Exp. Eye Res. (1991)

Polyol

52. 93-100

Accumulation L.-R. LIN, V. N. REDDY”,

in Cultured F. J. GIBLIN,

Human P. F. KADOR

Lens Epithelial AND

Eye Research Institute, Oakland University, Rochester, Ml 48309-4401. Bethesda, MD 20894, U.S.A.

Cells

J. H. KINOSHITA and National Eye Institute,

(Received 9 April 1990 and accepted in revised form 18 June 7990) Human lensepithelial(HLE)ceilsin tissueculture accumulatedsignificantlevelsof gala&o1 when they werecultured for 72 hr in mediumcontaining30 mMn-galactose.Polyol accumulationwasaccompanied by the appearanceof vacuolesas seenby transmissionelectron microscopy.The number and size of intracellular vacuolesincreasedwhen the culture period was extendedto 7 days. In addition. polyol accumulationwasaccompaniedby lossof myoinositol.Noneofethesechangesoccurred in cellsexposed to 30 M L-galactosewhich is not a substratefor aldosereductase.The accumulation of galactitol, intracellular vacuole formation and lossof myoinositol observedin n-galactose-exposed cells were preventedby the inclusionof the aldosereductaseinhibitor. sorbinil,in the culture medium.Comparison of the relative efficaciesof two aldosereductaseinhibitors indicatethat AL 1576 is nearly 20 timesmore potent than sorbinilin inhibiting the human lensenzyme.It is concludedthat vacuoleformation in HLE cellsis due to the osmoticeffectof polyol formation brought about by the action of aldosereductaseand that the etiology of human diabetic cataract may alsoinvolve the polyol pathway. K~I.Iwords : human lenseuithelium: tissueculture ; ultrastructure ; aldosereductase; polyol pathway ; diabt%ccataract, sorbinil,AL1 576.

1. Introduction

Aldose reductase (AR) has been implicated in the etiology of diabetic and galactosemic cataracts (Kinoshita, 1965, 1974; Varma and Kinoshita, 1974; Kinoshita, Kador and Datiles, 1981). Studies on experimental animals have provided ample evidence

that the intracellular accumulation of polyols in lens fibers leads to hypertonicity with resulting hydration and changes in membrane permeability. These changes are believed to be the initiating factors in ‘sugar cataracts’ (Kinoshita, 1965). In contrast to welldocumented studies with experimental animals, the

role of the polyol pathway in the development of human diabetic cataracts is unclear at the present time. Demonstration of the polyol pathway in human

lenses was based on the presence of sorbitol in diabetic cataracts (Pirie and van Heyningen, 1964) and the accumulation of this polyol in organ cultured human lenses in high glucose medium (Chylack, Henriques and Tung, 1978 ; Varma, Shocket and Richards,

1979). However, the absence of a correlation between sorbitol level and hydration of the lens raised the question of whether an osmotic insult is responsible for the initiation of human diabetic cataracts. Despite this inconclusive evidence, it is possiblethat sufficient

polyol may accumulate in regions where AR activity is high, thus causing osmotic insult to localized areas. Since the highest activity of AR is present in the epithelium (Collins and Corder, 1977 ; Jedziniak et al., 1981; Akagi et al., 1984), it is conceivable that these

cells may be more susceptible to intracellular

2. Materials and Methods Primary human lens epithelial (HLE) cultures were

established from anterior capsule specimens obtained from 5-12-month-old infants who underwent surgery for retinopathy of prematurity as described previously (Reddy et al., 1988). The explants of anterior capsule fragments were cultured in Dulbecco’s modified Eagles medium (DME) supplemented with 20% fetal bovine serum (FBS), and the epithelial cells became confluent in 2 weeks. The cells were trypsinized and subcultured through three additional passages. For this study, cells in the third passage (P,) were routinely employed. Approximately 100 000 cells per well were transferred to 24-well plates (Falcon, Lincoln Park, NJ) and after 24 hr, when the cells were attached to the culture dish, the medium was replaced with DME containing either 30 mM D-galactose or 30 rnM L-galactose. The osmolarity of the medium was adjusted to 310-320

* Forcorrespondence. 00144835,‘91/010093+08

hyper-

tonicity than the rest of the lens. Our recent success in growing primary cultures of human lens epithelium (Reddy et al., 1988; Arita, Lin and Reddy, 1988) made it possible to study the polyol pathway in these cells. We present evidence that lens epithelia, when cultured in the presence of high galactose, accumulate significant levels of gala&to1 with the appearance of intracellular vacuoles and changes in ultrastructure as observed by transmission electron microscopy (TEM). The accumulation of polyols is accompanied by the loss of myoinositol, an indicator of increased cell permeability. In addition, inclusion of aldose reductase inhibitors (ARIs) in high galactose medium prevents these biochemical and morphological changes.

