[CANCER RESEARCH 38, 823-829, March 1978] y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1 Ross Cameron,2 John Kellen, Arnost Kolin, Aaron Malkin, and Emmanuel Farber Department of Pathology. University of Toronto. Toronto U5G 1L5 ¡R.C., A. K., E. F.], and Department of Clinical Biochemistry, Sunnybrook Medical Centre, Toronto M4N 3U5 [J. K., A. M.¡,Ontario Canada ABSTRACT the fetus but very low activity in the adult. In primary tumors induced by aflatoxin B,, 3'-Me-DAB, N-OH-2-AAF, and 2- The activity of y-glutamyltransferase, as measured AAF and in transplantable hepatomas of rats, a 30- to 50- quantitatively and by histochemical staining, was studied fold increase in GGT activity has been observed (8, 9, 11, in different cell populations during the induction of liver 34). GGT activity of whole liver showed a 5-fold increase as cancer with 2-acetylaminofluorene (2-AAF) or diethylni- early as 4 weeks after 2-AAF treatment, by 6 weeks after 3'- trosamine and compared with findings in fetal and in Me-DAB, and by 8 weeks after N-OH-2-AAF (9, 11). Since intact and regenerating adult liver. The enzyme activity is duct epithelial cells show the highest concentration of GGT 20-fold higher in 12-week nodules than in control livers in normal liver as judged by histochemistry and since and 30-fold higher in 20-week nodules than in controls. A proliferation of these cells ("oval cells"), often to striking similar 30-fold increase in activity relative to control is degrees, is a common reaction in the liver during carcino- present in hepatomas, induced by either 2-AAF or dieth- genesis with many chemicals, the interpretation of the ylnitrosamine, and in fetal hepatocytes. The enzyme increase in GGT activity in homogenates of whole liver is shows increases in activity in foci of very early putative complicated. Histochemically, GGT is demonstrable in pro preneoplastic hepatocytes induced by a single dose of liferating bile duct cells [oval cells (5)] after 4 weeks of N- diethylnitrosamine and selected by low doses of 2-AAF hydroxy-A/-2-fluorenylacetamide treatment (11) and in hy- plus partial hepatectomy. By 7 days, the foci show a 4- perplastic nodules induced by aflatoxin B, (15) and N-OH-2- fold increase in enzyme activity, and by 3 weeks they are AAF (11). These observations suggested that GGT may be a 40-fold higher than in the control liver. Histochemically, marker not only for neoplastic hepatocytes but also for the foci are strongly positive for y-glutamyltransferase, preneoplastic hepatocytes (15). especially in the bile canaliculi. By 21 days, the ductular A number of hepatocyte populations have been deline (oval) cells induced by 2-AAF have disappeared. When ated as possible precursor lesions for liver cancer induced stained for the enzyme activity, the foci stand out clearly by chemical carcinogens (7). Recently, in this laboratory, a against the negative background of the liver, allowing putative premalignant population of hepatocytes, "the fo easy quantitation. It appears that y-glutamyltransferase cus," has been identified morphologically within 1 week is a useful marker for preneoplastic hepatocytes. after carcinogen treatment (29). These proliferating foci of hepatocytes emerge as early as 30 hr after partial hepatec INTRODUCTION tomy in the livers of rats treated with a single dose of diethylnitrosamine i.p. to "initiate" and with a diet contain Tissue antigens and enzymes that will serve as pheno- ing a low level of 2-AAF to "select" the initiated 2-AAF- typic markers for malignant cells are becoming increasingly resistant hepatocytes during the regeneration period after important as diagnostic aids (2, 20, 27). Some of these hepatectomy (29, 30). These foci have been produced with markers may appear in the blood and other body fluids and a number of hepatocarcinogens as initiators, namely, dieth allow for early identification of the presence of neoplastic ylnitrosamine, A/-hydroxy-/V-2-fluorenylacetamide, dimeth- cells (11, 27). In the liver, a-fetoprotein, preneoplastic ylnitrosamine, and aflatoxin B,. The foci show a functional antigen, and GGT3 have been identified as possible positive resistance to the cytotoxic effects of carcinogens, a prop markers for preneoplastic hepatocytes (4). GGT has been erty found in hyperplastic nodules and hepatomas (1, 6, studied both biochemically and histochemically in many 10). In order to confirm the possibility that GGT could be a tissues. GGT is found in a number of epithelial cells includ marker for premalignant hepatocytes, putative premalig ing those located in jejunal villi, pancreatic acini, bile ducts, nant hepatocyte populations including early hyperplastic seminal vesicles, and proximal convoluted tubules (23, 25), nodules, late hyperplastic nodules, and foci were examined as well as in lymphocytes, the retina, and choroid plexus for GGT activity, both biochemically and histochemically. (19, 22). In the liver, hepatocytes show high GGT activity in The striking increase in activity of this enzyme in such hepatocytes in comparison with those in normal and regen 1 This research was supported in part by grants from the National Cancer erating liver is the subject of this communication. Institute of Canada and Medical Research Council of Canada and the Connaught Development Fund of the University of Toronto. 