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Tempol-H inhibits opacification of lenses in organ culture
Free Radical Biology & Medicine, Vol. 35, No. 10, pp. 1194 –1202, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter
doi:10.1016/S0891-5849(03)00505-7
Original Contribution TEMPOL-H INHIBITS OPACIFICATION OF LENSES IN ORGAN CULTURE J. SAMUEL ZIGLER, JR.,* CHUAN QIN,* TOSHIKAZU KAMIYA,*1 MURALI C. KRISHNA,† QIUFANG CHENG,*2 SANTA TUMMINIA,*3 and PAUL RUSSELL* *Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, Bethesda, MD, USA; and †Radiation Biology Branch, National Cancer Institute, Bethesda, MD, USA (Received 4 April 2003; Revised 3 July 2003; Accepted 17 July 2003)
Abstract—Cataract is the world’s leading cause of blindness and a disease for which no efficacious medical therapy is available. To screen potential anti-cataract agents, a lens organ culture model system was used. Opacification of lenses maintained in culture was induced by specific insults including H2O2 or the cataractogenic sugar xylose. Potential anti-cataract agents were added to the culture medium and their ability to inhibit opacification and certain biochemical changes associated with the opacification were assessed. Among the compounds tested, Tempol-H, the hydroxylamine of the nitroxide Tempol, gave the most promising results. It significantly inhibited opacification of rat lenses in an H2O2-induced cataract system as well as opacification of rhesus monkey lenses induced by xylose. Tempol-H inhibited the loss of glutathione, the leakage of protein, and decreases in the ability of cultured lenses to accumulate 3H-choline from the medium, all of which were associated with the development of lens opacification. The antioxidative activity of Tempol-H and its ability to re-dox cycle make it an attractive candidate as a therapeutic agent for the prevention of aging-related cataract. © 2003 Elsevier Inc. Keywords—Lens, Cataract, Cataract therapy, Organ culture, Oxidative stress, Tempol-H, Free radicals
INTRODUCTION
bly increase in the absence of efficacious medical therapy. A number of preparations purported to have anticataract activity are sold in Europe and Asia; however, clear evidence of their efficacy is lacking [2]. No anticataract drug has been licensed in the United States despite considerable effort by research laboratories and pharmaceutical companies to develop such agents. A number of factors contribute to the difficulty that has been encountered. Clearly, cataract is not a single disease with a single etiology. There are at least three different major forms of cataract (nuclear, cortical, and posterior sub-capsular), each of which is multifactorial in etiology and highly variable in severity and rate of progression. Further complicating the situation, the factors contributing to age-related cataractogenesis are a variable combination of pathological processes and normal aging processes, which at present cannot be clearly demarcated. Based on the available knowledge of the biology of the normal lens and the cataractogenic process, several approaches have been taken in the design of anti-cataract agents. Because chronic oxidative stress is widely believed to be a major factor, if not the major factor, in the
Although advances in cataract surgical techniques and the refinement of intraocular lens implants have greatly benefited cataract patients, a medical therapy capable of preventing or slowing cataract development remains highly desirable. In the developing world cataract is the leading cause of blindness largely because medical services are often unavailable or extremely limited. In the United States cataract extraction is the most common surgical procedure among Medicare recipients, accounting for over $3 billion in annual expenditures [1]. As the population in this country and elsewhere becomes progressively older, the prevalence of cataract will inevitaAddress correspondence to: Dr. J. Samuel Zigler, National Eye Institute, Laboratory of Mechanisms of Ocular Diseases, 6 Center Drive MSC-2735, Bethesda, MD 20892-2735, USA; Tel: (301) 4966669; Fax: (301) 496-1759; E-Mail: [email protected]. 1 Current affiliation: Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. 2 Current affiliation: Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA. 3 Current affiliation: The Foundation Fighting Blindness, Owings Mills, MD, USA. 1194
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etiology of aging-related cataract [3], agents with antioxidative properties have received the most attention. Such compounds range from antioxidant vitamins (E, C, -carotene), to functional mimics of antioxidant enzymes, to a wide variety of low molecular weight compounds with antioxidant activity (e.g., ␣-lipoic acid, N-acetylcysteine). A second approach to development of anti-cataract agents is based on the hypothesis that phase separation phenomena are integral to cataract development [4]. Phase separation results from noncovalent attractive interactions between proteins in concentrated solutions, creating protein-rich and protein-poor regions [5]. In the lens, formation of such domains creates light scattering, leading to cataract. After extensive study in vitro, two putative phase separation inhibitors, pantethine and the radioprotective phosphorothioate WR77913 have been tested in several acute animal models of cataract [6]. Although the specific mechanisms remain uncertain, delays in the onset of cataract and in its severity were reported. The studies reported here were based on the underlying premise that whereas human aging-related cataract may result from a variety of initiating factors, there are certain common pathways of cellular damage that may always be central to the progression of the disease process. Therapeutic agents aimed at inhibiting such common pathways should be effective in slowing or stopping disease progression. Even a modest delay in the rate of progression of the disease would eliminate the need for surgery in many elderly individuals [7,8]. Previous studies from this laboratory have established that the lens, which is avascular and noninnervated in vivo, can be maintained in a fully viable state in organ culture, that cataracts can be induced in cultured lenses by various chemical or environmental perturbations, and that prevention or inhibition of cataract formation can be observed after addition of appropriate agents to counteract the cataractogenic stresses [9 –11]. On the basis of our studies as well as those from other laboratories, it was decided to use the lens organ culture system to screen potential anti-cataract agents.
MATERIALS AND METHODS
Animals Sprague Dawley rats were obtained from Taconic Farms (Germantown, NY, USA). Animals of either sex were used in the weight range of 76 –100 grams. The animals were handled in adherence to the Guide for the Care and Use of Laboratory Animals (National Academy Press). Rhesus monkey (Macaca mulatta) eyes were obtained from animals of about 2 years of age used in the Vaccine Testing Program, Center for Biologic Evalua-
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tion and Research, U.S. Food and Drug Administration (Bethesda, MD, USA). Monkey eyes enucleated immediately after death were brought to the laboratory and the lenses dissected and placed into culture within 3 h of death. Materials Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl) was obtained from Sigma Chemical Company (St. Louis, MO, USA). Tempol-H, the hydroxylamine reduction product of Tempol was prepared from Tempol by hydrogenation as previously described [12]. Stock solutions of Tempol-H were prepared in culture medium immediately before use. Samples from lens homogenates or culture medium taken for analysis of Tempol/Tempol-H were prepared in 0.5 mM diethylenetriaminepentaacetic acid (DETAPAC) to prevent oxidation and were stored at 4°C. Lens organ culture Dissection of lenses from rat [13] and monkey [11] eyes was as described earlier. Lenses were immediately placed in modified TC-199 medium prepared as described previously [13]. Rat lenses were cultured in 2.0 ml medium in 24 well cluster dishes; monkey lenses in 5.0 ml medium in 12 well cluster dishes. All lenses were allowed to equilibrate in the incubator (37°C, 5% CO2) for 2 h; lenses that were damaged during dissection were identified during this period by measuring protein leakage into the medium as previously described [14] and were discarded before experimental groups were established. After the equilibration period was complete, additions were made to the medium for each lens or the medium was exchanged as required to initiate the experiment. Rat lenses incubated in the presence of H2O2 were subjected to a single bolus of H2O2 producing a final concentration as indicated in the description of each experiment. Exposure of monkey lenses to 30 mM xylose or cellobiose was as previously described [11]. Cultured lenses were photographed using a Nikon eclipse E800 Dissecting microscope. Analyses Lens glutathione levels were determined using Ellman’s Reagent (DTNB) by a previously published method [15]. H2O2 concentration in culture media was measured using a Model 2700 Biochemistry Analyzer (Yellow Springs Instrument Co., Yellow Springs, OH, USA). Studies on the accumulation of 86Rb and 3H-choline by the cultured lens were as previously described [9,10]. Lens to medium concentration ratios were calculated and
