Abstract
Long-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 hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity highcapacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site I DAY 4 targets (K D[APPARENT] ≈2-4×10 -6 M and B MAX[APPARENT] ≈6 pmol/10 6 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site II DAY 4 targets (K D[APPARENT] ≈1.5×10 -3 M and B MAX[APPARENT] ≈350 pmol/10 6 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I K D . Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II K D . These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.
Original language | English (US) |
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Pages (from-to) | 26-35 |
Number of pages | 10 |
Journal | Toxicological Sciences |
Volume | 163 |
Issue number | 1 |
DOIs | |
State | Published - May 1 2018 |
Bibliographical note
Funding Information:This work was supported by grants from the American Cancer Society (IN93R [to KSK]); the National Institutes of Health (CA29540, CA26851 [to Stewart Sell], and AM28215, AM28392 [to HLL]); and the UCSD Academic Senate (RP118B [to HLL and KSK]).
Funding Information:
We thank Hal Skelly for additional technical assistance. This work was supported by grants from the American Cancer Society (IN93R [to KSK]); the National Institutes of Health (CA29540, CA26851 [to Stewart Sell], and AM28215, AM28392 [to HLL]); and the UCSD Academic Senate (RP118B [to HLL and KSK]).
Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
Keywords
- Hepatocarcinogenesis
- N-acetyl-2-aminofluorene
- Primary hepatocytes
- Procarcinogen binding