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UV laser photodamage to whole lenses
Exp. Eye Res. (1989) 49, 959-966
UV L a s e r P h o t o d a m a g e
to W h o l e L e n s e s
J A M E S DILLON, DEBDUTTA ROY, ABRAHAM SPECTOR, M. L. W A L K E R * , L. B. H I B B A R D * AND R. F. BORKMAN*
Department of Ophthalmology, Columbia University, New York, N Y 10032, U.S.A. and * School of Chemistry, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A. (Received 26 August 1988 and accepted in revised form 17 July 1989) Lenses from rat or calf were exposed in vitro to UV radiation from a nitrogen laser operated at 337.1 nm or from an excimer laser operated at 308 nm. Visible light transmission was monitored during calf lens irradiations at 308 nm and found to decrease. Proteins were extracted from the irradiated rat or calf lenses, separated into water soluble and insoluble fractions, and analysed using SDS-PAGE. Comparison of these gels with dark controls showed that, following photolysis, there was loss of polypeptide material in the 20-30 kDa region and concomitant formation of polymers at 40 and 60 kDa, and at > 100 kDa in calf lenses (308 nm irradiation) and rat lenses (337.1 nm irradiation) in vitro. In addition, there was evidence for formation of lower molecular weight polypeptides at 10 kDa in the protein from irradiated rat lenses. The rat SDS-PAGE gels were challenged against anti-calf gamma crystallin serum, There was clear evidence that the polymeric material, in the water insoluble protein fraction from the 337-1 nm photolysed rat lenses was derived in part from gamma crystallin. The macromolecular changes detected in these photolysed rat and calf lens proteins were similar to those previously reported to accompany aging in the human lens. Biochemical changes of the type observed in UV irradiated rat and calf lenses may be responsible for the loss of visible light transmission seen in calf lenses. Key words: uv radiation; laser; cataracts; intact lenses; photopolymerization.
1. I n t r o d u c t i o n The p o l y p e p t i d e s of t h e h u m a n lens p r o t e i n s u n d e r g o a n u m b e r of a g e - r e l a t e d changes, including f o r m a t i o n of b o t h higher a n d lower m o l e c u l a r weight species. The crystallins in h u m a n fetal lens yield p o l y p e p t i d e s p r i m a r i l y in t h e 20-30 k D a range (Ringens, H o e n d e r s a n d Bloemendal, 1982) ; b u t b y t h e age of 20 yr, p o l y p e p t i d e s in t h e 40 to 50 k D a range a n d in t h e 10-12 k D a range begin to a p p e a r . The 20-30 k D a p o l y p e p t i d e s are the n o r m a l c r y s t a l l i n gene p r o d u c t s , while t h e 40-50 k D a p o l y p e p t i d e s h a v e been shown to derive from beta- a n d g a m m a - c r y s t a l l i n (Roy, Dillon, W a d a , Chaney a n d Spector, 1984) b y p o s t - t r a n s l a t i o n a l modification. T h u s it a p p e a r s t h a t some aging a n d c a t a r a c t o u s changes in lens p r o t e i n s are a t t r i b u t a b l e to modifications of t h e p r e - e x i s t i n g c r y s t a l l i n p o l y p e p t i d e s (Garner, G a r n e r a n d Spector, 1981; Russell, Smith, Carpo a n d K i n o s h i t a , 1979). E x p e r i m e n t a l studies in which lens p r o t e i n s in solution were e x p o s e d to U V r a d i a t i o n h a v e suggested t h a t lens p r o t e i n changes a c c o m p a n y i n g aging could be a c c o u n t e d for b y p h o t o c h e m i c a l l y i n d u c e d processes. Such processes m a y be d i r e c t (Zigman, 1979; Dillon a n d Spector, 1980; B o r k m a n , Tassin a n d L e r m a n , 1981; F u j i m o r i , 1982; Mandal, K o n o a n d Bose, 1988; W a l k e r a n d B o r k m a n , 1988) or sensitized (Goosey, Zigler, J e r n i g a n a n d K i n o s h i t a , 1980; A n d l e y a n d C h a p m a n . 1986). P h o t o l y s i s of i n t a c t lenses in v i t r o has also been r e p o r t e d , b o t h w i t h a d d e d sensitizer (Zigler, J e r n i g a n , P e r l m u t t e r a n d K i n o s h i t a , 1982) a n d w i t h o u t a d d e d sensitizer ( H i b b a r d , K i r k a n d B o r k m a n , 1985; Rao, B a l a s u b r a m a n i a n a n d C h a k r a b a r t i , 1987). I n m a n y cases, a m a j o r result of p h o t o l y s i s a p p e a r s to be p h o t o 0014-4835/89/120959+08 $03.00/0
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J. DILLON ET AL.
