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Some changes in the lens of the dimethylsulphoxide-fed rabbit
Exp. Eye Res. (1972) 14,91-98
Some Changes in the Lens of the Dimethylsulphoxide-fed RUTH ,Vu$feld
VAN HEYNINGEN
Laboratory
Rabbit*
AND J. J. HARDING
University of Oxford, England
of Ophthalmology,
(Received20 April 1972, London) We have confirmed that there is a loss of y-cry&&n and an increase in water-insoluble protein in the lens of the dimethylsulphoxide (DMSO)-fed rabbit (Wood, Sweet, van Dolah, Smith and Contaxis, 1967). These changes were confined to the lens nucleus. The waterinsoluble protein which results from DMSO feeding is soluble in 7 M urea, unlike the waterinsoluble fraction isolated from the human cataraotous lens, which is largely urea-insoluble. The ammo acid content of the urea-soluble protein from the lens of the DMSO-fed rabbit appears to be the same as that from the control lens. Glutathione and protein thiol, and lactic acid, were also measured in the lens of the control and DMSO-fed rabbit. There was no appreciable difference.
1. Introduction In a study of the effect of dimethylsulphoxide (DMSO) feeding upon the lens of the rabbit (Wood, Sweet, van Dolah, Smith and Contaxis, 1967) the most significant tinding was a loss of soluble protein and a corresponding increase of insoluble protein. This change appeared to be an effect on y-crystallin, which was the only component of the soluble protein to be decreasedin concentration (Wood et al., 1967). We have made a further study of the changesin lens proteins, and have also measuredglutathione and lactic acid concentrations. We used adult rabbits (aged 16 to 32 weeks), introduced the DMSO gradually into the drinking water, and supplemented the diet with cabbage, in the hope of producing gradual changesin the lens, resembling nuclear cataract in man. This aim was partly fulfilled, in that the changeswere slight, came on gradually and resembled in some respects early nuclear cataract in man; but after the first few months the condition remained static.
2. Materials
and Methods
Dutch rabbits were fed on pelleted Diet 18 (Oxoid Ltd., Southwark Bridge Road, London S.E.l), with about 60 g cabbagedaily and drinking water ad lib. Twenty rabbits in five litters were used; each litter was divided into control and experimental animals; all comparisonswere madebetweenlitter mates.At the age of 16 to 32 weeksan aqueous solution of DMSO (BDH ChemicalsLtd., Poole,Dorset, England) wassubstituted for the drinking water of the experimental animals. For two weeksthe concentration of DMSO was 1.70,d (v/v), for the next two weeks 3.3% (v/v) and from then on 5% DMSO was given.
Animals were weighedweekly and the lenseswere examined by slit-lamp and ophthalmoscopeevery 2-3 weeks, after dilatation of the pupil with Mydrilate cyclopentolate. Rabbits Separation
were killed
with intravenous
Nembutal.
of lensprotein by solubility in water and urea
Each lens was weighed and dissected into cortex and nucleus. Each part was weighed, ground with 9 volumes of water and centrifuged at 10,000 g for 90 min at 4°C. The concentration of protein in the supernatant was assayed by the biuret reaction (Layne, 1957), * Address for reprints: Nuffield Laboratory England.
of Ophthalmology, 91
Walton Street, Oxford OX2 6AW,
R.
92
VAN
HEYNINGEN
AND
J. J. HARDING
using freeze-dried rabbit lens protein as standard. The water-insoluble protein was washed four times on the centrifuge with 5 ml water each time. For the fifth washing, the protein was suspended in 5 ml water and left at 4°C overnight before centrifugation in a weighed polythene centrifuge tube. The wet precipitate was frozen and dried in vacua and the centrifuge tube reweighed to give the weight of the water-insoluble protein. Two ml 7 M urea was added to the water-insoluble protein and after thorough disintegmtion with a glass rod, the mixture wa,s left overnight at 4°C. After centrifugation the supernatant was removed, and the urea-insoluble precipitate was washed twice with 5 ml 7 M urea and six times with 5 ml water. It was then frozen, dried and weighed. The weight of the urea-soluble protein was taken as the difference between that of the water-insoluble and the urea-insoluble. Urea-soluble protein was precipitated from solution by the addition of 10 volumes of acetone. After 16 hr at 4°C the precipitate was collected by centrifugation, washed four times with acetone and dried. Precipitation
