A C T A O P H T H A L M O L O G I C A VOL. 5 6 1 9 7 8
Department of Ophthalmology (Head: F. Phinizy Calhoun, Jr.), Emory University and Chemistry Department (Head: J . A . Bertrand), Georgia Institute of Technology, USA
ULTRAVIOLET RADIATION IN THE AGING AND CATARACTOUS LENS A Survey* BY
SIDNEY LERMAN and RAYMOND BORKMAN
An increase in the insoluble protein levels, in fluorescence, and a decrease in the SH concentration are three specific aging parameters in the mammalian lens. These can be markedly accelerated in human, rat and mouse lenses by in uitro exposure (for at least 4 h) to UV radiation (above 300 nm) and 3-aminotriazole (AT) and in viuo by exposing mice to UV and parenteral A T for 4-6 weeks. UV, fluorescence, electron paramagnetic resonance (EPR) and Lasar Raman Spectroscopy were performed on normal and UV plus A T exposed 4-6 week old rat lenses and normal and experimental human lenses (1 day to 75 years of age). These data demonstrate the presence and/or induction of at least one fluorescent compound (fluorogen) (360 nm activation, 440 nm emission) derived from tryptophan by a free radical induced photooxidation reaction. Similar studies on purified lens proteins and peptides indicate that this fluorogen is tightly bound to at least one specific peptide. The age related increase in the concentration of fluorescent compounds enhances the yellowing of the lens core and accompanies polymerization of soluble protein precursors into the insoluble protein fraction. There is also a progressive fall in SH levels (with age or UV exposure) and an increase in SS bonds without any significant alteration in the secondary configuration of the lens proteins Received November 9, 1977.
*
Presented at the Second International Congress of Eye Research, Jerusalem, Israel, September, 1976.
139
Sidney Lerman and Raymond Borkman
(which remain mainly in the anti-parallel beta-pleated configuration). It is postulated that UV radiation (300-380 nm) plays a role in lenticular aging, particularly nuclear sclerosis and its extreme state characterized by the Brunescent Cataract. Key words: nuclear cataract - lens aging - fluorogen - ultraviolet radiation - tryptophan.
The fluorescent property of the ocular lens was first noted well over one hundred years ago (Regnauld 1858) but it was not until the last decade that this phenomenon was associated with the increasing yellow coloration of the lens core (or nucleus) with age. T h e increasing yellow to yellow brown coloration of the lens nucleus with age has been shown to be associated with and related to the presence of fluorescent compounds in specific protein fractions of the ocular lens (see review by Lerman 1976). This process is induced by prolonged exposure of the lens to ultraviolet radiation above 295 nm. The cornea filters out all the ultraviolet light below 295 nm but the ocular lens absorbs most of the ultraviolet light above 295 nm. It has been suggested that in the normal lens the yellow pigmentation might serve as an intraocular filter for blue light (which is the wavelength of the visible spectrum subject to the greatest dispersion). There is also evidence that the pigments function as a protective filter with respect to the underlying vitreous and retina thereby preventing UV radiation from reaching the interior of the eye (Lerman & Borkman 1976; Weale 1973; Wolbarsht 1976).
Lens Fluorescence (a) Intrinsic Prior to considering lenticular fluorescence it is important to note that most proteins are endowed with a n intrinsic ultraviolet fluorescence because they contain aromatic amino acids (particularly phenylalanine, tyrosine and tryptophan). Of these three aromatic amino acids, phenylalanine has the lowest fluorescence quantum yield as compared with tyrosine and tryptophan (approximately 1/10 to 1/20 of the latter two). Protein fluorescence spectra are thus generally considered with respect to tyrosine and tryptophan. I n those proteins where no tryptophan is present (for example, insulin or ribonuclease) it is possible to demonstrate intrinsic tyrosine fluorescence only (Teale 1960), however, the presence of even one tryptophan residue in a protein (for example, horse serum albumin which has 17 tyrosines and 1 tryptophan) will result in an 140
UV Radiation in Lens Aging
intrinsic fluorescence essentially that of tryptophan alone. It has also been shown that the tryptophan emission maxima in proteins can vary from 332 to 342 nm depending on the protein. It should be noted that free tryptophan has a characteristic fluorescence emission at approximately 350360 nm but this shifts to the blue side of the spectrum when tryptophan fluorescence is measured in a protein. Mammalian lens proteins, in general, have a considerably higher content of tyrosine as compared to tryptophan but the presence of the tryptophan residues results in a n intrinsic protein fluorescence in the lens due to tryptophan alone. (b) Non-intrinsic
Fransois et al. (1961) noted that the presence of a fluorescent peptide in primate lenses which could be activated by UV light. Specific fluorescent proteins in the human and other mammalian lenses have been demonstrated by several investigators (see review by Lerman 1976). I n addition to the fluorescence due to tryptophan these proteins contain fluorescent material with a n activation wavelength at 360 nm and an emission peak at 420-440 nm. This type of fluorescence has been shown to be present both in the soluble and insoluble fractions of the human lens (mainly in the latter) and it increases with age, particularly in the insoluble lens protein fraction. This fluorogen is tightly bound to one peptide derived from the insoluble lens protein and is a tricyclic compound, molecular weight approximately 300 (Lerman 1972; Lerman & Borkman 1976). The increasing yellow coloration of the lens core is due to the presence of these fluorogens associated with specific protein fractions in the ocular lens.
