* Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan;
Kyushu University of Nursing and Social Welfare, Kumamoto 865-0062, Japan; and
Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received August 16, 1999; accepted October 8, 1999
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ABSTRACT |
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Key Words: cadmium; pancreas; trypsin; chymotrypsin; carboxypeptidase; mice..
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INTRODUCTION |
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The appropriate functioning of pancreatic proteases such as trypsin and chymotrypsin, which are endopeptidases, and carboxypeptidases, which are exopeptidases, is critical to life in mammalian species (Elliott and Elliott, 1997). These enzymes are produced from clusters of cells in the pancreas and then secreted into the intestine as pancreatic juice, where they act in the digestion of proteins (Elliott and Elliott, 1997
). Trypsin, chymotrypsin, and carboxypeptidase enter the intestine in the form of the inactive proenzymes, trypsinogen, chymotrypsinogen, and procarboxypeptidases, respectively (Elliott and Elliott, 1997
). If these enzymes are activated prematurely in the pancreas, the disease pancreatitis ensues (Elliott and Elliott, 1997
). On the other hand, if these enzymes are inhibited, nutrient malabsorption occurs (Layer and Keller, 1999
). Furthermore, because the proteases in other organs are also involved in a great variety of biologic processes in the cells, including removal of damaged or obsolete proteins (David and Shearer, 1989
), blood coagulation, and fibrinolysis (Elliott and Elliott, 1997
), alterations of the proteases may cause systematic dysfunction and potentially cancer.
In vitro experiments have reported that cadmium can displace essential metal components such as zinc and copper from metalloenzymes and thereby alter activity. Such enzymes include superoxide dismutase, alcohol dehydrogenase, carbonic anhydrase, and -aminolevulinic acid dehydrogenase (Goering et al., 1987
; Vallee and Ulmer, 1972
; Vallee and Galdes, 1974). Evidence indicates that several pancreatic enzymes can also be altered by cadmium exposure in vitro. For instance, Vallee (1964) found that the peptidase activity of carboxypeptidase A from bovine pancreas was inhibited by cadmium in vitro. Similarly, Folk and Gladner (1961) reported that cadmium had a inhibitory effect on the peptidase activity of carboxypeptidase B isolated from porcine pancreas. Green et al. (1952) reported that cadmium enhanced trypsin activity in vitro. The activities of lipase from porcine pancreas and amylase isolated from bacteria were also inhibited by cadmium (Vallee and Ulmer, 1972
). However, these alterations in pancreatic enzyme activities were all obtained under in vitro exposure conditions, and there are minimal data on the interactions of cadmium with pancreatic enzymes in intact animal experiments. Furthermore, the effects of cadmium on pancreatic enzymes may well vary between in vivo exposure and in vitro conditions, making predictions based on in vitro results potentially suspect. Thus, the present study was performed to examine the pancreatic effects of low-dose cadmium in mice, including the effects on tissue damage, protease activities, and essential trace element homeostasis. As an extension of the in vivo work, the effects of cadmium on protease activities in vitro were also investigated.
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MATERIALS AND METHODS |
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Animals and treatments.
Male ddY mice were obtained at 8 weeks of age from Kyudo Co. (Kumamoto, Japan). Mice were maintained on a 12-h light/dark cycle and were given free access to diet (CE-2, Japan CREA, Osaka, Japan) and water ad libitum. The mice were injected sc with saline (10 ml/kg) or CdCl2 (1 mg Cd/kg). One or 5 days after administration of CdCl2 or its vehicle (control), mice were euthanized by CO2 asphyxia, then immediately processed as described below.
In vivo protease assay.
Pancreas was homogenized (1:10 w/v) in 50 mM TrisHCl, pH 7.9, containing 50 mM NaCl, and subjected to the determination of enzyme activities and protein content. The activities of trypsin and chymotrypsin were determined according to the fluorescence method of Morita et al. (1977) using Bz-Arg-MCA and Suc-Ala-Ala-Pro-Phe-MCA, respectively. The activities of carboxypeptidase A and carboxypeptidase B were determined according to the ninhydrin method of Lee and Takahashi (1966) using Z-Gly-Phe and Bz-Gly-Lys, respectively. The activities of amylase and lipase were determined with commercially available kits (Wako Pure Chemicals and Dainippon Pharmaceutical Co., Ltd., Osaka, Japan, respectively). Protein content was determined according to the method of Lowry et al. (1951) using bovine serum albumin as the standard.
