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Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats
Cancer Letters, 88 (1987196-99 Elsevier Scientific Publishers Ireland Ltd
95
EFFECTS OF CALCIUM GLUCARATE ON THE PROMOTION OF DIE’I’IIYLNITROSAMINEINI’IIATED ALTERED HEPATIC FOCI IN RATS
OLADIPO A. OREDIPE**, ROLF F. BARTH’, MALGORZATA HANAUSEK-WALASZEKb~*, IVETA SAUTINS, ZBIGNIEW WALASZEKb***and THOMAS E. WEBB” Departments of’Pathology and bPhysiological Chemistry, The Ohio State University, Columbus, OH 43210 (U.S.A.) (Received 10 August 1987) (Revised version received 7 September 1987) (Accepted 8 September 1987)
SUMMARY
Calcium glucarate (CGT), an inhibitor of /3-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.
INTRODUCTION
Promotion is one of the steps in the multi-stage process of hepatocarcinogenesis. It is a reversible event characterized by focal proliferations of initiated cells during the preneoplastic phase [ 1,2]. Preneoplastic hepatocytes can be identified as altered hepatic foci (AHF) in hematoxylin and eosin-stained liver sections during the early and promotion phases of hepatocarcinogenesis [3]. The present study describes the protective effect of dietary calcium glucarate (CGT) on phenobarbital-promoted rat *Address correspondence to: Dr. O.A. Oredipe at The Ohio State University, Department of Pathology, 4170 Graves Hall. 333 W. 10th Avenue, Columbus, Ohio 43201. **Present address: The University of Texas System Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, TX 78957, U.S.A.
96
hepatocarcinogenesis, using as a criterion the inhibition of induction of AHF. The latter was assessed histopathologically by means of light microscopy. This study was prompted by the marked protective effects exhibited by CGT and similar acting compounds on experimental mammary and lung carcinogens [4,5]. MATERIALS
AND METHODS
Female Sprague-Dawley rats, weighing l!W-200 g, were purchased from Harlan Sprague-Dawley, Indianapolis, IN. The ZV-diethylnitrosamine (DENA), phenobarbital and calcium glucarate (CGT) were purchased from the Sigma Chemical Co., St. Louis, MO. The rats were maintained on rat chow alone or fortified with additives as noted below. Under ether anesthesia, 64 rats were subjected to 70% partial hepatectomy (PH) to induce hepatocellular proliferation [6] according to the hepatocarcinogenic protocol of Pitot et al. [7]. At 24 h post-operation, the animals were initiated with non-necrogenic doses of DENA (10 mg/kg body wt.) administered by the intragastric route and were maintained thereafter on a chow diet without CGT for 2 months. At 2, 4 and 8 weeks, some animals were randomly selected and killed under ether anesthesia. Liver tissues representative of each lobe were fixed in 10% neutral buffered formalin, paraffin-embedded and cut at a thickness of 4 pm. The-4 pm sections were stained with hematoxylin and eosin (H&E), and evaluated histopathologically by means of light microscopy. The presence and severity of AHF in each section were graded from 0 to 4 + with the former being none or normal and the latter as the most severe. The remaining rats were divided into 2 treatment groups that were further studied for up to 6 months. One group received 4% CGT plus 0.05% phenobarbital in chow diet throughout the duration of promotion, while the other had only 0.05Vo phenobarbital and served as a control. At monthly intervals of promotion up to 6 months, 4 or more animals in each group were killed, livers removed, fixed in 10% buffered formalin and 4-l.trn sections were cut, stained with H&E, and histopathologically evaluated as described above. RESULTS
For the purpose of evaluating the effects of 4% dietary CGT on the promotion phase, rats initiated with DENA and then kept exclusively on a chow diet for 2 months, were divided into 2 groups. One group of 28 rats was fed chow plus 0.05% phenobarbital for varying time intervals up to 6 months, while the other group was fed chow plus 0.05% phenobarbital diet supplemented with 4% CGT during the same period. The results, shown in Fig. 1, indicate that dietary CGT delayed the attainment of maximum levels and severity of altered foci in the livers of experimental rats. Evaluations of the H&E stained liver sections showed scattered foci composed of large cells with abundant eosinophilic cytoplasm in the case of CGT-fed rats (Fig. 2a) while numerous foci composed of large pale-staining vacuolated hepatocytes or neoplastic hepatocytes were commonly observed in those of the controls (Fig. 2b).
