Effects of naphthalene metabolites on cultured cells from eye lens

Free Radical Biology & Medicine, Vol. 10, pp. 255-261, 1991 0891-5849/91 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1991 Pergamon Press plc

Original Contribution

EFFECTS OF NAPHTHALENE METABOLITES ON CULTURED CELLS FROM EYE LENS

PAUL RUSSELL, TAKAH1KO YAMADA, GUO-TONG X U , * DONITA GARLAND, and J. SAMUEL ZIGLER, Jr. National Eye Institute, National Institutes of Health, Bethesda, MD and *Alcon Research Laboratories, Fort Worth, TX

(Received 5 July 1990; Revised 13 September 1990; Accepted 30 November 1990)

Abstract- -Naphthalene is toxic to the eye and results in opacification of the lens. To investigate the metabolic events that may be occurring in the lens epithelial cells, a cell line of lens from a transgenic mouse was incubated with various metabolites of naphthalene. Naphthoquinone at 50 p~M was toxic to most cells with a depletion of glutathione levels noted within 6 h of incubation. At 10 ixM, naphthoquinone caused an increase in specific activity of the enzyme DT-diaphorase. This enzyme is thought to be a defense against quinones since semiquinone formation is thought to be lessened. Naphthalene-1,2-dihydrodiol at 50 IxM also caused an increase in the specific activity of the DT-diaphorase, while at 10 p.M no apparent change occurred in the cells. Although there was evidence of metabolic alterations in the cells with the metabolites of naphthalene, the protein profile by two-dimensional gel electrophoresis did not change and there was no indication of an increase in carbonyl formation in the soluble proteins of the cells. These experiments indicate that the metabolites of naphthalene can cause alteration in the metabolism of the lens cells but may not cause apparent changes in the major proteins within the lens epithelium.

Keywords--Naphthalene, Diaphorase, Glutathione, Glutathione reductase, Lens, Free radicals

INTRODUCTION

The eye and, in particular, the lens of the eye are se- verely affected following naphthalene ingestion, l These toxic effects have been reported in a variety of animals as well as man. 2-4 It is not clear which of the metabolites of naphthalene contribute to the toxic effect; however, it is thought that 1,2-naphthoquinone is probably responsible for much of the damage: Naphthoquinone toxicity may result from direct alkylation of the sulfhydryl groups of proteins or from free radical products generated following the one-electron reduction of the quinone to the semiquinone. 6 There is some indication that the metabolism of naphthalene starts in the liver 7 and metabolites are then carried in the blood to other tissues. In the eye, there is evidence that naphthalene- 1,2-dihydrodiol is present in the aqueous humor of the eye when rats are fed naphthalene, s The reason for the interest in this cataract is the similarity in clinical findings with subcapsular senile cataract in man. 9 Aging- related cataract in humans is thought to be the result of oxidative damage, lO,ll

To understand more fully the effects of naphthalene metabolites on epithelial cells, cell cultures were ex-

Address correspondence to Paul Russell, Bldg. 6, Rm. 228, Na- tional Institutes of Health, Bethesda, MD 20892.

255

posed to either naphthalene-l,2-dihydrodiol or to 1,2- naphthoquinone. Naphthalene-1,2-dihydrodiol is formed from the 1,2-epoxide and is subsequently converted by dihydrodiol dehydrogenase (EC 1.3.1.20) to naphthalene-l,2-diol. The 1,2-diol is then thought to autooxidize to 1,2-naphthoquinone. One of the primary cellular defenses against quinones is the enzyme DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2), which is gener- ally found in the cytoplasm of a cell and catalyzes the two electron reduction of various quinones using either NADH or NADPH as a cofactor. 12 Thus, naphthoquinone can be converted back to the 1,2-diol by the action of this enzyme, which avoids the semiquinone radical formed by one-electron reduction. The diol can then be eliminated by conjugating systems. DT-diaphorase is inducible by a number of substrates 13"14 and is inhibited by 3,3'-methylene-bis(4-hydroxycoumarine) (dicumarol).

