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Tj494-09.tex

Screening for Ocular Phototoxicity
Joan E. Roberts
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Screening for Ocular Phototoxicity
Joan E. Roberts
Department of Natural Sciences, Fordham University, New York, New York, USA radiation. This light serves to direct vision and circadian rhythm Normally light transmission through the eye is benign and serves
(Roberts 2000a) and, therefore, under normal circumstances to direct vision and circadian rhythm. However, with very intense
must be benign. However, chronic exposure to intense light light exposure, or with ambient light exposure to the aged eye
and/or age-related changes can lead to light-induced damage and/or young or adult eye in the presence of light-activated (photo-
to the eye. Roberts 2001a; Zigman 1993; Dillon 1991; Andley sensitizing) drugs or dietary supplements, cosmetics, or diagnostic
dyes, light can be hazardous, leading to blinding disorders. Light
damage to the human eye is avoided because the eye is protected by
There are many drugs, dietary supplements, and diagnostic a very efficient antioxidant system and the chromophores present
dyes, both topical and systemic, that absorb in the ultraviolet absorb light and dissipate its energy. After middle age, there is a de-
(UV) or visible range and can be excited by wavelengths of crease in the production of antioxidants and antioxidant enzymes
light transmitted to the lens and retina. This absorption can lead and an accumulation of endogenous chromophores that are photo-
toxic. The extent to which a particular exogenous photosensitizing
to dramatically enhanced ocular damage through the phototoxic substance is capable of producing phototoxic side effects in the eye
side effects of those dyes and drugs (Fraunfelder 1982; Roberts depends on several parameters, including (1) the chemical struc-
1996). The extent to which a particular photosensitizing drug ture; (2) the absorption spectra of the drug; (3) binding of the drug
will affect the human lens or retina in vivo depends upon (1) its to ocular tissue (lens proteins, melanin, DNA); and (4) the abil-
residence time in the lens or retina, which is determined by ity to cross blood-ocular barriers (amphiphilic or lipophilic). For
instance, compounds that have either a tricyclic, heterocyclic, or
its structure; (2) the photoefficiency of the particular sensitizer, porphyrin ring structure and are incorporated into ocular tissues
due to its ability to absorb the appropriate wavelengths of light are potentially phototoxic agents in the eye. The extent to which
that have been transmitted to the lenticular or retinal substrates; these substances might damage the eye (photoefficiency) can be
(3) the mechanism by which it causes that damage, including predicted using in vitro and photophysical techniques. With sim-
possible binding of the dye to ocular constituents. Binding can ple, inexpensive testing, compounds can be screened for their po-
tential ocular phototoxicity at the developmental stage. It may be
alter the photochemical mechanism and also increase the re- that a portion of the molecule can be modified to reduce phototoxi-
tention time of the sensitizer in the eye); and (4) the presence city while leaving the primary drug effect intact. Preclinical safety
of endogenous quenchers or free-radical scavengers that could testing may prevent ocular side effects that can range from mild,
stop or retard these photochemical reactions. All of these pa- reversible blurred vision to permanent blindness.
rameters play a role in the photosensitized oxidation of humanocular tissue and its importance as an underlying mechanism in cataractogenesis and retinal damage.
Toxicology, Photosensitization, Phototoxic Drugs Presented here are several screens that can be used to de- termine the potential of drugs to cause light damage to the The eye is the organ that is most vulnerable, after the skin, eye. These include a simple screen that takes into account the to light damage because it is constantly subjected to ambient optical properties of the eye and the structure and absorptionspectra of the various drugs that can be used to eliminatevarious drugs as potential photo-oxidants in the eye. In addi- Received 20 March 2002; accepted 19 June 2002.
tion, a second, more detailed screen is presented that can be This article is based on a presentation at the symposium entitled "Ocular Phototoxicity," held during the 22nd Annual Conference of the used to determine (a) the quantitative potential for a drug to American College of Toxicology, November 4–8, 2001, in Washington, cause photo-oxidative damage in the eye, (b) the mechanism by which it occurs, and (c) the in vivo verification of phototoxicity.
The author wishes to thank the Hugoton Foundation for its finan- cial support and Dr. Ann Motten, NIEHS, North Carolina, for help inpreparing this manuscript.
Structure of the Eye
Address correspondence to Joan E. Roberts, PhD, Professor of Chemistry, Fordham University, 113 West 60th Street, New York, NY The human eye is composed of several layers (Figure 1).
10003, USA. E-mail: [email protected] The outermost layer contains the sclera, whose function is to International Journal of Toxicology, 21:1–10, 2002Copyright c
2002 American College of Toxicology 1091-5818/02 $12.00 + .00DOI: The structure of the human eye.
protect the eyeball, and the cornea, which focuses incoming diation from the sun can contain varying amounts of UV-C (100 light onto the lens. Beneath this layer is the choroid contain- to 280 nm), UV-B (280 to 320 nm), UV-A (320 to 400 nm), and ing the iris, which is known as the uvea. This region contains visible light (400 to 760 nm). Most UV-C and some UV-B are fil- melanocytes containing the pigment melanin, whose function tered by the ozone layer. UV light contains shorter wavelengths is to prevent light scattering. The opening in the iris, the pupil, of light than visible; the shorter the wavelength, the greater the expands and contracts to control the amount of incoming light.
energy and the greater the potential for biological damage.
The iris and the lens are bathed in the aqueous humor, a fluid The primate/human eye has unique filtering characteristics that maintains intraocular pressure; this fluid also contains var- (Bachem 1956). The human cornea cuts off all light below ious antioxidants and supports transport to the lens. The lens is 295 nm, so that all UV-C and some UV-B are filtered from positioned behind the iris; its function is to focus light onto the reaching the lens. The adult human lens absorbs the remaining UV-B and all UV-A (295 to 400 nm). Therefore, only visible Behind the lens is the vitreous humor, a fluid that supports light reaches the adult human retina. However, the young hu- the lens and retina and that also contains antioxidants. The retina man lens transmits a small window of UV-B light (320 nm) is composed of the photoreceptor cells (rods and cones) that re- to the retina and the elderly lens filters out much of the short ceive light and the neural portion (ganglion, amacrine, horizon- blue visible light (Barker, Brainard, and Dayhaw-Barker 1991).
tal and bipolar cells) that transduces light signals through the Aphakia (removal of the lens) and certain forms of blindness retina to the optic nerve. Behind the photoreceptor cells are the may also change the wavelength characteristics of light reach- retinal pigment epithelial cells, Bruch's membrane, and the pos- ing the retina.
terior choroid. The photoreceptor cells are avascular and theirnutrient support (ions, fluid, and metabolites) is provided by theretinal pigment epithelial cells. There is transport to the retinal Direct Light Damage in the Eye
pigment epithelial cells across the Bruch's membrane by the Short UV light exposure to the cornea leads to an inflamma- tion reaction (Offord et al. 1999; Pitts, Cullen, and Parr 1976).