In vitro protease assay.
Effects of cadmium on trypsin and chymotrypsin were performed as follows: 10 ng of each purified trypsin or chymotrypsin and various levels of cadmium were incubated for 30 min at 37°C in 20 µl of 50 mM TrisHCl, pH 7.9, containing 50 mM NaCl. These samples were then incubated for an additional 30 min at 37°C with 50 µl of 0.6 mM Bz-Arg-MCA and Suc-Ala-Ala-Pro-Phe-MCA, respectively. The activities of trypsin and chymotrypsin were determined as described above and were expressed as the amounts of AMC formed per minute. Effects of cadmium on purified carboxypeptidase A and carboxypeptidase B were performed as follows: 5 µg of each purified carboxypeptidase A or carboxypeptidase B and various levels of cadmium were incubated for 30 min at 37°C in 100 µl of 50 mM TrisHCl, pH 7.9, containing 50 mM NaCl. These samples were then incubated for an additional 30 min at 37°C with 200 µl of 20 mM Z-Gly-Phe and Bz-Gly-Lys, respectively. The activities of carboxypeptidase A and carboxypeptidase B were determined as described above and were expressed as the amounts of L-phenylalanine and L-lysine, respectively, released per minute.
Plasma alanine aminotransferase (ALT), amylase, lipase, and blood urea nitrogen (BUN).
To assess the acute toxicity of cadmium, plasma enzyme indicators of hepatic, renal, and pancreatic toxicity were measured. ALT, amylase, BUN, and lipase were assayed with commercially available kits (Wako Pure Chemicals and Dainippon Pharmaceutical Co., Ltd., respectively).
Tissue metal content.
Cadmium, Ca, Zn, Fe, and Cu concentrations in pancreas were measured by atomic absorption spectrophotometry with a Hitachi Model Z-8000 spectrophotometer after pancreas was digested with nitric acid.
Effect of Cd/EDTA on trypsin activity.
Ten nanograms of purified trypsin and various levels of cadmium were incubated for 30 min at 37°C in 20 µl of 50 mM TrisHCl, pH 7.9, containing 50 mM NaCl. These samples were then incubated for an additional 30 min at 37°C with 20 µl of 1 mM EDTA. The activity was determined as described above.
Effect of Cd on Zn released from carboxypeptidase A and carboxypeptidase B.
Purified carboxypeptidase A or carboxypeptidase B (280 µg each) and various levels of cadmium were incubated for 30 min at 37°C in 2 ml 50 mM TrisHCl, pH 7.9, containing 50 mM NaCl. After the incubation, the sample solution was ultrafiltrated using a CENTRICUT (W-10, Kurabo Ind. Ltd., Osaka, Japan) to separate zinc released and the protein. Zinc concentration in the filtrate was measured by atomic absorption spectrophotometry.
Statistics.
All data represent the mean ± SD of sample sizes ranging from four to six determinations. Statistical analysis was performed either by analysis of variance (ANOVA) followed by Duncan's multiple range test for multiple comparisons or by Student's t-test for paired comparisons. Probability values 0.05 were considered statistically significant.