$03.00/O

0 1991 AcademicPressLimited

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LIN

ET AL

FIG. 1. Phase contrast photomicrographs of confluent HLE cells. A, Confluent culture of passage one. B, Confluent culture after third passage.

mOsm with appropriate amounts of amino acids and vitamins (Gibco DME supplement of amino acids and vitamins), and the cell cultures were usually carried out for 72 hr. In some experiments, the culture time was exter,ded to 7 days. ARIs (sorbinil or AL 1576) were included in the culture medium along with high galactose in some experiments. Sorbinil and AL 1576 were provided by Pfizer Central Research and Alcon Laboratories, respectively. For the assay of polyols, 400000 P, cells per well were transferred to six well plates. cultured for 72 hr in appropriate media, and harvested for the quantification of hexoses and polyols. At the end of the culture period the cells were scraped from the dish and sonicated for 30 set in 0.6 ml 2% ZnSO,. using a Cell Disrupter 200 (Bronson Sonic Power Co., Danbury, CN, power level 3, 70% pulse). The sonicated cells were neutralized with an equivalent amount of Ba(OH), and centrifuged at 3000 rpm for 10 min. Aliquots of supernatants were lyophilized and silylated using Tri-Sil Z (Pierce Chemical Co.. Rockford, IL) as the silylating agent. Sample volumes ranging from 0.5 to 3.0 ~1 were chromatographed using a Shimadzu GC-MINI 3 Gas Chromatograph, and the polyols quantified as previously described (Reddy, Chakrapani and Steen, 1971). Electron Microscopy At the end of the culture period the plates were washed three times with serum-free DME and pre-fixed with 1% osmium tetroxide in 0.05 M sodium cacodylate buffer for 5 min. They were then fixed in 2.5 % glutaraldehyde for 30 min followed by postfixation with 1 Y. osmium tetroxide for an additional 30 min. The specimens were dehydrated in graded ethanol and embedded in Epon (Polysciences, Inc., Warrington,

PA). Polymerization of Epon was carried out in two steps, first at 50°C for 24 hr and then at 60°C for 48 hr. En fas sections were cut with a diamond knife and stained with uranyl acetate and Reynold’s lead solution and examined with an ISI-LEM 2000 or Phillips 410 transmission electron microscope. 3. Results In previous studies with animal models in which cataracts were induced by galactose feeding. the earliest changes noted were the ultrastructural abnormalities of anterior lens epithelium and superficial cortex (Kuwabara, Kinoshita and Cogan, 1969: Datiles et al., 1982). Therefore, we elected to first examine the effect of high galactose on the ultrastructure of HLE cells by culturing them in 30 mM Dgalactose and comparing their morphology to those of cells in either normal media or media containing I,galactose. HLE cells in the third passage were employed in this study. Figure 1 is a phase contrast micrograph of confluent cultures in passages 1 and 3. In general, 2-3 x 1 O6 cells could be harvested after three passages from approximately 2-3 mm” initial explants. Exposure of these cells to 30 mM n-galactose for 72 hr resulted in the formation of small vacuoles as seen by TEM [Fig. 2(B)]; however, the cells exposed to either normal [Fig. 2(A)] or L-galactose-containing medium [Fig. 3(A)] did not form vacuoles. When the culture time with n-galactose was extended to one week the number and size of the vacuoles increased [Fig. 3(B)]. Nagata et al. (1989) reported that canine lens epithelial cells, when cultured for only 6 hr in 30 mM o-galactose media. began to accumulate small vacuoles in the perinuclear zone. Therefore, we examined the effect of high galactosc on primary cultures

