A Common Biochemical Pattern in Preneoplastic Hepatocyte Nodules Generated in Four Different Models in the Rat1

[CANCER RESEARCH 45. 564-571, February 1985]

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, phÃ©nobarbital 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, phÃ©nobarbital; 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

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 Â¡.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. The microsomal pellet was rehomogenized in 4-(2- weeks. hydroxyethyl)-1-piperazineethanesulfonic acid buffer and resedimented The RH model was also used to generate hepatocyte nodules that once at the original speed. Measured aliquots were removed from the were transplanted to the spleen. Fischer rats were initiated with DENA, whole homogenate and from the PMS. 200 mg/kg body weight, given i.p. After a recovery period of 2 weeks, GGT activity (EC 2.3.2.2) was determined in serum and whole homog- the animals were selected for resistant hepatocytes by dietary 2-AAF for enates by the method of Szasz (93) using 7-glutamyl-p-nitroanilide as 2 weeks plus partial hepatectomy midway at the end of 1 week. The substrate and glycylglycine as glutamyl receptor. Total soluble sulfhydryl content ("GSH") was determined by the method of Ellman (15) using the animals were then placed on the basal diet for 21 weeks at which time the liver contained from 5 to 10 persistent hepatocyte nodules. The supernatant after adding 0.2 ml of 25% trichloroacetic acid to 0.2 ml of grayish-white nodules were dissected cleanly from the surrounding liver 25% liver homogenate and centrifuging at 3000 x g for 10 min. and were coarsely minced. Aliquots were transplanted into the spleens APD activity (EC 1.14.14.1) was measured in the PMS in a total of normal F-344 rats without any exposure to initiating or selecting volume of 5 ml containing 0.02 M potassium phosphate buffer, pH 7.4, procedures and allowed to grow into nodules for 70 weeks. At this time, 0.2 rriM NADP+, 8 HIM glucose 6-phosphate, 10 mw nicotinamide, 5 ITIM the nodules measured from 2 to 4 cm, were uniformly grayish-white, and MgCI2,1 (TIMaminopyrine, 3 units of glucose-6-phosphate dehydrogen on microscopic examination were typical of hepatocyte nodules without ase, 20 to 25 mg of PMS protein and water to volume. After incubation evidence of hepatocellular carcinoma. at 37Â°for 30 min, the reaction was stopped by adding 4 ml of 10% (w/ CMD Model. In the CMD model (82-84), Sprague-Dawley rats were v) ZnCI2, and after mixing, 2 ml of saturated BafOHfe. Five-mi aliquots initiated in Group A with DENA, 200 mg/kg body weight, i.p. and in from the supernatant after centrifugation at 3000 x g for 10 min were Group B with DENA, 50 mg/kg body weight, given i.p. at 18 hr after used to measure formaldehyde by the method of Nash (58). A standard partial hepatectomy. Animals in Group C were not initiated. Two weeks curve for formaldehyde, run with every group of determinations, was later, animals of all 3 groups were placed on the CMD diet for 52 weeks. used to calculate nmol of formaldehyde generated. PB Model. In the Peraino model (65, 66), weanling Sprague-Dawley Cytochromes P-450 and cytochrome Â£>5weredetermined in the micro rats were fed the 0.02% 2-AAF diet for 14 days followed by the basal somal fraction. The total cytochrome P-450 was measured in microsomal diet for 7 days. They were then placed on the basal diet containing aliquots containing protein (2 mg/ml) by the method of Omura and Sato 0.05% PB for 50 weeks. Animals in the control group (Group B) were (62). Cytochrome bÂ¡was measured in microsomal aliquots of the aarne placed on the 0.05% PB diet for 50 weeks without prior initiation with 2- concentration by the method of Mazel (55). Total microsomal heme was AAF. determined by the method of Paul ef al. (64). In the Pilot model (67, 68), Fischer rats were initiated with a single Glutathione S-transferase (EC 2.4.1.18) activity was determined in the dose of DENA, 10 mg/kg body weight, injected i.p. at 20 hr after partial cytosolic fraction. The total cytosolic GST activity was assayed by the hepatectomy. After being fed the basal diet for 3 weeks, they were method of Habig et al. (41) using CDNB as indicator. placed on the basal diet containing 0.05% PB for 48 weeks or for 44 Protein concentrations in all fractions were determined by the method weeks plus basal diet without PB for 4 weeks. A control group of animals of Lowry et al. (54) as modified by Miller (57) with bovine serum albumin were fed the PB diet for 48 weeks or for 44 weeks plus 4 weeks basal as standard. All determinations were expressed as concentrations per diet without PB. mg of protein. Student's f test was used to assess the degree of significance and OA Model. In the OA model, (11, 52, 71), for OA nodules No. 1, Fisher's transformed correlation coefficient to assess the degree of Fischer weanling rats were given DENA, 10 mg/kg body weight, injected i.p.; five weeks later, during which they were fed the basal diet, they association between different biochemical procedures (85). Results were were placed on the basal diet containing 1% OA for 48 weeks. For OA obtained using the Hewlett-Packard programmable calculator (Hewlett- nodules No. 2, Fischer rats were initiated with 1,2-dimethyl-hydrazine- Packard, Loveland, CO).

