Experimental Eye Research 81 (2005) 639–646 www.elsevier.com/locate/yexer
In vitro glucose-induced cataract in copper–zinc superoxide dismutase null mice Eva M. Olofssona,*, Stefan L. Marklundb, Kurt Karlssonb, Thomas Bra¨nnstro¨mc, Anders Behndiga b
a Department of Clinical Sciences/Ophthalmology, Umea˚ University, SE-901 87 Umea˚, Sweden Department of Medical Biosciences, Clinical Chemistry, Umea˚ University, SE-901 87 Umea˚, Sweden c Department of Medical Biosciences, Pathology, Umea˚ University, SE-901 87 Umea˚, Sweden
Received 6 December 2004; accepted in revised form 31 March 2005 Available online 9 June 2005
Abstract The purpose of this study was to evaluate the involvement of the superoxide radical in glucose-induced cataract using lenses from mice lacking the cytosolic copper–zinc superoxide dismutase (SOD1). Lenses from wild-type mice and SOD1 null mice were kept in organ culture with either 5.6 or 55.6 mM glucose for 6 days. The cataract formation was followed with digital image analysis and ocular staging. The lens damage was further quantified by analysis of the leakage of lactate dehydrogenase into the medium by the uptake of 86Rb and by determining the water content of the lenses. The formation of superoxide radicals in the lenses was assessed with lucigenin-derived chemiluminescence. Immunohistochemical staining for SOD1 was also performed on murine lenses. The SOD1 null lenses exposed to high glucose developed more cataract showed an increased leakage of lactate dehydrogenase and developed more oedema compared to the control lenses. At 5.6 mM glucose there was no difference between the SOD1 null and wild-type lenses. Staining for SOD1 was seen primarily in the cortex of the wildtype lens. This in vitro model suggests an involvement of the superoxide radical and a protective effect of SOD1 in glucose-induced cataract formation. q 2005 Elsevier Ltd. All rights reserved. Keywords: superoxide dismutase; cataract; diabetes mellitus
1. Introduction Diabetic patients are at high risk of developing cataract by mechanisms not well understood (Bron et al., 1993). Oxidative stress is generally thought to contribute to diabetic complications and has been suggested to play a major role in diabetic cataract (Ansari and Srivastava, 1990; Ansari et al., 1994; Chand et al., 1982; Devamanoharan et al., 1999; Hegde et al., 2003a; Lee and Chung, 1999; Obrosova et al., 1999; Srivastava and Ansari, 1988; Zhao et al., 2000). Oxidative stress could be induced by multiple mechanisms in diabetes. Glucose itself (Wolff and Dean, 1987), as well as primary glycation products and advanced glycation end-products (AGEs) (Sakurai and Tsuchiya, * Corresponding author. E-mail address:
[email protected] (E.M. Olofsson).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.03.022
1988) may autoxidize, forming reactive oxygen species such as the superoxide radical. Also, cell surface receptors for AGEs, RAGEs, are expressed in cultured human lens epithelial cells from cataract patients (Hong et al., 2000), and binding of AGEs to RAGEs is known to trigger superoxide formation in cells (Yan et al., 1994). Copper bound to AGE cross-linked proteins may catalyse the oxidation of ascorbate under the formation of superoxide radicals (Stitt, 2001), and ascorbate oxidation products may further promote AGE formation (Saxena et al., 1996). Also, ascorbylated lens proteins can generate superoxide in vitro (Linetsky et al., 1999). Superoxide can also be formed in the lens by NADPH oxidase (Rao et al., 2004) and by xanthine oxidase and the latter may be activated in diabetes (Cekic et al., 1999). The polyol pathway may also cause oxidative stress through the depletion of NADPH, resulting in reduced regeneration of glutathione (GSH) (Lee and Chung, 1999), but this may be less important in human—as well as murine—lens owing to very low levels of aldose reductase (Gaynes and Watkins, 1989; Hegde et al., 2003a,b).
