- Email: [email protected]
The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)
Chemico-Biological Interactions 130 – 132 (2001) 617 – 625
www.elsevier.com/locate/chembiont
The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis) Masatoshi Murata, Nobuo Ohta, Shinichi Sakurai, Shahabuddin Alam, Jen-Yue Tsai, Peter F. Kador, Sanai Sato * Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Rm 10 /10B09, 10 Center Dri6e, MSC 1850, Bethesda, MD 20892 -1850, USA
Abstract Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco’s modified Eagle’s mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 mM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123 910 mg/106 cells (n=5) to 3.991.9 mg/106 cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aldose reductase; Lens epithelial cells; Apoptosis; Sugar cataract; Dog
* Corresponding author. Tel.: + 1-301-4029849; fax: +1-301-4022399. E-mail address: [email protected] (S. Sato). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 2 8 9 - 1
618
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
1. Introduction Sugar cataracts develop in both hyperglycemia and galactosemia. Experimental studies have well established that these sugar cataracts are linked to the accumulation of excess amount of sugar alcohols [1,2]. Recent studies with transgenic mice that overexpress aldose reductase in the lens also confirm that the presence of aldose reductase is the key for the development of sugar cataracts [3]. However, the site(s) of initial insult induced by sugar alcohol accumulation in the lens remains elusive. Although aldose reductase is primarily localized in lens epithelial cells [4], morphological changes predominantly occur in superficial lens fiber cells rather than epithelial cells [5]. Even in the advanced stages of sugar cataract where massive fiber cell degeneration occurs in cortex, epithelial cells maintain relatively normal morphology. However, several studies indicate the importance of epithelial cells in sugar cataract formation. When cultured in high sugar medium, both dog and human lens epithelial cells develop multiple intracellular vacuoles whose formation is inhibited by the presence of aldose reductase inhibitors [6,7]. The similar intracellular vacuole formation also occurs in epithelial cells in galactose-fed rats prior to the appearance of any cortical changes [8]. Like rats, dogs fed galactose diet develop sugar cataracts whose formation is reduced in a dose-dependent manner by the administration of aldose reductase inhibitors [9,10]. Aldose reductase represents the major NADPH-dependent reductase in dog lens [11]. Dog lens epithelial cells accumulate sugar alcohols when exposed to medium containing either high glucose or galactose [6] or their respective fluorinated analogs 3-fluoro-3-deoxy-D-glucose [12] or 3-fluoro-3-deoxyD-galactose [13]. Here, we report that apoptosis is induced in dog lens epithelial cells when cultured long-term in medium containing D-galactose and the induction of this apoptosis is prevented by the concomitant addition of the aldose reductase inhibitor AL 1576 to the culture medium.
2. Material and methods
2.1. Chemical All chemicals utilized were of reagent grade. Dulbecco’s modified Eagle’s mimimum essential medium (DMEM), fetal calf serum (FCS) and trypsin/versene (0.05% trypsin and 0.02% ethylene diaminetetraacetic acid (EDTA) in Hank’s balanced salt solution) were obtained from Biofluids Inc. (Rockville, MD). Molecular ruler (100 bp) and DNA ladder (1 kb) were products of Bio-Rad Laboratories (Hercules, CA). An in situ apoptosis detection kit was purchased from Genzyme Diagnostics (Cambridge, MA). Freshly enucleated dog eyes from beagles were commercially obtained from Marshall Farm USA Inc. (North Rose, NY).
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
619
2.2. Lens epithelial cell culture Primary dog lens epithelial cells were cultured as previously described by Nagata et al. [6] on 24 well tissue culture plates containing DMEM medium supplemented with 20% FCS at 37°C under a 5% CO2 atmosphere. After reaching confluence at the fifth passages, the medium was replaced with DMEM medium supplemented with 20% FCS and 50 mM galactose.
2.3. Gas chromatographic sugar analysis Cultured cells were harvested with 0.05% trypsin and 0.02% EDTA solution and washed with phosphate buffered saline (PBS). Methyl-a-D-mannopyranoside (0.2 mg) was added as an internal standard to suspensions containing approximately 1× 106 cells and the mixture was sonication with 100 ml of 0.3 M zinc sulfate and then deproteinated by addition of 100 ml of 0.3 M barium hydroxide. After centrifugation at 10 000×g for 10 min, the resulting supernatant was lyophilized and silylated at 60°C for 1 h with 100 ml of Tri-Sil ‘Z’ diluted with 100 ml of pyridine. The derivatized mixture was then analyzed on a Shimadzu GC-15A gas chromatography (Shimadzu Corp., Tokyo, Japan) equipped with a fused silica capillary column maintained at 155°C with helium as the carrier gas. Peak identities were established using known sugar standards.