of protein. with trichloroacetic
acid
(TCA)
Lenscortex or nucleuswasground with nine times the weight of 6% (w/v) TCA. Protein was allowed to precipitate at 4°C usually overnight, before collection by oentrifugation. The precipitate was used for the estimation of protein thiol and the supernatant for glutathione and lactic acid. Glutathione
This was measuredusing the 5,5’-dithiobis-(2-nitrobenzoic acid) reagent of Ellman (1959), as describedby Harding (1970). DMSO at concentrations of up to 5% doesnot interfere. Protein
thiol
The protein precipitated by trichloroacetic acid wasdissolved in 6 M guanidinium chloride. Portions of this solution were taken and assayedfor thiol in 7 M urea, using the method describedfor glutathione. Isolation of u-, /3- and y-crystal&s The crystallins were isolated from the nuclei of one pair of rabbit lenses(pooled), u crystallin wasseparatedby isoelectricprecipitation andj3-and y-crystallins by gel titration on Sephadex 675 (Pharmacia, Great Britain, Ltd., Uxbridge Road, London W5), as describedby Harding (1969).In other experimentsthe initial precipitation of a-orystallin wasomitted and only y-crystallin wascompletely separatedon the Sephadex675 column. AmiNo acid analysis
Analyses were performed with an amino acid analyser (Locarte CompanyLtd., Emperor’s Gate, London S.W.7), by the method of Spackman, Stein and Moore (1958). Samplesof protein (about 2 mg) were hydrolyzed in constant-boiling HCl(2 ml). Hydrolysis was conducted for 18 hr at 108”C,in vacua. digestions The tryptic susceptibility of the proteins of rabbit lens was studied by the method of Harding (1972), using either the whole nucleus (one experiment), half the nucleus (two experiments) or the cortex (one experiment). In each casematerial from a DMSO-fed rabbit was comparedwith that from a litter-mate control killed at the sametime. Tryptic
THE
LENS
OF
THE
DMSO-FED
RABBIT
93
3. Results General condition of animals The rabbits on DMSO lost weight for the first weeks on the diet. Subsequently they did not put on weight as fast as the controls and laid down lesssubcutaneousfat, but they appeared quite healthy. They had a bad smell, presumably of dimethyl sulphide (seeWood, 1971) and other metabolic products of DMSO. Refraction of eyes The refraction of the two eyes of an animal, control or experimental, measuredwith a retinoscope, differed little. Refraction was first measured after the experimental animals had been 12 to 17 weeks on DMSO. The control values were from +2-O to +3*7 dioptres and theexperimental values from -0.5 to+24 dioptres; theexperimental values were 1.3 to 4.2 dioptres lessthan the litter-mate controls. Six months or more later the refraction of surviving rabbits was little changed. Table I gives someof the values found. TABLE
I
DMSO-fed rabbits and litter-mate controls. Refraction of eyes and cmncen&ation of proteins in the lens nucleus First
experiment: Duration
12-17
wks
DMSO-fed 20, 28, 32 (3) 14*3 (3) +0.5-&0.2 (3) 497&34 (3)
Age at start of experiment (wks) Duration of experiment (wks) Refraction of eyes (dioptrea) Lens weight (mg) Lena nucleus Weight (mg) Total protein (g y. wet wt) Water-sol. protein (g y. wet wt) Water-insol. protein (g y. wet wt) Urea-sol. protein (g y. wet wt) Seed
experiment Duration
: 48-52
Control
196fll 44.6kl.5 33.0-J= 1.4 11*6f3 10.6, 14.4
Difference: DMSO-fed minus
20, 28, 32 (3) 14*3 (3) +3.0*0.3 (3) 494&35 (3)
(3) (3) (3) (3) (2)
220-&28 43.9k4.7 37*9&3 6.013 2.5, 9.4
(3) (3) (3) (3) (2)
16, 20, 27 49~2 +2.9*1.0 534, 570 one cortex
(3) (3) (3) (2) lost
-2.5f0.7
control
(3)
+0.7*3.7 (3) -4.953 (3) +5*6&2.6 (3) +8.1, +5*0(2)
wks 16, 20, 27 (3) wk2 (3) +1.0*0.4 (3) 562&32 (3)
Age at start of experiment (wks) Duration of experiment (wks) Refraction of eyes (dioptres) Lens weight (mg)
Lens nucleus Weight (mg) Total protein (g y0 wet wt) Water-sol. protein (g y. wet wt) Water-insol. protein (g y. wet wt) Urea-sol. protein (g y0 wet wt)
One of each pair of litter-mates weeks (second experiment). Result,8 are expressed as mean
261fll 56417.9 41.257.2 15.612.0 15.5-&2.0
was given f
standard
5% DMSO deviation
(3) (3) (3) (3) (3)
259*13 (3) 52*8f 10.0(3) 44.5f9.3 (3) 8.3&0.6 (3) 8.256 (3)
for 12-17
weeks
(first
(no. of determinations).
-1.9*1.0
(3)
+4*051.3 -3.3h2.2 +7.3&0*5 +7.3&0.6
(3) (3) (3) (3)
experiment)
or for 48-52
94
Ii.