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RESULTS |
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DISCUSSION |
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In contrast to our in vivo results, the activity of trypsin was actually increased by cadmium treatment in vitro. This is consistent with a previously reported study that showed high levels of cadmium (1 mM) increased the activity of trypsin by 30% (Green et al., 1952). In the present study, the increased trypsin activity in vitro by cadmium exposure was seen at concentrations as low as 100500 µM (4090% increase). The increased enzyme activity of trypsin returned to the control levels by the subsequent EDTA treatment, indicating inhibitory effect is due to a direct interaction of cadmium and is reversible. Epstein et al. (1974) found that the divalent ions Cd and Mn, and the trivalent lanthanide ions, such as terbium, can bind to the single binding site for Ca ion on the trypsin. It is known that autocatalytic conversion of trypsinogen to trypsin is markedly activated by Ca ion (McDonald and Kunitz, 1941
). Trypsinogen possesses two binding sites for Ca ion; one is on the hexapeptide cleaved upon the conversion and the other is on the trypsin moiety (Epstein et al., 1974
). Although there is no connection between the enzymatic activity of trypsin and the presence of Ca ion (Sipos and Merkel, 1970
), it has been observed that the binding of Ca to the enzyme inhibits its autodigestion (Buck et al., 1962
). Thus, Ca binding to trypsin is thought to have an important biologic function. Kojima et al. (1994) reported that terbium increased the activity of trypsin in vivo but had no effect on purified trypsin in vitro. In this case, as Ca concentrations in the pancreas were increased by terbium treatment, the increased activity of trypsin in vivo might be due to the increased pancreatic Ca (Kojima et al., 1994
). In the present study, Ca concentrations in pancreas were not altered by cadmium treatment. The increased activity of trypsin after cadmium exposure in vitro appears to be due to the binding of cadmium to the enzyme, possibly at the Ca binding sites, whereas the decreased activity of trypsin in vivo might be due to the inhibitory effects of cadmium on trypsinogen and/or the enzymes converting trypsinogen to trypsin.
It is also known that chymotrypsinogen is converted to the active enzyme chymotrypsin by trypsin (Wilcox, 1970). In this case, the presence of Ca ion has no effect on activation (Wilcox, 1970
). In the present study, chymotrypsin activity in pancreas was decreased by cadmium treatment in vivo but there was no such effect on purified chymotrypsin in vitro. These results suggest that a decrease in the activity of chymotrypsin in vivo might be, in part, a consequence of the decrease in the activity of trypsin in pancreas.
In the present study, the activity of carboxypeptidase A in pancreas was decreased by cadmium treatment in vivo, and cadmium also shows a strong inhibition of carboxypeptidase A activity in vitro. Contrary to the results with carboxypeptidase A activity, carboxypeptidase B activity was not effected by cadmium treatment in vivo. It has been reported that cadmium can replace the native Zn in the carboxypeptidase and shows inhibitory effects on protease activities of carboxypeptidase A and carboxypeptidase B (Vallee and Ulmer, 1972). Similarly, in the present study, the substitution of cadmium for Zn in carboxypeptidase A and carboxypeptidase B were observed. The amount of Zn released from carboxypeptidase B was much larger than that from carboxypeptidase A. Despite this release of zinc, there was no significant change in carboxypeptidase B activity in either the intact pancreas or in the purified enzyme. Brown et al. (1963) have shown that trypsin accelerates the conversion of procarboxypeptidase A to carboxypeptidase A. From these results, the decreased carboxypeptidase A activity after cadmium treatment in vivo might be due to the direct inhibitory effect of cadmium and, in part, due to the inhibition of trypsin by cadmium.
Pancreatic proteases, such as trypsin, chymotrypsin, and carboxypeptidase, play a critical role in the mammalian digestion (Elliott and Elliott, 1997). Because pancreatic proteases are so destructive to proteins, it is crucial that their activity is controlled (Elliott and Elliott, 1997
). These enzymes are synthesized in the pancreas and secreted into the small intestine as inactive precursors, or zymogens, namely trypsinogen, chymotrypsinogen, and procarboxypeptidase, respectively (Elliott and Elliott, 1997
). Regulating the activation of these enzymes is very important to prevent the enzymes from digesting the pancreatic tissue (Elliott and Elliott, 1997
). On the other hand, inhibition of these enzymes cause nutrient malabsorption (Layer and Keller, 1999
). Furthermore, proteases in other organs also play a critical role in a variety of biologic processes, including removal of damaged or obsolete proteins (David and Shearer, 1989
), blood coagulation, and fibrinolysis (Elliott and Elliott, 1997
). Thus, the inhibition of these pancreatic proteases by low doses of cadmium may have an impact on a variety of important physiologic functions. It is quite clear that cadmium can cause chronic toxic effects within the pancreas, based on histologic analysis. This includes the induction of pancreatic cancers (Poirier et al., 1983
) and cellular transdifferentiations (Konishi et al., 1990
).