This is very painful and similar to a sunburn and is known as keratitis. However, these corneal wounds usually heal and do Light Absorption by the Human Eye
not cause permanent damage. On the other hand, UV damage For light to damage the eye, it must be absorbed. Each wave- to the lens and visible light damage to the retina are painless, length of light will affect different areas of the eye. Ambient ra- accumulative, and permanent. It can lead to the formation of OCULAR PHOTOTOXICITY cataracts (clouding of the lens) (Dillon 1991; Balasubramanian Drug-Induced Ocular Phototoxicity
2000), which can only be corrected by surgery, and to macular Most of the damage to the eye caused by direct irradiation and retinal degeneration (Ham et al. 1982), leading to permanent from the sun or artificial sources is from UV radiation. However, blindness for which there is no treatment.
in the presence of a light-activated (photosensitized) diagnostic A number of factors suppress or enhance light damage to the dye or drug, patients are in danger of enhanced ocular injury eye, including oxygen, antioxidants, repair mechanisms, and from both UV and visible light. The extent to which a particular photosensitizer will affect the human cornea, lens, and/or retinain vivo depends upon the following factors: (1) Its residence Oxygen Tension in the Eye time in the eye, which is determined by its structure and binding The cornea is highly oxygenated. The retina is supplied by of the dye to ocular constituents. Binding can not only alter the the blood so it has varying but high oxygen content in different photochemical mechanism but also can increase the retention portions of the retinal tissues. The aqueous humor and the lens time of the sensitizer in the eye. (2) The photoefficiency of the have low oxygen content but it is sufficient for photo-oxidation particular sensitizer (i.e., its ability to absorb the appropriate to occur. (McLaren et al. 1998; Roberts et al. 1992a; Kwan, wavelengths of light that have been transmitted to the lenticular Niinikoske, and Hunt 1971). The higher the oxygen content of or retinal substrates). These transmission characteristics change the tissue, the easier it is for light damage to occur.
with age. (3) The oxygen content of the ocular tissue. (4) Thepresence of endogenous quenchers or free-radical scavengers Defense Systems and enzyme detoxification systems that could stop or retard these Because the eye is constantly subjected to ambient radia- tion, each portion of the eye contains very efficient defensesystems. There are antioxidant enzymes (superoxide dismu-tase [SOD] and catalase) and antioxidants (e.g., vitamins E and PREDICTING OCULAR PHOTOTOXICITY
C, lutein, zeaxanthin, lycopene, glutathione, and melanin) that Based on the theoretical considerations stated above, it is serve to protect against oxidative and photo-induced damage relatively easy to predict that a drug cannot cause ocular dam- (Handelman and Dratz 1986; Giblin 2000; Seth and Kharb 1999; age through a photo-induced event. The short screen given in Edge et al. 1998; Khachik, Bernstein, and Garland 1997). Un- Table 1 will dramatically reduce the number of potential sub- fortunately, most of these antioxidants and protective enzymes stances needed to be considered for ocular phototoxicity.
decrease beginning at 40 years of age (Sarna 1992; Khachik,Bernstein, and Garland 1997; Samiec et al. 1998; Sethna,Holleschau, and Rathbun 1983). With the protective systems Short Screen for Potential Ocular Phototoxicity
diminish with age, there is loss of protection against all light- Measure the Absorbance Spectrum induced damage to the eye and the induction of age related In order for a chemical compound (diagnostic dye, drug, en- blinding disorders.
dogenous sensitizer) to induce a phototoxic response in anybiological tissue, it must first absorb light. This absorption is limited by the filtering characteristics of the biological tissues Even if the eye is damaged, the damage does not have to involved. A comparison of the transmission characteristics of be permanent. The cornea and retina have very efficient repair the eye with the absorbance spectrum of the drug may be used systems. However, damage to the lens is cumulative and not as a quick screen for phototoxicity. To have the potential to dam- repairable (Andley 1987).
age the aqueous or the lens, a drug needs a UV spectrum that consists of absorption wavelengths longer than 295 nm (Bachem 1956). This includes drugs such as chlorpromazine (maximum: Ocular damage from light can occur through either an inflam- 310 nm), tetracycline (maximum: 365 nm), and the porphyrins matory response or a photo-oxidation reaction. In an inflamma- (maxima: 392 nm, 500–650 nm) (Roberts 1984).
tory response, an initial insult to the tissue provokes a cascade of events that eventually results in wider damage to the tissue (Busch et al. 1999; Wang et al. 1999). In photo-oxidation reac- Short screen for potential phototoxicity in the lens/retina tions, a sensitizing compound in the eye absorbs light, is excitedto a singlet, then a triplet, state, and from the triplet produces 1. Chemical structure free radicals and reactive oxygen species that in turn damage the a. Heterocyclic, tricyclic, porphyrin ocular tissues (Straight and Spikes 1985).
