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

[CANCER RESEARCH 29, 1028â€”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 72Â°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 180â€”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 5â€”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 Â±0.03â€•0.30 Â±0.011.32 Â±0.0410.0 Â± 0.1Unligated Â±0.010.04 Â±0.010.11 Â±0.013.1 Â± lobes from operated rats Total61.340.9Free0.10 Â±0.140.29 Â±0.031.34 Â±0.079.5 Â± 0.1Ligated Â±0.020.04 Â±0.010.11 Â±0.013.0 Â± lobes Total60.5102bFree0.34 Â±QQ4b0.22 Â±002bÂ± 0â€¢06b4.0 Â± Â±[email protected] Â±002b0.70 Â±002b2.5 Â±0.l@' Changes in acid hydrolase activities 16 hours after ligating a portion of the liver. aMean Â±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

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. In normal liver the free mors. After repeated intramuscular transplantation, the hepa activity represented from 8 to 31% of the total activity, but 16 toma had signfficantly lower cathepsin activity than either the hr after liver ligation the free activity was 60â€”80% of the total primary tumor or liver. activity. The free cathepsin activity was 8â€”10% of the total activity in the normal liver and in the liver of rats fed the basal diet. Hepatomas: Induction, Primary, and Transplanted The free cathepsin activity remained high throughout the feed ing period and in the primary tumor, but it also decreased Measurements of free and total acid hydrolase activity were during transplantation of the hepatoma. made biweekly on, groups of 4â€”10rats during the 12-week Chart 2 shows a similar pattern of change for 13-galactosidase period in which 3 -Me-DAB was fed and during the 6-week activity to that observed with cathepsin. The maximum in period when the animals were returned to their regular diet. crease in both free and total activity occurred at 4 weeks. The Since acid hydrolase activities in rats fed the basal diet (as free and total activities were not significantly different from sayed biweekly) remained essentially constant, only the aver normal liver at 2 weeks. From 4 weeks on and throughout the age value for the entire feeding period is presented (Charts period of tumor development and in one of the 2 transplanted 1â€”4).The acid hydrolase activities were also determined in tumors, the free and total $3-galactosidase was significantly 20â€”30 rats with primary hepatomas and in 2 different trans higher than that found in normal liver. The percentage of the planted hepatomas in their 20th to 40th transplant genera total activity that was free was highest at 6 weeks and re tions. The cathepsin activities obtained are shown in Chart 1. mained higher than normal throughout the feeding period and Total cathepsin activity was significantly increased after 2 in the primary tumors. weeks on the diet containing 3'-Me-DAB and reached a maxi Aryl sulfatase activity during carcinogenesis and in the pri mum at 4 weeks. The total catheptic activity remained ele mary and transplanted tumors is shown in Chart 3. The total vated throughout the feeding period when compared with the activity was significantly increased after 2 weeks on the diet livers of rats fed the basal diet. The activity decreased slightly containing 3'-Me-DAB and reached a maximum at 4 weeks.

3.0 CATHEPSIN

2.5 TOTAL

C 2.0 E

@ â€˜.5

E a,

4, .0 C â€˜I, 0 1@ >, 0.4

U, 4) 0.3 0 E 0.2

0. I

@ BASAL2 4 6 8 0 2 2 4 6 NOP WEEKSFEO [email protected] R@4@,AR TUMOR TRANSPLANTED TUMORS

Chart 1. The free and total cathepsin activity during the induction, growth, and transplantation of hepatomas. The results are expressed as Mmoles of product produced/gm tissue/mm, with the standard error indicated by the brackets. Four animals fed the basal diet were assayed biweekly throughout the feeding period, and the average of these values is presented in the Chart. Each point during the feeding of 3'-methyl-4.dimethylaminoazobenzene (3'-Me.DAB)and the regular diet is an average of 4â€”10rats. The bars for normal liver, adjacent liver, primary tumors, and the 2 transplanted tumors are averages obtained from 20â€”30rats. The shaded portion of these bars represents the free activity. Details for determining free and total activities are given in the Methods section.

1030 CANCER RESEARCH VOL.29

0.5 /3 -GALACTOSI DASE TOTAL

4, 0.3

U) U)

FREE

0 010

[email protected]

@006

@0.O4

@O.02

BASAL2 4 6 8 10 12 2 4 6 NORMAL LIVER PRIMARY LIVER ADJACENT TLIvIOR WEEKS FED 3'-Me-DAB REGULAR ToTumor

Chart 2. The free and total @3-galactosidase activity during the induction, growth, and transplantation of hepatomas. For further details see the legend for Chart 1.

