|Molecular Vision 2004;
Received 21 April 2004 | Accepted 1 August 2004 | Published 6 August 2004
UV light increases vitamin C uptake by bovine lens epithelial cells
Alessandro Corti, Silvia Martina Ferrari, Alessandra
Lazzarotti, Antonella Del Corso, Umberto Mura, Alessandro Francesco
Casini, Aldo Paolicchi
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia and Dipartimento di Fisiologia e Biochimica, Universitá di Pisa, Pisa, Italy
Correspondence to: Prof. Aldo Paolicchi, Dipartimento di Patologia Sperimentale BMIE, Universitá di Pisa, via Roma 55, I-56126 Pisa, Italy; Phone: +39 050 2218 533; FAX: +39 050 2218 557; email: email@example.com
Purpose: To establish whether the oxidation of ascorbic acid (AA) in the aqueous humor may contribute to maintain the high concentration of AA of the anterior eye tissues during oxidative stress.
Methods: Primary cultures of bovine lens epithelial cells (BLEC) were incubated in a medium with the concentration of AA, glutathione (GSH), and cysteine (Cys) found in the aqueous humor. The intracellular concentration of AA was measured over time and compared to that of cultures maintained in the same medium but exposed to UV light.
Results: The uptake of extracellular AA by BLEC was 0.09±0.01 nmol/mg protein/min, but rose to 0.53±0.02 nmol/mg protein/min when cell cultures were exposed to a dose of UV-C light (178μW/cm2) capable of oxidizing extracellular AA but not intracellular AA. Under the same conditions, intracellular AA was oxidized when cell monolayers were treated with 1,3-bis(2-Chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase and thioredoxin reductase. The uptake of the oxidized form of AA, dehydroascorbic acid (DHAA), was approximately 7 times quicker than that of AA; the uptake and intracellular reduction of DHAA to AA was inhibited by depleting intracellular GSH. Extracellula glucose inhibited the uptake of DHAA by BLEC, and the extent of this inhibition was dependent on the concentration of extracellular glucose.
Conclusions: During mild oxidative stress, AA contained in aqueous humor may provide tissues of the anterior eye with a source of DHAA which is readily taken up and converted to intracellular AA, while intracellular AA is maintained in the reduced status by intracellular antioxidant systems. Since glucose inhibited DHAA uptake, this protective mechanism could be impaired in diabetes.
Enhanced production of reactive oxygen species (ROS) within the eye lens and consequent oxidative damage to the eye lens proteins is thought to be a major mechanism leading to the onset of cataract, the most frequent cause of blindness .
ROS can be generated endogenously by several enzymatic systems, or derive from environmental sources. Endogenous sources of ROS include mitochondria, peroxisomes, lipoxygenases, NADPH oxidase, and cytochrome P450. Exogenous sources include ionizing radiation, chemotherapeutics, inflammatory cytokines and environmental toxins . In the eye, an additional source of ROS causally related with the development of cataract is represented by ultraviolet light .
The lens, like other organs, is endowed with a complex and efficient system of defense capable of neutralizing oxidative species and to repair, recover, or degrade damaged molecules. These systems include non-enzymatic (glutathione, ascorbic acid, tocopherol, and carotenoids) and enzymatic (superoxide dismutase, glutathione peroxidase, and catalase) antioxidants [1,4]. Ascorbic acid (AA) is present in millimolar concentration in the anterior eye and in the lens, and is thought to act as a filter against diurnal radiation [5-9]. During its antioxidant function, AA is oxidized , and its oxidation product, dehydroascorbic acid (DHAA), which is known for being readily absorbed by the lens [10,11], has been detected in normal aqueous humor and lens, and is thought to be toxic to the lens and to induce cataract formation . Within the lens cells, DHAA is known to be efficiently reduced by means of GSH and NADPH dependent mechanisms. These are responsible of preventing its intracellular accumulation and damaging effects [13-16]. Due to the low concentration of GSH and to the absence DHAA recycling enzymes in the aqueous humor, extracellular AA is more prone to oxidation to DHAA than intracellular AA [17,18].
DHAA is known for being transported by proteins of the GLUT family , which are expressed in the lens, thus oxidation of the AA contained in the aqueous humor might cause the transfer of DHAA to lens epithelium [20,21]. The present study was undertaken to test the hypothesis that the oxidation of extracellular AA to DHAA, rather than increasing the intracellular concentration of DHAA, provides the lens epithelium with a source of AA due to its rapid reduction within lens epithelial cells.
