During 2 -Acetylaminofluorene Feeding Dietary Induction of Some Enzymes of Amino Acid Metabolism

[CANCER RESEARCH 29, 464â€”469, February 1969]

Dietary Induction of Some Enzymes of Amino Acid Metabolism during 2 -Acetylaminofluorene Feeding 1

Lionel A. Poirier2 and Henry C. Pitot@ Departments of Oncology and Pathology, McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY ably not common to all neoplasms even in tissues of such a high degree of biochemical differentiation as liver. Rather, it is The dietary induction of the mat liver enzymes tyrosine now apparent that a generalization probably more significant a-ketoglutnrate transaminase, semine dehydratase, tryptophan to our understanding of the malignant process is that in all pyrmolase, histidase, and ornithine-5-transaminase by the neoplasms studied, none have been found to possess a normal forced feeding of casein hydmolysate to protein-depleted ani complement of mechanisms controlling genetic expression mals was studied in those rats consuming a diet containing (23). Rather, relatively few if any normal mechanisms regulat 0.03% acetylaminofluorene as compared with control animals. ing enzyme synthesis in neoplasms have been found (21). At the end of a 4- to 5-week period of feeding control and Therefore, if one is to argue that a major biochemical differ carcinogen-supplemented diets, the metabolic increases of ence between neoplasms and their cell of origin resides in al omnithine transaminase and histidase seen in control animals tered regulation of genetic expression, similar or at least ana were absent in animals fed 2-acetylaminofluorene. At the same logous alterations might be expected to appear in the tissue time the induction of tryptophan pyrrolase and serine dehy undergoing the carcinogenic process. dratase was greatly diminished in animals receiving the carcino To date, relatively few investigations concerned with the al gen. No significant alteration in the dietary induction of tyro temation of enzymatic control mechanisms during the carcino sine transaminase could be observed during the 5-week period genic process in liver have been reported. Studies on enzyme of acetylaminofluorene administration, although an absolute induction during hepatocarcinogenesis reported in the litera increase in the basal and induced levels of TAKG were seen ture have led to conflicting results. In chronic feeding experi after 5 weeks of 2-acetylaminofluorene feeding. In animals fed ments with 3'-methyl-DAB4 (6â€”8) and with N-nitrosomorph the 2-acetylaminofluorene supplement, the base level of histi oline (13), the induction of tryptophan pymmolase by trypto dase decreased when compared to base levels seen in control phan was found to be greatly diminished or lost in the livers of animals. These findings indicate that the response of these the carcinogen-fed rats. Other groups, however, reported no enzymes to the administration of casein hydrolysate is modi marked loss of tryptophan pyrmolase induction by tmyptophan fled in the livers of rats fed the carcinogen acetylaminofluo in the livers of rats (2, 3, 10) fed 3'-methyl-DAB or mice fed mene for periods of 4â€”5weeks in such a way as to bear some DAB (9). Similarly (13), the chronic administration of N-nitro resemblance to the altered control mechanisms seen in the somorpholine to rats appeared to have no effect on the corti ultimate product of liver cnrcinogenesis, the hepatocellulnr car sone induction of hepatic tryptophan pyrrolase, although such cinoma. induction was reported to be greatly diminished in the mats fed 3'-methyl-DAB (2). Recently, Ruddon (31) has shown that INTRODUCTION chronic dimethylnitrosomine administration to rats resulted in a loss of the phenobambital-induced metabolism of hexobarbi Beginning with the experiments of Wnrburg (32) and fol tal and a loss of the â€œparadoxical affectâ€• of actinomycin D on lowed by those of Greenstein (5), altered enzyme content has the cortisone induction of tryptophan pyrmolase. Substrate in been widely recognized as one of the outstanding differences duction of TAKG was diminished in the livers of mats fed between neoplastic and normal tissue. However, it has become N-nitmosomorpholine; hormonal induction of TAKG, on the apparent that the loss in activity of any single enzyme is prob other hand, appeared to be unaffected in the livers of mats fed