2.5

ARYL SULFATASE

C 2.0

4, TOTAL U) U, 1.5 I. E 0' @I.0

.@: 0.5 z 0.4

@0.3 :1 0.2

0. I

BASAL2 4 6 8 0 2 2 4 6 NORMAL LIVER PRIMARY N@:I 2 LIVER Adjacent TUMOR TRANSPLANTED WEEKS FED 3@Me-DAB REGULAR TO TUMOR TUMORS DIET

Chart 3. The free and total aryl sulfatase activity during the induction, growth, and transplantation of hepatornas. For further details see the legend for Chart 1.

MAY 1969 1031

ACID PHOSPHATASE 0 TOTAL C

a) U, U, P

0 C 4) 0. FREE z a.

4)2U, 0 E

BASAL 2 4 6 8 0 2 2 4 6 NORMAL LIVER PRIMARY NO: I 2 WEEKS FED 3'-Me-DAB R@GU@R LIVER Adjacent TUMOR TRANSPLANTED to umor TUMORS

Chart 4. The free and total add phosphatase activity during the induction, growth, and transplantation of hepatomas. For further details see the legend for Chart 1.

The total activity declined rapidly with continued feeding and caused an increase in free activity at 4 and 6 weeks and a slight was back to normal levels when the rats were returned to their increase in total activity at 6 weeks. Both free and total ca regular diet. A portion of this decrease during the feeding theptic activity were increased after feeding a-naphthol iso period might have been due to the basal diet rather than the thiocyanate ; however, the increase in total activity was less 3 -Me-DAB. There was a steady decline in total aryl sulfatase than that obtained after feeding 3'-Me-DAB. activity in rats fed the basal diet as shown in Chart 7. The 2 The effects of feeding the various diets on $3-galactosidase are transplantable tumors were significantly lower in total aryl shown in Chart 6. The total activity of (3-galactosidase was sulfatase activity than that found in normal liver. The free activ decreased after 6 weeks of feeding 4'-Me-DAB. All of the other ity was very high after 4 weeks on the 3'-Me-DAB diet but diets caused significant increases in both free and total (3-galac declined rapidly with continued feeding. The free activity was tosidase activity. also quite different in the 2 transplantable tumors. Chart 7 illustrates the effects of the various additives to the The acid phosphatase activity shown in Chart 4 responded diet on the aryl sulfatase activity. The results with the basal differently from the other 3 enzymes to the feeding of azo diet are listed separately since a significant decrease was noted dye. The total activity was slightly below normal during the when comparisons were made with normal liver. The total aryl feeding of 3'-Me-DAB, remained low in the primary tumor, sulfatase activity declined for the first 6 weeks of feeding the and was markedly decreased in the transplantable tumors. The basal diet and then remained at this lower activity. The values free activity was slightly above normal during the early period for 8, 10, and 12 weeks of feeding the basal diet were 0.86, of feeding, but it also decreased significantly below normal in 0.98, and 0.92 respectively. Feeding 4'-Me-DAB also produced the primary and transplantable tumors. a lowering in total aryl sulfatase activity. This lowering might have been caused by the basal diet rather than the 4'.Me-DAB. Various Additives to the Diet Feeding 3'-Me-DAB resulted in increases in both free and total aryl sulfatase activity throughout the 6-week feeding period. Chart 5 shows the effects of feeding a weak carcinogen, Thioacetamide increased the free activity throughout the en strong carcinogens, and a stimulant for the proliferation of tire feeding period, but the total activity was increased only at biliary epithelial cells on free and total cathepsin activity. The the first sampling period. The stimulation of proliferation of basal diet gave results throughout the feeding period similar to bile cells by feeding a-naphthyl isothiocyanate resulted in in those obtained with the Rockland diet. The weak carcinogen creased activities of both free and total aryl sulfatase at every 4'-Me-DAB was similar to the basal diet except for a slight but sampling interval. significant decrease in total activity at 6 weeks. Both the free No consistent pattern of change in acid phosphatase activity and total catheptic activities were significantly increased at all was observed with any of the additions to the diet (Chart 8). 3 sampling intervals after feeding 3'-Me-DAB. Thioacetamide There was some indication of an increased free acid phospha

1032 CANCER RESEARCH VOL.29

3.O@ CATHEPSIN in either total acid phosphatase or its distribution between cell sap and particles during the first 24 hr of liver regeneration. An increase in free activity and a decrease in total activity have been shown for a number of liver lysosomal enzymes 2.5 after ligation of the blood vessels (8), starvation (3), or carbon tetrachloride poisoning (1, 3, 22). We obtained similar results with our methods as shown in Table 1. The lysosomal enzyme

