C57 BL/6 mice consume alcohol voluntarily (McClearn, 1988
). A step wise increase in alcohol content in a balanced liquid diet is known to make C57 mice dependent on alcohol with some loss of body weight (Piano et al., 2001
). While trying to attenuate this body weight loss we serendipitously discovered that C57 male mice consumed higher amounts of alcohol when a known amount of solid food was provided with the alcohol diet. Here we show that increased intake of alcohol diet resulted in high blood alcohol levels with a moderate loss of body weight. Such mice experienced handling-induced seizures within one hour of alcohol withdrawal. Interestingly, mice that received the alcohol liquid diet supplemented with chow achieved higher handling induced withdrawal scores than animals that had only been exposed to the alcohol liquid diet. We believe that this phenomenon can be explained by the fact that the chow supplemented alcohol animals consumed more of the alcohol containing liquid diet. Similar observations were made in alcoholic patients undergoing alcohol withdrawal. Patients that had consumed larger amounts of alcohol experienced more severe symptoms of alcohol withdrawal (Saitz, 1998
During our initial experiments, we observed that alcohol treated animals experienced a greater loss of weight than their pair-fed controls. Necropsy of the alcohol-fed mice on the day of sacrifice revealed that the animals had experienced intestinal hemorrhage during the alcohol treatment phase. Consumption of alcohol is known to result in gastric hemorrhagic lesions as well as mucosal injury to the small intestine (Bhandare et al., 1990
; Bode and Bode, 2003
). However supplementing the alcohol containing liquid diet with chow abolished the intestinal hemorrhage. It is likely that the presence of chow alleviated some of the mucosal injury of the gastrointestinal tract that occurs with chronic alcohol consumption.
By providing a small restricted
amount of chow simultaneously with the alcohol containing liquid diet we observed that the weight loss of alcohol-exposed mice was attenuated. However, the loss of body weight in the pair-fed control mice was less than that seen in mice receiving the alcohol containing liquid diet (Group I). This phenomenon of ‘energy wastage’ is believed to develop, at least in part, as a result of hepatic induction of Cyp 2E1 (Lieber, 1991
). Body weight loss can affect the physiological function of many organ systems (Piano et al., 2001
) and may introduce non-experimental variables. In our chow and alcohol diet paradigm the weight loss experienced by chronic alcohol exposed animals (Group II) mirrors more closely the weight loss observed in control animals (Group IV) and may therefore attenuate potential experimental confounds arising from differential weight loss between treatment groups.
Surprisingly, the daily consumption of alcohol containing liquid diet was significantly increased in mice when chow was provided with the alcohol containing liquid diet (). This increase in alcohol intake was reflected by an increase in blood alcohol levels (449 ± 44 mg% in Group II mice as compared to 299.6 ± 34 mg% in Group I mice).
At this time it is not clear why animals in Group II consumed more of the alcohol containing liquid diet as compared to the animals in Group I. We speculate that perhaps the consumption of solid food (i.e. chow) increased the animal’s need for fluid. Since the alcohol containing liquid diet is the only source of liquid for the animals they consumed more of it. Alternatively, perhaps the presence of food slowed the absorption of alcohol, and as a result the animals consumed more alcohol. In light of this hypothesis it is interesting to note that free access to water during chronic administration of alcohol containing liquid diet to Long-Evans rats has no effect on the consumption of the alcohol containing liquid diet (de Fiebre and de Fiebre, 2003
Regardless of the underlying mechanism(s), it is important to note that supplementing alcohol containing liquid diet with a restricted amount of rodent chow enhances the daily consumption of alcohol containing liquid diet in mice
We also performed a number of experiments to confirm the medical consequences of chronic alcohol exposure in our alcohol/chow treatment paradigm. Chronic alcohol treatment in vivo
is known to up regulate NMDA receptor number with a concomitant increase in NMDA R1 polypeptide levels in several different regions of the brain (Trevisan et al.1994
; Snell et al., 1996a
; Kalluri et al., 1998
). Therefore we examined, by Western blotting, the effect of chronic alcohol exposure on the NMDA R1 polypeptide levels in hippocampus and cerebral cortex of mice. Our Western data showed a statistically significant increase in NMDA R1 polypeptide levels in hippocampus and cerebral cortex of both alcohol-exposed mice (Groups I and II) as compared to their respective controls (Groups III and IV). This data confirms the previously reported alcohol-mediated up regulation of NMDA R1 polypeptide levels in hippocampus and cerebral cortex of mouse. Also, this data demonstrated that by providing chow with alcohol does not interfere with alcohol’s effects on the brain.
To assess the effect(s) of chronic alcohol exposure on an organ other than brain we chose to examine liver function, specifically Cyp 2E1 enzyme activity and Cyp 2E1 apoprotein levels. Our data demonstrated a several fold increase in Cyp 2E1 enzyme activity () and Cyp 2E1 apoprotein levels () in the livers of both alcohol-exposed mice (Groups I and II as compared to their respective control groups (Groups III and IV). These results are in agreement with the observations of Forkert and colleagues who reported chronic alcohol-mediated induction of Cyp 2E1 in mice (Forkert et al., 1991
). Although Cyp 2E1 apoprotein levels were comparably increased in both alcohol – treated groups of animals, Cyp 2E1 enzyme activity was significantly greater in animals that were treated with alcohol and chow (Group II). Increase in enzyme activity without increase in protein levels can perhaps be explained by the fact that phosphorylation of Cyp 2E1 enzyme alters the activity of the Cyp 2E1 enzyme without altering the polypeptide content (Oesch-Bartlomowicz et al., 1998
). Taken together, biochemical analyses showed that feeding a known amount regular chow with alcohol containing liquid diet does not interfere with the effects of alcohol on various organs in the body.
In summary, we have provided evidence that supplementing the alcohol containing liquid diet with some amount of rodent chow increased the consumption of the alcohol containing liquid diet without a major loss of body weight. Increased consumption of alcohol containing liquid diet when provided with chow resulted in higher blood alcohol levels. Such mice experienced handling induced seizures within 1h of alcohol withdrawal. Consumption of chow with alcohol did not interfere with some of the known effects of alcohol on different organs in mice.