Photochemical Sensitization by Azathioprine and Its Metabolites. Part 3. A Direct 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 found in patients receiving 6‐mercaptopurine (PSH) in the form of its pro‐drug azathioprine. In this study we have used EPR spectroscopy in conjunction with the spin‐trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reactive free radicals upon UV irradiation. When an aqueous anaerobic solution (pH 5 or 9) of PSH (pK 2 = 7.7) and either 2‐methyl‐2‐nitrosopropane (MNP) or nitromethane (NM) were irradiated (λ > 300 nm) with a Xe arc lamp, the corresponding purin‐6‐thiyl (PS . ) radical adduct and the reduced form of the spin trap (MNPIH’or CH 3 N0 2 ) were observed. However, no radical adducts were detected when PSH and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) were irradiated (λ= 320 nrn) in oxygen‐free buffer. These findings suggest that PSH does not photoionize but that instead MNP and NM are reduced by direct electron transfer from 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 and trapped by DMPO. 6‐Mercaptopurine did photoionize when irradiated with 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 presence of N 2 O. 1,3 (6‐Mercap‐topurine)* oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO 2 .‐ or glutathiyl radicals. The photochemical behavior of 6‐thioxanthine and 6‐thiouric acid was similar to PSH. However, the excited states of these metabolic 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 SO 3 ′‐ and SO 2 − ‐ ‐ 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 that may be involved in the etiology of skin cancer induced by azathioprine.