Fig 1.
Combined loss of Rb and p53 suppresses protoporphyrin accumulation, ductular reaction and Cyp3a expression in livers of DDC-fed mice.
(A) Representative pictures of H&E and CK19 staining on liver sections from mice of indicated genotypes after 3 weeks of DDC feeding. Arrows indicate protoporphyrin accumulation. (B) Semi-quantitative analysis of percentage area of protoporphyrin accumulation from mice fed with normal chow or 3-weeks DDC diet (n = 5 per group). (C) (D) (E) Expression of CK19, Cyp3a11 and FECH was measured by qPCR in livers of indicated genotypes. -DDC, Rbf/ f; p53f/f, were normalized to 1. Data presented as average ± SEM. * P < 0.05 compared to +DDC, Rbf/ f; p53f/f, # P < 0.05 compared to +DDC, RbΔ/Δ.
Fig 2.
Loss of Rb and p53 results in increased hepatocellular injury, hepatocyte proliferation and polyploidization after DDC.
(A) ALT and AST serum levels (B) Representative pictures of TUNEL, Ki67 and PH3 staining on liver sections (C) Percentage of TUNEL positive hepatocyte nuclei (D) Percentage of PH3 positive hepatocyte nuclei (E) Mitotic index of hepatocytes (F) Multinucleated hepatocytes (G) Anisokaryosis score of hepatocytes from mice of indicated genotypes fed with normal chow or 3-weeks DDC diet. Data presented as average ± SEM. * P < 0.05 compared to +DDC, Rbf/ f; p53f/f, # P < 0.05 compared to +DDC, RbΔ/Δ, $ P < 0.05 compared to +DDC, p53Δ/Δ.
Fig 3.
Inactivation of Rb and p53 shortens life span and accelerates hepatocellular carcinoma formation after DDC exposure.
(A) Survival and time to tumor curves of mice of indicated genotypes after feeding with DDC diet for 3 weeks at young ages and aged with normal chow. (B) Representative macroscopic pictures of H&E staining of liver tumors of mice feeding with DDC diet for 3 weeks at young ages and aged with normal chow. White and black dotted lines indicate the borders between normal liver tissue (L) and tumor (T).
Table 1.
Percentage tumor incidence, mean time to tumors and tumor classifications of mice with the indicated genotypes.
Fig 4.
Scheme for the role of RB and p53 in DDC metabolism.
DDC interacts with Cyp in particular Cyp3a to form N-methyl protoporphyrin IX which inhibits ferrochelatase causing an accumulation of protoporphyrin in the bile ducts. Bile duct injury induces ductular reaction and inflammation. In addition, DDC induces reactive oxygen species (ROS) and oxidative stress causing hepatocellular injury. Protoporphyrin, on the other hand, acts as an antioxidant and protects hepatocytes. Rb and p53 are important for Cyp expression. Oxidative stress activates Rb and p53. Loss of Rb and p53 results in reduced expression of Cyp3a, therefore less production of the ferrochelatase inhibitor and thereby prevents the accumulation of its substrate protophorphyrin. Consequently, less bile injury and ductular reaction was observed. The reduction of protoporphyrin, an antioxidant, increases oxidative induced hepatocellular injury and later liver tumors develop.