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Comparative Enhancing Effects of Phenobarbital, Amobarbital

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Comparative Enhancing Effects of Phenobarbital, Amobarbital [CANCER RESEARCH 35,2884 2890, October 1975] Comparative Enhancing Effects of Phenobarbital, Amobarbital, Diphenylhydantoin, and Dichlorodiphenyltrichloroethane on 2-Acetylaminofluorene-induced Hepatic Tumorigenesis in the Rat Carl Peraino, R. J. Michael Fry, Everett Staffeldt, and John P. Christopher 2 Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 SUMMARY INTRODUCTION Earlier studies showed that phenobarbital feeding en- In earlier studies (27, 29), the prolonged feeding of hanced hepatic tumorigenesis in rats previously fed 2- phenobarbital to rats that had previously been fed AAF 3 for acetylaminofluorene for a brief period. As part of an a brief period markedly increased the subsequent incidence investigation of the mechanism of this enhancement, the of liver tumors. Using this sequential feeding regime, the present study evaluated the relative enhancing abilities of present study evaluated the relative tumorigenic enhancing amobarbital, diphenylhydantoin, and dichlorodiphenyltri- abilities of other compounds that, to varying degrees, chloroethane (DDT), agents that resemble phenobarbital to resemble phenobarbital in their effects on liver structure and varying degrees in their effects on liver structure and metabolism. The substances examined were amobarbital, metabolism. A comparison of hepatic tumor yields in rats diphenylhydantoin, and DDT. Table 1 compares relevant fed 2-acetylaminofluorene, followed by the test substance characteristics of these agents with those of phenobarbital, (sequential treatment), showed that amobarbital and di- reviewed previously (27, 29). Amobarbital is closest to phenylhydantoin had no enhancing activity, whereas the phenobarbital in structure and, like phenobarbital, alters enhancing effect of DDT was similar to that of phenobarbi- the metabolism of other drugs by the liver. Diphenylhydan- tal. These results show that the sequential treatment toin is less closely related to phenobarbital structurally but technique readily distinguishes among substances differing has analogous effects on DNA synthesis and drug metabo- in enhancing ability and should prove useful in screening lism. DDT and phenobarbital are structurally dissimilar but additional substances for this activity. The comparative are closely analogous in their effects on the liver. biochemical effects of these substances in the liver can then The major objective of this study was the identification of be correlated with their relative enhancing abilities to a series of compounds that differ in ability to enhance liver provide information on the molecular events specifically tumorigenesis. Correlations of these differing enhancing associated with enhancement. Such correlations were initi- abilities with the effects of these compounds on metabolic ated in this study by comparing the effects of the four test processes in the liver can then be undertaken. Such correla- substances on liver weight and DNA synthesis. The results tions should facilitate the identification of those molecular showed that the enhancers, phenobarbital and DDT, each events that are essential to enhancement. stimulated liver DNA synthesis and increased liver weight, whereas the nonenhancers, amobarbital and diphenylhydan- toin, had neither effect. MATERIALS AND METHODS Phenobarbital and DDT both increased the early tumor incidence rate and maintained an increment in tumor Tumor Incidence Study. Male Sprague-Dawley rats at 22 incidence over that in the other treatment groups through- days of age were fed either a control diet or one containing out the experiment, although it is not clear whether this 0.02% AAF for 18 days, after which they were all fed the increment would persist indefinitely. In addition, although control diet for 7 days. The control and AAF-fed rats were the spectrum of tumor types observed ranged from highly then separately divided into 5 groups. Of the rats not fed differentiated to poorly differentiated in all treatment AAF, each group contained 48 animals; of those fed AAF groups, DDT and phenobarbital selectively increased the each group contained 120 rats. Both sets of groups were incidence of highly differentiated tumors throughout most given the following diets for the duration of the experiment: of the experiment. Group 1, control; Group 2, phenobarbital (Mallinckrodt Chemical Works, St. Louis, Mo.); Group 3, amobarbital (Sigma Chemical Co., St. Louis, Mo.); Group 4, diphenyl- ~This work was supported by the United States Energy Research and Development Administration. 