Toxicological Sciences 53, 474-480 (2000)
Copyright © 2000 by the Society of Toxicology
Acute, Nontoxic Cadmium Exposure Inhibits Pancreatic Protease Activities in the Mouse
* 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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Toxic effects of cadmium on liver, kidney, lung, and testes have been well established in experimental animals and in cell model systems. However, little is known about the effect of cadmium on pancreas, though the pancreas has been reported to accumulate high concentrations of cadmium. Therefore, in this study we examined the effects of cadmium on the pancreas of mice. A single sc injection of 1 mg Cd/kg to mice had no obvious toxic effects on the liver, kidney, and pancreas at both 1 and 5 days after cadmium treatment. Within the pancreas, however, the activities of trypsin, chymotrypsin, and carboxypeptidase A were significantly decreased at 1 day after cadmium treatment, whereas the activity of carboxypeptidase B was not changed. All pancreatic enzyme activities returned to the control levels by 5 days after cadmium treatment. The concentrations of cadmium in pancreas were very similar at 1 and 5 days after cadmium treatment, indicating a stable deposition of the metal. The concentration of zinc in pancreas was markedly increased at 5 days after cadmium treatment. In order to more fully examine the inhibitory effects of cadmium on these protease activities in pancreas, the direct effects of cadmium on purified proteases were studied in vitro. Contrary to the results in vivo, cadmium increased the activity of purified trypsin in a concentration-dependent manner. Consistent with the in vivo results, the activity of purified carboxypeptidase A was decreased by cadmium treatment in a concentration-dependent fashion in vitro. The activities of chymotrypsin and carboxypeptidase B did not change by the cadmium exposure in vitro. The enhanced activity of trypsin by cadmium was returned to the control levels by subsequent treatment with EDTA, indicating that enhancement was reversible. In addition, the zinc normally contained in purified carboxypeptidase A and carboxypeptidase B was released by the cadmium treatment. These results indicate that cadmium inhibits protease activities within the pancreas in vivo at doses that do not induce overt hepatic, renal, or pancreatic toxicity. Based on in vitro study, the decreases seen in trypsin and chymotrypsin activities might be based on indirect effects of cadmium, whereas the decreases in carboxypeptidase A are probably due to the direct inhibition by the metal.
Key Words: cadmium; pancreas; trypsin; chymotrypsin; carboxypeptidase; mice..
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cadmium is an important inorganic environmental pollutant, and the potential for human exposure has generally increased with the increasing use of this metal (Andersson et al., 1986
; Waalkes et al., 1991). Acute exposure to cadmium can result in damage to numerous tissues such as liver, kidney, lung, gastrointestinal tract, central nervous system, ovaries, and testes (Goering et al., 1994; Waalkes et al., 1992). Parenteral administration of cadmium in rats causes a rapid accumulation of cadmium in the liver and at sufficient doses can give rise to severe hepatic injury in the form of hepatocellular necrosis (Dudley et al., 1982). In humans and rodents, the kidney is a primary target of chronic cadmium toxicity (Friberg et al., 1986). Pulmonary tumors form in both humans and rats after inhalation of cadmium (IARC, 1993). In rodents, the testes are also among the most sensitive tissues to cadmium, and acute toxic and chronic carcinogenic effects occur within this organ (Gunn and Gould, 1970; Waalkes and Oberdörster, 1990; Waalkes and Rehm, 1992). In contrast to the liver, kidney, lung, and testes, little is known about the toxicity of cadmium in the pancreas, though the pancreas has been reported to accumulate high concentrations of cadmium (Onosaka and Cherian, 1981; Shimada et al., 1991). Onosaka and Cherian (1981) reported that injection of CdCl2 to rats induced the highest amount of metallothionein (MT) in liver, kidney, and pancreas, and that these levels were much higher than in other tissues. Cadmium in pancreas of rats is mostly bound to MT (Suzuki et al., 1983). However, MT in the pancreas is highly susceptible to oxidative reactions compared to MT in the liver, kidney, and spleen (Suzuki et al., 1983). The reasons for the high susceptibility to oxidative reactions of pancreatic MT have not been defined.
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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Chemicals.
Anhydrous cadmium chloride (CdCl2) was purchased from Wako Pure Chemicals (Osaka, Japan). Trypsin (Type XIII, from bovine pancreas),
-chymotrypsin (Type II, from bovine pancreas), carboxypeptidase A (Type I-DFP), and carboxypeptidase B (Type I, from porcine pancreas) were obtained from Sigma Chemical Co. (St. Louis, MO). Benzyl-arginine-4-methyl-coumaryl-7-amide (Bz-Arg-MCA), succinyl-alanyl-alanyl-proline-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA), benzyl-glycyl-lysine (Bz-Gly-Lys), and carbobenzoxy-glycyl-phenylalanine (Z-Gly-Phe) were obtained from the Peptide Research Foundation (Osaka, Japan). All other chemicals were commercially available and of reagent grade.
