Chronic ethanol feeding accelerates hepatocellular carcinoma progression in a sex-dependent manner in a mouse model of hepatocarcinogenesis

Abstract

Background: Chronic ethanol consumption increases the risk of hepatic cirrhosis and hepatocellular carcinoma (HCC). While sex differences exist in susceptibility to ethanol-induced liver damage/HCC development, little is known about the effects of ethanol on tumor progression.

Methods: Neonatal male and female mice were initiated with a single dose of diethylnitrosamine (DEN). Sixteen or 40 weeks later, animals were placed on a 10/20% (v/v) ethanol-drinking water (EtOH-DW; alternate days) regime for 8 weeks. At study end, liver tissue and serum were analyzed for liver pathology/function and cytokine expression.

Results: DEN reproducibly induced hepatic foci/tumors in male and female mice. Ethanol diminished hepatic function and increased liver damage, but ethanol alone did not induce hepatic foci/HCC formation. In DEN-initiated EtOH-DW animals, ethanol significantly increased tumor incidence and burden, but only in male mice. Male and female mice (±DEN) demonstrated comparable blood alcohol content at necropsy, yet increased hepatic damage and diminished hepatic function/antioxidant capacity were significantly greater in males. Analysis of liver mRNA for Th1, Th2, or T-regulatory factors demonstrated significantly elevated SMAD3 in male compared to female mice in response to EtOH, DEN initiation, and DEN + EtOH-DW.

Conclusions: These data demonstrate male mice are more susceptible to HCC incidence and progression in the setting of chronic ethanol feeding than females. Differences in markers of hepatic immune response in male mice suggest that increased TGFβ-SMAD3 signaling may enhance promotion in this model of HCC progression, effects modulated by chronic ethanol feeding.

Figure 1. Ethanol feeding enhances DEN-induced hepatocarcinogenesis in male mice

Representative images of livers resected from male and female mice (48 weeks) initiated with DEN (D) or DEN with ethanol feeding (D+E).

Figure 2. Ethanol feeding promotes hepatic injury in a DEN model of hepatocarcinogenesis in male mice

A) Representative images (x200 magnification) of sections from control (C) male mice (48 weeks) and pair-matched male mice maintained on ethanol feeding (E), initiated with DEN (D), or initiated with DEN and ethanol fed (D+E). Sections were stained with H&E, Picrosirius Red or an antibody against Glutathione S-transferase placental isoform (GSTpi). B) Cumulative total liver injury score (TLIS) was blind scored from representative sections (2 lobes/mouse, 5 fields/lobe) from each experimental group (male and female, 24- and 48-weeks; C, E, D, D+E). *p<0.05 versus respective male or female controls (C), #p<0.05 D+E (male or female) versus D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group.

Figure 3. Ethanol feeding promotes hepatic tumor incidence and size preferentially in male mice

A) Mean number of altered hepatic foci (AHF)/field and mean foci area (MFA) were calculated from glutathione S-transferase placental isoform (GSTpi) positive cells in microscopic fields (x200 magnification, 2 lobes/mouse, 5 fields/lobe) from control (C) male and female mice, and mice undergoing ethanol feeding (E), DEN-initiation (D) or DEN initiation followed by 8 weeks of ethanol feeding (D+E). *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group. B) Mean AHF and mean area occupied by foci were calculated from GSTpi+ cells from microscopic fields (x200 magnification, 2 lobes/mouse, 5 fields/lobe) from control (C) male and female mice, and mice undergoing E, D, or D+E. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group.

Figure 4. Ethanol feeding promotes hepatic tumorigenesis preferentially in male mice

A) Representative proliferating cell nuclear antigen (PCNA) immunohistochmistry of hepatic sections (x200 magnification) from control (C) mice, or mice maintained on ethanol (E), DEN-initiation (D), or DEN and ethanol (D+E) at 24 or 48 weeks. B) Number of PCNA positive cells per microscopic field (x200 magnification) were measured in representative sections (2 lobes/mouse, 5 fields/lobe) from individual male and female mice on different treatment regimes (24 and 48 weeks) and mean values (± SEM) were calculated. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group. C) Representative immunoblots of cyclin D1 expression in hepatic tissue lysates from male and female mice on different treatment regimes at 24 and 48 weeks (upper panel). Optical integrated volume was calculated, corrected for sample loading (stripping-re-probing membranes with anti-β-actin (β-Act)) and expressed as mean values (arbitrary units) ± SEM (lower panel). *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 6 independent samples per group.