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used as a measure of the condition of the lens after experimental treatments. Electron paramagnetic resonance (EPR) was used to quantitate the levels of Tempol. Tempol-H, which is EPR silent, was measured in the same way after oxidation to Tempol by addition of sodium ferricyanide. Analyses were performed on a Varian E4 X-band spectrometer as previously described [12]. Protein oxidation studies Bovine lens water-soluble proteins were prepared by homogenizing a bovine lens in water and removing insoluble material by centrifugation (20,000 ⫻ g, 20 min, 4°C). The supernatant was dialyzed exhaustively against deionized water and lyophilized. The lyophilized protein was stored at ⫺20°C in a tightly closed tube. For exposure to oxidizing systems, aliquots of the lyophilized protein were dissolved in phosphate-buffered saline (PBS) at 2.5 mg/ml. To 0.8 ml of this solution was added 0.2 ml PBS or a total of 0.2 ml PBS containing Tempol or Tempol-H and components of the oxidizing systems. Samples were exposed to oxidizing systems as follows: H2O2 plus FeSO4/EDTA as previously described [16], 20 nM rose Bengal, or 50 M flavin mononucleotide (FMN) plus light from fluorescent “cool white” bulbs as previously described [17]. Aliquots were taken from these experimental samples and appropriate control samples at various times, and were analyzed by SDS-PAGE on 12% Bis-Tris NuPage gels (Invitrogen Corporation, Carlsbad, CA, USA). RESULTS
The initial approach in these studies was to use a cocktail of agents with different modes of action (i.e., antioxidants, phase separation inhibitors, protease inhibitors, etc.) in hopes of achieving broader and stronger anti-cataract activity. After considerable effort it was concluded that this approach did not produce results that could be effectively interpreted because some of the agents being tested had definite negative effects on the cultured lenses either individually or when combined with other agents. Thus, studies were done on individual compounds. Table 1 lists some of the compounds tested, all of which had been reported to have some protective effects with lenses or lens cell cultures, or had activities in other systems that seemed potentially relevant to the lens. Using either H2O2 or xylose as cataractogenic agents for cultured lenses, it was found that most of the compounds had no protective effect as judged by the primary screening criteria of lens transparency and the ability of the lens to accumulate choline and rubidium. Figure 1 shows the results of a typical screening experiment. The data for 3H-choline are shown; the data
Table 1. Compounds Tested for Anti-cataract Activity Tempol (5 mM) Tempol-H (5 mM) Pantethine (5 mM) Carnosine (2 mM) E-64 (0.5 mM) N-acetyl cysteine (1 mM) Mercapto-proprionyl glycine (1 mM)
N-propyl gallate (1 mM) Piperidinol (4 mM) Phenyl-N-tert-butylnitrone (1 mM) Ascorbic Acid (1 mM) Butylated Hydroxytoluene (1 mM) Captopril (2.5 mM) Tiron (2 mM)
Note: The value given for each compound was the maximal concentration tested.
for 86Rb showed the same pattern, although the deficit in accumulation after H2O2 exposure was less as has typically been the case in such analyses. Lenses were exposed in culture to a single bolus of 250 M H2O2, tracer amounts of 3H-choline and86Rb were added after 18 –20 h and the concentration of label inside the lens was determined 4 h later as described in Materials and Methods. Although 250 M H2O2 does not have detectable effect on lens transparency under the conditions used, it does impair the lens’ ability to accumulate these compounds, as has been shown in previous studies [9,10]. This particular experiment includes the two compounds that gave the strongest protective effects in these studies, Tempol-H and mercapto-proprionylglycine (MPG). These agents showed a statistically significant protection of the ability of the incubated lens to accumulate the radiolabeled compounds. Carnosine showed no such effect, a result typical of those found with most of the agents tested. Tempol-H is the reduced form (hydroxylamine) of the nitroxide Tempol, which itself was found to be toxic to the cultured lens at the concentration (4 mM) used in these studies (data not shown). The toxicity of Tempol to the cultured lenses was evidenced by the development of haziness within the first 12 h and, as noted below, by an apparent change in the redox status within the lens. Although exposure of rat lenses to 250 M H2O2 as described above does not cause opacification of the lens, a bolus of 1.0 mM H2O2 produces clouding of the lens within a few hours and within a day, frank opacification and complete loss of ability to accumulate choline above the concentration in the medium (data not shown). Figure 2 shows the effect of Tempol-H (4 mM) when added to the culture medium concomitantly with this higher concentration of H2O2. The lenses in Panel A were incubated in control medium, lenses in Panel B demonstrate the typical opacification after 24 h in the same medium to which a bolus of 1.0 mM H2O2 was added at time zero, and lenses in Panel C were incubated in 1 mM H2O2 with the addition of 4 mM Tempol-H. The protective effect of Tempol-H was not due to removal of H2O2 from the medium. Measured H2O2 concentration in the medium
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Fig. 1. Accumulation of 3H-choline from the medium by cultured rat lenses. All lenses were incubated in 2.0 ml TC-199 with or without a single bolus of 250 M H2O2 for 18 h. A tracer level of 3H-choline was added to all culture wells after 14 h. Lenses were harvested and analyzed as previously described [10]. The medium from each lens was also counted and lens water to medium concentration ratios calculated and reported as mean ⫾ standard deviation. All lenses received 250 M H2O2 except the control group. Carnosine (1 mM), mercaptoproprionyl glycine (MPG, 1 mM) or Tempol-H (4 mM) was added to individual groups as indicated at the same time the H2O2 was added.
was not different between Groups B and C (data not shown), consistent with the work of others demonstrating that Tempol/Tempol-H does not react with H2O2 [18].
Although Fig. 2 demonstrates that Tempol-H strongly inhibits opacification even in the presence of 1 mM H2O2, these lenses will opacify upon longer incubation.
Fig. 2. Appearance of rat lenses after 24 h exposure to a single bolus of 1 mM H2O2 with or without addition of 4 mM Tempol-H. (A) Control lenses not exposed to H2O2. (B) Lenses exposed to H2O2. (C) Lenses exposed to H2O2 with the addition of 4 mM Tempol-H.
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Fig. 3. Concentration of glutathione in lenses after 18 h incubation with 1.5 mM H2O2 with or without addition of Tempol or Tempol-H. Glutathione in the H2O2-exposed lenses was reduced by about 33%, a loss largely prevented by addition of 4 mM Tempol-H.
It should be noted that 1 mM is well above any reported H2O2 concentrations to which the human lens is exposed in vivo. The reduced glutathione (GSH) content of the lens is quite high and in virtually all cataracts drops precipitously. To test the ability of Tempol-H to inhibit GSH loss in stressed lenses, we incubated rat lenses with a single bolus of 1.5 mM H2O2 in the presence or absence of 4 mM Tempol-H. As seen in Fig. 3, Tempol-H prevented most of the loss of GSH seen in the H2O2exposed lenses after 18 h incubation. In contrast, Tempol at the nontoxic levels shown had no apparent benefit, but did not enhance GSH loss in H2O2-treated lenses. These results are a single “snapshot” of the effect of Tempol-H on GSH levels; we did not attempt to characterize effects at the early stage of oxidative stress when GSH has been shown to drop precipitously before recovering somewhat [19]. In order to better characterize the apparent antioxidative activity of Tempol-H in the organ culture studies, we tested its ability to prevent oxidative damage to solutions of lens proteins exposed to systems generating reactive oxygen species. Figure 4 shows the results of a representative experiment in which solutions of bovine crystallins were exposed to visible light and FMN as previously described [17]. Oxidative damage to the proteins caused aggregation as is evident in Lane 2. Addition of either Tempol (Lane 3) or Tempol-H (Lane 4) at 4 mM final concentration was equally effective in preventing
the oxidative damage resulting from free radical species generated directly or indirectly by this system. Although the rat lens has been widely used in organ culture studies and has provided considerable insight into the biology of the normal lens and cataract development, it is also understood that rodent lenses differ in important ways from the human lens. The availability of a limited number of lenses from freshly euthanized rhesus monkeys (Macaca mulatta) used in the vaccine testing program of the Center for Biologics Evaluation and Research, FDA allowed for the testing of Tempol-H in a primate lens cataract system. Sugar-induced cataract, which has been studied extensively and has been investigated in the rhesus monkey lens organ culture system in this laboratory [11,20], was used because presumably opacification resulted from a different mechanism than that induced by H2O2. Lenses were cultured in medium containing 30 mM xylose or 30 mM cellobiose (a nonmetabolized sugar used as control) with or without the addition of Tempol-H. Lenses exposed to xylose alone developed a slight haziness after 3– 4 d and became completely opaque after about 2 weeks in culture [11]. Figure 5 shows photographs of representative lenses after 9 d of culture. The control lens in the medium containing cellobiose is completely clear, whereas the xylose-exposed lens shows extensive opacification, particularly in the superficial cortical region. Addition of Tempol-H to the culture medium with the xylose largely prevented this opacification. Lenses exposed to Tem-
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Fig. 4. Effect of photochemical oxidation on solutions of bovine lens crystallins. Exposure for 5 h to flavin mononucleotide (FMN) plus light caused extensive protein cross-linking as seen in Lane 2. Lane 1 contained FMN, but was protected from light. In Lanes 3 and 4 the samples were as in Lane 2 with the addition of 10mM Tempol-H or 10 mM Tempol, respectively.
