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| ORIGINAL ARTICLE |
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| Year : 1999 | Volume
: 47
| Issue : 1 | Page : 25-29 |
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Influence of UV-irradiation on enzymes in mouse ocular lens: in vitro studies
Nayan K Jain, UM Rawal
Department of Zoology, School of Sciences, Gujarat University, Ahmedabad, India
Correspondence Address:
Nayan K Jain
Dept. of Zoology, School of Sciences, Gujarat University, Ahmedabad
India
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 16130281 
Purpose: In vitro study of the enzymes involved in aerobic, anaerobic and hexose monophosphate shunt in ultraviolet radiation exposed mice lenses.
Method: Of the selected enzymes, lactic dehydrogenase (LDH) was representative of anaerobic glucose oxidation, succinic dehydrogenase (SDH) of the aerobic oxidation, and Glucose-6-phosphate dehydrogenase (G-6-PDH) of the Hexose Monophosphate (HMP) shunt. Other enzymes studied were ATPase and glutathione reductase (GR).
Results: Experiments with mice lenses in vitro showed that transparent lens became opaque following UV-irradiation at 360 nm. Opacification of the lens was accompanied by a change in enzyme activities for energy metabolism.
Conclusion: These changes were progressive in a manner analogous to sequential morphological changes, which would be crucial in maintaining lens transparency. Keywords: UV-radiation, ocular lens, enzyme activity, lens opacity, mouse.
How to cite this article:
Jain NK, Rawal U M. Influence of UV-irradiation on enzymes in mouse ocular lens: in vitro studies. Indian J Ophthalmol 1999;47:25-9 |
The metabolism of the ocular lens would appear to be sluggish in comparison with many other tissues as it is avascular and lies in a fluid with an oxygen-carrying capacity far below that of blood. This is substantiated by the low rate of consumption of oxygen and the utilization of glucose.[1] The ocular lens is almost completely made up of lens fibres devoid of intracellular organelles except in the single anterior layer of epithelial cells, and is placed in a closed cavity.[2],[3] In general, repair mechanisms in the ocular lens readily come into play, but the greater portion of the lens, particularly the nucleus, is entirely dependent on anaerobic glycolysis for metabolism.[4]
The lens can be regarded as an accumulating site of oxidative insults from radiant energy including photo-oxidation by UV-radiation. Unlike the cornea and retina, one can predict that the lens could undergo damage even at low levels of irradiation. This damage to the lens leading to opacification by UV radiation is supported by many researchers and a few medical research laboratories.[5][6][7][8] The normal utilization of oxygen in the lens is maintained by glutathione. How it acts, however, is not known. Nicotinamide adenine dinucleotide phosphate (NADP) is involved in the hexose-monophosphate (HMP) shunt in the breakdown of glucose; the NADPH2 formed then is possibly re-oxidized by glutathione reductase (GR).[9],[10]
In the present investigation, the main objective was to study some enzymes involved in anaerobic glucose oxidation (Lactate dehydrogenase-LDH, with its substrate lactate), in aerobic oxidation (succinate dehydrogenase-SDH) and in the HMP shunt (Glucose-6-Phosphate dehydrogenase-G-6-PDH), in UV-exposed mice lens in vitro. Other enzymes, ATPase and GR (with its product glutathione-GSH), were also analysed.
| Materials and Methods | |  |
Ocular lenses from 3-week-old albino mice (Mus musculus), swiss strain, without any eye anomalies were carefully enucleated and cultured in culture cluster plates with culture medium TC-199 (Himedia) containing 10% fetal calf serum and the necessary bio-compounds (such as glucose) at 37°C in a humidified environment of 5% CO2 in a B.O.D. incubator so that the temperature raised by UV lamps was regulated to 37°C by the cooling system of the incubator. Penicillin-streptomycin (100 units/ml) was used to inhibit bacterial growth. The culture medium was changed on alternate days.[11] The cultured lenses were exposed to UV-A radiation by an ultraviolet source (125W Black Lamp, Phillips, which was placed inside the incubator) for 24 hours, 48 hours, and 72 hours. The control-cultured lenses were maintained in total darkness, in the same ideal conditions in a B.O.D. incubator. The control-cultured and UV-exposed lenses were periodically examined by stereomicroscope and trypan blue dye to observe morphological changes and viability.
