REACTION BETWEEN METABOLICALLY ACTIVATED ACETAMINOPHEN AND PHOSPHOROTHIOATE OLIGONUCLEOTIDES

Assessment of toxic or mutagenic risks associated with phosphorothioate oligonucleotides (PTO) is important. In vitro and in vivo data have shown that PTOs are nontoxic and nonmutagenic. However, these studies do not address interactions between PTOs and other compounds. The sulfur on PTOs may provide a novel reactive center on a DNA molecule for drug interactions. This study chose acetaminophen (ACAP) as a model drug because ACAP is oxidized to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which reacts with sulfur-containing compounds. Reaction of dCTP(S) with NAPQI or activated ACAP formed a product distinct from the reactants. Analysis of the product by fast atom bombardment mass spectroscopy gave a molecular weight consistent with NAPQI bound to a sulfur. Higher-molecular-weight products were seen on a polyacrylamide gel electrophoresis after incubation of fluorescein-labeled PTO with NAPQI. These products were not seen after incubation of a phosphodiester oligonucleotide with NAPQI. P-31 NMR analysis confirmed the existence of a heterogenous mixture of adducts between a PTO and NAPQI. Non-sequence-specific PTOs of various lengths were tested for their ability to reduce ACAP toxicity. Cell viability showed that larger PTOs provided greater protection. We evaluated the ability of NAPQI to cause mutations in the LacZ gene of pBluescript plasmid which contained phosphorothioate linkages at designed locations within the gene. In addition, the ability of ACAP to cause mutations in the HGPRT locus in cells grown in dATP(S)-containing medium was measured. No mutations were seen in either assay. Based upon these results, activated ACAP is reactive with PTOs in vitro, although this interaction is nontoxic and nonmutagenic. (C) 1995 Academic Press, Inc.