Urocanic Acid Photochemistry and Photobiology - …homepage.univie.ac.at/mario.barbatti/papers/urocanic_acid/review... · Photochemistry and Photobiology, 1999, 69(2): 1 15-1 35 Invited

Photochemistry and Photobiology, 1999, 69(2): 1 15-1 35

Invited Review

Urocanic Acid Photochemistry and Photobiology

Taj Mohammad*l, Harry Morrison*l and Harm HogenEsch* Departments of 'Chemistry and "Veterinary Pathobiology, Purdue University, West Lafayette, IN, USA

Received 9 September 1998; accepted 19 November 1998

INTRODUCTION Urocanic acid (2-propenoic acid, 3-[ lH-imidazol-4(5)-yl], UA)? is one of the smallest molecules to have stimulated global interest among biologists, environmentalists, photo- chemists, photobiologists, medicinal chemists and immunol- ogists. Its history can be traced back more than a century to when it was first found in the urine of dogs (1). Interest in the molecule remained dormant until the middle of the century but rekindled in the late 1940s when it was detected in animal skin and sweat. This led to the proposal that UA acts as a natural sunscreen, possibly as a specific photoprotecting agent for DNA because of the overlap of the UA and DNA absorption spectra (2). Since 1983 there has been an explo- sive growth in UA research, primarily as a consequence of the proposal by De Fabo and Noonan (3) that the cis photoisomer (cUA) of trans-UA (tUA) could be responsible for the phenomenon of photoimmunosuppression. In a recent literature survey we found that about 25 research papers have appeared every year over the last decade involving UA. Several comprehensive reviews of the molecule's photobiology, photochemistry and photophysics have appeared (4-

*To whom correspondence should be addressed at: Department of Chemistry, Purdue University. West Lafayette, IN 47907. USA. Fax: 765-494-0239 or 765-494- 1736; e-mail: taj @cv3.chem.purdue.edu or [email protected]

tAhbra~iatioiis: BCC, basal cell carcinoma; BSA, bovine serum albumin; CHS. contact hypersensitivity; CMM. cutaneous malignant melanoma; dAdo. 2'-deoxyadenosine; DHUA, dihydrourocanic acid; DMT, 1.3-dimethylthymine; dsDNA. double-stranded DNA: DTH, delayed-type hypersensitivity; 2-FAA, 2-furanacrylic acid: GABA. y-aminobutyric acid; HSA, human serum albumin; IA. indoleacrylic acid: i.d., inner diameter; IFN-y, interferon-y; IL, interleukin; ImCHO, imidazolecarboxaldehyde; IMMA, imidazole-4(5)-methylidene malonic acid; ISRP. internal surface reversed-phase; k, . rate of DNA inactivation in the presence of sensitizer; k , rate of DNA inactivation in the absence of sensitizer; LC, Langerhans cells; %MOP, 8-methoxypsoralen; MP, melting point: MU. methyl urocanate; 2-MUA, 2-methylurocanic acid; NBT'+, nitroblue tetrazolium; NMMU, N-(methy1)methyl urocanate: 2-PAA. 2-pyrroleacrylic acid; PGE?, prostaglandin E?: PUVA, psoralen plus UVA: SPF. sun protection factor; ssDNA. single-stranded DNA; TAA. 2- and 3-thiopheneacrylic acid; TLC. thin-layer chromatography; TNF, tumor necrosis factor: UVA. ultraviolet A radiation (320-400 nm); UVB. ultraviolet B radiation (280-320 nm): 2-UA, [ring-2-'JC]urocanic acid; (Y-UA. [a- ''C]urocanic acid; cUA, cis-urocanic acid; tUA, trans-urocanic acid.

0 1999 American Society for Photobiology 0031-8655/99 $5.00+0.00

lo), the majority of which emphasize the putative role of UA in (photo)immunosuppression (4-8). Our purpose with this paper is to provide an overview of recent results in each of these areas. Brief summaries of some information previ- ously reviewed are included in order to make this presentation maximally useful.

BIOSYNTHESIS OF UA

Urocanic acid is synthesized as the trans (E) isomer through deamination of histidine by the enzyme histidine-ammonia lyase (histidase) (1 1). Studies involving deuterium-labeled isotopomers of histidine have revealed that enzymatic elimination of ammonia from histidine proceeds through a step- wise mechanism via the intermediacy of a carbanion (12). Trans-urocanic acid is a substrate for the enzyme, urocanase, an enzyme absent in the skin but present in the liver. Be- cause there is considerable histidase activity in skin, but no urocanase, tUA is a substantial constituent of the srratuni corneurn, being 0.7% of the dry weight of epidermis (13). Trans-urocanic acid is also detected to a lesser extent in the liver due to the presence of histidase activity in this tissue. In the liver, urocanase catabolizes tUA to imidazolonepro- pionic acid and subsequently to glutamic acid (c j Fig. 1). In the skin, sunlight isomerizes tUA to cUA. The latter is not recognized by urocanase, which has been shown to be light activated in Pseudomonas putida (14). In the absence of urocanase, UA is excreted in human sweat at a concentration of - 100 p,g/mL (15). An alternate metabolic pathway for histidine is the reaction of histidine decarboxylase that produces histamine (cj Fig. 1).

SOME CHARACTERISTICS OF UA

Urocanic acid is a crystalline white solid (melting point [mp], 223-225°C [decl) (1 1) that can be recrystallized from water. Aqueous solutions up to 20 mM can be prepared at ambient temperature with long stirring in phosphate buffer (pH 7) (16), and concentrations as high as 144 mM have been employed in studies involving the interaction of UA with DNA (17). Its solubility is highly dependent on temperature; saturated solutions of the crystalline dihydrate are approximately 7, 43 and 430 mM at 0, 37 and 100°C, respectively. The protonated and deprotonated forms are ex- tremely soluble in water. Interestingly, at its isoelectric

116 Taj Moharnrnad et a/.

Table 1. ed skin (18)

Ratio of t U N c U A at various sites of exposed and shield-

1 HIJ:C('tT2CH2CHWII> jCO~11

Ciluiamic .+<id

Figure 1. Metaholisni uf histidine leading to the formation of tUA and histamine. The tUX photoisomerizes to cUA in the slratiii?~ c'or- / l c t m i n Tunlight. Crocanase catabolizes tUA only at a remote site (in liver) and tht. product. imidamlone propionic acid, undergoes further decomposition to ii number of small niolecules (adapted from Norval e r ti/. (41 and Noonan and De Fabo ( 5 I ) .

point, pH 4.6. tUA is nearly insoluble, and this property has been exploited to purify i t through crystallization (1 1).

The cis (Z) isomer is an order of magnitude more soluble than tUA in organic and aqueous solvents. The two isomers are stable as solids for an indefinite period. and as aqueous solutions for up to 4 months when stored at 4°C in the dark. Samples taken from the skin on a cellophane adhesive tape are stable for up t o 2 weeks in the dark at ambient temperature ( 18). High-performance liquid chromatographic analysis of 2.2 mM solutions of each isomer in the aprotic solvent, dimethylsulfoxide, which had been maintained in the dark for 2 weeks at room temperature, indicated -8 and 4% degradation of cUA and tUA. respectively. Solutions stored at -20°C showed 15% less degradation. Degradation is accelerated in protic solvents and upon exposure to ambient light (T. Mohammad, unpublished results). For brief periods, UA is stable up to -75°C as is evidenced from the absence of a change in the absorption spectrum (19). However, at elevated temperature and longer reaction time, UA is unstable in organic solvents: for example. refluxing a 0.31 M solution of tUA in pyridine. under inert atmosphere, for 21 h resulted in -75% loss of UA ( 2 0 ) .

Upon exposure to light. tUA is converted in vitro and in \Ti\w to the cis isomer (mp 178-180°C) (21 ), an isomer that is not metabolized by urocanase. The extent of cUA formation is dependent upon absorbed UV doses and, as a result, varies in vi\v at different skin sites receiving different amount of natural sunlight. Table 1 shows that cUA is mainly found on exposed sites (22). but the ratios of tUA/cUA are higher than those reported in a more recent study by Shibata rt ul. (18). The discrepancy could be due to the racial difference between the Japanese ( 1 8) and British (22) volunteers who participated in the studies. Higher ambient sunshine hours in Britain relative to Japan can also account for the higher ratio of tUA/cUA in the Japanese study (18). The accumulation of tUA in the skin of Africans and Asians is higher than in the skin of European Caucasians (23).

Site\ Ratio

Back of hand Cheek Armpit Inner thigh Lower abdomen

0.83 1.14 8.28 7.88 5.86

PROTOTROPIC EQUILIBRIA OF UA Urocanic acid contains three protonation sites that have been assigned pKa of 3.5, 5.8 and 13, corresponding to the carboxylic acid and the tertiary and secondary nitrogens, respectively (1 1). Based on the pH dependence of the I5N NMR chemical shifts, Roberts et al. have assigned pKa values of 3.3/7.0 and 4.0/6.1 for cUA and tUA, respectively (24). The corresponding structures for tUA at selected pH ranges are shown in Fig. 2. These consist of neutral (A), zwitterion (B), carboxylate anion (C) and dianion (D) species at pH <3.5, 3.5-5.8, 5.8-13 and >13, respectively. One can expect similar structures for the cUA. Thus, UA pre- dominately exists as the zwitterion at the physiological pH of the stratum corneum (pH gradient = 4-6) and the viable epidermis pH of 7.4 (25). An extensive theoretical analysis of UA in the gas phase has appeared utilizing semiempirical AM1 and ab initio MP2/6-31G* calculations (26). The protonated forms of the UA isomers are predicted to be planar due to the delocalization of the n-electron system. The pre- ferred conformation is s-trans with respect to the bond between the acrylic acid side chain and the imidazole ring. The neutral UA isomers have approximately the same energy, whereas the charged cUA structures have lower molecular energies than the corresponding tUA isomers. The cUA conformers are stabilized by the formation of an internal hydrogen bond between the ring nitrogen and the carboxylic group (1 3 4 2 kJ/mol). The cationic forms of both isomers are the most stable tautomers that possess lower energy (-2.5 X 10' and 1 X 10l kJ/mol) than the corresponding anionic and neutral forms, respectively. In turn, the neutral forms of cUA and tUA are stabilized by -0.2 X 10' kJ/mol relative to the zwitterionic forms (26). The ions each have different ab-

Figure 2. Prototropic equilibria of tUA as a function of pH (adapted from Mehler and Tabor ( 1 I ) ) .

Photochemistry and Photobiology, 1999, 69(2) 1 17

sorption properties with A,,,,, shifting to the red as the pH increases (1 1,27). They also exhibit distinct photophysical characteristics (vide infru).

CHROMATOGRAPHIC ASSAY METHODS FOR UA

As discussed in a comprehensive review by Norval et ul. (4), before the advent of HPLC, UA was analyzed by mp, thin-layer chromatography (TLC), absorption and NMR spectroscopic techniques. Among the chromatographic methods, the most cost-effective is semiquantitative TLC. Whatman no. 1 paper or a TLC sheet, with an eluent consisting of 25% of 0.2 M ammonium hydroxide in 1-propanol, have been employed to separate the UA isomers, with cUA migrating more rapidly than tUA by 0.15 R, units. Quanti- tation was done by eluting the spots in 50 mM potassium phosphate (pH 7.5) and measuring the absorbance at 277 nm (28). Derivatized UA analogs have been analyzed by gas- liquid chromatography on a 3% SP 2100 on Supelcoport (29) and a Durabond DB-5 (0.1 pm film thickness fused-silica capillary column, 40 m X 0.32 mm inner diameter [i.d.]) (30).

A number of additional convenient, fast and reliable HPLC methods have recently become available for the analysis of UA isomers in vitro and in extracts from biological matrices. These include an Ultrasil-NH, column with isocratic elution using 20% of 10 mM potassium phosphate (pH 6.8) in acetonitrile for the analysis of UA isomers extracted from the skin of irradiated animals (16) and C8 (both analytical and semipreparative) as well as PRP-1 columns, employing both isocratic and gradient elution with 50-100 mM phosphate buffer (pH 7) (31-33). Other examples include (i) a Hypersil APS glass column (100 X 3 mm i.d., 5 pm particle size) that provides a detection limit for both isomers at a signal-to-noise ratio of 3 of a 3 nM solution for the 60 fmol injected sample (34); (ii) a 200 X 3 mm i.d. column packed with Chromsphere C18 (5 pm particle size) eluted with 5% acetonitrile in 20 mM potassium dihydrogen phosphate (pH 3.7) containing 1 g/L of sodium heptanesulfonate (35); (iii) a Novapak C18 column (150 X 3.9 mm i.d., 4 pm particle size) with 10 mM acetic acid (pH 5) (36); (iv) a Tosoh ODS 8OTS column (250 X 4.6 mm i.d., 7 pm particle size) with a mobile phase consisting of 7% acetonitrile in 20 mM potassium dihydrogenphosphate containing 1 g/L sodium heptanesulfonate, pH 3.7 (adjusted with 14.7 mM phosphoric acid) (18) (the response of the two isomers is linear under these HPLC conditions in the range of 1.5 nmol to 2 pmol (0.276 ng) per injection and the detection limit for each isomer was 2 pmol at a signal-to-noise ratio of 5:l); (v) an ODS Hypersil column (100 mm X 2 mm i.d., 3 pm particle size) with the mobile phase consisting of 10 mM ammonium phosphate (pH 5) and 2 mM tetrabutylammo- nium bromide (37,38); (vi) a LiChrosorb NH, Hibar 250-4 column (250 X 4 mm i.d., 5 pm particle size) with 50% acetonitrile in 50 n M potassium phosphate (pH 5) with a detection limit of 0.1 g/mL (27); (vii) a LiChrosorb RP 18 column (250 X 4 mm i.d., 5 pm particle size) with 2% acetonitrile in 100 mM sodium perchlorate (pH 3) with reported capacity factors of 3.25 and 2.60 for cUA and tUA, respectively, a resolution of 1.25 and a detection limit of 0.1

ng of both isomers (39); (viii) a C l 8 analytical column using a gradient of acetonitrile in 20 mM acetate buffer (pH 6.5) with reported detection limits for the UA isomers of 0.1 pmoVmL and a quantification limit for UA isomers extracted from the skin of 0.3 pg (40) and (ix) a 250 X 4 mm i.d. reversed-phase p-Bondapak column with 50 mM phosphate buffer (pH 3.4-3.6) containing 1-3 mM sodium octane sul- fonate and 2 4 % methanol (41). Note that correction factors are often needed for the quantitation of the two UA isomers because they have different absorbancies in the UV. For example, a correction factor of 1.1 1 is needed for cUA at 264 nm due to the higher absorptivity of the rruizs isomer (18). However, only a 2% correction is required at 254 nm (42).

It has always been a challenge to analyze small organic molecules in the presence of polymeric biomolecules with- out removing them from the matrix prior to injection onto the column. Internal surface reversed-phase (ISRP) supports combine the properties of size-exclusion and reversed-phase chromatography and eliminate the need for preclean-up and extraction procedures for direct injection of protein, serum and plasma samples containing drugs and their metabolites (43). This column has been applied to the analysis of UA/ DNA samples (44) and more recently irradiated mixtures of photovirucidal sensitizers in the presence of DNA (45).

Mass spectrometry has also been employed to detect small amounts of isotopically labeled UA with a sensitivity of 50 pghnjection with a signal-to-noise ratio of 5 (30). Moody- cliffe et ul. have used enzyme-linked immunosorbent assay in combination with an anti-cUA monoclonal antibody to detect the cis isomer in serum (46). Radioisotopomers of UA have been very helpful to follow the metabolism, biodistri- bution and irreversible binding to biological molecules at nano- and subnanomole levels (vide infru).

PHOTOREACTIVITY OF UA

Photodecomposition and photopolymerization

Ammonia, carbon dioxide, aspartic acid, fumaric acid, glutamic acid, maleic acid, glycine and urea have been identified as products from the extended UV irradiation of UA in aqueous solution (47). The UA photodecomposition is strongly pH dependent, alkaline media being twice as effective as neutral or acidic media. The presence of air in the reaction medium accelerates UA photodecomposition by a factor of two (48). Urocanic acid is reported to photopoly- merize, producing a polymer of MW >12000 (48). The rate of polymerization is 13-fold less in the absence of air. The irradiation of UA chased by incubation in the dark, in either argon or oxygen, did not affect polymerization. indicating that oxygen is catalytic only in the presence of light and that photoproducts do not catalyze polymerization in the dark. The inability of 254 nm light to release UA from the polymer rules out the presence of any cyclobutane dimeric products (48). The mechanism of the photopolymerization may be related to the propensity of UA to reduce oxygen via electron transfer to form the superoxide anion (49). Hydra- tion of UA radical cation accompanied by proton loss would give a UA radical that could initiate polymerization. In the absence of oxygen, hydrated electrons are produced through electron transfer from UA to solvent (vide infra).