Effects of UV Radiation on Lens Protein Ultraviolet light and ionizing radiation are capable of generating free radicals and it is accepted by many investigators that aging itself must be due at least in part to damage caused by radical reactions within the tissues. It has also been shown that proteins can be significantly altered by radical reactions with the aromatic amino acid residues in the proteins being particularly responsible (Meybeck & Windle 1969; Shields & Hamrich 1970; Zirlin 1969). Thus prolonged exposure of the ocular lens to long UV radiation could result in free radical formation and the generation of one or more fluorogens in increasing concentration. The proposal of protein tryptophan photo-degradation resulting in protein pigmentation (Kurzel et al. 1973a,b) correlates with our findings regarding the inverse relationship between tryptophan fluorescence intensity and fluorescence 141
Sidney Lerman and Raymond Borkman
emission intensity in the whole lens (Borkman et al. 1977; Lerman K: Borkman 1976). Previous studies in our Laboratory have demonstrated a n in vitro and in vivo induction and acceleration of one fluorescent peak in the ocular lens of the mouse, rat and human species (Lerman & Borkman 1976; Lerman et al. 1976). Studies on normal human lenses ranging in age from one day to ninety years have demonstrated that this fluorescence is not present to any significant extent within the first year of life but then becomes manifest and increases in emission intensity with age as shown in Figs. 1 and 2. The progressive increase in this fluorescence is paralleled by an increase in relative concentration of the insoluble protein fraction. A similar relationship exists in the rat and mouse lens. In vitro studies with lens incubates (mouse, rat and human lenses) have demonstrated that this fluorescence can be induced and/or markedly accelerated in lens incubation systems in which the lenses are exposed to 3-aminotriazole (AT) and UV radiation and a similar effect occurs in vivo in mice exposed to UV and parenteral AT (Lerman et al. 1976). AT is a catalase (peroxidase)
I /I TRP332
250
300
k 250
300
350
34 Y R HUMAN
400 nm
450
550
500
: 350
400
450
500
550
nm
Fig. I. Flucrescence spectra of 34 and 75 year normal human lenses showing TRP 332 fluorescence and 440 nm fluorescence (FL 440) a t 10-6 gain. Spectra in 78 year lenses also demonstrate bathochromic shift with 400 nm excitation resulting in 500 nm emission due to 2nd fluorescence region which develops with age (see Fig. 4).
142
UV Radiation in Lens Aging
HUMAN LENS
01
AGE
YRS
Fig. 2. 360 I -ratios 290
representing fluorescence intensity at 440 nm (360 nm excitation) over TRP
360 fluorescence at 332 nm (290 nm excitation). I - ratio shows age related increase in 290 normal human lens (X); marked increase in brown nuclear cataracts (N); relatively normal levels in cortical cataracts (C); and high normal values in mixed cortical and nuclear cataracts (M). Each point represents a single lens.
inhibitor, thus more glutathione is being used up by the lens in such incubation systems, and a significant decrease in the concentration of glutathione in lenses incubated with A T has been demonstrated by several investigators (Bhuyan & Bhuyan 1975; Bhubyan et al. 1973, 1974; Kuck 1974; and Table I). The resultant depletion of a n important free radical scavenger in the lens increases its sensitivity to UV radiation, thereby accelerating the photo-induced generation of fluorescent material. Action spectra performed on 4-6 h in vitro lens incubation systems (rat and human lenses) have demonstrated that tryptophan depletion occurs specifically with the generation of the 360/440 fluorescent material (Borkman et al. 1977). A plot of the rate of fluorogen production (normalized to constant photon flux) versus the radiation wavelength showed relatively little action at 360, 340 and 320 nm but the action increased sharply at 300 nm and remained constant at 143
Sidney Lerman and Raymond Borkman
Av. lens wt.
Whole lens
mg
(mg Of01
Av. lens wt.
Whole lens
mg
(mg O/O)
Cortex (mg "0)
Nucleus (mg O/O)
290 and 280 nm. This indicates that the material is generated photochemically with tryptophan as the prime UV light absorbing species and this conclusion is supported by the observation that the photochemical production of fluorogen is associated with a decrease in tryptophan fluorescence.
Free Radicals in the Lens and their Relationship to Fluorescence
EPR (electron paramagnetic resonance) spectra on whole rat and mouse lenses and on the nuclear core from human lenses demonstrate that a significant tryptophan free radical can be demonstrated in the normal rat and human lens after ten minutes irradiation with UV light, but a marked decrease in the signal is noted in those lenses which have been previously exposed to UV plus AT for at least 4-6 h. There is also a direct relationship between the signal in144
UV Radiation in Lens Aging
h
I
Fig. 3.