â€˜Thisthe first paper of a series on the Metabolic Adaptions during â€˜Thefollowing abbreviations are used: 3'-methyl-DAB, 3'-methyl Hepatocarcinogenesis. This work was supported in part by grants from 4-dimethylaminoazobenzene (Chem. Abstracts nomenclature: N,N the National Cancer Institute of the USPHS (CA-07175) and from the dimethyl-p-(m-tolylazo)-aniline); DAB, 4-dimethylaminoazobenzene American Cancer Society (P-314). (Chem. Abstracts: N,N-dimethyl-p-(phenylazo)-aniline); 2-methyl 2Present address: Institut du Cancer de Montreal, Hopital Notre DAB, 2-methyl-4-dimethylaminoazobenzene (Chem. Abstracts: Dame et Department de Biochimie, Universite de Montreal, Montreal, N,N-dimethyl-p-(phenylazo)-m-touidine); AAF, 2-acetylaminofluorene P. Q., Canada. (Chem. Abstracts: N-fluoren-2-ylacetamide) ; OT, omnithine-S-transami 3Career Development Awardee of the National Cancer Institute. nate; TAKG, tyrosine-a-ketoglutarate transaminase; EDTA, ethylene Received March 14, 1968; accepted October 13, 1968. diaminetetraacetate.

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either 3'-methyl-DAB (2) or N-nitmosomorpholine (13). The BODY WEIGHTS studies reported in this series of papers were undertaken to extend and amplify those described above utilizing a standard method of dietary enzyme induction (25) in order to investi gate the regulation of a number of enzyme syntheses simulta neously.

MATERIALS AND METHODS

Young adult male rats (170â€”190 gm) (Holtzman Rat Co., Madison, Wisconsin) were used in these experiments. They U) were housed in Wahman galvanized wire-mesh cages and given 4 food and water ad libitum. Upon arrival at this laboratory the a: rats were placed on a modified commercial grain diet (1) for (D 12 days to minimize possible enzymatic alterations due to z dietary changes. At the end of this period the experimental Iâ€” mats were fed the same diet containing 0.03% AAF (Mann I Research Laboratories, Inc., New York) (m.p. 19 1â€”193Â°C); w control rats were fed the diet without the carcinogen. The response to dietary induction was determined in the livers of mats fed the diets for 0, 2, 4, and 5 weeks, exclusive of the initial 12-day period. Five days prior to sacrifice the rats to be killed were placed on a semisynthetic 0% protein diet to lower the hepatic levels of the amino acid-metabolizing enzymes (25); this diet consisted of 75% glucose monohydrate (Ceme lose, Corn Products Refining Co.), 4% salt mix (Salt Mixture P. H. 4, General Biochemicals, Chagrin Falls, Ohio), 5% corn oil, 15% nonnutritive fiber (rice hulks), and 1% Vitamin Fortifica tion Mixture (General Biochemicals). The 0% protein diet WEEKS ON DIET given to the AAF-fed rats also contained 0.03% AAF. The rats Chart 1. Body weights of rats fed a control and 0.03% 2-acetylamino were then fasted overnight for the period immediately preced fluorene (AAF) diet. The asterisks denote a statistically significant dif ing the dietary induction. At zero time (6:00 A.M .) 