118 Taj Moharnmad et a/.

2 3

Series B.RI = CHs Series b R j = CH2CH3

4 5

R 2 = CHzCH=CH2

Figure 3. Benzophenone-photosensitized ( A > 380 nm) dimerization of UA esters in solution (51).

Photodimerization

Two cyclobutane dimers of UA have been isolated from frozen media. Their formation is pH dependent with pH 5.9 favoring formation of a cis, trans, trans dimer and pH 8 leading to the formation of a trans, trans, trans dimer (50). Though the singlet or triplet origin of the photodimers is not yet known, the benzophenone-photosensitized (A > 280 nm) dimerization of UA esters has been observed (5 1). The yield of dimer 3 of tUA ester in acetonitrile solution is about twice that of the cUA ester dimer 2, and the methyl ester l a is dimerized twice as effectively as the corresponding ethyl ester l b (Fig. 3). Presumably both of these observations can be explained on the basis of steric effects. Interestingly, the higher homologous ester, ally1 urocanate 4 exclusively produces dimers of the trans isomer. Another noteworthy fea- ture of 4 is selective dimerization of the conjugated olefin in the presence of an allylic double bond (51).

Photoisomerization

The photoisomerization of UA is its most intensely studied photoreaction. The following sections deal first with the qualitative and quantitative aspects of the direct photolysis and then take up the sensitized isomerization (through energy transfer and radical additiodelimination mechanisms).

Direct photoisomerization

The cisltrans isomerization of UA upon exposure to light has been known for quite some time (21,52). At 313 nm, the isomerization is quite efficient in both directions, e.g. the quantum efficiencies are 0.52 and 0.47 for trans -+ cis and cis + trans, respectively, in water where UA exists as zwitterionic species (53). These values do not vary with UA concentration and are the same at pH 7.4 where UA is deprotonated to carboxylate anion. However, irradiation of UA as a dianion at pH 11 and at elevated temperature (-50°C) leads to a small diminution in the quantum efficiencies for isomerization. Fluorescent white light is quite effective in inducing UA isomerization (54) as are sunlight and incan- descent lamps (28), and neither quartz, Pyrex glass, soda lime glass, polycarbonate, polypropylene nor window glass prevents this chemistry. Amber glass prevents UA isomerization, as do commercial plastic diffusers used on fluorescent fixtures (28). A report on the sensitivity of patients with systemic lupus erythematosus to fluorescent light includes speculation that this effect might be a consequence of the

photoisomerization of tUA to cUA with the latter acting as an immunomodulator (55) (see also vide infra). Ultraviolet A (320-400 nm) lamps have been shown to cause isomerization of UA in BALB/c mouse skin (16). A recently available narrow-band lamp from Philips (TLOl) emits at 31 1- 313 nm and very efficiently isomerizes UA; the ratio of biological effectiveness of TLOl to the broad-band (270-350 nm) Philips TL12 lamp is reported to be 0.6 (56,57). The photostationary state favors the cis isomer (53,58). Interest- ingly, there is more than a three-fold greater amount of cUA in the sunburned stratum corneum than in normal skin (40). The dosage of UVA-I (340-400 nm) needed to reach this state is much higher than that for UVA-I1 (320-340 nm), presumably due to the diminished absorption of UA in the UVA-I region (16). As one would expect, tUA in cosmetics left in the dark for 1 month showed no evidence for the formation of cis isomer (39).

The photoisomerization of UA exhibits an unusual wavelength dependency, with isomerization being 10-fold more efficient at the red edge of the absorption band relative to higher frequency (e.g. 254 nm) irradiation (59). The absorption maxima for cUA and tUA are -270 nm in aqueous solutions so that the action spectrum for isomerization is not superimposable on the absorption spectrum. It is noteworthy that at 313 nm the sum of +,r~l,, ,+L,, and + ~ l , - - l , r ~ l , l , equals unity. This suggests that the primary photochemical event is cis/ trans isomerization, a conclusion supported from the small value of quantum efficiency for UA photodegradation (0.001). Both low and high energy triplet quenchers were unable to quench the photoisomerization, an indication that the isomerization involved a singlet or a very short-lived triplet excited state (53).

It was postulated early on that the mismatch between the absorption spectrum and the isomerization action spectrum could be due either to the presence of a mixture of ground- state rotamers having different absorption properties and isomerization efficiencies or to the existence of multiple electronic transitions within the absorption envelope (59). Analogies for the wavelength-dependent photochemistry do exist in the literature. The closest example is cinnamic acid that forms different products upon irradiation at varying wavelengths; the origin of this wavelength-dependent reactivity has been attributed to multiple electronic states (60). In other cases, for example, dihexatriene (61) and diphe- nylbutadiene (62) multiple ground-state rotamers contribute to different photochemistry at various wavelengths as a result of selective excitation of a particular rotamer at a specific wavelength. Some support for the rotamer hypothesis for UA was provided by Shukla and Misra (63). They have used semiempirical CNDO/S-CI, MNDO and AM 1 molecular orbital calculations to support the presence of different tautomers possessing different excited electronic and ground states close in energy. They observed that both the fluorescence and excitation maxima -365 and 280 nm, respectively, are pH independent, i.e. tautomers A, B and C ( c j Fig. 2) share the same maxima. This implies that the emitting species in all solutions is deprotonated UA (i.e. the UA anion). The excitation spectrum vanes with concentration and in a concentrated solution moves to the long wavelength (-300 nm). This trend is interpreted as being due to differences in the relative population of the ground-state conform-

Photochemistry and Photobiology, 1999, 69(2) 119

ers (63). In cUA, the formation of a bicyclic structure as a result of internal hydrogen bonding gives extra stabilization by 15.2 kJ/mol (64). This much energy difference between the rotamers, i.e. s-cis, s-trans and s-trans and s-cis should provide different absorption band shapes and this is not the case. Thus, the contribution of ground-state rotamers for the wavelength-dependent photochemical behavior of UA could be ruled out.

Recent studies using photoacoustic calorimetry and ultra- fast picosecond and femtosecond transient-absorption spectroscopy have unequivocally confirmed the presence of multiple electronic transitions within the broad tUA absorption band, i.e. excitation at 308 nm produces a lnT* excited state while 266 nm excitation populates a 'TT* state localized on the imidazole ring. The authors conclude that excitation of tUA in the 260 nm region produces a short-lived singlet state (7 < 7 ps) that undergoes rapid (1.4 X 10" s-I) intersystem crossing to a relatively long-lived excited triplet state (7 > 10 ns). The ultimate triplet state does not decay via isomerization so that the inefficient isomerization that is observed is attributed to the singlet state (19). The triplet-state energy for UA was determined to be 230 kJ/mol, a value that agrees well with a value (55 kcaymol) estimated from triplet sensitization experiments (53).

Excitation at the long wavelength generates an entirely different excited state. Thus, the intensity of the photoacoustic signal is independent of the argon and oxygen environment when UA is excited at 308 nm, whereas excitation at 266 nm leads to the release of 63% absorbed energy as heat under oxygen relative to about 50% in argon-saturated solution. A higher amplitude of photoacoustic signal in the presence of oxygen suggests quenching of the resultant excited state, the lifetime of which is estimated to be >1 ps. In contrast to the photoacoustic signal obtained from excitation at 266 nm, almost the entire absorbed energy is non- radiatively released upon excitation of UA at 308 nm (these results are observed at both pH 5.6 and 7.2). The conclusion is that excitation at 308 nm generates a singlet state (7 < 100 ps) that selectively undergoes isomerization to cUA with a limiting rate constant of 1.2 X 10'" s-' (65). Excitation at 308 nm gives emission that is pH dependent and is observed at 370 and 386 nm in pH 7.2 and 5.6 solution, respectively. The emission maximum at 386 nm varies with the excitation wavelength between 300 and 320 nm. The lifetime of the emitting singlet state is short and is estimated to be <40 ps at both pH as a consequence of rapid nonradiative decay (19).

These studies would appear to lay to rest the alternate view that the wavelength-dependent isomerization of tUA has its origin in the presence of different ground-state tautomers and rotamers (63). The low quantum yield of fluorescence and short singlet lifetime that have been observed also presumably account for a report that UA is nonemissive (27). These authors have used time-resolved laser flash photolysis using 308 nm excitation to study UA photoisomerization and to evaluate the quantum efficiencies for this process. Changes in the ground-state absorption spectra between 310 and 315 nm were used to monitor the reaction in buffered solutions of varying pH values (3-10). It is claimed that the initial rate constants for the reaction are in the range of 6-9 X lo6 s- ' and that these are followed by slower rate

constants (1.2 X lo5 s-I). Oxygen does not affect the transient absorption leading to isomerization, an indication that a singlet excited state is involved in the isomerization process (27). The quantum efficiencies show a dependence on the pH of the reaction medium; +,ru,ts+cII at 313 nm increases as the pH is raised from 3.0 to 7.4 and then follows a downward trend. This trend is less specific for I $ ~ , ~ ~ , ~ ~ ~ , , , . These quantum efficiency values were supported by flash photolysis data (27). Interestingly, the sum of the two-way isomerization exceeds unity at higher pH. A kinetic model for the two reactions has been proposed that predicts that the trans + cis and cis + trans reactions are derived from short- and long-lived components of the transient, respectively

In additional photophysical studies, it was observed that the absorption spectrum of tUA is independent of temperature ranging between ambient and 76.5"C but shifts to higher energy at pH 5.6 relative to that at pH 7.2 (19). The quantum yields for fluorescence are rather low and <lo-?) resulting from excitation at 266 nm (Ae,,, 354 nm) and above 300 nm (Aern 354 nm), respectively. Excitation at 310 nm provides an emission spectrum (Ae,, 370 nm), the maximum for which is red shifted relative to excitation at 266 nm, and this trend is pH independent. The emission maximum at 354 nm is insensitive to excitation wavelengths (260-285 nm). The molecular structure of UA is different at pH 5.6 and 7.2 but gives similar values for the quantum efficiencies for isomerization and similar emission and excitation spectra, suggesting that a common excited state is involved at both pH. Thus, at pH 5.6, UA undergoes rapid proton transfer (<200 fs) from the protonated tertiary imidazole nitrogen to the solvent to give the same excited state, which is directly created via 266 nm excitation of a pH 7.2 solution. These observations are in agreement that the excitation spectra are pH insensitive between 3.2 and 11, whereas the absorption spectra are highly dependent on a variation in solution pH (63).

(27).

PHOTOSENSITIZED ISOMERIZATION OF UA Triplet energy transfer

Momson et al. demonstrated that UA isomerization could be efficiently sensitized by triplet sensitizers varying in triplet energy (53). These included biacetyl (66), sodium naph- thalene-2,6-disulfonate (67), benzophenone and acetone (68), corresponding to triplet energies of 55, 60, 69 and 79 kcal/mol, respectively. Based on the observation that these sensitizers induce trans to cis isomerization, it was argued that the UA triplet lies in the vicinity of 55 kcal/mol (53). It is interesting that the UA triplet populated by direct photolysis at -260 nm (19) is less prone to isomerization (59). Both thymine and 1,3-dimethylthymine (DMT) photosensitize isomerization of tUA. The DMT-sensitized quantum efficiency for isomerization of tUA (0.6 mM) using 254 nm excitation is 0.0028 under inert conditions (69). The isomerization is not quenched by the triplet quencher, 2,4-hexadienol, thus excluding diffusional energy transfer from a DMT triplet state in the sensitization. Both resonance energy transfer and "chemical" sensitization have been considered to explain this chemistry. Arguments against the former include the poor overlap of the emission spectrum of DMT

120 Taj Mohammad et a/.

and UA and the short estimated (-1-20 ps) lifetime of the DMT singlet state (70-72). A preassociated ground-state complex of DMTAJA is therefore proposed to account for the sensitization process. This is supported by the observation of hyperchromicity in the absorption spectrum of a DMTAJA mixture at the red edge of the spectrum (69); ap- parently the complex is destabilized in organic media (73,74).

8-Methoxypsoralen (8-MOP), in combination with ultraviolet A light, is highly effective in the PUVA (psoralen plus UVA) therapy of skin diseases (75). It has been demonstrated that such PUVA treatment is associated with immunosuppression, and this phenomenon has been attributed to 8- MOP-photosensitized isomerization of tUA in the skin (37). More recent in virro studies support this proposal (76). The 8-MOP photosensitizes isomerization of tUA with the level of sensitization depending on the drug concentration and UV dose. The major chemical event for UA under the photosensitization conditions is isomerization. as the total UA remains constant during photolysis. Triplet cncrgy transfer is the likely mechanism for sensitization because the 8-MOP triplet energy of 59-61 kcal/mol (77) exceeds the UA triplet energy of -55 kcal/niol ( 5 3 ) . This conclusion is supported by the evidence that the sensitized isomerization is completely quenched by oxygen due to the efficient interception of the 8-MOP triplet by oxygen at a diffusion-controlled rate (kq - 1 - 4 X 10" M - ' s-I) (77.78). There is a contradictory report that 8-MOP-photoscnsitized isomerization of UA is not quenched by oxygen. presumably due to leakage of UVB radiation from the UVA light sourcc (37). Whether these in vitro studies of 8-MOP are responsible for the in vivo immunosuppression remains to be determined.

In addition to 8-MOP, other drugs that photosensitize tUA isomerization include ketoprofen and ciprofloxacin; the latter is a particularly strong sensitizer. On the other hand, amio- darone and doxycycline quench UA isomerization (76). It is suggested that the sensitizers possess triplet energies higher. and the quenchers triplet energies lower. than the triplet of UA (55 kcal/mol) (53). Some unidentified endogenous chromophores have also been proposed to sensitize tUA isonierization in the skin (36). However, a rcccnt rcport precludes UA isomerization by an endogenous photosensitizer. It is argued that weak but direct absorption of long wavelengths of UVA light by UA leads to its isomerization (16).

Photogenerated radical-catalyzed isomerization

Urocanic acid can be isomerized through sensitization by the electron affinic dye, nitroblue tetrazolium (NBT'-) (79). The proposed mechanism involves photooxidation of UA by the NBT? * excited state to form the UA radical cation (49). The chemistry exhibited by this species includes oxidation to imidazolecarboxaldehyde (ImCHO) (3 1 ). When sodium azide is present and oxygen is rigorously excluded from the reaction medium. NBT' preferentially oxidizes the azide anion and the resultant azidyl radical catalyzes a one-way isomerization of cUA to tUA (c$ Scheme 1) (79). The quantum yield for the sensitized isomerization with 1 mM cUA and 20 mM sodium azide using 355 nm excitation is -1 X 10 '. This one-way isomerization mediated by azidyl radical would appear to rule out the possibility for the involvement

(adapted from refs. 31.79)

N B T ~ + hv (NBT~+)'

(NBT2+)' + N 3 - - NBT+ + N 3 .

N3. + N 3 - - - Ng-.

CUA + N 3 . - ImiH-CHC02H I

1 N3 - tUA + N3. Argon 1

0 2

Y V

0 0

* ImCHO + [O=CHCO,H] ---- i

Im I C 0 2 H

UA-01

Scheme 1.

of radical processes to convert tUA to cUA in biological systems. Interestingly, a recent study demonstrates that Mi- crococcits Iittercs in the flora of skin converts cUA to tUA; however, the mechanism of this transformation is not known (80).