p o l y m e r i z a t i o n o f the n o r m a l c r y s t a l l i n p o l y p e p t i d e s to high m o l e c u l a r weight, c o v a l e n t l y b o n d e d a g g r e g a t e s ( B o r k m a n , 1984). I t was of i n t e r e s t to us to i n v e s t i g a t e p h o t o l y s i s of i n t a c t lenses w i t h o u t a d d e d exogenous photosensitizers (such as k y n u r e n i n e s , riboflavin, or m e t h y l e n e blue). Our e x p e r i m e n t s allowed us to m o n i t o r t h e optical consequences of UV e x p o s u r e in calf lenses (light s c a t t e r i n g a n d / o r t u r b i d i t y ) a n d also to characterize, i m m u n o l o g i c a l l y , t h e high m o l e c u l a r weight p o l y p e p t i d e s p r o d u c e d p h o t o c h e m i c a l l y in r a t lenses. I r r a d i a t i o n s were carried o u t a t 308 or 337 nm, w a v e l e n g t h s which can more r e a d i l y p e n e t r a t e t h e cornea t h a n t h e s h o r t e r w a v e l e n g t h s used in some p r e v i o u s p h o t o l y s i s studies (Dillon a n d Spector, 1980 ; H i b b a r d et al., 1985). T h u s t h e p r e s e n t studies are m o r e r e l e v a n t to the h u m a n s i t u a t i o n in t h a t t h e y do n o t rely on a d d e d photosensitizers, t h e y use m o n o c h r o m a t i c r a d i a t i o n r e a d i l y t r a n s m i t t e d b y the cornea, a n d t h e y e m p l o y i n t a c t lenses r a t h e r t h a n p r o t e i n e x t r a c t s as the t a r g e t of t h e UV r a d i a t i o n .
2. M e t h o d s R a t lenses from 3-6-week-old Sprague--Dawley albino rats were used immediately following excision. Eyes from 4-5-month-old calves were obtained from a local slaughterhouse ; the lenses were removed within 4-5 hr of death and were used immediately or were kept at - 4~ until needed. The lenses were placed in quartz tubes and were irradiated either dry, under air, or were immersed in Ringer solution during irradiation at ambient temperature of 22 • 2~ No differences in visible light scattering behavior or subsequent S D S - P A G E analysis patterns were found for dry vs. Ringer irradiations.
Excimer or nitrogen Iaser
Quortz plote
Sample
UV
cell
filter
He-Ne laser
FIo. 1. Apparatus for monitoring lens visible light transmission during UV irradiation.
The light transmission and irradiation apparatus which we used is shown in Fig. 1. Visible light from a Helium-Neon laser at 632"8 nm was made collinear with the UV output from the excimer laser by using a quartz plate as a beam splitter as shown in Fig. 1. The visible light passing through the sample was detected by a photomultiplier tube. UV radiation from the laser was prevented from entering the phototube by a Corning filter. The Visible beam intensity was monitored periodically and found to be constant to within • 2 %. Thus we were able to normalize the transmission data to constant light intensity. Output from the phototube was detected by an OMEGA DAS-16F interface which digitized the photovoltage signals for processing by Labtech Notebook software on an IBM PS/2 computer system. Hence the visible light transmission data could be plotted as a relative percent transmission or as a relative optical density. The Lumonics model 520 excimer laser was operated at an average power of 750 mW corresponding to a pulse energy of 15 m J and a repetition rate of 50 Hz. The beam dimensions at the sample position were 5 mm • 7 ram. Irradiation times
UV LASER PHOTODAMAGE TO WHOLE LENSES
961
with this source were 60 min or less. The nitrogen laser was a Molectron UV-12, operated at a pulse energy of 1"5 mJ and a repetition rate of 30 pps, corresponding to an average power of about 45 mW. The beam dimensions at the sample position were 2 mm • ram. Irradiation times with this source were up to 20 hr. UV-laser power output was checked periodically during irradiations with a Scientech model 365 power and energy meter. After photolysis, rat lenses were homogenized in 20 mM Tris HCI, I tara EDTA and 10 mM dithiothreitol at pH 7-4 (400/~l of this solution per lens) and centrifuged at 20000 rpm for 20 min. The samples were then analysed by SDS-PAGE (Laemmli, 1970), stained with Coomassie Blue and optically scanned using an accessory to a Perkin-Elmer MPF-3 fluorimeter. For immunological analysis, the previously described blot technique was used (Roy et al., 1984). The calf lenses were dissected so as to remove the outer 1 or 2 mm thickness from the anterior (irradiated) lens face. This material was then homogenized in 63 mM Tris HCI, 2 % SDS, and 0"7 M mercaptoethanol at pH 7"4 and analysed by SDS-PAGE with a 15% separating gel. 3. R e s u l t s
A plot of relative optical d e n s i t y (OD) a t 632"8 n m vs. time of exposure to 308 n m excimer laser r a d i a t i o n for a n i n t a c t calf lens is shown in Fig. 2. The relative OD is I 0-9 0.80"7 0"6 -~ 0"5 ~. 0"4 0
0"3 0-2 0-1 I
0
I0
20
50
I
40
I
I
50
J
60
Exposure time (min)
FIG. 2. Change in relative optical density of intact calf lens upon exposure to 308 nm laser radiation. seen to increase from a value of 0"0 at time zero to 0"6 after 20 m i n irradiation. F u r t h e r i r r a d i a t i o n after this time is seen to have little a d d i t i o n a l effect on the OD of the sample. Since there is essentially no light a b s o r p t i o n a t 632"8 n m , most of the 632"8 OD produced b y U V i r r a d i a t i o n arises from t u r b i d i t y / l i g h t scattering, a n d n o t from absorption. The p a t t e r n of t u r b i d i t y growth shown for whole calf lenses in Fig. 2~ parallels t h a t seen in similar e x p e r i m e n t s with single, isolated lens proteins. F o r example, W a l k e r a n d B o r k m a n (1989) a n d M a n d a l et al. (1988) m o n i t o r e d visible light scattering a n d t u r b i d i t y in the purified calf g a m m a crystallins, g a m m a - I I , I I I , a n d IV, a n d f o u n d similar increases in light scattering following UV t r e a t m e n t (with or w i t h o u t added sensitizers) to those observed here. I r r a d i a t e d r a t lenses w e r e subjected to S D S - P A G E after s e p a r a t i o n into water soluble a n d insoluble fractions. Figure 3 shows 470 n m d e n s i t o m e t e r scans of the Coomassie Blue stained S D S - P A G E gels of the w a t e r insoluble fraction at zero time a n d after 12 hr exposure to the n i t r o g e n laser at 337"1 nm. As expected, the strongest
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ET AL.