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The superoxide dismutases (SOD) comprise an important part of the defence against superoxide radicals (Bhuyan and Bhuyan, 1978; Varma, 1977), detoxifying superoxide into hydrogen peroxide (Spector, 1995), which is further detoxified by catalase, glutathione peroxidase (Reddy, 1990) and peroxiredoxin (Kang et al., 1998). Lenses from mice with low levels of glutathione peroxidase (Srivastava et al., 1980) and mice lacking catalase (Ho et al., 2004) did not show a reduced tolerance to oxidative stress, indicating that hydrogen peroxide may be less central in cataract formation than earlier postulated. SOD exists in three different isoforms in higher organisms but it is the cytosolic copper–zinc SOD (SOD1) that accounts for almost the entire SOD activity in the human (Behndig et al., 1998) and the murine lens (Behndig et al., 2001). In humans, SOD1 is mainly localized in the lens epithelium and cortex (Fujiwara et al., 1992), and the activity of SOD1 declines with age and with cataract development (Ohrloff and Hockwin, 1984). We have previously shown that lenses from mice lacking SOD1 develop more cataract upon photochemical stress (Behndig et al., 2001) and that these lenses also have an increased concentration of superoxide radicals (Behndig et al., 2001). In human diabetic cataract, the SOD1 activity of the lens is even lower than in non-diabetic senile cataract (Ozmen et al., 2002). Diabetic rats show reduced lens SOD activity (Cekic et al., 1999) and a time-dependent inactivation of SOD1 activity by different sugars in vitro has been demonstrated (Yan and Harding, 1997). Thus, there is both potentially increased superoxide formation and reduced SOD activity in the lens in diabetes. To which extent this altered oxidant/ antioxidant status in the lens contributes to cataract in diabetes is not known. To explore this issue, we here compared the cataract formation in lenses from SOD1 null mice to that of lenses from wild-type mice under in vitro conditions with high glucose (Creighton et al., 1980).
6 female) were used for the analyses of cataract formation and lens damage. The mean ages of the SOD1 null and the wild-type mice were 19.2G8.2 weeks and 20.3G 10.2 weeks, respectively (meansGS.D.). All animals were killed with cervical dislocation after which both eye globes from each mouse were removed. The lenses were dissected and kept in tissue culture in 1.0 ml of Medium 199 (Gibco/ BRL, Life Technologies, Inc., Gaithersburg, MD) supplemented with 0.9 g lK1 NaHCO3, 25 mM Hepes pH 7.2 and 50 U mlK1 benzylpenicillin. Generally, from pairs of lenses, one lens was kept in medium with 55.6 mM glucose (10 times the normal concentration in the medium), while the other lens was kept in medium with a normal (5.56 mM) glucose concentration. The high concentration of glucose was chosen according to Creighton et al. (1980). Lower concentrations were unsuitable in this model since they would prolong the time of cataract development (Creighton et al., 1980) and even the control lenses with normal glucose will eventually develop opacities when cultivated in vitro. The osmolalities of the high and normal glucose media were 348 and 306 mOsm kgK1, respectively. All lenses were incubated at 378C in a humidified atmosphere of 95% air/5% CO2 in 24-well culture plates and the culture medium changed daily. The clarity of the lenses was recorded both by ocular inspection and digital image analysis of lens photos taken on day 1 and opacified lenses were presumed damaged during dissection and therefore discarded (see below). In addition, lenses with high lactate dehydrogenase (LDH) leakage into the culture medium on day 1 were discarded (see below). After these exclusions, the remaining numbers of lenses from the SOD1 null mice were 16 in the high glucose group (GK/K) and 13 in the normal glucose group (NK/K). Of the control lenses from the wild-type mice, 14 lenses remained in the high glucose group (GC/C) and 12 in the normal glucose group (NC/C). 2.2. Analysis of lens opacification
