Harry Morrison

IL-12 Prevents the Inhibitory Effects of cis-Urocanic Acid on Tumor Antigen Presentation by Langerhans Cells: Implications for Photocarcinogenesis

UV radiation induces skin cancer primarily by its DNA-damaging properties, but also by its capacity to suppress the immune system. The photoisomer of urocanic acid (UCA), cis-UCA, is an important mediator of UV-induced immunosuppression and is involved in the inhibition of tumor immunity. The immunomodulatory cytokine IL-12 is known to counteract many of the immunosuppressive effects of UV radiation, including UV-induced immune tolerance. In this study, we addressed whether IL-12 also reverts the immunosuppressive activities of cis-UCA. Cis-UCA inhibits the ability of Langerhans cells to present tumor Ags for primary and secondary tumor immune responses. IL-12 treatment completely prevented the suppression by cis-UCA. IL-12 also protected mice from cis-UCA-induced suppression of contact hypersensitivity responses. To study the effects of cis-UCA on Ag-processing and Ag-presenting function in vitro, Langerhans cells were treated with UCA isomers and incubated with OVA or OVA peptide323–339 before exposure to OVA-specific transgenic T cells. Cis-, but not trans-UCA suppressed Ag presentation, which was completely reversed upon addition of IL-12. Since these findings suggest that cis-UCA may play an important role in photocarcinogenesis by inhibiting a tumor immune response, mice were chronically UVB irradiated to induce skin cancer. Whereas all mice in the control groups developed tumors, mice treated with a mAb with specificity for cis-UCA showed a significantly reduced tumor incidence. These data strongly indicate the importance of cis-UCA during photocarcinogenesis and support the concept of counteracting cis-UCA as an alternative strategy to prevent UV-induced skin cancer, possibly via the application of IL-12.

Z/E PHOTOISOMERIZATION OF UROCANIC ACID*

The E ⇆ Z photoisomerization of the title compound (UA) (a naturally occurring sunscreen) has been studied in aqueous solution. At a UA concentration of 6mM and using 313nm excitation, φE→z= 0.52, φZ→E= 0.47 and the photostationary state is 34% E. Under these conditions, loss of UA is minimal. Low energy triplet quenchers fail to impede the isomerization, but the reaction can be induced by several triplet sensitizers. The ET for UA is estimated to be approximately 55 kcal/mol.

Urocanic Acid Photochemistry and Photobiology

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, photochemists, photobiologists, medicinal chemists and immunologists. 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 explosive 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 molecules photobiology, photochemistry and photophysics have appeared (4-

A WAVELENGTH EFFECT ON UROCANIC ACID E/Z PHOTOISOMERIZATION*

Abstract E‐Urocanic acid exhibits a single, featureless, long‐wavelength absorption band with λmax˜258 nm in water. However, the quantum efficiency for E→Z photoisomerization is wavelength dependent in this region, with the maximum value at the low energy edge of the band (e.g. 313 nm) and appreciably lower efficiencies measured at ≦ 300 nm.

Formation of Singlet Oxygen by Urocanic Acid by UVA Irradiation and Some Consequences Thereof

Singlet oxygen–initiated decomposition of urocanic acid (UCA) (3‐(1H‐imidazol‐4(5)‐yl)‐2‐propenoic acid) was used to successfully confirm the report that UCA generates singlet oxygen when irradiated with ultraviolet A light (UVA). The UCA‐generated singlet oxygen converts UCA to one or more products that then catalyze the further destruction of the UCA with UVA light by singlet oxygen formation. Some nicking of the φX‐174 supercoiled plasmid DNA was observed when UCA was irradiated with UVA to complete destruction of the starting material, and the product mixture was then mixed with the plasmid in the dark. More extensive nicking was seen when the photoproduct mixture and the plasmid were irradiated with UVA light. An “aged” (4 days) solution of UCA photoproduct no longer caused nicking in the dark but retained the capability to nick the plasmid when irradiated. There is evidence for the presence of hydroperoxides in the UCA photolysis product mixture, and the quenching studies with 2‐propanol indicate that free radicals are involved in the plasmid‐nicking photochemistry. Singlet oxygen does not appear to play a role in the nicking of the plasmid.

Effect of ring methylation on the photophysical, photochemical and photobiological properties of cis-dichlorobis(1,10-phenanthroline)rhodium(III)Chloride.