~ II;I I .I;!1 ~'"I I/~'~l l
20 kD
45 kD
Top
Fie. 3. Densitometer scans at 470 n m of Coomassie Blue stained S D S - P A G E gels of the water insoluble protein fraction from rat lenses: zero time ( ), and after 12 hr photolysis at 337'1 n m
(---).
A
B
C
FIG. 4. S D S - P A G E gels of calf lens insoluble protein: lane A, dark control; lanes B and C, after 60 m i n exposure of the intact lens (in vitro) to 308 n m excimer laser radiation. Lane C is the front I m m a n d lane B is the front 2 m m section of the irradiated (anterior) lens face.
UV LASER
PHOTODAMAGE
TO WHOLE
LENSES
963
50
40 "E
j 2 o kD
5C
"6 ill
kD
3o
I
2
I
4
I
6 Time (hr)
I I0
8
I
12
FIG. 5. Relative percent of crystallin polypeptides in the 20-25 k D a and 11 k D a regions as a function of 337.1 n m laser irradiation time. D a t a are shown for the rat water insoluble fractions.
S
P
0
S
P
4
S
P
8
S
P
12 hr
FIe. 6. A n t i - g a m m a blot of proteins extracted from irradiated rat lenses and subjected to S D S - P A G E (S, soluble and P, insoluble fractions). The time of 337"1 n m radiation is shown at the b o t t o m of each lane.
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J. DILLON ET AL.
bands in the unirradiated lenses appeared in the range 20-30 kDa, Following photolysis there was a loss of 20-30 kDa material and formation of diffuse bands at approximately 45 kDa and higher molecular weight, and an even more diffuse band at low molecular w e i g h t - - a b o u t 10-15 kDa. Irradiated calf lenses were also analysed by S D S - P A G E following separation into water-soluble and insoluble fractions (Fig. 4). The insoluble fraction from calf lenses irradiated for 1 hr at 308 nm showed diffuse, new bands in the 40-60 kDa region as well as high molecular weight material which did not enter the gels. These features were not presented in unirradiated controls which showed only the normal bands in the 20-30 kDa region. The UV irradiated whole lens material gave S D S - P A G E patterns similar to those observed in our previous study of UV effects on isolated calf lens crystallins (Walker and Borkman, 1989). The S D S - P A G E data on UV-irradiated rat lenses revealed a loss of material in the 20 kDa region and an increase in polypeptides with molecular weights above and below those appearing in the normal crystallins. This trend is plotted in Fig. 5 which shows the loss of 20 kDa material and the growth of fragmented polypeptides as a function of rat lens irradiation time. In addition, we observed increased amounts of polymeric material as a function of UV irradiation time. These changes are similar to those noted previously for human lens crystallins as a function of aging (Garner et al., 1981). Since it had been previously demonstrated t h a t the human 43 kDa component contains g a m m a crystallin (Roy et al., 1984), the gels from photolysed rat lenses were blotted against anti-gamma serum. A s seen in Fig. 6, the formation of poiymers (dimers, trimers, etc.) containing g a m m a brystallin was clearly in evidence following photolysis of intact rat lenses, particularly in the w a t e r insoluble fraction: 4. D i s c u s s i o n
The direct (no added photosensitizer) photolysis of intact r a t and calf lenses using nitrogen or excimer laser UV radiation has been shown to lead to some of the same macromolecular changes detected in aging or cataractous human lenses. These include increased visible light scattering, and decreased visible light transmission, at the organ level, and photo crosslinking and photo fragmentation of lens crystallin proteins, at the molecular level. Previous workers h a v e noted the photo polymerization of lens proteins and have suggested a possible relationshi p to lens aging and eataractogenesis (Dillon and Spector, 1980; Goosey et al., 1980; Fujimori, 1982). But, the present work is the first to report photolytic peptide bond cleavage in whole lenses. In addition, our work suggests a possible correlation between loss of visible light transmission in UV irradiated whole calf lenses and the accompanying lens protein photo polymerization. Other mechanisms m a y also contribute to the reduced transmission of the lens following UV exposure. Ringens, Hoenders and Bloemendal (1982) reported t h a t the insoluble fraction of human fetal lens contains actin (43 kDa) and vimentin (65 kDa). By the age of 20, vimentin is no longer detectable and the 43 kDa region contains a more diffuse band. I t has recently been demonstrated that the 43 k D a polypeptide in older human lenses contains significant amounts of beta and g a m m a crystallin polypeptides (Roy et al., 1984). The formation of a diffuse band of crystallin origin was observed in the photochemical data reported here. In addition, we found that polymerization does not stop at the dimer stage but continues on to trimers, tetramers, etc. Similar larger polymers have been observed in aged human lens proteins (Roy et al., 1984).
