Activation Of 2-acetylaminofluorene Research Articles

Activity of 2-Acetylaminofluorene as a UDS inducting agent in B6C3F1 mouse hepatocytes in vitro

2-Acetylaminofluorene (2AAF) is shown to be reproducibly active in inducing UDS in cultured hepatocytes from a B6C3F1 mouse liver.

Effect of autoxidized linoleic acid on the contents of cytochrome P-450 and cytochrome b5, and drug-metabolizing enzyme activities in rat liver.

Autoxidized linoleic acid (AL) having 800 meq/kg of peroxide value and 1,700 meq/kg of carbonyl value was given in repeated oral doses for 1-15 days at a daily dose of 2.5 ml/kg to male Wistar rats. During the administration, the effect of AL on drug-metabolizing activity was investigated periodically in liver microsomes. The contents of cytochrome P-450 and b5 were increased by consecutive oral doses for 3-7 days. Thereafter, the amount of cytochrome P-450 decreased gradually, but the b5 decreased slightly. Thus, after administration for 11-15 days, the cytochrome P-450 content was significantly lower but the cytochrome b5 content was rather high in comparison with the control group. The aminopyrine-N-demethylase activity was not reduced even after repeated oral doses of AL for 15 days. The activation of 2-acetyl amino fluorene (2-AAF), which the authors termed S-9 activity, gradually decreased during administration for more than 3 days, and completely disappeared after administration for 9 days.

The importance of 2-aminofluorene in the mutagenic activation of 2-acetylaminofluorene

The mutagenic activation of 2-acetylaminofluorene (AAF) and its derivatives N-hydroxy-AAF and 2-aminofluorene (AF) by pulmonary and hepatic microsomal fractions from untreated rabbits was investigated using Salmonella strain TA98. The mutagenicity of AAF in the presence of hepatic microsomes followed typical saturation kinetics. However, in the presence of pulmonary microsomes, the mutagenic activity increased linearly with increasing substrate concentration and approximated that obtained with low concentrations of AF. N-Hydroxy-AAF was 1/10th as mutagenic as AF in the presence of pulmonary microsomes, but 2–2.5 times more mutagenic than AF in the presence of hepatic microsomes. The activation of AAF by both fractions was completely inhibited by the deacetylase inhibitor paraoxon. Although AAF does not appear to be a substrate for cytochrome P450 form 5, antibodies to this form inhibited the activation of AAF by pulmonary and hepatic microsomes by 90% and 60%, respectively. These results indicate that the mutagenic activation of AAF by these fractions primarily involves deacetylation to AF, followed by cytochrome P450 form 5-mediated activation of AF.

Enhancement of S9 activation by S105 cytosolic fraction.

The addition of supplementary cytosolic fraction greatly enhances the activation of 2-acetylaminofluorene (AAF) by uninduced 9,000 x g supernatant fractions (S9) in the Ames test. Uninduced S9 is poor at activating AAF in the Ames test (although it is effective in the liquid based fluctuation test) probably because cytosolic material diffuses into the bottom agar. An enhancing effect of cytosol supplementation was also observed with 2-aminoanthracene (AA) and 2-aminofluorene (AF) with uninduced S9. Using Aroclor-induced preparations, supplementation with cytosol enhanced the activation of benzo[a]pyrene and ethidium bromide. With AAF and Aroclor-induced preparations, supplementation with cytosol produces a slight but significant increase in activation, but interpretation is complicated by the fact that Aroclor 105,000 x g supernatant fraction (S105) alone efficiently activates AAF, AF and AA. Norharman potentiated the enhancing effect of Aroclor S105 on Aroclor-S9 activation of AAF but inhibited the activation of AAF by S105 fraction alone. The enhancing effect of S105 fraction may explain some, but not all, of the differences between liquid-based and agar overlay based activation.

Optimal levels of S9 fraction in the Ames and fluctuation tests: apparent importance of diffusion of metabolites from top agar.

For activation of 2-acetylaminofluorene (AAF) there is an optimal level of rat liver S9 fraction which is considerably lower in the fluctuation test than in the Ames test. The optimal level of S9 is not markedly affected by the dose of AAF used, nor by the ratio of S9 to bacteria, nor by the presence of soft agar. The difference between Ames and fluctuation tests appears to be due to diffusion of some substance or substances from the top agar layer in the Ames test. Diffusion of the co-factors NADP and glucose-6-phosphate is not responsible for the difference in S9 optima, nor is diffusion of soluble S9 constituents although this may considerably affect the performance of the S9 mix. We present evidence that diffusion of non-mutagenic metabolites of AAF from the Ames test top agar may be responsible for the difference in S9 optima. Our results are consistent with a model whereby lipophilic non-mutagenic metabolites accumulate in the microsomes and inhibit further activation. When the metabolites are able to diffuse away, a higher level of S9 will be optimal. The model is consistent with some other phenomena of S9 activation.