VAN
HEYNINGEN
AND
J. J. HARDING
Intraocular pressure The tension in both eyes of 5 experimental and 5 control rabbits was measured by applanation tonometry and the readings converted to pressure in mmHg (Moses, 1958). There was no significant difference. In the eyes of the experimental rabbits the average value was 15.4 mmHg (range 13.8 to 16.6) and in the control 16.5 mmHg (range 13.8 to 17.7). Visible changes in the lens of the DMSO-fed
rabbit
These were the same as those described by others (Rubin and Barnett, 1967 ; Wood et al., 1967), but did not advance beyond the stage at which the periphery of the lens nucleus could be clearly seen. After one year on DMSO the condition of the lenses of surviving rabbits had remained static for many months, and the nuclei of the lenses of the control rabbits were gradually becoming demarcated and clearly visible. The effect of ageing on the appearance of the lens nucleus was less marked, but similar to that of DMSO-feeding. Lens weight There was no difference in the wet weight of control and experimental lenses.The demarcation between cortex ad nucleus was fairly distinct, the cortex being soft and the nucleus hard and sticky. We could detect no difference between the size or consistency of the nucleus of the experimental and of the control lenses. One experimental and one control lens from three pairs of rabbits were freeze-dried and re-weighed. All these animals came from the samelitter, aged 72 weeks at death. There was no significant difference in the dry weight of the lens, all values falling between 213 and 225 mg. The percent dry weight of all the lenseswas close to 40.7 (range 39.4 to 41.7). Solubility of lensproteins In two comparisons between control and experimental lensesthere was verylittle difference in the concentration of water-soluble, urea-soluble and urea-insoluble proteins in the lens cortex. In further experiments, therefore, the proteins of the lens nucleus only were examined. Table I gives the percentage of water-soluble, water-insoluble and urea-soluble proteins of the lens nucleus. A lens nucleus of a DMSO-fed rabbit was compared with the nucleus of the lens of a litter-mate control killed at the sametime. In five of the six comparisonsthe weight of lens substance in the nuclei of the DMSO-fed and of control lensesdiffered by 5 mg or less. The comparisonsare divided into two groups, three pairs in which DMSO-feeding continued for 12-17 weeks, and three pairs in which DMSO was given to the experimental animals for about one year. The effect of DMSO-feeding was not accentuated by lengthening the experiment. Table I shows that the concentration of water-soluble protein in the lens nucleus decreasedfrom 37.9% to 33.0% and 44.5% to 41.2% when that of water-insoluble protein increased from 6.0% to 11.6% and 8.3% to 15.6%. The fact that the loss of soluble protein is not exactly the sameas the increaseof insoluble protein is probably due to experimental error, the two values being measuredby different methods. The water-insoluble protein prepared from the control lens was light and fluffy but
THE
LENS
OF
THE
DMSO-FED
RABBIT
95
that prepared from the experimental lens was granular and more lumpy. Both preparations were almost entirely soluble in 7 M urea. Table I shows that the waterinsoluble, urea-soluble fraction of the protein of the lens nucleus has approximately doubled as a result of DMSO-feeding. Water-soluble crystullins
in the lens nucleus
The proportions of a-, p- and y-crystallins isolated from lens nuclei of control and experimental rabbits are shown in Table II. In three comparisons the y-crystallin was 19-22% of the water-soluble protein in the control lens, and lo-16% in the experimental lens. Thus about one-third of the y-crystallin had disappeared from the watersoluble proteins of the experimental lens. II
TABLE
Percentage of a-, /3- and y-crystallin in the water-soluble protein from the lens nuclei DMSO-fed rabbits and litter-mute controls
5% DMSO in drinking water
+ -
cc
B
Y
55.3 54.0 L--
28.4 26.2 .-. -J
16.3 19.7
+ + -
90 81 87 78
of
10 19 13 22
Glutathione a& protein thiol in lens nucleus The amounts of glutathione and protein thiol in the Iens of the DMSO-fed and control rabbits are shown in Table III. Neither glutathione nor protein thiol was significantly changed as a result of DMSO feeding. TABLE
III
Glut&h&me, protein thiol and lactic acid in the lens of DMSO-fed rabbits and litter-mate controls
DMSO-fed
Control (~moles/g
Glutathione in nucleus I’rotein thiol in nucleus Lactic acid in cortex Lactic acid in nucleus
Results
are expressed
a8 average
wet wt)
3.21&@60(5) 44.3 kt3.0 (5) 15.9 ho.6 (5) 10.1 kO.5 (3)
values
& standard
deviation
3.63&1.09(S) 45.8 12.7 (6) 16.4 &0+3 (5) Il.5 kO.8 (3)
(number
of determinations).
96
R. VAN
HEYNINGEN
AND
Amino acid composition of water-insoluble
J. J. HARDING
urea-soluble proteins
The amino acid compositions of the water-insoluble urea-soluble protein from control and experimental rabbits are shown in Table IV. Each is the mean of three analyses. The apparent difference in lysine content is probably caused by the poor baseline in this region of the amino acid chromatogram. Otherwise only the difference in tyrosine content is marginally significant. TABLE
IV
Amino acid composition of the water-insoluble but urea-solubleprotein of the lens nucleus of the DMSO-fed and control rabbits
His
A% ASP Thr Ser GlU Pro G’Y Ala Val Met He Leu TV Phe Lp*
Results * Values
Lactic
ad
are mean of three analyses unreliable, see text.
DMSO-fed
Control
34.6 89.0 99.0 27.8 95.0 134 64.8 80.9 43.9 47.7 20.3 34.6 83.1 55.0 57.0 (33.1)
36.1 84.3 101 29.5 91.9 130 69.5 79.3 46.4 48.7 17.8 33.1 82.0 49.6 56.1 (43.8)
expressed
as residues/thousand
residues.
in the lens
Lactic acid was assayedin the nucleus and cortex of the lens of DMSO-fed rabbits and the corresponding controls. The results are given in Table III. There was no significant difference in either cortex or nucleus. Tryptic susceptibility of lens proteins The initial slopesof the progress curves for tryptic digestion were 0.77 to 1.05 ml 2 mu NaOH/g/min for the nucleus of DMSO-fed rabbit lens and 0.59 to 0.70 ml 2 mM NaOH/g/min for that of the litter-mate controls. The percentage increase in rate was 30, 34 and 50% for the three comparisons.The initial slopesof the progress curves for tryptic digestion of the rabbit lens cortex showed an increase from 0.40 ml 2 mu NaOH/g of cortex/min in the control to 0.50 ml 2 mM NaOH/g of cortex/min in the lens of DMSO-fed rabbit. Thus in all four experiments the proteins of DMSO-fed lens were digested more rapidly than litter-mate controls.