2.4. RNA preparation mRNA from the cultured cells was isolated using a quick prep micro mRNA purification kit (Pharmacia, Piscataway, NJ) which combines the disruptive and protective properties of guanidinium thiocyanate (GTC) with the selectivity of oligo (dT)-cellulose chromatography in a spun-column format.
2.5. Re6erse transcription-polymerase chain reaction (RT-PCR) Cell mRNA was reversely transcribed to cDNA using oligo (dT) as a primer from 200 ng of cell mRNA. Samples were incubated at 40°C for 60 min and the reaction was stopped by heating the samples for 2 min at 95°C and then cooling them to 4°C. cDNA (1 ml) reaction was amplified using PCR. For aldose reductase mRNA amplification, the sense primer utilized was 5%GGACCTCTACCTTATTCACTG-3% and the antisense primer was 5%-TTGGCCCAGGGCCTGTCAG-3%. The cDNA amplification product was predicted to be 350 bp in length. Each sample was also amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as an internal control using the sense primer 5%-CATCACCATCTTCCAGGAGC-3% and the antisense primer 5%TAAGCAGTTGGTGGTGCAGG-3%. These primers yielded a 253 bp fragment of the GAPDH cDNA. The amplification reactions contained 50 mM KCl, 10 mM Tris–HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 2 U of amplitaq
620
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
DNA polymerase and 0.4 mM of each 5% and 3% primers. Amplifications were carried out for 30 cycles at 94°C for 1 min (denaturing), at 60°C for 1 min (annealing), at 72°C for 1 min (extension) with a final extension at 72°C for 10 min. The PCR products were visualized by electrophoresing the reaction mixture in 2% agarose gels containing 0.5 mg/ml ethidium bromide.
2.6. DNA fragmentation assay The epithelial cells were suspended in 10 mM Tris–HCl buffer, pH 8.0 containing 1 mM EDTA and lysed by incubating at 55°C for 2 h in the same buffer with 20 mg/ml RNase, 100 mg/ml proteinase K and 0.5% (W/V) sodium dodecyl sulfate (SDS). DNA was extracted with phenol and precipitated with 70% ethanol. Electrophoresis was conducted on 0.8% agarose gels containing ethidium bromide at 15 V for 12 h. Molecular ruler (100 pb) and 1 kb DNA ladder were utilized as DNA size markers.
2.7. In situ apoptosis detection In situ detection of lens epithelial cells was conducted according to the method of Li et al. [14] using the Genzyme Apoptosis Detection Kit. With this procedure apoptotic cells stained brown or dark brown while normal cells stained light green.
3. Results Primary cultures of dog lens epithelial cells isolated from beagles grow well in DMEM medium supplemented with 20% FCS for five to six passages. To investigate whether the exposure to high galactose initiates the cell death (apoptosis) of dog lens epithelial cells, the medium was replaced with DMEM medium containing 50 mM D-galactose at the fifth passage and the culture was continued for an additional 2 weeks. Through the fifth passage essentially all cells cultured in the normal DMEM medium appear microscopically normal with few degenerated cells detectable. However, the exposure to galactose appears to accelerate the cell death. The fine, multiple intracellular vacuoles were occasionally observed in the cells cultured in the medium containing 50 mM galactose. Moreover, when examined by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL), significant positive staining of cells cultured in galactose medium was observed (Fig. 1). The TUNEL positive staining for apoptosis was completely prevented by the addition of 1 mM of the aldose reductase inhibitor AL 1576 to the galactose medium. To confirm apoptosis in cells grown in galactose medium, DNA fragmentation of these cells was also examined (Fig. 2). Obvious DNA fragmentation was observed in cells cultured for 2 weeks in the medium containing 50 mM galactose. This DNA change was not observed in similar cells cultured in the presence of 1 mM of the aldose reductase inhibitor AL 1576.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
621
Since the aldose reductase inhibitor AL 1576 appeared to completely prevent apoptotic changes in cells exposed to high galactose, sugar alcohol accumulation and its inhibition by AL 1576 were also examined (Fig. 3). Cells grown in normal medium contained essentially no galactitol while cells cultured in the medium
Fig. 1. In situ apoptosis detection of dog lens epithelial cells. Dog lens epithelial cells were cultured for 2 weeks in the control DMEM medium supplemented with 20% FCS (A), the same medium containing 50 mM D-galactose (B), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (C). With this technique, the nucleus of apoptotic cell is stained brown.
