[CANCER RESEARCH 45. 564-571, February 1985] A Common Biochemical Pattern in Preneoplastic Hepatocyte Nodules Generated in Four Different Models in the Rat1 M. W. Roomi,2 R. K. Ho,3 D. S. R. Sarma, and E. Farber Departments of Pathology [M. W. R., R. K. H., D. S. R. S., E. F.] and Biochemistry [E. F.], University ol Toronto, Toronto, Ontario, Canada M5S 1A8 ABSTRACT Because of the central and presumably critical role of hepa tocyte nodules in the development of liver cancer in the rat (28), Hepatocyte nodules, structures consistently seen in every it was considered important to understand much more about model of liver carcinogenesis well before the first appearance of their biochemical properties. Initial orientation was given to the cancer, were examined with respect to some Phase I and Phase search for a possible biochemical basis for the resistance phe II components considered to be important in the metabolism of notype that allows for the genesis of nodules in the resistant carcinogens and other xenobiotics. Phase I components are hepatocyte model of liver carcinogenesis (24,26). The resistance those related to the metabolism of xenobiotics and include phenotype is expressed in this model in an obvious manner by microsomal cytochromes P-450 and mixed-function oxygenase the ability of altered hepatocytes, induced during initiation by activities. Phase II components are those related to the conju carcinogens, to proliferate to form nodules in the presence of a gation and detoxification reactions of xenobiotics and their me dose of a carcinogen that inhibits the proliferation of the majority tabolites and include glutathione S-transferases and glutathione. of hepatocytes (87, 89). The new phenotype also consists of a Nodules were induced by the resistant hepatocyte, choline- resistance on the part of nodule hepatocytes to the necrogenic deficient, methionine-low diet, phénobarbital and orotic acid effects of CCI4 and DMNA4 in vivo (32) and of aflatoxin B, in models of liver carcinogenesis. Also, nodules generated by the vitro (44, 51, 74), a decrease in the metabolic generation of resistant hepatocyte model were examined after transplantation derivatives of DMNA and 2-AAF capable of binding to DNA, to the spleen of syngeneic animals. The hepatocyte nodules RNA, and protein (32) and in a decreased metabolic conversing show a common biochemical pattern, consisting of decreased of 2-AAF to mutagenic derivatives (92). microsomal cytochromes P-450, cytochrome b5, and aminopyr- Some components of the microsomal mixed-function oxygen ine A/-demethylase activity and increased glutathione and y- ase system, including cytochromes P-450 (phase I components) glutamyltransferase in whole homogenates and glutathione S- and of the conjugating and detoxification systems for xenobiotics transferase activity in the cytosol. This similarity, appropriate to and their metabolites including glutathione, GST, and GGT a resistance phenotype, adds additional support for the hypoth (Phase II components), were examined first as a possible basis esis that hepatocyte nodules may be a common step in liver for the resistance phenotype of the hepatocytes in the nodules. carcinogenesis in several different models. These components are considered to be related to the metabo lism of many carcinogens and other xenobiotics (8-10, 20, 42, INTRODUCTION 43,46, 56, 63, 98). The study was then extended to hepatocyte nodules generated in some other models. Included are data on Nodular proliferations of hepatocytes, variously designated as nodules transplanted to the spleen of syngeneic animals that adenomas, hyperplastic nodules, and neoplastic nodules and were never exposed to a carcinogen. Despite the differences in herein designated by the neutral noninterpretive term, hepato the nature of the models, the nodules show an unusual degree cyte nodules, are consistently seen in liver carcinogenesis with of commonality in respect to a biochemical pattern. The experi every model, well before the appearance of hepatocellular cancer mental basis for this tentative conclusion and some of its impli (4,16,19, 24-27, 30, 35, 36, 38, 47-49, 65-70, 73, 76, 77, 79, cations are the subjects of this communication. 