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Acid Hydrolase Activity During the Induction and Transplantation of Hepatomas in the Rat

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Acid Hydrolase Activity During the Induction and Transplantation of Hepatomas in the Rat [CANCER RESEARCH 29, 1028—1035, May 1969] Acid Hydrolase Activity during the Induction and Transplantation of Hepatomas in the Rat Ralph F. Kampschmidt and Dan Wells BiomedicalDivirion, The Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73401 SUMMARY Most of the information currently available about acid hydrolases during carcinogenesis has been provided by the Both free and latent enzymatic activities of cathepsin, (3- studies of Deckers-Passau et al. (9). They studied changes in galactosidase, aryl sulfatase, and acid phosphatase were deter acid phosphatase, (3-glucuronidase, cathepsin, acid ribonucle mined in normal liver and during various stages of the produc ase, and acid deoxyribonuclease activity in the liver of rats fed tion and progression of hepatomas in Fischer rats. After 4 diets containing aminoazobenzene or 4-dimethylaminoazo weeks of feeding 3'-methyl-4-dimethylaminoazobenzene, there benzene. Total acid phosphatase decreased rapidly when these was a significant increase in free and total activity of all the diets were fed, but this change seemed to be unrelated to enzymes except acid phosphatase that persisted throughout carcinogenesis. Total j3-glucuronidase activity did not show any the feeding period. Both the free and total activities declined consistent change. Cathepsin, acid ribonudease, and acid when the rats were returned to their regular diet at 12 weeks. deoxyribonuclease activities increased during the first month The enzymatic activity found in the primary tumors was gen of feeding 4-dimethylaminoazobenzene, and these higher activ erally quite similar to that observed in the liver at the end of ities persisted during the entire precancerous period. The free the feeding period. A still further decline in activity, especially or unsedimentable activities of all of the enzymes that were of cathepsin and acid phosphatase, occurred when the tumors studied by Deckers-Passau et al. (9) were found to be higher were repeatedly transplanted. Somewhat similar changes were during the precancerous period. observed during the feeding period with thioacetamide and Wagner and Roth (27) found less total (3-glucuronidase and a a-naphthyl isothiocyanate but were not found when rats were higher proportion of the activity not bound to the lysosomal fed the basal diet, 4'-methyl-4-dimethylaminoazobenzene, or particle in Morris 5123D and Novikoffhepatomas than in nor during liver regeneration after partial hepatectomy. There was mal liver. It was our purpose not only to extend these studies some indication that these changes may be related to destruc to other lysosomal enzymes, but also to study the changes in tion of hepatocytes and proliferation of bile and/or littoral activity during various stages of the induction, growth, and cells. transplantation of hepatomas. INTRODUCTION MATERIALS AND METhODS Shamberger and Rudolph (24) found that cathepsin, 13-glu Rats curonidase, and acid phosphatase activities were enhanced in mouse skin cancer when compared to normal mouse skin. All animals were from our inbred strain of Fischer rats. The Dzialoszynski et al. (1 1) reported that aryl sulfatase activities original stock for this colony was the CDF strain obtained increased in skin, stomach, colon, and breast tissues when they from the Charles River Breeding Laboratories. They were rou became cancerous. Most of the normal tissues that have been tinely maintained at 72°F with 12 hr of light and 12 hr of used for this type of study have rather low acid hydrolase darkness and fed Rockland mouse and rat diet and water ad activity. It therefore seemed desirable to compare the acid libitum. The animals used for tumor transplantation weighed hydrolase activities during the transformation to cancer of a 180—200 gm; those started on an experimental diet usually tissue with known high activities of these enzymes. weighed between 150 and 160 gm. The liver has been the tissue used most frequently for studies of the acid hydrolase enzymes that are bound to the lysosome Diet (7) and for studies in carcinogenesis (19). The use of azo dyes Animals on the experimental diets were fed the basal diet makes it possible to predict fairly accurately when hepatomas described by Farber (12) containing one of the following addi will appear. Closely related compounds that are weak carcino tives: 0.06% 3'-Me-DA& , 0.06% 4'-Me-DAB, 0.066% thioacet gens are known (19) and can be used as controls. 1Abbreviations used are: 3'-Me-DAB, 3'-methyl-4-dimethylaminoazo Received August 9, 1968;.accepted December 30, 1968. benzene ; 4'-Me-DAB, 4'-methyl-4-dimethylaminoazobenzene. 