© 1993 Oxford University Press
research-article |
Relationship between Hepatocyte Necrosis, Proliferation, and Initiation Induced by Diethylnitrosamine in the Male F344 Rat
Chemical Industry Institute of Toxicology P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received May 21, 1992; accepted September 6, 1992
Diethylnitrosamine (DEN) is commonly used as an initiator in rodent models of multistage carcinogenesis. Because the initiating activity of DEN has been attributed, in part, to its induction of regenerative cell proliferation, the temporal and quantitative relationships among necrosis, replication, and initiation were characterized in livers of male F344 rats subsequent to administration of a single dose of 10 or 150 mg DEN/kg. Following a dose of 150 mg DEN/kg body weight, maximal hepatocellular necrosis was observed 2 days postinjection and amounted to 9% of the hepatic volume being necrotic by light microscopic criteria. Changes in serum levels of alanine and aspartate aminotransferases, indicators of hepatocellular necrosis, paralleled changes in the necrotic volume fraction. Hepatocyte replication was estimated using nuclear labeling with bromodeoxyuridine (BrdU), which was constantly infused for 2 or 7 days by osmotic minipump. BrdU labeling was maximally increased at 4 days with 2-day infusion (26.1% in treated vs 0.5% in controls) and at 7 days with 7-day infusion (46% in treated vs 2% in controls). Initiation was quantitated by enumeration of hepatocytes which stained positive for placental glutathione-S-transferase (GST-P). Increased numbers of GST-P-positive hepatocytes were observed on Day 4 and increased to a maximum of 109/cm2 section area, or 0.077% of all hepatocytes. Thus, the temporal pattern changes following 150 mg DEN/kg body wt are consistent with the attribution of regenerative cell proliferation contributing to the yield of initiated cells. A comparison of the peak BrdU (2-day) labeling index and the peak GST-P staining frequency suggests a rate of initiation of roughly 10–3-10–4/cell division following 150mg DEN/kg body wt. In contrast, 10 mg/kg DEN did not cause necrosis at Day 2 or increase in BrdU labeling (7-day infusion, 3.1%); however, GST-P hepatocytes were slightly increased over control (1.7 vs 0.0 and 4.1 vs 0.3/cm2 at 14 and 28 days, respectively). Thus, cell replication and yield of initiated (GST-P-positive) hepatocytes in rats receiving 10 mg DEN/kg body wt were both approximately 100-fold lower than those in rats receiving 150 mg DEN/kg body wt. These results indicate the dependence of the frequency of GST-P-positive cells on hepatocyte replication in initiation by DEN and provide crucial response data for quantitative modeling of experimental hepatocarcinogenesis.