TY - JOUR
T1 - 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
AU - Koch, Katherine S.
AU - Moran, Tom
AU - Shier, W. Thomas
AU - Leffert, Hyam L.
N1 - Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
PY - 2018/5/1
Y1 - 2018/5/1
N2 - 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.
AB - 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.
KW - Hepatocarcinogenesis
KW - N-acetyl-2-aminofluorene
KW - Primary hepatocytes
KW - Procarcinogen processing
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U2 - 10.1093/toxsci/kfy006
DO - 10.1093/toxsci/kfy006
M3 - Article
C2 - 29319795
AN - SCOPUS:85047006205
SN - 1096-6080
VL - 163
SP - 36
EP - 44
JO - Toxicological Sciences
JF - Toxicological Sciences
IS - 1
ER -