5 rats from ference between the body weights of the control and AAF-treated (P < the AAF-fed and 5 from the control groups were killed by 0.01) animals. The vertical lines at each point denote the standard decapitation; their livers were quickly removed and homoge errors of the groups of animals. Each group consisted of from 6 to 10 nized in 4 volumes of 0.2 M KC1 containing 0.1 M Tris buffer animals. (pH 8.2), iOâ€”@M EDTA, and i0@ M dithiothmeitol with the use of an Ultmaturrax homogenizer (Janke and Kunkel, Stauf fen, Germany). The remaining rats were intubated by a Enzyme Assays stomach tube with a dose of a 33% suspension of enzymati cally hydrolyzed casein (2 ml/100 gm body weight); the intu Histidase (27), OT (17), serine dehydratase (27), and TAKG bations were repeated 6 and 12 hours later. Groups of 5 rats (28) were determinedby previouslydescribedprocedures,and were sacrificed at 6, 12, and 18 hours, and their livers were activities were calculated. All enzyme assays were performed treated as above. Approximately 3 ml of each homogenate was under conditions of zero order kinetics, i.e., the rates were stored at â€”70Â°Cfor the assay of OT; 10 ml portions of each proportional to time and tissue or protein concentration. homogenate were centrifuged at 100,000 X g for one hour, Tryptophan pyrrolase assays were performed using an auto and the individual clear supemnatants were collected and stored mated combination unit previously described (27) for the de at â€”70Â°Cfor the later assay of serine dehydmatase, TAKG, termination of enzyme rates. The components of the system tmyptophan pyrrolase, and histidase. The body and liver are as follows: weights of all rats used in these experiments were measured at Reservoir: 10â€”2 M L-tryptophan (General Biochemicals), the time of sacrifice ; the results are illustrated in Charts 1 and 5 x iÃ¸â€”@% (w/v) hemin (Sigma Chemical Co., St. Louis, @ 2 respectively. Since no significant differences were noted in Mo.), 2 X 10 M ascorbic acid (Nutritional Biochemical @ the body weights of the rats before and after casein hydroly Corp., Cleveland, Ohio), 10 M potassium phosphate buffer, sate intubation, only the initial or zero-time body weights are pH 7.5, 0.1 mg/ml catalase (Sigma Chemical Co.). listed in Chart 1. Since significant differences were noted in Sampler tubes: iO@ M L-tmyptophan, 10 1 M potassium the 0- and 18-hour liver weights, each was listed separately in phosphate buffer, pH 7.5, 0.15 ml tissue extract. Chart 2. The solid line refers to the initial liver weight; the Equal amounts of reservoir and sampler-tube solution were dotted line refers to the weight 18 hours after multiple intuba mixed to give a total reaction mixture of 6 ml per sample. As tions of casein hydrolysate. the reaction proceeded at 37.5Â°C, the increase in optical den