Glutathione is believed to be the major conjugating agent in the lens involved in the elimination of such xe- nobiotics and also can reduce disulfide bonds that have been formed by naphthoquinone or other free radicals that might be generated. ~5 Glutathione reductase (EC 1.6.4.2) is essential for the maintenance of glutathione levels in the lens. 16 To assess the relationships among glutathione, glutathione reductase, and DT-diaphorase, the mouse lens epithelial cell line aTN4 was incubated with the naphthalene metabolites. This cell line was de-

256 P. RUSSELL et al.

rived from the lens of a transgenic animal and has the T-antigen of SV-40 linked to the ~xA-crystallin pro- moter. 17 This cell line expresses all the mouse lens a- crystallins in culture and was used as a model system in an attempt to decipher the mechanism of lens cell damage.

MATERIALS AND METHODS

The cell line ~TN4 clone 1.1 was used for this study. The cells were incubated in Dulbecco's MEM with 4500mg/mL of glucose and 10% fetal bovine serum. Cultures were treated by adding either the dihydrodiol or naphthoquinone (in 20% ethanol) to fresh culture medium and then incubating the cells for various periods of time. The cells were harvested by scraping and were washed two times in phosphate-buffered saline. The cell pellet was resuspended in 200 I~L of water and sonicated by two short pulses on a W-380 sonicator set at tip setting 4, 50% duty cycle and continuous pulse (Heat Systems-Ultrasonics, Inc., Farmingdale, NY). The son- icate was centrifuged at 14,000 x g for 15 min and the supematant was recentrifuged for an additional 10 rain. The supematant was then assayed for enzyme activities and for glutathione. For DT-diaphorase, the assay system was a modification of the method used for liver samples.18 In the well of a 96-well microtiter plate was added 250 ~L of 25 mM Tris-HCl (pH 7.5), 0.2 mM 2,6 dichlorophenol-indophenol and 0.2% Tween-20. To the appropriate wells, 20 tLL of 1 mM dicumarol was added and 5 I~L of a 1 to 3 dilution of the sample was then added. Tris buffer was added to the wells so that the final volume in each well was 275 ~L. To start the assay, 25 ~L of NADH (2.68 mg/mL) was added. The change in optical density at 620 nm was followed for 4 min. Duplicates of each sample were run and averaged together. The DT-diaphorase activity was the difference between the total diaphorase activity and that obtained when dicumarol was present. Three separate sets of experiments were run with each set having its own control cells.

The glutathione was also assayed using the microtiter wells. The assay was identical to the method of Sed- lak and Lindsay 19 except the volumes of all components were scaled down so that the final volume was 200 ixL. Triplicates of each sample were run and averaged together. Three separate sets of experiments were done. The glutathione reductase was also scaled down from the method of Bayoumi and Rosalki. 2° Briefly, to each well of the microtiter dish was added 185 p.L of 0.1 M NaPO4 with 1 mM EDTA (pH 7.5), 20 p~L of oxidzed glutathione (16 mg/mL) and, where appropriate, 10 ixL of sample. The reaction was started with the addition of 25 p.L of NADPH (3 mg/mL). The change in optical density at 340 nm was followed for 4 min. Each sam-

pie was done three times and there were three separate sets of experiments.

Two-dimensional electrophoresis was done on the urea soluble fraction of the cell pellet. Instead of soni- cating in water, the cells were sonicated in 8 M urea. The two-dimensional electrophoresis was done on PhastSystem as previously described. 21 The protein con- tent of the supematants was measured according to the method of Bradford. 22 Cell counting was done on a Coulter counter (Coulter Electronics Inc., Hialeah, FL).