Figure 4. Ethanol feeding promotes hepatic tumorigenesis preferentially in male mice

A) Representative proliferating cell nuclear antigen (PCNA) immunohistochmistry of hepatic sections (x200 magnification) from control (C) mice, or mice maintained on ethanol (E), DEN-initiation (D), or DEN and ethanol (D+E) at 24 or 48 weeks. B) Number of PCNA positive cells per microscopic field (x200 magnification) were measured in representative sections (2 lobes/mouse, 5 fields/lobe) from individual male and female mice on different treatment regimes (24 and 48 weeks) and mean values (± SEM) were calculated. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group. C) Representative immunoblots of cyclin D1 expression in hepatic tissue lysates from male and female mice on different treatment regimes at 24 and 48 weeks (upper panel). Optical integrated volume was calculated, corrected for sample loading (stripping-re-probing membranes with anti-β-actin (β-Act)) and expressed as mean values (arbitrary units) ± SEM (lower panel). *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 6 independent samples per group.

Figure 5. Ethanol feeding promotes cell death in hepatic tumorigenesis preferentially in female mice

Immunohistochemistry was performed on representative sections of control male and female mice (C) and mice maintained on ethanol (E), DEN-initiated (D), or DEN-initiated followed by ethanol feeding (D+E) at A) 24 and B) 48 weeks. Number of TUNEL positive cells per microscopic field (x200 magnification) were measured in representative sections (minimum of 2 lobes/mouse, 5 fields/lobe) and mean values (± SEM) calculated. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 8 independent mice/group.

Figure 6. Ethanol induces cytochrome P4502E1 expression and activity preferentially in male mice

A) Representative immunoblots of cytochrome P4502E1 (CYP2E1) expression in hepatic tissue lysates from male and female mice from control (C) and pair-matched animals maintained on ethanol (E), DEN-initiated (D), or DEN-initiated followed by ethanol feeding (D+E) at 24 and 48 weeks (upper panels). Optical integrated volume was calculated, corrected for sample loading (stripping-re-probing membranes with anti-β-actin (β-Act)) and expressed as mean values (arbitrary units) ± SEM (lower panel). *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 6 independent samples per group. B) RT-PCR analysis of CYP2E1 mRNA expression from pooled total mRNA samples from male and female mice on the different treatment regimes indicated at 24 and 48 weeks. C) p-nitrophenol hydroxylation was measured as a marker of CYP2E1 activity in hepatic lysates from male and female mice on different treatment regimes at 24 and 48 weeks. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points.

Figure 6. Ethanol induces cytochrome P4502E1 expression and activity preferentially in male mice

A) Representative immunoblots of cytochrome P4502E1 (CYP2E1) expression in hepatic tissue lysates from male and female mice from control (C) and pair-matched animals maintained on ethanol (E), DEN-initiated (D), or DEN-initiated followed by ethanol feeding (D+E) at 24 and 48 weeks (upper panels). Optical integrated volume was calculated, corrected for sample loading (stripping-re-probing membranes with anti-β-actin (β-Act)) and expressed as mean values (arbitrary units) ± SEM (lower panel). *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points. Minimum n = 6 independent samples per group. B) RT-PCR analysis of CYP2E1 mRNA expression from pooled total mRNA samples from male and female mice on the different treatment regimes indicated at 24 and 48 weeks. C) p-nitrophenol hydroxylation was measured as a marker of CYP2E1 activity in hepatic lysates from male and female mice on different treatment regimes at 24 and 48 weeks. *p<0.05 versus respective male or female E, #p<0.05 D+E versus respective male or female D mice, §p<0.05 male versus female within same treatment group/time points.

Figure 7. Ethanol feeding differentially affects immune responses in male versus female mice in the setting of hepatocarcinogenesis

A) Quantitative real time PCR (qRT-PCR) analysis of T-bet, GATA3, and SMAD3 mRNA expression in total hepatic mRNA isolated from control (C) and pair-matched animals maintained on ethanol (E), DEN-initiated (D), or DEN-initiated followed by ethanol feeding (D+E) at 48-weeks. *p<0.05 versus C, #p<0.05 versus respective male or female E mice, §p<0.05 male versus female within same treatment group/time points. B) Custom BioPlex assay was used to determine cytokine expression in pooled serum samples from control male (left) and female (right) mice (C), and pair matched animals maintained on ethanol (E), DEN-initiated (D), or DEN-initiated followed by ethanol feeding (D+E) at 48 weeks. Minimum n = 8 independent samples per group.