Bovine lenses were obtained from freshly slaughtered animals at a local slaughterhouse (Consorzio Macelli, S. Miniato, Pisa, Italy). Lens capsules were removed and lens capsule explants 2-3 mm wide were placed in 6 well culture dishes, together with 1 ml of RPMI 1640 medium, supplemented with antibiotics (penicillin/streptomycin), glutamine 2 mmol/l, and 10% fetal calf serum. Cellular outgrowths appeared after 4-6 days, were trypsinized and subcultured after 2 weeks. Unless otherwise specified, experiments were done with cells at passage 7-8.
Glutathione reductase and glutathione transferase activities were determined according to Massey and Williams  and Habig et al. , respectively.
Preparation of DHAA
DHAA was prepared immediately before the assay by exposing a solution of AA (50 mmol/l) in water to liquid bromine; excess bromine was removed by bubbling the solution with N2 .
Determination of AA
AA was determined according to Rose and Bode . Samples to be analyzed were extracted with 50 mM H3PO4, centrifuged at 5000x g for 5 min at 4 °C, and then analyzed for their AA content by high pressure liquid chromatography. Aliquots of the supernatants were injected in a C-18 reverse phase column (Resolve, Waters, USA) and determinations were performed by a Beckman Gold HPLC apparatus equipped with electrochemical detector (ESA Coulochem III) with a Model 5011 analytical cell.
Determination of GSH
GSH was analyzed by the enzymic method of Tietze  as modified by Baker et al.  to adapt it to a microtiter plate reader. Glutathione disulfide was determined by the vinylpiridine method [27,28].
Oxidation of intracellular and extracellular AA by exposure to UV light
Cell cultures were incubated at 37 °C in Dulbecco's phosphate buffered solution (PBS), supplemented with AA, GSH, and cysteine at concentrations reproducing those of aqueous humor (1.1 mM, 10 μM and 10 μM, respectively)  and were exposed to a UV source (Osram HNS 5 W/U, emission 178 μW cm2 at 254 nm at a distance of 40 cm from the source according to manufacturer's information). The conditions of exposure were chosen to reproduce an accidental exposure to one of the occupational sources of UV-C (e.g., germicidal lamp, UV transilluminator).
Uptake of AA and of DHAA
cell cultures were incubated at 37 °C in Dulbecco's phosphate buffered solution (PBS) containing, unless otherwise specificed, glucose 1 g/l. In some experiments, glutathione reductase and thioredoxin reductase where inhibited by incubating BLEC with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) 0.3 mmol/l dissolved in ethanol , or intracellular GSH was depleted by pretreating cell monolayers with diethylmaleate 1 mmol/l for 60 min. Intracellular content of AA was assayed at time intervals and expressed as nmol/mg protein.
Protein content of the samples was determined by the Bio-Rad protein assay kit (Bio-Rad, Milan, Italy).
Comparison between different treatments was performed with two way ANOVA. In AA and DHAA uptake experiments, linear regression of the increase of intracellular AA was calculated and the result expressed as slope±standard error.
Culture of epithelial cells
After 3-4 days, cell outgrowth appeared around the original explants. Cell monolayers were trypsinized when reaching 2-3 cm diameter and maintained up to 10 passages. The intracellular content of AA was 3.3±1.4 nmol/mg protein in the explant at plating, and declined to 0.07±0.02 nmol/mg protein at the eighth passage, following a one phase exponential decay curve (half life 7.6, 95%CI -0.4 to 0.3, R2 0.989, Figure 1), this is in line with the absence of AA in the RPMI 1640 medium. At the eighth passage, in BLEC intracellular GSH was found to be 23±7 nmol/mg protein (18±4 nmol/mg protein in the explant); glutathione reductase and glutathione transferase activities were, respectively, 7.5±1.4 and 64.8±16.1 U/g protein (1.0±0.1 and 56.1±2.3 in the original explant, respectively).
Differential effect of UV light on intracellular and extracellular AA
When BLEC cultures were incubated in PBS containing AA, Cys, GSH, (1.1 mM, 10 μM and 10 μM, respectively), in the presence of glucose 1 mg/ml, the uptake of AA was modest (0.09±0.01 nmol/mg protein/min), unless the cultures were exposed to UV light (178 μW/cm2), which raised the uptake to 0.53±0.02 nmol/mg protein/min.
The same dose of UV light did not cause oxidation of intracellular AA to DHAA, unless glutathione reductase and thioredoxin reductase were inhibited by BCNU, thus preventing GSH as well as NADPH dependent DHAA reduction (Figure 2).