of dog lens epithelial

cells under the same

POLYOL

ACCUMULATION

IN

CULTURED

HUMAN

LENS

EPITHELIAL

CELLS

95

FIG. 2. The effect of 30 mM D-galactose on the ultrastructure of HLEcellsin tissueculture. Ceilsfrom third passage werecult1xed for 72 hr and en fas sectionsexaminedwith TEM, as describedunder Materials and Methods.A, Ceilscultured in nor.mal mediu m. B. Cells from the samebatch cultured in the presenceof 30 mMD-galactose. Note vacuoleformation in the galactose-

containing medium. conditions as HLE cells. In these cells, vacuoles could be observed after 4 hr of incubation (data not shown) thus confirming the findings of Nagata et al. (19 89 ). It remains to be established whether the higher sensitivity of dog lens epithelial cells to n-galactose is related to the differences in the AR activity of these cells compared to that of HLE cells. The vacuole formation in the cultured HLE cells exposed to high galactose was prevented by the addition of 4 x 10 -5 M sorbinil to the culture medium [Fig. 3(C)], suggesting that the vacuoles may have resulted from the accumulation of galactitol brought about by the action of AR. These findings are similar to those in galactose-fed rat lenses in which morphological changes were prevented with sorbinil (Datiles et al.. 1982; Unakar and Tsui. 1983). Further insight into the processof vacuole formation

and changes in ultrastructure of the cells was gained when HLE cells were cultured in media containing 30 mM n-galactose both in the presence and absence of sorbinil for 72 hr and the polyol content assayed using gas liquid chromatography. Because of the variability in the amount of polyol formed in different batches of these cells, standard deviations for galactitol and myoinositol levels were large. Therefore, the comparison of the effect of high galactose and the AR1 on polyol levels is best made on the cells of the same batch and passage number. Figure 4 illustrates a typical chromatogram from one such experiment. It may be seen that a substantial level of galactitol accumulated in cells incubated in the presence of Dgalactose (peak c) compared with those exposed to Lgalactose. Although L-galactose is not a substrate for AR. a significant peak corresponding to galactitol was

96

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ET A

FIG. 3. Th .e effc:ctof 30 mM D- and L-galactose and sorbinil on the ultrastructure of HLE cells. Cells from thir rd 1>assag e WC ere cult Ul3 :d fiDr 7 Iiay‘s and ultrastructure examined as in Fig. 2. A, Cells cultured in the presence of 30 mM Irga tlac‘tose. I3. cc :lls cult urf :d irn the w :sence of 30 mM n-galactose. Note large intracellular vacuoles. C. Cells cultured in the PI rice of 30 rnM Dgala ct1me and 40 P,M sorbinil. Note complete absence of vacuole formation.

POLYOL

ACCUMULATION

IN

CULTURED

HUMAN

LENS

EPITHELIAL

CELLS

97

a 30mY D-galactoso

normal control

b

c

cl

3OmM L-galactoso

Fxc. 4. Gas-liquidchromatographicprofilesof extracts of HLEcellscultured in 30 mu D- or L-galactose.Equalnumberof cells from the third passage were cultured for 72 hr and equivalent aliquotschromatographedasdescribedunder methods.Peaks correspondto (a) a- D- and L-galactose,(b) /3-D- and L-galactose,and (c) galactitol+sorbitol, (d) myoinositol.

d

normal

control

30mM D-gakctose

FIG. 5. Effect of sorbinil on galactitol formation in HLF cellscultured for 72 hr. Identity of the peaksis the sameasin Fig. 4. Note the decreasein the sizeof the galactitol peak and the increasein myoinositollevel in cellscultured in the presenceof sorbinil.Also, the peakheightsfor galactose(a and b) in cellscultured in the presenceof galactoseplusARIs are higher than thosecultured in galactosemediumalone indicating the suppressionof the conversionof the sugar to the polyol.

