Acetyl-L-Carnitine Prevents Selenite-Induced Cataractogenesis in an Experimental Animal Model

Current Eye Research, 32:961–971, 2007Copyright ©c Informa Healthcare USA, Inc.ISSN: 0271-3683 print / 1460-2202 onlineDOI: 10.1080/02713680701673470

Acetyl-L-Carnitine PreventsSelenite-Induced Cataractogenesis in an

Experimental Animal ModelR. Elanchezhian, E. Ramesh,M. Sakthivel, M. Isai,and P. GeraldineDepartment of Animal Science,School of Life Sciences,Bharathidasan University,Tamil Nadu, India

M. Rajamohan,C. Nelson Jesudasan,and P.A. ThomasInstitute of Ophthalmology,Joseph Eye Hospital,Tamil Nadu, India

ABSTRACT Purpose: To investigate whether acetyl-L-carnitine (ALCAR) retardsselenite-induced cataractogenesis in vivo. Methods: On postpartum day 10, groupI pups received intraperitoneal saline and group II and group III pups receivedsubcutaneous sodium selenite; Group III pups also received intraperitoneal AL-CAR once daily on postpartum days 9–14. Both eyes of each pup were examinedup to postpartum day 30. After sacrifice, extricated pup lenses were analyzedfor antioxidant and redox system components. Results: There was dense lentic-ular opacification in all group II pups, minimal opacification in 33% of groupIII pups, and no opacification in 67% of group III and in all group I pups.Group II lenses exhibited significantly lower values of antioxidant and redoxsystem components and higher malondialdehyde concentrations than group Ior group III lenses. Conclusion: ALCAR prevents selenite-induced cataractogen-esis in Wistar rat pups, possibly by inhibiting depletion of antioxidant enzymeand redox system components and inhibiting lipid peroxidation.

KEYWORDS acetyl-L-carnitine; antioxidant enzymes; cataractogenesis; oxidative stress;selenite cataract

INTRODUCTIONCataract blindness is the major cause of preventable blindness worldwide,

especially in the developing countries of Africa and Asia.1 Cataract formationis mainly an age-related phenomenon, although socioeconomic and lifestylefactors may also influence its occurrence. Free radical-induced oxidative stresshas been identified as one of the major triggering factors for senile cataractformation.2 There are several endogenous defense mechanisms which protectthe lens against oxidative damage; these include the antioxidant enzymes cata-lase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), andcomponents of the redox system.3,4

A great deal of research has been performed on the possibility of retard-ing cataractogenesis by administration of antioxidants or by supplementationof the diet with micronutrients possessing antioxidant capabilities.5–8 Clinicaland experimental trials on retardation of cataract have been performed using

Received 14 July 2007Accepted 9 September 2007

Correspondence: P. Geraldine,Department of Animal Science,School of Life Sciences, BharathidasanUniversity, Tiruchirappalli-620 024,Tamil Nadu, India. E-mail:[email protected]

961

antioxidant compounds such as lycopene,9 α-ketoglutarate,10 and Ocimum sanctum;11 in these studies,the putative anti-cataractogenic effects are believed tohave occurred by maintaining normal levels of antioxi-dant enzymes and by preventing alteration of proteinsin the lens.

L-carnitine and its short-chain derivatives are essen-tial cofactors in mitochondrial transport and oxidationof long-chain fatty acids; they also act as scavengers ofoxygen free radicals in mammalian tissues.12 Acetyl-L-carnitine (ALCAR) is a naturally occurring, short- chainderivative of L-carnitine which is synthesized endoge-nously in the human brain, liver, and kidney by the en-zyme acetyl carnitine transferase.13 ALCAR facilitatesthe uptake of acetyl-CoA into the mitochondria dur-ing fatty acid oxidation, enhances acetylcholine produc-tion, and stimulates synthesis of protein and membranephospholipids. ALCAR counteracts oxidative stress byinhibiting the increase in lipid hydroperoxidation.14

ALCAR was recently shown to significantly retardselenite-induced cataractogenesis in an in vitro system.15

In the present study, we sought to investigate whetherALCAR could also retard selenite-induced cataractoge-nesis in an in vivo model. To better comprehend the un-derlying mechanisms responsible for this putative anti-cataractogenic activity, certain key biochemical param-eters of components of the redox system and enzymesof the antioxidant system, as well as markers of lipidperoxidation, were assayed in the lenses of Wistar ratpups that had been exposed to selenite and treated withALCAR.