THE
LENS
OF
THE
DMSO-FED
RABBI7
97
4. Discussion The visible changes in the lens and the changes in refraction measured by retinoscopy were similar to those previously reported by Wood et al. (1967), Rubin and Barnett (1967) and others. The intraocular pressure was not affected by DMSOfeeding. The lens wet weight and dry weight were unaffected by DMSO-feeding and therefore the increase in refractive power is not caused by a higher protein concentration or larger lens; nor did slit-lamp examination reveal any change in lens shape. There was no significant change in lactic acid, reduced glutathione or protein thiol content. Wood et al. (1967) found no change in glutathione levels in older rabbits and only a small change in ten-week old rabbits. Our main positive findings concern the proteins of the lens. Wood et al. (1967) had shown that there was a loss of y-crystallin and an increase of insoluble protein in lenses from DMSO-fed rabbits. Our results (Table I) show that the increase of waterinsoluble protein is confmed to the urea-soluble fractions of this protein. This was unexpected because the urea-insoluble fraction increases in human cataract (Pirie, 1968). Unlike human lens or rat lens (Pirie, 1968; Harding, 1969) the normal rabbit lens contains relatively little water-insoluble urea-insoluble protein. In this respect it resembles cow lens (Dische, 1965). The different amounts of urea-insoluble protein isolated from lenses of different animals indicate that the lens proteins of some animals are more susceptible to oxidation than those of others. The small amount in rabbit and cow lens is probably largely composed of fragments of fibre membranes (Dische, 1965). The increased amount of urea-insoluble protein isolated from human cataractous lens is probably largely due to unfolding of the water-soluble proteins during cataractogenesis (Harding, 1972). Our results showed that the increase of urea-soluble water-insoluble protein occurs in the nucleus. As the amino acid composition of the water-insoluble urea-soluble protein from experimental lens is the same as that from control lens, it is probable that both consist of a similar mixture of lens crystallins, the extra urea-soluble protein of experimental lenses is thus a similar mixture to the original water-insoluble but, urea-soluble protein. The decreased proportion of y-crystallin found in the lenses of DMSO-fed rabbits by Wood et al. (1967) using cellulose electrophoresis was confirmed in the present work using gel filtration to isolate y-crystallin. Some y-crystallin may have aggregated to appear as higher-molecular-weight protein on gel filtration and/or it may have become water-insoluble but urea-soluble. A relatively small extra amount of y-crystallin in this fraction would be difficult to detect. Susceptibility to proteolysis has often been used to study conformational changes in proteins (Rupley, 1967) and has recently been used to confirm that protein unfolding occurs in human cataract (Harding, 1972). The results presented indicate that DMSO-feeding may cause unfolding of some lens proteins but insufficient lenses were available to establish such a conclusion. If the proteins had unfolded it is unlikely that this was caused directly by DMSO because DMSO is a poor denaturant of proteins (Tanford, 1969). Radioactivity, following cutaneous treatment of rabbits with 3H-DMS0, has been shown to reach all parts of the eye (Kolb, Jgnicke, Kramer and Schulze, 1967). Wood (1971) has recently summarized our current knowledge of the metabolic fate of DMSO. It is not known whether DMSO itself or metabolic product(s) reach the eye. The mechanism whereby the observed optical changes are induced in the lens by treatment of the animal with DMSO is also unknown.
98
R. VAN
HEYNINGEN
AND
J. J. HARDING
4CKNOWLEDGMENTS
We thank ProfessorDon C. Wood for reading the typescript, Mr T. H. Kirkham for the refraction and intraocular pressuremeasurements,Mr D. J. Potter for his excellent care of the animalsand Mr K. Rixon for skilled technical assistance. REFERENCES Dische, 2. (1965). Invest. Ophthal. 4, 759. Ellman, G. L. (1959). Arch. B&hem. Biophys. f&70. Harding, J. J. (1969). Exp. Eye Res. 8, 14’7. Harding, J. J. (1970). B&hem. J. 117,957. Harding, J. J. (1972). Biochem J. (In press). Kolb, K. H., Jiinicke, M., Kramer, M. and Schulze, P. E. (1967). Ann. N. Y. AC&. Sci. 141,85. Layne, E. (1957). In Methods in Enzymology, vol. III, p. 450. (Ede. Colowick, S. P. and Kaplan, N. 0.) Academic Press, London and New York. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. biol. Chem. 193,265. Moses, R. A. (1958). Amer. J. Ophthd. 46,865. Pirie, A. (1968). Invest. Ophthul. 7, 634. Rubin, L. F. and Barn&t, K. C. (1967). Ann. N. Y. Ad. Sci. 141, 333. Rupley, J. A. (1967). In Methods in Enzymology, vol. XI, p. 905. (Ed. Him, C. H. W.) Academic Press, London and New York. Spa&man, D. H., Stein, W. H. and Moore, S. (1958). Anal. Chem. 30, 1190. Tanford, C. (1969). Adv. Prot. Chem.23,211. Wood, D. C., Sweet, D., van Dolah, J., Smith, J. C. and Contaxis, I. (1967). Ann. N. Y. Acud. Sci. 141, 346. Wood, D. C. (1971). In Dimethyl Suljoxide, vol. 1, p. 133-145. (Eds. Jacob, S. W., Rosenbaum, E. E. and Wood, D. C.) Marcel Dekker, Inc., New York.
