REACTION OF MUTAGENIC PHENACETIN METABOLITES WITH GLUTATHIONE AND DNA - POSSIBLE IMPLICATIONS FOR TOXICITY

  • 1 January 1984
    • journal article
    • research article
    • Vol. 26  (2) , 342-347
Abstract
The direct-acting mutagens, N-hydroxy-p-phenetidine and p-nitrosophenetole, are metabolites of the analgesic phenactin and may be responsible for its carcinogenic activity. The potential detoxification of these metabolites by glutathione was examined. Glutathione reacted rapidly with p-nitrosophenetole, which was quantitatively converted to a single product as determined by high-pressure liquid chromatography. The analysis of the product by fast atom bombardment mass spectrometry and 500-MHz 1H-NMR spectroscopy established its structure as N-(glutathion-S-yl)-p-phenetidine. The same glutathione conjugate was also formed when N-hydroxy-p-phenetidine was incubated with glutathione. Since conjugate formation from N-hydroxyl-p-phenetidine occurred slowly and was decreased in the presence of an argon atmosphere as well as by higher levels of glutathione, the conjugate may result from oxidation of the N-hydroxy arylamine to the nitrosoarene, which subsequently reacted with glutathione. N-(Glutathion-S-yl)-p-phenetidine was semistable in water (half-life, 6-7 h) and very unstable in the presence of nucleophiles such as 10 mM glutathione (half-life, 7 min), quantitatively decomposing to p-phenetidine. The conjugate was also very unstable in acidic buffers (half-life, 17 min, pH 5). Radiolabeled N-hydroxy-p-phenetidine, but not p-nitrosophenetole, bound covalently to calf thymus DNa in vitro, and 4 times more binding was detected at pH 5 than at pH 7. Glutathione did not significantly decrease binding of the N-hydroxy derivative at either pH, nor did purified ring-radiolabled N-(glutathion-S-yl)-p-phenetidine significantly bind to DNA at either pH. An important detoxification pathway for phenacetin in vivo may involve the facile oxidation of N-hydroxy-p-phenetidine to P-nitrosophenetole, which then reacts rapidly with glutathione to form an excretable conjugate.