present. This is probably due to sorbitol, since the media contains 5 mM glucose ; sorbitol and galactitol closely comigrate and could not be resolved in the GLC column used. The level of myoinositol in n-galactoseexposed cells was also much lower (peak d) than in the cells cultured in the presence of L-galactose. These results suggest that loss of myoinositol from Dgalactose-exposed cells is due to increased permeability of the cells leading to accelerated efflux. Similar observations caused by osmotic changes have been made previously in galactose-exposed lens (Kinoshita et al., 1969) or dog lens epithelial cells that were incubated in high galactose- or glucose-containing media (Nagata et al., 1989). Galactitol formation was effectively reduced by the presence of 4 x lo-” M sorbinil in the culture medium 7

containing high levels of D-galactose (Fig. 5). Fur-

thermore, the data show that loss of cell myoinositol was concomitantly prevented with the inhibition of AR by sorbinil. It may also be noted that when cells were grown in 30 mM n-galactose medium, the level of galactose (peaks a and b) in the cells is lower compared with the cells grown in 30 mM n-galactose + ARI. These results suggest that in the absence of ARI, galactose is being utilized and converted to the sugar alcohol. The inverse correlation between galactitol and myoinositol levels is illustrated by the data in Fig. 6 in which HLE cells were cultured for 72 hr in 30 mM D-

galactose-containing media with varying concentrations of AL 1576. Galactitol formation was progressively inhibited with increasing dosesof the AR1 BER 52

L.-R.

0

0:03

006

concentration

0:12

0:25

of AL-1576

0:5

NORMAL

(uhf)

FIG. 6. The effect of concentration of Al 1576 on galactitol and myoinositol levels in HLI? cells cultured for 72 hr in the presence of 30 mM n-galactose. The cells from the third passage were used for all of the experiments. One standard deviation is indicated by the error bars.

while myoinositol concentrations gradually increased. In the absence of ARI, the galactitol concentration was more than 350 nmol per lo6 cells while myoinositol level was barely detectable. In the presence of 0.5 ,UM AL 1576, the galactitol level was reduced by 85 % and the cell myoinositol level reached 2 50 nmol per lo6 cells. Additional experiments were carried out to determine the relative efficacy of sorbinil and AL 15 76 in inhibiting AR by estimating the dose required to inhibit galactitol formation by 50% in cells cultured for 72 hr. The data in Fig. 7 show that the concentration of AL 1576 required for such inhibition was 3.6 x 10e8 M while the corresponding dose for sorbinil was 7.2 x lo-’ M. Thus, AL 15 76 is 20 times more effective than sorbinil in inhibiting the human lens enzyme. 4. Discussion

These studies demonstrate that HLE cells accumulate significant levels of galactitol when cultured in the presence of high galactose and that the accumulation of the polyol results in the appearance of vacuoles

concentration

of

AL-1576

(Uhf)

LIN

ET AL.

similar to those observed in epithelial cells of galactosefed rats in vivo. The accumulation of polyol apparently involves AR, since inhibitors of this enzyme effectively block polyol formation and prevent the formation of vacuoles which are presumably due to the osmotic effect of intracellular accumulation of galactitol. The loss of myoinositol from galactose-exposed cells and its retention with the inhibition of AR is also consistent with the view that polyol accumulation and the resulting hydration leads to permeability changes as reported in studies of lens cells of other species (Kinoshita, 1965; Kinoshita et al., 1969; Reddy et al., 1976). The exposure of human lens cells to high levels of n-galactose has an adverse effect on morphology and permeability characteristics brought about by the action of AR. In both diabetic and galactosemic cataracts, polyol accumulation has been related to lens hydration, especially in the outermost fibres and epithelial cells (Kinoshita, Merola and Satoh, 1962; Kinoshita and Merola, 1964 ; Kinoshita, 19 6 5 ; Datiles et al., 1982). Lens epithelium has a critical role in transport and permeability (Kinsey and Reddy, 1965) so that hydration of these cells will result in an increased efnux of those substances that are maintained in higher concentration in the lens than the surrounding intraocular fluids (Reddy. 19 6 5 ; Kinoshita, 1965; Reddy et al., 1976). The intracellular levels of many substances are affected by osmotic changes in the lens; myoinositol and free amino acids are particularly sensitive (Kawaba, Carper and Kinoshita, 1988). When the accumulation of galactitol in HLE cells is prevented by the inhibition of AR, the concentration of myoinositol is maintained at a normal level. The fact that vacuole formation, presumably by overhydration, is also suppressed by the AR1 is consistent with the view of an osmotic effect caused by the accumulated galactitol. The vacuole formation following tissue culture of HLE cells in high galactose medium, as observed by TEM. appears to be similar to the findings reported at the early stages in vivo in rat lens epithelium of

concentration

of sorbinil

(uM)

FIG. 7. Inhibition of galactitol formation in HLE cells by AL 15 76 and sorbinil. The same batch of cells from the third passage were cultured for 72 hr in the presence of varying concentrations of the ARI. and the values required for 50% inhibition of galactitol formation were determined. The values for 50% inhibition were: AL 1576, 3.6 x 10e8 M; sorbinil, 7.2 x lo-’ M.