UV LASER PHOTODAMAGE TO WHOLE LENSES
965
Bond cleavage, though not as obvious in our data as polymerization, is suggested by the S D S - P A G E gel scans in Fig. 3. Although it is apparent from the anti-gamma blots that rat lens g a m m a crystallin is involved in the photo polymerization process, the 10-15 kDa region of our rat lens gels did not stain positive for g a m m a crystallin. Hence the parentage of these fragments is open to question at this time. The actual photolytic mechanisms, in intact lenses, at the wavelengths used in our experiments are not known. Irradiation at 308 nm could result in excitation of Trp residues in lens proteins by direct light absorption. Alternatively, some or all of the incident 308 nm radiation could be absorbed by other chromophores in the lens, including the known photosensitizer N-formylkynurenine (Walrant and Santus, 1974), and lead to singlet oxygen production and subsequent attack of lens proteins (Ziglcr et al., 1982). On the other hand, irradiation at 337"1 nm is less likely to excite Trp by direct absorption, and photosensitization by unknown absorbers in the lens is also a possibility. However, it is interesting to note t h a t 337"1 nm radiation can cause polymerization in isolated lens crystallins (Walker and Borkman,: 1989). In addition, 337"1 nm photolysis of free Trp monomer in solution produces m a n y of the same products found in the 290 nm photolysis of Trp where it is clear that the mechanism involves direct absorption by Trp (Borkman et al., 1986). In conclusion, m a n y of the macromolecular changes associated with human lens aging and cataracts are also seen in the 308 or 337 1 nm UV laser photolysis of rat and whole calf lenses in vitro. ACKNOWLEDGMENTS The support of NIH grants EY-6800 (Georgia Tech), EY-2283 (Columbia), and NEI grants to A. Spector are gratefully acknowledged. REFERENCES Andley, U. and Chapman, S. (1986). Conformational changes of bovine lens crystallins in a photodynamic system. Photochem. Photobiol. 44, 67-74. Borkman, R. (1984). Cataracts and photochemical damage in the lens. Ciba Foundation Symposium 106, Human Cataract Formation, (Ed. Spector, A.) Pp. 88-99. Pitman: London. Borkman, R., Hibbard, L. and Dillon, J. (1986). The photolysis of tryptophan with 337'1 nm laser radiation. Photochem. Photobiol. 43, 13-19. Borkman, R., Tassin, J. and Lcrman, S. (1981). The rates of photodestruction of tryptophan residues in human and bovine ocular lens proteins. Exp. Eye Res. 32, 747-54. Dillon, J. and Spector, A. (1980). A comparison of aerobic and anaerobic ph0tolysis of lens proteins. Exp. Eye Res. 31,591-9. Fujimori, E. (1982). Crosslinking and blue fluorescence of photo-oxidized alpha crystallin. Exp. Eye Res. 34, 381-8. Garner, W., Garner, M. and Spector, A. (1981). Gamma crystallin, a major cytoplasmic polypeptide, disulfide linked to membrane proteins in human cataract. Biochem. Biophys. Res. Commun. 98, 439-43. Goosey, J., Zigler, J., Jernigan, H. and Kinoshita, J. (1980). Crosslinking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen. Science 208, 1278-2380. Hibbard, L., Kirk, N. and Borkman, R. (1985). The in vitro photolysis of whole rat lenses using focused 290 nm laser radiation. Exp. Eye Res. 40, 285-90. Laemmli, U.K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 680-6. Mandal, K., Kono, M., Bose, S., Thomson, J. and Chakrabarti, B. (1988). Structure and stability of gamma-crystallins. IV. Aggregation and structural destabilization in photosensitized reactions, Photochem. Photobiol. 47, 583-92.
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Rao, C., Balasubramanian, D. and Chakraharti, B. (1987). Monitoring light induced changes in isolated intact eye lenses. Photochem. Photobiol. 46, 511-15. Ringens, P., Hoenders, H. and Bloemendal, H. (1982). Effect of aging on the water-soluble and water-insoluble protein pattern in normal human lens. Exp. Eye Res. 34, 201-7. Roy, D., Dillon, J., Wada, E., Chaney, W. and Spector, A. (1984). Nondisulfide polymerization of gamma- and beta-crystallins in the human lens. Proc. Natl~ Acad. Sci. U.S.A. 81, 2878-81. Russell, P., Smith, S., Carper, D. and Kinoshita, J. (1979). Age and cataract related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens. Exp. Eye Res. 29, 245-55. Walker, M. and Borkman, R. (1988). Light scattering and photo crosslinking in the calf lens crystallins gamma-II, III, and IV. Exp. Eye Res. 48, 375-83. Walrant, P. and Santus, R. (1974). N-Formylkynurenine, a tryptophan photooxidation product, as a photodynamic sensitizer. Photochem. Photobiol. 19, 411-17. Zig[er, J., Jernigan, H., Perlmutter, N. and Kinoshita, J. (1982). Photodynamic crosslinking of polypeptides in intact rat lens. Exp. Eye Res. 35,239-49. Zigman, S. (1979). Near UV light and cataracts. Photoche.m. Photobiol. 26, 437-41.