622
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
Fig. 2. Agarose gel electrophoresis of DNAs of dog lens epithelial cells. Dog lens epithelial cells were cultured for 2 weeks in the control DMEM medium supplemented with 20% FCS (lane 2), the same medium containing 50 mM D-galactose (lane 3), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (lane 4). Lane 1, DNA ladder makers (1 kb).
containing 50 mM galactose rapidly accumulated galactitol. After 3 days of culture, the galactitol level was 123910 mg/106 cells (n=4) and this accumulation was decreased by the presence of 1 mM AL 1576 to only 3.991.9 mg/106 cells (n= 4). The presence of aldose reductase in dog lens epithelial cells was confirmed by RT-PCR (Fig. 4). With the RT-PCR technique, aldose reductase mRNA was clearly detected in dog lens epithelial cells, and cells cultured in the medium containing 50 mM galactose for 2 weeks continued to express aldose reductase mRNA. Compared with the mRNA level of GAPDH used as an internal standard, the expression of aldose reductase appeared higher in cells exposed to high galactose compared with those cultured in control medium. The expression of aldose reductase was reduced to normal levels in cells cultured in galactose medium containing the aldose reductase inhibitor AL 1576.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
623
Fig. 3. Galactitol accumulation in dog lens epithelial cells. Dog lens epithelial cells were cultured for 3 days in DMEM medium supplemented 20% FCS (Control), the same medium containing 50 mM D-galactose (galactose, light gray bar), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (galactose and AL 1576, dark gray bar).
4. Discussion It has recently been suggested that apoptosis of lens epithelial cells is a key process in the development of many types of cataracts [14]. Apoptosis of epithelial cells has been reported to be the earliest event in the formation of experimental cataracts associated with hydrogen peroxide or ultraviolet light-induced cataracts [15,16]. The earliest changes in sugar cataract formation also occur in lens epithelial cells [8] and Gonzalez et al. [17] have reported that apoptosis of lens epithelial cells is induced when lenses are cultured in vitro in medium containing either high glucose or galactose. The present study confirms that cultured dog lens epithelial cells, when exposed to high galactose environment, also undergo apoptosis. Moreover, this galactose-induced apoptosis is inhibited by the addition of aldose reductase inhibitor suggesting that the induction of apoptosis is linked to the formation and/or accumulation of the sugar alcohol galactitol. Intracellular vacuole formation in lens epithelial cells cultured in galactose medium has been previously reported by a number of investigators [6,7]. While a
Fig. 4. RT-PCR of aldose reductase and GAPDH. Lanes 1 – 3 represent dog lens epithelia cells cultured for 2 weeks in DMEM medium supplemented 20% FCS, the same medium containing 50 mM D-galactose, and the medium containing 50 mM D-galactose and 1 mM AL 1576, respectively.
624
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
similar formation of multiple small vacuoles was observed in dog lens epithelial cells cultured for 2 weeks in galactose medium, the present results further suggest that this vacuole formation is linked to the induction of apoptosis. Although some cells formed vacuoles, vacuoles were absent in many other cells at this stage and they appeared to be microscopically normal. Nevertheless, despite their normal microscopic appearance, obvious DNA changes were observed in these cells. Essentially all cells were positively stained for apoptosis with the TUNEL techniques. This staining was also confirmed by DNA fragmentation. These results indicate that DNA changes in the cultured dog lens epithelial cells precede the visible morphological change of vacuole formation. In the galactose-induced apoptosis of dog lens epithelial cells, aldose reductase appears to play an important role. Aldose reductase expression was easily detected in dog lens epithelial cells cultured in the regular DMEM medium. These cells rapidly accumulated galactitol when cultured in galactose-containing medium. While the abnormal accumulation of sugar alcohols may affect various cell functions, the expression of aldose reductase is slightly increased in cells cultured in galactose medium. Aldose reductase expression is normalized when similar cells are cultured in galactose medium containing the aldose reductase inhibitor AL 1576. At the same time, galactitol levels in these cells are reduced and staining for apoptosis is normalized. This suggests that apoptosis of lens epithelial cells may be linked to osmotic insults induced by abnormal accumulation of sugar alcohols. While the present study does not establish whether epithelial cell apoptosis is the key process that results in sugar cataract formation, the present results indicate that apoptosis precedes vacuole formation and is linked to osmotic changes that are induced by the abnormal accumulation of sugar alcohols. While the present culture conditions under which apoptosis and subsequent vacuole formation can be detected appear extreme (long-term 1–2 week exposure to 50 mM of galactose), similar morphological change (intracellular vacuole formation) has been documented to rapidly occur within 3 days in the lens epithelial cells of galactose-fed rats [8]. This indicates that the process of apoptosis may proceed prior to any morphological changes such as vacuole formation in vivo in lens epithelial cells. This may also suggest the importance of aldose reductase-induced apoptosis of lens epithelial cells in the formation of sugar cataracts.