pol-H (1 or 4 mM) in the absence of xylose remained completely clear (not shown).
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As a biochemical correlate of opacification, the cumulative leakage of protein from cultured lenses was monitored daily for 15 d by analyzing an aliquot of the medium each day. Figure 6 shows data for individual lenses in 4 groups: control (cellobiose), xylose (30 mM) and xylose plus two different concentrations of Tempol-H. The control lenses had no detectable protein leakage during this period of time. For the xylose-exposed lenses, leakage of protein became detectable at about 1 week in culture and increased dramatically thereafter, reaching a total of about 4 mg by 15 d of culture. Supplementation of the medium with Tempol-H (0.2 mM) seemed to reduce leakage, although statistical analysis could not be done because of the limited number of lenses available. However, 1.0 mM Tempol-H had a very strong protective effect against xylose-induced protein leakage, consistent with its inhibition of opacification. Because Tempol-H afforded strong protection to lenses exposed in vitro to cataractogenic stresses, the uptake of both Tempol-H and Tempol by the cultured rat lens was investigated. Lenses were cultured as above in 4 mM Tempol or Tempol-H for 1, 2, 4, or 7 h and the concentration in the lens water (62.5% of wet weight) determined by EPR spectroscopy. Tempol, which is a stable free radical, could be measured directly; Tempol-H was measured as Tempol after oxidation with ferricyanide [20]. Figure 7 demonstrates that Tempol enters the lens more readily than does Tempol-H, a finding consistent with its more hydrophobic character. No Tempol was detected in lenses incubated in Tempol-H. Tempol was rapidly and efficiently reduced to Tempol-H after entering the lens, as demonstrated by the fact that no Tempol was detected in lenses incubated in 4 mM Tempol for 1 or 2 h. There was a small amount of Tempol (⬃ 5% of total) in most lenses incubated 4 h or longer in 4 mM Tempol, which may be an indication of the toxicity that becomes evident from loss of transpar-
Fig. 5. Appearance of representative rhesus monkey lenses after 9 d in culture in medium containing 30 mM xylose or 30 mM cellobiose, a nonmetabolized sugar used as control. Addition of 1 or 4 mM Tempol-H to the media of xylose-exposed lenses strongly inhibited opacification.
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Fig. 6. Cumulative leakage of protein from rhesus monkey lenses incubated in 30 mM sugars. Each curve represents a single lens, except for the lenses incubated in cellobiose where all 3 lenses had no detectable protein leakage.
ency with longer times in culture. Analysis of samples from lenses dissected into capsule/epithelium, cortex, and nucleus after 4 h culture indicated that the Tempol-H
present after incubation in either Tempol or Tempol-H was distributed relatively evenly throughout the entire lens and that in the 4 h Tempol-incubated lenses the small amount of Tempol detected in the lens was present primarily in the epithelium (data not shown). DISCUSSION
Fig. 7. Concentration of total Tempol plus Tempol-H in rat lenses cultured in medium containing either 4 mM Tempol (diamond symbols) or 4 mM Tempol-H (square symbols). Groups of lenses were harvested after 1, 2, 4, and 7 h of culture and the concentration of Tempol ⫹ Tempol-H in the lens water determined by EPR spectroscopy. Lenses incubated in Tempol-H were found to contain no Tempol. Lenses incubated in Tempol for 1 or 2 h also had no detectable Tempol, indicating rapid reduction to the Tempol-H form. In lenses incubated in Tempol for 4 or 7 h Tempol-H remained the dominant form, but a small amount of Tempol (⬃ 5% of total) was detected. Data are reported as mean concentrations (total Tempol ⫹ Tempol-H) ⫾ SD.
A major impediment to progress in developing a safe and effective medical therapy for aging-related cataract has been the lack of a convenient animal model that adequately reflects the human disease. We elected to take an in vitro approach in which lenses in organ culture were used instead of animal models for the initial screening of potential anti-cataract agents. A wide variety of compounds were screened individually and in various combinations. The agent that gave the strongest and most consistent protection was the hydroxylamine of the nitroxide Tempol. Nitroxides are stable free radicals that have long been used as spin labels in EPR spectroscopy and as contrast agents in NMR imaging [21]. More recently they have been investigated because of their antioxidant properties. Tempol has been shown to be an effective radioprotective agent, an activity attributed to its ability to break radical chain reactions by reacting with free radical species, its activity as a superoxide dismutase mimic, and its ability to oxidize transition metals and thereby inhibit Fenton chemistry [21]. In one
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study Tempol injected intracamerally was shown to protect against DNA strand breaks and cataract in rabbits exposed to x-rays [22]. Thus, Tempol is capable of inhibiting many of the processes implicated in generating the oxidative stress thought to be central to cataractogenesis. Tempol is highly cell permeable and has been shown to penetrate the blood/brain and blood/aqueous barriers. However, Tempol in this study was found to exert toxic effects on lenses in organ culture. Although the reduced form (hydroxylamine) of Tempol had generally been found to have little, if any, radioprotective effects in vitro [23], it has been found to have antioxidant effects in cells exposed to H2O2 [24]. Lens epithelial cell cultures exposed to H2O2 have been shown to be protected by addition of Tempol to the medium [18]. Tempol-H was tried in the organ culture system because the hydroxylamines were known to have less toxicity than the nitroxides and because re-dox cycling between the two states can readily occur in living systems [24]. In the lens, which is a highly reducing environment, we demonstrated that Tempol is very rapidly reduced to Tempol-H, with the equilibrium so strongly favoring the hydroxylamine that the nitroxide is generally undetectable by EPR. Under conditions of oxidative stress, the redox status within affected cells would shift to a more oxidizing state and the Tempol/Tempol-H ratio would increase. In our hands, Tempol was detected in cultured lenses only after several hours of exposure to toxic levels of Tempol or (data not shown) in lenses exposed in culture to 1 mM H2O2. Our data support the hypothesis that Tempol-H acts through an antioxidant mechanism. Tempol-H ameliorated the effects of H2O2 on cultured lenses in terms of protection from loss of transparency as well as from decrements in crucial biochemical parameters including glutathione concentration and membrane transport capacity. With isolated lens crystallins, Tempol-H was highly effective in preventing oxidative damage resulting from light and riboflavin-mediated generation of free radical species. In contrast, oxidative damage to proteins caused by exposure to rose bengal and light (data not shown), a Type II photochemical system in which damage is caused by singlet oxygen rather than free radicals [17] was not affected by addition of Tempol-H or Tempol. These findings suggest that scavenging free radicals and/or preventing their formation is key to the protective effects of Tempol-H. As shown in Fig. 4, the effects of Tempol in these studies were indistinguishable from those of Tempol-H, presumably because of the redox cycling between the two forms. The comparable effects of Tempol and Tempol-H in this test tube system are not reflected in the organ culture studies where Tempol-H is protective and Tempol exhibits toxicity. These disparate effects are observed in