Photoisomerization of UA in vivo

Absorption of UV photons causes UA in skin to isomerize from the trans to the cis isomer. This isomerization occurs in a dose-dependent manner until a steady state (photostationary state) is reached. The tUA also isomerizes to cUA in human skin in vivn when irradiated with UVA lamps (emission maximum at 370 nm) and the level of isomerization increases with UV dose (81). The UA concentration of isomers in human skin varies with the seasonal variation of UV radiation reaching the earth. The maximum isomerization in the exposed body sites is observed in July/August though the total concentration of UA is lower in these months than the rest of the season (82). The topical application of sunscreens with a low sun protection factor (SPF) inhibits tUA isomerization in human skin, with cUA formation decreasing with increasing SPF. The skin type has minimum effect on the rate of isomerization (34). In agreement with the above report, several inorganic and organic sunscreens possessing varying degree of SPF (5-19) have also been shown to quench the UVB-induced isomerization of tUA from 50% to 9 and 13% in the in vivo and in vitro studies, respectively (83). While inhibition of UA isomerization increases with increasing SPF, there is not complete or total blockage of this isomerization. Gibbs and coworkers (84) have determined action spectra for the photoisomerization of UA in vitro and in vivo in hairless mouse skin to correlate them with the wavelength-dependent isomerization of UA (59). They found that both the in virro and in vivo action spectra displayed a maximum isomerization at -310 nm, extending into the UVA-I1 (320-340 nm) with decreasing efficiency. The action spectra are red-shifted relative to the absorption spectrum of tUA (84), as would be anticipated by the earlier report of wavelength-dependent photoisomerization (59). In less concentrated UA solution (15 FM vs 6


mM), the in vitro action spectrum for cUA production cen- tered at 280 nm and showed only -10 nm red shift relative to the absorption spectrum (36), an observation that agrees with a previous report using 145 pM tUA (85). For the in vivo data, the authors suggest that red shift is partially caused by the screening of shorter wavelength (<300 nm) light by stratum corneum proteins and the opaqueness at shorter wavelengths of UA solution at its natural concentration of 10-20 mM (36). The possibilities of sensitization by long- wavelength-absorbing endogenous chromophores and association with skin proteins are also considered to explain the in vivo red shift (36).

The photoisomerization of tUA to cUA has taken on particular significance because cUA became implicated as a potential causative agent for photoimmunosuppression (3). Ac- tion spectra are critical in establishing such a relationship and there are inconsistencies in the wavelength dependence of the two phenomena. The in virro photoisomerization action spectrum centers at 280 and near 310 nm (36,84,86), red shifted from the absorption maximum at 268 nm because of the higher quantum efficiency at longer wavelengths (see above). The 280 nm maximum lies close to the reported maximum at 270 nm for the immune suppression action spectrum based on the contact hypersensitivity (CHS) response (3). However, as noted above, the in vivo isomerization action spectrum is reported to have a maximum near 310 nm (84,86). The in vivo UA absorption spectrum likewise shows a red shift from 268 nm to 280 nm to a value near 310 nm (36). Factors such as the possible binding of UA to skin proteins, the photosensitized isomerization of UA by other epidermal chromophores andor the shielding of lower wavelength UV light by other skin chromophores or proteins have been proposed to explain the red-shifted in vivo photoisomerization action spectra (7,36). The effects of epidermal chrornophores, nevertheless, cannot explain the reported differences between the absorption and photoisomerization action spectra in vitro. However, the observed +,lu,I,--t( , , of 0.08 (59) at 254 nm is inefficient at this wavelength of high absorptivity of tUA and the immune suppression action spectrum maximum observed at 270 nm (33).

In a recent study, Hanson and Simon (87) have proposed a kinetic rate equation model to explain the observed inconsistencies between the in vivo versus in vitro photoisomerization action spectra (36,84,86). This model takes note of the fact that the photoisomerization action spectrum depends upon two factors: (i) initial concentration of tUA present at the outset of irradiation and (ii) irradiation dose to generate cUA. In a concentrated tUA solution (absorbance, A > l) , the extinction coefficient at the irradiation wavelength does not influence the photochemical rate constant so that the rate of isomerization and formation of cUA maximizes near 310 nm as a result of faster initial rate for cUA production. In a dilute tUA solution (absorbance, A < l), the population of molecules excited at a given wavelength is a function of the absorption cross section at that wavelength. The initial formation of cUA is faster at the absorption maximum (near 280 nm) that corresponds to the absorption maximum of tUA. Using the same kinetic model it is argued that at the photostationary state reached with the ratios of incident photon between 10: 1 and 100: 1, more cUA is produced in dilute

Table 2. mers to DNA in the dark*

Evidence for the lack of selective binding of a-UA iso-

Isomer Sample Inside bag? Outside bagt

cis Control 9168 921 1 UA 9108 9176

trrrris Control 8723 884 1 UA 8735 8909

*After 3 days of dialysis of calf thymus DNA (2.6 mM) and/or buffer alone inside the dialysis bag against I mM a-UA isomers in 50 mM phosphate buffer (pH 7) at 4°C in the dark. Samples were run in duplicates with standard deviation (<i3%).

?Radioactivity ( d p d 1 0 0 )LL) detected outside the dialysis hag and associated with DNA at equilibration (T. Mohammad. unpublished results).

solution near 310 nm relative to that at the absorption maximum at 280 nm (87).

INTERACTION OF UA WITH BIOMOLECULES Interaction of UA with DNA in the dark

It could be expected that UA might associate with DNA because of the planar heterocyclic structures of both isomers (64) and their ionizable amino and carboxylic acid functionalities. However, the absorption and emission spectroscopic techniques that are typically employed to probe for such an interaction are of limited use in this system because of the overlap of the UA and DNA absorption spectra and their low quantum yields of emission. Alternatively, equilibrium dialysis has been employed using radiolabeled UA with no indication of preassociation to native, heat-denatured or preirradiated DNA (88). An analogous experiment with pure cUA also gave no evidence for association with DNA, and, in fact, there is no evidence of selectivity by DNA for one of the two UA isomers (c$ Table 2, T. Mohammad, unpublished results).

Photochemical interaction of UA with nucleic acids

Among the molecules of life, i.e. carbohydrates, lipids, proteins and nucleic acids, perhaps the latter are the most irn- portant molecules. They serve as our biological library, en- coding all the cellular information. They store, transcribe and transmit a variety of biological information. The DNA is the focal point, of molecular biology and biotechnology because of the genetic code (the information transmission from DNA to RNA to proteins). Because UA and DNA co- exist in that part of the animal most exposed to sunlight, it is vital to understand the photochemical interaction of these two skin chromophores. This becomes even more important in view of the fact that environmental factors are causing depletion of ozone layer that allows penetration of more UVB radiation from the sun to the earth (89-91).

Photosensitized inactivation of viral DNA

The first evidence that UA creates damage in nucleic acids was provided by its ability to photoinactivate infectious phage G4 single-stranded (ss)DNA (5577 nucleotides); broadband light of A > 254 nm was used (92). It i s note-


worthy that treatment of viral ssDNA inactivated in the absence of UA with the photoreactivating enzyme photolyase and blacklight lamps efficiently (47%) repaired the DNA damage. an indication that the major lesions are cyclobutane pyrimidine dimers (because DNA photolyase is quite specific for the repair of c,is-syn cyclobutane pyrimidine dimers (93-95)). The DNA lesions created in the presence of UA at pH 6.8 were not repairable. thus confirming that these are not cyclobutane pyrimidine dimers (93). Interestingly. DNA inactivated in the presence of UA at pH 5 resulted in photoreactivation of -50C% of that observed in the absence of UA (92). It is argued that at the higher pH there is an elimination of cyclobutane pyrimidine dimer formation (perhaps through photosensitized cleavage of dimers when formed). There must also be an enhancement of UA/DNA damage that is not subject to photoreactivation. Preirradiation of UA for a short time. followed by mixing with DNA in the dark at ambient temperature. does not alter virus infectivity. suggesting that UA photoproduct (mainly cUA) are not responsible for DNA inactivation (92).

In follow-up studies ( 5 8 ) . a 308 nm laser was used. which. in the absence of UA. inactivated both G4 and S13 (5386 nucleotides) ssDNA in a dose-dependent manner with simple exponential kinetics. The measurcd rate constants for the DNA of the two viruses were comparable (7 X 10- Iq/photon), and it was shown, using photolyase and SOS transle- sion Weigle reactivation (96). that the primary lethal lesions are cyclobutane pyrimidine dimers. Irradiation of viral ssDNA in the presence of tUA showed accelerated DNA inactivation but with a nonexponential inactivation curve. The initial portion had a steep inactivation slope while the ultimate slope was more shallow. When pure cUA was used at the outset. a converse effect was observed, i.e. the initial portion of the hiphasic curve was concave downward and the ultimate slope was identical to the ultimate slope of the tUA curve. Preirradiation of the tUA produced a single exponential curve identical in slope to that obtained upon extended irradiation of the cis or tr-uns isomers. The ultimate inactivation slope ( k . , , / k ~ ,..\ = 0.10) was therefore attributed to a photostationary-state mixture of cUA and tUA. wherein tUA sensitizes the inacti\/ation of DNA (k-r ,4/k_, . . , = 1.6) while cUA protects the DNA from damage (kTrA/ k = 0.014). It is proposed that protection by cUA could be due either to cleavage of pyrimidine dimers or to the inhibition of their induction (see above). In both cases the photoreactivation of the DNA lesions virtually disappears (dropping from 0.54 to < 0.02), indicating that UA produces nondimer lesions (presumably nicks or UA-DNA photoadducts) (32.33,88). The two strains of viral DNA (G4 and 513 phage) behaved identically when irradiated in the absence and presence of UA.

The dramatic difference in the activity of the two UA isomers toward ssDNA is extraordinary and not yet well explained. The two isomers do not differ appreciably in their spectroscopic properties. and though one can spcculate on the possible role of UA photoionization in the observed reduction of thymidine photodirners (vide infru). in fact the two isomers exhibit similar photoionization efficiencies (8.35 and 8.30 CV for tUA and cUA. respectively; C. Lif- shitz. private communication). Likewise, the ionization potential for cinnamic acid isomers in the gas phase do not

differ significantly; 8.9 and 9.0 eV for cis- and truns-cinnamic acid. respectively (97). Equilibrium dialysis studies with calf thymus DNA in the dark, using the pure radiolabeled UA isomers, do not indicate a selective binding of either isomer to the biopolymers (cf. Table 2) .

In contrast with what was observed with ssDNA, quite different results were obtained when double-stranded (ds)DNA was irradiated with a 308 nm laser in the absence or presence of UA. In the absence of UA, dsDNA was 13 times less sensitive than ssDNA to UV radiation, in agreement with the 254 nm inactivation studies (98). Photoreac- tivation was 78% efficient. The greater degree of repair relative to ssDNA is due to a higher proportion of cyclobutane pyrimidine dimers in dsDNA and the formation of -14% syn-trans cyclobutane pyrimidine dimers in ssDNA that are not repaired by photolyase (93). When UA was present, there was marked photosensitization of dsDNA inactivation (k . , . , lk~, , ,A = 13) and, because the rate of inactivation is slow relative to UA isomerization, the plots observed with the cis and truns isomers were identical. Efficient photoreactivation (76%) was observed for the sensitized dsDNA in contrast with that seen with the sensitized ssDNA, where reactivation was minimal (58). Interestingly, it was shown that many cytosines were involved in the UA-sensitized formation of pyrimidine dimers. Cytosines present in dimers are prone to rapid deamination to uracil when heated at 37°C for -90 min (99.100). Such deamination, followed by photoreactivation with photolyase (101). provides mutant DNA (102). This mutagenic effect can be confirmed by the creation of abasic sites upon treatment with uracil-N-glycosylase. The dsDNA photoinactivated in the presence of UA gave evidence for a high frequency of mutations that were observed after deamination of cytosines and photoreactivation of the mutant DNA (58). The apparent ability of UA to sensitize formation of pyrimidine dimers in DNA may be related to the possible role of cUA in photoimmunosuppression. Re- cent expcriments have confirmed that such dimers play an important role in the photoimmunosuppression phenomenon (vide ir?fi-u).

The potential mechanisms by which UA sensitizes viral ssDNA and dsDNA inactivation, while protecting against pyrimidine dimerization in ssDNA, are quite interesting (58). In ssDNA, the predominant lesions appear to be nonrepair- able nicks that are probably initiated through electron transfer between DNA and a UA excited state. There is ample evidence that UA can be oxidized by either direct irradiation in the presence of oxygen (49) and/or by photosensitization by a combination of purines and a dye (31,49,79). Were a UA radical cation to be formed, it could initiate chain scis- sion either by oxidizing a DNA base or by hydrogen abstrac- tion from a deoxyribose ring. in either case ultimately to give strand breaks (103-106). Reactive oxygen species, e.g. hy- droxyl radical and singlet oxygen. are not likely to be responsible for strand breaks because the viral photoinactivation studies were carried out under an inert atmosphere. Likewise, it was argued that an electron transfer mechanism may be responsible for the lack of pyrimidine dimers observed in the sensitized ssDNA. as it is well recognized that both reductants and oxidants can cleave such dimers (107- 1 1 1 ). As for dsDNA. the UA-photosensitizcd formation of cyclobutane pyrimidine dimers could also be occurring via


an electron transfer mechanism because several papers have recently been issued that confirm that pyrimidine radical cations can react with the neutral base to form dimers (112- 114). In this regard one should note that the environment surrounding the radical ions plays an important role in the successful dimerization reaction. The dimerization of arylal- kene radical cations generated via photosensitization is fa- vored within constrained media due to stabilization from slow back electron transfer (1 15). Alternatively, a singlet energy transfer mode could explain the sensitization reaction. It is also known that triplet states do not produce cytosine-containing dimers (93). The low triplet energy for UA (55 kcal/mol) (53) relative to that of the DNA bases should preclude a triplet energy transfer mechanism, a more common mode of sensitized pyrimidine dimerization (58).

Yarosh et al. have used a plate incorporation procedure to assess UA-induced mutagenicity in five tester strains of Salmonella typhimurium and Escherichia coli W2 uvrA cul- tures in the absence and presence of unfiltered UVB (60% emission between 280 and 320 nm) (17). These genotoxicity assays can detect a chemical's ability to induce frameshift and base-pair substitution mutations. None of the six tester strains showed positive responses with UA, even at the highest dose of UA isomers (10 mg), both in the absence and presence of microsomal enzymes. It is noteworthy that the highest UA dose used in the mutagenicity test was 50 times higher than that which led to a UA-dependent increase in UV-induced skin tumors (1 16). The DNA damage induced by genotoxic chemicals can also be measured by the excision repair response using unscheduled DNA synthesis assay (1 17). At the UV doses used to cause photoisomerization of UA and with concentrations of the UA isomers ranging between 0.72 and 144 mM (10 times higher than that used in the mouse skin tumor studies (1 16)) there was no detectable DNA damage (17).

Photoinitiated covalent binding of UA to calf thymus DNA

Covalent binding of radiolabeled UA to calf thymus DNA has been observed upon irradiation of a mixture of the two compounds and isolation of the DNA (88). Several tests on DNA that had been isolated from such a mixture and purified by exhaustive dialysis and multiple precipitations confirmed that UA had become permanently affixed to the nucleic acid. For example, the radiolabel and the nucleic acid were found to coelute from a Sephadex (size-exclusion) column, and enzymatic degradation of the DNA gave an HPLC peak that matched that of an authentic UA/['H-methyllthymidine marker. The binding was found to be UV dose dependent and increased exponentially with irradiation time. As much as 80 nmol of radiolabeYmg DNA (1 UA or its equivalent for every 38 nucleotides or every 11 thymidines) was incor- porated. The binding was more efficient (>30 times) using shorter wavelengths of light (i.e. 22.3 nmol UNmg DNA at A > 270 nm vs 0.67 nmol UA/mg DNA at A > 289 nm). The data at the longer wavelength are in agreement with a recent study employing a UVB light source (0.54 nmol UA/ mg DNA) (1 18). The dependence of binding on irradiation wavelength could be due to a difference in reactivity of the two distinct excited states that are populated upon excitation

into the longer versus shorter wavelength regions of the UA absorption band (see above). Alternatively, it has been proposed that the reaction is generally most effective when the DNA is electronically excited (32,119,120); thus, long wavelength light should be less effective because DNA absorbs very little of such light in competition with UA (see also the mechanistic discussion below). There is evidence that a UA photoproduct generated upon extensive irradiation (and ultimate photodegradation) in the absence of DNA can also covalently bind to the nucleic acid in the dark (9). Isolation of cUA and tUA from such preirradiated UA mixture and subsequent mixing with DNA in the dark did not show binding to DNA (9). This supports the lack of dark reactivity of UA isomers with viral ssDNA (92). Heat-denatured calf thymus DNA was found to be a better substrate for UA incorporation, with the ratio of UA binding to denatured DNA versus dsDNA = 17 with A > 289 nm. This ratio is reduced to ca unity using A > 270 nm (88). This result indicates that preassociation of the UA with the DNA is not critical for binding, the association with dsDNA generally being expected to be far greater than for the less-organized ssDNA. This conclusion is supported by the equilibrium dialysis studies that give no evidence for the association of either cUA or tUA with DNA in the dark (see above). The extent of binding also depends upon the ratio of UA to DNA at the outset. In one experiment the DNA concentration was reduced 20-fold and significantly higher binding levels are observed, e.g. >600 nmol UA/mg DNA, both for native and heat-denatured DNA (9).

