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Scientists have assumed that because the liver detoxifies alcohol, the metabolism of ethanol is what causes the impairments that lead to alcohol-induced liver disease.

One researcher at the Alcohol Research Center at the Veterans Affairs Medical Center wants to make sure that is true.

Dahn Clemens, Ph.D., adjunct assistant professor of internal medicine at UNMC, is working to determine how and what molecular mechanisms are being used, and which enzymes and cell functions are being impaired by alcohol.

"The difficult question is: Is it ethanol that causes these impairments or is it the metabolism of ethanol that is responsible?" Dr. Clemens asked.

In the liver, hepatocytes oxidize the majority of alcohol, or ethanol. A liver-specific enzyme, alcohol dehydrogenase, is responsible for the majority of this oxidation, which results in the production of a highly reactive intermediate acetaldehyde. This is then metabolized by aldehyde dehydrogenase to acetate. Researchers think the production of acetaldehyde may be responsible for some of the hepatic impairments that lead to alcohol-induced liver disease.

Approaching this question is difficult in vivo because it is hard to separate the differences between actual exposure to alcohol and the metabolism of alcohol.

The best way to study these differences would be to investigate ethanol metabolism in vitro. Unfortunately, when hepatocytes are removed from the liver and placed in culture, they rapidly de-differentiate and lose the ability to metabolize ethanol.

To perform these studies, Dr. Clemens constructed a recombinant hepatic cell line that stably and constitutively expresses alcohol dehydrogenase. The recombinant cells were constructed using a hepatacelluar carcinoma cell line, HepG2, which is incapable of metabolizing alcohol. He then genetically engineered the cells with a eukaryotic expression plasmid designed to express alcohol dehydrogenase in vitro. He termed them HAD cells.

Cell lines derived from cancer are used because cultured hepatocytes rapidly lose their specific functions, including the ability to metabolize ethanol.

"There is something about being in the liver that signals these cells to do what they are supposed to do. When you take them out of the liver and put them in an external environment, they stop. Tumor cells, however, have the ability to grow in an environment away from the liver. That’s why researchers culture cell lines that are carcinogenic, or derived from tumors," he said.

The HepG2 cell line expresses some of the functions of a normal hepatocyte, such as the asialoglycoprotein (ASGP) receptor, and it secretes albumin as well as a number of other plasma proteins. But, it doesn’t express alcohol dehydrogenase or cytochrome P450IIE1, the two major enzymes responsible for ethanol oxidation.

"Now, we could measure the parameters to see if the impairment is caused by ethanol or the metabolism of ethanol," he said. "We’re looking at specific biochemical responses in the cells."

Using this in vitro system, Dr. Clemens repeated some of the research on the ASGP receptor conducted by colleague Carol Casey, Ph.D., associate professor, internal medicine, and director of the biochemistry core facility at the Alcohol Center .

"Our experiments have shown that if we increase the amount of levels of acetaldehyde, we can also increase the level of impairment in the receptor. You can’t do that in an animal. You can increase the amount of acetaldehyde, but you can’t accurately measure it," he said.

The cells were exposed to 25 millimolar ethanol, which is equivalent to a human being legally drunk, for three to seven days. Impairments were noted after three days, but were most pronounced after seven. About a 40 percent impairment in the binding, internalization and degradation of the receptor were measured.

"With the receptors, we have a significant biological finding," Dr. Clemens said.

"When you drink alcohol, even if you don’t get drunk, a certain amount of liver damage occurs. Those cells have to be replaced. We think that over a long period of time there are slight assaults on the liver. The liver has to replace the damaged cells to regenerate itself. The epidermal growth factor receptor and other growth factor receptors located on the surface of the cells are very important in terms of helping the cell regenerate — signaling the liver to replace damaged hepatocytes. Eventually, over time, there’s a slight aberration in the signal," he said.

The effects of ethanol and ethanol metabolism on some of these biologically significant receptors — epidermal growth factors — is important because it initiates the signal transduction cascades that tells the cell to do a number of things, Dr. Clemens said.

"We don’t know if ethanol metabolism is causing this receptor to give the wrong signal, no signal, or too weak of a signal. All these are possibilities. We’re teasing out at what level in that signal transduction cascade the impairments actually are," he said.

Dr. Clemens’ research was funded by a VA Alcohol Center core grant for $250,000. He is continuing his research with a five-year $350,000 grant that began in September from the National Institute of Alcohol Abuse and Alcoholism.