References [1] J.H. Kinoshita, Mechanisms initiating cataract formation, Proctor Lecture, Invest. Ophthalmol. 13 (1974) 713–724. [2] P.F. Kador, The role of aldose reductase in the development of diabetic complications, Med. Res. Rev. 8 (1988) 325–352. [3] A.Y. Lee, S.K. Chung, S.S. Chung, Demonstration that polyol accumulation is responsible for diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the lens, Proc. Natl. Acad. Sci. USA 92 (1995) 2780 – 2784. [4] Y. Akagi, P.F. Kador, J.H. Kinoshita, Immunohistochemical localization for aldose reductase in diabetic lenses, Invest. Ophthalmol. Vis. Sci. 28 (1987) 163 – 167.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
625
[5] M. Datiles, H. Fukui, T. Kuwabara, J.H. Kinoshita, Galactose cataract prevention with sorbinil, an aldose reductase inhibitor: a light microscopic study, Invest. Ophthalmol. Vis. Sci. 22 (1982) 174–179. [6] M. Nagata, T.C. Hohman, C. Nishimura, C.M. Drea, C. Okiver, W.G. Robison, Jr, Polyol and vacuole formation in cultured canine lens epithelial cells, Exp. Eye Res. 48 (1989) 667 – 677. [7] L.-R. Lin, V.N. Reddy, F.J. Giblin, P.F. Kador, J.H. Kinoshita, Polyol accumulation in cultured human lens epithelial cells, Exp. Eye Res. 52 (1991) 93 – 100. [8] W.G. Robison, Jr, N. Houlder, J.H. Kinoshita, The role of lens epithelium in sugar cataract formation, Exp. Eye Res. 50 (1990) 641 – 646. [9] S. Sato, Y. Takahashi, M. Wyman, P.F. Kador, Progression of sugar cataract in the dog, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1925 – 1931. [10] S. Sato, K. Mori, M. Wyman, P.F. Kador, Dose-dependent prevention of sugar cataracts in galactose-fed dogs by the aldose reductase inhibitor M79175, Exp. Eye Res. 66 (1998) 217 – 222. [11] S. Sato, P.F. Kador, NADPH-dependent reductases of the dog lens, Exp. Eye Res. 50 (1990) 629–634. [12] M.J. Lizak, E.F. Secchi, J.W. Lee, S. Sato, E. Kubo, Y. Akagi, P.F. Kador, 3-FG as substrate for investigating flux through the polyol pathway in dog lens by 19F-NMR spectroscopy, Invest. Ophthalmol. Vis. Sci. 39 (1998) 2688 – 2695. [13] E.F. Secchi, M.J. Lizak, S. Sato, P.F. Kador, 3-Fluoro-3-deoxy-D-galactose: a new probe for studies on sugar cataract, Curr. Eye Res. 18 (1999) 277 – 282. [14] W.C. Li, J.R. Kuszak, K. Dunn, R.R. Wang, W. Ma, G.M. Wang, A. Spector, M. Leib, A.M. Cotliar, M. Weiss, Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals, J. Cell Biol. 130 (1995) 169 – 181. [15] A. Spector, G.M. Wang, R.R. Wang, W.C. Li, N.J. Kleiman, A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action, Exp. Eye Res. 60 (1995) 483–493. [16] W.C. Li, A. Spector, Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract, Free Radic. Biol. Med. 20 (1996) 301 – 311. [17] K. Gonzalez, S. McVey, J. Cunnick, I.P. Udovichenko, D. Takemoto, Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry, Curr. Eye Res. 14 (1995) 269 – 273.