2 Doctoral candidate in the General Sciences Department, Oregon State 3The abbreviations used are: AAF, 2-acetylaminofluorene; DDT, University, Corvallis, Ore. 9733 I. dichlorodiphenyltrichloroethane or l,l,l-trichloro-2,2 bis(p-chlorophe- Received March 27, 1975; accepted July 9, 1975. nyl)ethane. 2884 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Enhancement of A A F-induced Hepatic Tumorigenesis Table 1 Comparison of known effects Of phenobarbital, amobarbital, diphenvlhvdantoin, and DDT in the liver The numbers in parentheses are references, including primary sources and review articles. Effect in liver Hypertrophy and smooth DNA synthesis endoplasmic Microsomal and hyper- RNA RNA reticular enzyme Agent Structure plasia synthesis degradation proliferation activity Phenobarbital H Increases (I, Increases Decreases Increases(1, Increases c%-c"e~lO.-f--"N"-yO /" 27, 29) (27, 29) (27, 29) 27, 29) (13, 27, 29) Amobarbital H Unknown Unknov~n Unknown Unknown Increases 0 .i.-'/N%.O CH3_CH2~ [ |. (21) CH3-CH-CH2-CH2~NH 0 CH3 Diphenylhydantoin ~[/~~H"~ Increases Unknown Unknown No effect Increases N ~o (36) (14) (7, 10 12, 14, 35, 37, NH 38, 44) DDT col3 Increases Increases Decreases Increases Increases ~ f'/,-~~--- ~ ----~,----,'"1A (16) (24) (24) (15, 25, 26, (34, 1 l) 3O) cI Ct hydantoin (Sigma); Group 5, DDT' (Matheson, Coleman, Beginning on Day 26, the rats were given single daily i.p. and Bell, Cincinnati, Ohio). The dietary concentration of injections (between 7 a.m. and 9 a.m.) of phenobarbital each agent was 0.05%. Based on measurements of food sodium (83 mg/kg), amobarbital (75 mg/kg), diphenyl- consumption, it was estimated that the average daily intake hydantoin sodium (44 mg/kg), or DDT (1 12 mg/kg). The of these agents ranged from approximately 50 mg/kg of DDT was dissolved in cottonseed oil; the other agents were body weight during the 1st 4 to 6 months of the experiment dissolved in distilled water. Control rats received equivalent down to approximately 20 mg/kg of body weight by the volumes of water or cottonseed oil. The dosages of pheno- 15th month. No symptoms of sedation or toxicity were barbital, amobarbital, and DDT were equimolar; the dosage observed in any of the rats throughout the experiment. of diphenylhydantoin was one-half that of the other agents, Rats were killed at intervals (see "Results"; Chart 1) owing to the mortality observed in rats given higher doses. throughout the experiment and examined for tumors. Liv- The rats given phenobarbital or amobarbital slept for 2 to 3 ers were sliced with a razor blade into sections that were 5 hr following treatment. mm thick to reveal tumors beneath the surface. The largest Twenty-four hr after receiving its last dose of test agent, diameter of each tumor was recorded, and samples of these each rat was given an i.p. injection of [aH]thymidine (500 and other grossly observable lesions were taken for light uCi/kg; specific activity, 0.36 Ci/mmole; Schwarz/Mann, microscopy. Additional experimental details (rats, diets, Orangeburg, N. Y.) and was killed by cervical dislocation 1 husbandry, and histological preparations) were described hr later. Liver nuclei were isolated by the method of Biobei previously (27, 29). and Potter (3), and were counted in a hemocytometer. Acute Exposure Study. Weanling rats were fed the control For the determination of acid-insoluble radioactivity, diet for 25 days before being exposed to the test substances. 0.5-ml aliquots of the nuclear suspensions were placed on 9-cm filter papers folded to fit into Coplin jars. The samples ' Technical grade, containing: p,pI-DDT (see Footnote 3), 70%; o,p I- were then dried under a heat lamp and given a series of DDT [ 1, I, I-trichloro-2,2-(o-chlorophenyl-parachlorophenyl)ethane],12~: 10-rain washes, 3 times with 10% trichloroacetic acid, then p,pt-DDT [i,l-dichloro-2,2-bis(p-chlorophenyl)ethane], 3% p,pl-DDE [I,l-dichioro-2,2-bis(p-chlorophenyljethylene], 15%. Analysis, courtesy o1 twice with 95% ethanol. The samples were dried again, the Department of Agricultural Chemistry, Oregon State University, combusted in a Packard sample oxidizer, and counted in a Corvallis, Ore. liquid scintillation spectrometer. OCTOBER 1975 2885 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. C. Peraino et al. RESULTS 9 CONTROL A i o PHENOBARBITAL Tumor Incidence Study. The observed hepatic lesions i AAF~ [] DDT were classified into the following types. (a) Hyperplasia and o i 0 AMOBARBITAL hypertrophy. While these lesions appeared to represent a ,oo - D,PHE.YL.YOANTO,N new cell population with an altered proliferation rate and glycogen metabolism, which are attributes of neoplasia, the 80- ~/~-- ---o- main characteristics of normal liver structure were main- 7o,- ~// tained, (b) Adenomas. The characteristics of these lesions ~: 60~ ~ I .4 -o
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