* patterns have been shown to be similar with a variety of differ ent methods of acute liver injury, suggesting that this may @2.O represent a standard reaction to injury. The changes in enzymatic activity which occurred in the 1; present studies and in the studies of Deckers-Passau et al. (9) during the early period of feeding a carcinogenic diet, how TOTAL E â€˜.5 ever, were somewhat different from those which occurred dur D@ NORMAL AND ing either liver regeneration or injury. One possible explana 4) BASAL C tion might be a degeneration of parenchymal cells and a prolif U) eration of bile duct and/or littoral cells. Price et al. (20) made e 1.0 a detailed histologic study of the changes that occur after >@ Iâ€”. feeding 3'-Me-DAB. They found hyaline droplets or inclusions in the cytoplasm of the parenchymal cells after only 7 days of U) 4) feeding; these inclusions increased in number and size up to @ 0.5 about the 25th day. The damage to the parenchymal cells might explain the increase in free activity that we observed :1% FREE after feeding 3'-Me-DAB. Price et a!. (20) also observed a -@ â€”.4â€”NORMAL AND BASAL WEEKS 246 246 /3-GALACTOSIDASE DIET 0.51

Chart 5. The effect of various additions to the diet on free and total cathepsin activities at 2, 4, and 6 weeks. The open bars represent total and the shaded area free enzyme activities. Each bar is the average value TOTAL NORMAL obtained on 4â€”12rats. The asterisk denotes those values that are AND different from the regular diet with a P value of 0.05 or less. The dotted BASAL line is the average value for rats fed the basal x the Rocidand diets with the standard error indicated by the bracket. 3'[or 4'J-Me-DAB, 3'[or 4']-methyl-4-dimethylaminoazobenzene.

tase activity during the feeding of the strong carcinogens, 3'-Me-DAB and thioacetamide. FREE - -- 4- NORMAL AND DISCUSSION BASAL WEEKS 246 246 246 246 The total activity of the 4 lysosomal enzymes of normal rat p liver reported in this paper correspond to previously reported DIET \ \ values, with the exception of aryl sulfatase where our values were lower (5). None of these activities were found to change significantly when measured daily during liver regeneration. Previously Becker and Lane (4) obtained histochemical cvi dence for increased acid phosphatase in â€œauto-phagosomesâ€•of Chart 6. The effect of various additions to the diet on free and total the hepatic cells during the early hours after partial hepatec @-ga1actosidaseactivitiesat 2, 4, and 6 weeks. For further details see the tomy. Yong and Terayama (28), however, observed no change legend for Uiart 5.

MAY 1969 1033

C 2.5@ ARYL SULFATASE during the feeding of azo dyes. Carr (6) has shown an increase in acid hydrolases of reticuloendotheial cells when they are sthnulated. 4, The membrane surrounding the acid hydrolases seems to be U, U) more readily ruptured after 4 weeks of feeding 3'-Me-DAB and throughout the rest of the feeding period. The free activity of E cathepsin and 13-galactosidase remained high after the dye feed a) ing was stopped and even in the primary tumor. Deckers 0 TOTAL Passauet a!. (9) observed increased free or unsedimentable .@ U activity of all of the liver enzymes they studied after feeding 4) 4- a 4-dimethylaminoazobenzene. They indicated that this change U might be characteristic of the precancerous state since it was 0 4- absent in rats receiving aminoazobenzene and in the azo dye z fed rats that were protected with riboflavin supplementation U) (9). In our studiesincreasedfreeenzymeactivitywasalso 4) found in the livers of rats fed thioacetamide or a-naphthyl 0 E isothiocyanate. :1% There was a significant difference in the total activities be FREE tween the 2 transplantable tumors. This may have been due to NORMAL different cells of origin, but this would require much more extensive study to establish. Dzialoszynski et a!. (1 1) found an increase in aryl sulfatase activity when either skin, stomach, DIET @V@$)