2. Materials and methods 2.1. Animals and preparation of lenses The ARVO Statement for the Use of Animals in Ophthalmic and Vision Research was followed in this investigation. The investigation was approved by the research ethics committee of Umea˚ University, Umea˚, Sweden. The SOD1 null mice (initial background 129/CD1), generated by Dr A.G. Reaume et al., at Cephalon, Inc., develop normally, have normal weights and survive to adulthood (Reaume et al., 1996), and in our laboratory they survive more than a year. They were bred and genotyped as detailed previously (Behndig et al., 2001) and backcrossed 10 times into a wild-type strain (C57BL/6J). C57BL/6J mice were used as wild-type controls. Twenty-one SOD1 null mice (11 male, 10 female) and 21 wild-type mice (15 male,
The lens opacification was quantified with digital image analysis as described previously (Behndig et al., 2001) with some modifications. Briefly, 256-level grey scale photographs of a steel grid were taken through the lens in retro-illumination (Fig. 1). The standard deviation (S.D.) and mean density (MD) of the values obtained for each pixel within a central circular 1-mm2 area of the lens were calculated. Development of cortical cataract will render more grey pixels and less black/white pixels and thereby lower the S.D. The MD is little affected by cortical cataract, but a nuclear opacification will act as a filter, making the pixels generally darker, and the MD higher. The highest S.D. obtained for a clear lens in the material (48.5) and the MD for a completely opaque lens (255) were used in constructing a formula to quantify the lens opacifications in arbitrary units (a.u.):
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To determine the total activity of LDH in the mouse lens, eight lenses from four SOD1 null mice and seven lenses from four wild-type mice were dissected, homogenized with an ultraturrax in 1 ml PBS pH 7.0 (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 1.4 mM KH2PO4), sonicated for 1 min and shaken for 30 min at 48C. The lysates were centrifuged at 20 000g for 20 min at 48C and the LDH activities of the supernatants were analysed. The percentage of daily accumulated leakage of LDH activity/lens wet weight compared to the total LDH activity in a lens/lens wet weight was calculated. 2.4. Analysis of superoxide by lucigenin-derived chemiluminescence (LDCL)
Fig. 1. Typical cataract development in lenses from SOD1 null mice and wild-type mice. Lenses incubated with either normal or high glucose were photographed daily on a steel grid. GZ55.6 mM glucose, NZ5.6 mM glucose, K/KZSOD1 null lens, C/CZwild-type lens.
SD MD Opacification ða:u:Þ Z 1 K ! !100 48:5 255 Lenses with more than 25 a.u. on day 1 were excluded. The lenses were also staged from the photographs in a masked fashion according to Calvin et al. (1992), and assigned to one of six stages: 0Zno cataract, 1aZbeginning radial cortical aberrations, 1bZlarger and more pronounced cortical aberrations, 2Zwell-defined floriform cortical cataract, 3Zbreakdown of the floriform pattern, 4Z amorphous pattern of light scattering and incipient dense opacification and 5Zmature, corticonuclear cataract. The medians of the values obtained from three independent observers were used. Lenses with a median of 1a or more on day 1 were excluded. 2.3. Analysis of lactate dehydrogenase (LDH) leakage into the culture medium To quantify the damage to the lenses, the accumulated leakage of the intracellular LDH was determined (Kilic and Trevithick, 1995) by establishing the LDH activity in the culture medium for every lens after each day of incubation. The LDH activity in the culture medium was analysed using a kit from Roche/Hitachi (Roche Diagnostics GmbH, D-68298, Mannheim). A 0.1 ml sample of the lens culture medium was added to 0.5 ml 68 mM NaHPO4 containing 0.73 mM pyruvate. 0.1 ml NADH was then added to start the reaction, which was followed at 340 nm. 0.1 ml fresh culture medium served as blank. If the LDH activity on day 1 exceeded 1.25 U gK1 of lens dry weight, the lens was presumed damaged during dissection and therefore excluded.