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spite of the data demonstrating that the lens very rapidly converts Tempol to Tempol-H. Although the mechanism of this toxicity is unknown, it likely relates either to the high external concentration of Tempol in the Tempolexposed organ cultures, or to effects produced very quickly after the entry of Tempol into the lens before it is reduced to Tempol-H. These findings are similar in some ways to studies by Hahn et al. [12,23] comparing Tempol and Tempol-H as radioprotectors. In studies on cell cultures, Tempol-H exhibited no radioprotective activity, whereas Tempol was quite effective [23]; however, both Tempol and Tempol-H functioned as antioxidants in cell cultures exposed to H2O2 [24]. The failure of Tempol-H to protect the cell cultures from ionizing radiation was attributed to the fact that Tempol-H was not appreciably oxidized to Tempol in that system. In contrast, an in vivo study reported Tempol and Tempol-H to be equally effective as radioprotective agents [12]. This result is consistent with the fact that circulating levels of Tempol were similar within 10 min in mice injected with either agent. As in our lens organ cultures, much greater toxicity was observed in the Tempol-injected mice as a result of the transient spike in Tempol concentration during the first few minutes after injection [12]. Recent studies have confirmed that nitroxides and hydroxylamines are equally effective in protecting against free radicals generated via Fenton chemistry, whereas only nitroxides are effective against radiation damage [24]. Rodent lenses do differ in important ways from primate lenses and behave somewhat differently in culture [10]. To test the efficacy of Tempol-H in preventing cataractous change in a primate lens, we used xyloseinduced cataract in the cultured rhesus monkey lens. Sugar-induced cataract has been extensively studied in various species, both in vivo and in vitro, and has been shown to be initiated by the reduction of sugars to their polyols by the enzyme aldose reductase [25]. In the cultured monkey lens the cataract is less severe than in the rat lens, in part because the concentration of aldose reductase is only about 10% of that present in rat lens [20]. Tempol-H was quite effective in inhibiting opacification of the monkey lenses exposed to xylose. We believe that the protective effect reflects a significant oxidative component in the etiology of sugar cataract in the monkey. In the rat lens where the osmotic effect resulting from the high concentration of aldose reductase is so great, the oxidative component in cataract formation is minor. Tempol-H was not effective in preventing opacification of rat lenses exposed to 30 mM xylose in organ culture. The protective effect of Tempol-H in the monkey lens system is important, not only because it is a primate lens, but also because the cataractogenic process is initiated by a completely different mechanism
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than in the H2O2 cataract. It should be noted that in preliminary studies, it was found that Tempol-H also seemed to have protective effects against H2O2-induced stress in monkey lenses (data not shown). These studies were limited due to lack of available lenses. Because monkey lenses are much more resistant to oxidative stress than rat lenses [10], 2 mM H2O2 was used instead of 250 M used with the rat lenses. The monkey lens results support the concept that inhibiting pathways common to the development of cataracts irrespective of the factors initiating cataractogenesis is a viable approach to medical therapy for the disease. In conclusion, our data suggest that Tempol-H, with its strong antioxidant activity and capacity for redox cycling, is a promising candidate for development as an anti-cataract agent. Based on previous studies in animals [12] it does not seem likely that toxicity should be a problem. Development of mechanisms for topical application to the eye and subsequent testing for efficacy in preventing cataract in vivo are needed.
REFERENCES [1] Congdon, N. G. Prevention strategies for age related cataract: present limitations and future possibilities. Br. J. Ophthalmol. 85:516 –520; 2001. [2] Kador, P. F. Overview of the current attempts toward the medical treatment of cataract. Ophthalmology 90:352–364; 1983. [3] Spector, A. Oxidative stress-induced cataract: mechanism of action. FASEB J. 9:1173–1182; 1995. [4] Benedek, G. B. The theory of transparency of the eye. Appl. Opt. 10:459 – 473; 1971. [5] Benedek, G. B.; Pande, J.; Thurston, G. M.; Clark, J. I. Theoretical and experimental basis for the inhibition of cataract. Prog. Retin. Eye Res. 18:391– 402; 1999. [6] Clark, J. I.; Livesey, J. C.; Steele, J. E. Phase separation inhibitors and lens transparency. Optom. Vis. Sci. 70:873– 879; 1993. [7] Kupfer, C. Bowman lecture. The conquest of cataract: a global challenge. Trans. Ophthalmol. Soc. UK 104:1–10; 1985. [8] Taylor, H. R.; Keeffe, J. E. World blindness: a 21st century perspective. Br. J. Ophthalmol. 85:261–266; 2001. [9] Zigler, J. S. Jr.; Jernigan, H. M. Jr.; Garland, D.; Reddy, V. N. The effects of “oxygen radicals” generated in the medium on lenses in organ culture: inhibition of damage by chelated iron. Arch. Biochem. Biophys. 241:163–172; 1985. [10] Zigler, J. S. Jr.; Lucas, V. A.; Du, X. Y. Rhesus monkey lens as an in vitro model for studying oxidative stress. Invest. Ophthalmol. Vis. Sci. 30:2195–2199; 1989.
[11] Kamiya, T.; Zigler, J. S. Jr. Long-term maintenance of monkey lenses in organ culture: a potential model system for the study of human cataractogenesis. Exp. Eye Res. 63:425– 431; 1996. [12] Hahn, S. M.; Krishna, M. C.; DeLuca, A. M.; Coffin, D.; Mitchell, J. B. Evaluation of the hydroxylamine Tempol-H as an in vivo radioprotector. Free Radic. Biol. Med. 28:953–958; 2000. [13] Zigler, J. S. Jr.; Hess, H. H. Cataracts in the Royal College of Surgeons rat: evidence for initiation by lipid peroxidation products. Exp. Eye Res. 41:67–76; 1985. [14] Tumminia, S. J.; Qin, C.; Zigler, J. S. Jr.; Russell, P. The integrity of mammalian lenses in organ culture. Exp. Eye Res. 58:367–374; 1994. [15] Lou, M. F.; Huang, Q. L.; Zigler, J. S. Jr. Effect of opacification and pigmentation on human lens protein thiol/disulfide and solubility. Curr. Eye Res. 8:883– 890; 1989. [16] Zigler, J. S. Jr.; Huang, Q. L.; Du, X. Oxidative modification of lens crystallins by H2O2 and chelated iron. Free Radic. Biol. Med. 7:499 –505; 1989. [17] Jernigan, H. M. Jr.; Fukui, H. N.; Goosey, J. D.; Kinoshita, J. H. Photodynamic effects of rose bengal or riboflavin on carriermediated transport systems in rat lens. Exp. Eye Res. 32:461– 466; 1981. [18] Reddan, J. R.; Sevilla, M. D.; Giblin, F. J.; Padgaonkar, V.; Dziedzic, D. C.; Leverenz, V.; Misra, I. C.; Peters, J. L. The superoxide dismutase mimic TEMPOL protects cultured rabbit lens epithelial cells from hydrogen peroxide insult. Exp. Eye Res. 56:543–554; 1993. [19] Spector, A.; Wang, G. M.; Wang, R. R.; Li, W. C.; Kleiman, N. J. A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of Action. Exp. Eye Res. 60:483– 493; 1995. [20] Jernigan, H. M. Jr.; Zigler, J. S. Jr.; Liu, Y.; Blum, P. S.; Merola, L. O.; Stimbert, C. D. Effects of xylose on monkey lenses in organ culture: a model for study of sugar cataracts in a primate. Exp. Eye Res. 67:61–71; 1998. [21] Mitchell, J. B.; DeGraff, W.; Kaufman, D.; Krishna, M. C.; Samuni, A.; Finkelstein, E.; Ahn, M. S.; Hahn, S. M.; Gamson, J.; Russo, A. Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol. Arch. Biochem. Biophys. 289:62–70; 1991. [22] Sasaki, H.; Lin, L. R.; Yokoyama, T.; Sevilla, M. D.; Reddy, V. N.; Giblin, F. J. TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit. Invest Ophthalmol. Vis. Sci. 39:544 –552; 1998. [23] Hahn, S. M.; Tochner, Z.; Krishna, C. M.; Glass, J.; Wilson, L.; Samuni, A.; Sprague, M.; Venzon, D.; Glatstein, E.; Mitchell, J. B.; Russo, A. Tempol, a stable free radical, is a novel murine radiation protector. Cancer Res. 52:1750 –1753; 1992. [24] Xavier, S.; Yamada, K.; Samuni, A. M.; Samuni, A.; DeGraff, W.; Krishna, C. M.; Mitchell, J. B. Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage. Biochim. Biophys. Acta 1573: 109 –120; 2002. [25] Kinoshita, J. H.; Kador, P.; Datiles, M. Aldose reductase in diabetic cataracts. JAMA 246:257–261; 1981.