ACID PHOSPHATASE

Chart 7. The effect of various additions to the diet on free and total T I TOTAL aryl SulfataSe activities at 2, 4, and 6 weeks. The dotted line is the â€” â€”I- NORMAL average value for rats fed the Rockland diet with the standard error C I AND indicated by the brackets. The values for the basal diet are shown BASAL separately, since they differed significantly from the results obtained on 4) C normal liver. For further details see the legend for Chart 5. U) U) .pâ€” 1 E marked proliferation of bile ducts starting on about the 25th C' day of feeding. Most of the bile duct cells disappeared between 0 6 and 8 weeks. This time of proliferation of bile duct cells C 4, corresponds quite well to the time when an increase in total 0. activity occurred for cathepsin, (3-galactosidase, and aryl sulfa 0 tue. 4- z C C It has been shown that feeding or injection of a-naphthyl FREE isothiocyanate will cause massive proliferation of biliary epi 0. .___t NORMAL. thelial cells in rat liver (10, 14, 25, 26). Feeding of a-naphthyl U) .1 AND 4) isothiocyanate in our experiments caused an increase in total BASAL activity of cathepsin, 13-galactosidase, and aryl sulfatase; but it also resulted in increases in free activity. The investigations of I Steiner and Carruthers (25) and Desmet et al. (10) indicated that, in addition to biliary proliferation, there was also consid. erable damage to the parenchymal cells aft@r a-naphthol iso WEEKS 2 4 6 246 246 thiocyanate. The feeding of a-naphthol isothiocyanate for 12 months did not produce neoplasms in the livers of rats (26), DIET which would suggest that the enzymatic changes we observed are not directly related to the carcinogenic process. Hyperplasia of the reticuloendothelial system and an in crease in littoral cells of the liver occurs during the first few weeks of azo dye feeding (16, 17, 21). It is possible that the Chart 8. The effect of various additions to the diet on the free and stimulation of the reticuloendothelial system might be respon. total acid phosphatase activities at 2, 4, and 6 weeks. For further. details sible for some of the changes observed in enzymatic activity see the legend for Chart 5.