UV L a s e r P h o t o d a m a g e
to W h o l e L e n s e s
J A M E S DILLON, DEBDUTTA ROY, ABRAHAM SPECTOR, M. L. W A L K E R * , L. B. H I B B A R D * AND R. F. BORKMAN*
Department of Ophthalmology, Columbia University, New York, N Y 10032, U.S.A. and * School of Chemistry, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A. (Received 26 August 1988 and accepted in revised form 17 July 1989) Lenses from rat or calf were exposed in vitro to UV radiation from a nitrogen laser operated at 337.1 nm or from an excimer laser operated at 308 nm. Visible light transmission was monitored during calf lens irradiations at 308 nm and found to decrease. Proteins were extracted from the irradiated rat or calf lenses, separated into water soluble and insoluble fractions, and analysed using SDS-PAGE. Comparison of these gels with dark controls showed that, following photolysis, there was loss of polypeptide material in the 20-30 kDa region and concomitant formation of polymers at 40 and 60 kDa, and at > 100 kDa in calf lenses (308 nm irradiation) and rat lenses (337.1 nm irradiation) in vitro. In addition, there was evidence for formation of lower molecular weight polypeptides at 10 kDa in the protein from irradiated rat lenses. The rat SDS-PAGE gels were challenged against anti-calf gamma crystallin serum, There was clear evidence that the polymeric material, in the water insoluble protein fraction from the 337-1 nm photolysed rat lenses was derived in part from gamma crystallin. The macromolecular changes detected in these photolysed rat and calf lens proteins were similar to those previously reported to accompany aging in the human lens. Biochemical changes of the type observed in UV irradiated rat and calf lenses may be responsible for the loss of visible light transmission seen in calf lenses. Key words: uv radiation; laser; cataracts; intact lenses; photopolymerization.
1. I n t r o d u c t i o n The p o l y p e p t i d e s of t h e h u m a n lens p r o t e i n s u n d e r g o a n u m b e r of a g e - r e l a t e d changes, including f o r m a t i o n of b o t h higher a n d lower m o l e c u l a r weight species. The crystallins in h u m a n fetal lens yield p o l y p e p t i d e s p r i m a r i l y in t h e 20-30 k D a range (Ringens, H o e n d e r s a n d Bloemendal, 1982) ; b u t b y t h e age of 20 yr, p o l y p e p t i d e s in t h e 40 to 50 k D a range a n d in t h e 10-12 k D a range begin to a p p e a r . The 20-30 k D a p o l y p e p t i d e s are the n o r m a l c r y s t a l l i n gene p r o d u c t s , while t h e 40-50 k D a p o l y p e p t i d e s h a v e been shown to derive from beta- a n d g a m m a - c r y s t a l l i n (Roy, Dillon, W a d a , Chaney a n d Spector, 1984) b y p o s t - t r a n s l a t i o n a l modification. T h u s it a p p e a r s t h a t some aging a n d c a t a r a c t o u s changes in lens p r o t e i n s are a t t r i b u t a b l e to modifications of t h e p r e - e x i s t i n g c r y s t a l l i n p o l y p e p t i d e s (Garner, G a r n e r a n d Spector, 1981; Russell, Smith, Carpo a n d K i n o s h i t a , 1979). E x p e r i m e n t a l studies in which lens p r o t e i n s in solution were e x p o s e d to U V r a d i a t i o n h a v e suggested t h a t lens p r o t e i n changes a c c o m p a n y i n g aging could be a c c o u n t e d for b y p h o t o c h e m i c a l l y i n d u c e d processes. Such processes m a y be d i r e c t (Zigman, 1979; Dillon a n d Spector, 1980; B o r k m a n , Tassin a n d L e r m a n , 1981; F u j i m o r i , 1982; Mandal, K o n o a n d Bose, 1988; W a l k e r a n d B o r k m a n , 1988) or sensitized (Goosey, Zigler, J e r n i g a n a n d K i n o s h i t a , 1980; A n d l e y a n d C h a p m a n . 1986). P h o t o l y s i s of i n t a c t lenses in v i t r o has also been r e p o r t e d , b o t h w i t h a d d e d sensitizer (Zigler, J e r n i g a n , P e r l m u t t e r a n d K i n o s h i t a , 1982) a n d w i t h o u t a d d e d sensitizer ( H i b b a r d , K i r k a n d B o r k m a n , 1985; Rao, B a l a s u b r a m a n i a n a n d C h a k r a b a r t i , 1987). I n m a n y cases, a m a j o r result of p h o t o l y s i s a p p e a r s to be p h o t o 0014-4835/89/120959+08 $03.00/0
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p o l y m e r i z a t i o n o f the n o r m a l c r y s t a l l i n p o l y p e p t i d e s to high m o l e c u l a r weight, c o v a l e n t l y b o n d e d a g g r e g a t e s ( B o r k m a n , 1984). I t was of i n t e r e s t to us to i n v e s t i g a t e p h o t o l y s i s of i n t a c t lenses w i t h o u t a d d e d exogenous photosensitizers (such as k y n u r e n i n e s , riboflavin, or m e t h y l e n e blue). Our e x p e r i m e n t s allowed us to m o n i t o r t h e optical consequences of UV e x p o s u r e in calf lenses (light s c a t t e r i n g a n d / o r t u r b i d i t y ) a n d also to characterize, i m m u n o l o g i c a l l y , t h e high m o l e c u l a r weight p o l y p e p t i d e s p r o d u c e d p h o t o c h e m i c a l l y in r a t lenses. I r r a d i a t i o n s were carried o u t a t 308 or 337 nm, w a v e l e n g t h s which can more r e a d i l y p e n e t r a t e t h e cornea t h a n t h e s h o r t e r w a v e l e n g t h s used in some p r e v i o u s p h o t o l y s i s studies (Dillon a n d Spector, 1980 ; H i b b a r d et al., 1985). T h u s t h e p r e s e n t studies are m o r e r e l e v a n t to the h u m a n s i t u a t i o n in t h a t t h e y do n o t rely on a d d e d photosensitizers, t h e y use m o n o c h r o m a t i c r a d i a t i o n r e a d i l y t r a n s m i t t e d b y the cornea, a n d t h e y e m p l o y i n t a c t lenses r a t h e r t h a n p r o t e i n e x t r a c t s as the t a r g e t of t h e UV r a d i a t i o n .
