N-Acetyl-2-Aminofluorene (AAF): In vivo temporal expression patterns of growth cycle-dependent macromolecular binding constants KD are revealed by primary cultures of premalignant hepatocytes derived from a multi-cyclic hepatocarcinogenic feeding regimen

Abstract

Biochemical investigations of the hepatoprocarcinogen N-acetyl-2-aminofluorene (AAF) have shown that normal adult rat hepatocytes in primary culture express two sets of pharmacokinetic constants - designated Systems I and II, and Sites I and II - associated respectively with the metabolism (System I [high-affinity Km[APPARENT] and low-velocity VMAX[APPARENT]] and System II [low-affinity Km APPARENT] and high-velocity VMAX[APPARENT]]), and the macromolecular binding (Site I [high-affinity KD[APPARENT] and low capacity BMAX[APPARENT]] and Site II ([low-affinity KD[APPARENT] and high-capacity BMAX[APPARENT]]) of AAF. Additional findings - that genomically saturating levels of AAF-DNA adducts form far below reported extracellular AAF concentrations required to block replicative and repair DNA synthesis; and, that biphasic Site I and Site II BMAX[APPARENT] and KD[APPARENT] expression curves varied inversely with respect to time and magnitude during hepatocyte growth - led us to wonder how macromolecular binding constants are expressed during chemical hepatocarcinogenesis. These questions were addressed by Scatchard analysis measurements through five consecutive AAF feeding cycles. Notably, cultured premalignant hepatocytes displayed reduced and elevated BMAX[APPARENT] and KD[APPARENT] levels, respectively, akin to the Site I and Site II expression curves observed during hepatocyte growth transitions in vitro. In contrast, prominent hepatocellular functions such as N-OH-AAF production, DNA replication, cell aggregation and resistance to AAF toxicity displayed different temporal trajectories. Impact StatementStriking similarities are observed between both Site I and Site II BMAX and KD expression curves during in vitro and in vivo premalignant growth transitions. These new findings fit earlier ones that hepatocytes growing during carcinogen exposure manifest fewer intranuclear AAF-DNA adducts. How this phenomenon leads to malignancy remains unclear.