Metabolism and Toxicity of 2-Aminofluorene in a Genetic Mouse Model.

Previous work in our laboratory has shown that liver cytosolic N-acetyltransferase (NAT) from C57BL/6J (B6) mice N-acetylated 2-aminofluorene (AF) 5-10 times faster than NAT from A/J (A) mice. This thesis describes experiments which characterize further the mouse as a genetic model to study the importance of N-acetylation and deacetylation in AF metabolism and toxicity. With respect to deacetylation, a strain survey of nineteen inbred mouse strains showed that differences in AAF and N-OH-AFF deacetylase activities were under genetic control. AAF deacetylation was 2 times faster in B6 than in A mice and was analyzed further to determine the hereditary basis for the difference and kinetic properties of the enzyme(s) responsible. In analysis of AF N-acetylation, a correlation was established between differences in AF NAT activity in B6 and A mice and differences in AF elimination rates in the whole animal. Studies in intact B6 and A mice injected with AF showed that B6 mice eliminated AF from blood 3-4 times faster than A mice. B6AF(,1)/J mice had intermediate elimination rates consistent with intermediate inheritance of the NAT gene as did the congenic mouse, A.B6-NAT('r), suggesting a modifying influence of the background genes on AF elimination by the rapid NAT gene product. Differences in AF NAT activity between B6 and A mice also correlated with differences in rates of AF disappearance and AAF appearance in primary hepatocyte cultures. In the presence of paraoxon, an AAF deacetylase inhibitor, AF disappeared and AAF appeared 2 times faster in B6 hepatocytes than A hepatocytes. Preliminary studies indicated that differences in N-acetylating capacity between B6 and A mice may influence susceptibility to AF-induced DNA damage since hepatocytes from the rapid acetylator B6 mouse showed more unscheduled DNA synthesis than hepatocytes from the slow acetylator A mouse. Studies with reversible inhibitors of NAT were also carried out. Liver cytosolic NAT activity was inhibited competitively by para-aminobenzoic acid (PABA) and non-competitively by folic acid and methotrexate. PABA inhibited AAF appearance and AF disappearance completely in B6 hepatocytes but inhibited AF disappearance only partially in A hepatocytes suggesting the presence of an alternate route of AF metabolism in A hepatocytes.Ph.D.PharmacologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/159753/1/8402297.pd