CANCER RESEARCH VOL. 45 FEBRUARY 1985 565

RESULTS PB models. Many if not the majority of hepatocytes in the nodules from the different models show nuclear changes in that there is a more "open" appearance with more obvious euchromatin and The animals in the RH model that received DENA, 200 mg/kg body weight, for initiation (Group A) each had one hepatocellular less obvious heterochromatin. Whether this is reflected in iso carcinoma at 52 weeks, as reported previously for other experi lated chromatin has not been reported. ments (86). Of these, 25% showed lung mÃ©tastases.Of the 5 The hepatocyte nodules seen in the livers of rats with the RH, animals in Group B, 2 had carcinomas with no mÃ©tastases.The CMD, PB, and OA models as well as those that grew in the carcinomas measured from 3.5 to 5 cm in greatest diameter and spleen after transplantation show a common pattern of change showed the typical gross appearance of necrosis, hemorrhage, in 6 different biochemical parameters (Table 1). The microsomal and umbilication. All the animals in Groups A and B had hepa- Phase I components, cytochromes P-450 and APD, show a tocyte nodules. These were grayish-white and fairly circum consistent low level of between one-fourth and one-third that in scribed and measured from 2 to 3 cm in diameter. These the appropriate controls. Another microsomal cytochrome, cy- persistent nodules (17, 95) were quite uniformly grayish-white tochrome os, is also well below the levels in the controls. The on section and showed no gross (or microscopic) evidence of total microsomal heme content was measured in only one of cancer. Each liver contained from 7 to 10 such nodules, which nodule, those in the spleen, and was 0.95 Â±0.23 (S.D.) as were easily distinguished from the cancers. One of the 5 control compared to 2.00 Â±0.20in the control host normal liver. These animals (Group C) contained a very small nodule measuring 2 values for the total heme agree well with the levels of the P-450 mm in diameter, while the others were without nodules. cytochromes and cytochrome 05 as the major heme components The animals in the CMD model showed only a very mild fatty in microsomes. liver with only minimal fibrosis. The animals initiated with DENA, The levels of the microsomal cytochromes P-450 as well as 200 mg/kg body weight (Group A), showed a 20% incidence of the activity of APD in hepatocyte nodules in the 5 model systems carcinoma (2 of 5) with no mÃ©tastases,while the animals initiated used in this study were compared with published values for with DENA, 50 mg/kg body weight (Group B), showed no liver nodules generated in quite different models, especially the cancer at 52 weeks. All animals in Group A and B had grayish- models that use periods of exposure to dietary 2-AAF or ethion- white nodules that ranged from 1.5 to 3 cm in diameter and ine alternating with exposure to control diets (3, 7, 22, 33, 39, numbered from 5 toi 2 per liver. 45, 61, 78). As recorded in Table 2, the mean value for the P- The liver cancers in Group A measured from 2.5 to 5 cm in 450 cytochromes in nodules in 8 published reports using the diameter and were typical for hepatocellular carcinomas with other models is 0.35 with a range of 0.20 to 0.50 as compared umbilication, necrosis, and hemorrhage. The nodules were read to 0.29 (0.20 to 0.42) in this study. For APD, the mean value ily distinguished from the cancers. from 5 published reports (3, 5, 33, 39, 78) is 0.39 (0.24 to 0.50) The animals in the PB model, both Peraino and Pilot types, as compared to 0.35 (0.12 to 0.50) in this study. had larger livers in the controls because of the inductive effects With respect to glutathione, all the nodule populations studied of the phÃ©nobarbital. All thÃ¨animals had grayish-white nodules show significant elevations of total soluble sulfhydryl content in that varied from 0.5 to 3.5 cm in diameter, but no liver cancers the nodules as compared to control livers. The range of eleva could be identified either grossly or microscopically. tions was from 2.3- to 5.8-fold. Although the method of Ellman The animals in the OA model, 6 per group, each showed one (15) measures all soluble sulfhydryl groups, the major contributor hepatocellular carcinoma and several grayish-white hepatocyte to this pool is GSH. In a comparison between the values with nodules. The carcinomas measured from 4 to 6 cm in diameter the Ellman method and with the more specific method of Griffith and showed the usual necrosis, umbilication, and hemorrhage. (40), the differences in various livers were small and not of major The nodules measured from 1 to 3.5 cm in diameter. Two to 10 magnitude (74). were present per liver. The surrounding liver showed only minimal The large elevations in GGT (Table 1) offer quantitative agree fatty change with no fibrosis. The control animals on AO without ment with the histochemical findings of increased GGT activity initiation had neither nodules nor cancer and were minimally in the nodules in each model studied (6, 11, 34, 67, 70, 71, 81, fatty. 82, 89, 97). Previous quantitative studies on nodules with other The histological and cytological appearances of the nodules in models (5, 6, 22,34) show an even larger increase in the activity the 4 different models and in the nodules generated in the RH of GGT (Table 2), but in these reports, particulate protein rather model and transplanted to the spleen were remarkably similar, than total homogenate protein was used as the reference value. as described previously for other models (24, 25), with one GGT is known to be located predominantly in liver cell mem distinctive feature for the PB models, the obvious excess prolif branes. eration of smooth endoplasmic reticulum in both the surrounding The hepatocyte nodules in each of the models studied showed and nodule hepatocytes. All the nodules showed a similar phon a significant elevation of GST activity using only one substrate, otype with respect to organization, architecture, and cytological CDNB. This substrate is useful as a general one for GST activity characteristics. Unlike normal liver with its predominant architec but has different activities with different transferases (42, 43). tural pattern as single-cell plates (72), the hepatocytes in the The range in increments in this study is somewhat lower than in nodules in the different models are arranged in plates usually 2 the previous published reports (Table 2) using other models but or 3 cells thick or in a glandular arrangement. The hepatocytes is in the same general direction (3, 22). often show the presence of glycogen and considerable eosino- An interesting finding in this study was the consistent elevation philic cytoplasm corresponding usually to abundant smooth en of serum GGT activity in every model (Table 3). In the liver doplasmic reticulum. As expected, this is particularly obvious in models, it is impossible to distinguish a major or only contribution the hepatocytes in both the nodules and surrounding liver in the of nodules to a change in concentration of a blood component

CANCER RESEARCH VOL. 45 FEBRUARY 1985 566

Table 1 Comparison between the activities or concentrations of some microsomal, cytosolic, and whole-liver homogenate components in hepatocyte nodules in rats induced by 5 different procedures or models Constituent Homogenate PMS Microsomes Cytosol

P-450 (milliunits/mg (nmol/mg Â£>5 ModelRH-control protein)4.5 (^g/mgprotein)5.0 (nmol/hr/mgprotein)15.8 protein)0.70 (nmol/mgprotein)0.41 protein)1.57 (Group C) (5)" Â±2.1" Â±0.8 Â±1.8 Â±0.05 Â±0.02 + 0.39 RH-GroupA-nodules (5) 60.0 Â±22.7 29.6 Â±8.9 1.8 Â±0.6 0.20 Â±0.08 0.26 Â±0.06 2.38 Â±0.20 RH-GroupA-SURRC(5) 16.2 Â±3.7 6.2 Â±1.2 15.7 Â±1.7 0.67 Â±0.07 0.47 Â±0.03 1.80 Â±0.23 RH-GroupB-nodules (5) 35.8 Â±12.6 28.1 Â±10.3 2.1 Â±0.4 0.16 Â±0.02 0.18 Â±0.06 2.67 Â±0.70 RH-Group B-SURR(5)GGT 11.4Â±5.5"GSH" 8.3 Â±2.5"APD" 15.8 + 2.9Cytochromes0.63 Â±0.08Cytochrome0.44 Â±0.03GST((imol/min/mg1.54 Â±0.15