doi:10.1016/S0891-5849(03)00505-7
Original Contribution TEMPOL-H INHIBITS OPACIFICATION OF LENSES IN ORGAN CULTURE J. SAMUEL ZIGLER, JR.,* CHUAN QIN,* TOSHIKAZU KAMIYA,*1 MURALI C. KRISHNA,† QIUFANG CHENG,*2 SANTA TUMMINIA,*3 and PAUL RUSSELL* *Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, Bethesda, MD, USA; and †Radiation Biology Branch, National Cancer Institute, Bethesda, MD, USA (Received 4 April 2003; Revised 3 July 2003; Accepted 17 July 2003)
Abstract—Cataract is the world’s leading cause of blindness and a disease for which no efficacious medical therapy is available. To screen potential anti-cataract agents, a lens organ culture model system was used. Opacification of lenses maintained in culture was induced by specific insults including H2O2 or the cataractogenic sugar xylose. Potential anti-cataract agents were added to the culture medium and their ability to inhibit opacification and certain biochemical changes associated with the opacification were assessed. Among the compounds tested, Tempol-H, the hydroxylamine of the nitroxide Tempol, gave the most promising results. It significantly inhibited opacification of rat lenses in an H2O2-induced cataract system as well as opacification of rhesus monkey lenses induced by xylose. Tempol-H inhibited the loss of glutathione, the leakage of protein, and decreases in the ability of cultured lenses to accumulate 3H-choline from the medium, all of which were associated with the development of lens opacification. The antioxidative activity of Tempol-H and its ability to re-dox cycle make it an attractive candidate as a therapeutic agent for the prevention of aging-related cataract. © 2003 Elsevier Inc. Keywords—Lens, Cataract, Cataract therapy, Organ culture, Oxidative stress, Tempol-H, Free radicals
INTRODUCTION
bly increase in the absence of efficacious medical therapy. A number of preparations purported to have anticataract activity are sold in Europe and Asia; however, clear evidence of their efficacy is lacking [2]. No anticataract drug has been licensed in the United States despite considerable effort by research laboratories and pharmaceutical companies to develop such agents. A number of factors contribute to the difficulty that has been encountered. Clearly, cataract is not a single disease with a single etiology. There are at least three different major forms of cataract (nuclear, cortical, and posterior sub-capsular), each of which is multifactorial in etiology and highly variable in severity and rate of progression. Further complicating the situation, the factors contributing to age-related cataractogenesis are a variable combination of pathological processes and normal aging processes, which at present cannot be clearly demarcated. Based on the available knowledge of the biology of the normal lens and the cataractogenic process, several approaches have been taken in the design of anti-cataract agents. Because chronic oxidative stress is widely believed to be a major factor, if not the major factor, in the
Although advances in cataract surgical techniques and the refinement of intraocular lens implants have greatly benefited cataract patients, a medical therapy capable of preventing or slowing cataract development remains highly desirable. In the developing world cataract is the leading cause of blindness largely because medical services are often unavailable or extremely limited. In the United States cataract extraction is the most common surgical procedure among Medicare recipients, accounting for over $3 billion in annual expenditures [1]. As the population in this country and elsewhere becomes progressively older, the prevalence of cataract will inevitaAddress correspondence to: Dr. J. Samuel Zigler, National Eye Institute, Laboratory of Mechanisms of Ocular Diseases, 6 Center Drive MSC-2735, Bethesda, MD 20892-2735, USA; Tel: (301) 4966669; Fax: (301) 496-1759; E-Mail: [email protected]. 1 Current affiliation: Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. 2 Current affiliation: Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA. 3 Current affiliation: The Foundation Fighting Blindness, Owings Mills, MD, USA. 1194
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etiology of aging-related cataract [3], agents with antioxidative properties have received the most attention. Such compounds range from antioxidant vitamins (E, C, -carotene), to functional mimics of antioxidant enzymes, to a wide variety of low molecular weight compounds with antioxidant activity (e.g., ␣-lipoic acid, N-acetylcysteine). A second approach to development of anti-cataract agents is based on the hypothesis that phase separation phenomena are integral to cataract development [4]. Phase separation results from noncovalent attractive interactions between proteins in concentrated solutions, creating protein-rich and protein-poor regions [5]. In the lens, formation of such domains creates light scattering, leading to cataract. After extensive study in vitro, two putative phase separation inhibitors, pantethine and the radioprotective phosphorothioate WR77913 have been tested in several acute animal models of cataract [6]. Although the specific mechanisms remain uncertain, delays in the onset of cataract and in its severity were reported. The studies reported here were based on the underlying premise that whereas human aging-related cataract may result from a variety of initiating factors, there are certain common pathways of cellular damage that may always be central to the progression of the disease process. Therapeutic agents aimed at inhibiting such common pathways should be effective in slowing or stopping disease progression. Even a modest delay in the rate of progression of the disease would eliminate the need for surgery in many elderly individuals [7,8]. Previous studies from this laboratory have established that the lens, which is avascular and noninnervated in vivo, can be maintained in a fully viable state in organ culture, that cataracts can be induced in cultured lenses by various chemical or environmental perturbations, and that prevention or inhibition of cataract formation can be observed after addition of appropriate agents to counteract the cataractogenic stresses [9 –11]. On the basis of our studies as well as those from other laboratories, it was decided to use the lens organ culture system to screen potential anti-cataract agents.