Characterization of the uptake of AA and DHAA by BLEC
When cell monolayers were exposed to DHAA in the presence of 1 mM extracellular glucose, a concentration dependent, linear increase of intracellular AA was observed (1.1±0.1 nmol/mg protein/min and 2.1±0.2 nmol/mg protein min, respectively, in the presence of extracellular DHAA 0.25 mmol/l and 0.50 mmol/l, p<0.05 by ANOVA for the significance of each treatment and for the difference between the two treatments).
The increase of intracellular AA was approximately 7 times slower in the presence of the same concentration of extracellular AA (0.26±0.04 nmol/mg protein/min in the presence of extracellular AA 0.25 mmol/l, p<0.05). Upon exposure to extracellular DHAA 0.25 mmol/l, the increase of intracellular AA was concentration-dependently inhibited by extracellular glucose (3.2±0.5 nmol/mg protein/min, 1.6±0.1 nmol/mg protein min, and 0.6±0.03 nmol/mg prorein/min, respectively, in the presence of extracellular glucose 0, 1, and 5 g/l, p<0.05 for the difference between treatments). In the presence of extracellular DHAA, the increase of intracellular AA was inhibited (from 2.1±0.3 nmol/min/mg protein to 1.2±0.1 nmol/min/mg protein, p<0.05) when intracellular GSH was depleted by diethylmaleate from 20.5 nmol/mg protein to 6.0 nmol/mg protein.
The present study shows that a mild oxidative stimulus, corresponding to a short accidental exposure to a UV-C source in an occupational environment, is capable of oxidizing extracellular, but not intracellular AA, and accelerates the transfer of AA from the extracellular to the intracellular compartment via its transient oxidation to DHAA. Considering the relatively large volume of aqueous humor, and the wide contact surface with surrounding tissues, this mechanism might contribute to enhance the content of AA of all the anterior eye tissues in response to oxidative stress. In agreement with Fernando et al.  who found that in human lens epithelial cells the intracellular reduction of DHAA to AA depends mostly on the activity of thioltransferase, experiments with diethylmaleate and BCNU showed that this mechanism was dependent on the intracellular concentration of GSH and on the activity of glutathione reductase. In this respect, although the intracellular concentration of GSH and GSH related enzyme in BLEC underwent adaptation to culture conditions, the content of GSH and the activity of glutathione reductase remained in the same range as the original explant, thus confirming the basic requirement to make the GSH dependent recycling of DHAA operative also in vivo.
Among other antioxidants, AA is particularly concentrated not only in the lens, but in other parts of the eye, including cornea, aqueous humor, tears . AA is thought to act as a scavenger of radical species and of oxidizing radiation, thus its oxidation to DHAA is the consequence of the antioxidant function of this compound within the eye .
In the presence of severe oxidative challenge or defect of intracellular antioxidant enzymes, the accumulation of DHAA within the lens is thought to be a crucial step in the development of oxidative stress related diseases, and in particular of cataract . In the present study, we found that extracellular AA is more prone to oxidation than intracellular AA; since aqueous humor contains an elevated concentration of AA (1.1 mmol/l in the bovine eye) , mild oxidative challenges brought to this fluid, which lacks efficient DHAA reducing systems, are expected to lead to the formation of a relatively large amount of DHAA. In fact, in the experimental conditions adopted here, approximately 20% of extracellular AA was oxidized without causing any oxidation of intracellular AA.
DHAA is transported easily across the plasmamembranes , thus intracellular DHAA reducing systems at the same time are responsible for preventing the oxidation of intracellular AA and for reducing DHAA deriving from the extracellular milieu.
Interestingly, the fact that extracellular glucose, within the range of concentrations that are attained in diabetes, concentration dependently impaired the uptake and intracellular reduction of DHAA suggests an additional mechanism by which diabetes may impair antioxidant lens defense and promote oxidative damage in the eye.
In conclusion, the unique feature of the anterior eye, which has a large extracellular compartment (i.e., aqueous humor) containing a high concentration of AA, but not of GSH or other antioxidants capable of maintaining its reduced status, may represent a specific and short term mechanism for providing the cells of AA trough the uptake of DHAA. While preventing oxidative damage due to the increase of intracellular AA, this is likely to represent a physiological mechanism for enhancing intracellular AA during the exposure to oxidizing agents. Due to the ability of glucose to competitively inhibit the uptake of DHAA, this mechanism might be impaired in diabetes and in conditions, such as aging, in which the decline of intracellular reducing systems based on NADPH and GSH may impair the intracellular reduction of the DHAA originated in the extracellular milieu.
This work was supported by the Italian Ministry of Education, University and Research (COFIN and FIRB grants).
1. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J 1995; 9:1173-82.
2. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 3rd ed. Oxford (UK): Clarendon Press; 1999.
3. McCarty CA, Taylor HR. A review of the epidemiologic evidence linking ultraviolet radiation and cataracts. Dev Ophthalmol 2002; 35:21-31.
4. Giblin FJ. Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther 2000; 16:121-35.
5. Varma SD, Bauer SA, Richards RD. Hexose monophosphate shunt in rat lens: stimulation by vitamin C. Invest Ophthalmol Vis Sci 1987; 28:1164-9.
6. Ringvold A. Aqueous humour and ultraviolet radiation. Acta Ophthalmol (Copenh) 1980; 58:69-82.
7. Blondin J, Baragi V, Schwartz E, Sadowski JA, Taylor A. Delay of UV-induced eye lens protein damage in guinea pigs by dietary ascorbate. J Free Radic Biol Med 1986; 2:275-81.
8. Koskela TK, Reiss GR, Brubaker RF, Ellefson RD. Is the high concentration of ascorbic acid in the eye an adaptation to intense solar irradiation? Invest Ophthalmol Vis Sci 1989; 30:2265-7.
9. Reddy VN, Giblin FJ, Lin LR, Chakrapani B. The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium. Invest Ophthalmol Vis Sci 1998; 39:344-50.
10. Winkler BS, Orselli SM, Rex TS. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic Biol Med 1994; 17:333-49.
11. Kern HL, Zolot SL. Transport of vitamin C in the lens. Curr Eye Res 1987; 6:885-96.
12. Sasaki H, Giblin FJ, Winkler BS, Chakrapani B, Leverenz V, Shu CC. A protective role for glutathione-dependent reduction of dehydroascorbic acid in lens epithelium. Invest Ophthalmol Vis Sci 1995; 36:1804-17.
13. Langham ME, Heald K. Factors affecting the concentration of ascorbic acid in the crystalline lens of cattle and rabbits. Biochem J 1956; 63:52-6.
14. Gloster J. Reaction between dehydroascorbic acid and dialysed lens extract in vitro. Br J Ophthalmol 1956; 40:536-44.
15. Rose C, Devamanoharan PS, Varma SD. Dehydroascorbate reductase activity in bovine lens. Int J Vitam Nutr Res 1995; 65:40-4.
16. Akatsuka I, Bando M, Obazawa H, Oka M, Takehana M, Kobayashi S. NADH-dependent dehydroascorbate reductase in the rabbit lens. Tokai J Exp Clin Med 2001; 26:25-32.
17. Riley MV. The chemistry of the aqueous humor. In: Anderson RE editor. Biochemistry of the eye. San Francisco (1833 Fillmore St., San Francisco 94115): Manuals Program, American Academy of Ophthalmology, 1983. p. 79-95.
18. Richer SP, Rose RC. Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide. Vision Res 1998; 38:2881-8.
19. Rumsey SC, Kwon O, Xu GW, Burant CF, Simpson I, Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J Biol Chem 1997; 272:18982-9.
20. Merriman-Smith BR, Krushinsky A, Kistler J, Donaldson PJ. Expression patterns for glucose transporters GLUT1 and GLUT3 in the normal rat lens and in models of diabetic cataract. Invest Ophthalmol Vis Sci 2003; 44:3458-66.
21. Rose RC, Bode AM. Ocular ascorbate transport and metabolism. Comp Biochem Physiol A 1991; 100:273-85.
22. Massey V, Williams CH Jr. On the reaction mechanism of yeast glutathione reductase. J Biol Chem 1965; 240:4470-80.
23. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249:7130-9.
24. Maellaro E, Del Bello B, Sugherini L, Santucci A, Comporti M, Casini AF. Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. Biochem J 1994; 301:471-6.
25. Rose RC, Bode AM. Analysis of water-soluble antioxidants by high-pressure liquid chromatography. Biochem J 1995; 306:101-5.
26. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969; 27:502-22.
27. Baker MA, Cerniglia GJ, Zaman A. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 1990; 190:360-5.
28. Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980; 106:207-12.
29. Frischer H, Ahmad T. Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU [1,3-bis(chloroethyl)-1-nitrosourea]. J Lab Clin Med 1977; 89:1080-91.
30. Fernando MR, Satake M, Monnier VM, Lou MF. Thioltranferase mediated ascorbate recycling in human lens epithelial cells. Invest Ophthalmol Vis Sci 2004; 45:230-7.
31. Ringvold A, Anderssen E, Kjonniksen I. Distribution of ascorbate in the anterior bovine eye. Invest Ophthalmol Vis Sci 2000; 41:20-3.