury was examined by comparing H2O2-induced DNA strand breaks in epithelium of lenses removed from heterozygote (half knockouts) with wild-type controls and transgenic mice in which the enzyme was over-expressed by the introduction of human cDNA into fertilized mouse oocytes, as described under Methods (Ho et al., 1998b). The enzyme level in the three groups of mice is shown in Fig. 4. The heart mitochondria from heterozygote had 44% Sod2 level compared to the wild-type controls and the expression of the enzyme in the transgenic mouse was 158% of that in
Fig. 4. Sod2 expression in the heart—mitochondria of heterozygote (half knockout),wild-type control and hemizygous mice over-expressing the enzyme. The enzyme level in heart-mitochondria of transgenic animals was nearly 3·6 £ that of heterozygote.
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the wild-type. As shown in Fig. 5, when the lenses from the three genotypes for Sod2 were exposed to 25 mM H2O2, DNA damage was highest in the epithelium of lenses from heterozygote while the lenses from transgenic mice showed the least DNA damage (p , 0·01 values), clearly demonstrating the protective effect of Sod2 on H2O2-induced DNA strand breaks. Thus, the mitochondrial enzyme (Sod2) also protects the lens epithelium against oxidation-induced injury. Furthermore, the protective effect is correlated with the cellular enzyme level. 3.3. Sod2 deficiency and apoptosis
Fig. 5. Effect of H2O2 on DNA strand breaks in lens epithelium from heterozygote, wild-type controls and transgenic mice over-expressing Sod2. Isolated lenses were exposed to 25 mM peroxide (single bolus) for 30 min. DNA migration was measured in 50 cells isolated from epithelium in each group. Data are means ^ SD (p-value between heterozygote and transgenic was p , 0·01).
There are a number of studies in which Sod2 has been implicated in cell death as a result of a decrease in the mitochondrial enzyme (Li et al., 1995; Lebovitz et al., 1996; Fujimura et al., 1999). Although young mutant mice (5 – 18 days old) lacking Sod2 have been observed to display widely ranging pathologies including cardiomyopathy and neurodegeneration (Lebovitz et al., 1996), ocular manifestation of this deficiency is not known. Also, it is not known if partial deficiency of the enzyme leads to apoptosis resulting from the accumulation of ROS. To get an insight
Fig. 6. Regulation of Sod2 in human lens epithelial cells. Western blot analysis of SDS-PAGE from cell cultures. (A) Cells were transfected with (lane S) sense MnSOD, (lane AS) anti-sense MnSOD cDNA and lane VEC, plasmid alone. Lane 01/04: non-transfected cell line. Extracts containing 25 mg of protein from each experiment were used for separation on SDS-PAGE. Transblots were immuno-stained with anti 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) using a computerized image analysis program (NIH image, ver. 1·61) with permission. Matsui et al., Invest Ophthal Vis Sci 44:3467–3475, 2003.
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into Sod2-associated apoptosis, we modulated the enzyme level in lens epithelial cell cultures with expression vectors for Sod2 as described previously (Matsui et al., 2003). Fig. 6 shows Sod2 levels in HLE cells transfected with sense and antisense expression vectors of Sod2 cDNA compared with controls. When challenged with O2 2 , cells deficient in Sod2 (down regulated enzyme) showed greater number of apoptotic cells compared to controls or enzyme-rich cells (upregulated) as observed by TUNEL assay (Fig. 8). While the mechanism of apoptosis is complex and involves a cascade of reactions (Hengartner, 2000; Rich et al., 2000; Parone et al., 2003), one of the key steps leading to apoptosis is the leakage of cytochrome C from the mitochondria and activation of caspase 3 (Hengartner, 2000). In view of our earlier observation that oxidative challenge with H2O2 leads to mitochondrial membrane damage in Sod2-deficient lens epithelial cells (Matsui et al., 2003), we measured the appearance of cytochrome C in the cytoplasm of these cells following exposure to O2 2 anions. Cytochrome C in the cytosol and mitochondrial fractions were assayed by western blotting. While cytochrome C levels in mitochondrial fractions from the four cell extracts were indistinguishable (data not shown) its leakage into
Fig. 7. Effect of oxidative stress (O2 2 ) on cytochrome C leakage from mitochondria of human lens epithelial cells in which Sod2 was up- and down-regulated. Following exposure of the cells to O2 2 as described under Methods, they were dissolved in hypotonic buffer and cytoplasmic and mitochondrial fractions were separated and analysed for cytochrome C by Western blot analysis using anti-cytochrome C antibody. The bar graph on top is the scan of the immunoblots of cytosol fractions. The leakage of cytochrome C from antisense transfected cells (Sod2 deficient) is twice as great as from sense transfected cells or controls (SRA01/04, transfected with vector alone).