Some Changes in the Lens of the Dimethylsulphoxide-fed RUTH ,Vu$feld
VAN HEYNINGEN
Laboratory
Rabbit*
AND J. J. HARDING
University of Oxford, England
of Ophthalmology,
(Received20 April 1972, London) We have confirmed that there is a loss of y-cry&&n and an increase in water-insoluble protein in the lens of the dimethylsulphoxide (DMSO)-fed rabbit (Wood, Sweet, van Dolah, Smith and Contaxis, 1967). These changes were confined to the lens nucleus. The waterinsoluble protein which results from DMSO feeding is soluble in 7 M urea, unlike the waterinsoluble fraction isolated from the human cataraotous lens, which is largely urea-insoluble. The ammo acid content of the urea-soluble protein from the lens of the DMSO-fed rabbit appears to be the same as that from the control lens. Glutathione and protein thiol, and lactic acid, were also measured in the lens of the control and DMSO-fed rabbit. There was no appreciable difference.
1. Introduction In a study of the effect of dimethylsulphoxide (DMSO) feeding upon the lens of the rabbit (Wood, Sweet, van Dolah, Smith and Contaxis, 1967) the most significant tinding was a loss of soluble protein and a corresponding increase of insoluble protein. This change appeared to be an effect on y-crystallin, which was the only component of the soluble protein to be decreasedin concentration (Wood et al., 1967). We have made a further study of the changesin lens proteins, and have also measuredglutathione and lactic acid concentrations. We used adult rabbits (aged 16 to 32 weeks), introduced the DMSO gradually into the drinking water, and supplemented the diet with cabbage, in the hope of producing gradual changesin the lens, resembling nuclear cataract in man. This aim was partly fulfilled, in that the changeswere slight, came on gradually and resembled in some respects early nuclear cataract in man; but after the first few months the condition remained static.
2. Materials
and Methods
Dutch rabbits were fed on pelleted Diet 18 (Oxoid Ltd., Southwark Bridge Road, London S.E.l), with about 60 g cabbagedaily and drinking water ad lib. Twenty rabbits in five litters were used; each litter was divided into control and experimental animals; all comparisonswere madebetweenlitter mates.At the age of 16 to 32 weeksan aqueous solution of DMSO (BDH ChemicalsLtd., Poole,Dorset, England) wassubstituted for the drinking water of the experimental animals. For two weeksthe concentration of DMSO was 1.70,d (v/v), for the next two weeks 3.3% (v/v) and from then on 5% DMSO was given.
Animals were weighedweekly and the lenseswere examined by slit-lamp and ophthalmoscopeevery 2-3 weeks, after dilatation of the pupil with Mydrilate cyclopentolate. Rabbits Separation
were killed
with intravenous
Nembutal.
of lensprotein by solubility in water and urea
Each lens was weighed and dissected into cortex and nucleus. Each part was weighed, ground with 9 volumes of water and centrifuged at 10,000 g for 90 min at 4°C. The concentration of protein in the supernatant was assayed by the biuret reaction (Layne, 1957), * Address for reprints: Nuffield Laboratory England.
of Ophthalmology, 91
Walton Street, Oxford OX2 6AW,
R.
92
VAN
HEYNINGEN
AND
J. J. HARDING
using freeze-dried rabbit lens protein as standard. The water-insoluble protein was washed four times on the centrifuge with 5 ml water each time. For the fifth washing, the protein was suspended in 5 ml water and left at 4°C overnight before centrifugation in a weighed polythene centrifuge tube. The wet precipitate was frozen and dried in vacua and the centrifuge tube reweighed to give the weight of the water-insoluble protein. Two ml 7 M urea was added to the water-insoluble protein and after thorough disintegmtion with a glass rod, the mixture wa,s left overnight at 4°C. After centrifugation the supernatant was removed, and the urea-insoluble precipitate was washed twice with 5 ml 7 M urea and six times with 5 ml water. It was then frozen, dried and weighed. The weight of the urea-soluble protein was taken as the difference between that of the water-insoluble and the urea-insoluble. Urea-soluble protein was precipitated from solution by the addition of 10 volumes of acetone. After 16 hr at 4°C the precipitate was collected by centrifugation, washed four times with acetone and dried. Precipitation