In conclusion, the results of the present study indicate that cadmium inhibits the activities of various pancreatic proteases such as trypsin, chymotrypsin, and carboxypeptidase A in vivo in mice. This inhibition occurred at low doses of cadmium that did not induce any overt hepatic, renal, and pancreatic tissue damage. Further studies are needed to clarify the mechanism by which cadmium alters the activities of protease in pancreas in vivo and the toxicologic consequences of this inhibition.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Brown, J. R., Yamasaki, M., and Neurath, H. (1963). A new form of bovine pancreatic procarboxypeptidase A. Biochemistry 2, 877886.[ISI]
Buck, F. F., Vithayathil, A. J., Bier, M., and Nord, F. F. (1962). Mechanism of enzyme action. LXXIII. Studies on trypsins from beef, sheep, and pig pancreas. Arch. Biochem. Biophys. 97, 417424.[ISI][Medline]
David, L. L., and Shearer, T. R. (1989). Role of proteolysis in lenses: A review. Lens Eye Toxic. Res. 6, 725747.[Medline]
Dudley, R. E., Svoboda, D. J., and Klaassen, C. D. (1982). Acute exposure to cadmium causes severe liver injury in rats. Toxicol. Appl. Pharmacol. 65, 302313.[ISI][Medline]
Elliott, W. H., and Elliott, D. C. (1997). Digestion and absorption of food. In Biochemistry and Molecular Biology, pp. 6170. Oxford University Press, New York.
Epstein, M., Levitzki, A., and Reuben, J. (1974). Binding of lanthanides and of divalent metal ions to porcine trypsin. Biochemistry 13, 17771782.[ISI][Medline]
Folk, J. E, and Gladner, J. A. (1961). Influence of cobalt and cadmium on the peptidase and esterase activities of carboxypeptidase B. Biochim. Biophys. Acta 48, 139147.[ISI][Medline]
Friberg, L., Elinder, C.-G., Kjellström, T., and Nordberg, G. F. (1986). Cadmium and health. In A Toxicological and Epidemiological Appraisal, Vol. II. Effects and Responses (L. Friberg, C.-G. Elinder, T. Kjellström, and G. F. Nordberg, Eds.), pp. 257287. Boca Raton, Florida.
Goering, P. L., Mistry, P., and Fowler, B. A. (1987). Mechanisms of metal-induced cell injury. In Handbook of Toxicology (T. J. Haley and W. O. Berndt, Eds.), pp. 384425. Hemisphere, Washington D.C.
Goering, P. L., Waalkes, M. P., and Klaassen, C. D. (1994). Cadmium toxicity. In Handbook of Experimental Pharmacology; Toxicology of Metals, Biochemical Effects (R. A. Goyer and M. G. Cherian, Eds.), pp. 189214. Springer-Verlag, New York.
Green, M. M., Gladner, J. A., Cunningham, L. W., and Neurath, H. (1952). The effects of divalent cations on the enzymatic activities of trypsin and of -chymotrypsin. J. Am. Chem. Soc. 74, 21222123.[ISI]
Gunn, S. A., and Gould, T. C. (1970). Cadmium and other mineral elements. In The Testes: Influencing Factors Vol. 3 (A. D. Johnson, W. R. Gomes, and N. L. VanDemark, Eds.), pp. 377481. Academic Press, New York.
IARC (1993). Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry. In IARC Monogram Evaluating the Carcinogenic Risk to the Human, Vol. 58, pp. 119237. IARC, Lyon, France.