MATERIALS AND METHODSNine-day-old rat pups (Wistar strain) were used in

this study. The pups were housed with parents in largespacious cages, and the parents were given food andwater ad libitum. The animal room was well-ventilated,and a regular 12:12-h light/dark cycle was maintainedthroughout the experimental period. These animalswere used in accordance with institutional guidelinesand with the Association for Research in Vision andOphthalmology statement for the use of animals in re-search. The rat pups were divided into three groups,each group comprising fifteen pups:

1. Group I, which received only saline (control)2. Group II, which received selenite alone (cataract-

untreated)

3. Group III, which received selenite and ALCAR(cataract-treated).

Each rat pup in groups II and III received a singlesubcutaneous injection of sodium selenite (19 µmol/kgbody weight) on postpartum day 10. In addition, pupsin group III received intraperitoneal injections of AL-CAR (200 mg/kg body weight); the first dose of ALCARwas administered one day prior to the selenite injection,and the ALCAR injection was repeated once daily forfive consecutive days thereafter.

Morphological ExaminationWhen the rat pups first opened their eyes (approxi-

mately 16 days after birth), a slit-lamp biomicroscopicexamination was performed in each eye of each rat toprovide a morphological assessment of any opacifica-tion. Prior to performing the examination, mydriasiswas achieved by using a topical ophthalmic solutioncontaining tropicamide with phenylephrine (MaxdilPlus, Hi-Care Pharma, and Chennai, India); one dropof the solution was instilled every 30 min for 2 hoursinto each eye of each rat, with the animals being keptin a dark room. After 2 hours, the eyes were viewed bya slit-lamp biomicroscope at 12× magnification. At theend of experimental period (postpartum day 30), thedegree of opacification was graded and photographed.The degree of opacification was graded as follows:

0 normal transparent lens+ initial sign of nuclear opacity involving tiny scatters++ partial nuclear opacity+ + + mature nuclear opacity

Quantitative Analysis of Enzymesof the Antioxidant System

Rat pups in all three groups were anesthetized withdiethylether and then sacrificed by cervical dislocationon postpartum day 30 and the lenses excised. Bothlenses of each individual rat were processed together toconstitute a single value. The lenses were homogenizedin 10 times their mass of 50 mM phosphate buffer (pH7.2) and centrifuged at 12,000 rpm for 15 min at 4◦C.The supernatant obtained was used for the analysis ofenzymatic and non-enzymatic parameters. To calculatethe specific enzyme activity, protein in each sample wasestimated by the method of Bradford.16

R. Elanchezhian et al. 962

Catalase (CAT)

CAT activity was determined by the method ofSinha.17 In this test, dichromatic acetic acid is reducedto chromic acetate when heated in the presence of hy-drogen peroxide (H2O2), with the formation of per-chloric acid as an unstable intermediate. In the test, thegreen color developed was read at 590 nm against blankon a spectrophotometer. The activity of catalase was ex-pressed as units/mg protein (one unit was the amountof enzyme that utilized 1 mmol of H2O2/min).

Glutathione Peroxidase (GPx)

The activity of GPx was determined essentially as de-scribed by Rotruck et al.18 The principle of this methodis that the rate of glutathione oxidation by H2O2, ascatalysed by the GPx present in the supernatant, is deter-mined; the color that develops is read against a reagentblank at 412 nm on a spectrophotometer. In the test, theenzyme activity was expressed as units/mg protein (oneunit was the amount of enzyme that converted 1 µmolof GSH to the oxidized form of glutathione [GSSH] inthe presence of H2O2/min).

Superoxide Dismutase (SOD)

SOD activity was determined by the method ofMarklund and Marklund.19 In this test, the degree of in-hibition of pyrogallol auto-oxidation by supernatant ofthe lens homogenate was measured. The change in ab-sorbance was read at 470 nm against blank every minutefor 3 min on a spectrophotometer. The enzyme activitywas expressed as units/mg protein.