of protein. with trichloroacetic
acid
(TCA)
Lenscortex or nucleuswasground with nine times the weight of 6% (w/v) TCA. Protein was allowed to precipitate at 4°C usually overnight, before collection by oentrifugation. The precipitate was used for the estimation of protein thiol and the supernatant for glutathione and lactic acid. Glutathione
This was measuredusing the 5,5’-dithiobis-(2-nitrobenzoic acid) reagent of Ellman (1959), as describedby Harding (1970). DMSO at concentrations of up to 5% doesnot interfere. Protein
thiol
The protein precipitated by trichloroacetic acid wasdissolved in 6 M guanidinium chloride. Portions of this solution were taken and assayedfor thiol in 7 M urea, using the method describedfor glutathione. Isolation of u-, /3- and y-crystal&s The crystallins were isolated from the nuclei of one pair of rabbit lenses(pooled), u crystallin wasseparatedby isoelectricprecipitation andj3-and y-crystallins by gel titration on Sephadex 675 (Pharmacia, Great Britain, Ltd., Uxbridge Road, London W5), as describedby Harding (1969).In other experimentsthe initial precipitation of a-orystallin wasomitted and only y-crystallin wascompletely separatedon the Sephadex675 column. AmiNo acid analysis
Analyses were performed with an amino acid analyser (Locarte CompanyLtd., Emperor’s Gate, London S.W.7), by the method of Spackman, Stein and Moore (1958). Samplesof protein (about 2 mg) were hydrolyzed in constant-boiling HCl(2 ml). Hydrolysis was conducted for 18 hr at 108”C,in vacua. digestions The tryptic susceptibility of the proteins of rabbit lens was studied by the method of Harding (1972), using either the whole nucleus (one experiment), half the nucleus (two experiments) or the cortex (one experiment). In each casematerial from a DMSO-fed rabbit was comparedwith that from a litter-mate control killed at the sametime. Tryptic
THE
LENS
OF
THE
DMSO-FED
RABBIT
93
3. Results General condition of animals The rabbits on DMSO lost weight for the first weeks on the diet. Subsequently they did not put on weight as fast as the controls and laid down lesssubcutaneousfat, but they appeared quite healthy. They had a bad smell, presumably of dimethyl sulphide (seeWood, 1971) and other metabolic products of DMSO. Refraction of eyes The refraction of the two eyes of an animal, control or experimental, measuredwith a retinoscope, differed little. Refraction was first measured after the experimental animals had been 12 to 17 weeks on DMSO. The control values were from +2-O to +3*7 dioptres and theexperimental values from -0.5 to+24 dioptres; theexperimental values were 1.3 to 4.2 dioptres lessthan the litter-mate controls. Six months or more later the refraction of surviving rabbits was little changed. Table I gives someof the values found. TABLE
I
DMSO-fed rabbits and litter-mate controls. Refraction of eyes and cmncen&ation of proteins in the lens nucleus First
experiment: Duration
12-17
wks
DMSO-fed 20, 28, 32 (3) 14*3 (3) +0.5-&0.2 (3) 497&34 (3)
Age at start of experiment (wks) Duration of experiment (wks) Refraction of eyes (dioptrea) Lens weight (mg) Lena nucleus Weight (mg) Total protein (g y. wet wt) Water-sol. protein (g y. wet wt) Water-insol. protein (g y. wet wt) Urea-sol. protein (g y. wet wt) Seed
experiment Duration
: 48-52
Control
196fll 44.6kl.5 33.0-J= 1.4 11*6f3 10.6, 14.4
Difference: DMSO-fed minus
20, 28, 32 (3) 14*3 (3) +3.0*0.3 (3) 494&35 (3)
(3) (3) (3) (3) (2)
220-&28 43.9k4.7 37*9&3 6.013 2.5, 9.4
(3) (3) (3) (3) (2)
16, 20, 27 49~2 +2.9*1.0 534, 570 one cortex
(3) (3) (3) (2) lost
-2.5f0.7
control
(3)
+0.7*3.7 (3) -4.953 (3) +5*6&2.6 (3) +8.1, +5*0(2)
wks 16, 20, 27 (3) wk2 (3) +1.0*0.4 (3) 562&32 (3)
Age at start of experiment (wks) Duration of experiment (wks) Refraction of eyes (dioptres) Lens weight (mg)
Lens nucleus Weight (mg) Total protein (g y0 wet wt) Water-sol. protein (g y. wet wt) Water-insol. protein (g y. wet wt) Urea-sol. protein (g y0 wet wt)
One of each pair of litter-mates weeks (second experiment). Result,8 are expressed as mean
261fll 56417.9 41.257.2 15.612.0 15.5-&2.0
was given f
standard
5% DMSO deviation
(3) (3) (3) (3) (3)
259*13 (3) 52*8f 10.0(3) 44.5f9.3 (3) 8.3&0.6 (3) 8.256 (3)
for 12-17
weeks
(first
(no. of determinations).
-1.9*1.0
(3)
+4*051.3 -3.3h2.2 +7.3&0*5 +7.3&0.6
(3) (3) (3) (3)
experiment)
or for 48-52
94
Ii.