2. M e t h o d s R a t lenses from 3-6-week-old Sprague--Dawley albino rats were used immediately following excision. Eyes from 4-5-month-old calves were obtained from a local slaughterhouse ; the lenses were removed within 4-5 hr of death and were used immediately or were kept at - 4~ until needed. The lenses were placed in quartz tubes and were irradiated either dry, under air, or were immersed in Ringer solution during irradiation at ambient temperature of 22 • 2~ No differences in visible light scattering behavior or subsequent S D S - P A G E analysis patterns were found for dry vs. Ringer irradiations.
Excimer or nitrogen Iaser
Quortz plote
Sample
UV
cell
filter
He-Ne laser
FIo. 1. Apparatus for monitoring lens visible light transmission during UV irradiation.
The light transmission and irradiation apparatus which we used is shown in Fig. 1. Visible light from a Helium-Neon laser at 632"8 nm was made collinear with the UV output from the excimer laser by using a quartz plate as a beam splitter as shown in Fig. 1. The visible light passing through the sample was detected by a photomultiplier tube. UV radiation from the laser was prevented from entering the phototube by a Corning filter. The Visible beam intensity was monitored periodically and found to be constant to within • 2 %. Thus we were able to normalize the transmission data to constant light intensity. Output from the phototube was detected by an OMEGA DAS-16F interface which digitized the photovoltage signals for processing by Labtech Notebook software on an IBM PS/2 computer system. Hence the visible light transmission data could be plotted as a relative percent transmission or as a relative optical density. The Lumonics model 520 excimer laser was operated at an average power of 750 mW corresponding to a pulse energy of 15 m J and a repetition rate of 50 Hz. The beam dimensions at the sample position were 5 mm • 7 ram. Irradiation times
UV LASER PHOTODAMAGE TO WHOLE LENSES
961
with this source were 60 min or less. The nitrogen laser was a Molectron UV-12, operated at a pulse energy of 1"5 mJ and a repetition rate of 30 pps, corresponding to an average power of about 45 mW. The beam dimensions at the sample position were 2 mm • ram. Irradiation times with this source were up to 20 hr. UV-laser power output was checked periodically during irradiations with a Scientech model 365 power and energy meter. After photolysis, rat lenses were homogenized in 20 mM Tris HCI, I tara EDTA and 10 mM dithiothreitol at pH 7-4 (400/~l of this solution per lens) and centrifuged at 20000 rpm for 20 min. The samples were then analysed by SDS-PAGE (Laemmli, 1970), stained with Coomassie Blue and optically scanned using an accessory to a Perkin-Elmer MPF-3 fluorimeter. For immunological analysis, the previously described blot technique was used (Roy et al., 1984). The calf lenses were dissected so as to remove the outer 1 or 2 mm thickness from the anterior (irradiated) lens face. This material was then homogenized in 63 mM Tris HCI, 2 % SDS, and 0"7 M mercaptoethanol at pH 7"4 and analysed by SDS-PAGE with a 15% separating gel. 3. R e s u l t s
A plot of relative optical d e n s i t y (OD) a t 632"8 n m vs. time of exposure to 308 n m excimer laser r a d i a t i o n for a n i n t a c t calf lens is shown in Fig. 2. The relative OD is I 0-9 0.80"7 0"6 -~ 0"5 ~. 0"4 0
0"3 0-2 0-1 I
0
I0
20
50
I
40
I
I
50
J
60
Exposure time (min)
FIG. 2. Change in relative optical density of intact calf lens upon exposure to 308 nm laser radiation. seen to increase from a value of 0"0 at time zero to 0"6 after 20 m i n irradiation. F u r t h e r i r r a d i a t i o n after this time is seen to have little a d d i t i o n a l effect on the OD of the sample. Since there is essentially no light a b s o r p t i o n a t 632"8 n m , most of the 632"8 OD produced b y U V i r r a d i a t i o n arises from t u r b i d i t y / l i g h t scattering, a n d n o t from absorption. The p a t t e r n of t u r b i d i t y growth shown for whole calf lenses in Fig. 2~ parallels t h a t seen in similar e x p e r i m e n t s with single, isolated lens proteins. F o r example, W a l k e r a n d B o r k m a n (1989) a n d M a n d a l et al. (1988) m o n i t o r e d visible light scattering a n d t u r b i d i t y in the purified calf g a m m a crystallins, g a m m a - I I , I I I , a n d IV, a n d f o u n d similar increases in light scattering following UV t r e a t m e n t (with or w i t h o u t added sensitizers) to those observed here. I r r a d i a t e d r a t lenses w e r e subjected to S D S - P A G E after s e p a r a t i o n into water soluble a n d insoluble fractions. Figure 3 shows 470 n m d e n s i t o m e t e r scans of the Coomassie Blue stained S D S - P A G E gels of the w a t e r insoluble fraction at zero time a n d after 12 hr exposure to the n i t r o g e n laser at 337"1 nm. As expected, the strongest
962
J. D I L L O N
ET AL.