The activity of the enzymes LDH (E.C.I.1.1.27), SDH (E.C.1.3.99.1), ATPase (E.C.3.6.1.4.), G-6-PDH (E.C.1.1.1.49), GR (E.C.1.6.4.2) and the level of Lactic acid, GSH were analysed. (The numbers in parentheses refer to the Enzyme Commission for scientific classification -IU 5-of enzymes.)
| Lactic Acid and LDH | | |
Lactic acid was estimated by the modified method of Hullin and Noble.[12] The deproteinized lens supernatant was treated with 20% copper sulphate solution and 1 gm calcium hydroxide, followed by three successive centrifugations. The final supernatant was mixed with 0.05 ml of 12% copper sulphate solution in each tube, which was immersed in an ice bath, followed by 6 ml of concentrated sulphuric acid. The tubes were vigorously agitated, stoppered and heated for 30 minutes in water bath at 60° C. Subsequently 0.1 ml of 1.5% p-hydroxydi-phenyl reagent (in 0.5% NaOH) was added in each pre-cooled assay tube. The tubes were further incubated for 20 minutes at 30°C followed by 90 seconds in a boiling water bath. The optical density of the developed violet coloured assay solution was read at 560 nm after cooling.
The activity of LDH was determined by the modified method of Bergmeyer, Bernt and Hess.[13] The aqueous lens supernatant after 3000 rpm centrifugation for 20 minutes, was mixed with 2.85 ml phosphate pyruvate solution (0.05M phosphate buffer pH 7.5; 3.1×10-4 M Na pyruvate) and 0.05 ml reduced nicotinamide adenine dinucleotide (8×l0-3M NADH - 1 mg/1.5 ml of phosphate pyruvate solution). The optical density difference of the reaction mixture was read immediately after mixing at 1-minute intervals for 5 minutes at 366 nm.
| SDH and ATPase | | |
The activity of SDH was determined by the method of Beatly et al.[14] The assay mixture contained 1 ml of 0.1 M sodium succinate, 1 ml 0.2 M phosphate buffer pH 7.5, 1 ml of INT (0.1% Tetrazolium Salt-INT) solution and 0.4 ml of lens aqueous homogenate. After thorough mixing, all the tubes were incubated at 37°C for 1 hour, and the reaction was stopped by adding 0.1 ml of 30% TCA (trichloro acetic acid) in assay mixture. Ethyl acetate, 7 ml was added to each tube and this was centrifuged for 4 minutes after thorough mixing to extract the colour. The optical density was read at 420 nm.
The activity of ATPase was assayed by the method of Quinn and White.[15] The assay mixture contained 0.3 ml of 3 mM substrate solution (ATP, Na2 8.267 mg/5ml of Tris-HCl buffer 0.05 M pH 7.4), 0.1 ml of 150 mM NaCl, 0.1 ml of 30 mM KC1,0.1 ml of 3 mM MgCl, 0.2 ml of 0.05 M Tris-buffered sucrose solution (85.5 mg sucrose/ml of 0.05 M Tris-HCl buffer pH 7.4), and 0.1 ml of aqueous lens homogenate. The assay mixture was incubated at 30°C for 30 minutes with constant shaking. The reaction was stopped by adding 0.2 ml of 10% cold TCA solution, and kept for 10 minutes at 4°C for complete precipitation. The supernatant, after centrifugation, was used to estimate released inorganic phosphate by the method of Fiske and Subba Row.[16] 0.1 ml of this supernatant was mixed with 0.2 ml of 2.5% acidic ammonium molybdate, 0.7 ml of glass distilled water and 0.1 ml of reducing agent ANSA (0.25%, l-amino-2-naphthol, 4-sulphonic acid, 1.25% sodium sulphite and 1.5 mg% sodium bisulphite). This was mixed thoroughly and allowed to develop colour for 10 minutes at room temperature. The optical density was read at 620 nm.