The photo-oxidation reaction is outlined below: 2. Absorbance spectra a. Longer than 295 nm (lens); 400 nm (retina) Drug + light → singlet → triplet → free radicals/ b. Binds to DNA, lens protein, melanin 3. Skin phototoxicity reactive oxygen → ocular damage In the older human, drugs/dietary supplements, dyes (i.e., hy- Measure Binding of the Drug to Ocular Tissues pericin), and diagnostic dyes (photodynamic therapy porphyrins) Binding of a drug to ocular tissues (DNA—cornea, lens; that absorb in the 400 to 600 nm region could produce phototoxic proteins—lens; melanin—retina) (Roberts and Dillon 1987; damage to both the lens (Schey et al. 2000) and the retina. In very Dayhaw-Baker and Barker 1986; Steiner and Buhring 1990; young humans all drugs/dyes absorbing at wavelengths longer Sarna 1992) would increase its retention time in the eye. Fur- than 295 nm are potential photosensitizers in both the retina as thermore, binding of a photosensitizing substance to macro- well as the lens. Drugs that do not absorb in these regions cannot molecules increases the lifetime of its triplet state. It is the triplet cause photodamage.
state of the dye or drug that leads to further oxidative and free-radical reactions. Therefore, drugs/dyes that bind to ocular tis-sues are very likely to induce phototoxic damage (Roberts et al.
Examine the Chemical Structure 1991a) in that tissue. Binding is determined by measuring the Most drugs must have a tricyclic, heterocyclic, or porphyrin absorption spectrum of the drug in the presence and absence of ring to fulfill the energy requirements to produce a stable, long- lens proteins, DNA, and/or melanin (Roberts and Dillon 1987; lived (triplet) reactive molecule that will go on to produce free Roberts, Atherton, and Dillon 1990; Steiner and Buhring 1990).
radicals and reactive oxygen species. The addition of a halide A red shift in the absorbance spectrum of the drug in the pres- group can enhance the amount of triplet produced (Turro 1978).
ence of any of these biomolecules is an indication of binding of As seen in Figure 2, fluorescein and rose bengal have similar the sensitizer to the biomolecule.
structures except for the attached iodine groups. Fluorescein is highly fluorescent (singlet state) but is not very efficient at reach- Note Reports of Skin Phototoxicity ing the triplet state (poor quantum yield, 0.03, for the triplet).
Finally, any reports of skin phototoxicity for a particular drug The singlet state of rose bengal would easily go through inter- should provide a clear warning of potential ocular phototox- system crossing to the triplet (good quantum yield for the triplet icity. Skin phototoxicity is more readily apparent than ocular (0.60). By simple inspection of the structures of these two di- phototoxicity, although it is induced by compounds with similar agnostic dyes, we would conclude that rose bengal has a much chemical features (Oppenlander 1988).
greater potential to produce phototoxic damage to the eye thanfluorescein.
Detailed Screen for Predicting Ocular Phototoxicity
The short screen can determine a drug's potential for pho- Test the Solubility Properties totoxicity. Once it has been determined that a drug/dye is a Dyes or drugs to be examined should be tested for their par- possible photosensitizing agent, the more detailed screen given titioning in protic and aprotic solvents. Their hydrophobicity below can determine the potential site of damage (in situ fluo- will indicate potential for crossing blood-ocular barriers. More rescence techniques), predict the type and efficiency of damage hydrophilic substances are less likely to cross blood-ocular bar- (in vitro assays), and determine the mechanism of damage (pho- riers. Compounds that are amphiphilic or lipophilic cross all tophysical studies of the short lived excited state intermediates).
blood-retinal and/or blood-lenticular barriers. The probable site Once the excited state intermediates produced by a particular of damage may also be determined by the hydrophobicity (mem- photosensitizing agent have been determined, the introduction branes) or hydrophilicity (cytosol) of the photosensitizing dye of specific quenching agents may stop the unwanted reactions.
(Roberts et al. 1991a).
Determine the Potential Sites for Ocular Damage The site of damage is determined by the penetration of the drug and the transmission of the appropriate wavelengths oflight to that site. At these sites there are numerous substrates forphototoxic damage in the eye.
Corneal epithelial and endothelial cells may be easily damaged leading to keratitis (Pitts, Cullen, and Parr 1976; Hull, Csukas, and Green 1983). However, these cells have a veryefficient repair mechanism and the damage is rarely permanent.
Uveal cells are highly melanized and are ordinarily protected against light induced damage. However, melanogen-esis may be modified with phototoxic reactions leading to a greater risk from UV radiation (Hu et al. 2000).
Epithelial cells of the lens have direct contact with The structures of fluorescein and rose bengal. They have the aqueous humor. Their function is to control transport to heterocyclic ring systems.
the lens. They are most vulnerable to phototoxic damage.
OCULAR PHOTOTOXICITY Damage to these cells would readily compromise the viability of the lens (Roberts et al. 1994). The lens fiber membrane can be Detailed screen for ocular technique photochemically damaged through damage to the lipids and/orthe main intrinsic membrane protein (Roberts, Roy, and Dillon 1. In vitro studies 1985). This will result in a change in the refractive index, causing DNA, RNA, protein an opacification. Phototoxic reactions can lead to a modification Antioxidant enzymes of certain amino acids (histadine, tryptophan, cysteine) (Roberts Endothelial, epithelial, 1984; McDermott et al. 1991) and/or a covalent attachment of photoreceptor cell damage sensitizer to cytosol lens proteins. In either case, this changes b. Gel electrophoresis the physical properties of the protein, leading to aggregation and Amino acid analysis finally opacification (cataractogenesis). The covalently bound c. Mass spectrometry chromophore may now act as an endogenous sensitizer, produc- ing prolonged sensitivity to light. Because there is little turnover of lens proteins, this damage is cumulative.
Phototoxic damage can occur in retinal pigment epithelial tissues, the choroid, and the rod outer segments, which Protein modification contain the photoreceptors. If the damage is not extensive, there f. Normalization for are repair mechanisms to allow for recovery of retinal tissues.
However, extensive phototoxic damage to the retina can lead to 2. Biophysical techniques permanent blindness (Dayhaw-Barker and Barker 1986; Ham et al. 1982).
