PHOTOCHEMICAL SENSITIZATION BY AZATHIOPRINE AND ITS METABOLITES .3. ADIRECT EPR AND SPIN-TRAPPING STUDY OF LIGHT-INDUCED FREE-RADICALS FROM 6-MERCAPTOPURINE AND ITS OXIDATION-PRODUCTS

Sunlight has been implicated in the high incidence of skin cancer foun d in patients receiving 6-mercaptopurine (PSH) in the form of its pro- drug azathioprine. In this study we have used EPR spectroscopy in conj unction with the spin-trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reac tive free radicals upon UV irradiation. When an aqueous anaerobic solu tion (pH 5 or 9) of PSH (pK(2) = 7.7) and either 2-methyl-2-nitrosopro pane (MNP) or nitromethane (NM) were irradiated (lambda > 300 nm) with a Xe are lamp, the corresponding purin-6-thiyl (PS') radical adduct a nd the reduced form of the spin trap (MNP/H-. or CH3NO2.-) were observ ed. However, no radical adducts were detected when PSH and 5,5-dimethy l-1-pyrroline-N-oxide (DMPO) were irradiated (lambda = 320 nm) in oxyg en-free buffer. These findings suggest that PSH does not photoionize b ut that instead MNP and NM are reduced by direct electron transfer fro m excited state PSH, (1,3)(PSH). In aerobic solution, oxygen can act as an electron acceptor and the O-2(.-) and PS. radicals are formed an d trapped by DMPO. 6-Mercaptopurine did photoionize when irradiated wi th a Nd:YAG laser at 355 nm as evidenced by the appearance of the DMPO /H-. (e(eq)(-), + H+) adduct, which decreased in intensity in the pres ence of N2O. (1,3)(6-Mercaptopurine) oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO2.- or glutathiyl radicals. The photochemical behavior of 6-thioxanthine and 6-thiouric acid was similar to PSH. However, the excited states of these metabol ic oxidation products exhibited stronger reducing properties than (1,3 )<(PSH). Photolysis of PSH photoproducts purine-6-sulfonate or purine -6-sulfinate resulted in homolysis of the C-S bond and the appearance of the SO3.- and SO2.- radicals, respectively, which were detected by direct EPR. These studies demonstrate that UV irradiation of PSH, its photoproducts and metabolites generates a variety of free radicals tha t may be involved in the etiology of skin cancer induced by azathiopri ne.