High-affinity low-capacity and low-affinity high-capacity N-acetyl-2-aminofluorene (AAF) macromolecular binding sites are revealed during the growth cycle of adult rat hepatocytes in primary culture

Abstract

2Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to

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

“…When quiescent 12-day-old cultures were incubated for 24 h with 2 Â 10 À5 M [ 3 H]-AAF and subsequently subjected to autoradiography, the resulting tritium grains (although individually indistinguishable given the length of the development time) localized preferentially to the cytoplasm of hepatocytes in monolayer aggregates ( Figure 6). Hepatocyte nuclei showed markedly fewer grains than the cytoplasm, consistent with roughly 210-fold reductions in the nuclear levels of covalently bound DNA adducts under these conditions (Koch et al, 2018); some of these [ 3 H]-grains were also detected in mitotic figures. Interspersed nonparenchymal cells showed scant cytoplasmic and far fewer nuclear grains than hepatocytes.…”

“…A series of experiments were conducted using a wellcharacterized primary adult rat hepatocyte culture system to quantify the early biochemical processing and biological effects of AAF, and to elucidate the intracellular properties of processing in relation to hepatocyte DNA synthesis and cell division. These investigations are described in this and the subsequent report (Koch et al, 2018).…”

“…[AAF] i reached steady states between 6 and 24 h at all [ 14 C]-AAF or [ 3 H]-AAF concentrations tested. The specific nature of the bound material and its relationships to [AAF] i are described in the second report (Koch et al, 2018).…”

“…After 24 h exposure to 2 Â 10 À5 M AAF, cultured hepatocytes (day 13) showed no significant cytotoxicity as revealed by the: (a) absence of detached floating cells (Howard et al, 1981); (b) levels of DNA replication that were statistically similar to control levels (Koch et al, 2018), and presence of mitotic hepatocytes ( Figure 6); and (c) significant conversion of extracellular levels of 2 Â 10 À4 M AAF to the soluble metabolite N-OH-AAF ( Figure 3), and of radiolabeled AAF to covalently bound macromolecular DNA and protein adducts (Koch et al, 2018). Hepatocytes were plated exactly as described in the Primary hepatocyte culture section.…”

“…This report focuses on AAF uptake and metabolism; the subsequent report (Koch et al, 2018) focuses on macromolecule binding properties of AAF in relation to hepatocyte proliferation. A third report (Koch et al, in preparation) will describe similar measurements of AAF properties in cultured hepatocytes obtained from livers of adult rats fed a cyclic carcinogenic diet of AAF (Teebor and Becker, 1971).…”