BindingQuantum yields b. Laser flash photolysis Triplet detection Determine the Location/Uptake of the Dye/Drug The traditional method of determining up- take into ocular tissues is in vivo radiolabeling. This method is time consuming and expensive, although it is effective in de- termining which ocular tissues have accumulated the drug in d. Pulse radiolysis Radical and oxyradical An alternative method to de- Radical and oxyradical termine uptake of a drug into ocular tissues is ocular fluorome- try. After a dye or drug has absorbed light and is excited to thesinglet state, it can decay to the ground state and is accompanied by the emission of light. The is known as fluorescence. Because The in vitro assays used in determining the phototoxic effi- most photosensitizers are fluorescent, transmitted or reflective ciency include the following: fluorescence provides an accurate means of measuring uptake ofa sensitizer into ocular tissue that is simpler, less expensive, and 1. Cell Culture/Whole Tissues. The first reported assay for
less arduous than using radiolabeled materials. This technique phototoxicity in human ocular cells (Roberts l981) measured may also be used noninvasively, in vivo, for instance using a slit changes in macromolecular synthesis in the presence and lamp, to detect uptake of sensitizers into the human, or scanning absence of a light-activated drug. Other studies have assessed or reflective fluorometry to determine the presence of endoge- damage to corneal, lenticular, and retinal cells by measuring nous and exogenous fluorescent materials in the retina (Docchio pump function and enzyme activities both in vitro and in 1989; Docchio et al. 1991; Cubeddu et al. 1999; Sgarbossa et al.
situ (Andley et al. 1994; Roberts et al. l994; Organisiak and Winkler 1994; Rao and Zigler 1992; Dayhaw-Barker 1987).
2. Gel Electrophoresis, Amino Acid Analysis. Gel electro-
Determine the Phototoxic Efficiency phoresis has been used to monitor polymerization of ocular The targets of photo-oxidative reactions may be proteins, proteins (Kristensen et al. 1995; Roberts et al. 1992a; Roberts lipids, DNA, RNA, and/or cell membranes (Straight and Spikes l984; Zigler et al. 1982). Photopolymerization is one of the l985). In vitro tests can be designed to determine the specific most apparent changes in ocular protein induced by photo- site(s) of damage to the various ocular compartments (i.e., lens sensitizing dyes and drugs. Quantitative changes can be mea- and retinal epithelial cells and photoreceptor cells) and the prod- sured by scanning the gel and determining relative reaction ucts of those reactions.
rates. Specific amino acid modifications can be determined Table 2 presents a summary of additional biochemical and using amino acid analysis (Roberts 1984, 1996). Zhu and photophysical techniques that can be performed to more accu- Crouch (1992) have illustrated the wide variety of classi- rately predict the potential for and extent of in vivo phototoxicity.
cal protein analysis techniques (gel electrophoresis, amino acid analysis, sequencing, isoelectric point determination, where I = the intensity of the lamp at various wavelengths ad- Western blot, enzyme-linked immunosorbent assay [ELISA]) justed for the transmission characteristics of the cornea or lens, that can be used to investigate phototoxic damage induced by AB is the absorbance of the dye/drug, and λ is the number of dyes and drugs.
photons at those wavelengths. The rates of each photo-oxidative 3. Mass Spectrometry. Recent innovations in the field of
event are then adjusted accordingly for each sensitizer. This in mass spectrometry (liquid secondary ion mass spectrome- turn can be corrected for the actual transmission characteristics try [LSIMS] and electrospray ionization [ESI]) have allowed of the cornea and/or lens and the output of the sun to predict in for the identification of specific amino acid modifications vivo effects.
within large proteins through molecular weight mapping.
In summary, in vitro techniques determine the potential dam- These techniques have been applied to determine the specific age done to an ocular substrate, which gives information about sites of photooxidative damage in corneal and lenticular pro- the photoefficiency of a drug should it be taken up into the teins (Schey et al. 2000; Roberts et al. 2001). These studies various compartments of the eye. Additional information about can serve as a model for defining damage from any potential the site of potential damage can be predicted based on which phototoxic agent in the eye.
ocular substrate (DNA, RNA, protein, lipid) is affected.
4. Thin-Layer Chromatography. This technique is particu-
larly effective at separating triacyclycerol, free fatty acid, Determine the Excited State Intermediates That Cause and phospholipids from lens (Fleschner l995) and retinal the Phototoxic Damage (Organisiak et al. 1992) membranes. Thin-layer chromo- Some photochemical reactions in tissues are summarized tography/gas chromotography/mass spectrometry (TLC/GC/ MS) may be used to measure lenticular or retinal lipid mod- EXCITED STATES → INTERMEDIATES ifications (Handelman and Dratz l986). Specific lipids maybe modified in the presence of photosensitizing agents and separated on TLC plates. The plates can then be scanned for quantitative analysis of these specific changes.
OH·, ROO· 5. High-Performance Liquid Chromatography (HPLC).
→ TARGET and DAMAGE HPLC is particularly effective at separating and identify-ing lipid peroxides from the retina (Akasaka, Ohrui, and proteins = polymers Meguro 1993). It has also been used to identify adducts lipid = peroxides formed between DNA nucleotides and phototoxic agents DNA, RNA = cross-links (Oroskar et al. 1994). HPLC has also been used to assess The biophysical techniques used in determining the photo- the rates of photo-oxidation of lens proteins in the presence toxic efficiency include the following: of a sensitizer. Using this technique, it is possible to deter-mine the induced amino acid modification within the protein 1. Quantum Yields. In predicting the phototoxicity of a dye
and their location and to detect possible binding of sensitizing or drug, it is important to determine what proportion of pho- drugs to specific lens crystallins (McDermott et al. 1991).
tons lead to a benign (fluorescence, singlet state) event and 6. Normalization for Photons Absorbed. Whatever the target
what proportion lead to a potentially destructive event (phos- tissue or extent of damage, the toxic effects of these dyes phorescence, triplet state). The efficiency of a photoinduced and drugs are the result of photochemical reactions. As such, process may be expressed as its quantum yield (Q).
their rate of efficiency is dependant on the number of photonsabsorbed by the sensitizer in the biological tissue. Therefore,in order to get an accurate comparison of the photosensitizing Q = the number of photons used to produce an event the number of photons originally absorbed potency of various dyes and drugs with different structuresand absorptive characteristics, it is essential to normalize for It gives a measure of how likely a photochemical event the number of photons absorbed by each drug in a particular is to occur. The quantum yield is often expressed as a per- centage. For instance, the quantum yield for fluorescence of This can be done with a simple computer-generated math- fluorescein is 0.92 and that of rose bengal is 0.08 (Figure 2).
