Progression of Hepatic Adenoma to Carcinoma in Ogg1 Mutant Mice Induced by Phenobarbital

Abstract

The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1) responsible for the repair of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Mmh homozygous mutant (Ogg1^-/-) and wild-type (Ogg1^+/+) male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs) were found in PB-treated Ogg1^-/- mice, while Ogg1^+/+ animals developed only hepatocellular adenomas (HCAs) at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1^-/- mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1^+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1^-/- mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1^-/- and Ogg1^+/+ animals. Treatment of Ogg1^-/- mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1^-/- mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

Figure 1

Survival curves for male (a) and female (b) Ogg1^−/− and Ogg1^+/+ mice.

Figure 2

Formation of 8-OHdG and alterations to cell proliferation and apoptosis (ssDNA) in the livers of Ogg1^−/− and Ogg1^+/+ mice treated with PB at 500 ppm for 4 weeks (opened squares: control groups; black squares: PB-treated groups).

Figure 3

(a) Results of H&E staining and immunohistochemical evaluation of several hepatocarcinogenesis biomarkers (A–H) and p-Nrf2 (I, J) in the livers, HCC (A, C, E, G, and I), and HCA (B, D, F, H, and J) of Ogg1^−/− and Ogg1^+/+ mice maintained on 500 ppm PB for 78 weeks. Note that the nuclear expression of p-Nrf2 in PB-treated Ogg1^+/+ mice HCAs (arrow), but no staining in Ogg1^−/− HCCs. (b) Immunohistochemistry for 8-OHdG (A–D) and PCNA (E–H) in the surrounding liver tissue of Ogg1^−/− and Ogg1^+/+ mice treated with PB for 78 weeks. Note the high expression of 8-OHdG (A) and PCNA (E) in the livers of Ogg1^−/− mice treated with PB (arrows).

Figure 4

Representative blots for phospho-Nrf2 (p-Nrf2 (S40)) (a) and total Nrf2 (b) of Ogg1^−/− and Ogg1^+/+ PB-treated and control mice are presented. PB induced elevation of total Nrf2 protein levels in the surrounding liver tissue and liver tumors of Ogg1 null and mostly in the wild-type mice. However, p-Nrf2 was observed only in the livers and HCAs of wild-type animals, indicating that sufficient Nrf2 activation occurs only in PB-administered Ogg1^+/+ mouse livers. HCC: hepatocellular carcinoma; HCA: hepatocellular adenoma; S: surrounding liver tissue; C: untreated control liver.

Figure 5

Flow chart on mechanisms of hepatocarcinogenesis in the Ogg1^−/− and Ogg1^+/+ mice. ↑: activation, overexpression, elevation; −: no change.