The 32PP-postlabeling technique (121) has been used to detect UA-photoinduced damaged bases in calf thymus DNA. The DNA was treated with a 1 : 1 mixture of cUA and tUA and irradiated with UVB light (60% emission between 280 and 320 nm) with an accumulated dose of 100 kJ/m'. The relative adduct labeling (frequency of adducts per nucleotide) values were 9.4 X and 8.9 X lo-' in the irradiated and dark controls, respectively, thus indicating the absence of binding of either isomer to the DNA (17). The UVB dose used in this experiment was 60 times that of the minimal erythema1 dose and as much as two-thirds of the dose found effective in augmenting UV-induced skin tumors in mice (1 16). The apparent discrepancy between these two studies is obscured by the marked difference in the experimental protocols used. The lack of covalent binding in this experiment could be a consequence of the overall low quantum efficiency for binding of UA to DNA, especially with wavelengths above 300 nm (32,88) (vide infru).

BASE SPECIFICITY FOR UA BINDING

Studies using radiolabeled UA and the four polyribonucle- otides indicate a strong preference for binding to the thymine analog, poly[U] (32). The suspicion that cyclobutane adducts were the predominant photoproducts was corroborated by the isolation and identification of such adducts upon the photolysis of UA with thymidine (33) (vide infra). Photolysis of these photoadducts with 254 nm light leads to the reforma- tion of UA and thymidine (33), a sufficiently typical phenomenon to have become a diagnostic test for the presence of cyclobutane adducts in complex systems (1 19,122,123). Thus, the 254 nm photolysis of radiolabeled poly[U] gave


Table 3. ids under aerobic and anaerobic conditions* (32)

Photoinduccd covalent binding of UA to polynucleic ac-

nmol UMmg Nucleic acid Air/ argon nucleic acid

POlY [Ul Air

Poly[A] Air

Argon

Argon

66 95

43 35

“Irradiation (A > 270 nm) of a mixture of 2-UA (2.2 mM) and polynucleic acid ( I .9 mg/rnL) in 100 niM phosphatc buffer (pH 7.0) at 10°C for 46.1 h followed by exhaustive dialysis and multiple prccipitations.

Table 4. cleic acids (32.33)

Quantum efficiencies for covalent binding of U A to nu-

Quantum efficiency

Nucleic acid A,, (nm) Argonlair x 10’

Native DNA Nativc DNA Denatured DNA Denatured DNA Native DNA Denatured DNA Poly[Al POIY IUl Thymidine* Thymidine

308 308 308 308 366 266 308 308 308 266

Argon Air < Argon Air Argon Argon Air Air Argon Argon

0.30 :0.30 0.70 0.27 15.0 13.0 0.3 1 0.98 4.601

t

back labeled UA as one would expect if cyclobutanes had been formed (32). As one might also anticipate, the photolysis of DNA with radiolabeled dihydrourocanic acid (DHUA. see structure in Fig. 7 ) gave much lower levels of label incorporation into the DNA (88). Such cyclobutane adducts have also been observed with other biologically important acrylic acids. e .g . indoleaciylic acid and p-methoxycinnamic acid, and they also undergo retrocleavage with 254 nm light (119,120). Among the purines, poly[A] is the pre- ferred target for UA binding, and as opposed to poly[U], binding to purine is enhanced i n air ( c j Table 3, and further discussion below) (32). This provides a strong indication that there are multiple mechanisms for covalent binding of UA to DNA.

IMPLICATION OF UA-DNA ADDUCTS IN 1MMUNOSUPPRESSlON

A review of the potential role of cUA in the phenomenon of photoimmunosuppression is presented in detail below. However, it may be noted here that there is elegant experimental evidence that indicates that the repair of thymine dimers by photolyase or T4 endonuclease V (delivered viu a liposome delivery technique) can reverse immunosuppression of systemic CHS caused by UVB exposures. These results suggest that thymine dimers are at least partially responsible for the immune effect (124). Though photolyase would appear to be highly specific for the repair of the pyrimidine cyclobutane dimers (125), the fact that UA is a putative target for immunosuppressive radiation required a test of whether UA-pyrimidine cycloadducts might also be substrates for this enzyme. Photolysis of DNA containing authenticated UA-thymine cyclobutane adducts in the presence of purified photolyase gave no release of UA (95). The efficiency for the enzyme to repair UA-thymine adducts in DNA is estimated to be 2900-fold less than that for cleavage of the cis-syn thymine-thymine dimers. The conclusion that UA/DNA adducts are not involved in immunosuppression is supported by a recent study showing that UVB doses capable of inducing immunosuppression do not lead to the covalent binding of UA to DNA ( 1 18) and by the fact that UA covalently binds to DNA with a low quantum efficiency (32) (note comments about UA-photosensitized dimerization of pyrimidines in viral DNA above).

*Urocanic acid absorbs 92% of the incident light. +Composite quantum efficiency for the formation of two UA-thy-

$The quantum efficiency for the formation of cyclobutanc adduct is midine cyclobutane adducts.

lower than its retrocleavage at this wavelength (see text).

NATURE OF THE EXCITED STATE RESPONSIBLE FOR THE BINDING OF UA TO DNA Because of the extensive overlap of the absorption bands of UA and DNA, both substrates are simultaneously excited with broadband light (32.88). In order to assign the excited state involved in the UA/DNA interaction, the quantum efficiencies for covalent binding of UA to DNA were measured at two different wavelengths using laser excitation. As has already been noted, 308 nm laser excitation predomi- nantly populates the UA excited state, whereas both reactants compete for 266 nm light. The observation is that the binding efficiencies are 20-50 times higher at 266 nm (cf. Table 4). These quantum efficiencies are lower than that measured for the well-known skin photosensitizer drug, 8- MOP, which intercalates into DNA and binds with a quantum efficiency of 3.7 X lo-? and with a saturation point of 1 8-MOP/36 nucleotides (126). Based on the data in Table 4, it was concluded that the photoinduced binding is mainly derived from a DNA excited state reacting with the UA ground state (DNA* + UA -+ UA-DNA photoadducts). However, the recent studies of UA photophysics and the demonstration that 308 and 266 nm light lead to different UA excited states (19) (see above) provide an alternative rational for the DNA results. Adduct formation could derive from a UA triplet formed at 266 nm and known to be relatively unreactive toward olefin isomerization (19), while the proposed readily isomerized singlet formed at 308 nm may be far less reactive with DNA.

for the loss of UA under argon was measured in the presence of 1.31 mg/mL calf thymus DNA ([UA]/[DNA] = 2) using a 308 nm laser (32). The quantum efficiency for UA loss under argon in the presence of 4.0 mM thymidine is reported as 4.8 X lo-‘ (33). A value of 1.0 X lo-’ measured for the loss of UA in the absence of DNA using a 313 nm light (53) is much higher than these values. Because it is unclear from this paper as to whether this last experiment was done under argon, one cannot say with certainty whether the nucleic acid in-

A quantum efficiency of 2.4 X


X=lm or COzH. Y = COZH or Im

DMT UA-DMT I UA-OMT II

P, nm

H H

cUA K I A

Figure 4. [2+2] Photocycloaddition of UA to DMT and retrocleavage of the resultant cyclobutane photoadducts back to the starting materials upon reirradiation with 254 nm light (69).

hibits UA photodegradation. A recent report provides support for ascribing the low quantum yields for UA degradation to the short-lived singlet excited state (27).

MECHANISTIC STUDIES RELATED TO THE BINDING OF UA TO DNA Perhaps the first mechanistic information for the photochemical interaction of UA with DNA came from the isolation of two UA/DMT photoadducts (69). The adducts result from the cycloaddition of the acrylic acid olefinic bond of UA with the 5,6-double bond of DMT. The overall quantum "utilization" for the formation of the adducts is 1.8 X lo-*, a value comparable to the quantum efficiency (1.4 X lo-?) reported for the photodimerization of DMT itself (73). The term quantum utilization reflects the uncertainty about which substrate excited state is involved in the photocycloaddition. The quantum efficiencies for such bimolecular reactions will also be substrate concentration dependent. As expected, the cyclobutane ring is cleaved to UA and DMT upon reirradiation at 254 nm, with one adduct exclusively giving cUA and the other giving tUA (see Fig. 4). There are ample prece- dents for the excited state of DMT to react through photocycloaddition, both to another DMT molecule (73) as well as to other olefins (127,128). However, the UA excited state is also known to photodimerize, albeit only in the solid state (50). A ground-state complex of UA and DMT is evident from the hyperchromicity observed at 320 nm in the absorption spectrum of the two reactants (69). Because a water- soluble triplet quencher (2,4-hexadienol-l-o1; & 59 kcal/ mol) (129) had no effect on the formation of the photoadducts, it was concluded that the excited singlet state of DMT is involved in the reaction. Adducts of UA and thymidine have also been isolated using reaction conditions that lead to the incorporation of UA into DNA (33). Their structures were determined by a complete spectral analysis that indicated that they were diastereoisomers (Fig. 5). The two adducts are formed with comparable quantum efficiencies, e.g. 0.21 X and 0.25 X for UA-thymidine adduct I and adduct 11, respectively (33).

ADDITIONAL MECHANISM FOR UA BINDING TO DNA In addition to pyrimidine cycloaddition, there is compelling evidence for a second mechanism of binding of UA to DNA

OH OH I CH Im+ ,;,p/

Y. ,&: &k7 Holy,. o.J,) H co2-

., tp OH Im+ = ?I7 OH

\N/

HovlR1 UA-thymidine adduct I UA-thymidine adduct I1

Figure 5. Structures of UA-thymidine cyclobutane photoadducts isolated from the reaction of UA and thymidine under the conditions that lead to incorporation of UA into DNA (33).

that involves purines. As already noted, UA efficiently binds to DNA when 266 nm light is used, but this wavelength of light has been shown to cleave cyclobutane adducts efficiently. Thus, the irradiation of an authentic cyclobutane photoadduct of indoleacrylic acid (1A)-thymidine (1 19) with 266 nm laser light effectively released the thymidine and IA isomers with a quantum efficiency of -6.0 X (T. Mo- hammad, unpublished results). In addition, substantial covalent binding of UA to single-stranded homopolymers of purines, especially poly[A], has been observed (cf: Table 3). No adduct of UA with a purine has yet been reported, but there is ample evidence for a photochemical interaction between UA and 2'-deoxyadenosine (dAdo). The direct and/or sensitized irradiation of either cis- or trans-UA at A > 270 nm leads to UA oxidation via electron transfer, with the resultant UA' trapped by oxygen and ultimately cleaving to ImCHO (cf: Eq. 1) (31,49).

UA ImCHO

The presence of dAdo accelerates the formation of the ImCHO by a factor of >4 (31). Both the UA and dAdo excited states can be formed under the conditions of the photolysis. It is proposed that excited dAdo photoionizes and that the resultant radical cation oxidizes UA. Interestingly, the presence of sodium azide in these reaction mixtures catalyzes the cleavage of the UA side chain (31). The azide

dAdo - UA.+ U A - 0 2

[dAdo ~sorner]

l OHCCOzH

Q f C H O

H

ImCHO

Figure 6. Structures of proposed intermediates/products resulting from the photochemical interaction of UA and dAdo (48).


Table 5. a-UA to dsDNA*

Quantum efficiencies for covalent binding of 2-UA and

H ti Dih)dmuroranir a ~ t d ( l ~ l l l ; ~ \ l 4deninr I Adel

Figure 7. Structures of the products resulting from the photoinduced single electron transfer (SET) reaction between UA and dAdo (48,130).

seems to serve multiple roles: as a U A - trap (thus facilitat- ing the generation of an intermediate dioxetane, UA-02, Fig. 6), as a source of azidyl radicals that can add to the UA and as a singlet oxygen quencher that serves to protect the UA and ImCHO from alternative degradative pathways (79).

In addition to the aldehyde, another UA product is formed in low yield when UA is photolyzed at A > 270 nm (48). This is the DHUA shown in Fig. 7. Two dAdo products have also been observed in these reactions ( c j Fig. 7). One is adenine (48). The other has a longer HPLC retention time than dAdo. and its proton NMR spectrum is very similar to that of dAdo. However, i t slowly reverts back to dAdo in the dark and has not yet been identified (130). Because the quantum efficiencies for the binding of UA to nucleic acids (32) and for its oxidative cleavage (31) are comparable, studies have been carried out to determine the potential role of cleavage chemistry in the conjugation process. The DNA was irradiated with two UA radioisotopomers ( c - Fig. 8). 2- UA labeled in the ring (88.131) and a-UA with the label in the side chain (20,132). If cleavage chemistry plays a role, one would anticipate a lower level of label incorporation with a-UA. The relevant data are presented in Table 5 and indicate that comparable levels of incorporation are observed with each of the radioisotopomers. The results are independent of the wavelength of excitation. i.e. at 266 nm and 308 nm (L. Paredes, unpublished results) though the overall quantum efficiencies are higher at shorter wavelength as has already been noted. One should note that the quantum efficiencies reported in Tables 4 and 5 at a given wavelength do not match because these values are dependent upon the concentration of UA and DNA that were not same in these measurements.

CO,H C 0 2 H N- ~ N *

, , , ? ~ . - , , " . > d , . . . J ) > 2 li , -, ".,,:an. ,.,J , I , Figure 8. Radioisotoponiers of CA used t o drteimine if photooxi- dative cleavage is in\olvcd in the plic~tocorijiigation o f UA to DNA.

TRANSIENT SPECTROSCOPY OF UA The UA radical cation ( U A . ) is a common, proposed intermediate in the above chemistry. There is other. indirect evidence f o r its formation. Thus. excitation of the electron- deficient dye, NBT". in the presence of UA leads to oxidation of the latter in a dose-dependent manner (49). As one would anticipate. oxidation of UA is most efficient in basic media where it exists as a carboxylate anion (structure C in Fig. 2). The direct excitation of UA also reduces the electron

Quantum efficiency

Radioisotopomer A,, (nni) Argodair x 1 0 5

2-UA WCIA 7-UA a - U A 2-UA (U-LTA 2-UA (U-UA

308 308 308 308 266 266 366 266

Argon Argon

Air Air

Argon Argon

Air Air

1.5 0.9 1.6 1.1 8.0 6.0 10.0 12.0

"Solutions of UA ( 1 mM. 50 pCi/mmol) and DNA (3.9 mM) in 100 mM phosphate buffer were irradiated for 60 min with a XeCI- charged excinier laser (308 nm. 1 .1 X lo'' photon/s) and/or frequency-quadrupled Nd:YAG laser (266 nm, 1.2 X 10'' photon/ s). The labeled DNA was purified through exhaustive dialysis followed by two rounds of precipitation. The quantum efficiencies were calculated from the binding levels in the second pre- cipitate multiplied by the amount of DNA recovered after dialysis tL. Paredcs. unpublished results).

affinic dye, NBT'*, viu the intermediate formation of superoxide anion (OY-) as a result of electron transfer from UA* to ground-state molecular oxygen (49). It appears that UA'* is very unstable and attempts to observe it through transient absorption by sensitization with triplet chloranil, in solution and in a matrix, have been unsuccessful (L. Johnston, private communication). Because the free amino and carboxyl groups were thought to be destabilizing the transient through proton transfer, flash photolysis studies have been carried out on derivatives containing a protecting group on one or both of these functionalities. Excitation of truns-methyl urocanate (tMU, see structure in Fig. 9 below) in aqueous buffered solution (pH 7) using 266 nm laser radiation in a flow- cell under anaerobic conditions led to the formation of a transient at 390 nm and the characteristic absorption at 700

H CHI

mOI-N-(M~thYllm~LhyI wocdnate (LNMMU) m n i Methyl U ~ L I D I I ~ IrMU)

Figure 9. Chemical structures of U A analogs (58.147.148).


nm for the hydrated electron. The 390 nm species is attributed to the MU radical cation (lifetime > 100 p). The appearance of a broad transient absorption between 340 and 400 nm is attributed to the presence of at least two additional species, one of which is thought to be the MU radical anion. The presence of oxygen cleanly wipes out the absorption of the hydrated electron. Flash photolysis data for trans-N- (methy1)methyl urocanate (tNMMU, c$ Fig. 9 below) are comparable to those for MU under nitrogen, i e . , the formation of hydrated electrons at 700 nm and the NMMU radical cation at 410 nm (lifetime > 100 ps). Again, nitrogen-saturated solutions give an additional transient absorbing between 350 and 380 nm that may be due to the NMMU radical anion created through reduction by hydrated electrons. As already noted, flash photolysis of tUA under these conditions generates hydrated electrons in poor yield and a weak signal for the UA radical cation (L. Johnston, private communication). These observations have been supplement- ed with sensitization studies using a mixture of 9,lO-dicy- anoanthracene and biphenyl in acetonitrile. The radical cations of tMU and tNMMU were observed upon excitation with 355 nm laser light. There was no evidence for the UA radical cation being formed under these conditions (L. John- ston, private communication). It is noteworthy that in struc- turally related compounds bearing an a$-unsaturated lac- tone moiety, radical cations have been observed for a series of skin-sensitizing posralens, both upon direct and sensitized photooxidation. In 50% aqueous (buffered) acetonitrile, radical cations are observed at 550, 650 and 600 nm for 5-MOP, 8-MOP and psoralen, respectively (133). The transients, liv- ing on the order of 5 ps, can be trapped by azide, GMP and AMP at diffusion- or near diffusion-controlled rates, i.e. 2.1 X lolo, 2.5 X lo9 and 3.4 X lo7 M-l s-I , respectively (133).

