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Depression of serum cholinesterase activity by cadmium
Toxicology, 36 (1985) 131--138 Elsevier Scientific Publishers Ireland Ltd.
DEPRESSION OF SERUM CHOLINESTERASE ACTIVITY BY CADMIUM
HIROSHI UEHARAa'b, YASUNOBU AOKIa, NOBUHIRO SHIMOJOb and KAZUO T. SUZUKIa,* aNational Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, and bInstitute of Community Medicine, The University of Tsukuba, Niihari, Ibaraki 305 (Japan) (Received February 20th, 1985) (Accepted April 22nd, 1985)
SUMMARY Two serum enzymes which originate from the liver under different circumstances were examined as potential biological indicators in serum for cadmium toxicity. The first of those is an enzyme that leaks from damaged liver cells. The second is an enzyme that is secreted by the normal functioning liver. Cadmium chloride was injected s.c. into male and female rats o f the Wistar strain (8, 15 and 22 weeks old), at doses of 1.0, 1.5 and 2.0 mg Cd/kg body weight (in total 18 groups). Cholinesterase (CHE; EC 3.1.1.8) activity in serum was f o u n d to decrease with time after the administration of a single injection of cadmium chloride and, in all experimental groups, was significantly lower than the control values on day 2 after the injection. Glutamic pyruvic transaminase (GPT; EC 2.6.1.2) activity in serum, however, increased only in the oldest group of males receiving the high dose levels of cadmium. A timecourse experiment in which male and female rats 15 weeks of age were administered 1.5 mg Cd/kg body weight showed t h a t the serum CHE activity started to decrease on day 1 after the injection, attained the lowest level on days 2 and 3, and then recovered almost to control levels on day 5. On the o t h e r hand, the GPT activity remained at or less than control values throughout the experimental period. The results indicate that CHE activity in serum is a sensitive biological indicator for cadmium toxicity.
K e y words: Cadmium; Cholinesterase; Liver injury; Secreted enzyme; Glutamic pyruvic transaminase *Address all correspondence to: Dr. Kazuo T. Suzuki, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan.
0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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INTRODUCTION Cadmium is a potent nephrotoxin and the chronic toxicity of this metal manifests itself primarily in the renal proximal tubules [1--5]. However, cadmium also causes hepatic injury, especially when a single large dose is administered w i t h o u t pretreatment via parenteral route [6]. The renal injury or dysfunction caused by cadmium has been estimated through various kinds of enzymes and other marker substances which are excreted into the urine as a result of impaired reabsorption at the renal tubules and/or leakage from damaged renal cells. On the other hand, hepatic injury has been diagnosed by plasma or serum levels of various enzymes. Currently, these markers are limited to those enzymes that leak into the circulating fluid from the damaged liver. The marker enzymes in plasma or serum which originate from the liver can be div}ded into 2 groups. The first group consists o f those enzymes which leak from damaged hepatic cells into the circulating fluid and hence whose concentration in the plasma o r serum increase proportionally with the extent of the damage. The second group of enzymes are secreted only by the normal liver and hence decrease when hepatic damage occurs. Members of the first group have already been widely used as indicators of the hepatic injury caused by cadmium. However, thus far, only a few of the enzymes belonging to the latter group have been examined as possible indicators of hepatic damage [7--9]. During the course of a general survey of indicators for cadmium toxicity, we noticed that cholinesterase (CHE; EC 3.1.1.8) activity in serum was decreased significantly by the repeated administration of cadmium [10]. The present study was intended to examine the extent of the effect of cadmium on CHE activity in serum by administrating single injections of various doses of cadmium to male and female rats of different ages and by tracing changes in CHE activity with time. The activities of glutamic pyruvic transaminase (GPT; EC 2.6.1.2) and glutamic oxaloacetic transaminase (GOT; EC 2.6.1.1) in the serum were determined concurrently. GPT and GOT were chosen as being representative of those enzymes which leak from damaged liver cells. The serum activity of these 2 enzymes following a cadmium insult was compared with that of CHE, chosen as being representative of those enzymes which are secreted by the normal functioning liver. METHODS