ematical formula (Roberts 1996; Kristensen et al. 1995), This means that most of the light absorbed by fluorescein which takes into account the absorption spectrum of the drug, is given off in the form of fluorescence energy, making it a the output of the lamp source used in the experiments, and relative safe diagnostic dye for the eye. On the other hand, the optical properties of the eye. The total relative number the triplet quantum yield for fluorescein is 0.03 and for rose of photons absorbed by a drug under particular experimental bengal it is 0.60. This indicates that very little of the singlet conditions is the area under the product curve: fluorescein energy is transformed into a triplet, whereas mostof the energy of rose bengal will be available for intersystem Photons absorbed = I × AB × λ crossing and will reach the triplet state from whence it can OCULAR PHOTOTOXICITY produce ocular damage. Therefore, flourescein is appropriate transfer (electron exchange) between the sensitizing drug in but rose bengal would be an inappropriate diagnostic dye for an excited state and a substrate from the ocular tissues. Al- though these radicals are very short lived, they can be ob- 2. Laser Flash Photolysis. This method uses a pulse of mono-
served with ESR in situ during their photogeneration. For chromatic light to promote a specific dye or drug to an excited instance, illumination of rose bengal (RB), an ophthalmic state (Rodgers 1985).
diagnostic dye, in the presence of an electron donor such as
NADH, affords a radical anion of the dye RB·− to be directly
i. Triplet Detection. Time resolved techniques (either ab-
measured using ESR (Sarna et al. 1991).
sorption spectroscopy or diffuse reflectance) allow for the Radicals that are too reactive to accumulate in detectable detection of the triplet state of the excited chromophore quantities can frequently be detected by ESR using spin even in intact tissues. This technique has been used to de- trapping techniques. In this approach, an agent called a spin termine the absence (Dillon and Atherton l990) of triplet trap reacts with a short-lived radical R· to give a spin adduct
formation by the endogenous 3-hydroxykynurenine, as R-T·, which has a much longer lifetime. The original rad-
well as the presence of triplets from sensitizing drugs in ical R· is identified by the characteristic ESR spectrum of
intact lenses (Roberts, Atherton, and Dillon l991b).
the R-T·. Carbon-, nitrogen-, and sulfur-centered radicals, as
ii. Lifetime/Binding. All ocular damage from photooxida-
well as all of the important oxygen-centered radicals, (hy- tion reactions occurs through the triplet state of the drug.
droxyl, superoxide, alkoxyl and peroxyl) can be identified The longer the lifetime, the greater the potential for dam- using ESR either directly or in combination with the spin age. The lifetimes of the triplets of sensitizing drugs were trapping technique.
found to be greater when bound to macromolecules or Using these techniques, the photosensitized generation in an intact organ than when free in solution (Roberts, of superoxide in protic (Reszka, Lown, and Chignell l992) Atherton, and Dillion 1991b). The presence of a triplet and aprotic (Reszka et al. 1993) media can be monitored.
and an increase in lifetime when bound to intact ocular These are model systems for the hydrophilic (aqueous) and tissue is predictive of a drug's causing photo-oxidative hydrophobic (rod outer segment membrane) portions of the damage to the eye in vivo (Roberts et al. 1991a).
eye. ESR has recently been used to define the photochemi-cal mechanisms involved in the light activation of endoge- 3. Luminescence—Singlet Oxygen. The presence and lifetime
nous pigments in the lens (Reszka et al. 1996) and the retina of singlet oxygen can be determined using time resolved in- (Reszka et al. 1995). With these systems defined, ESR can be frared luminescence measurements at 1270 nm (Rodgers and used to predict potential phototoxic events induced by exoge- Snowden l982). Using this technique, it can be determined nous photosensitizing dyes and drugs and natural pigments whether and how efficiently a dye or drug can produce singlet (Roberts et al. 2000; Motten et al. 1999).
oxygen (Roberts et al. 2000; Motten et al. 1999). Because sin-glet oxygen is the most powerful oxidant in a photooxidationreaction, a dye or drug that is an efficient producer of singlet In summary, the molecular mechanism involved in the pho- oxygen could be predicted to induce phototoxic damage if totoxic damage induced in the eye is photosensitized oxidation present in the eye.
reactions. This mechanism begins with the absorption of light by 4. Pulse Radiolysis—Hydroxyl, Peroxy Radicals and Super-
the sensitizing compound (endogenous pigment, dye, or drug), oxide. Pulse radiolysis consists of the delivery of a very short
which promotes the compound to an excited singlet state (short intense pulse of ionizing radiation to a sample, the resultant lived) and then, through intersystem crossing, goes to the triplet changes in light absorption of the sample being followed by a state (long lived). The excited triplet state of the drug/dye then very fast spectrophotometer (Land 1985). The technique may proceeds either via a type I (free radical) or type II (singlet be used to detect the formation of short-lived radical species oxygen) mechanism, causing the eventual biological damage of a dye or drug (Roberts, Hu, and Wishart 1998; Roberts (Straight and Spikes 1985). Therefore, information about the et al. 2000). In addition, the interaction of a dye or drug with efficiency and excited state intermediates for a phototoxic re- excited oxygen intermediates (hydroxyl radical, superoxide, action in the eye obtained by using photophysical techniques peroxy radicals) (Land et al. 1983), which are clearly gener- (fluorescence, flash photolysis, pulse radiolysis, esr) can be pre- ated in this system, allows for an understanding of a possible dictive of phototoxicity in vivo. We have confirmed that pho- mechanism of in vivo photo-oxidative ocular damage.
tophysical studies collate well with in vivo data (Roberts et al.
5. Electron
1991a). For instance, tetrasulphonatophenylporphyrin (TPPS), Carbon-Centered Radicals and Superoxide. Electron spin
which binds to lens proteins, shows a long-lived triplet in the resonance (ESR or EPR) spectroscopy detects and character- intact calf and human lens, and produces singlet oxygen effi- izes species containing an odd number of electrons, namely ciently, causes photo-oxidative damage in vivo in pigmented free radicals and paramagnetic metal ions. The photo- mouse eyes, whereas uroporphyrin (URO), which produces an oxidation reactions responsible for the phototoxic responses efficient triplet but does not bind to ocular tissues, does not cause in the eye involve free radicals that are formed via electron photooxidative damage in vivo.