Quantitative Analysisof Redox System

Reduced Glutathione (GSH)

The GSH content was estimated by the method ofMoron et al.20 The lens homogenate was centrifugedat 5000 rpm for 15 min at 4◦C. To the resulting super-natant, 0.5 ml of 10% trichloroacetic acid was addedand the mix was re-centrifuged. The resulting protein-free supernatant was allowed to react with 4 ml of 0.3 MNa2HPO4 (pH 8.0) and 0.5 ml of 0.04% (wt/vol) 5,5-dithiobis-2-nitrobenzoic acid. The absorbance of theresulting yellow color was read spectrophotometricallyat 412 nm. A parallel standard was also maintained. Theresults were expressed in µmol/g wet weight.

Glutathione Reductase (GR)

Glutathione reductase (GR), which utilizes NADPHto convert oxidized glutathione to the reduced form,was assayed by the method of Stall et al.21 The changein absorbance was read at 340 for 2 min at intervals of 30seconds in a UV-spectrophotometer. The activity of glu-tathione reductase was expressed as nmol of NADPHoxidized/min/mg protein.

Glutathione-S-Transferase (GST)

The activity of GST was determined by the methodof Habig and Jacoby.22 The conjugation of GSH with1 chloro, 2-4 dinitrobenzene (CDNB), a hydrophilicsubstrate, was observed spectrophotometrically at 340nm to measure the activity of GST; one unit of GST wasdefined as the amount of enzyme required to conjugate1 µmol of CDNB with GSH/min.

Determination of Lipid PeroxidationThe extent of lipid peroxidation was determined by

the method of Ohkawa et al.,23 the principle of thismethod being that malondialdehyde (MDA), an end-product of lipid peroxidation, reacts with thiobarbituricacid (TBA) to form a pink chromogen. For this assay,0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of20% acetic acid (pH 3.5), and 1.5 ml of 0.81% thiobar-bituric acid aqueous solution were added in successionin a reaction tube. To this reaction mixture, 0.2 ml ofthe lens homogenate was added, and the mixture wasthen heated in boiling water for 60 min. After coolingto room temperature, 5 ml of butanol: pyridine (15:1v/v) solution was added. The mixture was then cen-trifuged at 5000 rpm for 15 min following which theupper organic layer was separated, and the intensity ofthe resulting pink color was read at 532 nm. Tetram-ethoxypropane was used as an external standard. Thelevel of lipid peroxides was expressed as nmol of MDAformed/gm wet weight.

Qualitative Analysis of AntioxidantEnzymes Using Native Page

Non-denaturing polyacrylamide gel electrophoresis(native-PAGE) was performed on lens samples essen-tially as described by Laemmli,24 except that SDS wasomitted from all buffers and the samples were notboiled before electrophoresis. The enzymes were runon the basis of equal amounts of protein (70 µg) in a

963 Acetyl-L-Carnitine Prevents Selenite Cataract

10% gel for SOD and GPx. Electrophoretic separationwas performed at 4◦C with a constant power supply of50 V for the stacking gel and 100 V for the separating gel.Staining for the activity of each enzyme was performedseparately as follows:

SOD activity was identified by the method ofBeauchamp and Fridovich.25 The gel was soakedin 50 mM Tris HCl buffer (pH 8) containing 10mg NBT, 1 mg ethylene diamine tetra-acetic acid(EDTA), and 2 mg riboflavin (50 ml final volume)and kept in the dark for 30 min. The gel was thenplaced on an illuminated light box to permit local-ization of the area of SOD activity, which appearedas a clear zone against a violet background.

GPx isozymes were separated by the method of Linet al.26 The gel was first soaked in 50 ml of 50 mMTris HCl buffer (pH 8) containing 200 mg reducedglutathione and 8 µl of 30 % H2O2 for 20 min. Thegel was then transferred to 50 ml of 50 mM Tris HClbuffer (pH 8) containing 25 mg nitroblue tetrazolium(NBT) chloride and 25 mg phenazine methosulphate(PMS). The appearance of white bands in the gelwas interpreted as indicating the presence of GPxisozymes.