.
www.elsevier.com/locate/chembiont
The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis) Masatoshi Murata, Nobuo Ohta, Shinichi Sakurai, Shahabuddin Alam, Jen-Yue Tsai, Peter F. Kador, Sanai Sato * Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Rm 10 /10B09, 10 Center Dri6e, MSC 1850, Bethesda, MD 20892 -1850, USA
Abstract Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco’s modified Eagle’s mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 mM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123 910 mg/106 cells (n=5) to 3.991.9 mg/106 cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aldose reductase; Lens epithelial cells; Apoptosis; Sugar cataract; Dog
* Corresponding author. Tel.: + 1-301-4029849; fax: +1-301-4022399. E-mail address: [email protected] (S. Sato). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 2 8 9 - 1
618
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
1. Introduction Sugar cataracts develop in both hyperglycemia and galactosemia. Experimental studies have well established that these sugar cataracts are linked to the accumulation of excess amount of sugar alcohols [1,2]. Recent studies with transgenic mice that overexpress aldose reductase in the lens also confirm that the presence of aldose reductase is the key for the development of sugar cataracts [3]. However, the site(s) of initial insult induced by sugar alcohol accumulation in the lens remains elusive. Although aldose reductase is primarily localized in lens epithelial cells [4], morphological changes predominantly occur in superficial lens fiber cells rather than epithelial cells [5]. Even in the advanced stages of sugar cataract where massive fiber cell degeneration occurs in cortex, epithelial cells maintain relatively normal morphology. However, several studies indicate the importance of epithelial cells in sugar cataract formation. When cultured in high sugar medium, both dog and human lens epithelial cells develop multiple intracellular vacuoles whose formation is inhibited by the presence of aldose reductase inhibitors [6,7]. The similar intracellular vacuole formation also occurs in epithelial cells in galactose-fed rats prior to the appearance of any cortical changes [8]. Like rats, dogs fed galactose diet develop sugar cataracts whose formation is reduced in a dose-dependent manner by the administration of aldose reductase inhibitors [9,10]. Aldose reductase represents the major NADPH-dependent reductase in dog lens [11]. Dog lens epithelial cells accumulate sugar alcohols when exposed to medium containing either high glucose or galactose [6] or their respective fluorinated analogs 3-fluoro-3-deoxy-D-glucose [12] or 3-fluoro-3-deoxyD-galactose [13]. Here, we report that apoptosis is induced in dog lens epithelial cells when cultured long-term in medium containing D-galactose and the induction of this apoptosis is prevented by the concomitant addition of the aldose reductase inhibitor AL 1576 to the culture medium.
2. Material and methods
2.1. Chemical All chemicals utilized were of reagent grade. Dulbecco’s modified Eagle’s mimimum essential medium (DMEM), fetal calf serum (FCS) and trypsin/versene (0.05% trypsin and 0.02% ethylene diaminetetraacetic acid (EDTA) in Hank’s balanced salt solution) were obtained from Biofluids Inc. (Rockville, MD). Molecular ruler (100 bp) and DNA ladder (1 kb) were products of Bio-Rad Laboratories (Hercules, CA). An in situ apoptosis detection kit was purchased from Genzyme Diagnostics (Cambridge, MA). Freshly enucleated dog eyes from beagles were commercially obtained from Marshall Farm USA Inc. (North Rose, NY).
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
619
2.2. Lens epithelial cell culture Primary dog lens epithelial cells were cultured as previously described by Nagata et al. [6] on 24 well tissue culture plates containing DMEM medium supplemented with 20% FCS at 37°C under a 5% CO2 atmosphere. After reaching confluence at the fifth passages, the medium was replaced with DMEM medium supplemented with 20% FCS and 50 mM galactose.
2.3. Gas chromatographic sugar analysis Cultured cells were harvested with 0.05% trypsin and 0.02% EDTA solution and washed with phosphate buffered saline (PBS). Methyl-a-D-mannopyranoside (0.2 mg) was added as an internal standard to suspensions containing approximately 1× 106 cells and the mixture was sonication with 100 ml of 0.3 M zinc sulfate and then deproteinated by addition of 100 ml of 0.3 M barium hydroxide. After centrifugation at 10 000×g for 10 min, the resulting supernatant was lyophilized and silylated at 60°C for 1 h with 100 ml of Tri-Sil ‘Z’ diluted with 100 ml of pyridine. The derivatized mixture was then analyzed on a Shimadzu GC-15A gas chromatography (Shimadzu Corp., Tokyo, Japan) equipped with a fused silica capillary column maintained at 155°C with helium as the carrier gas. Peak identities were established using known sugar standards.