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LIVER WEIGHT _____â€”CaseinHydrolysotethe University of Wisconsin Computing Center. The programs â€”â€”â€”â€” + Cosein Hydrolysote utilized are available on request. CONTROL AAF RESULTS

Biologic

Cl) AAF fed at a level of 0.03% in the diet produces a high 4 incidence of hepatocellulnr carcinomas in the rat (33) after (9 10â€”12weeks of feeding. Histologic alterations and changes in z cellular population, although apparent at 3 weeks of feeding, I-. did not become marked until after 5 weeks of AAF feeding. Thus the responses of the hepatic cell population were tested during the initial 5 weeks of carcinogen administration. In accord with previous observations (15), the chronic adminis 2 tmation of AAF in the diet of rats produced signs of toxicity. WEEKSON DIET The AAF-fed rats gained only 85 gm over the 5-week experi Chart 2. Liver weights of rats fed a control and 0.03% 2-acetylamino mental period, whereas the control rats gained 148 gm (Chart fluorene (AAF) diet before (â€”)and after (+) intubation with casein 1). In addition, although the control rats consistently showed hydrolysate. The closed circles connected by a solid line denote the a 1 5% increase in liver weight 18 hours after the multiple average liver weight Â±the standard error of the mean (vertical line) of intubation of casein hydrolysate, this response was lost in the rats fed a control and AAF diet for the time period indicated. The open circles connected by a dash line denote the average liver weights Â± livers of rats fed AAF for as little as 2 weeks (Chart 2). Stom standard errors of a group of animals fed the designated diet for the ach weights in the two groups were identical at the end of the time period indicated and administered casein hydrolysate by intuba intubation periods, suggesting that no defect in absorption of tion over an 18-hour period as described in the Methods. The asterisks the casein hydmolysate had occurred in the AAF-fed group. indicate a statistically significant difference between corresponding The livers of the AAF-fed rats appeared to increase in weight points(P< 0.01). throughout the 5-week period but at a slower rate than those of the control mats. The liver weight as a percentage of body sity due to kynurenine formation was measured at 365 m@zin weight increased in the AAF-fed mats (Charts 1, 2). the Gilford model 2000 multiple-sample absorbance recorder equipped with a Beckman monochromatem; the calculations were based on a molar extinction coefficient at 4.54 X i0@ for Biochemical kynurenine (11). AAF feeding produced a loss in the adaptive responses of OT TREATMENT OF DATA and histidase to dietary induction by casein hydrolysate, marked decreases in the induced levels of tryptophan pyrrolase In general, the oral intubation of casein hydrolysate pro and serine dehydratase and a substantial increase in the in duced two types of enzyme response. The first, exhibited by duced level of TAKG. Chart 3 illustrates the effects of the serine dehydratase, OT, tmyptophan pyrrolase, and histidase, intubation of casein hydrolysate on the adaptive response of was characterized by a continuous increase in the hepatic OT in protein-depleted rats fed a control or AAF-containing enzyme level throughout the 18-hour experimental period. diet for 0 to 5 weeks. The solid line represents the initial or Although the levels of each of these enzymes were determined zero-time level of OT at each of the weekly intervals studied; at 0, 6, 12, and 18 hours, only the 0- and 18-hour time points the dotted line represents the hepatic OT level 18 hours after were presented in this paper. In all cases, however, the 6- and multiple intubations of casein hydrolysate. Throughout the 12-hour enzyme levels were found to be consistent with the ex@emimental period, multiple intubations of casein hydroly observations made at 18 hours. As has been reported prey sate produced approximately a 200% increase in the hepatic ously (16), a somewhat different type of change in enzyme levels of OT in the control rats (Chart 3); similar responses level was manifested by the response of TAKG to the oral were elicited in the AAF-fed rats until the fifth week, when no intubation of casein hydmolysate. The enzyme levels increased significant difference could be detected in the hepatic levels of up to 6 or 12 hours and then decreased after this time period. OT before and after casein hydrolysate intubation (P < 0.05). Since the period of greatest net increase in enzyme level al Although the induced level of OT at the start of the experi ways occurred between 0 and 6 hours, only the values at those ment was significantly higher than on any subsequent week, two time points are presented here. All points on the graphs and the base level in the rats maintained on the control diets reported here represent the average values obtaiiied from be for 5 weeks was significantly lower than on any previous week, tween 4 and 6 rats. The standard deviation and standard error the physiologic significance of these observations is unknown. of the mean was calculated for each point. All comparisons The 30â€”50% increase in hepatic histidase following 18 hours between groups and all conclusions drawn were based on the treatment with casein hydrolysate noted in the livers of the significance of statistical analysis by the t test with P values of control rats throughout the 5 week experimental period (P < less than 0.05. All statistical calculations were carried out 0.05) was found to be completely lost in the livers of the rats through use of a Control Data Corporation 1604 computer of fed AAF for 4 or 5 weeks (Chart 4). In addition, there was a

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â€” â€” Casein Hydrolysote ORNITHINE-Ã´-TRANSAMINASE â€” - Cosein HISTI ______â€”â€”â€”â€” +Casein Hydrolysote â€”â€”â€”- +Co@jn CONTROL______AAF CONTROL AAF 50