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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of cadmium in mice on plasma indicators of cadmium-induced hepatic (ALT), renal (BUN), and pancreatic (amylase and lipase) toxicity are shown in Table 1
. No significant changes occurred in these enzyme markers at either 1 or 5 days after cadmium treatment. These data indicate that this dose (1 mg Cd/kg, sc) did not cause overt acute toxic effects in the liver, kidney, or pancreas of treated mice.
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The effects of this overtly nontoxic dose of cadmium on the activities of trypsin and chymotrypsin within pancreas were then studied (Fig. 1
). The activities of trypsin and chymotrypsin were decreased by almost 32% and 63%, respectively, at 1 day after cadmium treatment. By 5 day post-treatment, these enzyme activities had been restored to normal levels.
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The effects of cadmium on the in vivo activities of carboxypeptidase A and carboxypeptidase B in pancreas are shown in Figure 2
. The activity of carboxypeptidase A was decreased by almost 64% 1 day after cadmium treatment. However, carboxypeptidase A activity had been restored to normal levels by 5 days after cadmium exposure. The activity of carboxypeptidase B was unaffected by cadmium treatment regardless of time point. In addition, the activities of amylase and lipase in pancreas were unaltered by cadmium treatment (not shown).
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Table 2
shows the concentration of cadmium and other metals in the pancreas of mice after cadmium treatment. Cadmium was not detectable in the pancreas of control mice. The concentration of cadmium did not change between 1 and 5 days after cadmium treatment. In order to examine whether cadmium deposited in the pancreas altered the essential element homeostasis, Ca, Zn, Fe, and Cu levels were also measured (Table 2). The pancreatic concentration of Zn was significantly increased by almost 120% at 5 days after cadmium treatment. No changes were observed in the concentrations of other essential elements in the pancreas after cadmium treatment.
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As cadmium inhibited the activities of trypsin, chymotrypsin, and carboxypeptidase A in pancreas of mice, in vitro experiments were performed to determine if the inhibitory effects were due to a direct effect of cadmium. Contrary to the results of in vivo studies, cadmium markedly increased the activity of purified trypsin in vitro in a concentration-dependent manner (Fig. 3
). On the other hand, the activity of purified chymotrypsin did not change in vitro by cadmium treatment at concentrations up to 500 µM (not shown). In order to examine whether increased trypsin activity by cadmium was reversible, the effect of EDTA on cadmium-increased trypsin activity was investigated (Fig. 3). Increased trypsin activity after cadmium exposure was completely abolished by treatment with EDTA.
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The effects of cadmium in vitro on the activities of purified carboxypeptidase A and carboxypeptidase B are shown in Figure 4
. Consistent with the in vivo experiment, the activity of purified carboxypeptidase A was decreased by cadmium treatment in a concentration-dependent manner and the activity of carboxypeptidase B did not change by cadmium treatment at concentrations up to 500 µM (Fig. 4).
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Substitution of cadmium for Zn in carboxypeptidase A and carboxypeptidase B was also investigated (Fig. 5
). Zinc in the both enzymes was released by cadmium treatment in a concentration-dependent manner. The levels of released Zn from carboxypeptidase B was much greater than that from carboxypeptidase A. The release of Zn did not, however, appear to alter enzyme activity (see Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study examined the effect of cadmium on pancreas of mice, with special emphasis on pancreatic protease activities in both in vivo and in vitro experiments. It is well known that increased plasma amylase and lipase are the indicators of acute pancreatitis. In this study, the selected dose of cadmium (1 mg Cd/kg) did not induce any significant changes in amylase and lipase in either the plasma or pancreas. From this it can be concluded that cadmium had no overt toxic effects on pancreas tissue that would result in the extrusion of pancreatic cell contents into the plasma. Similarly, serum enzymatic indicators of liver and kidney toxicity were not altered by this dose of cadmium. Thus, results of the present study indicate that in vivo cadmium can inhibit protease activities in pancreas at relatively low doses and in the absence of overt toxicologic changes in the pancreas as well as in the liver and kidney.
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 100–500 µM (40–90% 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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The authors thank Kumiko Kado for excellent technical assistance.
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NOTES |
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1 To whom correspondence should be addressed. Fax: +81-96-371-4639. E-mail: hshimada{at}gpo.kumamoto-u.ac.jp.
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