~ II;I I .I;!1 ~'"I I/~'~l l
20 kD
45 kD
Top
Fie. 3. Densitometer scans at 470 n m of Coomassie Blue stained S D S - P A G E gels of the water insoluble protein fraction from rat lenses: zero time ( ), and after 12 hr photolysis at 337'1 n m
(---).
A
B
C
FIG. 4. S D S - P A G E gels of calf lens insoluble protein: lane A, dark control; lanes B and C, after 60 m i n exposure of the intact lens (in vitro) to 308 n m excimer laser radiation. Lane C is the front I m m a n d lane B is the front 2 m m section of the irradiated (anterior) lens face.
UV LASER
PHOTODAMAGE
TO WHOLE
LENSES
963
50
40 "E
j 2 o kD
5C
"6 ill
kD
3o
I
2
I
4
I
6 Time (hr)
I I0
8
I
12
FIG. 5. Relative percent of crystallin polypeptides in the 20-25 k D a and 11 k D a regions as a function of 337.1 n m laser irradiation time. D a t a are shown for the rat water insoluble fractions.
S
P
0
S
P
4
S
P
8
S
P
12 hr
FIe. 6. A n t i - g a m m a blot of proteins extracted from irradiated rat lenses and subjected to S D S - P A G E (S, soluble and P, insoluble fractions). The time of 337"1 n m radiation is shown at the b o t t o m of each lane.
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J. DILLON ET AL.
bands in the unirradiated lenses appeared in the range 20-30 kDa, Following photolysis there was a loss of 20-30 kDa material and formation of diffuse bands at approximately 45 kDa and higher molecular weight, and an even more diffuse band at low molecular w e i g h t - - a b o u t 10-15 kDa. Irradiated calf lenses were also analysed by S D S - P A G E following separation into water-soluble and insoluble fractions (Fig. 4). The insoluble fraction from calf lenses irradiated for 1 hr at 308 nm showed diffuse, new bands in the 40-60 kDa region as well as high molecular weight material which did not enter the gels. These features were not presented in unirradiated controls which showed only the normal bands in the 20-30 kDa region. The UV irradiated whole lens material gave S D S - P A G E patterns similar to those observed in our previous study of UV effects on isolated calf lens crystallins (Walker and Borkman, 1989). The S D S - P A G E data on UV-irradiated rat lenses revealed a loss of material in the 20 kDa region and an increase in polypeptides with molecular weights above and below those appearing in the normal crystallins. This trend is plotted in Fig. 5 which shows the loss of 20 kDa material and the growth of fragmented polypeptides as a function of rat lens irradiation time. In addition, we observed increased amounts of polymeric material as a function of UV irradiation time. These changes are similar to those noted previously for human lens crystallins as a function of aging (Garner et al., 1981). Since it had been previously demonstrated t h a t the human 43 kDa component contains g a m m a crystallin (Roy et al., 1984), the gels from photolysed rat lenses were blotted against anti-gamma serum. A s seen in Fig. 6, the formation of poiymers (dimers, trimers, etc.) containing g a m m a brystallin was clearly in evidence following photolysis of intact rat lenses, particularly in the w a t e r insoluble fraction: 4. D i s c u s s i o n
The direct (no added photosensitizer) photolysis of intact r a t and calf lenses using nitrogen or excimer laser UV radiation has been shown to lead to some of the same macromolecular changes detected in aging or cataractous human lenses. These include increased visible light scattering, and decreased visible light transmission, at the organ level, and photo crosslinking and photo fragmentation of lens crystallin proteins, at the molecular level. Previous workers h a v e noted the photo polymerization of lens proteins and have suggested a possible relationshi p to lens aging and eataractogenesis (Dillon and Spector, 1980; Goosey et al., 1980; Fujimori, 1982). But, the present work is the first to report photolytic peptide bond cleavage in whole lenses. In addition, our work suggests a possible correlation between loss of visible light transmission in UV irradiated whole calf lenses and the accompanying lens protein photo polymerization. Other mechanisms m a y also contribute to the reduced transmission of the lens following UV exposure. Ringens, Hoenders and Bloemendal (1982) reported t h a t the insoluble fraction of human fetal lens contains actin (43 kDa) and vimentin (65 kDa). By the age of 20, vimentin is no longer detectable and the 43 kDa region contains a more diffuse band. I t has recently been demonstrated that the 43 k D a polypeptide in older human lenses contains significant amounts of beta and g a m m a crystallin polypeptides (Roy et al., 1984). The formation of a diffuse band of crystallin origin was observed in the photochemical data reported here. In addition, we found that polymerization does not stop at the dimer stage but continues on to trimers, tetramers, etc. Similar larger polymers have been observed in aged human lens proteins (Roy et al., 1984).