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2 3 4 4 5 @O I 2 3 4 5-0 I 2 3 4 5 WEEKS ON DIET WEEKS ON DIET Chart 3. The effect of 0.03% 2-acetylaminofluorene (AAF) in the Chart 4. The effect of 0.03% 2-acetylaminofluorene (AAF) in the diet on the induction of ornithine-6-transaminase by casein hydroly diet on the induction of histidase by casein hydrolysate. The format sate. The format of the graph is similar to that seen in Chart 2 in that and experimental details are identical to those in Chart 3. the points connected by the dash line represent the enzyme level after 18 hours of casein hydrolysate administration as described in the base level of serine dehydmatase was 28 Â±S units!gm liver; the Methods. The closed circks and solid line represent control values of corresponding value in the control mats was 77 Â±11 (P < 0.01). the enzyme. The vertical bars are the standard error of the mean. See text for further details. Both the basal (zero-time) and the induced (6-hour) levels of TAKG were increased during the 5 weeks of administration of AAF (Chart 7). The induced level of 399 Â±36 observed at 5 78% decrease in the zero-time levels in the AAF-fed mats, in weeks in the AAF-fed mats was significantly different from the marked contrast to the 48% increase noted in the zero-time corresponding value of 235 Â±60 noted in the controls (P < levels of the control rats over the same 5-week experimental 0.01). Similarly, the 5-week base levels of TAKG, 49 Â±8 and period (P < 0.06). A decrease in the induced level of histidase 100 Â±15 for the control and AAF-fed rats respectively, were was noted as early as 2 weeks following the start of carcinogen significantly different (P < 0.05). administration. The chronic administration of AAF to rats led to marked DISCUSSION decreases in the induced levels of hepatic tryptophan pyrmolase and semine dehydratase. At all weeks investigated, the multiple The overall impression gained from these experiments is intubation of casein hydrolysate for 18 hours led to hepatic that, with respect to the control of enzyme synthesis, theme is tryptophan pyrrolase levels of 12-15 enzyme units/gm wet a marked deviation from the normal responses to dietary pro weight of liver in the control mats but of only 7 enzyme units! tein in the livers of AAF-fed rats. OT and histidase, two en gm wet weight of liver in the control rats fed AAF for 5 weeks zymes whose metabolic response were lost in the AAF-tmeated (Chart 5). At all times investigated, however, both in the AAF mats, have also lost their adaptive responses to dietary altera fed and in the control rats, theme was a significant difference between the basal and the induced enzyme levels (P < 0.01).

The 125% increase produced by casein hydmolysate in rats fed â€” â€” Cosein Hydrolysote AAF for 5 weeks appeared to be quantitatively similar to the TRYPTOPHAN PYRROLASE â€”â€”â€”â€” + Casein Hydrolysote 188% increase noted in the control rats at this time. This apparent decrease in tryptophan pyrrolase inducibiity noted CONTROL AAF in the control rats was due more to a 127% increase in the zero-time tryptophan pyrrolase levels from 0 to 5 weeks than to a decrease in the induced levels; the zero-time level of the enzyme in the AAF-fed rats remained essentially constant throughout the experiment. The basal and induced levels of

serine dehydratase in the control mats remained constant (0

throughout the 5-week period (Chart 6). Although the base C/) levels of semine dehydratase stayed essentially constant in the I-z AAF-fed rats, the induced levels underwent a continuous de chine; at 5 weeks the induced level of semine dehydratase in the AAF-fed rats was significantly less than in the control rats (P < 0.05). At allweeksinvestigated,however,the inducedlevels WEEKSON DIET were significantly higher than the corresponding levels in the Chart 5. The effect of 0.03% 2-acetylaminofluomene (AAF) in the AAF-fed rats. The AAF diet also appeared to affect the base diet on the induction of tryptophan pyrrolase by casein hydrolysate. level of semine dehydratase ; after 5 weeks of AAF feeding, the The format and experimental details are identical to those in Chart 3.

FEBRUARY 1969 467

â€” â€” Casein Hydrolysate TYROSINE -@- KETOGLUTARATEâ€” - Casein SERINE DEHYDRATASE + Casein Hydrolysate TRANSAMINASE -â€”â€”â€”+CaseinHydrolysate

