The Regulation of Serine Dehydratase and Glucose-6-Phosphatase in Hyperplastic Nodules of Rat Liver During Diethylnitrosamine and N-2-Fluorenylacetamide Feeding'

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[CANCER RESEARCH 35, 1075-1084,April 1975] The Regulation of Serine Dehydratase and Glucose-6-phosphatase in Hyperplastic Nodules of Rat Liver during Diethylnitrosamine and N-2-Fluorenylacetamide Feeding'

Tomoyuki 2 and Henry C. Pitot

Departments of Oncology and Pathology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY oxygenase, tynosine and ornithine aminotransferase, serine dehydnatase, and histidase (1, 10, 32, 35), and ofenzymes of Changes in the levels of senine dehydratase and glucose carbohydrate metabolism, including glucose-6-phosphatase, 6-phosphatase induced by dietary stimuli on starvation in glucokinase, glucose-6-phosphate dehydrogenase, and hyperplastic nodules of rat liver during diethylnitrosamine malic enzyme (9, 33). In addition, impaired cholesterol or N-2-fluorenylacetamide feeding were studied by immu feedback (15â€”17)or hexobarbital (35) metabolism during no- and enzyme histochemical methods. The study was azo dye (1, 9, 10, 15), FAA3 (16, 32), dimethylnitrosamine performed during cancinogenesis through a combined (35), and ethionine (17) feeding has been reported. These method of enzyme histochemistry and radioautography. results were interpreted as an expression of a characteristic Senine dehydratase was observed diffusely in the cyto â€œpreneoplasticâ€•change. On the other hand, the same or plasm of the original hepatocytes in the peniportal zone and other investigators (26, 3 1) observed no significant altera was induced markedly during diethylnitrosamine feeding tion in the induction of tryptophan oxygenase and tyrosine but only slightly during N-2-fluorenylacetamide feeding. aminotnansferase during the early weeks of N-nitrosomor The enzyme was deficient and not ind@icible in hyperplastic pholine or azo dye feeding, respectively. The reason(s) for nodules during their developing phase. Later during the the discordance among these various studies has not yet feeding period, however, there was an elevation of the level been clarified. of senine dehydratase and its inducibility with time in the A significant problem in all these experiments is that they majority of the nodules. A good correlation was observed were carried out by treating the preneoplastic liven as a between senine dehydratase and glucose-6-phosphatase in homogeneous mass of cells. Histochemical studies have their elevated levels and response to environmental stimuli. shown that the preneoplastic liver is a mixture of degenerat There was a minor group of hyperplastic nodules in which ing hepatocytes from the original parenchyma and various the deficiencies of these enzymes persisted and enzyme kinds of hyperplastic lesions exhibiting different enzyme induction was not observed. A greaten number of hyperplas levels (12, 21, 25, 41). Teebor and Seidman (38), working tic nodules with persistent enzyme deficiency was seen with hyperplastic nodules induced by FAA, reported reten during diethylnitrosamine carcinogenesis. tion of metabolic regulation of several enzymes of amino These results provide further information about the acid on carbohydrate metabolism. In an attempt to reconcile changing biological nature of hyperplastic nodules with these various findings, a sequential study of preneoplastic respect to their metabolic adaptability and enzyme levels liven utilizing a combined method of enzyme histochemistry during hepatocancinogenesis. and nadioautography was made that demonstrated a consid erable elevation in the level of some marker enzymes with time in the majority of â€œenzymedeficientâ€• hyperplastic INTRODUCTION areas induced by FAA, and the study also demonstrated heterogeneity in the potentiality of their phenotypic altera The impaired metabolic adaptation of hepatomas to tion among hyperplastic areas (23). Studies involving a close substrate or hormonal stimuli has been well documented correlation between biochemical and morphological from this and other laboratories (3â€”5,20, 28, 42). Studies changes and a consideration of sequential alterations in have also been carried out on enzyme regulation in liven these parameters seemed to be an essential requisite for our during hepatocarcinogen feeding. Most of these studies understanding of the biochemistry of hepatocarcinogenesis. revealed an inhibition of the adaptive formation of some The present study reports the results of a sequential, enzymes of amino acid metabolism, such as tryptophan histochemical study on the level of senine dehydratase and glucose-6-phosphatase and their dietary induction in the preneoplastic Liver, especially in hyperplastic nodules in 1 This work was supported in part by grants from the National Cancer duced by 2 different carcinogens, diethylnitrosamine and Institute (CA-07175) and the American Cancer Society (E-588).