Quantification of the isozyme bands for each en-zyme studied was performed by a densitometer (Gs 300transmittance/reflectance scanning densitometer, Hoe-fer Scientific Instruments, San Francisco, CA, USA).The band area was measured in pixels.

Statistical AnalysisQuantitative data were reported as mean ± SD. The

statistical significance of observed differences betweenthe values in the different groups was determined bythe Student’s t-test (unpaired), and the chi-square testwas applied wherever relevant. P < 0.05 was regardedas statistically significant.

RESULTSMorphological Examination

to Determine Lens Opacificationin Rat Pups

Morphological examination of both eyes of each ratpup was done by slit-lamp examination on the 30thday after birth. All 15 rat pups in group II (receivedselenite alone) exhibited dense opacification of the lens

(grade +++). In contrast, only 5 of 15 (33.3%) rat pupsin group III (received selenite and ALCAR) exhibitedlenticular opacification (grade +), with the lenses ofthe other 10 pups appearing normal (grade 0). All 15rat pups in group I (received normal saline; control)exhibited maximum transparency (grade 0) of the lens(Fig. 1, Table 1). The difference between the value ingroup II and group III rats was statistically significant(χ2 [df = 1] = 15; P < 0.01).

Quantitative Analysis of Enzymesof the Antioxidant System in Lenses

of Rat PupsCAT

The mean activity of CAT in lenses of group IIrats was significantly (P < 0.001) lower than that inlenses of group I rats and also significantly (P < 0.001)lower than that in lenses of group III rats (Table 2).The activity of CAT in group III rat lenses was signifi-cantly (P < 0.001) lower than that in group I rat lenses(Table 2).

GPx

The mean activity of GPx in lenses of group II ratswas significantly (P < 0.001) lower than that in lensesof group I rats and that in lenses of group III rats(P < 0.001) (Table 2). The mean activity of GPx ingroup III rat lenses was significantly (P < 0.01) lowerthan that in group I rat lenses (Table 2).

SOD

The mean activity of SOD in lenses of group II ratswas significantly (P < 0.001) lower than that in lensesof group I rats and also significantly (P < 0.001) lowerthan that in lenses of group III rats (Table 2). The meanactivity of SOD in lenses of group III rats was signifi-cantly (P < 0.01) lower than that in lenses of group Irats (Table 2).

Quantitative Analysis of Componentsof the Redox System in Lenses

of Rat PupsGSH

The mean concentration of GSH in lenses of groupII rats was significantly (P < 0.001) lower than that inlenses of group I rats and also significantly (P < 0.001)lower than that in lenses of group III rats (Table 3). The


FIGURE 1 Slit-lamp appearance of lenses in eyes of 16-day-old Wistar rat pups. (A) Lenses in normal (Group I) pup eyeshave all remained transparent (Grade 0 opacification). (B) Lensesin cataract-untreated (Group II) eyes have all become denselyopaque (Grade +++ opacification). (C) Lenses in some cataract-treated (Group III) pup eyes have become mildly opaque (Grade +opacification).

mean concentration of GSH in group III rat lenses wassignificantly (P < 0.01) lower than that in group I ratlenses (Table 3).

GR

The mean activity of GR in lenses of Group II ratswas significantly (P < 0.05) lower than that in lenses ofGroup I rats. However, no significant differences wereobserved between the mean activity of GR in lenses ofgroup I and group III rats or between the mean activityof GR in group II and group III rat lenses (Table 3).

GST

The mean activity of GST in lenses of group II ratswas significantly (P < 0.001) lower than that in lensesof group I rats and also significantly (P < 0.001) lowerthan that in lenses of group III rats. The mean activity ofGST in group III rat lenses was significantly (P < 0.05)lower than that in group I rat lenses (Table 3).

Determination of Lipid PeroxidationThe mean concentration of MDA in lenses of group

II rats was significantly (P < 0.001) greater than that inlenses of group I rats and also significantly (P < 0.001)greater than that in lenses of group III rats. The meanconcentration of MDA in group III rat lenses was sig-nificantly (P < 0.001) greater than that in group I ratlenses (Table 4).