RH-spleen transplant-host liver 2.4 Â±0.9 5.0 Â±1.1 12.7 Â±3.1 0.77 + 0.07 0.50 Â±0.06 1.64 + 0.35 (5) RH-spleen transplant-splenic 18.7 Â±6.6" 13.4 + 4.2" 6.4 Â±2.6e 0.32Â±0.10" 0.35 Â±0.06" 1.01 Â±0.87o nodule (5)

CMD-control(5)CMD-Group (Group C) Â±0.350.0 Â±0.420.4 Â±1.73.7 Â±0.070.1 Â±0.040.20 Â±0.082.88 (4)CMD-GroupA-nodules Â±10.09.9 Â±2.06.7 Â±1.013.6 5Â±0.080.83 Â±0.060.56 Â±0.441.98 (5)CMD-GroupA-SURR Â±0.820.8 Â±2.116.1 Â±2.14.4 Â±0.110.1 Â±0.070.1 83.1 Â±0.1 (4)CMD-GroupB-nodules Â±5.93.5 Â±0.14.8 Â±0.56.9 2Â±0.020.64 4Â±0.050.49 6Â±0.921 (5)PB-control B-SURR Â±1.13.1 Â±1.06.1 Â±1.134.6 Â±0.040.71 +0.080.48 .79 Â±0.421.90

(4)PB-Per-nodules(+PB) +0.410.5 Â±8.135.6 +0.110.94 +0.322.10 (5)PB-Per-SURR (+PB) Â±4.03.8+1.718.3 Â±5.031 +0.101.50 Â±0.431.39 (9)PB-PI-nodules(+PB) .2Â±3.946.5 80.89Â±0.1 +0.204.46 (3)PB-Pi-SURR (+PB) Â±12.31.9Â± Â±5.124.1 41.06Â±0.1 Â±1.682.66 (5)PB-control (+PB) 1.02.4 Â±1.911.3 Â±0.100.59 Â±0.251.44 (2)PB-Pi-nodules(-PB) Â±0.222.2 Â±4.25.9 +0.160.22 +0.162.801 (3)PB-Pi-SURR (-PB) Â±19.61.9 Â±1.614.7 Â±0.060.96 (5)OA-control (-PB) Â±0.51.6 Â±2.415.1 Â±0.050.68 .66 Â±0.260.78

(6)OA-nodules Â±0.241 Â±0.913.3 Â±0.25.1 +0.080.16 +0.030.22 Â±0.093.25 (6)OA-nodulesNo. 1 .2 Â±12.030.7 Â±2.514.8 Â±1.34.1 +0.060.11 Â±0.060.32 Â±0.602.64 No. 2 (6)1.9 Â±9.56.4 Â±3.58.5 Â±1.80.69 Â±0.020.40 Â±0.070.77 Â±0.58 Numbers in parentheses, number of animals. None of the control animals had any gross or microscopic nodules. Measurements were made on representative allqyots of the liver. b Mean + S.D. c SURR, surrounding liver; this portion of liver consists of nodules remodeled to varying degrees (often very advanced) plus an unknown amount of the original liver. +PB, basal diet containing 0.05% PB for 48 weeks; -PB, basal diet for 44 weeks plus basal diet without PB for 4 weeks; Per, Peraino model; Pi, Pilot model. "p < 0.001 ; highly significant (nodules versus control). 8 p < 0.01 ; significant (nodules versus control).

Table 2 as compared to other cellular lesions in the liver. This complica Comparison between relative activities or concentrations of some microsomal, tion is avoided in animals with the spleen transplants in which cytosolic, and whole-liver homogenate components in hepatocyte nodules, the liver is seemingly normal and the most likely source of the relative to control values, in rats induced by 5 different procedures or models Relative activity or concentration in nodules8 serum enzyme would be the hepatocyte nodule in the spleen. Not shown is the finding that the spleen does not contain any in thisstudy013.8(7.8-22.5)"literature029-170"in ComponentGGT GGT that can be visualized histochemically (53). ("paniculate") (3, 4, (whole homogenate) The data on the livers and hepatocyte nodules in animals on 17,29f "GSH" (whole homogenate) 3.4 (2.3-5.8)" continuous exposure to PB until the time of sacrifice are included + histochemical only (14, 59f for completeness. Under these conditions, the differences in 0.35(0.12-0.50)" 0.39 (0.24-0.50)" (1,3, 28, APD(PMS)Cytochromes 34, 70)" activities between nodules and control livers are minimized or P-450 (micro 0.29(0.20-0.42)"0.56 0.35 (0.20-0.50)" (1,5, 17, may even disappear (Table 1). When exposure to PB is termi somal) 28, 34, 40, 53, 70)e nated 4 weeks before the termination of the experiment, the (0.43-0.70)" 0.30 (33)" Cytochrome t>5(microsomal) 2.74(1.61-3.92)"Mean 3.90 (3.00-4.70)" (1,17)" differences between nodules and controls now become as evi GST (cytosolic)Mean 8 Control = 1.0; the control or "normal" liver in each reported experiment was dent as in the other models. considered as 100% (1.0). The values for the nodules are the activities or concen Results from control tissue and nodules were pooled, and trations relative to this standard value. correlation coefficients were calculated. As recorded in Table 4, In this study, the RH, CMD, and OA models were used to generate hepatocyte excellent correlations were obtained between the levels of micro nodules. '' In the published studies, totally different models were used. These models somal cytochromes P-450 and aminopyrine A/-demethylase, the used continuous or intermittent long-term exposure to carcinogens such as 2-AAF 2 Phase I components and between GST versus GSH and GSH or ethionine. " Numbers in parentheses, range. versus GGT. The correlation coefficient was smaller between e Numbers in parentheses, references. GST and GGT. The correlation coefficients between the activat-