| Gsh and gr | | |
GSH was determined by the modified Sedlak and Linsay method[17] using Ellman's reagent. The 2.5 ml of 0.02 M EDTA lens homogenate was mixed with 2.0 ml of glass distilled water and 0.5 ml of 50% TCA. The mixture was shaken intermittently for 15 minutes and then centrifuged for 15 minutes at 3000 g. The supernatant of this centrifugation was mixed with double volume of 0.4 M. Tris buffer pH 8.9 (in 0.02 M EDTA) and 0.1 ml of 0.01 M DTNB (5-5' dithiobis 2-nitrobenzoic acid). The optical density was read at 412 nm within 5 minutes of the addition of DTNB.
The activity of GR (NADPH2 specific) was analysed by the method of Horn.[18] The lens was homogenized in 0.067 M phosphate buffer pH 6.6 and centrifuged at 9000 g for 15 minutes at 0°C. The assay mixture, containing 2.0 ml of 0.067 M phosphate buffer pH 6.6, 0.2 ml of 6×l0-3 M NADPH2 (6 mg /10 ml of 1% NaHCO3), 0.2 ml of 7.5×l0-3 M oxidized glutathione pH 6.6, and 0.6 ml of supernatant of lens homogenate was pipetted successively in a test cuvette and mixed thoroughly. The optical density of this assay mixture at 366 nm was read at 0, 1-, 2- and 3-minute intervals. The differences of the average values of optical density were used for calculation.
| G-6-PDH | | |
The activity of G-6-PDH was analysed by the method of Lohr and Waller.[19] The lens was homogenized in 0.9% physiological saline with 6.6×10-4 M EDTA at 0-4°C and then centrifuged for 20 minutes at 15000 rpm at 0°C. 2.4 ml of 0.05 M Triethanolamine buffer pH 7.5 (with 0.2% EDTA sodium salt), 0.5 ml of the above supernatant and 0.05 ml of 3×l0-2 M NADP (in 1% NaHCO3) were mixed and allowed to stand for 5 minutes at 25°C. Then 0.05 ml of 4×10-2 M glucose-6-phosphate solution was added. The optical density of the test mixture was read at 366 nm.
Blank tubes (substrate-free for enzymes and sample-free for GSH and lactic acid) were run simultaneously and their optical densities were read. The data were analysed statistically using the Student's 't' test, and p values were calculated as shown in the tables.
| Results | | |
Experiments with mice lenses in vitro showed that transparent lens became opaque following UV irradiation at 360 nm. The cultured lens after 24 hours of UV irradiation has shown initial nuclear opacity, while after 48 hours of UV irradiation the sub-capsuled region of the lens also became diffusely opaque. Mature cataract was observed after 72 hours of UV irradiation in cultured mice lens. Unexposed cultured mice lenses, i.e., the control lenses, were clear and transparent.
| Lactic Acid and LDH | | |
The lactic acid output and the activity of LDH in UV-exposed cultured mice lenses and their controls are given in [Table - 1] and their percentage of differences are given in [Table - 3]. It was found that the concentration of lactic acid and the LDH activity in all stages of UV irradiation decreased progressively as the opacity became severe [Tables:1] and [Table - 3].
| SDH and ATPase | | |
A significant decrease in the SDH activity was found associated with the progressive decreased activity of ATPase in all stages of opacity [Table - 1]. There was highly significant loss of SDH activity, 30.5% and ATPase activity, 27.06% in the mature opacity [Table - 3].