1028 CANCER RESEARCH VOL.29 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1969 American Association for Cancer Research. AcidHydrolases during Carcinogenesis amide, or 0.08% a-naphthol isothiocyanate. The animals re Triton X-100, at a final concentration of 0.2%, and homoge ceiving 3'-Me-DAB were fed this diet for 12 weeks and were nizing an additional 20 strokes. then returned to the Rockland diet until the hepatomas de veloped, usually 5—8weeks later. Enzymatic Activity Surgical Operations Cathepsin was determined using a 4% w/v hemoglobin sub strate in 0.2 M acetate buffer at pH 3.6. The reaction was Partial hepatectomies were done by the method of Higgins stopped with 5% trichloracetic acid (2). Cathepsin activity was and Anderson (13). In one group of rats the same liver lobes obtained by subtracting the value of the zero time control were ligated but not removed. Enzyme analyses were made 16 containing all the reaction components from the value of the hr later on the ligated lobes and the unligated lobes from the incubated sample. Catheptic activity was expressed as pmoles same animal. of tyrosine/gm tissue/mm. Aryl sulfatase activity was mea sured by the method of Roy (22), using nitrocatechol sulfate Prhnary and Transplantable Tumors as the substrate in 0.5 M acetate buffer at pH 5.4. The results were expressed as pmoles of nitrocatechol/gm tissue/min. A The animals fed 3'-Me-DAB were allowed to develop large slight modification of the method of Sellinger et a!. (23) was tumor nodules, with an average total tumor weight of 25 gm, used for (3-galactosidase. The substrate o-nitrophenylgalactopy before an attempt was made to separate the tumor from the ranoside was buffered in 0.2 M acetate at pH 5.0, and the adjacent liver tissue. Only the nonnecrotic regions of these reaction was stopped by adding 0.4 M glycine adjusted to pH nodules were used for enzyme analyses. The samples of adja 10.8 with sodium hydroxide. The method of Lowry et al. (15) cent liver tissue also contained some small tumor nodules. was used to determine acid phosphatase. The substrate was Primary tumors from 2 different animals were transplanted p-nitrophenylphosphate, and the buffer was 0.2 M acetate at by injecting 0.2 ml of a 50% suspension of tumor cells in the pH 5.5. Results were expressed as @imolesof p-nitrophenol/gm rectus femoris. The tumors were assayed at varying periods tissue/mm. between the 20th and 40th transplant generations. The transplants from Tumor Number 1 were slow growing, RESULTS and the tumors used for enzymatic assay averaged 35 gm at 24 days after transplantation. Tumor Number 2 grew at a faster PartialHepatectomyor Ligationof the Blood Supply rate and averaged 43 gm at 15 days of tumor growth. Partial hepatectomies were performed on 36 rats, and the Tissue Preparation free and total acid hydrolase activities were measured daily for 9 days after the operation. The activity of the 4 acid hydro The liver or tumor tissue was diluted 1:10 with ice cold 0.25 lases did not differ significantly from normal liver at any time M sucrose and mixed with 3 strokes of the Potter-Elvehjem during liver regeneration. tissue homogenizer, consisting of a smooth-walled glass tube The effects of ligating several lobes of the liver on the free fitted with a Teflon pestle. An aliquot of this homogenate was and total acid hydrolase activity 16 hr later are shown in Table centrifuged at 25,000 X g for 30 miii, and the free activity was 1. The unligated lobes of the operated rats were not signifi measured in the supernatant, with no attempt to release latent candy different in acid hydrolase activity from normal liver. or bound activity. Total activity was measured in the superna The ligated lobes had signfficantly lower total activities of tant of another aliquot of the homogenate after treating with cathepsin, (3-galactosidase, aryl sulfatase, and acid phosphatase. 1TissueNo Table liver/mmCathepsin@-gs1actosidaseArylproduct/gm of phosphataseNormal rats@tmo1es sulfataseAcid liver33Total1.49 0.2Free0.13 ±0.03―0.30 ±0.011.32 ±0.0410.0 ± 0.1Unligated ±0.010.04 ±0.010.11 ±0.013.1 ± lobes from operated rats Total61.340.9Free0.10 ±0.140.29 ±0.031.34 ±0.079.5 ± 0.1Ligated ±0.020.04 ±0.010.11 ±0.013.0 ± lobes Total60.5102bFree0.34 ±QQ4b0.22 ±002b± 0•06b4.0 ± ±[email protected] ±002b0.70 ±002b2.5 ±0.l@' Changes in acid hydrolase activities 16 hours after ligating a portion of the liver. aMean ±standard error. bSig@ijfi@itly different from the mean of the normal liver at a P value of 0.05 or less. MAY 1969 1029 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1969 American Association for Cancer Research. Ralph F. Kampschmidt and Dan Wells When the liver was ligated, a much higher percentage of the when the animals were returned to their regular diet, but it activity was found free in the supernatant fraction after centri was still significantly above normal even in the primary tu fuging at 25,000 X g for 30 min.
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