UV LASER PHOTODAMAGE TO WHOLE LENSES
965
Bond cleavage, though not as obvious in our data as polymerization, is suggested by the S D S - P A G E gel scans in Fig. 3. Although it is apparent from the anti-gamma blots that rat lens g a m m a crystallin is involved in the photo polymerization process, the 10-15 kDa region of our rat lens gels did not stain positive for g a m m a crystallin. Hence the parentage of these fragments is open to question at this time. The actual photolytic mechanisms, in intact lenses, at the wavelengths used in our experiments are not known. Irradiation at 308 nm could result in excitation of Trp residues in lens proteins by direct light absorption. Alternatively, some or all of the incident 308 nm radiation could be absorbed by other chromophores in the lens, including the known photosensitizer N-formylkynurenine (Walrant and Santus, 1974), and lead to singlet oxygen production and subsequent attack of lens proteins (Ziglcr et al., 1982). On the other hand, irradiation at 337"1 nm is less likely to excite Trp by direct absorption, and photosensitization by unknown absorbers in the lens is also a possibility. However, it is interesting to note t h a t 337"1 nm radiation can cause polymerization in isolated lens crystallins (Walker and Borkman,: 1989). In addition, 337"1 nm photolysis of free Trp monomer in solution produces m a n y of the same products found in the 290 nm photolysis of Trp where it is clear that the mechanism involves direct absorption by Trp (Borkman et al., 1986). In conclusion, m a n y of the macromolecular changes associated with human lens aging and cataracts are also seen in the 308 or 337 1 nm UV laser photolysis of rat and whole calf lenses in vitro. ACKNOWLEDGMENTS The support of NIH grants EY-6800 (Georgia Tech), EY-2283 (Columbia), and NEI grants to A. Spector are gratefully acknowledged. REFERENCES Andley, U. and Chapman, S. (1986). Conformational changes of bovine lens crystallins in a photodynamic system. Photochem. Photobiol. 44, 67-74. Borkman, R. (1984). Cataracts and photochemical damage in the lens. Ciba Foundation Symposium 106, Human Cataract Formation, (Ed. Spector, A.) Pp. 88-99. Pitman: London. Borkman, R., Hibbard, L. and Dillon, J. (1986). The photolysis of tryptophan with 337'1 nm laser radiation. Photochem. Photobiol. 43, 13-19. Borkman, R., Tassin, J. and Lcrman, S. (1981). The rates of photodestruction of tryptophan residues in human and bovine ocular lens proteins. Exp. Eye Res. 32, 747-54. Dillon, J. and Spector, A. (1980). A comparison of aerobic and anaerobic ph0tolysis of lens proteins. Exp. Eye Res. 31,591-9. Fujimori, E. (1982). Crosslinking and blue fluorescence of photo-oxidized alpha crystallin. Exp. Eye Res. 34, 381-8. Garner, W., Garner, M. and Spector, A. (1981). Gamma crystallin, a major cytoplasmic polypeptide, disulfide linked to membrane proteins in human cataract. Biochem. Biophys. Res. Commun. 98, 439-43. Goosey, J., Zigler, J., Jernigan, H. and Kinoshita, J. (1980). Crosslinking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen. Science 208, 1278-2380. Hibbard, L., Kirk, N. and Borkman, R. (1985). The in vitro photolysis of whole rat lenses using focused 290 nm laser radiation. Exp. Eye Res. 40, 285-90. Laemmli, U.K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 680-6. Mandal, K., Kono, M., Bose, S., Thomson, J. and Chakrabarti, B. (1988). Structure and stability of gamma-crystallins. IV. Aggregation and structural destabilization in photosensitized reactions, Photochem. Photobiol. 47, 583-92.
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Rao, C., Balasubramanian, D. and Chakraharti, B. (1987). Monitoring light induced changes in isolated intact eye lenses. Photochem. Photobiol. 46, 511-15. Ringens, P., Hoenders, H. and Bloemendal, H. (1982). Effect of aging on the water-soluble and water-insoluble protein pattern in normal human lens. Exp. Eye Res. 34, 201-7. Roy, D., Dillon, J., Wada, E., Chaney, W. and Spector, A. (1984). Nondisulfide polymerization of gamma- and beta-crystallins in the human lens. Proc. Natl~ Acad. Sci. U.S.A. 81, 2878-81. Russell, P., Smith, S., Carper, D. and Kinoshita, J. (1979). Age and cataract related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens. Exp. Eye Res. 29, 245-55. Walker, M. and Borkman, R. (1988). Light scattering and photo crosslinking in the calf lens crystallins gamma-II, III, and IV. Exp. Eye Res. 48, 375-83. Walrant, P. and Santus, R. (1974). N-Formylkynurenine, a tryptophan photooxidation product, as a photodynamic sensitizer. Photochem. Photobiol. 19, 411-17. Zig[er, J., Jernigan, H., Perlmutter, N. and Kinoshita, J. (1982). Photodynamic crosslinking of polypeptides in intact rat lens. Exp. Eye Res. 35,239-49. Zigman, S. (1979). Near UV light and cataracts. Photoche.m. Photobiol. 26, 437-41.