2 Research Fellow of the National Cancer Institute of Canada (1974 to MATERIALS AND METHODS 1976). 3 The abbreviations used are: GGT, -y-glutamyltransferase (EC 2.3.2.2); 3'- Me-DAB, 3'-methyldimethylaminoazobenzene; N-OH-2-AAF. N-hydroxy-2- Animals and Carcinogenic Diet Regimens. For produc acetylaminofluorene; 2-AAF, 2-acetylaminofluorene. tion of foci, male Fischer 344 rats (Charles River, Wilming Received September 6. 1977; accepted December 13, 1977. ton, Mass.) weighing 100 to 200 g were given 1 dose of MARCH 1978 823 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. R. Cameron et al. diethylnitrosamine, 100 or 200 mg/kg i.p. Two weeks later, to 10 min. Protein was determined according to the method the rats were fed a standard basal diet (24% protein) of Lowry ef al. (18). containing 0.02% 2-AAF (Bioserv, Frenchtown, N. J.) for 1 •y-Glutamyl-p-nitroanilide, glycylglycine, and bovine se week and were then subjected to two-thirds partial hepatec- rum albumin were obtained from Sigma Chemical Co., St. tomy (12). 2-AAF was continued 1 week after hepatectomy Louis, Mo. All other chemicals were from other commercial (Fig. 1), at which time multiple foci are visible grossly in the sources except as specified. liver as small, grayish white nodules about 1 mm in diameter GGT Histochemistry. The GGT was detected according (29). Essential controls were treated as shown in Chart 1. to the method described by Rutenberg ef al. (26). Slices of For chronic feeding experiments, the basal diet containing liver with foci, nodules, hepatomas, or control liver were 0.02% 2-AAF was fed to rats for 20 weeks continuously, and serially cut in a cryostat (5 nm), air dried, fixed in ice-cold then the rats were given Purina rat chow. For diethylnitros- acetone for 30 min, and incubated in freshly prepared amine-induced hepatomas, diethylnitrosamine was given in medium containing y-glutamyl-4-methoxy-2-naphthylamide the drinking water continuously (50 ppm) for 36 weeks. (Vega-Fox Biochemicals, Tucson, Ariz.) as substrate and Biochemistry. All animals were sacrificed by cervical Fast Blue BB salt (GURR-Searle Diagnostic, High Wy- fracture after a 24-hr period of fasting. Grossly visible combe, Buckinghamshire, England) as coupling agent (26). nodules and foci were shelled out. Where sufficient tissue Spare sections were stored at 4°and retained their enzy (about 0.5 g) could be obtained, biochemical analysis was matic activity unchanged for at least 6 months. Control performed on tissue from each animal separately; other sections incubated in a similar medium without substrate wise, nodules or foci and surrounding nonnodular liver were negative. tissue were pooled. Careful histological and histochemical studies were performed on all tissues used for biochemical assay. The tissue was fixed in Carnoy's solution for histo RESULTS logical examination and stained with hematoxylin and eosin. Histology. Animals fed the continuous 0.02% 2-AAF regi Gray-white hyperplastic nodules were identified as early men develop grossly visible gray-white nodules within 6 to as 6 to 8 weeks in livers of animals fed the 0.02% 2-AAF diet 8 weeks after beginning the diet. Such nodules, microscop continuously, and at 12 weeks they were sufficiently large ically, are typical hyperplastic liver nodules (3). Seven for gross isolation for discrete biochemical and morpholog months after the beginning of 2-AAF feeding, a few rats ical study. Late liver-colored hyperplastic nodules develop develop hepatocellular carcinomas and by 1 year most rats by 16 to 20 weeks on a continuous 2-AAF diet (3, 6). have hepatomas. With diethylnitrosamine given continu Assay for GGT. Tissue was homogenized in 10 volumes ously in the drinking water, rats first develop hepatomas at of ice-cold 0.25 M sucrose with 10 strokes of a Thomas about 10 months following the beginning of carcinogen homogenizer. The homogenate was centrifuged at 1,000 x treatment, and by 1.5 years most rats have hepatomas. With g for 10 min, and the pellet containing nuclei and debris a single dose of diethylnitrosamine (250 to 300 mg/kg ¡.p.), was discarded. The supernatant was further centrifuged at some rats develop hepatomas by 12 to 15 months. All the 100,000 x g for 60 min (International IEC B60 centrifuge hepatomas were typical trabecular hepatocellular carcino with Rotor A169) to pellet the nonnuclear particulate frac mas. tion. This pellet was resuspended in 5 volumes of 0.1 M Foci appear very early following initiation with a single Tris-HCI (pH 7.6) containing 0.01 M MgCL and used for dose of diethylnitrosamine and selection with 2-AAF com assay.