Qualitative Analysis of AntioxidantSystem Enzymes by Native Page

SOD

Two isoforms of SOD (SOD1 and SOD2) weredetected following native-PAGE of lens homogenatesfrom all three groups of rats (Fig. 2). The isoform SOD1appeared as a band of essentially similar staining inten-sity in lenses of group I and group III rats (band areas112.44 and 105.27, respectively), whereas the band ingroup II rat lenses exhibited decreased staining inten-sity (band area 87.51). The staining intensity of the iso-form SOD2 band was also essentially similar in lensesof groups I and III rats (band areas 92.23 and 90.11,respectively); however, the SOD2 isoform was detectedas a band of very minimum intensity (band area 23.32)in lenses of group II rats (Fig. 2).


TABLE 1 Morphological examination of lenses of rat pups

Number of pups withdifferent degrees of

lenticular opacification

Experimental groups No. of pups 0 + ++ + + +

Number of pupsin which lenticular

opacification occurred

Group I (normal) 15 15 — — — 0Group II (cataract-untreated) 15 — — — 15 all 15Group III (cataract-treated) 15 10 5 — — 5 of 15 (33%)

Group I rat pups received only saline; Group II rat pups received only selenite; Group III rat pups received selenite and acetyl-L-carnitine.The degree of opacification was graded as follows: 0 normal transparent lens; + initial sign of nuclear opacity involving tiny scatters; ++ partial nuclear

opacity; +++ mature nuclear opacity.

TABLE 2 Quantitative analysis of antioxidant system enzymes in lenses of rat pups

Enzymes analyzed (unit of activity) Group I (Normal) Group II (cataract-untreated) Group III (cataract-treated)

1. Catalase (µmol H2O2 7.40 ± 0.22 3.86 ± 0.17a,b 6.77 ± 0.08a

consumed/mg protein/min)2. Glutathione peroxidase (µmol 34.17 ± 2.01 24.89 ± 1.16a,b 30.05 ± 1.29a

glutathione oxidized/mg protein/min)3. Superoxide dismutase 2.29 ± 0.26 1.00 ± 0.11a,b 1.71 ± 0.11a

(units/mg protein)

Group I rat pups received only saline; Group II rat pups received only selenite; Group III rat pups received selenite and acetyl-L-carnitine. Each valuerepresents the mean (±SD) of observations made on samples from five animals from the same group. Statistical analysis was performed by the Studentt-test.

aValue significantly different (P < 0.05) from Group I value.bValue significantly different (P < 0.05) from Group III value.

TABLE 3 Quantitative analysis of Redox system components in the lenses of rat pups

Component analyzed (unit of activity) Group I (normal) Group II (cataract-untreated) Group III (cataract-treated)

1. Reduced glutathione 8.36 ± 0.37 5.06 ± 0.61a,b 7.63 ± 0.51a

(µmol/gm tissue)2. Glutathione reductase (nmol of NADPH 0.18 ± 0.035 0.13 ± 0.035a 0.17 ± 0.035

oxidized/min/mg protein)3. Glutathione-S-transferase (µmol of 5.49 ± 0.68 3.02 ± 0.38a,b 4.47 ± 0.41a

CDNB conjugated with GSH/ min)



TABLE 4 Quantitative analysis of malondialdehyde in the lenses of rat pups

Lipid Peroxidation (unit of activity) Group I (normal) Group II (cataract-untreated) Group III (cataract-treated)

Malondialdehyde (nmol/gm wet weight) 60.16 ± 3.33 92.84 ± 3.92a,b 74.98 ± 3.35a




FIGURE 2 Qualitative analysis of superoxide dismutase (SOD)in lenses of Wistar rat pups. L1—Group I (normal); L2—Group II(cataract-untreated); L3—Group III (cataract-treated).

GPx

Three isoforms of GPx (GPx1, GPx2 and GPx3) weredetected in the lenses of group I and group III rats.The isoforms GPx1 (band areas 85.13, 82.35, and 84.05,respectively) and GPx2 (band areas 78.43, 76.21, and77.40, respectively) appeared as bands of essentially sim-ilar staining intensity in the lenses of all three groups ofrats. Similarly, the band corresponding to isoform GPx3was essentially similar in staining intensity in lenses ofrats of groups I and III (band areas 50.80 and 48.12,respectively); however, the GPx3 isoform was not de-tected in lenses of group II rats (Fig. 3).