EPR spectra of normal human 8, 47, and 83 years, 3 mm central portion of lens nucleus: See Lerman & Borkman 1976, for details of preparation of lens material; spectroscopy conditions: 77'K, 75 millimwatts power, 9.2 GHz microwave frequency, 500 g sweep at 435 values for each lens derived from 295 fluorescence spectra on whole lens. (See Figs. 2 and 4).
2.5 min, 0.1 sec time constant. I
360
- and I - ratio 290
tensity of the UV induced tryptophan free radical and the age of the human lens. T h e highest signal intensity is seen in the young lens and the signal decreases significantly with age particularly after the fifth or sixth decade (Fig. 3). This correlates with the evidence previously cited that tryptophan depletion is associated with generation of fluorescence as aging proceeds (some of the fluorogens being generated as a U V induced photodegradation product of tryptophan). T h e specific site of the UV-induced action thus appears to be on one or more protein-bound tryptophan residues in which a photochemical degradation occurs. Weiter & Finch (1975) have demonstrated the tryptophan triplet state in U V exposed human lens material as well as the tryptophan free radical. This excited triplet state may be dissipated by a photochemical reaction which ultimately generates fluorogens by intersystem crossing to the ground singlet state as suggested by Kurzel et al. (1973a,b) or by other routes. The photochemical degradation is initiated by the photoionization of the excited tryptophan molecule and results in the cleavage of the Indole ring at C2-c3 position. Laser flash photolysis experiments on aqueous tryptophan have recently shown that U V degradation of aqueous tryptophan occurs via such a photoionization mechanism (Bryant et al. 1975). 145 Acta ophthal. 5(i, I
10
Sidney Lerman and Raymond Borkman
Conclusion The preceding discussion has demonstrated that fluorescence spectroscopy can be utilized to measure an aging parameter in the ocular lens and that one major fluorescent region appears to be derived from tryptophan on the basis of prolonged exposure of the lens to ultraviolet light above 295 nm. This fluorescence which has an activation peak of 360 nm and an emission peak at 420-440 nm is definitely age-related, as is the increase in the water insoluble proteins and the decline in SH and concomitant increase in the level of SS bonds particularly in the lens nucleus. It is postulated that ultraviolet radiation above 295 nm plays a significant role in the increasing yellow colour of the lens nucleus and in lenticular aging, particularly nuclear sclerosis and its extreme state characterized by the brunescent (brown) cataract. The yellow pigmentation is confined mainly to the lens nucleus since there is a n insufficient concentration of free radical scavenger in this region to abort the photochemical degradation reaction. It is interesting to note that D-penicillamine which is an excellent free radical scavenger is capable of preventing the induction of the fluorescent material in young rat or human lenses in vitro and mouse lenses both in vitro and in vivo (Lerman & Borkman 1976; Lerman et al. 1976). Thus the lens nucleus is particularly susceptible to UV radiation since it has a relative lack of the normal free radical scavenger present in the lens (glutathione) as compared with the lens cortex; this becomes particularly pronounced as the lens ages (Table I). The relative excess of glutathione in the lens cortex would therefore be sufficient to abort any photo-degradation of tryptophan that could theoretically occur in that portion of the lens. While the whole lens nucleus derived from the brown cataract may contain a relatively similar level of tryptophan as compared with the lens nucleus in the normal individual, there may be a decreased amount of tryptophan in the insoluble protein fraction of the lens nucleus, particularly in the urea insoluble portion in the brown nuclear cataract. Although this would represent only a few of the total tryptophan residues in the lens, they would be sufficient to account for the findings regarding tryptophan depletion related to tryptophan free radical generation and increasing fluorescence as measured by EPR and fluorescence spectroscopy. T he latter two methods are more sensitive compared with amino acid composition data and would pick up much smaller fluctuations in relative concentrations of specific amino acids. Thus only a relatively limited number of tryptophan residues are actually susceptible to ultraviolet induced photo-degradation because of their specific location and immediate environment so that following absorption of UV light, and excited tryptophan molecule can not dissipate its excitation by the various mechanisms available to it (Feitelson 146
UV Radiation in Lens Aging
1% HUMAN
..
O.bj 0.5 1
1'0
2'0
4b 50~6'0 7'0 8b
3'0
AGE
9b
YRS
Fig. 4 . 295 I - ratios representing fluorescence intensity of 2nd fluorescence region at 500435 520 nm (435 nm excitation) over TRP 332 nm fluorescence (295 nm excitation). 295 and 435 nm interference filters employed since cortical cataracts were also examined and 435
regular spectroscopy resulted in marked light scattering. I - values in normal lens 295 (X), in nuclear cataract (N), cortical cataract (C) and mixed cortical and nuclear cataract (M). Each point represents a single lens.