Abstract Methylated analogues of cis-dichlorobis(1,10-phenanthroline)rhodium(III)chloride (BISPHEN) have been prepared in order to increase the hydrophobicity of the parent compound, and thus create octahedral rhodium (III) complexes suitable for use as anticancer and antiviral agents that can be photoactivated. The parent complex has been shown in earlier work to be unable to cross through cell membranes. Octamethylation, as in the case of cis-dichlorobis(3,4,7,8-tetramethyl-1,10-phenanthroline)rhodium(III)chloride (OCTBP), provides enough hydrophobicity to be taken up by KB tumor cells. It also provides a higher level of ground-state association with double-stranded DNA and increases the quantum efficiency of photoaquation by greater than 10-fold, relative to BISPHEN. OCTBP forms covalent bonds to deoxyguanosine when irradiated with the nucleoside, as has been seen with the parent complex. Irradiation of OCTBP in the presence of the KB or M109 tumor cell lines using narrow-band UVB (λ = 311 nm) irradiation initiates a considerable amount of phototoxicity. There is evidence that OCTBP acts as a prodrug (i.e. after passing through the cell membrane the metal complex is photolyzed to cis-chloro aquo OCTBP, which may be the active phototoxic agent). OCTBP and the tetramethyl analogue cis-dichlorobis(4,7-dimethyl-1,10-phenanthroline)rhodium(III)chloride (47TMBP) also show photoaquation upon excitation with visible light (λ > 500 nm), and indeed, some phototoxicity of KB cells is observed at these wavelengths as well. This is attributed to direct population of photoactive triplet-excited states. These results, together with our earlier studies of cis-dichloro[dipyrido(3,2-a: 2′,3′-c)phenazine (1,10-phenanthroline)rhodium(III)chloride (DPPZPHEN) demonstrate that such octahedral rhodium complexes are viable “photo-cisplatin” reagents.

Photochemistry and photobiology of urocanic acid.

: E-Urocanic acid is a metabolite of histidine which accumulates in the skin and is excreted in sweat. It has been of interest to photobiologists and photodermatologists because of its intense absorption band at approximately 270 nm, a feature suggestive of a role as a natural photoprotecting agent for DNA. Early work concentrated on the E----Z isomerization resulting from UV excitation. Recent studies have revealed additional, potentially significant, photobiological properties, i.e. photochemical binding to DNA and an apparent involvement of the Z isomer in the phenomenon of photoimmunosuppression.

Photochemical inactivation of single-stranded viral DNA in the presence of urocanic acid.

Abstract— Urocanic acid (UA) has previously been shown to react photochemically in vitro with N,N‐dimethylthymine. In this study, mixtures of UA and phage G4 single‐stranded DNA have been irradiated with UV light (λ≥ 254 nm) and the DNA assayed for infectivity. At the concentrations of UA employed (typically 5.4 × 10‐3M) there is extensive absorption of the incident light by the UA. The DNA is inactivated at rates greater than that predicted from the calculated shielding by UA, indicating that photosensitization is occurring. Photosensitization is also indicated by the fact that at high UA concentrations the inactivation rate does not decrease to zero but approaches a residual value. Furthermore, the ability to photoreactivate DNA that has been photolyzed in the presence of UA is much reduced relative to that observed upon photolysis of the DNA alone. UA is therefore responsible for the production of UV‐induced DNA lesions, which are resistant to photoreactivation.

Photochemical covalent binding of urocanic acid to polynucleic acids

Photolysis of E-[ring-2-14C]urocanic acid (UA) with native or denatured calf thymus DNA leads to covalent binding of the radiolabel to the nucleic acid. A similar observation is made upon photolysis of the labeled UA with the polyribonucleotides, in which case a strong preference is observed for binding to poly[U]. DNA or poly[U], which had been reacted with UA and purified by dialysis and multiple precipitations, releases UA upon further irradiation with 254 nm light (as expected for cyclobutane adducts). Quantum efficiencies for binding of the UA to native DNA have been measured at 308 and 266 nm and are 0.30 x 10(-5) and 1.3 x 10(-4), respectively, at comparable reactant concentrations. The large increase at the shorter wavelength (where DNA absorption is more competitive) is taken as evidence for the primary role of a DNA excited state in initiating the binding reaction(s).

PHOTOCHEMICALLY INDUCED BINDING OF Rh(phen)2Cl2+ TO DNA†

Abstract— The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 times 10‐4 mol Rh per mol photons and 1.6 times 10‐3 mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.