Molecular Pharmacology, Vol 18, 117-127, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Mechanism of N-Hydroxyacetylarylamine Mutagenicity in the Salmonella Test System: Metabolic Activation of N-Hydroxyphenacetin by Liver and Kidney Fractions from Rat, Mouse, Hamster, and Man

¹ Biochemical Pharmacology Section, Laboratory of Chemical Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
² Department of Environmental Toxicology, National Institute of Public Health, Oslo 1, Norway
³ Department of Clinical Pharmacology, Huddinge Hospital, Karolinska Institutet, S-141 86 Huddinge, Sweden

The mutageicity of phenacetin and acetaminophen and their respective N-hydroxylated metabolites was determined in Salmonella typhimurium tester strains TA 98 and TA 100 in both the presence and the absence of liver and kidney metabolic activating systems from rat, mouse, hamster, and man. In TA 98 neither phenacetin, acetaminophen, nor N-hydroxyacetaminophen was mutagenic in either the presence or the absence of liver S-9 fractions from the 3-methylcholanthrene (3-MC)-induced rat or untreated hamster liver microsomes. The inclusion of NADPH with either the S9 or the microsomal fractions had no effect on the mutageicity of any of these compounds. Although N-hydroxyphenacetin was not directly mutagenic in TA 100, it was mutagenic in the presence of either the liver microsomal or the S-9 fractions from all three animals and in the presence of human liver microsomes. N-Hydroxyphenacetin was also mutagenic in TA 98 in the presence of liver microsomes from either the hamster or the mouse. Neither phenacetin, acetaminophen, nor N-hydroxyacetaminophen was mutagenic in TA 100 even in the presence of various metabolic activating systems. Pretreatment of the animals with either 3-MC or Arochior 1254 or the addition of NADPH had no effect on the mutagenic activation of N-hydroxyphenacetin or any of the other compounds by liver and kidney fractions. N-Hydroxyphenacetin was also mutagenic to TA 100 in the presence of both rat and hamster kidney S-9 fractions and mouse kidney microsomes. p-Nitrosophenetole was mutageic to TA 100 in both the presence and the absence of subcellular liver fractions and only slightly mutagenic to TA 98 even in the presence of metabolic activating systems. Ascorbic acid inhibited the mutation frequency (80-90%) of N-hydroxyphenacetin in both TA 100 and TA 98 and of p-nitrosophenetole in TA 100 but caused a slight increase in the mutation frequency of N-hydroxy-2-aminofluorene and 2-nitrosofluorene in both TA 98 and TA 100. In the presence of 1 mM ascorbic acid, p-nitrosophenetole rapidly decomposed with a half-life of 45 s to form azoxyphenetole and azophenetole. Paraoxon, at a 1 µM concentration, markedly inhibited both the mutagenicity of N-hydroxyphenacetin in TA 100 mediated by subcellular liver fractions from rat, hamster, mouse, and man as well as rat kidney S-9 fractions by 90% and the in vitro deacetylation of N-hydroxyphenacetin mediated by hamster liver microsomes. These data indicate that the initial step in the mutagenic activation of N-hydroxyphenacetin in the Salmonella system proceeds via the same mechanism as that of the known carcinogen N-hydroxy-2-acetylaminofluorene.

Note:
ACKNOWLEDGMENT The authors would like to thank Dr. John Strong, Laboratory of Chemical Pharmacology, NCI, for his assistance in obtaining the mass spectra.