| Gsh and gr | | |
A significant loss of glutathione, 29.27% at initial nuclear opacity and highly significant loss, 42.03% in mature cataract was noted [Tables:2] and [Table - 3]. These changes, which are interdependent are associated with reduction in GR activity. A highly significant (p<0.001) decrease was noted in all types of opacity.
| G-6-PDH | | |
The data shown in [Table - 2] demonstrate a decline in the level of G-6-PDH activity with progression of opacity which is dose dependent on UV exposure (showing reduction in the level of G-6-PDH activity in the lens due to UV irradiation). The G-6-PDH activity was noted to reduce significantly, by 12.24% to 29.16% (p<0.001), with progression of opacity [Table - 3].
| Discussion | | |
Alteration in lens metabolism and in biochemical indices were progressive in a manner analogous to sequential morphological changes, which would be crucial in maintaining the transparency. As LDH and G-6-PDH are loosely linked to the tissue, they may be targets for extensive oxidative insults. The progressive decline in the level of lactate in the mice lens during the progression of lens opacity shows a considerable disturbance or inhibition in the anaerobic glycolytic pathway by UV radiation. Iwig et al[20] reported that LDH activity depends on the growth rate of cultivated lens cells. The differentiation of lens epithelial cells into fibre cells is connected with a decrease in LDH activity.[21] The significant decrease in the activity of LDH during the initiation of lens opacification may be due to the involvement of the epithelial cells in the lens opacification brought about by UV radiation.
There is considerable loss of SDH activity in the lens during the initiation and progression of UV-induced lens opacity. SDH is an essential enzyme of citric acid cycle and aerobic energy flow. But the citric acid cycle is not very active in the ocular lens metabolism. Although 3 to 5% of glucose[22] is catabolized within the citric acid cycle, this is of importance only in the lens epithelium.[23] Histological studies[8] in rats reveal that UV radiation causes cellular damage and stratification of the lens epithelial layers. This might be the reason for the disturbances in the citric acid cycle, leading to alterations in the SDH activity. The loss of ATPase activity in the cataractous lenses in turn retards the energy flow and its dependent metabolism. The ATPase enzymes have highly reactive -SH groups. The oxidation of these SH groups by the interaction of UV radiation, could be a possible influence on the interaction of ATPase enzymes.
It is interesting to note that the activity of other sulfhydryl-contaming enzymes like G-6-PDH in the lens were also lowered after UV irradiation. One of the main functions of G-6-PDH is the maintenance of lens protein sulfhydryl groups in the reduced state. Another possible mechanism for inactivation or loss of activity of the enzyme may be the degradation of protein and depletion of reduced glutathione. The oxidized glutathione is reduced by GR in the presence of NADPH[2] produced by HMP shunt.[24],[25] GSH concentration and GR activity decreased significantly with progression of opacity. Similar results for GR were obtained by Roger and Augusteyn[26] in human senile cataract.
GR alteration suggests that with the decreased GR activity reduced glutathione cannot be maintained in the lens, as UV causes the photo-oxidation of GSH, converting it into the oxidised form (GSSG). This may also be due to the interaction of GSH with protein, giving rise to mixed disulphide formation. A considerable body of evidence, however, supports the view that oxidation of sulphur group in the lens protein, i.e., few enzymes is the key step in cataractogenesis.[27]
Significant disturbances in lens metabolism through changes in enzyme activity lead to a disruption in energy flow, which in turn causes the alterations of its conformation. These changes might lead to disturbances in lens transparency and lens biomolecules which became more susceptible to further oxidative insults.
| Acknowledgement | | |
This investigation has been supported by grants from the UGC (264/27911/93-95).
