Differential changes in MAP kinases, histone modifications, and liver injury in rats acutely treated with ethanol

Abstract

Background: Acute ethanol is known to affect cells and organs but the underlying molecular mechanisms are poorly explored. Recent developments highlight the potential importance of mitogen-activated protein kinases, MAPKs (i.e., ERK1/2, p38, and JNK1/2) signaling, and histone modifications (i.e., acetylation, methylation, and phosphorylation) in the actions of ethanol in hepatocytes. We have therefore investigated significance of these molecular steps in vivo using a model in which rats were acutely administered ethanol intraperitoneally (IP).

Methods: Ethanol was administered IP (3.5 gm/kg body weight) to 12-week-old male Sprague-Dawley rats. Liver was subsequently removed at 1 and 4 hours. Serum was used for alcohol and ALT assays. At the time of the removal of liver, small portions of each liver were formalin-fixed and stained with hematoxylin and eosin (H&E) and used for light microscopy. Western blot analysis was carried out with specific primary antibodies for various parameters.

Results: There were clear differences at 1 and 4 hours in blood ethanol, ALT, steatosis, and cleaved caspase 3. Apoptosis at 1 hour was followed by necrosis at 4 hours. Acute alcohol elicited a marked increase in the phosphorylation of ERK1/2 and moderate increases in the phosphorylation of p38 MAPK and JNK. Temporally different phosphorylation of histone H3 at ser-10 and ser-28 occurred and acetylation of histone H3 at lys 9 increased progressively.

Conclusions: There were distinct differences in the behavior of the activation of the 3 MAP kinases and histone modifications after acute short exposure of liver to ethanol in vivo. Although all 3 MAPKs were rapidly activated at 1 hour, the necrosis, occurring at 4 hours, correlated to sustained activation of ERK1/2. Transient activation of p38 is associated with rapid phosphorylation of histone H3, whereas prolonged activation of ERK1/2 is correlated to persistent histone H3 acetylation.

Fig. 1. Serum ethanol levels after acute ethanol administration

Rats were given IP a single ethanol binge dose (3.5 gm/kg) and the levels of serum ethanol were determined at 1 hr and 4 hr as described under materials and methods. Control represents animals given saline for binge control. Values are mean ± SE (n=5 rats).

Fig. 2. Serum ALT, cleaved caspase 3, and steatosis after acute ethanol administration

Rats were given IP a single ethanol binge dose (3.5 gm/kg) and the levels of serum ALT were determined at 1 hr and 4 hr as described under materials and methods. Cleaved caspase 3 levels were determined by western immunoblot at 1 hr and 4 hr after ethanol administration. Steatosis was monitored at 1hr and 4 hr after ethanol administration. Sections of liver samples were stained with hematoxylin and eosin. Liver triglycerides were determined at 4 hr by colorimetric method. Control represents animals given saline for binge control. Values are mean ± SE (n=4 to 5 rats) * significant compared to control group (p<0.05).

Fig. 3. Levels of phosphorylated ERK1/2, p38 MAPK and JNK1/2 in cytosolic extracts

Ethanol binge was administered as described in Fig. 1 The levels of phosphorylated ERK1/2, p38 MAPK and JNK1/2 in cytosolic cell extracts were determined at 1 hr and 4 hr. Control represents animals given saline for binge control. Values are mean ± SE (n= 3 to 5 rats) * significant compared to control group (p<0.05)

Fig. 4. Levels of phosphorylated ERK1/2, p38 MAPK and JNK1/2 in nuclear extracts

Ethanol binge was administered as described in Fig. 1. The levels of phosphorylated ERK1/2, p38 MAPK and JNK1/2 in nuclear extracts were determined at 1 hr and 4 hr as described under materials and methods. Control represents animals given saline for binge control. Values are mean ± SE (n=3 to 4 rats) * significant compared to control group (p<0.05).

Fig. 5. Levels of phosphorylated histone and acetylated histone after acute ethanol administration

Rats were given IP a single ethanol binge dose (3.5 gm/kg) and levels of phosphorylated H3 ser-10 & ser-28 and acetylated H3 Lys 9 in nuclear extracts were determined at 1 hr and 4 hr as described under materials and methods. Values are mean ± SE (n=4 to 5 rats) *significant compared to control group (p<0.05).