HPLC analyses were performed on a LKB HPLC system using a Beckman Ultrasphere C-18 reversed phase column. A linear gradient of 5-50% acetonitrile in H20 was used to elute the naphthalene metabolites. Samples were prepared from the culture medium. The medium was centrifuged to remove cells and the proteins were precipitated with 20% trichloroacetic acid. Aliquots of the acid soluble fraction were partially neu- tralized to about pH 6 with NaOH, filtered through a 0.22 micron filter, and injected onto the column. The column was eluted at a flow rate of 1.0 mL/min and the eluate was monitored continuously at 250 nm. Extracts were made of the cells, and protein carbonyl derivatives were determined by reaction of the soluble proteins with 2,4 dinitrophenyl hydrazine and subsequent separation of the protein hydrazone derivatives. 23

Long-Evans rats weighing 125 gms were fed 0.5mg/ kg of naphthalene for two days and then the dosage was increased to 1 mg/kg/day. The rats were sacrificed at days 3, 7, and 14 and the livers were dissected. Weighed portions of liver were washed twice in phosphate-buffered saline. The diaphorase assay was done with the soluble liver sample exactly as described earlier. 18

RESULTS

The dihydrodiol did not appear to be toxic to the cells and doubling times remained identical with control cells. The cells retained their normal morphology throughout the entire 72 h. Therefore, for both the dihydrodiol and the naphthoquinone incubations, the levels of glutathione, glutathione reductase and DT-diaphorase were measured at 6, 24, 48, and 72 h after a bolus of the metabolite. Since induction of the enzyme aldose reductase in this cell line in another model system that inves- tigated hyperosmotic conditions was not pronounced until 24--48 h, it was hoped that the time points chosen would give an indication of any enzyme change. The lens cells, however, were markedly altered when either 10 p,M or 50 p~M naphthoquinone was added into the medium. In both cases, many of the cells rounded up. There were some cells that did survive the initial bolus at both concentrations of naphthoquinone. Although considerably changed from the normal cuboidal mor-

Lens cells and naphthalene metabolites 257

20O

~ 160 t

l

CONTROL lO/,di 50/~it WITH DICUMAROL ~ DT DIAPHORbSE

Fig. 1. Total diaphorase activity of cells incubated for 72 h with naphthalene- 1,2-dihydrodiol. The diaphorase activity that was not inhibitable by dicumarol is shown with the filled bars. The DT-diaphorase activity, that is, the diaphorase activity inhibited by dicumarol, is shown with the open bars. Total diaphorase activity was the sum of these two activities.

phology, these cells were present even at the end of the 3-day incubation period. The control cells increased in number about 15 fold in 3 days. At the same time point, the number of cells in the 10 ixM naphthoquinone were only 3% more than the number plated initially. With the higher concentration of the metabolite, fewer cells remained than were initially present.

Pellets of cells incubated with 50 ~xM naphthoquinone taken at 6 h had a dark brown color. The 10 IxM naphthoquinone pellet was tan, but still much darker than the control pellet. There was no indication of any brown color with the dihydrodiol treatment. The brown material from the 50 IxM naphthoquinone-treated material was not soluble in water; however, there was some slight color associated with the urea soluble material. Chloroform-methanol or acetone extraction of the water insoluble material did not change the color of the pellet.

HPLC of the cell medium showed that for the 50 ~M dihydrodiol sample, 41 I~M remained at the 6-h time point. The level of dihydrodiol remained at about this level for the rest of the experiment. HPLC of the spent culture medium for the naphthoquinone incubations as well as medium incubated without cells indicated that the level of this compound was very low at the end of 6 h, suggestive of the very reactive nature of the naphthoquinone. Although there was an interfering peak in the vicinity of the naphthoquinone peak partially ob- scuring the data, at most, only about 10% of the naphthoquinone could have remained at the 6-h time point.

The activity of diaphorase was measured in the cells. In control cells, the DT-diaphorase activity was about one-third of the total diaphorase activity (Fig. 1). The total diaphorase activity increased with dihydrodiol in-

cubation, but the increase was inhibitable by dicumarol, that is, only the DT-diaphorase was elevated. The activity that was not inhibited by dicumarol was not signifi- cantly changed with either this metabolite or with the naphthoquinone incubation.

