Mature male Sprague-Dawley rats were obtained from the Genetic Engineering Research Institute(GERI in KIST, Taejeon, Korea). All animals were maintained with standard laboratory food for rats and sterilized water. The animal quarters were maintained at 21–24°C and 40–60% relative humidity. A 12-hour light and dark circadian cycle was repeated.

Collagenase (Type 1), EGTA, Trypan blue, PG, DMSO (dimethyl sulfoxide), 2-thiobarbituric acid (TBA) and 1,1,3,3,-tetramethoxypropane (TMP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Insulin and penicillin were obtained from E.R. Squibb & Sons, Inc. (Princeton, NJ, USA). Strepytomycin was purchased from Eli Lilly (Indianapolis, In, USA). Dulbecco’s MEM was obtained from Flow Laboratories, Inc. (McLean, VA, USA). Vitrogen 100 purified bovine dermal collagen (type 1) was purchased from Collagen Corporation (Palo Alto, CA, USA). Bio-Rad, as reagents utilized for protein assays, were obtained from Bio-Rad Laboratories (Cat. 500–0006 Hercules, Richmond, CA, USA). [³H]-thymidine was purchased form NEN Corporation (New England, UK).

Hepatotoxic agents (ethanol) and therapeutic agents (PG) were used for toxicological and pharmacological studies. All agents were added to the culture medium at 4 hour intervals after the initial plating, and the medium was changed every 24 hours for 3 days. Agents were dissolved in DMSO or distilled water. The concentration of DMSO to which the cultures were exposed (0.1% of culture medium) did not affect the cell response.

Experimental groups were divided into normal controls, a PG treated group, an ethanol treated group and an ethanol with PG combine treated group (ethanol+PG group). For the study of DNA synthesis, we used several kinds of PG concentration, such and 10⁻⁸M, 10⁻⁷M, 10⁻⁶M, 10⁻⁵M. PG was changed consecutively everyday for 3 days in the PG-treated group. In the case of ethanol & PG combine treated group, the concentration of PG was 10⁻⁶M, 10⁻⁵M and the ethanol was 100 mM, 200 mM. For the study of malondialdehyde production, ethanol was treated with 50 mM, 100 mM, or 200 mM. Also, ethanol in combination with PG was treated in the measurement of malondialdehyde.

Hepatocytes were isolated through the collagenase perfusion method proposed by Dickins et al.¹⁰⁾ Rats were anesthetized with urethan (1 g/kg body weight), and a strict aseptic technique was maintained throughout the following procedure: the abdomen was opened by a midline incision and the intestines were displaced to the left. Then the hepatic portal vein was cannulated with an 18 gauge catheter placement unit and perfusion of the liver in situ was initiated at a flow rate of 15ml/min with al Masterflex pump (Cole Parmer, Chicago. IL, USA). To allow the blood to escape and perfusate from the liver, the inferior vena cava was cut. After about 100–150ml of buffer had been perfused through the liver, the thoracic cavity was opened, the superior vena cava was cannulated with a 14 gauge catheter placement unit, and the inferior vena cave was ligated. This diverted the outflow of the liver perfusate to the superior vena cava cannula which was returned to the perfusion bottle for recirculation through the liver of the animal. Throughout the entire procedure, the perfusion buffer used was Ca²⁺-free Hanks’ balanced salt solution (HBSS) supplemented with insulin (10⁻⁷ M) and gentamycin sulfate (50 μg/ml), 0.5 mmol/l EGTA. It was maintained at 37°C and gassed continuously with 95% O₂ and 5% CO₂. After recirculation of the perfusion buffer was established, collagenase was added via a 0.45μm filter (Millipore) to the bottle of recirculation perfusion buffer (about 100ml), and perfusion was continued for 15–20 min by which time the liver had swollen. The liver was removed to a beaker containing warmed (37°C) perfusion buffer (50ml) to which collagenase had not been added, and the capsule was ruptured with sterilized scissors. Hepatocytes were released by gentle swirling of the liver and pipetting with a large bore pipette. The cell suspension was filtered through a 210μm nylon mesh into a beaker placed on ice. The filtrate was transferred to ice-cold, sterile 50ml centrifuge tubes, and the hepatocytes were sedimented by centrifugation at 50g for 4 minutes. The pellets were washed two to three times and the final sediment was resuspended in the perfusion buffer (about 20ml) and stored on ice until further use. The viability of the cells was assessed in the final cell suspension with 900μl of 0.4% (w/v) trypan blue in 0.95% (w/v) NaCl by allowing the mixture to stand for 5 minutes on ice. A sample was transferred to a chamber for determination of the viable and nonviable cells.

The cell suspension was diluted to 0.4×10⁶ cell/ml in hormone-supplemented complete AB media¹¹⁾ and 2ml were pipetted into dishes precoated with 100μg of rat tail collagen. After hepatocytes were inoculated into collagen-coated culture dishes, they were incubated at 37°C in a humidified 5% CO₂/95% air incubator for 72 hours and the medium was changed every 24 hours.

The rate of DNA synthesis was determined by pulse-labelling cultured cells with [³H]-thymidine at 37°C for 2 hours. After labelling, hepatocytes were washed two times each with cold saline. The cells were harvested by rubber policemen and solubilized with NaOH(0.2 N, 1ml). The cell solution was neutralized with an equal of 0.2 N HCl and precipitated with 10% trichloroacetic acid (TCA). After precipitating, TCA precipitated material were each washed twice with 5% TCA. The final pellet was solubilized with 1 ml of 0.2 N NaOH. Aliquots of this solution were used for measuring radioactivity, DNA amount and protein level. Incorporation of [³H]-thymidine was determined by liquid scintillation spectrophotometer. Results were presented as mean±SD of triplicate cultures from representative experiments. Each experiment was carried out at least three times with cells. DNA content was measured by the method of Labarca et al.¹²⁾ using the fluorescence spectrophotometer(Hitachi). All DNA determinations using Hoechst 33258 reagent were performed in a phosphate-saline (2M NaCl, 50 mM Na₂HPO₄, and 2 mM EDTA, pH 7.4) buffer containing 1μg of Hoechst 33258 reagent per milliliter. Aliquots of sample solution (0.1ml) were slowly diluted with 2.4ml of phospha-tesaline buffer. Following incubation for 5 min at 20°C, the fluorescence intensity was measured at the excitation wave length of 355 nm and the emission wave length of 460 nm. Calf Thymus DNA was used to generate a standard DNA curve.

The assay methods proposed by Guidet et al.¹³⁾ were used to assess malondialdehyde (a measure of lipid peroxidation). Final cultured hepatocytes were weighed, minced and homogenized immediately in 0.02 M sodium phosphate buffer, pH 7.4 (1:10 w/v). Immediately, 1ml of 17.5% trichloroacetic acid (TCA) was added to 1ml of the homogenate and the specimen was then placed on ice. After adding 1ml of 0.6% thiobarbituric acid and pH 2, the homogenates were placed in a boiling water bath for 15 minutes and then allowed to cool. One millimeter of 70% TCA was added and the mixture was allowed to incubate for 20 minutes. The sample was then centrifuged for 15 minutes at 2,000 rpm and optical density of the supernatant read at 534 nm against a reagent blank with a spectrophotometer. The amount of malonidialdehyde, expressed in nanomoles, was calculated with a molar extinction coefficient of 1.56×10⁵ M¹cm⁻¹.

All results were expressed as the mean±SD (standard deviation). The significance of the difference between mean values was assessed by unpaired Student’s t-test, and p value less than 0.05 was accepted.