In Vivo Testing
tion of exogenous photosensitizing dyes, drugs, and herbal sup- The use of either the short or more detailed (Tables 1 and plements in the eye, which may potentially harm the eye. Also, 2) screens for ocular phototoxicity will not totally eliminate the for those drugs that must be continued, in spite of their phototox- need for accurate in vivo experiments. The function of these stud- icity (i.e., antimalarial drugs; Motten et al. 1999), appropriate ies is to limit the need for in vivo testing for ocular phototoxicity protection against light (sunglasses) and specific supplementary of large numbers of drugs. Those drugs found in screening to be antioxidants may be prescribed to retard or eliminate the most highly likely to produce phototoxic side effects in the eye should severe blinding side effects (Roberts and Mathews-Roth 1993).
be tested further with animal studies to determine the exact site and extent of damage to be expected in humans (Roberts et al.
1991a).
Prolonged use of a phototoxic drug is most probably of greater Akasaka, K., H. Ohrui, and H. Meguro. 1993. Normal-phase high-performance long-term risk to the eye than short-term dosage because of cu- liquid chromatography with a fluorimetric postcolumn detection system forlipid hydroperoxides. J. Chromatog. 628:31–35.
mulative damage. Because there is an active repair system in the Andley, U. P. 1987. Photodamage to the eye. Photochem. Photobiol. 46:l057– cornea, there should be little or no long-term side effects of pho- totoxicity there. However, because there is no turnover in the lens Andley, U. P., J. S. Hebert, A. R. Marrison, J. R. Reddan, and A. P. Pentland.
constituents, any modification in that tissue will tend to stay and 1994. Modulation of lens epithelial cell proliferation by enhanced accumulate with age. Thus cataractogenesis may not develop prostaglandin synthesis after UVB exposure. Invest. Ophthalmol. Vis. Sci.
35:374–381.
until much later than the initial insult. In addition, phototoxic Bachem, A. 1956. Ophthalmic action spectra. Am. J. Ophthal. 41:969–975.
damage to the lens may not only cause direct damage to cell via- Balasubramanian, D. 2000. Ultraviolet radiation and cataract. J. Ocul. Pharma- bility but may undermine its defense system, so that gross mor- col. Ther. 16:285–297.
phological effects may appear much later than the original insult.
Barker, F. M., G. C. Brainard, and P. Dayhaw-Barker. 1991. Transmission of the The susceptibility of both the lens and retina to light-induced human lens as a function of age. Invest. Ophthalmol. Vis. Sci. 32S:1083.
Busch, E. M. ,T. G. Gorgels, J. E. Roberts, and D. van Norren. 1999. The effects damage increases with the age-related changes in chromophores of two stereoisomers of N-acetylcysteine on photochemical damage by UVA (Roberts et al. 2000, 2001, 2002), with concurrent decrease in and blue light in rat retina. Photochem. Photobiol. 70:353–358.
antioxidant status (Giblin 2000; Samiec et al. 1998; Sarna 1992).
Cubeddu, R., P. Taroni, D. N. Hu, N. Sakai, K. Nakanishi, and J. E. Roberts.
Therefore the elderly may be particularly sensitive to drugs and 1999. Photophysical studies of A2-E, putative precursor of lipofuscin, in other agents that induce phototoxic side effects. Sight may be re- human retinal pigment epithelial cells. Photochem. Photobiol. 70:172– gained after cataract surgery; however, damage to the retina that Dayhaw-Barker, P. 1987. Ocular photosensitization. Photochem. Photobiol. is not repaired leads to permanent blindness. The environmental lighting, particularly the constant presence of intense ambient Dayhaw-Barker, P., and F. M. Barker. l986. Photoeffects on the eye. In Photo- light, must always be taken into account when assessing the biology of the skin and the eye, ed. E. M. Jackson, 117–147. New York, NY: potential in vivo ocular toxicity of a drug.
Marcel Dekker.
Dillon, J. 1991. Photophysics and photobiology of the eye. J. Photochem. Photobiol. B Biol. 10:23–40.
Dillon, J., and S. J. Atherton. 1990. Time resolved spectroscopic studies on the Even if a drug has the potential to produce phototoxic side intact human lens. Photochem. Photobiol. 51:465–468.
effects in the eye, no damage will be done if the specific wave- Docchio, F. 1989. Ocular fluorometry: Principles, fluorophores, instrumentation and clinical applications. Lasers Surg. Med. 9:515–532.
lengths of optical radiation absorbed by the drug are blocked Docchio, F., M. Boulton, R. Cubeddu, R. Ramponi, and P. Dayhaw-Barker. 1991.
from transmittance to the eye. This can be easily done with Age-related changes in the flourescence of melanin and lipofuscin granules wrap-around eyeglasses (Gallas and Eisner 2001; Sliney 1999; of the retinal pigment epithelium: A time-resolved fluorescence spectroscopy Merriam 1996) that incorporate specific filters. Furthermore, study. Photochem. Photobiol. 54:247–253.
nontoxic quenchers and scavengers could be given in conjunc- Edge, R., E. J. Land, D. McGarvey, L. Mulroy, and T. G. Truscott. 1998. Rel- ative one-electron reduction potentials of carotenoid radical cations and the tion with the phototoxic drug to negate its ocular side effects interactions of carotenoids with the vitamin E radical cation. J. Am. Chem. while allowing for the primary effect of the drug (Roberts 1981; Roberts et al. 1991a; Roberts and Mathews-Roth 1993).
Fleschner, C. R. 1995. Fatty acid composition of triacylglycerols, free fatty acid and phospholipids from bovine lens membrane fractions. Invest. Ophthal. Vis.
Sci. 36:261–264.
Fraunfelder, F. T. 1982. Drug-induced ocular side effects and drug interactions, 2nd ed., Philadelphia: Lea & Febiger.
With simple, inexpensive in vitro testing, compounds can be Gallas, J., and M. Eisner. 2001. Eye protection from sunlight damage. In Sun- monitored at their developmental stage for potential ocular pho- light protection in man, ed. P. U Giacomoni, 437–455. Amsterdam, Holland: totoxicity. It may be that a portion of the molecule can be modi- fied to reduce phototoxicity while leaving the primary drug effect Giblin, F. J. 2000. Glutathione: A vital lens antioxidant. J. Ocul. Pharmacol. intact. This may reduce the necessity of later, more costly, drug Ham, W. T., H. A. Mueller, J. J. Ruffolo, D. Guerry, and R. K. Guerry. 1982.
recalls. In the future, the more effective use of ocular fluorometry Action spectrum for retinal injury from near ultraviolet radiation in the aphakic will allow for a more accurate assessment of the uptake and loca- monkey. Am. J. Ophthal. 93:299–305.