CONTROL______AAF CONTROL AAF

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O__ I 2 3 4 5@O I 2 3 4 5 2 3 4 5@0 I 2 3 4 WEEKS ON DIET WEEKSON DIET Chart 6. The effect of 0.03% 2-acetylaminofluorene (AAF) in the Chart 7. The effect of 0.03% 2-acetylaminofluorene (AAF) in the diet on the induction of serine dehydratase by casein hydrolysate. The diet on the induction of tymosine-a-ketoglutarate transaminase by casein format and experimental details are identical to those in Chart 3. hydrolysate. The format and experimental details are identical to those in Chart 3. tion in the Morris 5123, 7800, and Reubem H-35 hepatomas (un published observations). In addition, the decreased base levels of so, whether they are essential to that process. Recent expemi histidase noted in the AAF-fed rats were reflected in the 5123, ments conducted in this laboratory indicate that the loss of 7316, 7777, and H-35 hepatomas (26). As in the livers of the enzyme inducibility is at least associated with the carcinogenic AAF-fed rats, the dietary induction of tryptophan pyrrolase process; the chronic administration of the hepatocnrcinogen has been found to be markedly diminished or completely 3'-methyl-DAB caused a loss in the inducibiity of OT and absent in virtually all tumors investigated, including 5123 (19, HIS; the chronic administration of the noncnrcinogenic isomer 20), 7800 (19), 7316 (19), 7793 (19), H-35 (4), Novikoff (3), 2-methyl-DAB did not affect the adaptive responses of any of and primary tumors induced by 3'-methyl-DAB (9). In the the 5 amino acid-metabolizing enzymes investigated (29). The AAF-fed rats, the base level and the dietary induction of semine mechanism by which the carcinogen inhibited the metabolic dehydmatase are both diminished. These observations correlate increases in enzyme levels remains to be demonstrated. Recent with the levels and adaptive responses of semine dehydmatase observations have shown that the dietary induction of semine found in tumors, but numerous exceptions exist. Thus, the dehydratase (25) and tryptophan pymrolase (21) reflect true levels of semine dehydmatase are high in the 5123 (22) and 7793 protein synthesis and not solely the activation or solubilization (26), normal in the 7316, 7795, and 7800 (26), and low in the of preexisting enzyme. Our results thus suggest that dietary Novikoff (20), Dunning (20), H-35, 7794, and 7777 (26) hepa AAF is affecting the protein synthetic machinery of the cell. tomas. The dietary induction of serine dehydratase does occur Recent developments on the binding of activated amine car in the 7800 hepatoma (19), but is virtually absent in the 5123, cinogens to DNA (12, 14) both in vivo and in vitro (30) sug 7316, 7794, and 7795 hepatomas (unpublished observations). gest that metabolic products of carcinogens may be acting as TAKG presents an anomolous picture. In AAF-fed mats both mutagens. Experiments are currently in progress in this laboma its basal and induced levels are considerably higher than in the tory to determine whether the altered enzyme inducibility is a control rats, but its adaptive response appears to be normal. reflection of direct gene alteration or cytoplasmic changes Similarly, the TAKG levels of hepatomas have been reported which may indirectly affect the regulation of genetic expres to be either high, as in the H-35, 7800, 7793, 7777 (21), and sion. 5123 (24) hepatomas, or normal, as in the 7795, 7316 (19), or 5123 tumors implanted in adrenalectomized hosts (24). Unlike ACKNOWLEDGMENTS the livers of the AAF-fed rats, however, no hepatomas investi The authors wish to express their sincere gratitude to Drs. James and gated, including the 5123, H-35, and 7800 hepatomas, have Elizabeth Miller for their help and suggestions during the course of this been found to possess a normal dietary induction ofTAKG; all work. The technical assistance of Mrs. Christine Shannon and Mrs. possessed a diminished adaptive response to tyrosine or to Annabelle Cutler is gratefully acknowledged. casein hydrolysate when compared to normal liver (19, 25). The general conclusion that the livers of mats fed AAF under go a loss of metabolic adaptability of several enzymes is in REFERENCES good agreement with previous observations on tryptophan 1. Andersen, R. A., Enomoto, M., Miller, E. C., and Miller, J. A. pyrrolase in the livers of rats fed either 3'-methyl-DAB or Carcinogenesis and Inhibition of the Walker 256 Tumor in the Rat dimethylnitrosamine. However, the induction of TAKG is an by trans-4-Acetylamino-stilbene. Its N-hydroxy Metabolite and obvious exception to this in liver and hepatomas. The enzy Related Compounds. Cancer Res., 24: 128â€”140,1964. matic resemblance between the preneoplastic and the neo 2. Andersen, R. A., Raina, P. N., and Milholland, R. J. Altered plastic livers raises the questions of whether the enzymatic Responses to Coritisol in Precancerous Liver. Onocologia, 20: alterations are at all related to the carcinogenic process and, if 153â€”166,1966.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. Dietary Induction of Some Enzymes of Amino Acid Metabolism during 2-Acetylaminofluorene Feeding

Lionel A. Poirier and Henry C. Pitot

Cancer Res 1969;29:464-469.

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