MATERIALS AND METHODS
Animals Sprague Dawley rats were obtained from Taconic Farms (Germantown, NY, USA). Animals of either sex were used in the weight range of 76 –100 grams. The animals were handled in adherence to the Guide for the Care and Use of Laboratory Animals (National Academy Press). Rhesus monkey (Macaca mulatta) eyes were obtained from animals of about 2 years of age used in the Vaccine Testing Program, Center for Biologic Evalua-
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tion and Research, U.S. Food and Drug Administration (Bethesda, MD, USA). Monkey eyes enucleated immediately after death were brought to the laboratory and the lenses dissected and placed into culture within 3 h of death. Materials Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl) was obtained from Sigma Chemical Company (St. Louis, MO, USA). Tempol-H, the hydroxylamine reduction product of Tempol was prepared from Tempol by hydrogenation as previously described [12]. Stock solutions of Tempol-H were prepared in culture medium immediately before use. Samples from lens homogenates or culture medium taken for analysis of Tempol/Tempol-H were prepared in 0.5 mM diethylenetriaminepentaacetic acid (DETAPAC) to prevent oxidation and were stored at 4°C. Lens organ culture Dissection of lenses from rat [13] and monkey [11] eyes was as described earlier. Lenses were immediately placed in modified TC-199 medium prepared as described previously [13]. Rat lenses were cultured in 2.0 ml medium in 24 well cluster dishes; monkey lenses in 5.0 ml medium in 12 well cluster dishes. All lenses were allowed to equilibrate in the incubator (37°C, 5% CO2) for 2 h; lenses that were damaged during dissection were identified during this period by measuring protein leakage into the medium as previously described [14] and were discarded before experimental groups were established. After the equilibration period was complete, additions were made to the medium for each lens or the medium was exchanged as required to initiate the experiment. Rat lenses incubated in the presence of H2O2 were subjected to a single bolus of H2O2 producing a final concentration as indicated in the description of each experiment. Exposure of monkey lenses to 30 mM xylose or cellobiose was as previously described [11]. Cultured lenses were photographed using a Nikon eclipse E800 Dissecting microscope. Analyses Lens glutathione levels were determined using Ellman’s Reagent (DTNB) by a previously published method [15]. H2O2 concentration in culture media was measured using a Model 2700 Biochemistry Analyzer (Yellow Springs Instrument Co., Yellow Springs, OH, USA). Studies on the accumulation of 86Rb and 3H-choline by the cultured lens were as previously described [9,10]. Lens to medium concentration ratios were calculated and
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used as a measure of the condition of the lens after experimental treatments. Electron paramagnetic resonance (EPR) was used to quantitate the levels of Tempol. Tempol-H, which is EPR silent, was measured in the same way after oxidation to Tempol by addition of sodium ferricyanide. Analyses were performed on a Varian E4 X-band spectrometer as previously described [12]. Protein oxidation studies Bovine lens water-soluble proteins were prepared by homogenizing a bovine lens in water and removing insoluble material by centrifugation (20,000 ⫻ g, 20 min, 4°C). The supernatant was dialyzed exhaustively against deionized water and lyophilized. The lyophilized protein was stored at ⫺20°C in a tightly closed tube. For exposure to oxidizing systems, aliquots of the lyophilized protein were dissolved in phosphate-buffered saline (PBS) at 2.5 mg/ml. To 0.8 ml of this solution was added 0.2 ml PBS or a total of 0.2 ml PBS containing Tempol or Tempol-H and components of the oxidizing systems. Samples were exposed to oxidizing systems as follows: H2O2 plus FeSO4/EDTA as previously described [16], 20 nM rose Bengal, or 50 M flavin mononucleotide (FMN) plus light from fluorescent “cool white” bulbs as previously described [17]. Aliquots were taken from these experimental samples and appropriate control samples at various times, and were analyzed by SDS-PAGE on 12% Bis-Tris NuPage gels (Invitrogen Corporation, Carlsbad, CA, USA). RESULTS
The initial approach in these studies was to use a cocktail of agents with different modes of action (i.e., antioxidants, phase separation inhibitors, protease inhibitors, etc.) in hopes of achieving broader and stronger anti-cataract activity. After considerable effort it was concluded that this approach did not produce results that could be effectively interpreted because some of the agents being tested had definite negative effects on the cultured lenses either individually or when combined with other agents. Thus, studies were done on individual compounds. Table 1 lists some of the compounds tested, all of which had been reported to have some protective effects with lenses or lens cell cultures, or had activities in other systems that seemed potentially relevant to the lens. Using either H2O2 or xylose as cataractogenic agents for cultured lenses, it was found that most of the compounds had no protective effect as judged by the primary screening criteria of lens transparency and the ability of the lens to accumulate choline and rubidium. Figure 1 shows the results of a typical screening experiment. The data for 3H-choline are shown; the data
Table 1. Compounds Tested for Anti-cataract Activity Tempol (5 mM) Tempol-H (5 mM) Pantethine (5 mM) Carnosine (2 mM) E-64 (0.5 mM) N-acetyl cysteine (1 mM) Mercapto-proprionyl glycine (1 mM)
N-propyl gallate (1 mM) Piperidinol (4 mM) Phenyl-N-tert-butylnitrone (1 mM) Ascorbic Acid (1 mM) Butylated Hydroxytoluene (1 mM) Captopril (2.5 mM) Tiron (2 mM)
Note: The value given for each compound was the maximal concentration tested.
for 86Rb showed the same pattern, although the deficit in accumulation after H2O2 exposure was less as has typically been the case in such analyses. Lenses were exposed in culture to a single bolus of 250 M H2O2, tracer amounts of 3H-choline and86Rb were added after 18 –20 h and the concentration of label inside the lens was determined 4 h later as described in Materials and Methods. Although 250 M H2O2 does not have detectable effect on lens transparency under the conditions used, it does impair the lens’ ability to accumulate these compounds, as has been shown in previous studies [9,10]. This particular experiment includes the two compounds that gave the strongest protective effects in these studies, Tempol-H and mercapto-proprionylglycine (MPG). These agents showed a statistically significant protection of the ability of the incubated lens to accumulate the radiolabeled compounds. Carnosine showed no such effect, a result typical of those found with most of the agents tested. Tempol-H is the reduced form (hydroxylamine) of the nitroxide Tempol, which itself was found to be toxic to the cultured lens at the concentration (4 mM) used in these studies (data not shown). The toxicity of Tempol to the cultured lenses was evidenced by the development of haziness within the first 12 h and, as noted below, by an apparent change in the redox status within the lens. Although exposure of rat lenses to 250 M H2O2 as described above does not cause opacification of the lens, a bolus of 1.0 mM H2O2 produces clouding of the lens within a few hours and within a day, frank opacification and complete loss of ability to accumulate choline above the concentration in the medium (data not shown). Figure 2 shows the effect of Tempol-H (4 mM) when added to the culture medium concomitantly with this higher concentration of H2O2. The lenses in Panel A were incubated in control medium, lenses in Panel B demonstrate the typical opacification after 24 h in the same medium to which a bolus of 1.0 mM H2O2 was added at time zero, and lenses in Panel C were incubated in 1 mM H2O2 with the addition of 4 mM Tempol-H. The protective effect of Tempol-H was not due to removal of H2O2 from the medium. Measured H2O2 concentration in the medium
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Fig. 1. Accumulation of 3H-choline from the medium by cultured rat lenses. All lenses were incubated in 2.0 ml TC-199 with or without a single bolus of 250 M H2O2 for 18 h. A tracer level of 3H-choline was added to all culture wells after 14 h. Lenses were harvested and analyzed as previously described [10]. The medium from each lens was also counted and lens water to medium concentration ratios calculated and reported as mean ⫾ standard deviation. All lenses received 250 M H2O2 except the control group. Carnosine (1 mM), mercaptoproprionyl glycine (MPG, 1 mM) or Tempol-H (4 mM) was added to individual groups as indicated at the same time the H2O2 was added.
was not different between Groups B and C (data not shown), consistent with the work of others demonstrating that Tempol/Tempol-H does not react with H2O2 [18].
Although Fig. 2 demonstrates that Tempol-H strongly inhibits opacification even in the presence of 1 mM H2O2, these lenses will opacify upon longer incubation.
Fig. 2. Appearance of rat lenses after 24 h exposure to a single bolus of 1 mM H2O2 with or without addition of 4 mM Tempol-H. (A) Control lenses not exposed to H2O2. (B) Lenses exposed to H2O2. (C) Lenses exposed to H2O2 with the addition of 4 mM Tempol-H.
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Fig. 3. Concentration of glutathione in lenses after 18 h incubation with 1.5 mM H2O2 with or without addition of Tempol or Tempol-H. Glutathione in the H2O2-exposed lenses was reduced by about 33%, a loss largely prevented by addition of 4 mM Tempol-H.