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Fig. 8. Effect of oxidative stress (O2 2 ) on apoptosis as determined by TUNEL assay in human lens epithelial cells with up- and down-regulation of Sod2. Cells cultured on cover slips, exposed to O2 2 and fixed as described in Section 2 were subjected to terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining using ApopTag apoptosis detection kit, and analysed with fluorescence microscopy. It may be noted that there are many more TUNEL positive cells in which the enzyme is downregulated (anti-sense) compared to controls or those in which Sod2 is upregulated (sense).
the cell cytoplasm was greater from Sod2 deficient cells compared with those in which enzyme was upregulated or from non transfected or cells tranfected with vector alone (Fig. 7); the bar graph illustrates the relative scanned density of cytochrome C present in the cytosol fractions from the four cell extracts; the leakage of cytochrome C into cell cytoplasm is twice as large from Sod2-deficient cells compared with controls and those in which the enzyme level was up-regulated. TUNEL staining of sense- and anti-sense transfected HLE cells compared with nontransfected and those transfected with vector alone are shown in Fig. 8. It may be noted that there are very few tunel positive cells in the two controls and sense transfected cells whereas there is a marked increase in tunel positive staining in antisense transfected cells which are deficient in Sod2. Thus the cells with lower level of Sod2 undergo greater apoptosis. Since cytochrome C is known to activate caspase-3 enzyme, which is thought to initiate apoptosis, experiments were carried out to determine the activation of this enzyme in cells with up- and down-regulated Sod2 following exposure to O2 2 . The data in Fig. 9 demonstrate increased caspase-3 activity in Sod2-deficient cells compared to controls and up-regulated cells and are consistent with the enhanced leakage of cytochrome C; they also correlate with the level of Sod2 in the respective cell cultures.
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To further confirm and quantify the effect of ROS on apoptosis in cultured cells with Sod2-deficiency, the cells exposed to O2 2 were stained with annexin V-FITC and analysed by flow cytometry (Fig. 10). Fluorescence intensity, a measure of apoptosis, is greater in cells in which the enzyme was down-regulated compared to non-transfected controls or cells in which the enzyme was up-regulated. Increase in fluorescence intensity of the cells is indicated by the shift of the curve to the right. Based on the log scale for X-axis it is estimated that fluorescence intensity of antisense transfected cells is approximately 1·4 times that of upregulated cells. The value for non transfected controls is intermediate to the up- and down regulated cells. Thus, Sod2 plays a critical role in oxidationinduced apoptosis.
4. Discussion
Fig. 9. Effect of oxidative stress (O2 2 ) on the activation of caspase-3 in human lens epithelial cells with up- and down-regulation of Sod2. Cells cultured on cover slips, exposed to O2 2 and fixed as described in Section 2 were reacted first with antibody to cleaved caspase-3. They were then incubated with TRITC-conjugated secondary antibody and analysed by fluorescence microscopy. It may be noted that there are greater number of fluorescent cells in Sod2-deficient cells (anti-sense) compared to controls or in cells with up-regulated enzyme (sense).
Fig. 10. Effect of oxidative stress on apoptosis of cultured lens epithelial cells with up- and down-regulation of Sod2; Annexin V staining was performed in these cells after exposure to photochemically generated 5 oxidative stress (O2 2 ). Cells (5 £ 10 ) were incubated with riboflavin for 15 hr and then irradiated with a 15-W circular daylight fluorescent lamp for 1 hr. After reincubation of 4 hr at 378C, cells were collected and subjected to Annexin V-FITC staining. The Intensity of Annexin VFITC signal in cells was analysed by flow cytometry (FACS, Ex ¼ 448 nm; Em ¼ 530 nm) using FITC signal detector. Annexin V-FITC signal in Sod2-deficient cells was more intense (red line) than in control cells (black line), or cells in which the enzyme was up regulated (green line). Increase in fluorescence intensity is reflected by the shift in curves to the right (see text).