VAN
HEYNINGEN
AND
J. J. HARDING
Intraocular pressure The tension in both eyes of 5 experimental and 5 control rabbits was measured by applanation tonometry and the readings converted to pressure in mmHg (Moses, 1958). There was no significant difference. In the eyes of the experimental rabbits the average value was 15.4 mmHg (range 13.8 to 16.6) and in the control 16.5 mmHg (range 13.8 to 17.7). Visible changes in the lens of the DMSO-fed
rabbit
These were the same as those described by others (Rubin and Barnett, 1967 ; Wood et al., 1967), but did not advance beyond the stage at which the periphery of the lens nucleus could be clearly seen. After one year on DMSO the condition of the lenses of surviving rabbits had remained static for many months, and the nuclei of the lenses of the control rabbits were gradually becoming demarcated and clearly visible. The effect of ageing on the appearance of the lens nucleus was less marked, but similar to that of DMSO-feeding. Lens weight There was no difference in the wet weight of control and experimental lenses.The demarcation between cortex ad nucleus was fairly distinct, the cortex being soft and the nucleus hard and sticky. We could detect no difference between the size or consistency of the nucleus of the experimental and of the control lenses. One experimental and one control lens from three pairs of rabbits were freeze-dried and re-weighed. All these animals came from the samelitter, aged 72 weeks at death. There was no significant difference in the dry weight of the lens, all values falling between 213 and 225 mg. The percent dry weight of all the lenseswas close to 40.7 (range 39.4 to 41.7). Solubility of lensproteins In two comparisons between control and experimental lensesthere was verylittle difference in the concentration of water-soluble, urea-soluble and urea-insoluble proteins in the lens cortex. In further experiments, therefore, the proteins of the lens nucleus only were examined. Table I gives the percentage of water-soluble, water-insoluble and urea-soluble proteins of the lens nucleus. A lens nucleus of a DMSO-fed rabbit was compared with the nucleus of the lens of a litter-mate control killed at the sametime. In five of the six comparisonsthe weight of lens substance in the nuclei of the DMSO-fed and of control lensesdiffered by 5 mg or less. The comparisonsare divided into two groups, three pairs in which DMSO-feeding continued for 12-17 weeks, and three pairs in which DMSO was given to the experimental animals for about one year. The effect of DMSO-feeding was not accentuated by lengthening the experiment. Table I shows that the concentration of water-soluble protein in the lens nucleus decreasedfrom 37.9% to 33.0% and 44.5% to 41.2% when that of water-insoluble protein increased from 6.0% to 11.6% and 8.3% to 15.6%. The fact that the loss of soluble protein is not exactly the sameas the increaseof insoluble protein is probably due to experimental error, the two values being measuredby different methods. The water-insoluble protein prepared from the control lens was light and fluffy but
THE
LENS
OF
THE
DMSO-FED
RABBIT
95
that prepared from the experimental lens was granular and more lumpy. Both preparations were almost entirely soluble in 7 M urea. Table I shows that the waterinsoluble, urea-soluble fraction of the protein of the lens nucleus has approximately doubled as a result of DMSO-feeding. Water-soluble crystullins
in the lens nucleus
The proportions of a-, p- and y-crystallins isolated from lens nuclei of control and experimental rabbits are shown in Table II. In three comparisons the y-crystallin was 19-22% of the water-soluble protein in the control lens, and lo-16% in the experimental lens. Thus about one-third of the y-crystallin had disappeared from the watersoluble proteins of the experimental lens. II
TABLE
Percentage of a-, /3- and y-crystallin in the water-soluble protein from the lens nuclei DMSO-fed rabbits and litter-mute controls
5% DMSO in drinking water
+ -
cc
B
Y
55.3 54.0 L--
28.4 26.2 .-. -J
16.3 19.7
+ + -
90 81 87 78
of
10 19 13 22
Glutathione a& protein thiol in lens nucleus The amounts of glutathione and protein thiol in the Iens of the DMSO-fed and control rabbits are shown in Table III. Neither glutathione nor protein thiol was significantly changed as a result of DMSO feeding. TABLE
III
Glut&h&me, protein thiol and lactic acid in the lens of DMSO-fed rabbits and litter-mate controls
DMSO-fed
Control (~moles/g
Glutathione in nucleus I’rotein thiol in nucleus Lactic acid in cortex Lactic acid in nucleus
Results
are expressed
a8 average
wet wt)
3.21&@60(5) 44.3 kt3.0 (5) 15.9 ho.6 (5) 10.1 kO.5 (3)
values
& standard
deviation
3.63&1.09(S) 45.8 12.7 (6) 16.4 &0+3 (5) Il.5 kO.8 (3)
(number
of determinations).
96
R. VAN
HEYNINGEN
AND
Amino acid composition of water-insoluble
J. J. HARDING
urea-soluble proteins
The amino acid compositions of the water-insoluble urea-soluble protein from control and experimental rabbits are shown in Table IV. Each is the mean of three analyses. The apparent difference in lysine content is probably caused by the poor baseline in this region of the amino acid chromatogram. Otherwise only the difference in tyrosine content is marginally significant. TABLE
IV
Amino acid composition of the water-insoluble but urea-solubleprotein of the lens nucleus of the DMSO-fed and control rabbits
His
A% ASP Thr Ser GlU Pro G’Y Ala Val Met He Leu TV Phe Lp*
Results * Values
Lactic
ad
are mean of three analyses unreliable, see text.
DMSO-fed
Control
34.6 89.0 99.0 27.8 95.0 134 64.8 80.9 43.9 47.7 20.3 34.6 83.1 55.0 57.0 (33.1)
36.1 84.3 101 29.5 91.9 130 69.5 79.3 46.4 48.7 17.8 33.1 82.0 49.6 56.1 (43.8)
expressed
as residues/thousand
residues.
in the lens
Lactic acid was assayedin the nucleus and cortex of the lens of DMSO-fed rabbits and the corresponding controls. The results are given in Table III. There was no significant difference in either cortex or nucleus. Tryptic susceptibility of lens proteins The initial slopesof the progress curves for tryptic digestion were 0.77 to 1.05 ml 2 mu NaOH/g/min for the nucleus of DMSO-fed rabbit lens and 0.59 to 0.70 ml 2 mM NaOH/g/min for that of the litter-mate controls. The percentage increase in rate was 30, 34 and 50% for the three comparisons.The initial slopesof the progress curves for tryptic digestion of the rabbit lens cortex showed an increase from 0.40 ml 2 mu NaOH/g of cortex/min in the control to 0.50 ml 2 mM NaOH/g of cortex/min in the lens of DMSO-fed rabbit. Thus in all four experiments the proteins of DMSO-fed lens were digested more rapidly than litter-mate controls.
