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Mechanism of in Vitro Mutagenic Activation and Covalent Binding of N-Hydroxy-2-acetylaminofluorene in Isolated Liver Cell Nuclei from Rat and Mouse¹

Biochemical Pharmacology Section, Laboratory of Chemical Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20014

The in vitro mutagenic activation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in the Salmonella test system (strain TA 1538) by isolated rat and mouse liver cell nuclei and the in vitro covalent binding of N-OH-AAF to nuclear nucleic acids and proteins were studied. Mutagenic activation of N-OH-AAF and 2-acetylaminofluorene by liver cell nuclei from both rat and mouse was observed. The mutagenic activation of N-OH-AAF by the mouse nuclei was 6 to 7 times greater than in the rat nuclei (2300 versus 350 revertants per 10 µg N-OH-AAF). The mutagenicity of N-OH-AAF was increased 2-fold when NADPH was included with the nuclei in the assay mixture, and this increase in N-OH-AAF mutagenicity was inhibited by -naphthoflavone. The carboxyesterase:amidase inhibitor paraoxon (10^-5 M) completely inhibited the mutagenic activation of N-OH-AAF in both mouse and rat liver nuclei. Paraoxon (10^-4 M) had no effect on the mutagenic activation of N-OH-AAF in the 100,000 x g supernatant fraction from rat liver, whereas 50% inhibition of N-OH-AAF mutagenesis was observed when adenosine 3'-phosphate 5'-phosphosulfate was added to the supernatant fraction. The addition of rat liver 100,000 x g supernatant to mouse nuclei resulted in decreased mutagenicity of N-OH-AAF that could be further inhibited by paraoxon (10^-5 M). On the other hand the addition of rat liver 100,000 x g supernatant to rat liver nuclei resulted in an increase in N-OH-AAF mutagenicity that was insensitive to inhibition by paraoxon. Antioxidants such as vitamin E and butylated hydroxytoluene at 10^-2 M concentration inhibited the mutagenic activation of N-OH-AAF by 20 to 30% in the mouse nuclear preparation, whereas vitamin C at the same concentration increased the N-OH-AAF mutagenesis 2- to 3-fold. Addition of cysteamine and methionine (10^-2 M) to the mouse nuclear preparation resulted in 60 and 30% inhibition, respectively, of N-OH-AAF mutagenesis.

The in vitro covalent binding of N-OH-AAF to nuclear nucleic acids and proteins was observed with both rat and mouse nuclei. The degree of covalent binding to nuclear protein and RNA was similar in both nuclei, but the binding to DNA in the rat nuclei was twice that observed in mouse DNA (21 versus 10 pmol/mg/10 min). Paraoxon (10^-4 M) inhibited by more than 70% the covalent binding of N-OH-AAF to nuclear nucleic acid and proteins in both mouse and rat nuclei. Similar inhibition by paraoxon of the covalent binding of N-OH-AAF to microsomal protein was observed, whereas paraoxon had no effect on the binding of N-OH-AAF to protein in 100,000 x g supernatant fraction from rat liver.

Combining rat liver nuclei with the 100,000 x g supernatant had no significant effect on the covalent binding of N-OH-AAF to nuclear nucleic acid and protein except when adenosine 3'-phosphate 5'-phosphosulfate was included, which resulted in a significant (p < 0.05) decrease in the binding to nuclear RNA and proteins. Cysteamine and vitamin C (10^-2 M) significantly (p < 0.05) inhibited the covalent binding of N-OH-AAF to nuclear RNA and protein, whereas methionine at the same concentration had no effect.

The first step in the in vitro mutagenic activation of N-OH-AAF by isolated rat and mouse liver cell nuclei and/or the 100,000 x g supernatant fraction from rat liver and the in vitro covalent binding to nuclear nucleic acids and protein occur primarily via deacetylation by either the membrane-bound amidase or the N,O-acyltransferase in the supernatant fraction.

¹ Parts of this work were presented at the annual meetings of the American Society for Pharmacology and Experimental Therapeutics in New Orleans, La., August 15 to 19, 1976, and Columbus, Ohio, August 21 to 25, 1977 (35, 37).

² Present address: Pennsylvania State University, College of Medicine, Hershey Medical Center, Hershey, Pa. 17033.

³ To whom requests for reprints should be addressed.

Received 12/28/77. Accepted 4/ 4/78.

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