Binge Alcohol Is More Injurious to Liver in Female than in Male Rats: Histopathological, Pharmacologic, and Epigenetic Profiles

Abstract

Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ-coactivator 1α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase-α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes.

Fig. 1.

Histologic and pathologic changes in ethanol binge treated rats. Ethanol three-repeat binge treatments (each 5 g/kg, 12 hours apart) in male and female rats (groups of four) were performed as described under Materials and Methods. Four hours after the last binge, blood samples and liver tissues were collected. (A) Blood ethanol concentration. (B) Histology image showing differences in lipid vesicles. (C) Count of lipid vesicles. (D) Triglyceride levels. (E) Serum ALT levels. (F) Cleaved caspase-3. Values are mean ± S.E.M. (n = 4 rats). For (A and C–E), the values are shown on y-axis. For (F), values are presented as fold increase over respective sex controls. FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge. a,Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05).

Fig. 2.

Levels of CYP2E1 (A) and catalase (B). After binge treatments (as in Fig. 1), the whole-cell extracts were prepared (see Materials and Methods). The protein levels of these components were determined by Western blotting. Fold change over respective male or female control values are presented as mean ± S.E.M. (n = 4 rats). a, Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05). FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge.

Fig. 3.

Changes in MAPK signaling components. Levels of total ERK 1/2, phospho-ERK 1/2, and ratio of phospho- to total ERK 1/2 (A); levels of total p38, phospho-p38, and ratio of phospho- to total p38 (B); and levels of Total JNK, phospho-JNK, and ratio of phospho- to total JNK (C) are presented. The experimental details were as described in Fig. 2. The protein levels of these components in whole-cell extracts were determined by Western blotting. As in Fig. 2, the values are mean ± S.E.M. (n = 4 rats). a, Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05). FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge.

Fig. 4.

Changes in transcription related factors. Protein levels of total CREB (A); phospho-CREB (B); ratio of phospho- to total CREB (C); PGC-1α (D); c-FOS (E); and c-MYC (F) are shown. The protein levels of these components were determined by Western blotting. As in Fig. 2, values are fold change over respective sex controls and are mean ± S.E.M. (n = 4 rats). a, Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05). FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge.

Fig. 5.

Histone H3 acetylation and phosphorylations. Levels of H3AcK9 (A); H3AcK9PS10 (B); H3PS10 (C); and H3PS28 (D) are presented. The experimental details were as described in Fig. 2. The levels of these components were determined by Western blotting. Values are fold change over respective sex control and are mean ± S.E.M. (n = 4 rats). a, Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05). FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge.

Fig. 6.

Analysis of PEth species. Lipids were extracted from liver samples and PEth analyzed by ESI-MS/MS on a triple-quadrupole instrument using shotgun lipidomics method (see Materials and Methods). Values (mean ± S.D.) are from four male or female rats and are presented as picomoles per milligram of tissue.

Fig. 7.

DGKα protein levels. In whole-cell extracts, DGKα protein levels in WCE were determined by Western blotting, followed by densitometric quantification. Increases in the protein levels after binge ethanol are presented as fold increases over respective sex controls. The experimental details were as described in Fig. 2. Values are mean ± S.E.M. (n = 4 rats). a, Significant compared with control (P < 0.05); b, significant compared with the male ethanol binge group; (P < 0.05). FC, female control; FE, female ethanol binge; MC, male control; ME, male ethanol binge.