N-acetyl-2-aminofluorene (AAF) processing in adult rat hepatocytes in primary culture occurs by high-affinity low-velocity and low-affinity high-velocity AAF metabolite-forming systems

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Itsmetabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primarymonolayer adult rat hepatocyte cultures and high specific-activity [ring G ^-3 H]-N-acetyl-2- aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-termcultures. AAFmetabolismproceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 lg AAF/10 ⁶ cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culturemedia. Extracellularmetabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes ofmetabolism: System I (high-affinity and lowvelocity), K _m[APPARENT] =1.64×10 ^-7 Mand V _{MAX[APPARENT]} =0.1nmol/1 ⁶ cells/day and System II (low-affinity and highvelocity), K _m[APPARENT] =3.25×10 ^-5 Mand V _{MAX[APPARENT]} =1000nmol/10 ⁶ cells/day. A third system ofmetabolism of AAF to ×AF, with K _m[APPARENT] and V _{MAX[APPARENT]} constants of 9.6×10 ^-5 Mand 4.7nmol/1 ⁶ cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of themolecules andmechanisms responsible for multi-system processing remain to be fully defined.