2.4. RNA preparation mRNA from the cultured cells was isolated using a quick prep micro mRNA purification kit (Pharmacia, Piscataway, NJ) which combines the disruptive and protective properties of guanidinium thiocyanate (GTC) with the selectivity of oligo (dT)-cellulose chromatography in a spun-column format.
2.5. Re6erse transcription-polymerase chain reaction (RT-PCR) Cell mRNA was reversely transcribed to cDNA using oligo (dT) as a primer from 200 ng of cell mRNA. Samples were incubated at 40°C for 60 min and the reaction was stopped by heating the samples for 2 min at 95°C and then cooling them to 4°C. cDNA (1 ml) reaction was amplified using PCR. For aldose reductase mRNA amplification, the sense primer utilized was 5%GGACCTCTACCTTATTCACTG-3% and the antisense primer was 5%-TTGGCCCAGGGCCTGTCAG-3%. The cDNA amplification product was predicted to be 350 bp in length. Each sample was also amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as an internal control using the sense primer 5%-CATCACCATCTTCCAGGAGC-3% and the antisense primer 5%TAAGCAGTTGGTGGTGCAGG-3%. These primers yielded a 253 bp fragment of the GAPDH cDNA. The amplification reactions contained 50 mM KCl, 10 mM Tris–HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 2 U of amplitaq
620
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
DNA polymerase and 0.4 mM of each 5% and 3% primers. Amplifications were carried out for 30 cycles at 94°C for 1 min (denaturing), at 60°C for 1 min (annealing), at 72°C for 1 min (extension) with a final extension at 72°C for 10 min. The PCR products were visualized by electrophoresing the reaction mixture in 2% agarose gels containing 0.5 mg/ml ethidium bromide.
2.6. DNA fragmentation assay The epithelial cells were suspended in 10 mM Tris–HCl buffer, pH 8.0 containing 1 mM EDTA and lysed by incubating at 55°C for 2 h in the same buffer with 20 mg/ml RNase, 100 mg/ml proteinase K and 0.5% (W/V) sodium dodecyl sulfate (SDS). DNA was extracted with phenol and precipitated with 70% ethanol. Electrophoresis was conducted on 0.8% agarose gels containing ethidium bromide at 15 V for 12 h. Molecular ruler (100 pb) and 1 kb DNA ladder were utilized as DNA size markers.
2.7. In situ apoptosis detection In situ detection of lens epithelial cells was conducted according to the method of Li et al. [14] using the Genzyme Apoptosis Detection Kit. With this procedure apoptotic cells stained brown or dark brown while normal cells stained light green.
3. Results Primary cultures of dog lens epithelial cells isolated from beagles grow well in DMEM medium supplemented with 20% FCS for five to six passages. To investigate whether the exposure to high galactose initiates the cell death (apoptosis) of dog lens epithelial cells, the medium was replaced with DMEM medium containing 50 mM D-galactose at the fifth passage and the culture was continued for an additional 2 weeks. Through the fifth passage essentially all cells cultured in the normal DMEM medium appear microscopically normal with few degenerated cells detectable. However, the exposure to galactose appears to accelerate the cell death. The fine, multiple intracellular vacuoles were occasionally observed in the cells cultured in the medium containing 50 mM galactose. Moreover, when examined by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL), significant positive staining of cells cultured in galactose medium was observed (Fig. 1). The TUNEL positive staining for apoptosis was completely prevented by the addition of 1 mM of the aldose reductase inhibitor AL 1576 to the galactose medium. To confirm apoptosis in cells grown in galactose medium, DNA fragmentation of these cells was also examined (Fig. 2). Obvious DNA fragmentation was observed in cells cultured for 2 weeks in the medium containing 50 mM galactose. This DNA change was not observed in similar cells cultured in the presence of 1 mM of the aldose reductase inhibitor AL 1576.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
621
Since the aldose reductase inhibitor AL 1576 appeared to completely prevent apoptotic changes in cells exposed to high galactose, sugar alcohol accumulation and its inhibition by AL 1576 were also examined (Fig. 3). Cells grown in normal medium contained essentially no galactitol while cells cultured in the medium
Fig. 1. In situ apoptosis detection of dog lens epithelial cells. Dog lens epithelial cells were cultured for 2 weeks in the control DMEM medium supplemented with 20% FCS (A), the same medium containing 50 mM D-galactose (B), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (C). With this technique, the nucleus of apoptotic cell is stained brown.