THE
LENS
OF
THE
DMSO-FED
RABBI7
97
4. Discussion The visible changes in the lens and the changes in refraction measured by retinoscopy were similar to those previously reported by Wood et al. (1967), Rubin and Barnett (1967) and others. The intraocular pressure was not affected by DMSOfeeding. The lens wet weight and dry weight were unaffected by DMSO-feeding and therefore the increase in refractive power is not caused by a higher protein concentration or larger lens; nor did slit-lamp examination reveal any change in lens shape. There was no significant change in lactic acid, reduced glutathione or protein thiol content. Wood et al. (1967) found no change in glutathione levels in older rabbits and only a small change in ten-week old rabbits. Our main positive findings concern the proteins of the lens. Wood et al. (1967) had shown that there was a loss of y-crystallin and an increase of insoluble protein in lenses from DMSO-fed rabbits. Our results (Table I) show that the increase of waterinsoluble protein is confmed to the urea-soluble fractions of this protein. This was unexpected because the urea-insoluble fraction increases in human cataract (Pirie, 1968). Unlike human lens or rat lens (Pirie, 1968; Harding, 1969) the normal rabbit lens contains relatively little water-insoluble urea-insoluble protein. In this respect it resembles cow lens (Dische, 1965). The different amounts of urea-insoluble protein isolated from lenses of different animals indicate that the lens proteins of some animals are more susceptible to oxidation than those of others. The small amount in rabbit and cow lens is probably largely composed of fragments of fibre membranes (Dische, 1965). The increased amount of urea-insoluble protein isolated from human cataractous lens is probably largely due to unfolding of the water-soluble proteins during cataractogenesis (Harding, 1972). Our results showed that the increase of urea-soluble water-insoluble protein occurs in the nucleus. As the amino acid composition of the water-insoluble urea-soluble protein from experimental lens is the same as that from control lens, it is probable that both consist of a similar mixture of lens crystallins, the extra urea-soluble protein of experimental lenses is thus a similar mixture to the original water-insoluble but, urea-soluble protein. The decreased proportion of y-crystallin found in the lenses of DMSO-fed rabbits by Wood et al. (1967) using cellulose electrophoresis was confirmed in the present work using gel filtration to isolate y-crystallin. Some y-crystallin may have aggregated to appear as higher-molecular-weight protein on gel filtration and/or it may have become water-insoluble but urea-soluble. A relatively small extra amount of y-crystallin in this fraction would be difficult to detect. Susceptibility to proteolysis has often been used to study conformational changes in proteins (Rupley, 1967) and has recently been used to confirm that protein unfolding occurs in human cataract (Harding, 1972). The results presented indicate that DMSO-feeding may cause unfolding of some lens proteins but insufficient lenses were available to establish such a conclusion. If the proteins had unfolded it is unlikely that this was caused directly by DMSO because DMSO is a poor denaturant of proteins (Tanford, 1969). Radioactivity, following cutaneous treatment of rabbits with 3H-DMS0, has been shown to reach all parts of the eye (Kolb, Jgnicke, Kramer and Schulze, 1967). Wood (1971) has recently summarized our current knowledge of the metabolic fate of DMSO. It is not known whether DMSO itself or metabolic product(s) reach the eye. The mechanism whereby the observed optical changes are induced in the lens by treatment of the animal with DMSO is also unknown.
98
R. VAN
HEYNINGEN
AND
J. J. HARDING
4CKNOWLEDGMENTS
We thank ProfessorDon C. Wood for reading the typescript, Mr T. H. Kirkham for the refraction and intraocular pressuremeasurements,Mr D. J. Potter for his excellent care of the animalsand Mr K. Rixon for skilled technical assistance. REFERENCES Dische, 2. (1965). Invest. Ophthal. 4, 759. Ellman, G. L. (1959). Arch. B&hem. Biophys. f&70. Harding, J. J. (1969). Exp. Eye Res. 8, 14’7. Harding, J. J. (1970). B&hem. J. 117,957. Harding, J. J. (1972). Biochem J. (In press). Kolb, K. H., Jiinicke, M., Kramer, M. and Schulze, P. E. (1967). Ann. N. Y. AC&. Sci. 141,85. Layne, E. (1957). In Methods in Enzymology, vol. III, p. 450. (Ede. Colowick, S. P. and Kaplan, N. 0.) Academic Press, London and New York. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. biol. Chem. 193,265. Moses, R. A. (1958). Amer. J. Ophthd. 46,865. Pirie, A. (1968). Invest. Ophthul. 7, 634. Rubin, L. F. and Barn&t, K. C. (1967). Ann. N. Y. Ad. Sci. 141, 333. Rupley, J. A. (1967). In Methods in Enzymology, vol. XI, p. 905. (Ed. Him, C. H. W.) Academic Press, London and New York. Spa&man, D. H., Stein, W. H. and Moore, S. (1958). Anal. Chem. 30, 1190. Tanford, C. (1969). Adv. Prot. Chem.23,211. Wood, D. C., Sweet, D., van Dolah, J., Smith, J. C. and Contaxis, I. (1967). Ann. N. Y. Acud. Sci. 141, 346. Wood, D. C. (1971). In Dimethyl Suljoxide, vol. 1, p. 133-145. (Eds. Jacob, S. W., Rosenbaum, E. E. and Wood, D. C.) Marcel Dekker, Inc., New York.