CANCER RESEARCH VOL. 45 FEBRUARY 1985 567

Table 3 supply (12,13,24,25), several histochemical characteristics (26, Activities of GGTin serum of rats with different models of hepatocarcinogenesis 31 ), and gross appearance (25). Even though all properties have GGT activity not been examined in nodules from every model, there is a Model (units/liter) RH-control (Group C)a(5)" ND1- sufficient spread to indicate the likelihood of an unusual degree RH-GroupA" (5) 67.8 Â±15.9e of similarity among the nodule population from several different RH-GroupB' (5) 18.2 Â±2.7 models. This conclusion is reinforced by the recent finding that a M, 21,000 polypeptide is present in nodules from 6 different RH-spleentransplant (5) 10.1 Â±4.2 models but not in fetal, neonatal, regenerating, or mature liver CMD control (Group Cf (5) ND CMD Group A" (10) (21). 14.7 Â±6.2 Does the suggested commonality in phenotypic expression CMD Group B' (5) 13.1 Â±4.5 pertain not only to each nodule population but also to each OA control (5) ND individual nodule? This important question was not the objective OA experimental(10) 48.5 Â±2.8 of the current study and, therefore, no data are available. Such " Initiated with DENA,50 mg/kg body weight, plus partial hepatectomy and then a study with different models is under way. However, there are fed basal diet without any selection. b Numbers in parentheses,number of animals. some data from other studies. Even though individual micro c ND, not detectable. " Initiated with DENA,200 mg/kg body weight, selected with dietary 2-AAF plus scopic foci of altered cells, the precursors for nodules, do vary CCUand then fed the basal diet. considerably in some histochemical properties, such as GGT, * Mean Â±S.D. glucose-6-phosphatase, ATPase, and microsomal epoxide hy- 'initiated with DENA, 50 mg/kg body weight, plus partial hepatectomy and drolase (18, 60, 67, 70), the patterns in grossly visible nodules, selected and fed basal diet as for Group A. 9 Fed the CMD diet without prior initiation. both early and persistent, are much more uniform. For example, h Initiated with DENA,200 mg/kg body weight, and then fed CMD diet. well over 90% of hepatocytes in nodules in the RH model show ' Initiated with DENA, 50 mg/kg body weight, plus partial hepatectomyand then increased staining for epoxide hydrolase (18), GGT, and DT- fed CMD diet. diaphorase (quinone reducÃase)(60). Also, with the same model, Table 4 over 90% of hepatocyte nodules show increased staining for Correlationof phase I and II drug-metabolizing components in hepatic nodules GSH (1, 2). Using a different model, intermittent exposure to 2- generated in RH model AAF, AstrÃ¶mei a/. (3) found that "small" (1 to 3 mm in diameter), Results of "medium" (3 to 5 mm in diameter), and "large" (>5 mm in correlation diameter) all showed the same levels of the P-450 cytochromes, Variableanalyzed analysis p NADPH-cytochrome c reducÃase,epoxide hydrolase, and GST. PhaseI enzymes Cytochrome P-450 vs. aminopyrineN-demethylase 0.952 <0.001 Also, individual nodules in the same livers were quite similar in their biochemical patterns. Soil ef a/. (88) and Conway ef a/. (12, PhaseII components GST vs. GSH 0.807 <0.001 13) found that individual nodules above 1 mm in diameter were GSH vs. -GT 0.864 <0.001 similar in respect to the decrease in their portal blood supply. GST vs. -GT 0.622 <0.001 Clearly, Ihere is a need for further comparative studies in different PhaseI vs. Phase II components models and with more markers. However, the available evidence Cytochrome P-450 vs. GST -0.749 Cytochrome P-450 vs. GSH -0.889 points lo Ihe visible hepatocyte nodules being quite similar in at Cytochrome P-450 vs. -GT -0.799 leasl several phenolypic characteristics including the organiza a Phase I components are involved in the metabolism of xenobiotics, including tion and arrangement of their contained hepatocytes and their the metabolic activation of chemical carcinogens to active metabolites. Phase II blood supply. II should be emphasized that the normal liver components are involved in the conjugation and detoxification of xenobiotics shows quite a range of phenotypic expressions in different zones includingcarcinogens and their metabolites. 6 Similar correlations have been calculated for the data from the CMD and OA (72), and il is doubtful whelher Ihe range among differenl nodules models. The correlations are very similar in all 3 models. is any larger. Al leasl in Ihe RH model, nodules arise randomly Ihroughoul Ihe liver acinus (89) and may in part reflect Iheir sile ing system for xenobiotics, the Phase I components, and the of origin. conjugation and detoxification system for xenobiotics and their The findings wilh respecl lo Ihe hepalocyle nodules, gener metabolites, the Phase II components, were quite large and ated in Ihe RH model and growing in Ihe spleen, are particularly negative. interesting wilh regard to the origin of Ihe common phenolypic pattern. Allhough, in Ihe RH model, Ihe nodules were examined DISCUSSION many weeks after Ihe termination of exposure lo an external It is evident from the results of this study that hepatocyte agenl lhal could induce a new pattern of componente in Ihe nodules in 4 different models of liver carcinogenesis, the RH, nodules, the hepatocyte nodules in the spleen are growing in CMD, PB, and OA models, have a common biochemical pattern animals with no exposure to any known xenobiotic. Since Ihe with respect to some microsomal, cytosolic, and other compo nodules in Ihe spleen were examined more lhan 1 year after nents. These results are in unusually good agreement with iransplanlalion, Â¡Iis virtually Impossible to ascribe the nodule studies of hepatocyte nodules generated in quite different models phenolypic pattern lo an induction phenomenon by exogenous such as continuous or intermittent exposure to 2-AAF or ethio- xenobiotics. This is made even more improbable in view of Ihe nine (3, 5, 6, 22, 33, 34, 39, 61) (Table 2). Thus, as recently results in Ihe hosl liver. The values in Ihe latter were in Ihe same summarized (29), the hepatocyte nodules can now be considered general ranges when compared lo Ihe conlrols for Ihe various as having at least 14 biochemical components in common. To experimental groups (Tables 1 and 2). Thus, it appears highly these can be added cellular organization and architecture, blood likely thai Ihe new biochemical pattern in the hepalocyle nodules