LDCL was used to analyse the amount of superoxide anion radicals in the lenses during the cultivation. At the end of day 5, the lenses were transferred to vials containing 0.5 ml of M199 with either 5.56 mM glucose or 55.6 mM glucose. 25 mM lucigenin (Li et al., 1998) (Sigma, St Louis, MO) was added and the vials placed in a luminometer (TD 20/20, Turner Designs, Inc., Sunnyvale, CA). The resulting luminescence was measured after a 90 sec delay during 60 sec and was divided by the lens wet weight (see below) and presented as relative light units (RLU)/mg of wet weight. LDCL was not analysed in all of the lenses in the first set of lenses. Therefore, to increase the number of lenses in each group a second set of lenses was prepared for LDCL analysis from six SOD1 null mice (age 40G11.3 weeks; 4 male, 2 female) and nine wild-type controls (age 36G3.1 weeks; 6 male, 3 female). These lenses were subject to the same treatments and exclusion criteria as described above. After exclusions, 5 GK/K, 6 NK/K, 7 GC/C and 8 NC/C of these lenses remained. To validate that the LDCL was generated by superoxide formation within the lenses, the LDCL was quantified in eight freshly dissected lenses from four SOD1 null mice (age 19G2.3 weeks; 3 male, 1 female) and eight lenses from four wild-type controls (age 21G2.5 weeks; 3 male, 1 female) before and after addition of 50 mM of the SOD mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP). The LDCL was measured during six consecutive 1-min periods after the addition of MnTMPyP. 2.5. Analysis of the water content and uptake
86
Rubidium (86Rb)
The lenses were transferred on day 6 into wells containing M199, with either 5.56 mM glucose or 55.6 mM glucose, with 75 ng mlK1 86Rb (Amersham Biosciences) for 10 min, as previously detailed (Behndig et al., 2001). The initial specific activity of the 86Rb was 62 MBq mgK1. The lenses were then rinsed and blotted, placed on small stainless steel plates, and weighed before and after drying at 608C for 24 hr. The results were used for water content
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Table 1 Opacification of wild-type and SOD1 null lenses exposed to high or normal levels of glucose assessed by ocular staging Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Wild-type High glucose nZ14 Normal glucose nZ12
0 (0–0) 0 (0–0)
1a (1a–2) 1b (1a–1b)
1a (0–1b) 1b (2–2)
1a (1a–2) 1b (1b–2)
2 (1b–3) 1b (1a–2)
3 (2–4) 1b (1b–2)
SOD1 null High glucose nZ16 Normal glucose nZ13
0 (0–0) 0 (0–0)
3 (2–4)* 1a (1a–1a)
5 (3–5)* 1a (1a–1a)
5 (4–5)* 1a (1a–1b)
5 (4–5)* 1b (1a–1b)
5 (5–5)* 1b (1b–1b)
The results are presented as median values and interquartile ranges (first to third quartiles). High glucoseZ55.6 mM, normal glucoseZ5.56 mM. *Significantly (p!0.05) higher ocular staging compared to all other groups on that day.
determination. The dried specimens were then soaked in 1.0 ml of 10% trichloroacetic acid at 378C for 24 hr. After centrifugation at 10 000g for 10 min, 0.9 ml of the supernatant was mixed with scintillation liquid and placed in a scintillation counter. The initial radioactivity in the incubation medium was determined and the uptake of 86Rb per ml of lens H2O was calculated. 2.6. Immunohistochemistry Immunohistochemical staining of an eye from a wildtype mouse, age 24 weeks, was performed using a rabbit polyclonal antibody raised against a synthetic polypeptide corresponding to amino acids 24–36 in the murine SOD1 sequence. The antibody was purified on protein A Sepharose followed by adsorption–desorption to the peptide immobilized on SulfoLink coupling gel (Pierce, Inc., Rockford, IL). The whole eye globe was dissected and fixed in buffered formaldehyde solution and processed for standard immunohistochemical staining according to the peroxidase-antiperoxidase (PAP) technique. 4-mm-thick serial sections were photographed in a Zeiss photomicroscope with a digital microscope camera (DKC-5000, Sony Corp., Tokyo, Japan). An eye from a SOD1 null mouse, age 23 weeks, was used as a negative control to confirm the specificity of the staining.
parametric testing due to the presence of zeros in the material. When comparing only two independent samples, Student’s t-test or Mann–Whitney U-test was used. Correlations between the different analyses were tested with Pearson correlations. In all of the tests a level of p%0.05 was considered statistically significant.