PHOTOINITIATED COVALENT BINDING OF UA TO PROTEIN IN VITRO In addition to binding to nucleic acids, UA also targets proteins. Irradiation of a mixture of radiolabeled 2-UA (cJ: Fig. 8) and bovine serum albumin (BSA) at A > 270 nm leads to the covalent attachment of UA to the protein (134). Coelu- tion of UA and BSA from a Sephadex G-100 (size-exclusion) column confirmed the irreversible binding of the label. The binding is dependent on the absorbed UV dose and reached 58 nmol of radiolabel per mg of BSA after 48 h of irradiation. Slightly lower binding levels were observed under aerobic conditions, probably due to the degradation of UA under these conditions in the dark (134). There was no evidence for preassociation of UA to the BSA. Though the biological significance of binding of UA to BSA is not yet clear, the level of incorporation of UA into the protein is comparable to that observed for the binding of other small molecules to BSA and human serum albumin (HSA), e.g. 8- MOP, 22 nmol/mg BSA (135); anthracene, 2.7 nmol/mg HSA (136), nitrofurfural, 50 nmol/mg HSA (137) and 3,3',4',5-tetrachlorosalicylanilide, 15 nmol/mg BSA (138).

THE ROLE OF IRREVERSIBLE PHOTOBINDING OF UA TO BIOMOLECULES IN VIVO There are contradictory reports on the UA photocytotoxicity resulting from its damage of biomolecules. Neither cUA nor

tUA is taken up by primary mouse keratinocytes. As a result, UA isomers do not reduce cell viability of keratinocyte culture in the dark (1 18). A recent report supports such a lack of interaction of either cUA or tUA with cells in the dark using DNA binding, unscheduled DNA synthesis and induc- tions of mutations assays, even at very high doses (144 mM) of UA (17). However, there is some evidence that 2-UA (ctf: Fig. 8) penetrates L5178Y TK6+/- mouse lymphoma cells when incubated at 37°C. Irradiation of the frozen mixture of UMcells followed by cell lysis and phenol extraction indicates binding levels of 3.1 nmol UNmg nucleic acid (48). These binding levels may be biologically significant; lower levels of DNA lesions induced by other chemicals are known to cause mutations in L5178Y TK6+/- cells (139). The binding to DNA is both wavelength and dose dependent (32,88). The observation that 2-UA enters the mouse Iym- phoma cells is corroborated by an observation that administration of tUA produces cytotoxic effects in mice suffering from intestinal carcinoma, leukemia neoplasms, Lewis pul- monary and Ehrlich ascites (140). It was noted above that at the low UVB doses capable of initiating immunosuppression in mice, no binding of UA to DNA of BALB/c mice is observed (1 18). One possible reason for the lack of UA binding to mouse DNA could be a reduced bioavailability of UA and its rapid (1944% in <30 min) clearance in blood when it is topically applied (1 18).

It is interesting that the isolation of protein from the 2- UA photolysis with the L5178Y TK6+/- lymphoma cells in the frozen matrix also gave evidence of label incorporation at a level of 55 nmol label/mg protein (48). Topically applied radiolabeled UA has also been observed to bind to serum protein of Skh:HR mice after UVB irradiation of the dorsal skin (141).

UA REACTIONS WITH, AND GENERATION OF, SINGLET OXYGEN AND SUPEROXIDE ANION

There are reports that DNA bases (142,143) and UA (144) photosensitize singlet oxygen formation. Urocanic acid has been shown to be reactive with riboflavin-generated superoxide anion (49). There is both chemical and spectroscopic evidence (vide infra) that UA can sensitize the formation of singlet oxygen (lo?, 'Ag). The irradiation of UA in the presence of oxygen leads to UA decomposition that is minimized by the presence of the singlet oxygen quencher, sodium azide (31,79). This observation indicates that UA both generates singlet oxygen and also reacts with it. The latter has also been observed through studies of UA reactivity with singlet oxygen independently generated by rose bengal photosensitization (10). Reactivity of UA with singlet oxygen is not surprising because of the presence of an imidazole ring analogous to its precursor, histidine. The latter is a well- known singlet oxygen trap and reacts with near diffusion- controlled rate constants (0.32-54.3 X lox M-l s-I ) in aqueous solutions (145). Recently, Hanson and Simon have presented spectroscopic evidence that UA sensitizes the formation of excited-state singlet oxygen, the characteristic emission ([Ag + 3Zg) of which is detected at 1270 nm (146). This was supported from the photoacoustic data when they observed less retained heat in the presence of oxygen. The

128 Taj Mohamrnad et a/.

authors therefore propose that oxygen quenches the UA triplet state via energy transfer and generates electronically excited singlet oxygen (lo2). It is argued that there is a good correlation between the action spectra of UA triplet formation and the photosagging of mouse skin. The authors con- jecture that the latter physiological response may be due to the generation of ‘0, by UA (146).

UA ANALOGS

Several UA analogs have been synthesized and studied to uncover the mechanism for DNA photoinactivation and understand the molecular basis for the immunosuppression by cUA. Norval et ( I / . ( 147) have carried out a fairly extensive structure-activity relationship study on several analogs of UA i n which the primary structure of UA has been modified (see Fig. 9). The authors have investigated the synthetic analogs. ?--methylurocanic acid (ZMUA), 2-furanacrylic acid (2-FAA). 2-pyrroleacrylic acid (2-PAA). 2- and 3-thiopheneacrylic acid (TAA). both as cis and trans isomers. Hista- mine (c$ Fig. 1 ) and DHUA (cJ Fig. 7 ) were also tested. A delayed-type hypersensitivity (DTH) response of mice to Herpes sinzplex virus type 1 infection was used to evaluate the immunosuppressive abilities of the UA analogs. Ring methylation, as in 2-MUA. resulted in the disappearance of immunosuppressive activity. possibly a consequence of an increase in hydrophobicity relative to UA. Both the cis and truns isomers of ‘-FAA, 2-PAA and 2-TAA were effective in the induction of immunosuppression; the activities of the cis isomers generally exceeded that of the corresponding truns isomers. On the other hand. the 3-TAA isomers were found to be poor immunosuppressants. The cis isomer of 2- PAA was the most potent at a dose of 50 pglmouse, indicating that substitution of the tertiary nitrogen with carbon in UA did not affcct the biological activity. Substitution of the ring nitrogen in 2-PAA with oxygen or sulfur led to retention of the immunosuppressive activity. It seems that the intact acrylic acid side chain of UA does not play a significant role in the (unidentified) receptor binding because both DHUA and histamine were found active in the suppression of the DTH response. It was deduced that a five- mernbcred heterocyclic ring i h necessary for suppressor activity (147).

Imidazole-4(5 )-mcthylidene malonic acid (IMMA) (20.131) can be viewed as a composite of both cis- and trans-UA. Its absorption spectrum in 50 mM phosphate buffer (pH 7) resembles that of cUA and tUA in the sense that there is a single broad and structureless band with a slight red shift relative to the UA isomers ( 148). The molecule is mildly photoactive. with decarboxylation leading to the generation of a mixturc of cUA and tUA as the primary photochemical reaction. The reaction is of low quantum efficiency (1.2 X 10 ’) and is independent of excitation wavelength ( I 48). The IMMA possesses photobiological properties that are the average of the UA isomers (148). Irradiation in the presence o f phage S13 ssDNA with a 308 nm laser leads to inactivation of the bacteriophage DNA in a dose- dependent manner. providing a ratio of k-lMh,Alk lhls,A - 0.43. This ratio falls between the values for pure cUA and tUA (0.014 and 1.6, respectively) (58). An absence of photoreactivation in the sensitized virus confirms that IMMA,

-

like UA, protects ssDNA against the formation of cyclobutane pyrimidine dimers. However, unlike UA that produces biphasic inactivation curves (58) , IMMA gives a DNA survival curve that is linear. For dsDNA the ratio kTIMMA/k-lMhlA is 10.3 relative to the value of 13 observed for a photostationary-state mixture of UA. As with UA, the inactivated DNA can be repaired with an 80% efficiency, thus suggesting the sensitized lesions to be mainly cyclobutane pyrimidine dimers. The IMMA irreversibly binds to calf thymus DNA with a quantum efficiency of 2 X

The methyl ester of UA (tMU) has an absorption spectrum in aqueous solution (pH 6.8) that is red shifted by -15 nm relativc to UA (58). It undergoes cisltrans photoisomerization, both in organic and aqueous solvents. Isomerization is the primary photochemical event at low conversions. In dichloromethane 85% cMU is formed after irradiation of a 20 mM solution of tMU with 254 nm light (29). The photostationary state is highly wavelength and pH dependent. Irra- diation of a dichloromethane solution with 3 13 nm radiation gives only 16% cMU while the photostationary state in buffered solution (pH 6.8) at 308 nm is 46% cMU (58). The photostationary state is >95% cMU in the presence of the Lewis acid, BF,.OEt,, mainly due to one-way trans + cis isomerization (29). This is attributed to strong intramolecular hydrogen bonding between the imidazole NH and carbonyl oxygen, the strength of which is increascd in the presence of BF,.OEt, (29). The INDOlS-CI calculations have been used to predict precisely the relative energies of the electronic transitions of the cis and truns isomers and their dependence on the mode of hydrogen bonding (149). The quantum yields with 313 nm excitation are 0.63 and 0.33 for trans + cis and cis + truns isomerization, respectively (29). An extended irradiation of long chain alkyl esters of UA in organized media leads to deesterification to yield UA ( 150).

Irradiation of tMU and viral ssDNA provides results similar to those seen with UA. The inactivation curve with tMU is biphasic. There is an early portion involving sensitized inactivation (ktMUlk-Ml: = 5.4) and an ultimate portion, at- tributable to the photostationary state, which gives evidence for protection of the virus from UV lesions ( k , , , , k M U = 0.31 ). Subjecting the inactivated virus to DNA photolyase or Weigle reactivation (<0.02) did not reverse the virus infectivity, an indication that MU, like UA, practically elimi- nates the creation of pyrimidine dimers (58). As is the case with UA itself, the ethyl ester is oxidized by direct excitation or via sensitized reaction with NBT2+ (49). The ester oxidation appears to be about half as efficient as that of the acid. There is evidence for the participation of superoxide (formed by electron transfer from the ester to oxygen) in the reduction of the chemical probe (49).

Truns-N-(methyl) methyl urocanate is an example of UA with fully protected acid and amine functionalities. Its absorption band in 100 mM phosphate buffer (pH 6.8) has a maximum at 290 nm and is broad and structureless. It undergoes cisltrans isomerization upon photolysis in phosphate buffer (pH 6.8) with 308 nm light. The photostationary state is rich in cNMMU (62%), and the major photochemical event is isomerization about thc acyclic ester double bond. Photodegradation under an inert atmosphere is minimal at 308 nm, &,, -0.3 X 10-3 (T. Mohammad, unpublished re-

(148).


sults). The tNMMU photosensitizes inactivation of S 13 ssDNA when excited at 308 nm. The inactivation curve is biphasic and provides values of k+NMMUIk-NMMU = 7.2 and 0.3 for the initial and ultimate slope, respectively. A lack of photoreactivation suggests the predominant lesions to be other than cyclobutane pyrimidine dimers (I. Tessman, private communication).

EFFECTS OF UA ON THE IMMUNE SYSTEM

Although the relative contribution of cUA to UV-induced immunosuppression is uncertain, the immunosuppressive properties of this compound are well established. It is suggested that UVB-induced DNA damage and cUA follow different pathways to initiate cascades of chemical mediators in the skin and lymphoid cells (15 1). Experimental treatment of laboratory animals with cUA suppresses CHS and DTH, (3,152), delays the rejections of allografts (37,153-155) and suppresses the onset of graft-versus-host disease (37). The mechanisms by which cUA induces immunosuppression are not well understood as discussed in detail in previous reviews (5,7). This section will focus on the recent studies that attempt to elucidate the mechanisms of cUA-induced immunosuppression.

The cUA affects the function of antigen-presenting cells in mice. In an early study, systemic administration of cUA to mice suppressed the antigen-presenting function of spleen dendritic cells (156). This effect was only observed 7 days following the administration of cUA and not when cUA was added to dendritic cells in vitro. The lack of a direct effect on antigen-presenting cells was confirmed in a recent study in which cUA had no effect on either the maturation or function of bone marrow-derived dendritic cells in v i ~ o (157). The cUA suppressed the mixed epidermal-lymphocyte reaction by a maximum of only 27% following 3-6 day pre- incubation of human epidermal cells with cUA (158). No effect was seen in another study in which human epidermal cells were incubated for 18 h with cUA (159), and cUA had no effect in a mixed lymphocyte reaction (158). In aggre- gate, these in vitro studies suggest, but do not prove, that cUA has an indirect, rather than a direct effect on dendritic cells.

In contrast, results of two recent studies suggest that cUA may directly affect the function of Langerhans cells (LC). Incubation of epidermal cells with cUA in vitro for 2 or 3 h inhibited the ability of these cells to sensitize mice for a CHS or DTH reaction (1 60,16 1). In addition, whereas injection of mice with epidermal cells pulsed with tumor-associated antigens prevented the outgrowth of tumors following challenge, cUA-treated LC failed to do so (161). Because the epidermal cells in these experiments contained only 5- 15% LC, it is possible that cUA induced the secretion of mediators from other cells, in particular keratinocytes, that affect the function of LC. However, cUA has no effect on the expression of interleukin (1L)-10 or tumor necrosis factor (TNF)-(w by mouse keratinocytes (161), nor on the secretion of various cytokines, including TNF-(w, by human keratinocytes (162).

The mechanism by which cUA interferes with the function of antigen-presenting cells is also unclear. The cUA has no effect in vivo or in virro on the expression of major his-

tocompatibility complex I1 or various costimulatory molecules by dendritic cells (156, 157, 161). Intradermal injection of cUA reduces the length of the dendritic processes of LC within 2 h, accompanied by reduced expression of the intermediate filament vimentin (163). Irradiation of the skin with UVB and intradermal injection of TNF-a and vinblas- tine, an alkaloid that interferes with microtubule assembly. had similar effects. This suggests that cUA may interfere with the function of LC by disrupting the cytoskeleton (163).

Several soluble mediators seem to have a role in cUA- induced immunosuppression. These include histamine, prostaglandin E2 (PGE?) and TNF. Immunosuppression by cUA is partially blocked by histamine receptor H, and H? antagonists (164). This has led to the hypothesis that cUA acts via histamine receptors. However, recent studies in which the function of cUA and histamine were compared indicate that these molecules bind to different receptors ( 165,166). Indeed, one report provides direct evidence that cUA does not bind to histamine receptors, but instead can bind to y- aminobutyric acid (GABA) receptors (167). Because histamine receptor antagonists partially block cUA-induced immunosuppression, it is likely that cUA causes the secretion of histamine (168). The source of the histamine has not been identified, but the two most likely sources are mast cells and keratinocytes. Mast cells store histamine in their granules. and cUA can cause degranulation of mast cells (169). Hu- man keratinocytes contain and secrete histamine at a low level, and this is enhanced by UV irradiation in vifro ( 170).