0
1
2 3 Months After
4 5 6 Diethylnitrosa~ine
7
6
Fig. 1. Effects of dietary CGT on the promotion phase of DENA-initiated, phenobarbital-promoted hepatocarcinogenesis. Phenobarbital (0.05%) alone and 0.05% phenobarbital plus 4% CGT in chow diets were initiated 2 months (arrow point) after initiation with DENA. Control rats ( l 0) were maintained on chow diet supplemented with 0.05% pheobarbital, while experimental rats (O-----O) received the promoter plus 4% CGT throughout the intervals of promotion. The liver sections were evaluated histopathologically and graded as described in the text. Each point represents the mean f SE. of 4 or more rats.
DISCUSSION
D-Glucaro-1 ,Clactone is the natural regulator of /3-glucuronidase in vivo [8], but is rapidly excreted from the body. Endogenous levels can be maintained through the use of sustained release forms. Dietary 2,5-di-O-acetyl-D-glucaro-l ,rl-dilactone, which is converted at the acid pH of the stomach to D-glucaro-1,4-lactone, was initially shown to inhibit 7,12-dimethyl-benzanthracene-induced rat mammary tumorigenesis [9]. This same compound also inhibited N-methyl-ZV-nitrosoureainduced mammary carcinogenesis, but in this case it may have reduced the sensitivity of the mammary gland to the carcinogen through increased clearance of estrogens [lo]. Dietary CGT, one third of which can be converted to D-glucaro-1 ,Qlactone in the stomach [5,8], has been shown to inhibit the promotion phase of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis [4] and benzo[a]pyreneinduced lung tumorigenesis in mice [5]. In these instances, any effect of calcium ion was ruled out. Dietary CGT at a concentration of 4% appears to be the minimal amount needed for maximal tumor inhibition [4,5]. The results of the present study suggest that dietary CGT had a marked inhibitory effect on the emergence of DENA-initiated preneoplastic lesions in rat livers, and caused a significant delay in
Fig. 2. Photomicrographs of livers from control and experimental rats. Sections were cut at4pms md stained with hematoxylin and eosin. Livers from rats that had received DEN A, followed by phenobarbital and CGT diet (a) showed scattered areas composed of large acidophilic cells i or cells Hrith abundant eosinophilic cytoplasm ( X 128). Livers from animals that had received DEINA followed by phenobarbital alone (b) showed numerous foci composed of large pale-staining, vacuoleated hepatocp tes, or neoplastic hepatocytes ( x 200).
99
the development of AHF. This delay without a permanent reduction in the expression or severity of AHF, could be attributed to enhanced clearance of phenobarbital since the metabolism and excretion of this promoting agent are known to be influenced by factors affecting net glucuronidation [l I]. Although dietary CGT has been shown to inhibit /%glucuronidase in vivo, mechanisms other than increased clearance of the promoting agent due to increased net glucuronidation cannot be ruled out. Based on the present findings, it would be of great interest to study the effects of dietary CGT on the appearance of selected histochemical enzymatic markers of hepatocarcinogenesis. Experiments currently in progress may provide answers to these questions. ACKNOWLEDGMENTS
We are very grateful to Drs. MS. Rao and J.K. Reddy, Department of Pathology, Northwestern University Medical School, Chicago, IL and Dr. H.C. Pitot, McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison, WI and Dr. C.W. Johnson of Monsanto/EHL Corporation, St. Louis, MO., for their helpful review of selected H&E sections and the discussion that followed. This work was supported by grants 5 ROl CA 3812-3 and P-30-DA16058-14 from the National Cancer Institute, and American Cancer Society Institutional Research Grant IN- 16W. REFERENCES 1