3. Results SOD1 null lenses exposed to high glucose (GK/K) developed significantly more cataract compared to the SOD1 null lenses with low glucose (NK/K) and the wildtype lenses with high (GC/C) and low (NC/C) glucose on ocular staging (p!0.05) (Table 1, Fig. 1) and on digital image analysis (p!0.02) (Fig. 2). The results for ocular staging and digital image analysis for all groups combined showed a high correlation (rZ0.898, p%0.005). The GK/K lenses also showed a significantly higher leakage of LDH compared to the controls (p%0.009) (Fig. 3). There was no significant difference in total intralenticular LDH activity between freshly dissected SOD1 null and wildtype lenses (SOD1 null lens; 27.2G1.5 U gK1 of lens wet weight, wild-type lens; 26.3G1.0 U gK1 of lens wet weight (meansGSE, pZ0.63)). Based on this, the GK/K group had
2.7. Statistical analysis Statistical analyses were performed using SPSS statistical software. Digital image analysis, accumulated leakage of LDH, water content and uptake of 86Rb were analysed with a 2-way factorial analysis of variance (2-way ANOVA). Data for LDH and 86Rb were transformed by logarithms in base 10 (log 10) and data for digital image analysis and water content by the arcsine of the square root of a proportion (digital image analysis; ARCSINEO (a.u./60), water content; ARCSINEO (%/100)). After transformation, all data fulfilled the demands of equal variance for the statistical tests. The ocular staging data and the LDCL results were analysed using Kruskal–Wallis and post-hoc testing according to Dunn (Zar, 1974), since the former were categorical data, and the latter did not fulfil the assumptions of
Fig. 2. Digital image analysis of lens opacification (meansGSE). *Significant interaction between SOD1 null mice and high glucose (2way ANOVA, p!0.02). Black trianglesZSOD1 null lenses with 55.6 mM glucose, black circlesZwild-type lenses with 55.6 mM glucose, white circlesZwild-type lenses with 5.6 mM glucose, white trianglesZSOD1 null lenses with 5.6 mM glucose.
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Fig. 3. Accumulated leakage of LDH from lenses (meansGSE). *Significant interaction between SOD1 null mice and high glucose (2-way ANOVA, p%0.009). Black trianglesZSOD1 null lenses with 55.6 mM glucose, black circlesZwild-type lenses with 55.6 mM glucose, white circlesZwild-type lenses with 5.6 mM glucose, white trianglesZ SOD1 null lenses with 5.6 mM glucose.
lost 12% of its total LDH activity into the medium on day 6, the GC/C group had lost 8%, the NK/K group had lost 3% and the NC/C group had lost 5%. The results for LDH leakage correlated significantly (p%0.05) with the results for digital image analysis, ocular staging, water content and LDCL (rZ0.605, 0.721, 0.540, and 0.355). The combination of high glucose and SOD1 null lenses gave a significantly higher water content (pZ0.001) compared to the other combinations (Table 2). High glucose gave a significantly lower 86Rb uptake (p!0.001) in both SOD1 null lenses and wild-type lenses when comparing the concentration of rubidium in the lens water in the early influx phase. However, when instead comparing the amount of rubidium per lens, there was no significant difference although high glucose showed a slightly lower 86Rb uptake for both SOD1 null and wild-type lenses (Table 2). The reason for this discrepancy may be the differences in the degree of oedema between the lenses in this study. Uptake of 86Rb was the variable least correlated to the other variables, its best correlation being rZK0.543 (p%0.005) with the lens water content.