Injection o f cadmium Male and female rats of the Wistar strain {JCL, Clea Japan Co., Tokyo) were purchased from a breeder at 4 weeks of age and were fed standard laboratory chow {MF diet, Oriental Yeast Co., Tokyo) and distilled water ad libitum. The animals were injected with a single s.c. dose of cadmium chloride in saline {0.1 ml/rat) at dose levels of 1.0, 1.5 or 2.0 mg Cd/kg body weight at ages of 8, 15 and 22 weeks for the dose-response experiment in different ages and sexes. Blood samples were obtained from each animal by cutting
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the tail twice {once before the injection and again 1 day after the injection). Animals were killed by exsanguination from the carotid artery under light ether anaesthesia 2 days after the injection. Serum was separated from blood by centrifuging at 2300 g for 10 min after allowing blood to stand for 30 min. Five rats were used per data point and each datum before the injection was used as the respective control because activity of CHE in serum was shown n o t to be dependent on routes of blood collecting (tail vein or carotid artery) in preliminary experiments. For the recovery experiment, blood samples were obtained from 25 rats of each sex (15 weeks old) b y cutting tails before the injection of cadmium (1.5 mg Cd/kg b o d y wt) and these data served as the control. Groups of 5 animals were then killed by exsanguination at 12 h, 2, 3, 5 and 7 days after the injection. Serum was separated as mentioned above. Determinations o f enzyme activities in serum Serum was separated by centrifuging at 2300 g for 10 min. The activities of CHE, GOT and GPT in serum were determined on a GEMSAEC Fast Analyzer using Cholinesterase-Colour-Test [11], GOT Monostest [12] and GPT opt. [13] (Boehringer-Mannheim Co., Mannheim) according to the manufacturer's monographs. Control serum (Precinorm EA} was used as the reference sample. Statistical analysis Statistical evaluation of alterations from the controls were made by Welch's t-test. A value of P < 0.05 was accepted as significant and marked with a star.
RESULTS Four-week-old male and female rats were purchased at the same time and then maintained until 8, 15 and 22 weeks of age. The b o d y weights of females and males at these ages were 147.1 + 5.2 and 219.1 + 8.8, 221.6 +- 10.3 and 351.6 + 18.4, and 219.9 -+ 10.3 and 388.5 + 42.0 g, respectively. The CHE activity in the control serum of the male rats was low and remained at a relatively constant level throughout (Figs. 1A--C). Control CHE activity for female rats was a b o u t 3--7 times higher than that o f the males and increased with age {Figs. 1D--F). A single injection of cadmium caused a time
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Fig. 1. Changes in serum CHE activity with dose, age and sex after a single s.c. injection o f cadmium. Male (Panels A, B, and C) and female (Panels D, E, and F) rats were injected s.c. with cadmium chloride at doses o f 1.0 (a), 1.5 (o) and 2.0 (=) mg Cd/kg b o d y weight. Blood samples were obtained by cutting the tail of each rat twice; once before the injection (the day 0) and a second time 1 day after the injection. All rats were killed by exsanguination on day 2 after the injection. Ages of the rats were as follows: 8 (Panels A and D), 15 (Panels B and E) and 22 weeks old (Panels C and F). The data have been expressed as means _+ S.D. o f 5 samples.
one post-injection and attained the lowest level (60% less than the control value) on days 2 and 3 after the injection. CHE activity recovered almost to control levels on day 5 after the injection (Fig. 2A). The time-course of CHE activity for the female rat (Fig. 2B) was similar to that for the male. Again, enzymic activity started to decrease on day 1, was the lowest level on days 2 and 3 after the injection (60% less than the control value), and returned to the control level on day 5 after the injection (Fig. 2B). In contrast to the marked effect of cadmium on CHE activity, the metal did not cause an increase in GPT activity except for in the oldest group of males (Fig. 3). Some of the data in Fig. 3 showed rather unexplainable decreases in view of the fact that GPT activity in serum is known to increase
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Fig. 2. Changes in serum CHE activity with time after a single s.c. injection o f cadmium (1.5 mg Cd2*/kg body wt). Tail-vein blood samples obtained from male (Panel A) and female (Panel B) rats (15 weeks old) prior to the injection of cadmium (data are for 25 sera (mean ~ S.D.)) served as controls. The data for 12 h, 3, 3, 5 and 7 days correspond to those of sera obtained by killing 5 rats at the respective times. The data points for day 1 are the same as those used in Figs. 1B and 1E.