POLYOL

ACCUMULATION

IN CULTURED

HUMAN

LENS

galactose-fed animals (Kuwabara et al., 1969). Present findings are also consistent with those reported recently by Nagata et al. (1989) in a study of dog lens epithelial cells cultured in the presence of high galactose or glucose. However, the adverse effect of high sugar in dog lens epithelial cells was found to occur within 6 hr of culture (also confirmed in the present study) whereas in the HLE cells, 72 hr of culture were required to demonstrate similar vacuole formation, suggesting lower AR activity in human lens cells. A number of previous studies have shown that the extent of polyol formation is related to the activity of AR in lenses of different species. Unlike the rat, rabbit, degu or guinea-pig lens, the young calf lens has limited AR activity so that the level of polyol accumulation upon galactose exposure was insufficient to cause an increase in hydration (Kawaba et al., 1988). This may be the case with human lenses in which sorbitol and fructose synthesis was documented but no correlation between sorbitol level and water gain could be made (Chylack et al., 1978 ; Varma et al., 1979). In view of the present findings that intracellular vacuoles were seen in HLE cells only after 72 hr, even though the osmotic stress of galactitol which is not further metabolized is much greater than sorbitol. it is not surprising that the osmotic effect of sorbitol could not be detected in human lenses that were cultured for only 24 hr. Although the overall tissue polyol level in intact human lenses exposed to high glucose does not appear to have been osmotically significant in the aforementioned studies (Chylack et al., 1978; Varma et al., 1979), it may indeed have a profound effect if much of the osmolyte is confined to the single layer of epithelium. It is, therefore, reasonable to conclude that despite the low AR activity in the whole lens, the osmotic stress in the epithelium of human diabetic lens may be a significant factor in initiating cataractous changes analogous to those in animal models. A practical aspect of this investigation, which may prove helpful in the future, is the use of lens epithelial cell cultures in determining the efficacy of ARIs for possible clinical use in human diabetic disease. Kador et al. (1980) have shown that ARs from different tissues vary in their susceptibility to inhibition by different ARIs. For example, sorbinil was most effective in inhibiting the rat lens enzyme compared with either human lens or human placental enzyme. Thus, an AR1 which is most potent in an animal model may not necessarily be as effective against the human lens enzyme. They pointed out that the evaluation of ARIs for potential clinical use may require the use of AR from the appropriate target tissue. It is apparent that the lens epithelial cells used in the present study serve as an excellent model for evaluating various ARIs as possible therapeutic agents against human diabetic cataracts. The results of such an investigation will be reported in a subsequent publication.

EPITHELIAL

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Acknowledgments This study was supported by NE1 Grants EY00484, EY02027, EY05230 (Core Grant for Vision Research), and Alcon Awards. We are grateful to Dr Michael Trese of the Eye Research Institute of Oakland University and William Beaumont Hospital, Royal Oak, MI, for his cooperation in obtaining specimens of HLE.