622
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
Fig. 2. Agarose gel electrophoresis of DNAs of dog lens epithelial cells. Dog lens epithelial cells were cultured for 2 weeks in the control DMEM medium supplemented with 20% FCS (lane 2), the same medium containing 50 mM D-galactose (lane 3), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (lane 4). Lane 1, DNA ladder makers (1 kb).
containing 50 mM galactose rapidly accumulated galactitol. After 3 days of culture, the galactitol level was 123910 mg/106 cells (n=4) and this accumulation was decreased by the presence of 1 mM AL 1576 to only 3.991.9 mg/106 cells (n= 4). The presence of aldose reductase in dog lens epithelial cells was confirmed by RT-PCR (Fig. 4). With the RT-PCR technique, aldose reductase mRNA was clearly detected in dog lens epithelial cells, and cells cultured in the medium containing 50 mM galactose for 2 weeks continued to express aldose reductase mRNA. Compared with the mRNA level of GAPDH used as an internal standard, the expression of aldose reductase appeared higher in cells exposed to high galactose compared with those cultured in control medium. The expression of aldose reductase was reduced to normal levels in cells cultured in galactose medium containing the aldose reductase inhibitor AL 1576.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
623
Fig. 3. Galactitol accumulation in dog lens epithelial cells. Dog lens epithelial cells were cultured for 3 days in DMEM medium supplemented 20% FCS (Control), the same medium containing 50 mM D-galactose (galactose, light gray bar), and the medium containing 50 mM D-galactose and 1 mM AL 1576 (galactose and AL 1576, dark gray bar).
4. Discussion It has recently been suggested that apoptosis of lens epithelial cells is a key process in the development of many types of cataracts [14]. Apoptosis of epithelial cells has been reported to be the earliest event in the formation of experimental cataracts associated with hydrogen peroxide or ultraviolet light-induced cataracts [15,16]. The earliest changes in sugar cataract formation also occur in lens epithelial cells [8] and Gonzalez et al. [17] have reported that apoptosis of lens epithelial cells is induced when lenses are cultured in vitro in medium containing either high glucose or galactose. The present study confirms that cultured dog lens epithelial cells, when exposed to high galactose environment, also undergo apoptosis. Moreover, this galactose-induced apoptosis is inhibited by the addition of aldose reductase inhibitor suggesting that the induction of apoptosis is linked to the formation and/or accumulation of the sugar alcohol galactitol. Intracellular vacuole formation in lens epithelial cells cultured in galactose medium has been previously reported by a number of investigators [6,7]. While a
Fig. 4. RT-PCR of aldose reductase and GAPDH. Lanes 1 – 3 represent dog lens epithelia cells cultured for 2 weeks in DMEM medium supplemented 20% FCS, the same medium containing 50 mM D-galactose, and the medium containing 50 mM D-galactose and 1 mM AL 1576, respectively.
624
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
similar formation of multiple small vacuoles was observed in dog lens epithelial cells cultured for 2 weeks in galactose medium, the present results further suggest that this vacuole formation is linked to the induction of apoptosis. Although some cells formed vacuoles, vacuoles were absent in many other cells at this stage and they appeared to be microscopically normal. Nevertheless, despite their normal microscopic appearance, obvious DNA changes were observed in these cells. Essentially all cells were positively stained for apoptosis with the TUNEL techniques. This staining was also confirmed by DNA fragmentation. These results indicate that DNA changes in the cultured dog lens epithelial cells precede the visible morphological change of vacuole formation. In the galactose-induced apoptosis of dog lens epithelial cells, aldose reductase appears to play an important role. Aldose reductase expression was easily detected in dog lens epithelial cells cultured in the regular DMEM medium. These cells rapidly accumulated galactitol when cultured in galactose-containing medium. While the abnormal accumulation of sugar alcohols may affect various cell functions, the expression of aldose reductase is slightly increased in cells cultured in galactose medium. Aldose reductase expression is normalized when similar cells are cultured in galactose medium containing the aldose reductase inhibitor AL 1576. At the same time, galactitol levels in these cells are reduced and staining for apoptosis is normalized. This suggests that apoptosis of lens epithelial cells may be linked to osmotic insults induced by abnormal accumulation of sugar alcohols. While the present study does not establish whether epithelial cell apoptosis is the key process that results in sugar cataract formation, the present results indicate that apoptosis precedes vacuole formation and is linked to osmotic changes that are induced by the abnormal accumulation of sugar alcohols. While the present culture conditions under which apoptosis and subsequent vacuole formation can be detected appear extreme (long-term 1–2 week exposure to 50 mM of galactose), similar morphological change (intracellular vacuole formation) has been documented to rapidly occur within 3 days in the lens epithelial cells of galactose-fed rats [8]. This indicates that the process of apoptosis may proceed prior to any morphological changes such as vacuole formation in vivo in lens epithelial cells. This may also suggest the importance of aldose reductase-induced apoptosis of lens epithelial cells in the formation of sugar cataracts.