CANCER RESEARCH VOL. 45 FEBRUARY 1985 568

metabolizing systems in hyperplastic nodules from the livers of rats receiving is constitutive, not induced. 2-acetylaminofluorene in their diet. Carcinogenesis (Lond.), 4: 577-581,1983. This probability is further increased by the pattern of compo 4. Bannasch, P., Mayer, D., and Hacker, H. J. Hepatocellular glycogenesis and nents measured. As indicated previously (20), the pattern seen hepatocarcinogenesis. Biochim. Biophys. Acta, 605:217-245,1980. 5. Bock, K. W., Lilienblum, W., Pfeil, H., and Eriksson, L. C. Increased uridine is not that of any single type of chronic exposure to an inducing diphosphate-glucuronyltransferase activity in preneoplastic liver nodules and agent. The closest similarity is between the initial pattern of Morris hepatomas. Cancer Res., 42: 3747-3752,1982. induction in the liver with butylated hydroxyanisole or butylated 6. Cameron, R., Kellen, J., Kolin, A., Malkin, A., and Farber, E. 7-Glutamyltrans- ferase in putative premalignant liver cell populations during hepatocarcinogen hydroxytoluene and the pattern in the nodules (8, 9, 98). How esis. Cancer Res., 38: 823-829, 1978. ever, the patterns are by no means the same. 7. Cameron, R., Sweeney, G. D., Jones, K., Lee, G., and Farber, E. A relative deficiency of cytochrome P-450 and aryl hydrocarbon (benzo(a)pyrene] hy- The biochemical pattern observed in the hepatocyte nodules, droxylase in hyperplastic nodules induced by 2-acetylaminofluorene in rat liver. consisting of low levels of microsomal activating enzyme com Cancer Res., 36: 3888-3893,1976. ponents (cytochromes P-450, some mixed-function oxygen- 8. Cha, Y-N., and Bueding, E. Effects of 2(3)-tert-butyl-4-hydroxyanisole admin ases), high levels of epoxide hydrolase, or-diaphorase, (quinine istration on the activities of several hepatic microsomal and cytoplasme enzymes in mice. Biochem. Pharmacol., 28: 1917-1921,1979. reducÃase),glutathione, GST, and one UDP-glucuronyltransfer- 9. Cha, Y-N., and Heine, H. S. Comparative effects of dietary administration of ase (I) is very appropriate for a resistance phenotype (9,10, 20, 2(3)-iert-butyl-4-hydroxyanisole and 3,5-di-fert-4-hydroxytoluene on several hepatic enzyme activities in mice and rats. Cancer Res., 42:2609-2615,1982. 24, 30, 32, 42, 43, 46, 50, 56, 63, 74, 80, 92, 98). Decreased 10. Chasseaud, L. F. The role of glutathione and glutathione-S-transferase in the ability to generate reactive moieties from some xenobiotics and metabolism of chemical carcinogens and other electrophilic agents. Adv. increased ability to inactivate whatever becomes activated are Cancer Res., 29: 175-274,1979. 11. Columbano, A., Ledda, G. M., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. an unusually effective combination for the expression of resist R. Dietary orotic acid, a new selective growth stimulus for carcinogen altered ance by hepatocyte nodules, as evidenced by: (a) more efficient hepatocytes in rat. Cancer Lett., 76:191-196,1982. excretion of one carcinogen, 2-AAF, by animals with many 12. Conway, J. G., Popp, J. A., Ji, S., and Thurman, R. G. Effect of size in portal circulation of hepatocyte nodules from carcinogen-treated rats. Cancer Res., hepatocyte nodules in their liver (90); (b) a resistance of nodules 43:3374-3378,1983. to induction of cell death in vivo by CCU and DMNA (32); (c) 13. Conway, J. G., Popp, J. A., and Thurman, R. G. Fluorescence of dye infused diminished "binding" of DMNA and 2-AAF to DNA, RNA, and via the hepatic artery (H) and portal vein (P) in hepatic nodules from diethylnitrosamine-treated rats. Fed. Proc., 43: 590,1984. protein in nodules (32); (d) resistance of isolated nodule hepato- 14. Demi, E., and Cesterie, D. Histochemical demonstration of enhanced glutathi cytes to some hepatotoxins such as aflatoxin BT and 2-AAF (39, one content in enzyme-altered islands induced by carcinogens in rat liver. Cancer Res., 40: 490-491,1980. 46, 66); (e) resistance to some cytotoxic effects of DENA (37); 15. Ellman, G. L. Tissue sulfhydryl group. Arch. Biochem. Biophys., 82: 70-77, and (f) most importantly, ability to grow in an environment that 1959. severely inhibits cell proliferation of the bulk of the liver cells (87). 16. Emmelot, P., and Scherer, E. The first relevant cell stage in rat liver carcino genesis. A quantitative approach. Biochim. Biophys. Acta, 605: 247-304, However, the finding of a common pattern in nodules in at 1980. least one model, the OA model, in which differential resistance 17. Enomoto, K., and Farber, E. Kinetics of phenotypic maturation of remodeling to inhibition of cell proliferation seems to have been ruled out of hyperplastic nodules during liver carcinogenesis. Cancer Res., 42: 2330- 2335, 1982. (62, 71), raises a basic question concerning the nodules. The 18. Enomoto, K., Ying, T. S., Griffin, M. J., and Farber, E. Immunohistochemical "resistance phenotype" of the hepatocyte nodules must be re study of epoxide hydrolase during experimental liver carcinogenesis. Cancer Res., 47:3281-3287,1981. lated to something other than its role in the expansion of an 19. Epstein, S., Ito, N., Merkow, L., and Farber, E. Cellular analysis of liver initiated resistant hepatocyte population (26, 30). Conceivably, carcinogenesis. The induction of large hyperplastic nodules in the liver with W- this phenotype is a reflection of an even more basic type of 2-fluorenylacetamide or ethionine and some aspects of their morphology and glycogen metabolism. Cancer Res., 27; 1702-1711,1967. adaptation to xenobiotics as represented by the hepatocyte 20. Ericksson, L., Ahluwalia, M., Spiewak, J., Lee, G., Sarma, D. S. R., Roomi, M. nodules. It is possible that the hepatocyte nodule represents a W., and Farber. E. Distinctive biochemical pattern associated with resistance form of physiological response to a hazardous environment in of hepatocytes in hepatocyte nodules during liver carcinogenesis. Environ. Health Perspect., 49:171-174,1983. which a sensitive hepatocyte population is replaced by a resistant 21. Ericksson, L. C., Sharma, R. N., Roomi, M. W., Ho, R. K., Farber, E., and one that enables the host organism to cope more efficiently with Murray, R. F. A characteristic electrophoretic pattern of cytosolic polypeptides some forms of xenobiotics (28, 30). The redifferentiation of the from hepatocyte nodules generated during liver carcinogenesis in several models. Biochem. Biophys. Res. Commun., 777: 740-745,1983. hepatocyte nodule to a normal-appearing mature liver (95) could 22. Ericksson, L. C., Torndal, U-B., and Andersson, G. N. Isolation and character conceivably be a part of this fundamental response to exposure ization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male Wistar rats. Cancer Res., 43: 3335-3347,1983. to xenobiotics. If this viewpoint is valid, the response of the liver 23. Farber, E. Ethionine carcinogenesis. Adv. Cancer Res., 7: 383-474, 1963. to an initiator and a promoting environment, with hepatocyte 24. Farber, E. Hyperplastic liver nodules. Methods Cancer Res., 7:345-375,1973. nodules as a common end point during the first steps in hepa- 25. Farber, E. The pathology of experimental liver cancer. In: H. M. Cameron, D. A. Unsell, and G. P. Warwick (eds.), Liver Cell Cancer, pp. 243-277. Amster tocarcinogenesis, can be interpreted as a physiological adapta dam: Elsevier Press, Inc., 1976. tion to toxic agents. The occasional occurrence of persistence 26. Farber, E. The sequential analysis of liver cancer induction. Biochim. Biophys. in the nodule and its high probability of further cellular evolution Acta, 605: 149-176,1980. 27. Farber E. The biology of carcinogen-induced hepatocyte nodules and related to cancer may now be viewed in a somewhat different perspec liver lesions in the rat. Toxicol. Pathol., 70: 197-201,1982. tive than given by the dominant current paradigm of carcinogen- 28. Farber, E. Chemicals, evolution and cancer development. Rous-Whipple Award esis as a biological abnormality or aberration. lecture. Am. J Pathol., 108: 270-275,1982. 29. Farber, E. The biochemistry of preneoplastic liver: a common metabolic pattern in hepatocyte nodules. Can. J. Biochem. Cell Biol., 62: 486-494, 1984. 30. FÃ¤rber,E., and Cameron, R. The sequential analysis of cancer development. REFERENCES Adv. Cancer Res., 35:125-226, 1980. 31. Farber, E., Cameron, R. G., Laishes, B., Un, J-C., Mediine, A., Ogawa, K., and 1. Ahluwalia, M. B. Glutathione metabolism in early hepatocyte nodules during Soil, D. B. Physiological and molecular markers during carcinogenesis. In: A. liver carcinogenesis. M.Sc. Thesis, University of Toronto, 1984. C. Griffin and C. R. Shaw (eds.), Carcinogens: Identification and Mechanisms 2. Ahluwalia, M., and FÃ¤rber,E. Alterations in glutathione status in early hyper- of Action, pp. 319-335. New York: Raven Press, 1978. plastic liver nodules. Proc. Am. Assoc. Cancer Res., 25:15, 1984. 32. Farber, E., Parker, S., and Gruenstein, M. The resistance of putative premalig 3. AstrÃ¶m, A., De Pierre, J. W., and Eriksson, L Characterization of drug- nant liver cell populations, hyperplastic nodules, to the acute cytotoxic effects