References Akagi, Y., Yajima, Y., Kador, P. F.. Kuwabara, T. and Kinoshita, J. H. (1984). Localization of aldose reductase in the human eye. Diabetes 33, 562-6. Arita. T., Lin. L.-R. and Reddy, V. N. (1988). Differentiation of human lens epithelial cells in tissue culture. Elcp. Eye Res. 47, 905-10. Chylack, Jr., L. T., Henriques, H. and Tung, W. (1978). Inhibition of sorbitol production in human lenses by an aldose reductase inhibitor. Invest. Ophthalmol. Vis. Sci. 17 (Suppl.), 300. Collins, J. G. and Corder, C. N. (1977). Aldose reductase and sorbitol dehydrogenase distribution in substructures of normal and diabetic rat lens. Invest. Ophthalmol. Vis. Sci. 16, 242-6. Datiles, M., Fukui, H., Kuwabara. T. and Kinoshita. J. H. (1982). Galactose cataract prevention with sorbinil, an aldose reductase inhibitor: a light microscopic study. Invest. OphthaImoZ. Iris. Sci. 22, 174-9. Jedziniak, J. A., Chylack, Jr., L. T., Cheng, H. M., Gillis, M. K.. Kalustian, A. A. and Tung, W. H. (1981). The sorbitol pathway in the human lens: aldose reductase and polyol dehydrogenase. Invest. Ophthalmol. Vis. Sci. 20, 314-26. Kador. P. F.. Kinoshita, J. H., Tung. W. H. and Chylack, Jr., L. T. (1980). Differences in the susceptibility of various aldose reductases to inhibition. II. Invest. OphthaimoI. Vis. Sci. 19, 980-2. Kawaba, T.. Carper, D. A. and Kinoshita. J. H. (1988). Polyol-induced changes in the calf lens. In Polyol Pathway and Its Role in Diabetic Complications (Eds Sakamoto, N., Kinoshita, J. H., Kador, P. F. and Hotta, N.). Pp. 164-9. Excerpta Medica: Amsterdam, The Netherlands. Kinoshita, J. H. (1965). Cataracts in galactosemia. The Jonas Friedenwald Memorial Lecture. Invest. Ophthalmol. 4. 786-99. Kinoshita, J. H. (1974). Mechanisms initiating cataract formation. Proctor Lecture. Invest. Ophthalmol. Vis. Sci. 13, 713-24. Kinoshita, J. H., Barber, G. W., Merola. L. 0. and Tung, W. (1969). Changes in the levels of free amino acids and myo-inositol in the galactose-exposed lens. Invest. Ophthalmol. 8, 625-32. Kinoshita, J. H., Kador, P. F. and Datiles, M. (198 1). Aldose reductase in diabetic cataracts. J. Am. Med. Assoc. 246. 257-61. Kinoshita, J. H. and Merola. L. 0. (1964). Hydration of the lens during the development of galactose cataract. Invest. Ophthalmol. 3, 577-83. Kinoshita, J. H., Merola, L. 0. and Satoh, K. (1962). Osmotic changes caused by the accumulation of dulcitol in the lenses of rats fed with galactose. Nature 194. 1085-7. Kinsey, V. E. and Reddy, D. V. N. (1965). Studies on the crystalline lens-XI. The relative role of the epithelium and capsule in transport. Invest. Ophthalmol. 4. 104-16. Kuwabara, T., Kinoshita, J. H. and Cogan. D. G. (1969). Electron microscopic study of galactose-induced cataract. Invest. Ophthalmol. 8, 13349. 7-2

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Nagata, M.. Hohman, T. C., Nishimura, C., Drea, C. M., Oliver, C. and Robinson, W. G. (1989). Polyol and vacuole formation in cultured canine lens epithelial cells. Exp. Eye Res. 48, 667-77. Pirie, A. and van Heyningen, R. (1964). The effect of diabetes on the content of sorbitol, glucose, fructose. and inositol in the human lens. Exp. Eye Res. 3, 124-31. Reddy, D. V. N. (1965). Amino acid transport in the lens in relation to sugar cataracts. Invest. Ophthalmol. 4. 700-8. Reddy, V. N., Chakrapani, B. and Steen, D. (1971). Sorbitol pathway in the ciliary body in relation to accumulation of amino acids in the aqueous humor of alloxandiabetic rabbits. Invest. Ophthalmol. 10, 870-S. Reddy, V. N., Lin, L.-R., Arita, T., Zigler, Jr., J. S. and Huang,

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ET AL.

Q. L. (1988). Crystallins and their synthesis in human lens epithelial cells in tissue culture. Exp. Eye Res.47, 465-78. Reddy, V. N., Schwass,D., Chakrapani, B. and Lim, C. P. (1976). Biochemical changes associatedwith the developmentand reversalof galactosecataracts. Exp. EyeRes.23, 483-93. Unakar, N. J. and Tsui, J. Y. (1983). Inhibition of galactoseinduced alterations in ocular lens with sorbinil. Exp. Eye Res.36, 685-94. Varma, S.D. and Kinoshita.J. H. (1974). Sorbitolpathway in diabeticand galactosemicrat lens. Biochim.Biophys. Acta 338. 632-640. Varma, S. D., Shocket, S.S. and Richards, R. D. (1979). Implicationsof aldosereductasein cataractsin human diabetes.Invest.Ophthalmol.Vis. Sci. 18, 23741.