5
10
11
Farber, E. and Sarma, D.S.R. (1987) Hepatocarcinogenesis: A dynamic cellular perspective. Lab. Invest., 56,4-22. Farber, E. (1982) Sequential events in chemical carcinogenesis. In: Cancer: A Comprehensive Treatise, pp. 485-509. Editor: F.F. Becker. Plenum Publishing Corporation, New York. Squire, R.A. and Levitt, M.H. (1975) Report of a workshop on classification of specific hepatocelhtlar lesions in rats. Cancer Res., 35, 3214-3223. Walaszek, Z., Hanausek-Walaszek, M., Minton, J.P. and Webb, T.E. (1986) Dietary glucarate as anti-promoter of 7.12-dimethylbenz(a)anthracene-induced mammary tumorigenesis. Carcinogenesis, 7, 1463-1466. Walaszek, Z., Hanausek-Walaszek, M. and Webb, T.E. (1986) Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis. Cancer Letters, 33,25-32. Higgins, GM. and Anderson, R.M. (1931) Experimental pathology of the liver. Restoration of liver of white rats following partial surgical removal. Arch. Pathol., 12, 186-202. Pitot, H.C., Barsnenss, L., Goldsworthy, T. and Kitagawa. T. (1978) Biochemical characterisation of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature, 27 1,456-458. Levvy, GA. and Conchie, J. (1%) /3-Glucurononidase and the hydrolysis of glucuronides. In: Glucuronic Acid, pp. 301-364. Editor: G.J. Dutton. Academic Press, New York. M. and Webb, T.E. (1984) Inhibition of 7,12Walaszek, Z., Hanausek-Walaszek, dimethylbenzanthracene-induced mammary tumorigenesis by 2,5-di-0-acetyl-D-glucaro-1,4:6,3dilactone, an in vivo &glucuronidase inhibitor. Carcinogenesis, 5.767-772. Walaszek, Z., Hanausek-Walaszek, M. and Webb, T.E. (1986) Inhibition of N-methyl-Nnitrosourea-induced mammary tumorigenesis in the rat by a g-glucuronidase inhibitor. I.R.C.S. Med. Sci., 14,671-678. Marselos, M., Dutton, G. and Hanninen, 0. (1975) Evidence that D-glucaro-1,4-lactone shortens the pharmacological actions of drugs being disposed via the bile as glucuronides. Biochem. Pharmacol., 24,1855-1858.
95
EFFECTS OF CALCIUM GLUCARATE ON THE PROMOTION OF DIE’I’IIYLNITROSAMINEINI’IIATED ALTERED HEPATIC FOCI IN RATS
OLADIPO A. OREDIPE**, ROLF F. BARTH’, MALGORZATA HANAUSEK-WALASZEKb~*, IVETA SAUTINS, ZBIGNIEW WALASZEKb***and THOMAS E. WEBB” Departments of’Pathology and bPhysiological Chemistry, The Ohio State University, Columbus, OH 43210 (U.S.A.) (Received 10 August 1987) (Revised version received 7 September 1987) (Accepted 8 September 1987)
SUMMARY
Calcium glucarate (CGT), an inhibitor of /3-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.
INTRODUCTION
Promotion is one of the steps in the multi-stage process of hepatocarcinogenesis. It is a reversible event characterized by focal proliferations of initiated cells during the preneoplastic phase [ 1,2]. Preneoplastic hepatocytes can be identified as altered hepatic foci (AHF) in hematoxylin and eosin-stained liver sections during the early and promotion phases of hepatocarcinogenesis [3]. The present study describes the protective effect of dietary calcium glucarate (CGT) on phenobarbital-promoted rat *Address correspondence to: Dr. O.A. Oredipe at The Ohio State University, Department of Pathology, 4170 Graves Hall. 333 W. 10th Avenue, Columbus, Ohio 43201. **Present address: The University of Texas System Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, TX 78957, U.S.A.