With the dihydrodiol, the 10 I~M level did not signif- icantly change any of the parameters measured (Fig. 2). At 50 txM dihydrodiol, the glutathione was decreased after 72 h and the activity of the DT-diaphorase was increased about three fold over the control levels. In contrast, the naphthoquinone affected the cells at both the high and low dosages (Fig. 3). At 50 ~xM after 24 h, neither the level of glutathione nor the activities of glutathione reductase nor DT-diaphorase was measur- able. The failure to detect these parameters may be the result of the very low number of cells that survived, The sensitivity of the assay systems may not have been ade- quate to measure such small amounts of material. The DT-diaphorase increased about three fold over the control level at the lower dose of naphthoquinone after only 24 h and remained elevated for the next 48 h.

In order to compare the change in activity of DT-diaphorase in the cell line to changes in the whole animal, rats were fed naphthalene and the specific activity in the liver of this enzyme was measured after 3 days, 1 week, and 2 weeks of naphthalene feeding. The initial specific activity of DT-diaphorase was about 10 times higher in the rat liver than in the lens cells. Nevertheless, both the liver and the cells showed a similar increase of about seven fold in specific activity at the 3-day time point (Fig. 4).

There were some lens cells which survived the high dose of the naphthoquinone and although the number was not very large, cells were collected and run on two- dimensional gel electrophoresis. The pattern of the cellular proteins and polypeptides that were separated first by urea isoelectric focusing and then by SDS PAGE appeared to be similar to the control pattern (Fig. 5). No striking differences were found at 48 h among the control, dihydrodiol-treated, or the naphthoquinone-treated cells that survived the initial bolus. There was no indication of any protein that might be induced by these metabolites although a polypeptide that was only a mi- nor component of the total cellular protein could easily escape detection with this system. The lack of response to the naphthalene metabolites can be compared to the rapid and characteristic effect seen when the cells are incubated in high osmolarity medium. 24 In that case, the enzyme aldose reductase is induced and can be seen easily in both one- and two-dimensional electrophoresis patterns.

Because naphthoquinone is thought to cause oxidative stress by free radical mechanisms, the levels of protein carbonyl derivatives were measured. The carbonyl levels are thought to be measures of oxidative


INCUBATION WITH N A P H T H A L E N E - 1,~--DIHYDRODIOL

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i 0.600 0.800.

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GLUTATHIONE REDUCTASE

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Fig. 2. Results of measurements on etTN4 cells. The top graph shows the glutathione levels in nmol/l~g protein in the control cells (open bars), cells incubated with 10~M (single hatched bars) and 501zM (cross-hatched bars) naphthalene-l,2-dihydrodiol. The grouping of results are for the 6, 24, 48, and 72-h time points. The middle graph shows the activity of naphthalene reductase (units/mg protein) in the cells under the same experimental conditions. The bottom graph shows the DT-diaphorase activity (units/mg protein). The single asterisk indicates a significant difference of p<.05 by Student's t test.

damage. 25'26 No consistent increases were found in the

carbonyl derivatives of the soluble proteins when the cells were incubated with the naphthalene metabolites. While the absolute values varied slightly between experiments , control and experimental results were the same within experimental error.

DISCUSSION

The el iminat ion of reactive compounds and free radicals in the lens is extremely important since cells can- not be removed from within the capsule of the lens throughout life. The lens cells must min imize the dele-


INCUBATION WITH NAPHTHOQUINONE

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Fig. 3. Results of measurements on ctTN4 cells incubated with naphthoquinone. The graphs have identical layouts as in Fig. 2. The double asterisks indicate significant differences of p<.01 by Student's t test.

terious effects of such agents or risk eventual cataract formation. The metabolism of naphthalene can result in the formation of very reactive semiquinone radicals as well as other possibly toxic intermediates, but naphthalene metabolites can be removed from cells and the lens by conjugation with other compounds. Naphthoquinone can undergo two electron reduction mediated by DT-diaphorase thereby avoiding formation of the semiquinone radical and producing the 1,2-diol which can be conju-

gated and eliminated. Thus, DT-diaphorase is believed to ameliorate damage from quinones by competing with the one-electron reduction mechanisms. The findings with the lens cells in culture are consistent with this hypothesis and suggest that the cells react to the presence of metabolites of naphthalene by increasing the specific activity of an enzyme that has the potential of detoxify- ing intermediates in this pathway. This enzyme is also increased in a similar fashion in the livers of animals


5.