FIGURE 3 Qualitative analysis of glutathione peroxidase (GPx)in lenses of Wistar rat pups. L1—Group I (normal); L2—Group II(cataract-untreated), L3—Group III (cataract-treated).

DISCUSSIONHuman cataract is currently treated by surgical

means. However, due to the huge cataract backlog andproblems associated with cataract surgery, efforts arebeing made to formulate a medication that will aid inprevention of cataractogenesis and, perhaps, treatmentof established cataract. Thus far, no single compoundhas found widespread acceptance for these indications,although many compounds have been evaluated. Thus,there is a vital need for further research in this area.

Administration of selenite to experimental animalsresults in increased lipid peroxidation in the lens andhydrogen peroxide (H2O2) formation in the aqueoushumor of the eye.27 Selenite also decreases the level ofreduced glutathione (GSH) in the lens,27 possibly due toactive oxygen generation as a result of selenite reactingwith reduced GSH.28 The selenite cataract, which isan extremely rapidly induced and convenient modelof cataractogenesis, shows a number of similarities tothe human senile nuclear cataract, such as formationof vesicles, increased levels of calcium, the presence ofinsoluble protein and decreased water-soluble protein,the occurrence of proteolysis, and diminished amountsof GSH.29 These features may explain the popularity ofthe rodent model of selenite cataract for rapid screeningof possible anticataract agents.

The lens in vitro is highly susceptible to damage byreactive oxygen species (ROS); there is loss of trans-parency, decreased active transport of cations, GSHand ATP, protein insolubilization, and generation oflipid peroxides.30–32 In vivo, however, even when ROSoccur, the appearance of cataracts is delayed due tothe presence of various enzymatic (superoxide dismu-tase [SOD], catalase [CAT] and glutathione peroxi-dase [GPx]) and non-enzymatic (e.g., ascorbic acid)mechanisms30,33 that lessen the deleterious effects ofROS. With advancing age, these antioxidant mecha-nisms tend to decline in efficacy and hence need to bereplenished. Thus, different compounds with knownantioxidant properties have been evaluated for putativeeffects in retarding or preventing experimental selenite-induced cataractogenesis; in this model, nutrients suchas Vitamin C,5 pantethine,34 aqueous extract of greentea,6,8 aqueous extract of black tea,6 an extract of Gingkobiloba,7 and a procyanidin-rich extract of grape seed35

have all been found to prevent the progression ofcataract formation. However, other compounds needto be evaluated as well.


Acetyl-L-carnitine (ALCAR) is a compound that hasbeen found to reverse age-related alterations in fattyacid profiles and declines in metabolic rates and car-diolipin levels.36 ALCAR plays an important role inthe production of energy in the mitochondria by con-trolling the influx of long-chain fatty acids into themitochondria for β-oxidation and by stabilizing thefluidity of cell membranes via regulation of sphin-gomyelin levels; it also provides a reservoir of substratefor production of cellular energy. The antioxidant po-tential of ALCAR has been highlighted by studies show-ing that ALCAR can inhibit xanthine oxidase-induceddamage to human diploid fibroblasts37 and rat skele-tal muscle,38 lipid peroxidation in the periphery of thebrain38,39 and pro-oxidant-induced DNA single-strandbreaks in human peripheral blood lymphocytes;40

ALCAR has also been shown to act as a scavenger ofoxygen free radicals in mammalian tissues.12 With ref-erence to the ocular lens, ALCAR was shown to amelio-rate diabetic cataractogenesis41 and to significantly re-tard selenite-induced cataractogenesis,15 while its parentcompound, L-carnitine, was reported to prevent H2O2-induced lenticular opacification;42 however, these stud-ies were only performed on in vitro systems. In thepresent study, we sought to investigate whether the invitro antioxidant potential of ALCAR translated into ef-ficacy in retarding selenite-induced cataractogenesis inan in vivo model. Toward this goal, certain key biochem-ical parameters of components of the redox system andenzymes of the antioxidant system, as well as markers oflipid peroxidation, were assayed in the lenses of Wistarrat pups that had been exposed to selenite and treatedwith ALCAR.