A second mediator that has been identified is PGE?, because treatment of mice with indomethacin, a cyclooxygen- ase inhibitor, partially inhibits cUA-induced immunosuppression (168). The cUA induced the release of PGE, from human monocytes in vitro (165) and synergized with histamine in the release of PGE, from keratinocytes (166). Pros- taglandin E, is also a mediator of UV-induced immunosuppression (171) and initiates a cytokine cascade by inducing the secretion of IL-4, followed by the secretion of IL-10 (172). The latter is a potent inhibitor of antigen-presenting cells ( 1 73.174), and neutralization of IL- 10 with monoclonal antibodies in vivo prevents UV-induced suppression of DTH. In addition, PGEz inhibits the production of IL-12 by lipo- polysaccharide-stimulated human blood monocytes ( 175). Interleukin-12 is a critical mediator of CHS, and neutralization of IL-12 almost completely inhibits CHS (176). How- ever, it remains to be proven if IL-12 has a direct effect or acts via stimulation of interferon-? (IFN-7). A recent study has presented strong evidence for the protection against cUA-induced immunosuppression by IFN-y. The IFN-y- gene knockout mice expressed -40% higher immunosuppression than control mice after treatment with cUA (177). Taken together, these data suggest the following pathway of cUA-induced immunosuppression. The cUA induces degranulation of mast cells resulting in the release of histamine or stimulates the secretion of histamine by keratinocytes. The cUA and histamine cause the release of PGE, from keratinocytes, and cUA by itself may cause additional release of PGE? from monocytes. Prostaglandin E2 induces the production of IL-4 and IL- 10 and simultaneously suppresses the synthesis of IL-12 and/or IFN-y, resulting in suppression of DTH and CHS reactions.

Tumor necrosis factor-a has been identified as another


mediator of UV- and cUA-mediated suppression of CHS reactions (178,179). Because mast cells store preformed TNF-a in their granules. cUA-induced mast cell degranulation may not only release histamine but also cause a rapid increase of TNF-(U in the skin.

It should be pointed out that not all the data fit this model of cUA-mediated immunosuppression. Treatment of mice with histamine antagonists and indomethacin only partially prevented cUA-induced suppression of CHS, and a com- bined treatment with indomethacin and histamine antagonists had no additive effect (168). Furthermore, the suppression of the DTH to tumor-associated antigens by cUA was not inhibited by histamine antagonists or indomethacin (161). This suggests that other mechanisms also contribute to cUA-induced immunosuppression. The mechanisms may differ between acute and chronic treatment with cUA because these treatments seem to have different effects on the immune responses. When mice were treated for 4 weeks with cUA, the CHS and DTH responses were not suppressed. However, other immune responses. including the mixed epidermal cell-lymphocyte reaction and lymphopro- liferative response of spleen cells, were suppressed in cUA- treated mice ( 180).

ADDENDUM The total UA concentration and the relative (percent) and absolute (nmoVcm') cUA concentration are reported ( I 81 ) to be the same in basal cell carcinoma (BCC) and cutaneous malignant melanoma (CMM) patients as in the healthy controls before irradiation. However. the cUA concentration after UV exposure of the forehead and the upper back was significantly higher in both cancer patients relative to the control group. Interestingly, both the BCC and the CMM patients showed comparable levels of cUA formation after exposure to UV radiation.

In a recent meeting abstract ( 182). i t has been shown that DNA damage (mainly pyrimidine dimers) is responsible for some, but certainly not all, immunosuppression caused by UV exposure. The photoisomerization of tUA is the major factor in the initiation of UV-induced melanoma.

CONCLUSIONS Urocanic acid continues to be an intriguing molecule. A wealth of information has accumulated over the past two decades on its photochemical. photophysical, photobiological and (photo )immunosuppressive properties. The photophysics of UA is now well understood and recent studies confirm that the wavelength-dependent photochemistry orig- inates from close-lying multiple electronic transitions under the broad absorption band. The singlet excited state acces- sible at longer wavelengths undergoes isomerization more efficiently than the short-lived triplet state that is populated at shorter wavelengths. Urocanic acid isomerization can be induced vitr triplet sensitization and radical additiodelimination mechanisms. Urocanic acid sensitizes and reacts with singlet oxygen. Electronically excited UA reacts with biomolecules including proteins and DNA. The quantum efficiencies of these processes are relatively low. The reactivity with DNA is supported by two independent and comple- mentary studies: covalent binding to calf thymus DNA and

inactivation of viral nucleic acid. The mechanisms for DNA damage are partially identified; the former involves, predom- inantly if not exclusively, cyclobutane adducts of UA and thymine and the latter the sensitization of cyclobutane pyrimidine dimers and strand breaks. It is now well established from studies emerging from several laboratories that cUA is immunosuppressive. However, its mode of action is not clear at present. The proposals that cUA affects the function of LC and other antigen-presenting cells and induces the secretion of several cytokines await further verification. There are contradictory reports on the possible role of histamine receptors for cUA. The GABA receptors have also been in- voked as targets for cUA binding. Future studies are needed to determine the relative contributions of these mechanistic models for cUA-caused immunosuppression.

REFERENCES 1 .

2.

3 .

3.

5.

6 .

7.

8.

9.

10.

1 I .

12.

13.

14.

IS

16

17

18

Jaffe. M. (1874) Concerning a new constituent in the urine of dogs. Brr. Deiir. Clirni. Ges. 7. 1669-1673. Parrish. J . A. ( 1982) Therapeutic in vi\w photochemistry: photochemical toxicity studies in humans. J . Nafl . Cmcer Disf. 69. '73-278. De Fabo. E. C. and F. P. Noonan (1983) Mechanism of immune suppression by ultraviolet irradiation in vivo. I. Evidence for the existence of a unique photoreceptor in skin and its role in photoimmunology. J. E.rp. Med. 158, 83-98. Norval. M.. T. J. Simpson and J . A. Ross (1989) Urocanic acid and immunosuppression. Phoroclierti. Phorobiol. 50. 267-275. Noonan, F. P. and E. C. De Fabo (1992) Immunosuppression by ultraviolet B radiation: initiation by urocanic acid. Iinniirnol. Tootliij, 13, 250-754. Noonan. F. P. and E. C. De Fabo (1993) UV-induced immunosuppression: relationships between changes in solar UV spectra and immunologic responses. In Eni,irotinirntttl UV Pliorobiolog? (Edited by A. R. Young, L. 0. Bjom. J . Moan and W. Nultsch), pp. 113-148. Plenum Press, New York. Norval. M., N. K. Gibbs and J . Gilmour (1995) The role of urocanic acid in UV-induced immunosuppression: recent ad- vances ( 1992- 1994). Pliorochrm. Phorobiol. 62, 209-2 17. Norval. M. ( I 996) Chromophore for UV-induced imrnunosup- pression: urocanic acid. Phorochc~rn. Phofr~hiol . 63, 386-390. Morrison. H. (1985) Photochemistry and photobiology of urocanic acid. PIiorodrritirr/olo~~ 2, 158-165. Morrison. H. and R. M. Deibel (1986) Photochemistry and photobiology of urocanic acid. Pliotochern. Phorobiol. 43, 663-665. Mehler. A. H. and H. Tabor (1953) Deamination of histidine to form urocanic acid in liver. J. Biol. Chrrii. 201. 775-784. Furuta. T.. H. Takahashi and Y. Kasuya (1990) Evidence for a carbanion intermediate in the elimination of ammonia from 1.-histidine catalyzed by histidine ammonia-lyase. J. Am. Chem. Soc. 112. 3633-3636. Tahachnick, J . ( 1957) Urocanic acid, the major acid soluble. UV-absorbing compound in guinea pig epidermis. Arch. Biiiclier~r. Biop1iy.s. 70, 395-298. Hug. D. H. and R. C . Venema (1985) Photoinactivation of urocanase in P.seirdotfionus puritltr. Activation by a biochemi- cally generated excited state. Ariri. N.Y. Ac-lid. Sci. 453, 388- 389. Zenisek. A. and J. A. Kral ( 1953) The occurrence o f urocanic acid i n sweat. Biocliitii. Biopkys. Acto 12, 479-480. Webbcr. L. J. . E. Whang and E. C. De Fabo (1997) The effects of UVA-I (340400 nm), UV-11 (320-330 nm) and UVA-I + I1 on the photoisomeriration of urocanic acid i t i i ivo . Photo- cii~~rn. Pliorobiol. 66. 484192. Yarosh. D. B.. S. D. Gellings. L. G. Alas. J. T. Kibitel, R. H. C. San. V. 0. Wagner. 111 and G. N. McEwcn. Jr. (1992) The biological interaction of c i s and frtii7s-urocanic acid with DNA. Phofoc/c,r~~irttol . Phnfoirnrrztriiol. Pliolomrd 9, I 2 1 - 126. Shibata. K.. Y. Nishioka. T. Kawada, T. Fushiki and E. Sug-


imoto (1997) High-performance liquid chromatographic measurement of urocanic acid isomers and their ratios in naturally light-exposed skin and naturally shielded skin. J . Chroniutogr. B 695. 434438.

19. Hanson. K. M.. B. Li and J. D. Simon (1997) A spectroscopic study of the epidermal ultraviolet chromophore trans-urocanic acid. J. Am. Chem. Soc. 119, 2715-2721.

20. Mohammad, T. and H. Morrison (1991) A general approach to the synthesis of “C-labeled photoactive acrylic acids. J. La- belled Compd. & Rndiopharm. 29. 1019-1026.

21. Edlbacher, S. and F. Hertz (1943) Urocanic acid. Z. Physiol. Chem. 279, 63-65.

22. Kavanagh. G.. J. Crosby and M. Norval (1995) Urocanic acid isomers in human skin-analysis of site variation. Br. J . Der- niatol. 133. 728-73 1 ,

23. Stab, F., U. Hoppe and G. Sauermann (1994) Urocanic acid and its function in endogenous antioxidant defense and UV- protection in human skin. J . Invest. Dermatol. 102, 666. [Ab- stract]

24. Roberts, J. D.. C. Yu. C. Flanagan and T. R. Birdseye (1982) A nitrogen-I5 nuclear magnetic resonance study of the acid- base and tautomeric equilibria of 4-substituted imidazoles and its relevance to the catalytic mechanism of a-lytic protease. J. Am. Chem. Soc. 104. 3945-3949.

25. Ohman. H. and A. Vahlquist (1994) I n vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. Actn Dennuto- Venereal. (Stockh.) 74, 375-379.

26. Lahti. A,, M. Hotokka, K. Neuvonen and P. Ayras (1997) Quantum-chemical gas-phase calculations on the protonation forms of trans- and cis-urocanic acid. Struct. Chem. 8, 331- 342.

27. Laihia, J. K.. H. Lemmetyinen, P. Pasanen and C. T. JansCn (1996) Establishment of a kinetic model for urocanic acid photoisomerization. J. Photochem. Photobiol. B Biol. 33, 21 1-2 17.

28. Hug, D. H. and J. K. Hunter (1994) Adventitious interconver- sion of cis- and trans-urocanic acid by laboratory light. Pho- tochem. Photobiol. 59, 303-308.

29. Lewis, F. D., D. K. Howard. J. D. Oxman, A. L. Upthagrove and S. L. Quillen (1986) Lewis acid catalysis of photochemical reactions. 6. Selective isomerization of P-furylacrylic and urocanic esters. J. Am. Chern. Soc. 108, 5964-5968.

30. Furuta. T., M. Katayama, H. Shibasaki and Y. Kasuya (1992) Simultaneous determination of stable isotopically labelled L- histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry. J. Chroniatogr. B 576. 213-219.

31. Mohammad, T., A. Kasper and H. Morrison (1994) Urocanic acid photobiology. Purine-assisted photooxidation to 1H-imidazole-4(5)-carboxaldehyde. Tetrahedron Lett. 35, 4903- 4906.

32. Farrow, S. J., T. Mohammad, W. Baird and H. Morrison (1990) Photochemical covalent binding of urocanic acid to polynucleic acids. Chem.-Biol. Interact. 75, 105-1 18.

33. Farrow. S. J . , C. R. Jones. D. L. Severance. R. M. Deibel, W. M. Baird and H. Morrison (1990) Urocanic acid photobiology. Identification and characterization of the major photoadducts formed between urocanic acid and thymidine. J. Org. Chem. 55, 275-282.

34. Krien, P. M. and D. Moyal (1994) Sunscreens with broad- spectrum absorption decrease the trans to cis photoisomerization of urocanic acid in the human strutum corneum after multiple UV light exposures. Phorochern. Photobiol. 60, 280-287.

35. Hemelaar, P. J. and G. M. J. Beijersbergen van Henegouwen ( 1996) The protective effect of N-acetylcysteine on UVB-induced immunosuppression by inhibition of the action of cis- urocanic acid. Photochem. Phorobiol. 63, 322-327.

36. Jones. C . D., A. K. Barton. J. Crosby, M. Norval and N. K. Gibbs (1996) Investigating the red shift between in vitro and in ijivo urocanic acid photoisomerization action spectra. Pho- tochem. Photobiol. 63, 302-305.

37. Gruner, S., W. Diezel. H. Stoppe, H. Oesterwitz and W. Henke (1992) Inhibition of skin allograft rejection and acute graft- host disease by cis-urocanic acid. J. Invest. Dermutol. 98,459- 462.

38. Morrison, H., D. Avnir and T. Zarrella (1980) Analysis of Z

and E isomers of urocanic acid by high-performance liquid chromatography. J. Chromatogr. 183, 83-86.

39. De Orsi. D., L. Gagliardi, F. Chimenti and D. Tonelli ( 1995) High-performance liquid chromatographic determination of urocanic acid isomers in cosmetic products. ~ t i r o } i i ~ ~ t o ~ ~ r ~ ~ ~ / i i ( ~

40. Takahashi, M. and T. Tezuka (1997) Quantitative analysis of histidine and cis and trans isomers of urocanic acid by high- performance liquid chromatography: a new assay method and its application. J. Chroniatogr. B 688, 197-203.

41. Kammeyer. A., S. Pavel, S. S. Asghar. J. D. Bos and M. B. M. Teunissen (1997) Prolonged increase of cis-urocanic acid levels in human skin and urine after single total-body ultraviolet exposures. Photochem. Photohiol. 65. 593-598.

42. Simmonds, S. W. (1987) Studies of the photochemical inactivation of double stranded-viral DNA in the presence of urocanic acid. M.S. Dissertation, Purdue University, West Lafay- ette, IN.

43. Pinkerton, T. C. , T. D. Miller. S. E. Cook, J. A. Perry. J. D. Rateike and T. 3. Szczerba (1986) The nature and use of internal surface reversed-phase columns: a new concept in high performance liquid chromatography. Biochr-ornutognr/~h~ 1.

44. Mohammad, T. and H. Morrison (1990) Rapid analysis of small molecules in the presence of DNA by high-performance liquid chromatography using internal surface reversed-phase silica. J. Chroniatogr. B 533. 195-200.

45. Mohammad, T. and H. Morrison (1997) Simultaneous determination of methylene violet, halogenated methylene violet and their photoproducts in the presence of DNA by high-performance liquid chromatography using an internal surface reversed-phase column. J . Chromatogr. B 704, 265-275.

46. Moodycliffe, A. M., M. Norval, I. Kimber and T. J. Simpson (1993) Characterization of a monoclonal antibody to cis-urocanic acid: detection of cis-urocanic acid in the serum of irradiated mice by immunoassay. Inimirnology 79. 667-672.

47. Hasegawa, H. (1960) Photolysis of urocanic acid. Nngcisuki Igukkai Zasshi 35, 1669-1676; c$ Chem. Ahstr. 1961. 55. 95 19h.

48. Farrow, S. J. (1988) The photochemistry and photobiology of urocanic acid and indoleacrylic acid. Ph.D. Dissertation, Pur- due University, West Lafayette, IN.

49. Morrison. H. and R. M. Deibel (1988) Urocanic acid photobiology. Photooxidation and superoxide formation. Photo- chern. Photobiol. 48, 153-156.

50. Anglin, J. H., Jr. and W. H. Batten ( 1970) Structure of urocanic acid photodimers. Photochem. Photohiol. 11. 27 1-277.

51. D’Auria, M. and R. Racioppi (1998) Photochemical dimerization of esters of urocanic acid. J. Phorochern. Photohiol. A Chem. 112, 145-148.

52. Baden, H. P. and M. A. Pathak (1967) The metabolism and function of urocanic acid in skin. J. Invest. Derrnurol. 48, I I - 17.