This study describes the effect of variation of Sod1 and Sod2 both in vivo and in cultured lens epithelial cells and their response to oxidative stress in lens epithelium. A major objective of this investigation was to assess the relative role of the two enzymes by making use of transgenic animals in which a specific enzyme was over-expressed by gene manipulation or made deficient through gene knockout. While both isoenzymes serve to protect the cells against O2 2 induced injury, it is expected that their function may differ since they are localized in different compartments of the cell. In contrast to Sod1, which is present in the cytoplasm, Sod2 is sequestered in mitochondria so that oxidative injury may involve apoptosis. The studies with transgenic mice over-expressing Sod1, or its knockout model clearly demonstrate that this enzyme plays a critical role in protection against H2O2-induced DNA damage to lens epithelium. Lens epithelium from animals lacking Sod1 show greater DNA strand breaks and thus more sensitive to oxidative challenge as compared to lenses from wild-type control mice. Furthermore, up-regulation of the enzyme in transgenic animals results in a greater protection against H2O2-induced DNA damage. Thus, the ability of the lens epithelium to protect itself against oxidation-induced damage appears to be directly correlated with Sod1 levels (Fig. 1). Another significant finding in this study is that near absence or lack of the enzyme in knockout animal results in lens opacities approximately at 12 months of age (Fig. 2). This is the first report of cataract development in Sod1 knockout mice and point to the critical role of this enzyme in defense against oxidative stress. Previous studies (Ho et al., 1998a) have shown that mice null to Sod1 are phenotypically normal except that the female mice are less fertile. Therefore, cataract development in these animals suggests that lens may be more sensitive to oxidation-induced damage compared to other tissues. Since animals null to Sod1 do not appear to affect other antioxidant enzymes,
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including Sod2, one is tempted to ascribe the protective effect of Sod1 against H2O2-induced DNA strand breaks to this cytosolic enzyme. The delayed onset cataracts observed in Sod1 knockout mice is similar to cataracts observed in GPX-1 knockout mice (Reddy et al., 2001), which also grow and reproduce normally (Ho et al., 1997; Spector et al., 2001). At present, it is unclear whether the mechanisms leading to cataract formation in the two animal models are similar or distinct and remain to be investigated further. In GPX-1 knockout mice, cataract development was gradual and appear to involve changes in nuclear fiber membranes. In any case, both GPX-1 and Sod1-deficient mice serve as useful animal models for age-onset cataracts (Spector et al., 1996; Reddy et al., 1997; Ho et al., 1998c; Reddy et al., 2001; Spector et al., 2001). The investigations on the antioxidant role of Sod2 involve genetically altered (Sod2) mice with deficiency or over-expression of the enzyme as well as up- and downregulation of the enzyme in cultured HLE cells. Unlike the Sod1 model, it was not possible to use intact lenses from homozygous mice since these animals did not survive beyond a few days after birth. Although there are previous reports (Li et al., 1995; Lebovitz et al., 1996) in which Sod2 knockout mice survived from 5 to 18 days after birth, in our hands Sod2-deficient mice did not live beyond 3 days after birth. However, using heterozygote (half-knockout) containing 50% of the enzyme compared to wild-type controls, we have demonstrated that lenses from these animals are much more sensitive to oxidative insult compared to wildtype controls. Furthermore, the protective effect of Sod2 on DNA damage to epithelium from in situ lenses of mice in which the enzyme was genetically elevated by the introduction of human Sod2 cDNA was significantly enhanced compared to lenses from heterozygote ðp ,0·01Þ: The enzyme level in transgenic mice was nearly three times higher than in heterozygote (relative levels, 158 to 44%—Fig. 4). Thus, the ability of Sod2 to protect against oxidative damage is also directly related to the enzyme levels in the respective genotypes. These findings are consistent with our previous studies (Matsui et al., 2003) which showed that cells with elevated enzyme levels were more resistant to the cytotoxic effect of H2O2, photochemically generated O2 2 or UV-B irradiation. While the results of this study demonstrate the critical role of Sod2 (as well as Sod1) in protection against oxidation-induced DNA damage in lens epithelial cells, they do not reveal the specific effect of Sod2 deficiency in the mitochondria. Since mutant mice lacking Sod2 do not survive, it is difficult to separate its effect on oxidative insult from other forms of SOD. The direct role of Sod2 in antioxidant defense of mitochondria was therefore studied by varying the level of Sod2 in cultured HLE cells. Mitochondria are especially sensitive to oxidative damage and it has been reported that oxidative damage can cause mitochondrial membrane damage (Zhang et al., 1990,
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Kabrowski et al., 1999) and cell death (Zhang et al., 1990; Karbowski et al., 1999; Liu and Keefe, 2000). Consistent with such reports, our results have shown that cells deficient in Sod2 display mitochondrial membrane damage (Matsui et al., 2003) and undergo apoptosis (Figs. 8 –10). In the model system we have employed in which Sod2 was up- and downregulated by sense-and antisense expression vectors, oxidative challenge with photochemically generated O2 2 results in greater apoptotic cell death. Additonally, caspase 3 activation (Fig. 9) and apoptosis correlate with cytochrome C leakage from the mitochondria into the cytoplasm. Although the mechanism by which cytochrome C leaks from mitochondria is complex, in the present study, its leakage into the cell cytoplasm may be due to membrane damage, as seen by the swelling of mitochondria (Matsui et al., 2003). Thus, it appears that in lens epithelium as in many other cell types, Sod2 deficiency plays a critical role in apoptosis. Finally, present approach to modulate the Sod2 enzyme level in cultured cells may provide a model system to explore the mechanism of Sod2-associated apoptosis in a wide variety of mammalian cells.
Acknowledgements Supported in part by National Institutes of Health grants EY-00484 (V.N.R.), Core center grant EY07003, and HL56421 (Y.-S.H.). This investigation is part of US Cooperative Cataract Research Group (CCRG) Program.
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