Isolation and Partial Characterizations of Oval and Hyperplastic Bile Ductular Cell-enriched Populations from the Livers of Carcinogen and Noncarcinogen-treated Rats 1

An oval cell-enriched population was isolated using two isopyknic centrifugation steps in Percoli gradients from the livers of young adult male rats maintained for 6 to 12 weeks on a cholinedeficient diet containing 0.05% DL-ethionine. This cell population equilibrated sharply at densities ranging between 1.07 and 1.08 g/ml, possessed a mean cell diameter in fixed-cell smears of 13.6 pm, and showed viabilities of >95% as judged by trypan blue dye exclusion. Contamination of this population by hepatocytes and Kupffer cells was determined to be <1 % and between 4 and 14%, respectively. 7-Glutamyl transpeptidase activity was demonstrated both biochemically and histochemically to be the most constant marker for evaluating the oval cell-enriched pop ulation isolated at various times over the 6 to 12 weeks of the choline-deficient/DL-ethionine dietary regimen. In contrast, the percentages of nonhepatocytic cells showing labeling for DNA synthesis and for Â«-fetoprotein were both found to be the highest in the oval cell-enriched population isolated at 6 weeks and lowest in that obtained at 12 weeks of dietary treatment. Fur thermore, at 10 to 11 weeks, 19.2% of the nonhepatocytic cells in this population were positive for albumin, while 2.1% were positive for glucose-6-phosphatase activity, indicating some cells to be intermediate in function between the oval cell and the hepatocyte. In comparison, hyperplastic bile ductular epithelial cells in tissue preparations isolated from the livers of rats previ ously subjected to 13 weeks of chronic feeding of the noncarcinogenic cholestatic agent, 1-naphthyl isothiocyanate, or at 8 to 13 weeks following bile duct ligation were found to be strongly positive for 7-glutamyl transpeptidase activity, as well as to be positive for alkaline phosphatase activity, but to be essentially negative for glucose-6-phosphatase activity, glycogen content, and albumin production. However, an occasional bile ductular cell in these preparations was found to exhibit a strong cytoplasmic binding of [6,7-3H]estradiol, an indirect measure of Â«fetoprotein production. Also, a low, but demonstrable amount of DNA synthesis was noted in the bile ductular cells present in these preparations. Furthermore, a viable cell population highly enriched in bile ductular epithelial cells was isolated by isopyknic centrifugation in Percoli following enzymatic dissociation of the 1This investigation was supported by USPHS Grant R01 CA30102 awarded to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services. This study was presented in part at the 67th annual meeting of the Federation of American Societies for Experimental Biology, April 1983, Chicago, IL (39), and in part at the 74th annual meeting of the American Association for Cancer Research, May 1983, San Diego, CA (40). 2To whom requests for reprints should be addressed, at Department of Pathol ogy, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298. 3 In partial fulfillment of the requirements for the degree of Ph.D. in Pathology and Laboratory Medicine. Received September 1, 1983; accepted April 26, 1984. hyperplastic tissue preparation from bile duct ligated rats. The peak densities of this cell population ranged between 1.06 and 1.09 g/ml, and it was found to be composed essentially of small basophilic cells having a mean diameter of ~15 /Â¿m, as well as some intact bile ductules. Greater than 70% of these isolated cells were histochemically positive for 7-glutamyl transpeptidase activity, and this cell population exhibited a specific activity for this enzyme that was at least 3 to 4 times higher than that shown by the oval cell-enriched population from the carcinogentreated rats.