1971; Lerman & Borkman 1976). While the majority of the tryptophan residues in the lens proteins can absorb and dissipate the ultraviolet radiation without undergoing any significant alteration, an adequate amount of free radical scavengers are also available within their immediate environment so. that UV photo-degradation will be aborted in some of the tryptophan residues which do enter the excited state, particularly in the lens cortex. Recent experiments in our laboratory have also demonstrated the presence of a second fluorescent region (approximately 420-435 nm activation and 500520 nm emission) which becomes apparent in the lens after the first decade of life and increases with age but becomes particularly pronounced in the brown cataract as compared with the normal lens from a comparable age group and with the cortical opacities or cortical cataract in which the lens nucleus remains relatively unaffected (Fig. 4 and Lerman & Borkman 1976). It is still not clearly 147
Sidney Lerman and Raymond Borkman
defined whether this second fluorescence emission peak is characteristic of a separate species or if it derives from the previously discussed fluorescent material, possibly as a degradation product.
Acknowledgments This work was supported by NIH Grants EY-01575 and EY-01138. Th e assistance of the Atlanta Lions Eye Bank in obtaining human lenses is gratefully acknowledged. Th e authors also acknowledge Professor Allen K. Garrison’s advice in the EPR experiments and for the use of his spectrometer.
References Bhuyan D. K. & Bhuyan K. C. (1975) Regulation of hydrogen percxide leve!s in eye humors. IRCS Med. Sci. 3, 415. Bhuyan K. C., Bhuyan D. K. & Katzin H. M. (1973) Amizol induced cataract and inhibition of lens catalase in rabbit. Ophthalmic Res. 5 , 236-247. Bhuyan K. C., Bhuyan D. K. & Turtz A. (1974) Aminotriazole - effect on the ocular tissue in rabbit. IRCS Med. Sci. 2, 1994. Borkman R. F., Dalrymple A. & Lerman S. (1977) Ultraviolet action spectrum for production of a fluorogen in the ocular lens. Photocliem. Photobiol. 26, 129-132. Bryant F. D., Santus R. & Grossweiner L. I. (1975) Laser flash photolysis of aqueous tryptophan. J . Pliys. CIiem. 79,27 11-27 16. Feitelson J. (1971) T h e formation of hydrated electrons from the excited state of indole derivatives. Photochem. Photobiol. 13, 87-96. Fransois J,, Rabaey M. & Recoules N. (1961) A fluorescent substance of low molecular weight in the lens of primates. Arch. Ophthal. (Chicago) 65, 118-126. Kuck J. F. (1974) Effect of long wave ultraviolet light on the lens in vitro with 3-aminotriazole (abstract). A R V O Annual Meefing, p. 84. Kurzel R., Wolbarsht M. L. & Yamanashi B. S. (1973a) Spectral studies on normal and cataractous intact human lenses. Exp. Eye Res. 1 7 , 65-71. Kurzel R., Wolbarsht M. L., Yamanashi B. S., Station G. W. & Borkman R. F. (1973b) Tryptophan excited states and cataracts in the human lens. Nature (Lond.) 241, 132-133. Lerman S. (1972) Lens proteins and fluorescence. Israel /. Med. Sci. 8, 1583-1589. Lerman S. (1976) Lens fluorescence in aging and cataract formation. Docum. ophthal. 8, 241-260. Lerman S. & Borkman R. F. (1976) Spectroscopic evaluation and classification of the normal, aging and cataractous lens. Oplithalmic Res. 8, 335-353. Lerman S., Kuck J. F., Borkman R. & Saker E. (1976) Induction, acceleration and prevention (in vitro) of a n aging parameter in the ocular lens. Ophthalmic Res. 8, 213-226. Meybeck A. & Windle J. J. (1969) Electron paramagnetic resonance study of X-ray and gamma-ray irradiated peptides in the solid state. Radiat. Res. 40, 263-275.
148
UV Radiation in Lens Aging Regnauld J. (1858) Sur la fluorescence des milieux de I’oeil chez I’homme et quelques mammiferes. L’lnstitut 26, 410. Shields H. & Hamrich P. J., Jr. (1970) Relative stability of the characteristic sulfur and doublet resonances in x-irradiated native proteins as measured with ESR. Radial. Reg. 41, 259-267. Teale F. W . J. (1960) The ultraviolet fluorescence of proteins in neutral solution. Biochem. J. 76, 381-388. Weale R. R. (1973) T h e efrects of the aging lens on vision. In: Ziba Symposium X I X . The Human Lens in Relation to Cataract, pp. 7-19. Elsevier/Amsterdam. Weiter J. J. & Finch E. D. (1975) Paramagnetic species in cataractous human lens. Nature (Lond.) 254, 536-537. Wolbarsht M. L. (1976) The function of intraocular color filters. Fed. Proc. 55, 44-50. Zirlin A. & Karel M. J. (1969) Oxidation effects in a freeze dried gelatin-methyl linoleate system. J. Food Sci. 34, 160-164.
Author’s address: Sidney Lerman, M. D., Laboratory for Ophthalmic Research, Emory University, Atlanta, Georgia 30322, USA.