53. Morrison, H.. D. Avnir. C. Bernasconi and G. Fagan (1980) Z/E photoisomerization of urocanic acid. Photoche~n. Photo- biol. 32, 71 1-714.

54. McGrath. H.. Jr.. J. M. Bell and J. W. Haycock (1994) Fluo- rescent light activates the immunomodulator cis-urocanic acid in vitro: implications for patients with systemic lupus erythematosus. Ann. Rheum. Dis. 53. 396-399.

55. Rihner. M. and H. McGrath (1992) Fluorescent light photo- sensitivity in patients with systemic lupus erythematosus. Ar- thritis Rheum. 35, 949-952.

56. El-Ghorr, A. A. and M. Norval (1997) Biological effects of narrow-band (311 nm TLOl) UVB irradiation: a review. J. Photochem. Photobiol. B Biol. 38, 99-1 06.

57. Gibbs. N. K., M. Norval, N. J. Traynor, J. C. Crosby. G. Lowe and B. E. Johnson (1993) Comparative potency of broad-band and narrow-band phototherapy to induce edema, sunburn cells and urocanic acid photoisomerization in hairless mouse skin. Photochem. Photobiol. 58. 643-647.

58. Mohammad. T., I. Tessman. H. Morrison, M. A. Kennedy and S. W. Simmonds (1994) Photosensitized inactivation of infec-

41, 370-372.

96-1 05.


trous DNA by urocanic acid. indoleacrylic acid and rhodium complexes. Photochem. P hotohiol. 59. 189- 196.

59. Morrison, H.. C. Bernasconi and G. Pandey (1984) A wavelength elfect on urocanic acid pliotoisomerizatioii. Phoroclierii. Photobiol. 40. 5-19-550.

60. Ullman, E. F.. E. Babad and M. Sung (1969) Wavelength- dependent photochemistry of u.P-unsaturated carboxylate aii- ions. J . Am. Clicni. Soc. 91, 5791-5797.

61. Saltiel. J . . D. F;. Sears. Jr.. Y.-P. Sun and J . -0 . Choi (1991) Evidence for ground-state s-c.;.~ conformers in the fluorescence spectra of alI-trat i , i - 1 .h-diphciiyl- 1.3.5-hexatriene, J . Am. Chum. SOC. 114. 3607-36 13.

63. Wallace-Williams, S . E.. S . hloller. R . A. Goldbeck. K. M. Hanson. I. W. Lewis. W. A. Yee and D. S . Kliger (1993) Excited-state s -c i s retainers produced by extreme edge excitation of all-tr[ifr\- 1.4-diphcnyl- I .3-hutadiene. J . Phys. Clievi. 97. 9587-9592.

63. Shuhla. M. K . and P. C. Misra ( 1995) Electronic spectra. structure and ph~,toi~omerizatioii of urocanic acid. Spwtrochiiii. AC.If/ 51A. 831 -X.38.

hl. Lahti. A,. M. Hotokha. K. Ncuvonen and P. Ayras (1995) A theoretical \tud> of the conformers of f rc im- and cis-urocanic acid. J . Mcil. .Strrrc,t. f Tlicwcl ir t t i . 1 331. 169-1 79.

6s. Li. B.. K. M. Hnn\on and J . D. Simon (1997) Primary processes of the electronic excited states of /rmA-urocanic acid.

66. Lamola. A. .4. ( 1968) Applications of electronic energy traiis- ter i n mlution. Phoroc~hrrii. Phorobio/. 8. 601-616.

67. Lamola. A. A. ( 1966) Molecular mechanisms in nucleic acid photochemi\try. 1. Sen\itired photochemical splitting of thymine dimer. .I. Am. C11cvti. So(.. 88. 813-819.

68. Murov. S. I . . . I. Carmichael and G. L. Hug (1993) Htiritlbod vf P/rotoc.lic,.rii.sirr?.. Marcel Dekker, New York.

69. Morrison. H.. C . Berna\coni and G. Pandey (1983) Urocanic acid photohiology. Photocycloaddition of N.N-dimethylthymine to urocanic acid. P / i o r o ~ ~ / i ~ ~ i 1 1 . Photohiol. 38. 23-27.

70. Vipny. P. and M. Duquesne i 1976) On the fluorescence prop- crtie5 of nucleotide\ and polynucleotides at room temperature. I n E.\rired Stcife 5 of HioloCyicrri hlolrcirles. Proceedin,q.s of (111

Iiirermrriomil Cor!frrrricr (Edited by J. 8 . Birks). pp. 167-177. John Wiley & Sons. N e w York.

71. Oraevshy. A. A,. A. V. Sharkob and D. N. Nikogosyan (1981) Picosecond study o f electronically excited singlet states of nucleic acid components. Clrrm. P/iy.s. Lerr. 83. 376-280.

72. Nikogosyan. D. N.. D. A. Angelov and A. A. Oraevsky (1983) Determination o f parameters of excited states of DNA and KNA bases hy laser LJV photolysis. P h ~ ~ t n c - h e ~ . Phorohiol. 35. 627-635.

73. Kleopfer. R . and H. Morrison (1972) Organic photochemistry. XVII. The solution-phase photodimerization of dimethylthymine. J . A m C l i ~ i . So<.. 94. 155-364.

74. Otten. J. G.. C. S. Yeh. S . Byrn and H. Morrison (1977) So- lution phase photodiiiirrization of tetramethyluracil. Further ctudies on the photochemistry of ground-state aggregates. J. h i . Chem. .%K. 99. 6353-6359.

75. Kodighiero. G.. F. Dall'Acqua and D. Averbeck (1988) New psoralen and angelicin derivatives. In P.wrci/e~i DNA Photo- hidOgI', Vol. I (Edited hy F. P. Gasparro). pp. 37-1 14. CRC Press. Boca Raton. FL.

76. Gibbs. N. K.. G . Toir and B. E. Johnson ( 1996) Evidence that certain phototoxic drugs photosensitize urocanic acid isomer-

77. Craw. M.. R. V. Bensasson. J . C. Ronfard-Haret, M. T. SaeMelo and T. G. Truscott ( 1983) Some photophysical prop- ertie\ of 3~carhcthosypsoralen. 8-niethoxypsoralen and 5- metlioxypsorslcii triplet states. Phcirocliem. Photobiol. 37.

78. Poppe. W. and 1.. I>. Grosaweiiier i 1975) Photodynamic sen- 5itiLation bj, 8-methoxypsoralen I , ~ L / singlet oxygen niecha- nism. P/iotoc.liftrr. P l i ~ t ~ ~ h i ~ l . 22. 2 17-219.

79. Kpissay. A,. C . N. Kuhl. T. Mohaniniad. K. Habei- and H. Morrison i lW7) Evidence for azidyl radical initiated olefin isomci-ization. One-way isonierization of (Z)-urocanic acid. Tr.truhcdrori Lrtt . 38, 8435-8438.

.I. Phy.v. ChOl7l. '4 101, 969-971

ization. J . ~ h V ~ ~ K ' h ~ ' 7 i i . P/io/(JhiO/. B RiV/. 34, 63-66.

6 1 1-615.

80. Hug, D. H. and J. K. Hunter (1995) Microorganisms from skin metabolize cis-urocanic acid. Photocheni. P/zotobiul. 61, 2 IS. [Abstract]

81. Schwarz. W.. H. Schell, G. Huettinger, H. Wasmeier and T. Diepgen ( 1987) Effects of UVA on human strcrficni corrierrrii histidine and urocanic acid isomers. Pliorodentintolog~~ 4. 269- 271.

81. Olivarius, F. de Fine, H. C. Wulf, J. Crosby and M. Norval i 1997) Seasonal variation in urocanic acid isomers in human shin. Pliotocher~. Plrorohiol. 66. 119-123.

83. van der Molen, R. G.. C . Out-Luiting, A. M. Weerheim, H. K. Koerten and A. M. Mommaas (1998) Efficacy of sunscreem in protection against UV-induced isomerization of urocanic acid in human skin. Photochmi. Photohiol. 67, 7 IS. [Abstract]

84. Gibbs, N. K., M. Norval. N. J. Traynor, M. Wolf, B. E. John- son and J . Crosby (1993) Action spectra for the froi is to cis photoisomerization of urocanic acid in v i r ro and in mouse skin. Photocherti. Phorobiol. 57. 584-590; correction. Photoclieni. Phorohiol. ( 1993) 58, 769.

85. Laihia. J. K. and C. T. JansCn ( 1994) Urocanic acid photocon- version in relation to erythematogenicity of radiation from different types of phototherapy equipment. Phorodenrintol. Pho-

86. Kamrneyer. A,, M. B. M. Teunissen, S. Pawl. M. A. De Rie and J . D. Bos (1995) Photoisomcrization spectrum of urocanic acid in human skin and in 1.itr-o: effects of simulated solar and artificial ultraviolet radiation. Br. J. Dermutol. 132, 884-89 I .

87. Hanson. K. M. and J. D. Simon (1997) The photochemical isomerization kinetics of urocanic acid and their effects upon the iji 1,irro and i n viiu photoisoincrization action spectra. Pho- r ~ h ~ i i . Pfiofobiol. 66, 8 17-820: correction. Photocl?e~i. Plio- tobiol. (1998) 67, 473.

88. Morrison. H., B. Mauclair, R. M. Deibel, G. Pandey and W. M. Baird ( 1985) Urocanic acid photobiology. Photochemical hinding to calf-thymus DNA. Photochrrii. Photohiol. 41, 25 1- 257.

89. De Fabo. E. C . and F. P. Noonan ( 1992) Urocanic acid, photoimmunology and ozone depletion: human health implications. In Biologicul Effects oj Lighr. Proceediizg.r of LI Syni- posiitrii (Edited by M. F. Holick and A. M. Kligman). pp. 387- 396. de Gruyter. Berlin, Germany.

90. Young. A. R. ( 1997) Chromophores in human skin. Phys. Med. B id . 42. 789-802.

91. Garssen. J . , M. Norval, A. El-Ghorr, N. K. Gibbs. C . D. Jones. D. Cerimele C. De Simone. S. Caffieri, F. Dall'Acqua, F. R. De Gruijl, Y. Sontag and H. Van Loveren (1998) Estimation of the effect of increasing UVB exposure on the human immune system and related resistance to infectious diseases and tumours. J . Pliotoclieni. Photobiol. B Riol. 42, 167-1 79.

92. Tessman. I.. H. Morrison. C. Bernasconi, G. Pandey and L. Ekanayake ( 1983) Photochemical inactivation of single-stranded viral DNA in the presence of urocanic acid. Photochetn.

93. Patrick. M. H. and R. 0. Rahn ( 1976) Photochemistry of DNA and polynucleotides: photoproducts. In Photochemistn/ cirit l Photobiology qf Nircleic Acids, Vol. I1 (Edited by S. Y. Wang). pp. 35-95. Academic Press, New York.

94. Sancar. A. (1994) Structure and function of DNA photolyase. Biochrrnistn 33. 2-9.

95. Terrian, D. L.. C. N. Kuhl. I . Tessman and H. Morrison i 1996) On the role of urocanic acid in photoimmunosuppression: at- tempted photorepair of urocanic acid-DNA cyclobutane adducts with DNA photolyase. Plrorochem. Phorobiol. 63, 898- 900.

96. Tessman. I. (1990) SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so. .I. Rrrcteriol. 172, 5503-5505.

97. Lias. S. G.. J. E. Bartrness. J . F. Liebman. J . L. Holmes. R. D. Levin and W. G. Mallard (1988) Gas-phase ion and neutral thermochemistry. J. P/?!s. Chrnz. Ref: Durn 17, iSuppl. 1 ) . 388.

98. Sinsheimer. R. L.. B. Starman, C. Nagler and G . Guthrie (1962) The process of infection with bacteriophage +X174. I. Evidence for a "replicative form." 1. Mol. Biol. 4, 142-160.

99. Tessman. 1.. S.-K. Liu and M. A. Kennedy (1992) Mechanism

t ~ i t ~ i ~ i ~ ~ d . P hoto/?i~d. 10. 13-1 6.

PhVrVfll~J/. 38, 29-35.


of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine. Proc. Natl. Acad. Sci. USA 89, 1159-1163.

100. Tessman, I. (1992) Bypass of UV lesions by E. coli DNA polymerase in vivo. Photochem. Photobiol. 55, 80s. [Abstract]

101. Sancar, G. B., F. W. Smith, R. Reid, G. Payne, M. Levy and A. Sancar (1987) Action mechanism of Esrherichia coli DNA photolyase. 1. Formation of the enzyme-substrate complex. J. B id . Chem. 262, 478-485.

102. Tessman, I. and M. A. Kennedy (1991) The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutanc dimers as the likely source of the mutations associated with photoreactivation. Mol. Gett. Genet. 227, 144-148.

103. Armitage, B. (1998) Photocleavage of nucleic acids. Chem. Ret,. 98. 1171-1200.

104. Paillous, N. and P. Vicendo (1993) Mechanisms of photosensitized DNA cleavage. J. Photochem. Photobiol. B Biol. 20, 203-209.

105. Kochevar, I. E. and D. A. Dunn (1990) Photosensitized reactions of DNA: cleavage and addition. In Bioorganic Photo- chemistry, Vol. I (Edited by H. Morrison), pp. 273-315. John Wiley & Sons. New York.

106. Stubbe. J., J . W. Kozarich, W. Wu and D. E. Vanderwall (1996) Bleomycins: a structural model for specificity, binding, and double strand cleavage. Ace. Chem. Res. 29, 322-330.

107. Begley. T. (1994) Photoenzymes: a novel class of biological catalysts. Arc. Chern. Res. 27, 394-401.

108. Dandliker, P. J., M. E. Nliiiez and J. K. Barton (1998) Oxi- dative charge transfer to repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metal- lointercalators. Biochemistni 37, 6491-6502.

109. Hartzfeld, D. G. and S. D. Rose (1993) Efficient pyrimidine dimer radical anion splitting in low polarity solvents. J. Am. Chem. Soc. 115, 850-854.

110. Heelis. P. F.. R. F. Hartman and S. D. Rose (1993) Detection of radical ion intermediates in flavin-photosensitized pyrimidine dimer splitting. Photochent. Photobiol. 57, 442-446.

11 I . Yang, D.-Y. and T. P. Begley (1993) Mechanistic studies on DNA photolyase VIII: studies on the fragmentation of the radical anion and cation of a uracil-alkene photoadduct. Tetra- hedron Lett. 34. 1709-1712.

112. Pouwels, P. J. W., R. F. Hartman, S. D. Rose and R. Kaptein ( 1994) CIDNP evidence for reversibility of the photosensitized splitting of pyrimidine dimers. J. Am.-Chem. Soc. 116, 6967- 6968.

113. Pouwels, P. J. W.. R. F. Hartman, S. D. Rose and R. Kaptein ( 1 995) Photo-CIDNP study of pyrimidine dimer splitting I: reactions involving pyrimidine radical cation intermediates. Photochern. Photobiol. 61, 563-574.

114. Pouwels, P. J. W., R. F. Hartman, S. D. Rose and R. Kaptein ( 1995) Photo-CIDNP study of pyrimidine dimer splitting 11: reactions involving pyrimidine radical anion intermediates. Photocheni. Photobiol. 61, 575-583.

1 15. Brancaleon. L.. D. Brousmiche, V. Jayathirtha Rao, L. J. John- ston and V. Ramamurthy (1998) Photoinduced electron transfer reactions within zeolites: detection of radical cations and dimerization of arylalkenes. J . Am. Chem. Soc. 120, 4926- 4933.

116. Reeve. V. E., G. E. Greenoak. P. J. Canfield. C. Boehm-Wil- cox and C. H. Gallagher (1 989) Topical urocanic acid enhanc- es UV-induced tumor yield and malignancy in the hairless mouse. Photochem. Phorobiol. 49, 459-464.

117. Cleaver, J. and G. Thomas (1981) Measurement of unscheduled synthesis by autoradiography. In DNA Repair: A Labo- rutor?, Manual of Research Procedures, Vol. 1B (Edited by E. C. Friedberg and P. Hanawalt). pp. 277-287. Marcel Dekker, New York.

118. IJland, S. A. J., F. P. Noonan, S. Ceryak, D. P. T. Steenvoor- den, B. Bouscarel, D. Hug, 0. M. J. Beijersbergen van He- negouwen and E. C . De Fabo (1998) Urocanic acid does not photobind to DNA in mice irradiated with immunosuppressive doses of UVB. Photochein. Photobiol. 67, 222-226.