96
hepatocarcinogenesis, using as a criterion the inhibition of induction of AHF. The latter was assessed histopathologically by means of light microscopy. This study was prompted by the marked protective effects exhibited by CGT and similar acting compounds on experimental mammary and lung carcinogens [4,5]. MATERIALS
AND METHODS
Female Sprague-Dawley rats, weighing l!W-200 g, were purchased from Harlan Sprague-Dawley, Indianapolis, IN. The ZV-diethylnitrosamine (DENA), phenobarbital and calcium glucarate (CGT) were purchased from the Sigma Chemical Co., St. Louis, MO. The rats were maintained on rat chow alone or fortified with additives as noted below. Under ether anesthesia, 64 rats were subjected to 70% partial hepatectomy (PH) to induce hepatocellular proliferation [6] according to the hepatocarcinogenic protocol of Pitot et al. [7]. At 24 h post-operation, the animals were initiated with non-necrogenic doses of DENA (10 mg/kg body wt.) administered by the intragastric route and were maintained thereafter on a chow diet without CGT for 2 months. At 2, 4 and 8 weeks, some animals were randomly selected and killed under ether anesthesia. Liver tissues representative of each lobe were fixed in 10% neutral buffered formalin, paraffin-embedded and cut at a thickness of 4 pm. The-4 pm sections were stained with hematoxylin and eosin (H&E), and evaluated histopathologically by means of light microscopy. The presence and severity of AHF in each section were graded from 0 to 4 + with the former being none or normal and the latter as the most severe. The remaining rats were divided into 2 treatment groups that were further studied for up to 6 months. One group received 4% CGT plus 0.05% phenobarbital in chow diet throughout the duration of promotion, while the other had only 0.05Vo phenobarbital and served as a control. At monthly intervals of promotion up to 6 months, 4 or more animals in each group were killed, livers removed, fixed in 10% buffered formalin and 4-l.trn sections were cut, stained with H&E, and histopathologically evaluated as described above. RESULTS
For the purpose of evaluating the effects of 4% dietary CGT on the promotion phase, rats initiated with DENA and then kept exclusively on a chow diet for 2 months, were divided into 2 groups. One group of 28 rats was fed chow plus 0.05% phenobarbital for varying time intervals up to 6 months, while the other group was fed chow plus 0.05% phenobarbital diet supplemented with 4% CGT during the same period. The results, shown in Fig. 1, indicate that dietary CGT delayed the attainment of maximum levels and severity of altered foci in the livers of experimental rats. Evaluations of the H&E stained liver sections showed scattered foci composed of large cells with abundant eosinophilic cytoplasm in the case of CGT-fed rats (Fig. 2a) while numerous foci composed of large pale-staining vacuolated hepatocytes or neoplastic hepatocytes were commonly observed in those of the controls (Fig. 2b).
0
1
2 3 Months After
4 5 6 Diethylnitrosa~ine
7
6
Fig. 1. Effects of dietary CGT on the promotion phase of DENA-initiated, phenobarbital-promoted hepatocarcinogenesis. Phenobarbital (0.05%) alone and 0.05% phenobarbital plus 4% CGT in chow diets were initiated 2 months (arrow point) after initiation with DENA. Control rats ( l 0) were maintained on chow diet supplemented with 0.05% pheobarbital, while experimental rats (O-----O) received the promoter plus 4% CGT throughout the intervals of promotion. The liver sections were evaluated histopathologically and graded as described in the text. Each point represents the mean f SE. of 4 or more rats.
DISCUSSION
D-Glucaro-1 ,Clactone is the natural regulator of /3-glucuronidase in vivo [8], but is rapidly excreted from the body. Endogenous levels can be maintained through the use of sustained release forms. Dietary 2,5-di-O-acetyl-D-glucaro-l ,rl-dilactone, which is converted at the acid pH of the stomach to D-glucaro-1,4-lactone, was initially shown to inhibit 7,12-dimethyl-benzanthracene-induced rat mammary tumorigenesis [9]. This same compound also inhibited N-methyl-ZV-nitrosoureainduced mammary carcinogenesis, but in this case it may have reduced the sensitivity of the mammary gland to the carcinogen through increased clearance of estrogens [lo]. Dietary CGT, one third of which can be converted to D-glucaro-1 ,Qlactone in the stomach [5,8], has been shown to inhibit the promotion phase of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis [4] and benzo[a]pyreneinduced lung tumorigenesis in mice [5]. In these instances, any effect of calcium ion was ruled out. Dietary CGT at a concentration of 4% appears to be the minimal amount needed for maximal tumor inhibition [4,5]. The results of the present study suggest that dietary CGT had a marked inhibitory effect on the emergence of DENA-initiated preneoplastic lesions in rat livers, and caused a significant delay in
Fig. 2. Photomicrographs of livers from control and experimental rats. Sections were cut at4pms md stained with hematoxylin and eosin. Livers from rats that had received DEN A, followed by phenobarbital and CGT diet (a) showed scattered areas composed of large acidophilic cells i or cells Hrith abundant eosinophilic cytoplasm ( X 128). Livers from animals that had received DEINA followed by phenobarbital alone (b) showed numerous foci composed of large pale-staining, vacuoleated hepatocp tes, or neoplastic hepatocytes ( x 200).