References [1] J.H. Kinoshita, Mechanisms initiating cataract formation, Proctor Lecture, Invest. Ophthalmol. 13 (1974) 713–724. [2] P.F. Kador, The role of aldose reductase in the development of diabetic complications, Med. Res. Rev. 8 (1988) 325–352. [3] A.Y. Lee, S.K. Chung, S.S. Chung, Demonstration that polyol accumulation is responsible for diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the lens, Proc. Natl. Acad. Sci. USA 92 (1995) 2780 – 2784. [4] Y. Akagi, P.F. Kador, J.H. Kinoshita, Immunohistochemical localization for aldose reductase in diabetic lenses, Invest. Ophthalmol. Vis. Sci. 28 (1987) 163 – 167.
M. Murata et al. / Chemico-Biological Interactions 130–132 (2001) 617–625
625
[5] M. Datiles, H. Fukui, T. Kuwabara, J.H. Kinoshita, Galactose cataract prevention with sorbinil, an aldose reductase inhibitor: a light microscopic study, Invest. Ophthalmol. Vis. Sci. 22 (1982) 174–179. [6] M. Nagata, T.C. Hohman, C. Nishimura, C.M. Drea, C. Okiver, W.G. Robison, Jr, Polyol and vacuole formation in cultured canine lens epithelial cells, Exp. Eye Res. 48 (1989) 667 – 677. [7] L.-R. Lin, V.N. Reddy, F.J. Giblin, P.F. Kador, J.H. Kinoshita, Polyol accumulation in cultured human lens epithelial cells, Exp. Eye Res. 52 (1991) 93 – 100. [8] W.G. Robison, Jr, N. Houlder, J.H. Kinoshita, The role of lens epithelium in sugar cataract formation, Exp. Eye Res. 50 (1990) 641 – 646. [9] S. Sato, Y. Takahashi, M. Wyman, P.F. Kador, Progression of sugar cataract in the dog, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1925 – 1931. [10] S. Sato, K. Mori, M. Wyman, P.F. Kador, Dose-dependent prevention of sugar cataracts in galactose-fed dogs by the aldose reductase inhibitor M79175, Exp. Eye Res. 66 (1998) 217 – 222. [11] S. Sato, P.F. Kador, NADPH-dependent reductases of the dog lens, Exp. Eye Res. 50 (1990) 629–634. [12] M.J. Lizak, E.F. Secchi, J.W. Lee, S. Sato, E. Kubo, Y. Akagi, P.F. Kador, 3-FG as substrate for investigating flux through the polyol pathway in dog lens by 19F-NMR spectroscopy, Invest. Ophthalmol. Vis. Sci. 39 (1998) 2688 – 2695. [13] E.F. Secchi, M.J. Lizak, S. Sato, P.F. Kador, 3-Fluoro-3-deoxy-D-galactose: a new probe for studies on sugar cataract, Curr. Eye Res. 18 (1999) 277 – 282. [14] W.C. Li, J.R. Kuszak, K. Dunn, R.R. Wang, W. Ma, G.M. Wang, A. Spector, M. Leib, A.M. Cotliar, M. Weiss, Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals, J. Cell Biol. 130 (1995) 169 – 181. [15] A. Spector, G.M. Wang, R.R. Wang, W.C. Li, N.J. Kleiman, A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action, Exp. Eye Res. 60 (1995) 483–493. [16] W.C. Li, A. Spector, Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract, Free Radic. Biol. Med. 20 (1996) 301 – 311. [17] K. Gonzalez, S. McVey, J. Cunnick, I.P. Udovichenko, D. Takemoto, Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry, Curr. Eye Res. 14 (1995) 269 – 273.
.