CANCER RESEARCH VOL. 45 FEBRUARY 1985 569

of some hepatocarcinogens.Cancer Res., 36: 3879-3887,1976. metabolizingenzyme activity and in the formation of reactive drug metabolites 33. Feo, F., Canuto, R. A., Garcea.R., Brossa, O., and Caselli,G. C. PhÃ©nobarbital in the liver. Ciba Found. Symp., 76: 25-42,1980. stimulation of cytochrome P-450 and aminopyrine N-demethylase in hyper- 64. Paul, K. G., Theorell, H., and Akeson, A. The molar light absorption of pyridine plastic liver nodules during OL-ethioninecarcinogenesis.Cancer Lett., 5: 25- ferroprotoporphyrin(pyridinehaemochromogen).ActaChem.Scand.,7:1284- 30, 1978. 1287, 1953. 34. Fiala, S., Mohindru, A., Kettering, W. G., Fiala, A. E., and Morris, H. P. 65. Peraino, C., Fry, R. J. M., and Grube, D. D. Drug-induced enhancementof glutathione and gamma glutamyltranspeptidase Â¡nrat liver during chemical hepatic tumorigenesis. In: T. J. Slaga, A. Sivak, and R. K. Boutwell (eds.), carcinogenesis.J. Nati. Cancer Inst., 57: 591-598,1976. Carcinogenesis:Mechanismsof Tumor Promotion and Cocarcinogenesis,pp. 35. Firminger, H. I. Hlistopathology of carcinogenesis and tumors of the liver in 421-432. New York: Raven Press, 1978. rats. J. Nati. Cancer inst., 75. 1427-1445, 1955. 66. Peraino, C., Fry, R. J. M., and Staffeldt, E. Reduction and enhancement by 36. Foulds,L. NeoplasticDevelopment,Vol. 2, pp. 205-237. New York: Academic phÃ©nobarbitalofhepatocarcinogenesisinduced in the rat by 2-acetylaminoflu- Press, Inc., 1975. orene. Cancer Res., 37:1506-1512,1971. 37. Gans, J. H. Diethylnitrosamine-inducedchanges in mouse liver morphology 67. Pilot, H. C., Bareness, L., Goldsworthy, T., and Kitagawa, T. Biochemical and function. Proc. See. Exp. Biol. Med., 153: 116-120,1976. characterization of stages of hepatocarcinogenesis after a single dose of 38. Goldfarb,S. A morphologicaland histochemicalstudy of carcinogenesisof the diethylnitrosamine.Nature (Lond.),277: 456-458,1978. liver in rats fed 3'-methyl-4-dimethylaminoazobenzene. Cancer Res., 33: 68. Pitot, H. C.. and Sirica, A. E. The stages of initiation and promotion in 1119-1128,1973. hepatocarcinogenesis.Biochim. Biophys. Acta, 605:191-215,1980. 39. GrÃ¡vela,E.,Feo, F., Canuto, R. A., Garcea, R., and Gabriel, L. Functionaland 69. Popper, H., Stemberg, S. S., Oser, B. C., and Oser, M. The carcinogeniceffect structural alterations of liver ergastoplasmic membranes during DL-ethionine of aramite in rats. A Study of hepatic nodules.Cancer (Phila.),73:1035-1046, hepatocarcinogenesis.Cancer Res., 35: 3041-3047, 1975. 1960. 40. Griffith, O. W. Determination of glutathione and glutathione disulfide using 70. Pugh, T. D., and Goldfarb, S. Quantitative histochemicaland autoradiographic glutathione reducÃaseand 2-vinylpyridine. Anal. Biochem., 706: 207-212, studies of hepatocarcinogenesisin rats fed 2-acetylaminofluorenefollowed by 1980. phÃ©nobarbital.CancerRes., 38: 4450-4457,1978. 41. Habig,W. H., Pabst, M. J., and Jakoby, W. B. Glutathione-S-transferases.The 71. Rao, P. M., Nagamine,K., Ho, R. K., Roomi, M. W., Laurier, C., Rajalakshmi, first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249: 7130- S., and Sarma, D. S. R. Dietaryorotic acid enhancethe incidenceof vglutamyl 7139, 1974. transferase positive foci in rat liver induced by chemical carcinogens. Carcin 42. Jakoby, W. B. (ed.). Enzymatic Basis of Detoxification, Vol. 2. New York: ogenesis(Lond.),4: 1541-1545,1983. Academic Press, Inc., 1980. 72. Rappaport, A. M. Physioanatomicalbasis of toxic liver injury. In: E. Farberand 43. Jakoby, W. B. (ed.).Detoxicationand drug metabolism:conjugationand related M. M. Fisher (eds.),Toxic Injury of the Liver, Part A, pp. 1-57. Basel: Marcel systems. Methods Enzymol., 77: 1-476, 1981. Dekker, 1979. 44. Judah, D. J., Legg, R. F., and Neal, G. E. Development of resistance to 73. Reuber, M. D. Developmentof preneoplasticand neoplastic lesionsof the liver cytotoxicity during aflatoxin carcinogenesis. Nature (Lond.), 265: 343-345, in male rats given 0.025 percent W-2-fluorenyldiacetamide. J. Nati. Cancer 1977. Inst, 34: 697-723,1965. 45. Kaneko, A., Dempo, K., Kaku, T., Yokoyama, S., Satoh, M., Mori, M., and 74. Roberts, E., Ahluwalia, M. B., Lee, G., Chan, C., Sarma, D. S. R., and Farber, Onoe,T. Effect of phÃ©nobarbitaladministrationof hepatocytes constituting the E. Resitance to hepatotoxins acquired by hepatocytes during liver regenera hyperplastic nodules induced in rat liver by 3'-methyl-4-dimethylaminoazoben- tion. Cancer Res., 43: 28-34,1983. zene. Cancer Res., 40: 1658-1662, 1980. 75. Roomi, M. W., Ho, R. K., Cameron, R. G., Sarma, D. S. R., and Farber, E. A 46. Ketterer, B., Coles, B., and Myer, D. J. The role of glutathione in detoxification. novel biochemicalpattern in hepatocyte nodules generated during liver carcin Environ. Health Perspect.,49: 59-69,1983. ogenesis in five different models. Proc. Am. Assoc. Cancer Res., 25: 112, 47. Kinosita, R. Studies on the carcinogenic chemical substances. Trans. Jpn. 1984. Pathol. Soc., 27: 665-729,1937. 76. Roomi, M. W., Ho, R. K., and Farber, E. Resistant hepatocytesâ€”auniquecell 48. Kitagawa, T. Histochemicalanalysisof hyperplastic lesions and hepatomasof during chemicalcarcinogenesis.Proc. Can. Fed. Biol. Soc., 26: 59,1983. the liver of rats fed N-2-fluorenylacetamide.Gann, 62:207-216,1971. 