99
the development of AHF. This delay without a permanent reduction in the expression or severity of AHF, could be attributed to enhanced clearance of phenobarbital since the metabolism and excretion of this promoting agent are known to be influenced by factors affecting net glucuronidation [l I]. Although dietary CGT has been shown to inhibit /%glucuronidase in vivo, mechanisms other than increased clearance of the promoting agent due to increased net glucuronidation cannot be ruled out. Based on the present findings, it would be of great interest to study the effects of dietary CGT on the appearance of selected histochemical enzymatic markers of hepatocarcinogenesis. Experiments currently in progress may provide answers to these questions. ACKNOWLEDGMENTS
We are very grateful to Drs. MS. Rao and J.K. Reddy, Department of Pathology, Northwestern University Medical School, Chicago, IL and Dr. H.C. Pitot, McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison, WI and Dr. C.W. Johnson of Monsanto/EHL Corporation, St. Louis, MO., for their helpful review of selected H&E sections and the discussion that followed. This work was supported by grants 5 ROl CA 3812-3 and P-30-DA16058-14 from the National Cancer Institute, and American Cancer Society Institutional Research Grant IN- 16W. REFERENCES 1
5
10
11
Farber, E. and Sarma, D.S.R. (1987) Hepatocarcinogenesis: A dynamic cellular perspective. Lab. Invest., 56,4-22. Farber, E. (1982) Sequential events in chemical carcinogenesis. In: Cancer: A Comprehensive Treatise, pp. 485-509. Editor: F.F. Becker. Plenum Publishing Corporation, New York. Squire, R.A. and Levitt, M.H. (1975) Report of a workshop on classification of specific hepatocelhtlar lesions in rats. Cancer Res., 35, 3214-3223. Walaszek, Z., Hanausek-Walaszek, M., Minton, J.P. and Webb, T.E. (1986) Dietary glucarate as anti-promoter of 7.12-dimethylbenz(a)anthracene-induced mammary tumorigenesis. Carcinogenesis, 7, 1463-1466. Walaszek, Z., Hanausek-Walaszek, M. and Webb, T.E. (1986) Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis. Cancer Letters, 33,25-32. Higgins, GM. and Anderson, R.M. (1931) Experimental pathology of the liver. Restoration of liver of white rats following partial surgical removal. Arch. Pathol., 12, 186-202. Pitot, H.C., Barsnenss, L., Goldsworthy, T. and Kitagawa. T. (1978) Biochemical characterisation of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature, 27 1,456-458. Levvy, GA. and Conchie, J. (1%) /3-Glucurononidase and the hydrolysis of glucuronides. In: Glucuronic Acid, pp. 301-364. Editor: G.J. Dutton. Academic Press, New York. M. and Webb, T.E. (1984) Inhibition of 7,12Walaszek, Z., Hanausek-Walaszek, dimethylbenzanthracene-induced mammary tumorigenesis by 2,5-di-0-acetyl-D-glucaro-1,4:6,3dilactone, an in vivo &glucuronidase inhibitor. Carcinogenesis, 5.767-772. Walaszek, Z., Hanausek-Walaszek, M. and Webb, T.E. (1986) Inhibition of N-methyl-Nnitrosourea-induced mammary tumorigenesis in the rat by a g-glucuronidase inhibitor. I.R.C.S. Med. Sci., 14,671-678. Marselos, M., Dutton, G. and Hanninen, 0. (1975) Evidence that D-glucaro-1,4-lactone shortens the pharmacological actions of drugs being disposed via the bile as glucuronides. Biochem. Pharmacol., 24,1855-1858.