The slit-lamp observations made in the morpholog-ical phase of the present study appear to suggest thatALCAR is able to significantly retard selenite-inducedcataractogenesis in Wistar rat pups. By the end of thestudy period, all rat pups that had received only selen-ite (Group II) were found to have developed a densenuclear opacity in the lens of each eye, whereas onlyone-third of pups that had received selenite and beentreated with ALCAR were found to have mild lenticu-lar opacification in each eye (Table 1; Fig. 1A, 1B, and1C). These in vivo observations corroborate previouslyreported in vitro findings.15

CAT, GPx, and SOD are important components ofthe innate enzymatic defences of the lens. SOD, achain-breaking antioxidant enzyme that occurs in redblood cells43 and the lenses of different species,44 exists

in two forms: one containing Mn2+, restricted to the mi-tochondria, and a cytosolic form containing Zn2+andCu2+; SOD converts superoxide to H2O2. The enzymeGPx, first demonstrated in the lens by Pirie,45 maintainsthe integrity of the phospholipid bilayer of membranesby putting a brake on the lipid peroxidation initiated bysuperoxide. The CAT enzyme, which has been clearlydemonstrated in the lens,46 is a hemoprotein that re-quires the reduced form of nicotinamide adenine dinu-cleotide phosphate (NADPH) for regeneration to its ac-tive form.47 Both CAT and GPx catalyse the transforma-tion of H2O2 within the cell to harmless by-products,thereby curtailing the quantity of cellular destructioninflicted by products of lipid peroxidation.48

A high concentration of GSH has been found toprotect the lens from oxidative damage and toxicchemicals;49 thus, depletion of GSH seriously affectsGSH-dependent enzymes such as GPx, glutathionereductase (GR), and glutathione S-transferase (GST),and also leucotriene C4 synthetase and the glutare-doxin system, rendering the cells susceptible to a toxicchallenge.50 GR maintains the intracellular level ofGSH by preserving the integrity of cell membranes andby stabilizing the sulfhydryl groups of proteins. Ad-ministration of carnitine and lipoic acid to aged ratshas been found to increase the activity of GR by in-creasing the levels of GSH and the reducing equivalentNADPH.51,52

In the present study, the mean activities of CAT, GPx,and SOD and the mean level of GSH were found tobe significantly lower in the lenses of cataract-untreated(Group II) rats than that in normal control (Group I)rat lenses (Tables 2 and 3). A reduction in the activ-ities of enzymes such as SOD, GPx, CAT, GST, andGR29 has been found to accompany an increase of freeradical species in the aqueous humor and a significantdecrease in NADPH and GSH content in the lens.53 Aprogressive decrease of GSH in the lens was found to beassociated with experimental cataract formation or hu-man senile cataract formation.54,55 A depletion in GSHcontent in post-mitotic tissues has also been found tooccur during aging,56 possibly due to enhanced oxida-tive damage due to free radicals. In the present study,the mean activities of GR and GST in lenses were foundto be lower in Group II rats (exposed to selenite only)than in Group I (normal) rats (Table 3). These loweredactivities were possibly due to depletion of the lenticularGSH pool (observed in Group II rats) that occurred asa consequence of exposure to selenite; such a depletion


of GSH has been reported to occur in selenite-inducedoxidative stress.53,57 In the lenses of Group III rats (ex-posed to selenite but treated with ALCAR), the meanlevel of GSH was found to be increased (relative toGroup II rat lenses), with an accompanying increase inthe mean activities of GR and GST (Table 3).

Lipid peroxidation is considered to be the ba-sic mechanism of cellular damage caused by freeradicals;58,59 during oxidative stress, there is an increasein lipid peroxidation, resulting in membrane damageand a consequent rise in the level of malondialde-hyde (MDA). In the present investigation, the meanlevel of MDA was found to be significantly higher inthe lenses of Group II (cataract-untreated) rats thanthat in Group I (normal) rat lenses, a finding simi-lar to that reported by others;8,9,60 however, in lensesof Group III rats (cataract-treated), the mean level ofMDA was significantly lower than that in Group II ratlenses (Table 4). Thus, in the lenses of rats that had beenexposed to selenite alone (Group II rats), there was asignificant depletion of GSH (Table 3) and increasedmembrane damage (as indicated by the increased levelsof MDA) (Table 4); such changes in GSH and MDAlevels in the presence of selenite have previously beenreported.8 ALCAR appeared to prevent the occurrenceof such changes in Group III rats.