77. Sasaki, T., and Yoshida, T. Experimentelle Erzeugung des Lebercarcinoms 49. Kitagawa, T. Sequential phenotypic changes in hyperplastic areas during durch Futterung mit 0-Amidoazotoluol. Virchows Arch. Pathol. Anat. Physiol. hepatocarcinogenesisin the rat. Cancer Res., 35:1075-1084,1975. Klin. Med., 295:175-200,1935. 50. Kitahara, A., Yamazaki, T., Ihikawa, T., Camba, E. A., and Sato, K. Changes 78. Sato, M., Yoshida, T., Suzuki, Y., Degawa, M., and Hashimoto, Y. Selective in activities of glutathione peroxidase and glutathione reducÃaseduringchem induction of mfcrosomal 2-acetylaminofluoreneN-hydroxylation by dietary 2- ical hepatocarcinogenesisin the rat. Gann, 74: 649-655,1983. acetylaminofluorenein rats. Carcinogenesis(Lond.),2: 571-574,1981. 51. Laishes, B., Roberts, E., and Farber, E. In vitro measurement of carcinogen 79. Scherer, E., and Emmelot, P. Foci of altered liver cells by a single dose of resistant liver cells during hepatocarcinogenesis.Int. J. Cancer, 27: 186-193, diethylnitrosamineand partial hepatectomy: their contribution to hepatocarcin 1978. ogenesis in the rat. Eur. J. Cancer, 77: 145-154,1975. 52. Laurier, C., Tatematsu, M., Rao, P. M., Rajalakshmi,S., and Sarma, D. S. R. 80. Schor, N. A., Ogawa, K., Lee, G., and Farber, E. The use of OT-diaphorasefor Promotion by orotic acid of liver carcinogenesis in rats initiated by 1,2- the detection of foci of early neoplastictransformationin rat liver. Cancer Lett., dimethylhydrazine.Cancer Res., 44: 2186-2191, 1984. 5:167-171, 1978. 53. Lee, G., Tatematsu, M., and Makowka, L. The development of hepatocellular 81. Schulte-Hermann,R., Ohde, G., Schuppler, J., and Timmermann-Trosiener,I. carcinoma from carcinogen altered hepatocytes implanted in the spleen. Iden Enhanced proliferation of putative preneoplastic cells in rat liver following tification of a new cell population with relevance to cancer. Proc. Am. Assoc. treatment with the tumor promoters phÃ©nobarbital,hexachlorocyclohexane, Cancer Res., 24: 106,1983. steroid compounds, and nafenopin.Cancer Res., 41: 2556-2563,1981. 54. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein 82. Sells, M. A., Katyal, S. L., Sell, S., Shinozuka, H., and Lombardi, B. Induction measurement with the folin phenol reagent. J. Biol. Chem., 793: 265-275, of foci of altered -y-glutamyl-transpeptidasepositivehepatocytesin carcinogen- 1951. treated rats fed a choline deficient diet. Br. J. Cancer, 40: 274-283,1979. 55. Mazel, P. Experiments illustrating drug metabolismin vitro. In: B. M. La Du, G. 83. Shinozuka, H., Lombardi, B., Sell, S., and lammarino, R. M. Early histotogical H. Mandel, and E. L. Way (eds.), Fundamentalsof Drug Metabolismand Drug and functional alterationsof ethionineliver carcinogenesisin rats fed a choline- Disposition, pp. 546-582. Baltimore, Williams & Wilkins, 1971. deficient diet. Cancer Res., 38:1092-1098,1978. 56. Meister, A., and Anderson, M. E. Glutathione. Annu. Rev. Biochem., 52: 711- 84. Shinozuka, H., Lombardi, B., Sell, S., and lammarino, R. M. Enhancementof 760, 1983. DL-ethionine-inducedlivercarcinogenesis in rats fed a choline-devoiddiet. J. 57. Miller, G. L. Proteindetermination for large numbers of samples. Anal. Chem., Nati. Cancer Inst., 67: 813-817,1978. 37:964,1959. 85. Snedecor, G. W., and Cochran, W. S. In: Statistical Methods ed. 6, pp. 172- 58. Nash, T. The colorimetrie estimation of formaldehyde by means of Hantzsch 198. Ames, IA: Iowa State UniversityPress, 1967. reaction. Biochem.J., 55: 416-421, 1953. 86. Solt, D. B., Cayama, E., Tsuda, H., Enomoto, K., Lee, G., and Farber, E. 59. Neal, G. E., Metcalfe, S. A., Legg, R. F., Judah, D. J., and Green, J. A. Promotionof livercancerdevelopmentby briefexposureto dietary 2-acetylami Mechanism of the resistance to cytotoxicity which precedes aflatoxin B, nofluorene plus partial hepatectomy or carbon tetrachtoride.Cancer Res., 43: hepatocarcinogenesis.Carcinogenesis(Lond.),2: 457-461,1981. 188-191,1983. 60. Ogawa, K., Solt, D. B., and Farber,E. Phenotypicdiversity as an early property 87. Solt, D. B., and Farber, E. New principle for the analysis of chemicalcarcino of putative preneoplastic hepatocyte populations in liver carcinogenesis.Can genesis. Nature (Lond.),263: 702-703,1976. cer Res., 40: 725-733, 1980. 88. Solt, D., Hay, J. B., and Farber, E. Comparison of the Wood supply to 61. Okita, K., Noda, K., Fukumoto, Y., and Takemoto, T. Cytochrome P-450 in diethylnitrosamine-inducedhyperplastic nodules and hepatomas and to the hyperplastic liver nodules during hepatocarcinogenesiswith W-2-fluorenylacet- surrounding liver. Cancer Res., 37:1686-1691,1977. amide in rats. Gann,67: 899-902, 1976. 89. Solt, D. B., Mediine,A., and Farber, E. Rapidemergenceof carcinogen-induced 62. Omura, T., and Sato, R. The carbon monoxide binding pigment of liver hyperplastic lesions in a new model for the sequentialanalysis of liver carcin microsomes. J. Biol. Chem., 239: 2370-2378,1964. ogenesis. Am. J. Pathol.,88: 595-618,1977. 63. Orrenius, S., Thor, H., and Jemstrom, B. The influence of inducers on drug- 90. Spiewak, J., and Eriksson, L. C. Pharmacokineticsof 2-acetylaminofluorene