It should be noted that 1 mM is well above any reported H2O2 concentrations to which the human lens is exposed in vivo. The reduced glutathione (GSH) content of the lens is quite high and in virtually all cataracts drops precipitously. To test the ability of Tempol-H to inhibit GSH loss in stressed lenses, we incubated rat lenses with a single bolus of 1.5 mM H2O2 in the presence or absence of 4 mM Tempol-H. As seen in Fig. 3, Tempol-H prevented most of the loss of GSH seen in the H2O2exposed lenses after 18 h incubation. In contrast, Tempol at the nontoxic levels shown had no apparent benefit, but did not enhance GSH loss in H2O2-treated lenses. These results are a single “snapshot” of the effect of Tempol-H on GSH levels; we did not attempt to characterize effects at the early stage of oxidative stress when GSH has been shown to drop precipitously before recovering somewhat [19]. In order to better characterize the apparent antioxidative activity of Tempol-H in the organ culture studies, we tested its ability to prevent oxidative damage to solutions of lens proteins exposed to systems generating reactive oxygen species. Figure 4 shows the results of a representative experiment in which solutions of bovine crystallins were exposed to visible light and FMN as previously described [17]. Oxidative damage to the proteins caused aggregation as is evident in Lane 2. Addition of either Tempol (Lane 3) or Tempol-H (Lane 4) at 4 mM final concentration was equally effective in preventing
the oxidative damage resulting from free radical species generated directly or indirectly by this system. Although the rat lens has been widely used in organ culture studies and has provided considerable insight into the biology of the normal lens and cataract development, it is also understood that rodent lenses differ in important ways from the human lens. The availability of a limited number of lenses from freshly euthanized rhesus monkeys (Macaca mulatta) used in the vaccine testing program of the Center for Biologics Evaluation and Research, FDA allowed for the testing of Tempol-H in a primate lens cataract system. Sugar-induced cataract, which has been studied extensively and has been investigated in the rhesus monkey lens organ culture system in this laboratory [11,20], was used because presumably opacification resulted from a different mechanism than that induced by H2O2. Lenses were cultured in medium containing 30 mM xylose or 30 mM cellobiose (a nonmetabolized sugar used as control) with or without the addition of Tempol-H. Lenses exposed to xylose alone developed a slight haziness after 3– 4 d and became completely opaque after about 2 weeks in culture [11]. Figure 5 shows photographs of representative lenses after 9 d of culture. The control lens in the medium containing cellobiose is completely clear, whereas the xylose-exposed lens shows extensive opacification, particularly in the superficial cortical region. Addition of Tempol-H to the culture medium with the xylose largely prevented this opacification. Lenses exposed to Tem-
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Fig. 4. Effect of photochemical oxidation on solutions of bovine lens crystallins. Exposure for 5 h to flavin mononucleotide (FMN) plus light caused extensive protein cross-linking as seen in Lane 2. Lane 1 contained FMN, but was protected from light. In Lanes 3 and 4 the samples were as in Lane 2 with the addition of 10mM Tempol-H or 10 mM Tempol, respectively.
pol-H (1 or 4 mM) in the absence of xylose remained completely clear (not shown).
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As a biochemical correlate of opacification, the cumulative leakage of protein from cultured lenses was monitored daily for 15 d by analyzing an aliquot of the medium each day. Figure 6 shows data for individual lenses in 4 groups: control (cellobiose), xylose (30 mM) and xylose plus two different concentrations of Tempol-H. The control lenses had no detectable protein leakage during this period of time. For the xylose-exposed lenses, leakage of protein became detectable at about 1 week in culture and increased dramatically thereafter, reaching a total of about 4 mg by 15 d of culture. Supplementation of the medium with Tempol-H (0.2 mM) seemed to reduce leakage, although statistical analysis could not be done because of the limited number of lenses available. However, 1.0 mM Tempol-H had a very strong protective effect against xylose-induced protein leakage, consistent with its inhibition of opacification. Because Tempol-H afforded strong protection to lenses exposed in vitro to cataractogenic stresses, the uptake of both Tempol-H and Tempol by the cultured rat lens was investigated. Lenses were cultured as above in 4 mM Tempol or Tempol-H for 1, 2, 4, or 7 h and the concentration in the lens water (62.5% of wet weight) determined by EPR spectroscopy. Tempol, which is a stable free radical, could be measured directly; Tempol-H was measured as Tempol after oxidation with ferricyanide [20]. Figure 7 demonstrates that Tempol enters the lens more readily than does Tempol-H, a finding consistent with its more hydrophobic character. No Tempol was detected in lenses incubated in Tempol-H. Tempol was rapidly and efficiently reduced to Tempol-H after entering the lens, as demonstrated by the fact that no Tempol was detected in lenses incubated in 4 mM Tempol for 1 or 2 h. There was a small amount of Tempol (⬃ 5% of total) in most lenses incubated 4 h or longer in 4 mM Tempol, which may be an indication of the toxicity that becomes evident from loss of transpar-
Fig. 5. Appearance of representative rhesus monkey lenses after 9 d in culture in medium containing 30 mM xylose or 30 mM cellobiose, a nonmetabolized sugar used as control. Addition of 1 or 4 mM Tempol-H to the media of xylose-exposed lenses strongly inhibited opacification.
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Fig. 6. Cumulative leakage of protein from rhesus monkey lenses incubated in 30 mM sugars. Each curve represents a single lens, except for the lenses incubated in cellobiose where all 3 lenses had no detectable protein leakage.
ency with longer times in culture. Analysis of samples from lenses dissected into capsule/epithelium, cortex, and nucleus after 4 h culture indicated that the Tempol-H
present after incubation in either Tempol or Tempol-H was distributed relatively evenly throughout the entire lens and that in the 4 h Tempol-incubated lenses the small amount of Tempol detected in the lens was present primarily in the epithelium (data not shown). DISCUSSION
Fig. 7. Concentration of total Tempol plus Tempol-H in rat lenses cultured in medium containing either 4 mM Tempol (diamond symbols) or 4 mM Tempol-H (square symbols). Groups of lenses were harvested after 1, 2, 4, and 7 h of culture and the concentration of Tempol ⫹ Tempol-H in the lens water determined by EPR spectroscopy. Lenses incubated in Tempol-H were found to contain no Tempol. Lenses incubated in Tempol for 1 or 2 h also had no detectable Tempol, indicating rapid reduction to the Tempol-H form. In lenses incubated in Tempol for 4 or 7 h Tempol-H remained the dominant form, but a small amount of Tempol (⬃ 5% of total) was detected. Data are reported as mean concentrations (total Tempol ⫹ Tempol-H) ⫾ SD.