149
Department of Ophthalmology (Head: F. Phinizy Calhoun, Jr.), Emory University and Chemistry Department (Head: J . A . Bertrand), Georgia Institute of Technology, USA
ULTRAVIOLET RADIATION IN THE AGING AND CATARACTOUS LENS A Survey* BY
SIDNEY LERMAN and RAYMOND BORKMAN
An increase in the insoluble protein levels, in fluorescence, and a decrease in the SH concentration are three specific aging parameters in the mammalian lens. These can be markedly accelerated in human, rat and mouse lenses by in uitro exposure (for at least 4 h) to UV radiation (above 300 nm) and 3-aminotriazole (AT) and in viuo by exposing mice to UV and parenteral A T for 4-6 weeks. UV, fluorescence, electron paramagnetic resonance (EPR) and Lasar Raman Spectroscopy were performed on normal and UV plus A T exposed 4-6 week old rat lenses and normal and experimental human lenses (1 day to 75 years of age). These data demonstrate the presence and/or induction of at least one fluorescent compound (fluorogen) (360 nm activation, 440 nm emission) derived from tryptophan by a free radical induced photooxidation reaction. Similar studies on purified lens proteins and peptides indicate that this fluorogen is tightly bound to at least one specific peptide. The age related increase in the concentration of fluorescent compounds enhances the yellowing of the lens core and accompanies polymerization of soluble protein precursors into the insoluble protein fraction. There is also a progressive fall in SH levels (with age or UV exposure) and an increase in SS bonds without any significant alteration in the secondary configuration of the lens proteins Received November 9, 1977.
*
Presented at the Second International Congress of Eye Research, Jerusalem, Israel, September, 1976.
139
Sidney Lerman and Raymond Borkman
(which remain mainly in the anti-parallel beta-pleated configuration). It is postulated that UV radiation (300-380 nm) plays a role in lenticular aging, particularly nuclear sclerosis and its extreme state characterized by the Brunescent Cataract. Key words: nuclear cataract - lens aging - fluorogen - ultraviolet radiation - tryptophan.
The fluorescent property of the ocular lens was first noted well over one hundred years ago (Regnauld 1858) but it was not until the last decade that this phenomenon was associated with the increasing yellow coloration of the lens core (or nucleus) with age. T h e increasing yellow to yellow brown coloration of the lens nucleus with age has been shown to be associated with and related to the presence of fluorescent compounds in specific protein fractions of the ocular lens (see review by Lerman 1976). This process is induced by prolonged exposure of the lens to ultraviolet radiation above 295 nm. The cornea filters out all the ultraviolet light below 295 nm but the ocular lens absorbs most of the ultraviolet light above 295 nm. It has been suggested that in the normal lens the yellow pigmentation might serve as an intraocular filter for blue light (which is the wavelength of the visible spectrum subject to the greatest dispersion). There is also evidence that the pigments function as a protective filter with respect to the underlying vitreous and retina thereby preventing UV radiation from reaching the interior of the eye (Lerman & Borkman 1976; Weale 1973; Wolbarsht 1976).
Lens Fluorescence (a) Intrinsic Prior to considering lenticular fluorescence it is important to note that most proteins are endowed with a n intrinsic ultraviolet fluorescence because they contain aromatic amino acids (particularly phenylalanine, tyrosine and tryptophan). Of these three aromatic amino acids, phenylalanine has the lowest fluorescence quantum yield as compared with tyrosine and tryptophan (approximately 1/10 to 1/20 of the latter two). Protein fluorescence spectra are thus generally considered with respect to tyrosine and tryptophan. I n those proteins where no tryptophan is present (for example, insulin or ribonuclease) it is possible to demonstrate intrinsic tyrosine fluorescence only (Teale 1960), however, the presence of even one tryptophan residue in a protein (for example, horse serum albumin which has 17 tyrosines and 1 tryptophan) will result in an 140
UV Radiation in Lens Aging
intrinsic fluorescence essentially that of tryptophan alone. It has also been shown that the tryptophan emission maxima in proteins can vary from 332 to 342 nm depending on the protein. It should be noted that free tryptophan has a characteristic fluorescence emission at approximately 350360 nm but this shifts to the blue side of the spectrum when tryptophan fluorescence is measured in a protein. Mammalian lens proteins, in general, have a considerably higher content of tyrosine as compared to tryptophan but the presence of the tryptophan residues results in a n intrinsic protein fluorescence in the lens due to tryptophan alone. (b) Non-intrinsic
Fransois et al. (1961) noted that the presence of a fluorescent peptide in primate lenses which could be activated by UV light. Specific fluorescent proteins in the human and other mammalian lenses have been demonstrated by several investigators (see review by Lerman 1976). I n addition to the fluorescence due to tryptophan these proteins contain fluorescent material with a n activation wavelength at 360 nm and an emission peak at 420-440 nm. This type of fluorescence has been shown to be present both in the soluble and insoluble fractions of the human lens (mainly in the latter) and it increases with age, particularly in the insoluble lens protein fraction. This fluorogen is tightly bound to one peptide derived from the insoluble lens protein and is a tricyclic compound, molecular weight approximately 300 (Lerman 1972; Lerman & Borkman 1976). The increasing yellow coloration of the lens core is due to the presence of these fluorogens associated with specific protein fractions in the ocular lens.