4.

3.

2-

1.

0

• , \',~

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3 7 14 DAYS

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Fig. 4. DT-diaphorase activities for samples of rat liver from control animals (open bars) and animals fed naphthalene (hatched bars) for 3 days, 1 week, and 2 weeks. The activity is in units/l~g protein. The double asterisks indicate significant differences of p < .01 by Student's t test.

fed naphthalene. The results suggest that when the level of dihydrodiol

was at 10 p~M, the conjugation and elimination of the 1,2-diol by the cells may have been rapid enough to prevent cellular damage. The diaphorase activity did not increase above the normal value. At the higher concentration, the DT-diaphorase activity did increase suggest- ing at least some of 1,2-diol might autoxidize to naphthoquinone before it could be conjugated and de- toxified by the cell. With the 10 txM naphthoquinone, the specific activity of the DT-diaphorase rapidly increased. Higher levels of this compound resulted in rapid death of most of the cells in the culture, perhaps indi- cating that the cell could not sufficiently recover from this challenge.

Additional experiments were done using the naphthalene-l,2-diol. At low concentrations, the cells contin-

ued to divide normally although the DT-diaphorase increased in about the same manner as with the 50 txM dihydrodiol (data not shown). A higher concentration resulted in an increase in doubling time for these cells to 36 h compared to the normal 17 h and more rapid increases in the DT-diaphorase activity. The naphthalene-l,2-diol is thought to autooxidize to the naphthoquinone; therefore, the data from these experiments is difficult to assess since the exact concentration of start- ing material is unknown. HPLC data indicated that the commercially available compound contained several peaks, so that results from these experiments although suggestive, are not definitive since the initial concentration of metabolite might have varied from one experiment to the next.

One of the more curious findings was the apparent lack of major changes in the protein pattems that were obtained on the two dimensional gels. The lack of clear differences with all the compounds tested suggests that either the DT-diaphorase was such a minor component that the change in protein amount was not detectable or that the increase in specific activity might not be an ac- tual induction of this enzyme. Additional experimenta- tion is needed to define the reasons for these findings.

It has been demonstrated in other systems that the oxidation of proteins in vivo 25 and in vitro 26 is accom- panied by the generation of protein carbonyl derivatives. The lack of carbonyl in the soluble protein of the lens cells contrasts with the presence of these compounds in the aged brunescent lens.l° One of the possible expla- nations for this discrepancy might be the level of proteolysis. Higher level of proteases are present in the epithelial cells compared with the lens fiber cells in vivo. 2v'2s Because of higher levels of proteolysis, epithelial cells might be more resistant to the oxidative stress of the naphthalene metabolites than the fiber cells even though the epithelium would presumably be exposed to

A B C

68 kD •

46 kD -

30 kD -

20 kD -

Fig. 5. Two-dimensional gel electrophoresis of the urea soluble proteins from control cells (A), cells incubated with 50 p.M naphthalene-1,2-dihydrodiol (B), and cells incubated with 50 IxM naphthoquinone (C) for 2 days. The direction of the isoelectric focusing gradient in the first dimension is shown as are the positions of the molecular weight markers for the second dimension.


a h igher level o f these compounds entering the lens f rom

the aqueous humor o f the eye.

These results show conc lus ive ly that lens cells in cul-

ture will increase DT-diaphorase act ivi ty when chal-

lenged with metabol i tes o f naphthalene. These cultured

cells appear to be s imilar to the l iver cells in this re-

spect. The data are consistent with the hypothesis that

this enzyme acts in cells to detoxify these quinones. In

contrast, the act ivi ty o f glutathione reductase does not

increase in this system and appears to maintain cel lular

glutathione as long as the cells remain viable. The lens

epithelial culture system seems to be a very good one to

study short- term changes in the general metabol i sm of

the lens cells.

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Effects of naphthalene metabolites on cultured cells from eye lens