Int. J. Environ. Res. Public Health 2010, 7 1081 7. Dynamic Contribution of ADH3 to Alcohol Metabolism through Regulation of Activity by Ethanol Administration When a small dose (1 g/kg) of ethanol is administered to mice, the blood alcohol concentration (BAC) reaches around 20 mM, at which ADH1 shows the maximum activity. This level of BAC is also observed in people who become slightly drunk after imbibing about two 633-mL bottles of beer. A medium dose (3 g/kg) causes mice to lose the righting reflex for several minutes just after administration, and the BAC is around 60 mM. A large dose (5 g/kg) produces acute alcohol intoxication with a BAC of over 100 mM, and the righting reflex is lost for several hours [24,25]. To investigate how the activities of ADH1 and 3 change during alcohol metabolism and what their relative contributions are, we periodically measured total liver ADH activity and the content of the two ADHs after various doses of ethanol were administered to mice [24]. When measured by the standard ADH assay using 15 mM ethanol as a substrate, the total liver ADH activity involved not only ADH1 but also ADH3, from the data that the total activity correlates more strongly with the sum of two ADH contents than with just the ADH1 content. When the ethanol dose was small, the contents of both ADHs in the liver increased, resulting in about a 41% increase in total liver ADH. Thus, both ADHs are acutely induced in the liver and actively participate in alcohol metabolism at moderate drinking. The acute induction of ADH isozymes, which are due to increases in mRNA [38], are known to occur in yeast [39] and plant [40] cells in hypoxic conditions. These data suggest that the acute induction of liver ADHs observed in mouse by a small dose of ethanol is also due to hypoxic conditions of the liver cell, where NADH/NAD ratio is increased by ethanol metabolism [41]. ADH3 may be related to brain disorders due to hypoxia, because it distributes in the brain in areas that are vulnerable to ischemia or hypoxic cerebropathy [23] as the sole ADH isozyme in the brain [42]. When the ethanol dose was medium [24], the liver ADH1 content decreased from the early stages to the end of alcohol metabolism. On the other hand, the liver ADH3 content increased in the early stages, but decreased in later stages, which is similar to the time-wise change found in the total ADH activity of the liver. For a large dose [24], the content of both ADHs in the liver markedly decreased, starting just after ethanol administration, which resulted in a marked decrease in total ADH activity. However, the ADH3 content recovered and increased in the later stages of alcohol metabolism, although that of ADH 1 kept decreasing to the end. Thus, at intoxicating doses of ethanol, ADH3 makes a positive contribution to alcohol metabolism, as shown by the time-wise change in its content in the liver during alcohol metabolism, whereas the contribution of ADH 1 decreases because its content in the liver decreases. Another point is that the ADH3 activity of liver extract can be measured by using hexenol as a substrate and 4-methylpyrazole to inhibit ADH1 [43]. Even though the ADH3 content exhibited a time-dependent change at intoxicating doses of ethanol (3, 5 g/kg), the ADH3 activity assayed by this method remained high during alcohol metabolism, as observed for the small dose [44]. Furthermore, the catalytic efficiency (Vmax/Km) of the ADH activity of liver extract for ethanol increased dose- dependently [45]. These data suggest that at intoxicating doses of ethanol, ADH3 activity remains high by regulation of both enzyme composition and enzyme kinetics to compensate for the reduced role of ADH1 in alcohol metabolism. Thus, both ADH1 and ADH3 contribute to alcohol metabolism, dynamically changing the enzyme composition and kinetics.