Effects of UV Radiation on Lens Protein Ultraviolet light and ionizing radiation are capable of generating free radicals and it is accepted by many investigators that aging itself must be due at least in part to damage caused by radical reactions within the tissues. It has also been shown that proteins can be significantly altered by radical reactions with the aromatic amino acid residues in the proteins being particularly responsible (Meybeck & Windle 1969; Shields & Hamrich 1970; Zirlin 1969). Thus prolonged exposure of the ocular lens to long UV radiation could result in free radical formation and the generation of one or more fluorogens in increasing concentration. The proposal of protein tryptophan photo-degradation resulting in protein pigmentation (Kurzel et al. 1973a,b) correlates with our findings regarding the inverse relationship between tryptophan fluorescence intensity and fluorescence 141
Sidney Lerman and Raymond Borkman
emission intensity in the whole lens (Borkman et al. 1977; Lerman K: Borkman 1976). Previous studies in our Laboratory have demonstrated a n in vitro and in vivo induction and acceleration of one fluorescent peak in the ocular lens of the mouse, rat and human species (Lerman & Borkman 1976; Lerman et al. 1976). Studies on normal human lenses ranging in age from one day to ninety years have demonstrated that this fluorescence is not present to any significant extent within the first year of life but then becomes manifest and increases in emission intensity with age as shown in Figs. 1 and 2. The progressive increase in this fluorescence is paralleled by an increase in relative concentration of the insoluble protein fraction. A similar relationship exists in the rat and mouse lens. In vitro studies with lens incubates (mouse, rat and human lenses) have demonstrated that this fluorescence can be induced and/or markedly accelerated in lens incubation systems in which the lenses are exposed to 3-aminotriazole (AT) and UV radiation and a similar effect occurs in vivo in mice exposed to UV and parenteral AT (Lerman et al. 1976). AT is a catalase (peroxidase)
I /I TRP332
250
300
k 250
300
350
34 Y R HUMAN
400 nm
450
550
500
: 350
400
450
500
550
nm
Fig. I. Flucrescence spectra of 34 and 75 year normal human lenses showing TRP 332 fluorescence and 440 nm fluorescence (FL 440) a t 10-6 gain. Spectra in 78 year lenses also demonstrate bathochromic shift with 400 nm excitation resulting in 500 nm emission due to 2nd fluorescence region which develops with age (see Fig. 4).
142
UV Radiation in Lens Aging
HUMAN LENS
01
AGE
YRS
Fig. 2. 360 I -ratios 290
representing fluorescence intensity at 440 nm (360 nm excitation) over TRP
360 fluorescence at 332 nm (290 nm excitation). I - ratio shows age related increase in 290 normal human lens (X); marked increase in brown nuclear cataracts (N); relatively normal levels in cortical cataracts (C); and high normal values in mixed cortical and nuclear cataracts (M). Each point represents a single lens.
inhibitor, thus more glutathione is being used up by the lens in such incubation systems, and a significant decrease in the concentration of glutathione in lenses incubated with A T has been demonstrated by several investigators (Bhuyan & Bhuyan 1975; Bhubyan et al. 1973, 1974; Kuck 1974; and Table I). The resultant depletion of a n important free radical scavenger in the lens increases its sensitivity to UV radiation, thereby accelerating the photo-induced generation of fluorescent material. Action spectra performed on 4-6 h in vitro lens incubation systems (rat and human lenses) have demonstrated that tryptophan depletion occurs specifically with the generation of the 360/440 fluorescent material (Borkman et al. 1977). A plot of the rate of fluorogen production (normalized to constant photon flux) versus the radiation wavelength showed relatively little action at 360, 340 and 320 nm but the action increased sharply at 300 nm and remained constant at 143
Sidney Lerman and Raymond Borkman
Av. lens wt.
Whole lens
mg
(mg Of01
Av. lens wt.
Whole lens
mg
(mg O/O)
Cortex (mg "0)
Nucleus (mg O/O)
290 and 280 nm. This indicates that the material is generated photochemically with tryptophan as the prime UV light absorbing species and this conclusion is supported by the observation that the photochemical production of fluorogen is associated with a decrease in tryptophan fluorescence.
Free Radicals in the Lens and their Relationship to Fluorescence
EPR (electron paramagnetic resonance) spectra on whole rat and mouse lenses and on the nuclear core from human lenses demonstrate that a significant tryptophan free radical can be demonstrated in the normal rat and human lens after ten minutes irradiation with UV light, but a marked decrease in the signal is noted in those lenses which have been previously exposed to UV plus AT for at least 4-6 h. There is also a direct relationship between the signal in144
UV Radiation in Lens Aging
h
I
Fig. 3.