643
For both the high and low glucose concentration the LDCL was higher in the SOD1 null lenses as compared to the wild-type lenses (p!0.05). However, exposure to high glucose did not increase the LDCL significantly in neither the SOD1 null nor the wild-type lenses (Table 2). LDCL correlated significantly (p%0.05) with every other method of analysis, the best correlation being with digital image analysis (rZ0.440) and ocular staging (rZ0.436) of cataract. The two sets of lenses used for LDCL analysis did not differ on digital image analysis (pO0.17) or on ocular staging on day 5 (pO0.14). To test the specificity for superoxide radicals, the LDCL of lenses was determined before and 5 min following the addition of the low-molecular weight SOD mimic, MnTMPyP. In eight SOD1 null lenses, LDCL was reduced from 1.6G0.4 RLU mgK1 lens wet weight to 0.4G 0.2 RLU mgK1 (pZ0.046). In the wild-type lenses, the reduction from 0.3G0.1 to 0.1G0.1 RLU mgK1 was not significant (pZ0.273). The small residual LDCL in the SOD1 null lenses 5 min after the addition of MnTMPyP did not differ significantly from the LDCL in the wild-type lenses at the same time point (pZ0.195). Immunohistochemistry showed staining for SOD1 in the lens epithelium and cortex of the wild-type eye, but no staining in the nucleus (Fig. 4A). In the SOD1 null lens, the staining was weak to negligible (Fig. 4B).
4. Discussion In this in vitro model of glucose-induced cataractogenesis, we have shown that lenses from mice lacking SOD1 are more prone to develop cataract compared to lenses from wild-type mice. These control lenses, when exposed to high glucose, also showed a slight increase in cataract development and cell damage on most of the tested variables. However, the cataractogenic effect was not as pronounced as for the SOD1 null lenses with high glucose, which showed pronounced increased measures of injury or cataract in all tested variables. This was especially evident when
Table 2 Uptake of 86Rb, H2O content and lucigenin-derived chemiluminescence (LDCL) from superoxide in wild-type and SOD1 null lenses exposed to high or normal levels of glucose 86
Rb
H2O (%)
LDCL (RLU/mg of lens wet weight)
572.9G31.8 628.3G58.5
61.7G1.4 (nZ14) 61.0G1.8 (nZ12)
0.8G0.2 (nZ17) 0.4G0.2 (nZ17)
616.5G40.3 687.6G76.0
71.6G1.3 (nZ16) 61.2G1.1 (nZ13)
1.8G0.2 (nZ16) 1.4G0.2 (nZ16)
(ng/ml of lens H2O)
(pg/lens)
Wild type High glucose Normal glucose
137.5G11.8 (nZ14) 163.9G16.2 (nZ12)
SOD1 null High glucose Normal glucose
99.4G5.7 (nZ16) 173.1G24.2 (nZ13)
The results are presented as meansGSE. High glucoseZ55.6 mM, normal glucoseZ5.56 mM. High glucose gave a significantly lower 86Rb uptake in ng/ml of lens water (p!0.001) in both SOD1 null lenses and wild-type lenses (2-way ANOVA, p!0.001). The interaction of SOD1 null mice and high glucose was significant for water content (2-way ANOVA, pZ0.001). SOD1 null lenses showed higher LDCL than wild-type lenses regardless of glucose concentration (Kruskal–Wallis, p!0.001).
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Fig. 4. Immunohistochemical staining of the mouse lens with a polyclonal rabbit antibody against murine SOD1. (A) adult wild-type lens. (B) adult SOD1 null lens. (C) Adult wild-type retina, a tissue known to be rich in SOD1, for comparison. (D) Adult SOD1 null retina. Note cortical staining in A, strong staining in C and weak or negligible staining in B and D. Scale bar, 100 mm.