when liver damage occurs and that is increased by repeated injections of cadmium [10]. Time-dependent changes in GPT activity were also followed subsequent to the administration of 1.5 mg Cd/kg body weight to 15-week-old male and female rats (Fig. 4). Most of the changes were not significant compared to the control and some values lower than the controls were again observed. Direct effect of cadmium on CHE activity in serum was examined in vitro by the following experiment. Cadmium chloride was added in vitro to serum o f 10-week-old male rats to a concentration of up to 15 #g/ml serum. CHE activity in serum was measured right after the addition or after incubation for 60 min at 37°C. The activity was n o t depressed in any experiments compared to a control value before addition of cadmium (around 600 mU/ml serum). DISCUSSION Enzyme activities in serum or plasma are used clinically to diagnose abnormal functioning of a wide variety of organs. Enzymes that exist in the soluble fraction of organs leak into the body fluid when these organs are injured, thus, their serum or plasma activities increase with the extent of injury. On the other hand, the serum or plasma activity of enzymes that are secreted into the body fluid by normal organs decreases when these organs are injured. GPT and GOT belong to the former group of enzymes and are
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Fig. 3. Changes in serum GPT activity with dose, age and sex after a single s.c. injection o f c a d m i u m . The same sera used for Fig. 1 were used in this experiment. Male (Panels A, B, and C) and female (Panels D, E, and F) rats o f 8 (Panels A and D), 15 (Panels B and E) and 22 weeks old (Panels C and F) were injected with c a d m i u m at ~oses of 1.0 (A), 1.5 0 ) and 2.0 ( - ) mg Cd/kg b o d y weight. The data for day 1 in the panels C and F were not obtained due to a lack o f serum. The data for the highest dose in the panel C were 31.4 ± 4.8 (day 0) and 301 *- 233 m U / m l serum (day 2), and were not s h o w n because t h e y were off-scale. The data were expressed as means ± S.D. o f 5 samples.
generally used as markers diagnostic of hepatic function. GPT is known clinically to be a more specific marker to liver function than is GOT [14]. The target organ for the toxicity of cadmium is widely recognized to be the kidneys, especially in the case of chronic cadmium toxicity. Therefore, various kinds of marker substances including urinary enzymes have been used to diagnose renal dysfunction and injury. On the other hand, marker substances for the toxicological effect of cadmium on the liver are mostly limited to those that are leaked from the damaged liver. Although CHE activity in serum or plasma has previously been determined in an epidemiological survey
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2 ~4 Time after injection ( day ) Fig. 4. Changes in serum GPT activity with time after a single s.c. injection of cadmium. Panels A (male) and B (female) for GPT activity correspond to those in Fig. 2.
of workers exposed to cadmium [8] and in 2 experimental studies of cadmium toxicity [7,9], those studies made no a t t e m p t to explore differences between these 2 types of enzymes in sensitivity to the effects of cadmium on the liver. The present study revealed that the CHE activity in serum is markedly reduced by a single injection of a relatively low dose of cadmium. Although cadmium concentration in serum is k n o w n to increase right after the injection of cadmium, the metal is rapidly transferred mainly to the liver and the serum concentration was decreased: to a marginally detectable level within several hours [15]. Therefore, result of the time-course experiment shown in Fig. 2 suggests that the depression of CHE activity in serum is not due to a direct effect of cadmium on CHE in serum. The in vitro experiment further confirmed that the depression of CHE activity in serum was n o t due to a direct effect on CHE but was due to a decrease in a m o u n t of CHE. A signififcant dose-response relationship was not observed for the CHE activity by the doses used in the present study (Fig. 1). This m a y indicate that the doses were too high to observe a dose-response relationship irrespective of too low doses to observe significant alterations in the conventional indicators, leaked enzymes from liver. As the activity of CHE in serum is t h a t of a pseudo-cholinesterase and because it originates from the liver, reductions in CHE activity indicate that at least a part of the secretory functions of the liver and/or the biosynthesis of the secretory enzyme are damaged by cadmium. On the other hand, the GPT activity in serum was not increased by the cadmium doses used in the present study suggesting that the liver was n o t injured to the extent that intracellular soluble enzymes were leaked into the body fluid. The activity of serum GOT was also determined in this study (data not shown}. However, no significant increase in the activity of this enzyme was observed. These results suggest that both GPT and GOT activities in serum are less sensitive markers of cadmium toxicity than is serum CHE activity.