CANCER RESEARCH VOL. 45 FEBRUARY 1985 570

(2-AAF) secretion in normal rats and rats with 2-AAF-inducednodules. Proc. 95. Tatematsu, M., Nagamine,Y., and Farber, E. Redifferentiationas a basis for Am. Assoc. Cancer Res., 24: 76,1983. remodeling of carcinogen-induced hepatocyte nodules to normal-appearing 91. Stewart, H. L., and Snell, K. C. The histopathology of experimental tumors of liver. Cancer Res., 43: 5049-5058,1983. the liver in the rat. In: F. Homburger (ed.), Physiopathologyof Cancer, Ed. 2, 96. Teebor, G. W., and Becker, F. F. Regression and persistence of hyperpiastic pp. 85-126. New York: Paul B. Hoeber, Inc., 1959. hepatic nodules induced by N-2-fluorenylacetamideand their relationship to 92. Stout, D. L., and Becker, F. F. Alteration of the ability of liver microsomes to hepatocarcinogenesis.Cancer Res., 37:1-3,1971. activate A/-2-fluorenylacetamideto a mutagen of Salmonellatyphimurium dur- 97. Tsuda, H., Lee,G., and Farber,E. Inductionof resistant hepatocytes as a new ing hepatocarcinogenesis.Cancer Res., 38: 2274-2278,1978. principle for a possible short-term in vivo test for carcinogens. Cancer Res., 93. Szasz, G. Reaction-rate method for -y-glutamyltransferase activity in serum. 40:1157-1164,1980. Clin. Chem., 22:2051-2055,1976. 98. Wattenberg, L. W. Inhibitors of chemical carcinogens. Adv. Cancer Res., 26: 94. Tatematsu, M., Murasaki, G., Nakinashi,K., Miyata, Y., Shinohara,Y., and Ito, 197-226,1978. N. Sequential quantitative studies on hyperpiastic nodules in the liver of rats 99. Williams, G. M. The pathogenesis of rat liver cancer caused by chemical treated with carcinogenicchemicals. Gann, 70:125-130,1979. carcinogens. Biochim. Biophys. Acta, 605:167-189,1980.

CANCER RESEARCH VOL. 45 FEBRUARY 1985 571

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. A Common Biochemical Pattern in Preneoplastic Hepatocyte Nodules Generated in Four Different Models in the Rat

M. W. Roomi, R. K. Ho, D. S. R. Sarma, et al.

Cancer Res 1985;45:564-571.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/2/564

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/2/564. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.