Kojima, S., Makihira, T., Funakoshi, T., and Shimada, H. (1994). Effect of terbium on protease activity in pancreas of mice. Res. Commun. Mol. Pathol. Pharmacol. 85, 227235.[ISI][Medline]
Konishi, N., Ward, J. M., and Waalkes, M. P. (1990). Pancreatic hepatocytes in Fischer and Wistar rats induced by repeated injections of cadmium of cadmium chloride. Toxicol. Appl. Pharmacol. 104, 149156.[ISI][Medline]
Layer, P., and Keller, J. (1999). Pancreatic enzymes: Secretion and luminal nutrient digestion in health and disease. J. Clin. Gastroenterol. 28, 310.[ISI][Medline]
Lee, Y. P., and Takahashi, T. (1966). Improved colorimetric determination of amino acids with the use of ninhydrin. Anal. Biochem. 14, 7177.[ISI]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
McDonald, M. R., and Kunitz, M. (1941). Effect of calcium and other ions on the autocatalytic formation of trypsin from trypsinogen. J. Gen. Physiol. 25, 5373.
Morita, T., Kato, H., Iwanaga, S., Takada, K., Kimura, T., and Sakakibara, S. (1977). New fluorogenic substrates for -thrombin, factor Xa, kallikreins, and urokinase. J. Biochem. 82, 14951498.[Abstract]
Onosaka, S., and Cherian, M. G. (1981). The induced synthesis of metallothionein in various tissues of rat in response to metals. I. Effect of repeated injection of cadmium salts. Toxicology 22, 91101.[ISI][Medline]
Poirier, L. A., Kasprzak, K. S., Hoover, K., and Wenk, M. L. (1983). Effects of calcium and magnesium on the carcinogenicity of cadmium chloride in Wistar rats. Cancer Res. 43, 45754581.[Abstract]
Shimada, H., Kawagoe, M., Kamenosono, T., Kiyozumi, M., Funakoshi, T., and Kojima, S. (1991). Comparative effects of N,N-disubstituted dithiocarbamates on excretion and distribution of cadmium in mice. Toxicology 68, 157167.[ISI][Medline]
Sipos, T., and Merkel, J. R. (1970). An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry 9, 27662775.[ISI][Medline]
Suzuki, K. T., Ohnuki, R., Yaguchi, K., and Yamada, Y. K. (1983). Accumulation and chemical forms of cadmium and its effect on essential metals in rat spleen and pancreas. J. Toxicol. Environ. Health 11, 727737.[ISI][Medline]
Vallee, B. L. (1964). Active center of carboxypeptidase A. Fed. Proc. 23, 817.[ISI][Medline]
Vallee, B. L., and Ulmer, D. D. (1972). Biochemical effects of mercury, cadmium and lead. Annu. Rev. Biochem. 41, 91128.[ISI][Medline]
Vallee, B. L., and Galdes, A. (1984). The metallobiochemistry of zinc enzymes. In Advances in Enzymology and Related Areas of Microbiology (A. Meister, Ed.), Vol. 56, pp. 283430. Wiley Interscience, New York.
Waalkes, M. P., and Oberdörster, G. (1990). Cadmium carcinogenesis. In Biological Effects of Heavy Metals, Vol. II, Mechanisms of Metal Carcinogenesis (E. D. Foulkes, Ed.), pp. 129158. CRC Press, Boca Raton, FL.
Waalkes, M. P., Wahba, Z. Z., and Rodriguez, R. E. (1991). Cadmium. In Hazardous Materials Toxicology. Clinical Principles of Environmental Health (J. B. Sullivan and G. R. Krieger, Eds.), pp. 845852. Williams and Wilkins, Baltimore.
Waalkes, M. P., and Rehm, S. (1992). Carcinogenicity of oral cadmium in the male Wistar (WF/NCr) rat: Effect of chronic dietary zinc deficiency. Fundam. Appl. Toxicol. 19, 512520.[ISI][Medline]
Waalkes, M. P., Coogan, T. P., and Barter, R. A. (1992). Toxicological principles of metal carcinogenesis with special emphasis on cadmium. Crit. Rev. Toxicol. 22, 175201.[ISI][Medline]
Wilcox, P. E. (1970). Chymotrypsinogens-chymotrypsins. In Methods in Enzymology (G. E. Perlmann and L. Lorand, Eds.), Vol, XIX, pp. 64108. Academic Press, New York.