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A l t h o u g h m o r e e x a m p l e s are n e c e s s a r y b e f o r e a d e f i n i t i v e c o n c l u s i o n c a n be m a d e , t h e p r e s e n t r e s u l t s s u g g e s t t h a t s e r u m e n z y m e s w h i c h are s e c r e t e d f r o m t h e n o r m a l f u n c t i o n i n g l i v e r m a y b e m o r e s e n s i t i v e i n d i c a t o r s o f cadm i u m t o x i c i t y t h a n t h o s e e n z y m e s w h i c h are r e l e a s e d f r o m s e v e r e l y d a m a g e d liver cells. ACKNOWLEDGEMENT K . T . S . a n d Y . A . e x p r e s s t h e i r t h a n k s t o Dr. I c h i r o W a k i s a k a f o r his e n couragement. REFERENCES 1 E. Aughey, G.S. Fell, R. Scott and M. Black, Histopathology of early effects of oral cadmium in rat kidney. Environ. Health Perspect., 54 (1984) 153. 2 L. Friberg, Cadmium and the kidney. Environ. Health Perspect., 54 (1984) 1. 3 R.R. Lauwerys, A. Bernard, H.A. Roels, J.-P. Buchet and C. Viau, Characterization of cadmium proteinuria in man and rat. Environ. Health Perspect., 54 (1984) 147. 4 K. Nogawa, Biological indicators of cadmium nephrotoxicity in persons with low-level cadmium exposure. Environ. Health Perspect., 54 (1984) 163. 5 Z.A. Shaikb and C. Tobyama, Urinary metallothionein as an indicator of cadmium body burden and of cadmium-induced nephrotoxicity. Environ. Health Perspect., 54 (1984) 171. 6 R.E. Dudley, D.J. Svoboda and C.D. Klaassen, Acute exposure to cadmium causes severe liver injury in rats. Toxicol. Appl. Pbarmacol., 65 {1982) 302. 7 M.S. Dubale and P. Shah, Biochemical alterations induced by cadmium in the liver of Channa punctatus. Environ. Res., 26 (1981) 110. 8 R.R. Lauwerys, J.P. Bucbet, H.A. Roles, J. Brouwers and D. Stanescu, Epidemiological survey of workers exposed to cadmium. Arch. Environ. Health, 28 (1974) 145. 9 S.V.S. Rana, V.P. Agrawal and N.G. Bhardwaj, A qualitative study of few enzymes in the liver of cadmium fed rats. Ind. H~alth, 21 (1983) 137. 10 H. Uehara, Y, Aoki, R. Kawamura, H. Sunaga, M. Yamamura, M. Nishikawa, N. Shimojo and K.T. Suzuki, E[fects oi diet on tissue accumulation of cadmium and its toxicological indicators. Eisei Kagaku, 31 (1985) P-24. 11 G.L. Ellman, K.D. Courtney, V. Andres Jr. and R.M. Featherstone, A new and rapid colorimetric determination of acetbylcbolinesterase activity. Biochem. Pbarmacol., 7 (1961) 88. 12 A. Karmen, A note on the spectropbotometric assay of glutamic oxaloacetic transaminase in b u m a n blood serum. J. Clin. Invest., 34 (1955) 131. 13 F. Wroblewski and J.S. LaDue, Serum glutamic-pyruvic transaminase in cardiac and hepatic disease. Proc. Soc. Exp. Biol. Med., 91 {1956) 569. 14 H. Suzuki, Selection of liver function tests. Saishin Igaku, 35 (1980) 2471. 15 K.T. Suzuki and M. Yamamura, Changes of metal contents and isometallothionein levels in rat tissues after cadmium loading. Biochem. Pharmacol., 29 (1980) 2407.