analysing the cell leakage of LDH, which clearly showed increased cell leakage in the SOD1 null lenses with high glucose compared to the wild-type lenses with high glucose. Therefore, the results of this study suggest that the two separate stresses, high glucose and absence of SOD1, in synergy have a great influence on cataract development. The function of SOD1 is superoxide scavenging, and we here found that SOD1 is expressed in the lens epithelium and cortex, where the oxidative damage leading to cataract occurs (Spector, 1995). It has been demonstrated that SOD1 plays a critical role in the protection against damage induced by ROS in cultured lens epithelial cells and also that there is a spontaneous development of lens opacification with advanced age in the SOD1 null genotype (Reddy et al., 2004). Although SOD1 is the dominating SOD isoenzyme in the lens as a whole (Behndig et al., 2001), the other SOD isoenzymes may also exert significant anti-oxidative effects in the microenvironment of the lens epithelium, as demonstrated for SOD2 (Matsui et al., 2003; Reddy et al., 2004). The LDCL method used for detection of superoxide in the present study has been the subject of some debate, since lucigenin in itself can act as a source of superoxide generation, a reaction favoured at higher concentrations. In the present study, we used a low concentration to reduce this risk (Li et al., 1998). Some chemiluminescent probes also have a limited specificity, and may require the addition of a specific scavenger of the ROS in question, as a control, to validate the results. An increase in SODquenchable luminol luminescence with age has been demonstrated in the rat lens (Trevithick and Dzialoszynski, 1997). SOD1, however, may be less suitable to validate the origin of the LDCL as being superoxide, owing to poor penetration through the lens capsule of intact cultured lenses. In a previous study, we found only a small (about 10%), insignificant lowering of the LDCL in intact SOD1 null lenses with the addition of SOD1 (Behndig et al.,
2001). In contrast, we here demonstrate a 75% reduction in LDCL in SOD1 null lenses 5 min after adding the lowmolecular weight SOD mimic MnTMPyP. Together with the reported high specificity of lucigenin for superoxide (Myhre et al., 2003) and the high specificity of a knockout mouse model, this finding strongly implicates elevated superoxide levels as the source of the augmented LDCL found in the SOD1 null lenses, both at normal and increased glucose levels. Superoxide radicals may be damaging to the lens through Fenton chemistry yielding hydroxyl radicals (Spector, 1995), which can cause damage to cell membranes through lipid peroxidation (Barber and Thomas, 1978), DNA damage (Breen and Murphy, 1995) or protein aggregation (Dean et al., 1997). This may be facilitated in diabetic conditions due to increased copper (Lin, 1997) and iron (Cekic and Bardak, 1998) contents in the lens. Recent studies have shown that lens cells exposed to high glucose develop cataract and show raised biochemical markers of oxidative damage (Hegde et al., 2003a; Jain et al., 2002). Also the formation of AGEs may be augmented in diabetes through metal catalysed oxidative reactions with unstable Amadori products. Ascorbate might also take part in oxidative reactions, promoting further formation of AGEs (Saxena et al., 1996). Ascorbate, also an antioxidant, might on the other hand protect against oxidative damage (Hegde and Varma, 2004; Varma et al., 1979), but the role of ascorbate in this in vitro model is likely minor, since it is unstable and may be close to zero in the culture medium here used, despite its presence in the formula of the medium (Stralin et al., 2003). In fact, the low levels of ascorbate may increase the relative importance of SOD1 as a superoxide scavenger in this type of in vitro cataract model. The superoxide radical is also known to react readily with nitric oxide (NO), generating the highly reactive and cytotoxic compound peroxynitrite (ONOOK) (Beckman et al., 1990; Wu et al., 1997). The oxidative stress cascade initiated by RAGE binding can enhance nuclear factorkappa B transcription (Stitt, 2001), and indirectly increase the formation of NO through the induction of inducible nitric oxide synthetase (iNOS). It is known that both iNOS mRNA and iNOS protein are expressed in cataractous lenses from rats before the onset of lens opacification (Inomata et al., 2001). Furthermore, selenite-induced cataract, shown to be prevented by many different antioxidants (Devamanoharan et al., 1991; Shearer et al., 1997; Varma et al., 1995), can also be prevented in rats by treatment with NOS inhibitors (Ito et al., 2001), indicating an involvement of NO in this type of cataract. Little is known about the functions of NO in the lens, however, and further studies are needed to evaluate the possible role of NO in diabetic cataract. We here show that the combined effect of SOD1 deficiency and high glucose markedly enhances lens damage. We suggest that this may contribute to the pathogenesis of diabetic cataract where the lowered SOD
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levels in the lens may exacerbate the vulnerability of the lens, accelerating cataract development.
Acknowledgements This study was funded by grants from the Swedish Research Council, Synfra¨mjandets Research Fund and the KMA fund.
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