OCULAR PHOTOTOXICITY Handelman, G. J., and E. A. Dratz. l986. The role of antioxidants in the retina Roberts, J. E. 1981. The effects of photooxidation by proflavin in HeLa cells I.
and retinal pigment epithelium and the nature of prooxidant induced damage.
The molecular mechanisms. Photochem. Photobiol. 33:55–60.
Adv. Free Radic. Biol. Med. 2:1–89.
Roberts, J. E. 1984. The photodynamic effect of chlorpromazine, promazine Hu, D. N. 2000. Regulation of growth and melanogenesis of uveal melanocytes.
and hematoporphyrin on lens protein. Invest. Ophthalmol. Vis. Sci. 25:746– Pigment Cell Res. 13:81–86.
Hull, D. S., S. Csukas, and K. Green. l983. Trifluoperazine: Corneal endothelial Roberts, J. E. 1996. Ocular phototoxicity. In Dermatotoxicology, 5th ed., eds. F.
phototoxicity. Photochem. Photobiol. 38:425–428.
Marzulli and M. Maiback, 307–313. Washington, DC: Taylor and Francis.
Khachik, F., P. S. Bernstein, and D. L. Garland. 1997. Identification of lutein and Roberts, J. E. 2000. Light and immunomodulation. Ann. N.Y. Acad. Sci. 917:435– zeaxanthin oxidation products in human and monkey retinas. Invest. Ophthal. Vis. Sci. 38:1802–1811.
Roberts, J. E. 2001. Ocular phototoxicity. J. Photochem. Photobiol. B Biol. Kristensen, S., R. H. Wang, H. Tonnesen, J. Dillon, and J. E. Roberts. 1995. Pho- toreactivity of biologically active compounds. VII. Photosensitized polymer- Roberts, J. E., S. J. Atherton, and J. Dillon. 1990. Photophysical studies ization of lens proteins by antimalarial drugs in vitro. Photochem. Photobiol.
on the binding of tetrasulphonatophenylporphyrin (TPPS) to lens proteins.
Photochem. Photobiol. 52:845–848.
Kwan, M., J. Niinikoske, and T. K. Hunt. l971. Oxygen tension in the aqueous Roberts, J. E., S. J. Atherton, and J. Dillon. 1991b. Detection of porphyrin excited and the lens. Invest. Ophthal. 11:108–111.
states in the intact bovine lens. Photochem. Photobiol. 54:855–857.
Land, E. J. 1985. Pulse radiolysis. In Primary photoprocesses in biology and Roberts, J. E., and J. Dillon. 1987. In vitro studies on the photosensitized medicine, eds. R. V. Bensasson, G. Jori, E. J. Land, and T. J. Truscott, 35–44.
oxidation of lens proteins by porphyrins. Photochem. Photobiol. 46:683– Amsterdam, Holland: Elsevier.
Land, E. J., T. Mukherjee, A. J. Swallow, and J. M. Bruce. 1983. One-electron Roberts, J. E., E. L. Finley, S. A. Patat, and K. L. Schey. 2001. Photooxidation reduction of Adriamycin: Properties of the semiquinone. Arch. Biochem. of lens proteins with xanthurenic acid: A putative chromophore for catarac- togenesis. Photochem. Photobiol. 74:740–744.
McLaren, J. W., S. Dinslage, J. P. Dillon, J. E. Roberts, and R. F. Brubaker.
Roberts, J. E., A. Harriman, S. J. Atherton, and J. Dillon. 1992a. A noninvasive 1998. Measuring oxygen tension in the anterior chamber of rabbits. Invest. method to detect oxygen tensions and other environmental factors in the lens.
Ophthalmol. Vis. Sci. 39:1899–1909.
In Proceedings of the International Society of Ocular Phototoxicity, Abstract McDermott, M., R. Chiesa, J. E. Roberts, and J. Dillon. 1991. Photooxida- 64, Sedona, Arizona.
tion of specific residues in a-crystallin polypeptides. Biochemistry 30:8653– Roberts, J. E., D. N. Hu, and J. F. Wishart. 1998. Pulse radiolysis studies of melatonin and chloromelatonin. J. Photochem. Photobiol. B. Biol. 42:125– Merriam, J. C. 1996. The concentration of light in the human lens. Trans. Am. Ophthalmol. Soc. 94:803–918.
Roberts, J. E, J. Kinley, A. Young, G. Jenkins, S. J. Atherton, and J. Dillon. 1991a.
Motten, A.G., L. J. Martinez, N. Holt, R. H. Sik, K. Reszka, C. F. Chignell, In vivo and photophysical studies on photooxidative damage to lens proteins H. H. Tonnesen, and J. E. Roberts. 1999. Photophysical studies on antimalarial and their protection by radioprotectors. Photochem. Photobiol. 53:33–38.
drugs. Photochem. Photobiol. 69:282–287.
Roberts, J. E., B. M. Kukielczak, D.-N. Hu, D. S. Miller, P. Bilski, R. H. Sik, A. G.
Offord, W. A., N. A. Sharif, K. Mace, Y. Tromvoukis, E. A. Spillare, O. Avanti, Motten, and C. F. Chignell. 2002. The role of A2E in prevention or enhance- W. E. Howe, and A. M. A. Pfeifer. 1999. Immortalized human corneal ep- ment of light damage in human retinal pigment epithelial cells. Photochem. ithelial cells for ocular toxicity and inflammation studies. Invest. Ophthalmol. Vis. Sci. 40:1091–1101.
Roberts, J. E., and M. Mathews-Roth. 1993. Cysteine ameliorates photosensi- Oppenlander, T. 1988. A comprehensive photochemical and photophysical assay tivity in erythropoietic protoporphyria. Arch. Dermatol. 129:1350–1351.
exploring the photoreactivity of drugs. Chimia 42:331–342.