1034 CANCER RESEARCH VOL.29

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1969 American Association for Cancer Research. Acid Hydmlases during Carcinogenesis colon, or breast tissue became cancerous. Shamberger and duced Cholestasis. Am. J. PathoL, 52: 401â€”422, 1968. Rudolph (24) measured cathepsin, 13-glucuronidase, and acid 11. Dzialoszynski, L. M., Frohlich, A., and Kroll, J. Cancer and Aryl phosphatase and found all of them had higher activity in suiphatase Activity. Nature, 212: 733â€”734,1966. mouse skin cancer than in mouse skin. We found the cathepsin 12. Farber, E. Similarities in the Sequence of Early Histological and j3-galactosidase activity slightly higher in primary hepato Changes Induced in the Liver of the Rat by Ethionine, 2-Acetyl mas when compared to normal liver, but these activities de aminofluorene, and 3'-Methyl-4-dimethylaminoazobenzene. Cancer Res., 16: 142â€”148,1956. creased upon transplantation. Aryl sulfatase and acid phospha 13. Higgins, G. M., and Anderson, R. M. Experimental Pathology of tue were lower in both primary and transplanted hepatomas the Liver. I. Restoration of the Liver of the White Rat Following than they were in normal liver. Wagner and Roth (27) found Partial Surgical Removal. Arch. Pathol., 12: 186â€”202,1931. less total 13-glucuronidase in the Morris 5123D and Novikoff 14. Lopez, M., and Mazzanti, L. Experimental Investigation on Alpha hepatoma than in normal liver. They also observed that a high Naphthyl-Isothiocyanate, as a Hyperplastic Agent of the Biliary er proportion of the activity was recovered as free activity in Ducts in the Rat. J. Pathol. Bacteriol., 69: 243â€”250,1955. the high-speed supernatant fraction of tumor tissue. 15. Lowry, 0. H., Roberts, N. R., Wu, M., Hixon, W. S., and Crawford, E. J. The Quantitative Histochemistry of Brain. II. Enzyme Mea ACKNOWLEDGMENTS surements. J. Biol. Chem., 207: 19â€”38,1954. 16. Lozzio, B. B. Mechanisms of Reticuloendothelial Alteration Pro The authors are indebted to Dr. Donald E. Kizer and his Carcinogene duced by Azo Dye. J. Reticuloendothelial Soc., 4: 85â€”108,1967. sis Section for mixing the diets and feeding the animals. We also thank 17. Lozzio, B. B., Machado, E., Lew, V., and Royer, M. Catabolisme de Mrs. Faye Jones for technical assistance. l'hemoglobine darn l'hyperfonction du Systeme Reticuloendo theliale Produite par le p-dimethylaminoazobenzene. J. Reticulo endot@lial Soc., 1: 293â€”305,1964. REFERENCES 18. Martini, E., and Dianzoni, M. U. Activation of Cathepsin in Fatty Liver. Experientia, 14: 285â€”286, 1958. 19. Miller, J. A., and Miller, E. C. The Carcinogenic Aminoazo Dyes. 1. Alpers, D. H., and Isselbacher, K. J. The Effect of Carbon Tetra Advan. Cancer Res., 1: 339â€”396, 1953. chloride on Rat-Liver Lysosomes. Biochim. Biophys. Acta, 137: 20. Price, J. M., Harman, J. W., Miller, E. C., and Miller, J. A. Progres 33â€”42,1967.â€¢ sive Microscopic Alterations in the Livers of Rats Fed the Hepatic 2. Anson, M. L. The Estimation of Pepsin, Trypsin and Cathepsin Carcinogens 3'-Methyl-4.dimethylaminoazobenzene and 4'-Fluoro with Hemoglobin. J. Gen. Physiol., 22: 79â€”89, 1938. 4-dimethylaminoazobenzene. Cancer Res., 12: 192â€”200,1952. 3. Beaufay, H., van Campenhout, E., and de Duve, C. Tissue Fraction 21. Richardson, H. L., and Barsos-Nachtnebel, E. Study ofLiver Tu ation Studies. 11. Influence of Various Hepatotoxic Treatments on mors Development and Histology Changes in Other Organs in Rats the State of Some Bound Enzymes in Rat Liver. Biochem. J., 73: Fed Azo Dye 3'-Methyl-4-dimethylaminoazobenzene. Cancer Res., 617â€”623,1959. 11: 398â€”403, 1951. 4. Becker, F. F., and Lane, B. P. Regeneration of the Mammalian 22. Roy, A. B. The Suiphatase of Ox Liver. I. The Complex Nature of Liver. I. Auto-Phagocytosis During Dedifferentiation of the Liver the Enzyme. Biochem. J., 53: 12â€”15, 1953. Cell in Preparation for Cell Division. Am. J. Pathol., 47: 783â€”801, 23. Sellinger, O.Z., Beaufay, H., Jacques, P., Doyen, A., and de Duve, 1965. C. Tissue Fractionation Studies. 15. Intracellular Distribution and 5. Bowers, W. E., Finkenstaedt, J. T., and de Duve, C. Lysosomes in Properties of@3-N-Acetylglucosaminidase and fI-Galactosidase in Rat Lymphoid Tissue. 1. The Measurement of Hydrolytic Activities in Liver. Biochem. J., 74: 450â€”456,1960. Whole Homogenates. J. Cell Biol., 32: 325â€”338, 1967. 24. Shamberger, R. J., and Rudolph, G. Increase of Lysosomal En 6. Carr, I. The Cellular Basis of Reticulo-endothelial Stimulation. J. zymes in Skin Cancer. Nature, 213: 617â€”618, 1967. Pathol. BacterioL, 94: 323â€”330, 1967. 25. Steiner, J. W., and Carruthers, J. S. Electron Microscopy of Hyper. 7. de Duve, C. The Lysosome Concept. In: A. V. S. de Reuck and M. plastic Ductular Cells in a.Naphthyl Isothiocyanate-induced Cir p. Cameron (ccli.), Lysosomes. Ciba Foundation Symposium, pp. rhosis. Lab. Invest., 12: 471â€”498,1963. 1â€”31. London: J. & A. Churchill Ltd., 1963. 26. Ungar, H., Moran, E., Eisner, M., and Eliakim, M. Rat Intrahepatic 8. de Duve, C., and Beaufay, H. Tissue Fractionation Studies. 10. Biliary Tract Lesions from Alpha-Naphthyl Isothiocyanate. Arch. Influence of Ischaemia on the State of Some Bound Enzymes in Pathol., 73: 427â€”435,1962. Rat Liver. Biochem. J., 73: 610â€”616, 1959. 27. Wagner, R. L., and Roth, J. S. Some Properties of@3-Glucuronidase 9. Deckers-Passau, L., Maisin, J., and de Duve, C. The Influence of Activity in Normal Rat Liver and Several Transplantable Rat Hepa Azodycs on Lysosomal Enzymes in Rat Liver. Acta Unio Intern. tomas. Cancer Res., 27: 2053â€”2059, 1967. Contra Cancrum, 13: 822â€”836, 1957. 28. Yong, H., and Terayama, H. Acid Phosphatase Activity and Its 10. Desmet, V. J., Krstalovic, B., and von Damme, B. Histochemical Distribution Between Particles and Cell Sap at Early Stages of Rat Study of Rat Liver in Alpha-Napthyl Isothiocyanate (ANIT) In Liver Regeneration. Gann, 57: 291â€”294,1966.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1969 American Association for Cancer Research. Acid Hydrolase Activity during the Induction and Transplantation of Hepatomas in the Rat

Ralph F. Kampschmidt and Dan Wells

Cancer Res 1969;29:1028-1035.

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