EPR spectra of normal human 8, 47, and 83 years, 3 mm central portion of lens nucleus: See Lerman & Borkman 1976, for details of preparation of lens material; spectroscopy conditions: 77'K, 75 millimwatts power, 9.2 GHz microwave frequency, 500 g sweep at 435 values for each lens derived from 295 fluorescence spectra on whole lens. (See Figs. 2 and 4).
2.5 min, 0.1 sec time constant. I
360
- and I - ratio 290
tensity of the UV induced tryptophan free radical and the age of the human lens. T h e highest signal intensity is seen in the young lens and the signal decreases significantly with age particularly after the fifth or sixth decade (Fig. 3). This correlates with the evidence previously cited that tryptophan depletion is associated with generation of fluorescence as aging proceeds (some of the fluorogens being generated as a U V induced photodegradation product of tryptophan). T h e specific site of the UV-induced action thus appears to be on one or more protein-bound tryptophan residues in which a photochemical degradation occurs. Weiter & Finch (1975) have demonstrated the tryptophan triplet state in U V exposed human lens material as well as the tryptophan free radical. This excited triplet state may be dissipated by a photochemical reaction which ultimately generates fluorogens by intersystem crossing to the ground singlet state as suggested by Kurzel et al. (1973a,b) or by other routes. The photochemical degradation is initiated by the photoionization of the excited tryptophan molecule and results in the cleavage of the Indole ring at C2-c3 position. Laser flash photolysis experiments on aqueous tryptophan have recently shown that U V degradation of aqueous tryptophan occurs via such a photoionization mechanism (Bryant et al. 1975). 145 Acta ophthal. 5(i, I
10
Sidney Lerman and Raymond Borkman
Conclusion The preceding discussion has demonstrated that fluorescence spectroscopy can be utilized to measure an aging parameter in the ocular lens and that one major fluorescent region appears to be derived from tryptophan on the basis of prolonged exposure of the lens to ultraviolet light above 295 nm. This fluorescence which has an activation peak of 360 nm and an emission peak at 420-440 nm is definitely age-related, as is the increase in the water insoluble proteins and the decline in SH and concomitant increase in the level of SS bonds particularly in the lens nucleus. It is postulated that ultraviolet radiation above 295 nm plays a significant role in the increasing yellow colour of the lens nucleus and in lenticular aging, particularly nuclear sclerosis and its extreme state characterized by the brunescent (brown) cataract. The yellow pigmentation is confined mainly to the lens nucleus since there is a n insufficient concentration of free radical scavenger in this region to abort the photochemical degradation reaction. It is interesting to note that D-penicillamine which is an excellent free radical scavenger is capable of preventing the induction of the fluorescent material in young rat or human lenses in vitro and mouse lenses both in vitro and in vivo (Lerman & Borkman 1976; Lerman et al. 1976). Thus the lens nucleus is particularly susceptible to UV radiation since it has a relative lack of the normal free radical scavenger present in the lens (glutathione) as compared with the lens cortex; this becomes particularly pronounced as the lens ages (Table I). The relative excess of glutathione in the lens cortex would therefore be sufficient to abort any photo-degradation of tryptophan that could theoretically occur in that portion of the lens. While the whole lens nucleus derived from the brown cataract may contain a relatively similar level of tryptophan as compared with the lens nucleus in the normal individual, there may be a decreased amount of tryptophan in the insoluble protein fraction of the lens nucleus, particularly in the urea insoluble portion in the brown nuclear cataract. Although this would represent only a few of the total tryptophan residues in the lens, they would be sufficient to account for the findings regarding tryptophan depletion related to tryptophan free radical generation and increasing fluorescence as measured by EPR and fluorescence spectroscopy. T he latter two methods are more sensitive compared with amino acid composition data and would pick up much smaller fluctuations in relative concentrations of specific amino acids. Thus only a relatively limited number of tryptophan residues are actually susceptible to ultraviolet induced photo-degradation because of their specific location and immediate environment so that following absorption of UV light, and excited tryptophan molecule can not dissipate its excitation by the various mechanisms available to it (Feitelson 146
UV Radiation in Lens Aging
1% HUMAN
..
O.bj 0.5 1
1'0
2'0
4b 50~6'0 7'0 8b
3'0
AGE
9b
YRS
Fig. 4 . 295 I - ratios representing fluorescence intensity of 2nd fluorescence region at 500435 520 nm (435 nm excitation) over TRP 332 nm fluorescence (295 nm excitation). 295 and 435 nm interference filters employed since cortical cataracts were also examined and 435
regular spectroscopy resulted in marked light scattering. I - values in normal lens 295 (X), in nuclear cataract (N), cortical cataract (C) and mixed cortical and nuclear cataract (M). Each point represents a single lens.
