Metallothionein Protection against Alcoholic Liver Injury through Inhibition of Oxidative Stress

ZHANXIANG ZHOU,* XIUHUA SUN,* AND Y. JAMES KANG1,*,†

*Departments of Medicine, Pharmacology, and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40202; and †Jewish Hospital Heart and Lung Institute, Louisville, Kentucky 40202

Summary:

Antioxidants have been shown to be an effective treatment of alcoholic liver disease. Metallohthionein (MT) is a cysteine-rich protein that has antioxidant properties. The goal of the study was to see if MT can offer protection against acute alcohol-induced hepatotoxicity and to find the connection between oxidative stress and alcoholic liver injury. Alcohol was given to wild-type mice and MT-overexpressing mice. They were observed to see liver injury, oxidative stress and ethanol metabolism associated changes. It was concluded that MT is effective in cytoprotection against alcohol induced liver injury, and that hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.

Procedures and Results:

The animals used were MT-TG mice and the wild-type C57BL/6 controls. The mice were divided into a control group and ethanol group. To simulate blood alcohol levels, behavioral effects, and physiological changes similar to human binge drinking, they followed a previously developed model in which the mice are given three doses of 25% ethanol. The amounts were based on body weight (5g ethanol / 1 kg body weight). 4 hours after the last dose, the mouse were anesthetized and then studied.

Liver tissues were homogenized in 4 volumes of 10 mM Tris-HCl buffer, pH 7.4, at 4°C. The homogenate was centrifuged then 200 μL the supernatant was transferred to microtubes to measure MT levels via a cadmium-hemoglobin affinity assay. Results showed that MT concentration was normally ~10 times higher in MT-TG mice. After given ethanol, MT concentrations increase in both mice, but the MT-TG mice still remained ~8 times higher than the wild type.

Serum alanine aminotransferase (ALT) activity was colorimetrically measured using a Diagnostic Kit from Sigma Chemical. It was found that ALT levels increased after ethanol consumption in wild type mice, but remained constant for MT-TG mice.

To determine hisopathological effects, the liver tissues were cut into 3mm thick slices, fixed with 10% neutral formalin, stained with hematoxylin and eosin then photographed. It was seen that the primary effect of ethanol in wild type mice was microvesicular steatosis. Some hepatocyte necrosis was also observed by cell enlargement and nuclear dissolution. In MT-TG mice, ethanol only caused minor microvesicular steatosis.

A and B are normal livers of wild type and MT-TG mice, respectively. C and D are the livers of wild type and MT-TG mice and received ethanol. Arrows indicate microvesicular steatosis and arrowheads indicate necrosis. CV represents the location of the central vein

To observe ethanol induced ultrastructural changes, the livers were fixed by vascular perfusion, and observed using a transmission electron microscope. It was observed that wild type mice who received ethanol had glycogen and fat accumulation, organelle abnormality, focal cytoplasmic degeneration, mitochondrial abnormalities and endoplasmic reticulum degeneration. In MT-TG mice, there was much less glycogen and fat accumulation and no change to the organelles.

A and B are normal liver samples from wild type and MT-TG mice, respectively. (C and E) and (D and F) are livers from wild type and MT-TG mice that received ethanol. M, mitochondria; RER, rough endoplasmic reticulum; L, lipid droplets; G, glycogen; FCD, focal cytoplasmic degeneration

Oxidized protein levels were based on carbonyl concentrations. Liver tissues homogenized, then 1.0 ml of homogenate was combines with w ml of 10 mM DNPH and 2 N HCL. The mixture was incubate for 1 hour at room temp, then the protein was precipitated out. The protein was dissolved again and insoluble debris was removed by centrifuge. The DNPH derrivated were measured at 360 nm. The concentration of carbonyl groups was calculated by using an absorbance coefficient 22 mM⁻¹ cm⁻¹. It was determined that protein carbonyl levels increased 2.7x in wild type mouse liver and that there was no significant increase in MT-TG mice.

Comments on the Experiment:

Over all, I believe these researchers had an excellent experiment. Rather than focusing on one particular outcome, they did multiple types of test to examine MT’s effect at protecting against alcohol. However one issue I had with this experiment was when they visualized liver samples. They provide example photos but do not discuss in detail how they compared images between wild type and MT-TG mice. Their lack of description in their protocol makes me assume they just eyed it, without taking significant measurements. Although their examples show a clear difference, I would be more comfortable if they did some analysis with ImageJ.

URL: http://ebm.rsmjournals.com/cgi/reprint/227/3/214.pdf