The expression of multiple isoforms of enzymes mayrepresent one of the primary mechanisms regulatingcellular metabolism,61 but little is known about theexpression of isoforms of antioxidant enzymes duringoxidative stress. Hence, we evaluated the patterns ofexpression of isozymes of the SOD and GPx enzymesduring selenite cataractogenesis and following adminis-tration with ALCAR (Figs. 2 and 3). In general, the num-ber and staining intensity of isozyme bands were foundto be greater in lenses of cataract-treated (Group III) andnormal control (Group I) rats than in cataract-untreatedrats (Group II); there was a decreased intensity of stain-ing of the SOD1 isozyme and complete absence ofSOD2 and GPx 3 isozymes in lenses of group II rats,suggesting that the lenses of these rats were particularlyvulnerable to oxidative stress (Figs. 2 and 3). Differentialexpression of enzyme isoforms has been noted in barleyshoot and root exposed to saline stress,61 and possiblyreflects a shift in gene expression.62 In the present inves-tigation, alteration in gene expression of the SOD andGPx enzymes possibly occurred in lenses of Group IIrats following exposure to selenite. However, adminis-tration of ALCAR in Group III rats possibly restored the

pattern of gene expression to normal, so that the lensesof Group III rats and Group I (normal) rats exhibitedessentially similar isozyme patterns of SOD and GPx.Interestingly, supplementation with melatonin duringaging has been proposed as a means of increasing theactivity of the antioxidant defense system (ADS) genewith a view to promoting the synthesis of antioxidantenzymes.63 In the present study, a similar phenomenonpossibly occurred following administration of ALCAR.

In the present investigation, ALCAR possibly pre-vented or retarded oxidative damage to sulphydrylgroups in the lens epithelium (the initial event inselenite cataractogenesis) by direct radical scaveng-ing activity64 or indirectly by stabilizing mitochon-drial membranes and chelating metals; such propertieshave previously been shown to attenuate toxic cation-induced generation of ROS through the electron trans-port chain.65 L-carnitine and its acyl esters (which in-clude ALCAR) are known to be involved in repair ofmembrane phospholipids that are damaged by oxida-tive insult;66 ALCAR can also inhibit oxidant-inducedDNA single-strand breaks.40 These mechanisms proba-bly retarded selenite-induced oxidative damage to lensmembranes in the present study.

In conclusion, administration of ALCAR appearedto prevent cataractogenesis in the lenses of selenite- in-jected rat pups (rats subjected to oxidative stress) byreducing the intensity of lipid peroxidation and byenhancing the activities of antioxidant enzymes andthe functions of the redox system. It remains to beseen whether a similar anti-cataractogenic effect can bedemonstrated in humans.

The relevance of our results to the actual process ofcataract formation in humans requires careful evalua-tion. We used a model of acute induction of selenitecataract and found ALCAR to have anti-cataractogenicpotential; whether a similar protective effect wouldbe obtained in a chronic, low-dose model of selenitecataractogenesis,57 which may more closely resemblethe evolution of age-related human cataract, requiresevaluation. Moreover, experimental selenite cataractsdiffer from actual human cataracts in not exhibitingany high molecular-weight covalent aggregates or in-creased disulfide formation, and in being dominatedby rapid calpain-induced proteolytic precipitation;29

therefore, the results obtained in experimental selen-ite cataractogenesis cannot be automatically extrapo-lated to the evolution of human cataracts. Nonetheless,we believe that the results of the present investigation


strongly suggest that ALCAR is able to prevent ex-perimental selenite-induced cataractogenesis in an ex-perimental animal model, which may ultimately haverelevance to the management of cataract blindness inhumans.

ACKNOWLEDGEMENTSThe authors thank the Department of Biotechnology,

Government of India, for the financial assistance pro-vided. Instrumentation facility provided by DST-FISTis also acknowledged.

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Acetyl-L-Carnitine Prevents Selenite-Induced Cataractogenesis in an Experimental Animal Model