2 Eleanor Roosevelt International Fellow in Cancer Research. Received August 28, 1974; accepted January 10, 1975. S The abbreviation used is: FAA, N-2-fluorenylacetamide.

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FA A. Hyperplastic nodules, which present as focal prolifer [Hayashi et a!. ( 13)], and for histological observation (he ations of parenchymal cells in liven tissue in relatively early matoxylin and eosin). For the combined enzyme histo phases of every type of hepatocancinogenesis, including both chemical and radioautographic study, frozen sections areas and nodules of hyperplasia described by Reuben (34) made of liver tissues fixed with Baker's cold formol cal or the enzyme-deficient islands of Friedrich-Freksa et al. cium were also used together with the unfixed serial sec (12), have been described as probably representing early tions. preneoplastic changes in liven parenchymal cells (6, 8, 24, Senine dehydratase was stained by an indirect immuno 34). fluorescent technique. The preparation of specific anti-rat A new immunohistochemical method has been utilized liver senine dehydratase rabbit serum was described previ for the demonstration of senine dehydnatase, an enzyme that ously (19). The immunohistochemical staining method was has been extensively studied by quantitative analysis in described in detail elsewhere (22). Briefly, sections 6 @m enzyme induction experiments. in thickness were mounted on slides, air dried, fixed for 30 seconds in cold methanol, dried again, and briefly rinsed MATERIALS AND METHODS with phosphate-buffered saline (phosphate-buffered 0.01 M NaC1 solution, pH 7.0). The sections were covered Male Sprague-Dawley rats weighing 140 to 160 g with a few drops of anti-senine dehydratase serum, incu (Sprague-Dawley Co., Madison, Wis.) were used. They bated for 30 mm at room temperature, and then rinsed with were housed in an air-conditioned room at 21â€”25Â°in phosphate-buffered saline for 15 mm. After this a few drops wine-mesh cages with alternating 12-hr periods of light and of fluorescein isothiocyanate-conjugated goat anti-rabbit dark. The diethylnitrosamine group was given chow and â€˜y-globulin (Hyland Laboratories, Los Angeles, Calif.) water containing 0.01% diethylnitnosamine (Eastman Or were placed on the section, and it was incubated for 30 mm ganic Chemicals, Rochester, N. Y.). The diethylnitnosamine at room temperature, rinsed with phosphate-buffered solution was prepared weekly and kept in dark bottles. The saline for 15 mm and mounted under cover slips with glyc FAA group was given chow containing 0.03% of FAA erol. (Mann Research Laboratories, Inc., N. Y.) and water. The The presence or absence of induction on repression was rats were given the carcinogens either for the initial 9 weeks judged by comparative study of staining patterns and their on continuously throughout the experimental period. The intensities between tissues from treated (induced or ne dietary induction of senine dehydratase was observed in the pressed) and nontreated (control) rats. These different tis livers of rats fed a 90% protein diet containing 0.03% FAA sues were put together, cut simultaneously, and stained (FAA group) or a 90% protein diet (diethylnitrosamine on the same slide. Comparative studies of enzyme staining group) for 5 days during 4, 6, 9, 12, and 15 weeks of between different sections were also done. carcinogenesis. The effect of dietary glucose repression of serine dehydratase was observed in the livers of rats fed a RESULTS 0% protein diet for 5 days during carcinogen feeding. By feeding 90 or 0% protein diet for 5 days, 10-fold differences The morphological and histochemical changes occurring in the level of senine dehydratase are seen in normal rat liven in the liven of rats drinking water containing 0.01% diethyl (18). The composition of the 90 and 0% diets were described nitrosamine or fed a diet containing 0.03% FAA were previously (29). The induction on activation of glucose similar to those described previously (2 1, 24). In both 6-phosphatase was observed in the liven of rats fasted for 24 diethylnitrosamine and FAA carcinogenesis, numerous hy hr during carcinogenesis. A significant increase of activity, perplastic nodules developed in the middle or peniportal up to 190% of the value for unfasted animals, is seen in areas of hepatic lobules around the 6th week, at the earliest normal rat liver after 24 hr of fasting (27). To confirm the during the 4th week. The nodules consisted of collections of sequential phenotypic change in hyperplastic nodules in proliferating hepatocytes, monphologically similar to nor duced by diethylnitrosamine feeding, a combined method of mal hepatocytes but biochemically deficient in some marker fi-glucuronidase staining and radioautography was used in enzymes, including glucose-6-phosphatase and f3-glucunoni the same manner as was originally applied to the analysis of dase (Figs. I to 3), in their developing phase. The hyperplas hyperplastic areas during FAA feeding (23). Ten rats from tic nodules rapidly increased in size and number, while the the diethylnitrosamine group were partially hepatectomized original hepatocytes degenerated almost completely by the (14) at the 9th week of feeding, and a total of 400 sCi 12th week of carcinogen feeding. The fate of the original [3H]thymidine was injected every 3 hr from 18 to 27 hr after hepatocytes was followed either by sequential morphologi the operation (100 @.iCi/injection). Four rats were killed 1 hr cal and histochemical observation or, more directly, by after injection of the label to see the specific labeling of following, through radioautography, sequential changes in areas and nodules of hyperplasia. Liven tissues were ob the labeling of the original hepatocytes, which had been tamed from the remaining 6 rats at the 12th and 15th weeks labeled with [3H]thymidine before starting the administrat by biopsy and autopsy. tion of carcinogen (23). In diethylnitrosamine carcinogene Small pieces of the liver tissue were frozen on Dry Ice. sis, at the 12th week several, and at the 15th week multiple, Serial sections were made in a cnyostat and stained for â€œnodular lesionsâ€• were microscopically carcinomas, re senine dehydratase (22), glucose-6-phosphatase [the flected by a positive serum a-fetoprotein, as determined by method of Wachstein and Meisel (40)], fi-glucuronidase the double-diffusion technique in agar in 25 and 100% of the