| References | | |
| 1. | Kinoshita JH. Pathway of glucose metabolism in the lens. Invest Ophthalmol 1965;4:619-28.  [ PUBMED] |
| 2. | Bloemendal H. The vertebrate eye lens. Science 1977;197:127-38. [ PUBMED] |
| 3. | Raffery NS. Lens morphology. In: Maisel, editor. The Ocular Lens. New York: Mercel Dekker Inc; 1985. p 1-59. |
| 4. | Kinoshita JH, Merola LO. The utilization of pyruvate and its conversion to glutamate in calf lens. Exp Eye Res 1961;l:53-59. |
| 5. | Zigman S, Yulo T, Schultz J. Cataract induction in mice exposed to near UV-light. Ophthalmol Res 1974;6:259-70. |
| 6. | Lerman S. Radiant Energy and the Eye. New York: Macmillan Publications; 1980. p 115-86. |
| 7. | Jain NK, Rawal UM, Khamar BM. Alteration in the sulhydryl profile of rat lens by UV radiation. Proc 46 AH India Ophthal Conf, Bombay; 1988. p 185-88. |
| 8. | Jain NK, Rawal UM, Khamar BM. Scanning electron microscopy of UV-induced cataract. Proc 50 All India Ophthalmol Conf, New Delhi; 1992. p 197-207. |
| 9. | Kinoshita JH. Annual review: selected topics in ophthalmic biochemistry. Arch Ophthalmol 1964;72:554-71. [ PUBMED] |
| 10. | Van Heningen R, Linklater J. The metabolism of the bovine lens in air and nitrogen. Exp Eye Res 1975;20:393-96. |
| 11. | Tiansheng H, Russell P, Kinoshita JH. In vitro incubation paralleling changes occurring during mouse cataract formation. Exp Eye Res 1982;35:521-33. |
| 12. | Hullin RP, Noble RL. The determination of lactic acid in microgram quantities. Biochem 1953;55:289-91. [ PUBMED] [ FULLTEXT] |
| 13. | Bergmeyer HU, Berht E, Hess B. Determination of lactic dehydrogenase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. New York: Academic Press; 1965. p 736-43. |
| 14. | Beatly CM, Basinger GM, Dully CS, Bocek RM. Comparison of red and white voluntary skeletal muscles of several species of primates. J Histochem Cytochem 1966;14:590-600. |
| 15. | Quinn PJ, White IG. Distribution of adenosine triphosphatase activity in ram and bull spermatozoa. J Reprod Fertil 1968;15:449-52. [ PUBMED] |
| 16. | Fiske CH, Subba Row Y. The colorimetric determination of phosphorus. J Biol Chem 1925;66:375-79. |
| 17. | Sedlak J, Lindsay RH. Estimation of total, protein-bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968;25:192-205. [ PUBMED] |
| 18. | Horn HD. Determination of glutathione reductase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. New York: Academic Press; 1965. p 875-84. |
| 19. | Lohr GW, Waller HD. Determination of Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. New York: Academic Press; 1965. p 744-51. |
| 20. | Iwig M, Glasser D, Hieka H. Reactivation of nucleus containing lens fibre cells to mitotic growth biochemical and immunochemical analysis. Cell Differ 1978;7:159-69. |
| 21. | Iwig M, Glasser D. Investigations on mitotic activity, cell density, and enzyme activities in the bovine eye during cell differentiation. Ophthalmic Res 1972/73;4:328-42. |
| 22. | Hockwin O, Ohrloff C. Ageing of lens metabolism. Ophthalmic Res 1979;11:389-407. |
| 23. | Hockwin O, Korte I. In Nordmann J, Dardanne MU, editors. Biochemistry of the Eye. Basel: S. Karger; 1968. p 216-21. |
| 24. | Reddy VN. Metabolism of Glutathione in the lens. Exp Eye Res 1971;11:310-28. [ PUBMED] |
| 25. | Magaw JM. Glutathione and ocular photobiology. Curr Eye Res 1984;3:83-87. |
| 26. | Roger KM, Augusteyn RC. Glutathione reductase in Normal and cataractous human lenses. Exp Eye Res 1978;27:719-21. |
| 27. | Garner MH, Spector A. Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc Nat Acad Sci 1980;77:1274-77. [ PUBMED] [ FULLTEXT] |
[Table - 1], [Table - 2], [Table - 3]
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