1971; Lerman & Borkman 1976). While the majority of the tryptophan residues in the lens proteins can absorb and dissipate the ultraviolet radiation without undergoing any significant alteration, an adequate amount of free radical scavengers are also available within their immediate environment so. that UV photo-degradation will be aborted in some of the tryptophan residues which do enter the excited state, particularly in the lens cortex. Recent experiments in our laboratory have also demonstrated the presence of a second fluorescent region (approximately 420-435 nm activation and 500520 nm emission) which becomes apparent in the lens after the first decade of life and increases with age but becomes particularly pronounced in the brown cataract as compared with the normal lens from a comparable age group and with the cortical opacities or cortical cataract in which the lens nucleus remains relatively unaffected (Fig. 4 and Lerman & Borkman 1976). It is still not clearly 147
Sidney Lerman and Raymond Borkman
defined whether this second fluorescence emission peak is characteristic of a separate species or if it derives from the previously discussed fluorescent material, possibly as a degradation product.
Acknowledgments This work was supported by NIH Grants EY-01575 and EY-01138. Th e assistance of the Atlanta Lions Eye Bank in obtaining human lenses is gratefully acknowledged. Th e authors also acknowledge Professor Allen K. Garrison’s advice in the EPR experiments and for the use of his spectrometer.
References Bhuyan D. K. & Bhuyan K. C. (1975) Regulation of hydrogen percxide leve!s in eye humors. IRCS Med. Sci. 3, 415. Bhuyan K. C., Bhuyan D. K. & Katzin H. M. (1973) Amizol induced cataract and inhibition of lens catalase in rabbit. Ophthalmic Res. 5 , 236-247. Bhuyan K. C., Bhuyan D. K. & Turtz A. (1974) Aminotriazole - effect on the ocular tissue in rabbit. IRCS Med. Sci. 2, 1994. Borkman R. F., Dalrymple A. & Lerman S. (1977) Ultraviolet action spectrum for production of a fluorogen in the ocular lens. Photocliem. Photobiol. 26, 129-132. Bryant F. D., Santus R. & Grossweiner L. I. (1975) Laser flash photolysis of aqueous tryptophan. J . Pliys. CIiem. 79,27 11-27 16. Feitelson J. (1971) T h e formation of hydrated electrons from the excited state of indole derivatives. Photochem. Photobiol. 13, 87-96. Fransois J,, Rabaey M. & Recoules N. (1961) A fluorescent substance of low molecular weight in the lens of primates. Arch. Ophthal. (Chicago) 65, 118-126. Kuck J. F. (1974) Effect of long wave ultraviolet light on the lens in vitro with 3-aminotriazole (abstract). A R V O Annual Meefing, p. 84. Kurzel R., Wolbarsht M. L. & Yamanashi B. S. (1973a) Spectral studies on normal and cataractous intact human lenses. Exp. Eye Res. 1 7 , 65-71. Kurzel R., Wolbarsht M. L., Yamanashi B. S., Station G. W. & Borkman R. F. (1973b) Tryptophan excited states and cataracts in the human lens. Nature (Lond.) 241, 132-133. Lerman S. (1972) Lens proteins and fluorescence. Israel /. Med. Sci. 8, 1583-1589. Lerman S. (1976) Lens fluorescence in aging and cataract formation. Docum. ophthal. 8, 241-260. Lerman S. & Borkman R. F. (1976) Spectroscopic evaluation and classification of the normal, aging and cataractous lens. Oplithalmic Res. 8, 335-353. Lerman S., Kuck J. F., Borkman R. & Saker E. (1976) Induction, acceleration and prevention (in vitro) of a n aging parameter in the ocular lens. Ophthalmic Res. 8, 213-226. Meybeck A. & Windle J. J. (1969) Electron paramagnetic resonance study of X-ray and gamma-ray irradiated peptides in the solid state. Radiat. Res. 40, 263-275.
148
UV Radiation in Lens Aging Regnauld J. (1858) Sur la fluorescence des milieux de I’oeil chez I’homme et quelques mammiferes. L’lnstitut 26, 410. Shields H. & Hamrich P. J., Jr. (1970) Relative stability of the characteristic sulfur and doublet resonances in x-irradiated native proteins as measured with ESR. Radial. Reg. 41, 259-267. Teale F. W . J. (1960) The ultraviolet fluorescence of proteins in neutral solution. Biochem. J. 76, 381-388. Weale R. R. (1973) T h e efrects of the aging lens on vision. In: Ziba Symposium X I X . The Human Lens in Relation to Cataract, pp. 7-19. Elsevier/Amsterdam. Weiter J. J. & Finch E. D. (1975) Paramagnetic species in cataractous human lens. Nature (Lond.) 254, 536-537. Wolbarsht M. L. (1976) The function of intraocular color filters. Fed. Proc. 55, 44-50. Zirlin A. & Karel M. J. (1969) Oxidation effects in a freeze dried gelatin-methyl linoleate system. J. Food Sci. 34, 160-164.
Author’s address: Sidney Lerman, M. D., Laboratory for Ophthalmic Research, Emory University, Atlanta, Georgia 30322, USA.
149