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rats at the 12th and 15th weeks, respectively (24, 25). In change the level of senine dehydratase or glucose-6-phospha FAA carcinogenesis, overt carcinoma developed usually tase, where there had been no original enzyme level, but did later than the 24th week, with positive serum a-fetopno change the level where there seemed to have been some tein in 25% of the rats at the 30th week (25). original enzyme level (Figs. 9 and 10). Later, in the majority At the 9th week ofdiethylnitrosamine carcinogenesis, the of these hyperplastic nodules, both in DEN and FAA hyperplastic nodules were rather specifically labeled by feeding there was a considerable elevation of these enzymes, injecting [3H]thymidine after partial hepatectomy (Figs. 2 and increased staining of senine dehydratase or glucose and 3). The original hepatocytes with @-glucuronidase 6-phosphatase activity was clearly observed in these lesions activity were barely labeled, probably because their prolifer after dietary induction (Figs. 9, 11, 12, and 13). ative activity was inhibited by the toxic action of the In the nodules with persistent glucose-6-phosphatase carcinogen. A study with liver tissue obtained at the 12th deficiency there was also senine dehydratase deficiency. and 15th weeks from rats labeled in this manner at the 9th Induction of the enzymes was not observed in these lesions week showed that a considerable elevation of enzyme at the later phase of carcinogenesis on even 6 weeks after activity occurred in the majority of the labeled hyperplastic withdrawal of the carcinogen in both groups (Fig. 11). lesions within 3 to 6 weeks (Figs. 4, 5, and 6). The elevated Generally, there was good correlation between the elevated level of enzyme varied individually, and there was a minor levels of senine dehydratase and glucose-6-phosphatase in group of labeled nodules with persistent enzyme deficiency the hyperplastic nodules. The level of induction seemed and growth in size during the entire observation period. generally proportional to the original level of the enzymes. These observations concerning the phenotypic change in In the liver of rats fed a 0% protein diet for 5 days during hyperplastic nodules induced by diethylnitrosamine were either DEN or FAA feeding, there was almost no specific similar to the previous results obtained with FAA car fluorescence of senine dehydnatase throughout the liven cinogenesis (23). In diethylnitrosamine carcinogenesis, how tissue. ever, the number of nodules with persistent enzyme defi Generally, a significant increase in the enzymes was ciency and growth in size was much larger than that seen in clearly indicated by the presence of remarkable fluorescence the FAA feeding and often, after the 12th week, major or the lead sulfide, compared with that of noninduced livers. portions of a section were occupied by enzyme-deficient Since there was a general parallelism in the original enzyme large nodules and hepatocellular carcinomas in which levels between senine dehydratase and glucose-6-phospha labeled cells were hardly detected. tase, correlative observations between the 2 different stains The staining pattern of serine dehydratase in normal rat were also helpful in estimating the original enzyme levels in liver before and after dietary induction is shown in Fig. 7b. the nodules after environmental perturbations. Senine dehydratase was demonstrated by specific fluores cence diffusely in the cytoplasm of hepatocytes in the peniportal zone. At the 4th and 6th weeks of diethylnitro DISCUSSION samine cancinogenesis, when there was centrolobular de generation associated with loss of glucose-6-phosphatase The present results have shown another aspect of the activity but when original hepatocytes were still abundant phenotypic changes occurring in hyperplastic nodules dun (Fig. 8a), senine dehydratase was demonstrated in the pen ing diethylnitrosamine and FAA feeding by demonstrating portal cells at a level and pattern similar to that of normal the â€œmaturationâ€•in their metabolic adaptability as well as liver. After feeding the diethylnitrosamine group a 90% their endogeneous enzyme levels with time of feeding. This protein diet, there was a remarkable increase of senine phenomenon may also be seen in the elevation of glucose dehydnatase specific fluorescence in the original hepato 6-phosphatase and f3-glucunonidase levels in hyperplastic cytes (Fig. 8b). Since there was degeneration and loss of nodules during azo dye feeding as has been described (23). hepatocytes in the centrolobulan zone, where there is The enzymic deficiency on impaired metabolic adaptation in neither an endogenous level nor any induction of senine a hyperplastic nodule at a given time during cancinogenesis dehydratase normally (22), a proportionately larger portion should not necessarily be regarded as a fixed feature of the of the hepatic lobuli was occupied by hepatocytes with cells in the nodule and need not be interpreted as a stronger fluorescence than was seen in the normal rat liver characteristic preneoplastic change. after induction. In the liver of fasted rats during diethylni As pointed out in the â€œIntroduction,â€•the preneoplastic trosamine feeding, there was a marked elevation of glucose liver is a mixture of degenerative original hepatocytes and 6-phosphatase in the original hepatocytes (Fig. 9), as is seen hypenplastic regions with variable enzyme features. The in the normal rat liver (27). In the FAA group, however, the metabolic adaptability of the original hepatocytes, for their original level of senine dehydratase was low and its induc part, differs according to the type of carcinogen, in this tion by high-protein diet or induction of glucose-6-phospha study diethylnitnosamine and FAA. FAA, in the dose given, tase by fasting was much less remarkable. may be a much stronger inhibitor of RNA and protein In the hyperplastic nodules in their developing phase at synthesis in the original hepatocytes than is diethylnitrosa the 6th and 9th weeks, both in diethylnitrosamine and FAA mine. All the biochemical studies concerning the metabolic feeding, senine dehydratase, glucose-6-phosphatase, and regulation of the preneoplastic liven, cited in the â€œIntroduc $-glucuronidase were absent or slightly positive only in focal tion,â€•may be more properly interpreted on the basis of the cells within a nodule. The dietary stimuli or fasting did not present results. For example, Teebon and Seidman (38)

