Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice

Abstract

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.

Figure 1.

Gross (A–D) and microscopic (E–L) lesions in GSTA3 KO mice treated with weekly (12 or 24) AFB1 injections (0.5 mg/kg). (A) Normal (control) liver, male mouse, 24 injections of vehicle (DMSO) (L, M, R, C – left, middle, right and caudate lobes). (B) Cysts (short arrows in middle lobe) and tumor (long arrow in left lobe), male, 12 injections. (C) Multiple nodules (short arrows) and tumors (long arrows in left and right), male, 24 injections. (D) Entire liver composed of coalescing nodules (arrows), female, 24 injections. (E and F) Multifocal cholangiomas (arrows), male, 24 injections, H&E (×40). Cholangioma in F is a higher magnification (×200) of a rectangle in E. (G) Cholangiocarcinoma (arrows), female, 24 injections, H&E (×200). (H and I) Becker type 1 hepatocellular nodule (arrows), female, 24 injections, H&E (×40). I is a higher magnification (×200) of a rectangle in H. (J) Pan-CK positive cholangioma cells (arrows), female, 24 injections (×200). (K). Mucicarmine stained mucin in cholangiocarcinoma, male, 24 injections (×200). (L) GGT positive focus of hepatocytes (arrow), female, 12 injections (×40). (M) GST-P stained small focus of hepatocytes (arrow) and individual cells, female, 24 injections (×200).

Figure 1.

Gross (A–D) and microscopic (E–L) lesions in GSTA3 KO mice treated with weekly (12 or 24) AFB1 injections (0.5 mg/kg). (A) Normal (control) liver, male mouse, 24 injections of vehicle (DMSO) (L, M, R, C – left, middle, right and caudate lobes). (B) Cysts (short arrows in middle lobe) and tumor (long arrow in left lobe), male, 12 injections. (C) Multiple nodules (short arrows) and tumors (long arrows in left and right), male, 24 injections. (D) Entire liver composed of coalescing nodules (arrows), female, 24 injections. (E and F) Multifocal cholangiomas (arrows), male, 24 injections, H&E (×40). Cholangioma in F is a higher magnification (×200) of a rectangle in E. (G) Cholangiocarcinoma (arrows), female, 24 injections, H&E (×200). (H and I) Becker type 1 hepatocellular nodule (arrows), female, 24 injections, H&E (×40). I is a higher magnification (×200) of a rectangle in H. (J) Pan-CK positive cholangioma cells (arrows), female, 24 injections (×200). (K). Mucicarmine stained mucin in cholangiocarcinoma, male, 24 injections (×200). (L) GGT positive focus of hepatocytes (arrow), female, 12 injections (×40). (M) GST-P stained small focus of hepatocytes (arrow) and individual cells, female, 24 injections (×200).

Figure 2.

Histologic effects of AFB1 treatment (0.5 mg/kg) in GSTA3 KO mice. (A–H) H&E stained images. (A) Liver of a male mouse given DMSO once a week for 13 weeks (×200). (B–H) livers after weekly injections of 0.5 mg/kg AFB1. (B) Grade 2 OC proliferation extends to mid-zone (×200). (C) Grade 4 OC proliferation extends from portal (P) to central (C) zone (×200X). (D) Microvesicular fatty change (×200). (E) Megahepatocytes (examples shown with arrows) (×200). (F) Nuclear inclusions (examples shown with arrows) (×400). (G) Cholangiomas (×200). (H) Nodule (Becker grade I) (×40).

Figure 3.

Oval cell proliferation in liver in response to acute AFB1 exposure (1 mg/kg). (A–F and I) H&E staining. (G and H) A6 and Pan-CK immunostaining, respectively. (A) KO female mouse liver, untreated. P = portal vein branch, C = central vein branch (×200). (B) KO female mouse, 5 days after AFB1. Acute injury with cell necrosis and swelling visible (×100). (C and D) KO female mouse, 10 days after AFB1. Numerous oval cells are visible (arrows in D). D is a higher magnification (×400) of the dashed line rectangle in C (×200). (E) KO female mouse, 15 days after AFB1. Oval cells are seen throughout the lobule (arrows) (×200). (F) KO female mouse, 30 days after AFB1. Numerous oval cells are still present (arrows) (×200). (G) KO female mouse, 15 days after AFB1. Oval cells are A6-positive (arrows) (×200). (H) KO female mouse, 30 days after AFB1. Oval cells are Pan-CK-positive (arrows) (×200). (I) KO male mouse, 10 days after AFB1. No oval cells are seen at any time after AFB1 (×200).

Figure 4.

AFB1-DNA adduct formation in liver in response to acute AFB1 exposure. Mice were given 0.87 mg/kg AFB1 in DMSO or DMSO only (control) by gavage, and livers collected 2 h later. WT-F, wild type female; WT-M, wild type male; KO-F, knockout female; KO-M, knockout male. Data are expressed as mean ± SEM (N = 5). *Significant difference using a two-tailed Student’s t-test at P < 0.05.

Figure 5.

Gender and genotype comparison of GSTs and aflatoxin metabolizing proteins. Four groups of 3 month old C57Bl/6J mice representing WT males, WT females, KO males and KO females (seven animals for each group) were sacrificed and livers removed and extracts prepared. For GST isoform analyses, these extracts were further chromatographed on a glutathione affinity column. Equal amounts of purified GST-containing proteins (A–E) or extracts (F–H) were then electrophoresed and analyzed by Western immunoblotting. (A) GSTA3; (B) GSTP1; (C) GSTM1; (D) GSTM5; (E) GSTA4; (F) CYP1A2; (G) CYP3A11; (H) epoxide hydrolase. Blots were then stripped and reprobed with- and data normalized to- VDAC1 loading control. Quantitation of blot signals was carried out using ImageJ. (I) GST activity in WT and KO mouse livers. Samples were prepared as described above and GST enzyme activity measured in the affinity purified extracts. White plot, wild type mice; grey plot, knockout mice. Data are expressed as mean ± SEM (N = 7). *Significant difference using a two-tailed Student’s t-test at P < 0.05.

Figure 5.

Gender and genotype comparison of GSTs and aflatoxin metabolizing proteins. Four groups of 3 month old C57Bl/6J mice representing WT males, WT females, KO males and KO females (seven animals for each group) were sacrificed and livers removed and extracts prepared. For GST isoform analyses, these extracts were further chromatographed on a glutathione affinity column. Equal amounts of purified GST-containing proteins (A–E) or extracts (F–H) were then electrophoresed and analyzed by Western immunoblotting. (A) GSTA3; (B) GSTP1; (C) GSTM1; (D) GSTM5; (E) GSTA4; (F) CYP1A2; (G) CYP3A11; (H) epoxide hydrolase. Blots were then stripped and reprobed with- and data normalized to- VDAC1 loading control. Quantitation of blot signals was carried out using ImageJ. (I) GST activity in WT and KO mouse livers. Samples were prepared as described above and GST enzyme activity measured in the affinity purified extracts. White plot, wild type mice; grey plot, knockout mice. Data are expressed as mean ± SEM (N = 7). *Significant difference using a two-tailed Student’s t-test at P < 0.05.