82-84, 87, 89, 91, 94, 96, 97, 99). Their possible importance as a step in the carcinogenic process is indicated not only by their regular occurrence but more definitively by their role as a site of MATERIALS AND METHODS origin for liver cell cancer with 5 different carcinogens in several Animals models (19, 23, 38, 69, 70,79,89). When studied in some detail, the nodules show consistent and characteristic patterns of cell Young male Fischer F344 rats, either weanling or weighing from 130 organization and structure, architecture, histochemistry, and to 150 g and 5 to 6 weeks old, and male Sprague-Dawley rats, weanling physiology and are phenotypically quite different from liver at or young adults weighing from 150 to 170 g (both from Charles River any stage in its normal development (24, 25, 28, 30, 31). Breeding Laboratories, Wilmington, MA), were used. The rats were maintained on a semisynthetic moderately high-protein (26%) basal diet 1This research was supported by grants from the National Cancer Institute of (Dyets, Bethlehem, PA) and a 12-hr light and 12-hr dark daily cycle. They Canada, the National Cancer Institute, NIH (CA 21157, CA37077), and the Medical Research Council of Canada (MT-5594). A preliminary report of some of this were given food and water ad libitum and were acclimatized to their research was presented at the Annual Meeting of the American Association for environment for at least 1 week before their use in any experiment. Cancer Research in Toronto in May 1984 (75). 2 To whom requests for reprints should be addressed, at Department of Pathol ' The abbreviations used are: DMNA, dimethylnitrosamine; 2-AAF, 2-acetylami- ogy, University of Toronto, Medical Science Building, Toronto, Ontario, Canada nofluorene; RH, resistant hepatocyte; CMD, choline-deficient, low-methionine diet; M5S 1A8. PB, phénobarbital; OA, orotic acid, DENA, diethylnitrosamine; GSH, glutathione 3 Present address: Department of Pathology, Guangxi Medical School, Nanning, (reduced); GST, glutathione S-transferases; PMS, postmitochondrial supernatant; Guangxi, China. GGT, 7-glutamyltransferase; APD, aminopyrine-W-demethylase; CDNB, 1-chloro- Received June 8, 1984; accepted November 2, 1984. 2,4-dinitrobenzene; i.g.. intragastrically. CANCER RESEARCH VOL. 45 FEBRUARY 1985 564 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. BIOCHEMICAL PATTERN OF HEPATOCYTE NODULES Diets and Chemicals 2HCI, 100 mg/kg body weight, injected i.p. at 18 hr after partial hepatec tomy. The animals were placed on the basal diet containing 1% OA for The CMD, and 0.02% 2-AAF diets were from Dyets (Bethlehem, PA) 48 weeks. All animals received a single necrogenic dose of CCU in com (Nos. 100670 and 101121, respectively). The CMD diet was the same oil at 7 weeks (52, 71). A control group received the same dietary as that used by Sells et al. (82) and Shinozuka et al. (83, 84). The PB regimen without partial hepatectomy plus 1,2-dimethylhydrazine, i.e., diet was the same basal diet as used for the 2-AAF diet and contained without initiation. 0.05% PB. For harvesting of nodules, the animals were anesthetized with ethyl The following chemicals were purchased from Sigma Chemical Co., ether, and the livers were rapidly removed, chilled, and weighed. The St. Louis, MO: o-glucose 6-phosphate (disodium salt); NADP+; glucose- hepatocyte nodules were easily identified by their grayish-white color 6-phosphate dehydrogenase (catalogue No. G8875; approximately 600 and sharp demarcation, from the surrounding reddish-brown liver. The units/ml); nicotinamide; L-7-glutamyl-p-nitroanilide; glycylglycine; GSH; nodules ranged from 0.5 to 3.5 cm in diameter. No obvious hepatocellular CDNB; 5,5'-dithiobis(2-nitrobenzoic acid); and bovine serum albumin. carcinomas were included. This was checked by histological examination Sodium dithionite and aminopyrine were from Aldrich Chemical Co., of a portion of each nodule. The nodules were rapidly separated from Milwaukee, Wl. All chemicals were of the highest reagent grade. the surrounding liver, and all the nodules (5 to 20 per liver) from each animal were pooled. Each such pool was considered as a single speci Experimental Design men. Hepatocyte nodules were generated in rats with 4 different regimens. Preparation of Serum and Tissue Fractions and Determinations RH Model. Fischer rats were initiated in Group A with DENA, 200 mg/ kg body weight, given ¡.p.;and in Groups B and C, with DENA, 50 mg/ Blood was withdrawn by cardiac puncture or from the aorta from kg body weight, given i.p. at 18 hr after partial hepatectomy. Animals of anesthetized animals. Serum was collected for determination of GGT all 3 groups were placed on the basal diet for 2 weeks. Those in the activity. Carefully dissected hepatocyte nodules and surrounding liver control group, Group C, were continued on the basal diet for an additional and livers from control animals were rinsed twice with ice-cold 0.09% 52 weeks. For selection of resistant hepatocytes, those in Groups A and NaCI solution, weighed, and homogenized in 3 volumes of 0.05 M 4-(2- B were placed on the 0.02% 2-AAF diet for 2 weeks and received CCU hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.0) containing 0.2 ml/100 g body weight, diluted 1:1 with com oil i.g. at the midpoint, 0.2 M sucrose and 1 HIM EDTA. Homogenates were centrifuged at at the end of 1 week (87, 89,97). The animals were placed on the basal 10,000 x g for 30 min, and the PMS removed and centrifuged at 100,000 diet after the end of the 2-week 2-AAF diet and remained so for 52 x g for 60 min.