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observed a gradual decrease of endogenous glucose-6-phos hyperplastic nodules might be understood as a similar type phatase levels and its induction by starvation in male rat of phenomenon based on a similar mechanism in molecular liver from the 4th to 8th weeks of FAA feeding, followed by order. Probably, repression and/or derepression of multiple an elevated endogenous level and proportionally normal gene expressions occurs when an altered cell develops. induction either in â€œnonnodulanâ€•portionsof the liver or in Such states may not be stable and may be followed by van nodules at the 12th week. They mayhave observed the toxic ous phenotypic changes. The direction of the change may effect of the carcinogen on the original hepatocytes at the or may not tend toward normalcy. 4th week, the average enzyme level and the induced level of The real nature of these individually heterogeneous hy enzymes in the degenerative original hepatocytes and â€œim perplastic nodules, with or without phenotypic changes, in matureâ€• hyperplastic nodules in their developing phase at their relation to the development of carcinoma is still ob the 8th week, and the enzyme regulation in hyperplastic scune. nodules in certain steps of maturation at the 12th week. Since they used a very high dose of the carcinogen, the ACKNOWLEDGMENTS sequential changes in the preneoplastic liver must have proceeded faster than that of our experiments, i.e., the liver The authors wish to thank Lona Barsnessfor her excellenttechnical must have been almost totally replaced with hyperplastic assistance. areas and nodules by the 12th week. 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Fig. I. The liver of a rat at the 9th week of diethylnitrosamine feeding stained for glucose-6-phosphatase. There are numerous hyperplastic nodules, some of which are the size of a hepatic lobule (arrows). Note the marked enzyme deficiency in most cells comprising the nodules at this phase. However, also note cells with some enzyme activity within nodules, suggesting the tendency of maturation of the nodules. x 10. Fig. 2. A section from the liver of a rat at the 9th week of diethylnitrosamine feeding labeled with [â€˜H]thymidine after partial hepatectomy, stained for @-glucuronidase and exposed to film for radioautography. The dark portion indicates the site of the original hepatocytes with enzyme activity. Note that the hyperplastic nodules are pale with a deficiency of enzyme and that the cells of the nodules are specifically labeled in contrast to the original hepatocytes. x 25. Fig. 3. A higher magnification ofa part ofthe same section shown in Fig. 2 demonstrates more clearly the enzyme deficiency and specific labeling of the hyperplastic nodule. x 150. Fig. 4. A section from the liver ofa rat at the 12th week ofdiethylnitrosamine feeding and 3 weeks after labeling, shows a nodule with elevated enzyme level (left) and a nodule with persistent enzyme deficiency. The cells of both nodules are labeled. fl-Glucuronidase staining and radioautography. x 75. Fig. 5. A higher magnification of a part of the nodule with elevated enzyme activity in Fig. 4. Note the pericanalicular arrangement of the enzyme activity and silver grains over the nuclei of parenchymal cells. x 300. Fig. 6. The liver of a rat at the 15th week of diethylnitrosamine feeding stained for @-glucuronidase (a) and glucose-6-phosphatase (b) (serial sec tions). By this time the whole liver is replaced by