A major impediment to progress in developing a safe and effective medical therapy for aging-related cataract has been the lack of a convenient animal model that adequately reflects the human disease. We elected to take an in vitro approach in which lenses in organ culture were used instead of animal models for the initial screening of potential anti-cataract agents. A wide variety of compounds were screened individually and in various combinations. The agent that gave the strongest and most consistent protection was the hydroxylamine of the nitroxide Tempol. Nitroxides are stable free radicals that have long been used as spin labels in EPR spectroscopy and as contrast agents in NMR imaging [21]. More recently they have been investigated because of their antioxidant properties. Tempol has been shown to be an effective radioprotective agent, an activity attributed to its ability to break radical chain reactions by reacting with free radical species, its activity as a superoxide dismutase mimic, and its ability to oxidize transition metals and thereby inhibit Fenton chemistry [21]. In one
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study Tempol injected intracamerally was shown to protect against DNA strand breaks and cataract in rabbits exposed to x-rays [22]. Thus, Tempol is capable of inhibiting many of the processes implicated in generating the oxidative stress thought to be central to cataractogenesis. Tempol is highly cell permeable and has been shown to penetrate the blood/brain and blood/aqueous barriers. However, Tempol in this study was found to exert toxic effects on lenses in organ culture. Although the reduced form (hydroxylamine) of Tempol had generally been found to have little, if any, radioprotective effects in vitro [23], it has been found to have antioxidant effects in cells exposed to H2O2 [24]. Lens epithelial cell cultures exposed to H2O2 have been shown to be protected by addition of Tempol to the medium [18]. Tempol-H was tried in the organ culture system because the hydroxylamines were known to have less toxicity than the nitroxides and because re-dox cycling between the two states can readily occur in living systems [24]. In the lens, which is a highly reducing environment, we demonstrated that Tempol is very rapidly reduced to Tempol-H, with the equilibrium so strongly favoring the hydroxylamine that the nitroxide is generally undetectable by EPR. Under conditions of oxidative stress, the redox status within affected cells would shift to a more oxidizing state and the Tempol/Tempol-H ratio would increase. In our hands, Tempol was detected in cultured lenses only after several hours of exposure to toxic levels of Tempol or (data not shown) in lenses exposed in culture to 1 mM H2O2. Our data support the hypothesis that Tempol-H acts through an antioxidant mechanism. Tempol-H ameliorated the effects of H2O2 on cultured lenses in terms of protection from loss of transparency as well as from decrements in crucial biochemical parameters including glutathione concentration and membrane transport capacity. With isolated lens crystallins, Tempol-H was highly effective in preventing oxidative damage resulting from light and riboflavin-mediated generation of free radical species. In contrast, oxidative damage to proteins caused by exposure to rose bengal and light (data not shown), a Type II photochemical system in which damage is caused by singlet oxygen rather than free radicals [17] was not affected by addition of Tempol-H or Tempol. These findings suggest that scavenging free radicals and/or preventing their formation is key to the protective effects of Tempol-H. As shown in Fig. 4, the effects of Tempol in these studies were indistinguishable from those of Tempol-H, presumably because of the redox cycling between the two forms. The comparable effects of Tempol and Tempol-H in this test tube system are not reflected in the organ culture studies where Tempol-H is protective and Tempol exhibits toxicity. These disparate effects are observed in
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spite of the data demonstrating that the lens very rapidly converts Tempol to Tempol-H. Although the mechanism of this toxicity is unknown, it likely relates either to the high external concentration of Tempol in the Tempolexposed organ cultures, or to effects produced very quickly after the entry of Tempol into the lens before it is reduced to Tempol-H. These findings are similar in some ways to studies by Hahn et al. [12,23] comparing Tempol and Tempol-H as radioprotectors. In studies on cell cultures, Tempol-H exhibited no radioprotective activity, whereas Tempol was quite effective [23]; however, both Tempol and Tempol-H functioned as antioxidants in cell cultures exposed to H2O2 [24]. The failure of Tempol-H to protect the cell cultures from ionizing radiation was attributed to the fact that Tempol-H was not appreciably oxidized to Tempol in that system. In contrast, an in vivo study reported Tempol and Tempol-H to be equally effective as radioprotective agents [12]. This result is consistent with the fact that circulating levels of Tempol were similar within 10 min in mice injected with either agent. As in our lens organ cultures, much greater toxicity was observed in the Tempol-injected mice as a result of the transient spike in Tempol concentration during the first few minutes after injection [12]. Recent studies have confirmed that nitroxides and hydroxylamines are equally effective in protecting against free radicals generated via Fenton chemistry, whereas only nitroxides are effective against radiation damage [24]. Rodent lenses do differ in important ways from primate lenses and behave somewhat differently in culture [10]. To test the efficacy of Tempol-H in preventing cataractous change in a primate lens, we used xyloseinduced cataract in the cultured rhesus monkey lens. Sugar-induced cataract has been extensively studied in various species, both in vivo and in vitro, and has been shown to be initiated by the reduction of sugars to their polyols by the enzyme aldose reductase [25]. In the cultured monkey lens the cataract is less severe than in the rat lens, in part because the concentration of aldose reductase is only about 10% of that present in rat lens [20]. Tempol-H was quite effective in inhibiting opacification of the monkey lenses exposed to xylose. We believe that the protective effect reflects a significant oxidative component in the etiology of sugar cataract in the monkey. In the rat lens where the osmotic effect resulting from the high concentration of aldose reductase is so great, the oxidative component in cataract formation is minor. Tempol-H was not effective in preventing opacification of rat lenses exposed to 30 mM xylose in organ culture. The protective effect of Tempol-H in the monkey lens system is important, not only because it is a primate lens, but also because the cataractogenic process is initiated by a completely different mechanism
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than in the H2O2 cataract. It should be noted that in preliminary studies, it was found that Tempol-H also seemed to have protective effects against H2O2-induced stress in monkey lenses (data not shown). These studies were limited due to lack of available lenses. Because monkey lenses are much more resistant to oxidative stress than rat lenses [10], 2 mM H2O2 was used instead of 250 M used with the rat lenses. The monkey lens results support the concept that inhibiting pathways common to the development of cataracts irrespective of the factors initiating cataractogenesis is a viable approach to medical therapy for the disease. In conclusion, our data suggest that Tempol-H, with its strong antioxidant activity and capacity for redox cycling, is a promising candidate for development as an anti-cataract agent. Based on previous studies in animals [12] it does not seem likely that toxicity should be a problem. Development of mechanisms for topical application to the eye and subsequent testing for efficacy in preventing cataract in vivo are needed.
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[11] Kamiya, T.; Zigler, J. S. Jr. Long-term maintenance of monkey lenses in organ culture: a potential model system for the study of human cataractogenesis. Exp. Eye Res. 63:425– 431; 1996. [12] Hahn, S. M.; Krishna, M. C.; DeLuca, A. M.; Coffin, D.; Mitchell, J. B. Evaluation of the hydroxylamine Tempol-H as an in vivo radioprotector. Free Radic. Biol. Med. 28:953–958; 2000. [13] Zigler, J. S. Jr.; Hess, H. H. Cataracts in the Royal College of Surgeons rat: evidence for initiation by lipid peroxidation products. Exp. Eye Res. 41:67–76; 1985. [14] Tumminia, S. J.; Qin, C.; Zigler, J. S. Jr.; Russell, P. The integrity of mammalian lenses in organ culture. Exp. Eye Res. 58:367–374; 1994. [15] Lou, M. F.; Huang, Q. L.; Zigler, J. S. Jr. Effect of opacification and pigmentation on human lens protein thiol/disulfide and solubility. Curr. Eye Res. 8:883– 890; 1989. [16] Zigler, J. S. Jr.; Huang, Q. L.; Du, X. Oxidative modification of lens crystallins by H2O2 and chelated iron. Free Radic. Biol. Med. 7:499 –505; 1989. [17] Jernigan, H. M. Jr.; Fukui, H. N.; Goosey, J. D.; Kinoshita, J. H. Photodynamic effects of rose bengal or riboflavin on carriermediated transport systems in rat lens. Exp. Eye Res. 32:461– 466; 1981. [18] Reddan, J. R.; Sevilla, M. D.; Giblin, F. J.; Padgaonkar, V.; Dziedzic, D. C.; Leverenz, V.; Misra, I. C.; Peters, J. L. The superoxide dismutase mimic TEMPOL protects cultured rabbit lens epithelial cells from hydrogen peroxide insult. Exp. Eye Res. 56:543–554; 1993. [19] Spector, A.; Wang, G. M.; Wang, R. R.; Li, W. C.; Kleiman, N. J. A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of Action. Exp. Eye Res. 60:483– 493; 1995. [20] Jernigan, H. M. Jr.; Zigler, J. S. Jr.; Liu, Y.; Blum, P. S.; Merola, L. O.; Stimbert, C. D. Effects of xylose on monkey lenses in organ culture: a model for study of sugar cataracts in a primate. Exp. Eye Res. 67:61–71; 1998. [21] Mitchell, J. B.; DeGraff, W.; Kaufman, D.; Krishna, M. C.; Samuni, A.; Finkelstein, E.; Ahn, M. S.; Hahn, S. M.; Gamson, J.; Russo, A. Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol. Arch. Biochem. Biophys. 289:62–70; 1991. [22] Sasaki, H.; Lin, L. R.; Yokoyama, T.; Sevilla, M. D.; Reddy, V. N.; Giblin, F. J. TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit. Invest Ophthalmol. Vis. Sci. 39:544 –552; 1998. [23] Hahn, S. M.; Tochner, Z.; Krishna, C. M.; Glass, J.; Wilson, L.; Samuni, A.; Sprague, M.; Venzon, D.; Glatstein, E.; Mitchell, J. B.; Russo, A. Tempol, a stable free radical, is a novel murine radiation protector. Cancer Res. 52:1750 –1753; 1992. [24] Xavier, S.; Yamada, K.; Samuni, A. M.; Samuni, A.; DeGraff, W.; Krishna, C. M.; Mitchell, J. B. Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage. Biochim. Biophys. Acta 1573: 109 –120; 2002. [25] Kinoshita, J. H.; Kador, P.; Datiles, M. Aldose reductase in diabetic cataracts. JAMA 246:257–261; 1981.