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To determine the threshold dose of β-Naphthoflavone (BNF) that induces hepatocellular tumor promoting effects, reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substance (TBARS) formation, and drug-metabolizing enzymes that protect against ROS generation, two-stage liver carcinogenesis model was used. Partial hepatectomized rats (n = 11 to 12) were fed diets containing 0, 0.03, 0.06, 0.125 or 0.25% BNF for 6 weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, glutathione S-transferase placental form (GST-P)-positive foci significantly increased in rats given 0.25% BNF. No marked changes in ROS production and TBARS contents were observed between the BNF treated and DEN alone groups. Real-time RT-PCR showed that the expression of Cyp1a1, Cyp1a2, Cyp1b1 and Nqo1 significantly increased in the groups given 0.03% BNF or more, but Ugt1a6, Akr7a3 and Gstm1 significantly increased in the groups given 0.125% BNF or more. Gpx2 and Yc2 significantly increased in the groups given 0.06% BNF or more and 0.25% BNF, respectively. Inflammation-related genes such as Ccl2, Mmp12, Serpine1 and Cox-2 significantly increased in the 0.25% BNF group. In immunohistochemistry, the number of cyclooxygenase-2 (COX-2)-positive cells increased in rats given 0.25% BNF. These results suggest that 0.25% BNF is the threshold dose for liver tumor promotion, and the fact that inflammation-related genes and COX-2 protein increased in the 0.25% BNF group strongly suggests that inflammation is involved in the liver tumor promoting effect of BNF in rats.

Threshold dose of liver tumor promoting effect of β-naphthoflavone in rats