hyperplastic nodules of various sizes and with various enzyme levels. The enzyme level is generally higher in smaller nodules, in some even exceeding the level of normal hepatocytes. x 6. Fig. 7. Serial sections from normal adult rat liver with (left portion of demarcating line) or without (right portion) dietary induction, stained for glucose-6-phosphatase (a) and serine dehydratase (b). The 2 different tissue samples were frozen together and then cut and stained simultaneously. The serine dehydratase specific fluorescence is demonstrated at a high level in the periportal and midzonal hepatocytes of the induced rat liver. The serine dehydratase level of the noninduced rat liver was very low, especially in this case, and is hardly demonstrable. The enzyme was virtually absent in the cells around the central veins (cv), even after induction. x 40. Fig. 8. Serial sections from the liver of a rat after dietary induction at the 6th week of diethylnitrosamine feeding (lower portion ofdemarcating line) and I section from a noninduced rat (upperportion), stained for glucose-6-phosphatase (a) and serine dehydratase (b). The 2 different tissue samples were frozen together and then cut and stained simultaneously. The enzyme staining in the original hepatocytes induced by dietary casein during the diethylnitrosamine feeding is comparable to that seen in normal liver in Fig. 7. x 40. Fig. 9. Changes in glucose-6-phosphatase staining induced by fasting in the liver of a rat during the 10th week of diethylnitrosamine feeding. The lower portion of the section is a biopsy sample of liver taken when the rat was on chow as a control, and the upperportion is that taken from the same rat 24 hr after biopsy and fasting. Both specimens were put together and cut and stained for glucose-6-phosphatase simultaneously. There are many hyperplastic nodules in this section (arrows). Note the remarkable elevation ofthe enzyme level both in original hepatocytes and several ofthe hyperplastic nodules, the largest of them denoted by thick arrows, while in some other nodules (thin arrows), no remarkable enzyme elevation is seen. x 8. Fig. 10. Sections from a rat after dietary induction at the 9th week of diethylnitrosamine feeding showing the enzyme deficiencies of glucose-6-phosphatase (a) and serine dehydratase (b) in a hyperplastic nodule (arrows) during its developing phase. x 40. Fig. I I . Sections from a rat after dietary induction at the 15th week of diethylnitrosamine feeding showing marked increase of serine dehydratase antigen with high fluorescence (b) in the matured hyperplastic nodule (MN), asjudged from the glucose-6-phosphatase level (a), while almost no change is seen in the nodule with the enzyme deficiency (EDN). x 40. Fig. 12. The liver from a rat after dietary induction at the 12th week of diethylnitrosamine feeding showing a marked increase of serine dehydratase antigen including a hyperplastic nodule (left upper portion). x 80. Fig. 13. The liver from a rat after dietary induction at the 15th week of FAA feeding showing marked increase of serine dehydratase antigen in a matured hyperplastic nodule. x 60.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1975 American Association for Cancer Research. The Regulation of Serine Dehydratase and Glucose-6-phosphatase in Hyperplastic Nodules of Rat Liver during Diethylnitrosamine and N-2-Fluorenylacetamide Feeding

Tomoyuki Kitagawa and Henry C. Pitot

Cancer Res 1975;35:1075-1084.

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