Roberts, J. E., C. Reme, M. Terman, and J. Dillon. 1992b. Exposure to bright Organisciak, D. T., M. A. Darrow, Y.-L. Jiang, G. E. Marak, and J. C. Blanks.
light and the concurrent use of photosensitizing drugs. New. Engl. J. Med. 1992. Protection by dimethylthiourea against retinal light damage in rats.
Invest. Ophthalmol. Vis. Sci. 33:1187–1192.
Roberts, J. E., D. Roy, and J. Dillon. 1985. The photosensitized oxidation of the Organisiak, D. T., and B. S. Winkler. 1994. Retinal light damage: Practical and calf lens main intrinsic protein (MP26) with hematoporphyrin. Curr. Eye Res. theoretical considerations. Prog. Retinal Res. 13:1–29.
Oroskar, A., G. Olack, M. J. Peak, and F. P. Gasparro. 1994. 4-Aminomethyl- Roberts, J. E., S. Schieb, W. H. Garner, and M. Lou. 1994. Development of a 4,5,8-trimethylpsoralen photochemistry: The effect of concentration and new photo-oxidative induced cataract model using TPPS in an intact lens.
UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA.
Invest. Ophthal. Vis. Sci. 35:2137.
Photochem. Photobiol. 60:567–573.
Roberts, J. E., J. F. Wishart, L. Martinez, and C. F. Chignell. 2000. Pho- Pitts, D. G., A. P. Cullen, and W. H. Parr. 1976. Ocular ultraviolet effects in the tochemical studies on xanthurenic acid. Photochem. Photobiol. 72:467– rabbit eye. DHEW (NIOSH) Publication 77:130–138.
Rao, D. M., and J. S. Zigler, Jr. 1992. Levels of reduced pyridine nucleotides Rodgers, M. A. J. 1985. Instrumentation for the generation and detection of and lens photodamage. Photochem. Photobiol. 56:523–528.
transient species. In Primary photoprocesses in biology and medicine, eds.
Reszka, K., P. Bilski, C. F. Chignell, and J. Dillon. 1996. Photosensitization by R. V. Bensasson, G. Jori, E. J. Land, and T. J. Truscott, 1–24. North Holland: the human lens component kynurenine: An EPR and spin trapping investiga- tion. Free Radic. Biol. Med. 20:23–34.
Rodgers, M. A. J., and P. T. Snowden. 1982. Lifetime of O2 (singlet delta) in Reszka, K., P. Bilski, R. H. Sik, and C. F. Chignell. 1993. Photosensitized gen- liquid water as determined by time resolved infra-red luminescence measure- eration of superoxide radical in aprotic solvents: An EPR and spin trapping ments. J. Am. Chem. Soc. 104:5541–5543.
study. Free Radic. Res. Commun. 19:33–44.
Samiec, P. S., C. Drews-Botsch, E. W. Flagge, J. C. Kurtz, P. Sternberg, R. L.
Reszka, K., G. Eldred, R. H. Wang, C. F. Chignell, and J. Dillon. 1995. Pho- Reed, and D. P. Jones. 1998. Glutathione in human plasma decline in associ- tochemistry of human lipofuscin as studied by EPR. Photochem. Photobiol. ation with aging, age-related macular degeneration and diabetes. Free Radic. Biol. Med. 24:699–704.
Reszka, K., J. W. Lown, and C. F. Chignell. 1992. Photosensitization by ani- Sarna, T. 1992. Ocular melanins. J. Photochem. Photobiol. 12:215–258.
cancer agents. 10. Ortho-semiquinone and superoxide radicals produced dur- Sarna, R., J. Zajac, M. K. Bowman, and T. G. Truscott. 1991. Photoinduced ing anthapyrazole-sensitized oxidation of catechols. Photochem. Photobiol. electron transfer reactions of rose bengal and selected electron donors.
J. Photochem. Photobiol. A Chem. 60:295–310.
Schey, K. L., S. Patat, C. F. Chignell, M. Datillo, R. H. Wang, and J. E. Roberts.
Straight, R., and J. D. Spikes. 1985. Photosensitized oxidation of biomolecules.
2000. Photooxidation of lens proteins by hypericin (active ingredient in In Singlet O, Vol IV. Polymers and Biopolymers, ed. A. Frimer, 91–143.
St. John's wort). Photochem. Photobiol. 72:200–207.
Boca Raton, FL: CRC Press.
Sethna, S. S., A. M. Holleschau, and W. B. Rathbun. 1983. Activity of glutathione Turro, N. 1978. Modern molecular photochemistry. Menlo Park, CA: synthesis enzymes in human lens related to age. Curr. Eye Res. 2:735–742.
Seth, R. K., and S. Kharb. 1999. Protective function of alpha-tocopherol against Wang, X. C., C. Jobin, J. B. Allen, W. Roberts, and G. J. Jaffe. 1999. Suppres- process of cataractogenesis in humans. Ann. Nutr. Metab. 43:286–289.
sion of NF-kappaB-dependent proinflammatory gene expression in human Sgarbossa, A., N. Angelini, D. Gioffre, T. Youssef, F. Lenci, and J. E. Roberts.
RPE cells by a proteasome inhibitor. Invest. Ophthalmol. Vis. Sci. 40:477– 2001. The uptake, location and fluorescence of hypericin in bovine intact lens.
Curr. Eye Res. 21:597–601.
Zhu, L., and R. K. Crouch. 1992. Albumin in the cornea is oxidized by hydrogen Sliney, D. H. 1999. Geometrical assessment of ocular exposure to environmen- peroxide. Cornea 11:567–572.
tal UV radiation—implications for ophthalmic epidemiology. J. Epidemiol. Zigler, J. S. Jr., H. M. Jernigan, Jr., N. S. Perlmutter, and J. H. Kinoshita. 1982.
Photodynamic cross-linking of polypeptides in intact rat lens. Exp. Eye Res. Steiner, K., and K. U. Buhring. l990. The melanin binding of bisoprolol and its toxicological relevance. Lens Eye Toxicol. Res. 7:319–333.
Zigman, S. l993. Ocular light damage. Photochem. Photobiol. 57:1060–1068.

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