119. Farrow, S. J., T. Mohammad. W. Baird and H. Morrison

(1990) Photolytic covalent binding of indoleacrylic acid to DNA. Photochem. Photobiol. 51. 263-27 I .

120. Mohammad, T., W. M. Baird and H. Morrison (1991) Photo- chemical covalent binding of p-methoxycinnamic acid to calf thymus DNA. Bioorg. Chem. 19, 88-100.

121. Reddy, M. V. and K. Randerath (1986) Nuclease P,-mediated enhancement of sensitivity of 32P-postlabeling test for struc- turally diverse DNA adducts. C~zrcinogenesis 7, 1543- 1548.

22. Herbert. M. A.. J. C. LeBlanc. D. Weinblum and H. E. Johns ( 1969) Properties of thymine dimers. Photochrm. Photobiol. 9, 33-43.

23. Kanne, D., K. Straub, J. E. Hearst and H. Rapoport (1981) Isolation and characterization of pyrimidine-psoralen-pyrim- dine photoadducts from DNA. J. Am. Chem. Soc. 104. 6754- 6764.

24. Kripke. M. L.. P. A. Cox, L. G. Alas and D. B. Yarosh (1992) Pyrimidine dimers in DNA initiate systemic immunosupprrs- sion in UV-irradiated mice. Proc. N d . Acad. Sci. USA 89.

125. Kim, S.-T. and A. Sancar (1991) Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase. Bio- chemistry 30, 8623-8630.

126. Ou. C. N., C. W. Tsai. K. J. Tapley, Jr. and P.-S. Song (1978) Photobinding of 8-methoxypsoralen and 5,6-dimethoxycou- marin to DNA and its effect on template activity. Bioclternisrr~ 17, 1047-1053.

127. Maleski, R. and H. Morrison (1972) Organic photochemistry. XXI. Photocycloaddition of 1.3-dimethylthymine to in situ generated ethylene. Mol. Photoehenr 4. 55-59.

128. Wexler. A. J., J. A. Hyatt. P. W. Raynolds, C. Cottrell and J. S. Swenton (1978) A comparison of the photoaddition reactions of nucleic acid nitrogen bases and cyclohexenones with isobutylene. The role of rigidity in product formation. J. Am. Chem. Soc. 100. 512-520.

129. Kellogg, R. E. and W. T. Simpson (1965) Perturbation ofsiti- glet-singlet transition energies. J. Am. Chem. Soc. 87. 4230- 4234.

130. Kasper. A. M. (1991) The photochemistry of urocanic acid with 2'-deoxyadenosine and a study of arylkster orbital inter- actions in the excited state. Ph.D. Dissertation, Purdue Uni- versity, West Lafayette, IN.

131. Peterkofsky, A. (1962) The mechanism of action of histidase: aminoenzyme formation and partial reactions. J. Biol. Chem.

132. Kraml, M. and L. P. Bouthillier (1955) The conversion of urocanic acid to glutamic acid in the intact rat. Can. .I. Biocheni.

133. Wood, P. D. and L. J. Johnston (1997) Generation and characterization of psoralen radical cations. Photocheni. Photobiol. 66, 642-648.

134. Deibel, R. M., H. Morrison and W. M. Baird (1987) Urocanic acid photobiology. Photochemical binding of urocanic acid to bovine serum albumin. Phorochem. Phorobiol. 45. 42 1-423.

135. Yoshikawa. K., N. Mori, S. Sakakibara. N. Mizuno and P.-S. Song ( 1979) Photoconjugation of 8-methoxypsoralen with proteins. Phorochem. Photobiol. 29, 1 127-1 133.

136. Sinha, B. K. and C. F. Chignell (1983) Binding of anthracene to cellular macromolecules in the presence of light. Photo- chem. Photobiol. 37, 33-37.

137. Busker, R. W.. G. M. J. Beijersbergen van Henegouwen, G. J. H. Vaassen and R. F. Menke (1989) Irreversible photobinding of nitrofurantoin and nitrofurfural to plasma proteins in titro. J. Photochem. Photobiol. B Biol. 4. 207-2 18.

138. Kochevar. I. E. and L. C. Harber (1977) Photoreactions of 3,3',4'.5-tetrachlorosalicylanilide with proteins. 1. Invest. Der- matol. 68, 151-156.

139. Clive, D., R. McCuen, J. F. S. Spector, C. Piper and K. H. Mavournin (1983) Specific gene mutations in LS178Y cells in culture. Mutat. Res. 115. 225-25 1.

140. Burobin, V. A,, 0. V. Ponomareva, T. G . Nikolaeva and N. Y. Yurchenko (1985) Biological activity of urocanic acid. Vopr. Med. Khitn. 31, 102-106; c$ Chem. Abs. 102, 1601 19y.

141. Reeve, V. E., M. Bosnic, W. G. Reilly and R. D. Ley (1991)

75 16-7520.

237, 787-795.

Physiol. 33, 590-598.

134 Taj Moharnrnad et a/.

Urocanic acid photobiology i n thc hairless mouse. Photochcw1. Photobi~~l . 53. 88s. [Abitract]

1-12, Bishop. S. M.. M. Malone. D. Phillips. A. W. Parker and M. C. R. Symons ( 1994) Singlet oxygen sensitimtion by excited state DNA. J . Cheiti. .So(,. Chriii. Coi i i i r i tui . , 87 1-872.

143. Mohammad. T. rind H. Morrison ( 1996) Evidence for the photosensitized formation of singlet oxygen by UVB irradiation of 2'-deoxyguanosine 5'-monophosphate. J. Aim Chriir. Sol.. 118, 1221-1222.

144. Hanson, K. M. and 1. D. Simon ( 1998) The origin o f the wavelength-dependent photoreactivity o f trctii v-urocanic acid. Pho-

145. Wilkinson. F. and J . G. Brununer (1981) Rate constants for the decay and reactions of the lowest electronically excited singlet state of molecular oxygen in rolution. ./. Phys. C'hcw. Rej: Drrtu 10, 809-999.

1-16, Hanson. K. M. and J . I). Simon i 1998) Epidermal ti-m.v-urocanic acid and the I-&-A-induced photoaging of skin. Proc. Nut/. Acrrd. sci. USA 95. 10576-10.578.

147. Norval. M.. T. J. Simpson. E. Bardshiri and S. E. M. Howie (1989) Urocanic acid analogues and the suppression of the delayed type hypersensitivity response to Herpes sitiydex virus. P horoc.lwni. P l t o r d ~ i o l , 19. 633-639.

148. Houghtaling, M. A, . M. A . Kennedy. T. Mohaminad. 1. Tess- inan and H. Morrison ( 1996) Photochemistry and photobiology of iniidazole-4(5)-methylidene malonic acid: an analog of both E- and Z-urocanic acid. Phon~heit l . Photobiol. 64, 7 16-2 19.

149. 1,ewis. F. D. and A. B. Yoon (1995) The influence of intramolecular hydrogen bonding on the structure and E H Z photoisomerization of iirociinic acid derivati\es. Res. Chrtii. Irrter- tried. 21. 749-763.

150. Franceschi, S. . \?. Andreu, N. de Viguerie. M. Riviere. A. Lat- tes and A. Moiwnd (1998) Synthesis and aggregation beha\.- iour 0 1 two-headed surfactant\ containing the urocanic acid moiety. N t w . .I. <'hem., 2 - 2 3 I .

151. Kripke. M. L. ( 1998) PreGdent's lecture: photoimniunology- a personal odyssey. Pliorocheii~. Pho toh id . 67. 83s. [Abstract]

1.53. ROSS. J . A.. S. E. Houie, M. Norval. J. Maingay and T. J. Simpson ( Ic)861 Ultraviolet-irradiated urocanic acid suppresses dclayed-type hypersensitivity t o herpes simplex virus in mice. .I. / i i iw\ t . Dri-irrrrtol. 87. 6 3 6 6 3 3 .

153. Guyniei-. R. H. iind T. E. Mandel (1993) Urocanic acid as an immunosuppressant in allotransplantation in mice. Trtriisp/tti~-

154. Gieseler. R K.. F. Klemp. M. Brcdt. R. Mentlein. R. Kuhn. B. von Gaudecber. J . H. Peters and R. Schlcmminger (1994) Prolongation of survival of small bowel transplant recipients after treatment with cis-urocanic acid. Troti.vp/ctiit. Proc. 26. I601 - 1 603.

159. Filipec. M., E. I.etl\o. %. Haskova. D. Jenickova, P. Holler. A. Jancarek and V. Holan i 1998) The effect of urocanic acid on graft rqjection i n an experimental model of orthotopic corneal transplantation in rabhits. Grwfi.s Arch. Cliii. E.rp. Ophthol- tiiol. 236. 65-68.

156. Noonan, F. P.. E. C. De Fabo and H. Morrison (1988) Ci.\- urocanic acid. a product formed by ultraviolet B irradiation of the skin, initiates an antigen presentation defect in splenic dendritic cells i n vivo. ./. Iiiivst. Do.iirtrro1. 90. 92-99.

157. Lappin, M. B.. .J. M. Weisa. E. Scopf. M. Norval and J . C. Simon (1997) Physiologic doses of urocanic acid do not alter the allostiniulatory lunction or the development of murine dendritic cells in vitro. P ~ i ( i / ( j ( l ( , i - i i i c t t [ j / . P/iotoiiiiiiirii~ol. Pliotoiirerl. 13. 163-168.

158. Hurks. H. M.. <.'. Out-Luiting. R. G. Van den Molen. B. J . Vernicer. 1:. I f . C'laa\ a n d A. M. Mommaas (1997) Differential \uppression of the human mixed epidermal cell lymphocyte reaction (MEC1.R) and mixed lymphocyte reaction (MLR) by c.i.\-urocanic acid. Photochciiz. Photohiol. 65. 6 16-62 1 .

1.59. Rattis. F.-M.. J . Peguet-Navarro, P. Courtellemsnt, G. Redzin- iak and D. Schrnitt ( 1995) c.i.\--Urocanic acid failed to affect i i l

r i r i - o human Langerhans cell allostimulatory function. P / I ( J / w chrt?i. Photohtol. 62. 9 14-9 16.

160. Dai. R. and J . W. Streilein (1997) Liltraviolet B-exposed and roluble factor-pre-incubated epidermal Langerhans cells fail

toc,hei?f. Photobid. 67. 538-540.

tfft;<Jll 55. 3 6 4 3 .

to induce contact hypersensitivity and promote DNP-specific tolerance. J . Inivst. Deriiirrtol. 108. 721-726.

161. Beissert. S., T. Mohammad, H. Torri, A. Lonati, Z . Yan. H. Morrison and R. D. Granstein ( 1997) Regulation of tumor antigen presentation by urocanic acid. J. Inlmunol. 159, 92-96.

162. Redondo. P., J. Garcia-Foncillas. F. Cuevillas. A. Espana and E. Quintanilla ( 1996) Effects of low concentrations of cis- and trans-urocanic acid on cytokine elaboration by keratinocytes.

163. Bacci. S., T. Nakamura and J. W. Streilcin (1996) Failed antigen presentation after UVB radiation correlates with modifi- cations of Langerhans cell cytoskeleton. J . Irivest. Deriiiurol. 107, 838-813.

164. Norval, M.. J . W. Gilmour and T. J . Simpson (1990) The effect of histamine receptor antagonists on immunosuppression induced by the cis-isomer of urocanic acid. Phototleriricltol. Pho- toiirinritiiol. Photonied. 7. 243-248.

165. Hart, P. H.. C. A. Jones, K. L. Jones. C. J. Watson, 1. Santucci. L. K. Spencer and J . J . Finlay Jones (1993) Cis-urocanic acid stimulates human peripheral hlood monocyte prostaglandin E, production and suppresses indirectly tumor necrosis factor-alpha levels. J . Ii i i i?~itno/. 150, 45 144523.

166. Jaksic. A, , J . J . Finlay Jones, C . .I. Watson, L. K. Spencer. I. Santucci and P. H. Hart (1995) Cis-urocanic acid synergizes with histamine for increased PGEL production by human keratinocytes: link to indomethacin-inhibitable UVB-induced immunosuppression. Photochetn. Photobiol. 61. 303-309.

67. Laihia, J . K.. M. Attila. K. Neuvonen, P. Pasanen, L. Tuomisto and C. T. Jansen (1997) Binding of urocanic acid to GABA,. but not to histamine H I receptors. J . Iiiivst. Dcwnntol. 107. 664. [Abstract]

68. Hart. P. H.. A. Jaksic. G. Swift, M. Norval, A. A. El-Ghorr and J. J . Finlay Jones (1997) Histamine involvement in UVB- and c.is-urocanic acid-induced systemic suppression of contact hypersensitivity responses. Ii?zi~iuiio/ogy 9 1, 601-608.

69. Wille. J. J. and A. Kydonieus (1995) Abrogation of contact hypersensitivity in mice by topically-applied mast cell degran- ulating agents. J . /iii>es/. Drritiotol. 104. 679. [Abstract]

170. Malaviya. R.. A. R. Morrison and A. P. Pentland (1996) His- tamine in human epidermal cells is induced by ultraviolet light injury. J . Itii.c~s/. Derimtol. 106, 785-789.

171. Jun, B. D.. L. K. Roberts. B. H. Cho, B. Robertson and R. A. Dayncs ( 1988) Parallel recovery of epidermal antigen-presenting cell activity and contact hypersensitivity responses in mice exposed to ultraviolet irradiation: the role of a prostaglandin- dependent mechanism. J . Itwest. Dermntol. 90, 3 1 1-3 16.

172. Shreedhar. V.. T. Giese. V. W. Sung and S. E. Ullrich (1998) A cytokine cascade including prostaglandin E,. IL-4, and IL- 10 is responsible for UV-induced systemic immune suppression. J . Inlitwiol. 160, 3783-3789.

173. Enk, A. H., V. L. Angeloni. M. C. Udey and S. I. Katr (1993) Inhibition of Langerhans cell antigen-presenting function by

174. Beissert. S.. J . Hosoi, S. Grabbc. A. Asahina and R. 1). Cran- stein (1995) IL-I0 inhibits tumor antigen presentation by epidermal antigen-presenting cells. J. Iini?ii/ii~~/. 54, 1280-1286.

175. Van der Pouw Kraan. T. C. T. M., L. C. M. Boeije, R. J. T. Smeenk. J . Wijdenes and L. A. Aarden ( 1995) Prostaglandin- E2 is a potent inhibitor of human interleukin 12 production. ./. E.\-p. Med. 181, 775-779.

176. Muller. G. , J. Saloga. T. Germann, G. Schulcr, J. Knop and A. H. Enk (1995) IL-I2 as mediator and adjuvant for the induction of contact sensitivity in i%itw. J . Iini?iitnol. 155, 4661-4668.

177. Reeve. V. E.. M. Bosnic and N. Nishimura (1998) Role of interferon-y in protection by UVA (320-400 nm) radiation against UVB (280-320 nm)-induced immunosuppression. Pho- tochriri. Photobio~. 67, 16s. [Abstract]

78. Rivas. J. M. and S. E. Ullrich (1994) The role of IL-4. IL-10, and TNF-CY in the immune suppression induced by ultraviolet radiation. J . Leitkocyte B i d . 56. 769-775.

79. Kurimoto. I . and J. W. Streilein ( 1992) C'i.7-urocanic acid suppression of contact hypersensitivity induction is mediated via tumor necrosis factor-alpha. J . Itiiiiiutml. 148, 3072-3078.

80. El-Ghorr. A. A. and M. Norval (1997) The effect of chronic

P / f ~ J / ( ~ t l f ~ t t l l l f o / . P h O / ~ i l ? / l ? l ~ l i l ~ ~ . P h O t ~ l l l ~ d . 12, 237-243.

1L- 10. J . I t i i ~ ~ t t i z o l . 151. 2390-2398.


treatment of mice with urocanic acid isomers. Photochern. Photobiol. 65. 866-872.

181. Olivarius, F. D. F., J. Lock-Andersen, F. G . Larsen, H. C. Wulf. J. Crosby and M. Norval ( 1 998) Urocanic acid isomers in patients with basal cell carcinoma and cutaneous malignant melanoma. Br. J . Dermurol. 138, 986-992.

182. Donawho, C. K.. M. Norval and D. B. Yarosh (1998) Molec- ular target of UV-B radiation in the melanoma enhancing effect of UV irradiation in a murine model. Phorocheni. Photo- bid. 67, 64s. [Abstract]

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