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DEPRESSION OF SERUM CHOLINESTERASE ACTIVITY BY CADMIUM
HIROSHI UEHARAa'b, YASUNOBU AOKIa, NOBUHIRO SHIMOJOb and KAZUO T. SUZUKIa,* aNational Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, and bInstitute of Community Medicine, The University of Tsukuba, Niihari, Ibaraki 305 (Japan) (Received February 20th, 1985) (Accepted April 22nd, 1985)
SUMMARY Two serum enzymes which originate from the liver under different circumstances were examined as potential biological indicators in serum for cadmium toxicity. The first of those is an enzyme that leaks from damaged liver cells. The second is an enzyme that is secreted by the normal functioning liver. Cadmium chloride was injected s.c. into male and female rats o f the Wistar strain (8, 15 and 22 weeks old), at doses of 1.0, 1.5 and 2.0 mg Cd/kg body weight (in total 18 groups). Cholinesterase (CHE; EC 3.1.1.8) activity in serum was f o u n d to decrease with time after the administration of a single injection of cadmium chloride and, in all experimental groups, was significantly lower than the control values on day 2 after the injection. Glutamic pyruvic transaminase (GPT; EC 2.6.1.2) activity in serum, however, increased only in the oldest group of males receiving the high dose levels of cadmium. A timecourse experiment in which male and female rats 15 weeks of age were administered 1.5 mg Cd/kg body weight showed t h a t the serum CHE activity started to decrease on day 1 after the injection, attained the lowest level on days 2 and 3, and then recovered almost to control levels on day 5. On the o t h e r hand, the GPT activity remained at or less than control values throughout the experimental period. The results indicate that CHE activity in serum is a sensitive biological indicator for cadmium toxicity.
K e y words: Cadmium; Cholinesterase; Liver injury; Secreted enzyme; Glutamic pyruvic transaminase *Address all correspondence to: Dr. Kazuo T. Suzuki, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan.
0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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INTRODUCTION Cadmium is a potent nephrotoxin and the chronic toxicity of this metal manifests itself primarily in the renal proximal tubules [1--5]. However, cadmium also causes hepatic injury, especially when a single large dose is administered w i t h o u t pretreatment via parenteral route [6]. The renal injury or dysfunction caused by cadmium has been estimated through various kinds of enzymes and other marker substances which are excreted into the urine as a result of impaired reabsorption at the renal tubules and/or leakage from damaged renal cells. On the other hand, hepatic injury has been diagnosed by plasma or serum levels of various enzymes. Currently, these markers are limited to those enzymes that leak into the circulating fluid from the damaged liver. The marker enzymes in plasma or serum which originate from the liver can be div}ded into 2 groups. The first group consists o f those enzymes which leak from damaged hepatic cells into the circulating fluid and hence whose concentration in the plasma o r serum increase proportionally with the extent of the damage. The second group of enzymes are secreted only by the normal liver and hence decrease when hepatic damage occurs. Members of the first group have already been widely used as indicators of the hepatic injury caused by cadmium. However, thus far, only a few of the enzymes belonging to the latter group have been examined as possible indicators of hepatic damage [7--9]. During the course of a general survey of indicators for cadmium toxicity, we noticed that cholinesterase (CHE; EC 3.1.1.8) activity in serum was decreased significantly by the repeated administration of cadmium [10]. The present study was intended to examine the extent of the effect of cadmium on CHE activity in serum by administrating single injections of various doses of cadmium to male and female rats of different ages and by tracing changes in CHE activity with time. The activities of glutamic pyruvic transaminase (GPT; EC 2.6.1.2) and glutamic oxaloacetic transaminase (GOT; EC 2.6.1.1) in the serum were determined concurrently. GPT and GOT were chosen as being representative of those enzymes which leak from damaged liver cells. The serum activity of these 2 enzymes following a cadmium insult was compared with that of CHE, chosen as being representative of those enzymes which are secreted by the normal functioning liver. METHODS
Injection o f cadmium Male and female rats of the Wistar strain {JCL, Clea Japan Co., Tokyo) were purchased from a breeder at 4 weeks of age and were fed standard laboratory chow {MF diet, Oriental Yeast Co., Tokyo) and distilled water ad libitum. The animals were injected with a single s.c. dose of cadmium chloride in saline {0.1 ml/rat) at dose levels of 1.0, 1.5 or 2.0 mg Cd/kg body weight at ages of 8, 15 and 22 weeks for the dose-response experiment in different ages and sexes. Blood samples were obtained from each animal by cutting
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the tail twice {once before the injection and again 1 day after the injection). Animals were killed by exsanguination from the carotid artery under light ether anaesthesia 2 days after the injection. Serum was separated from blood by centrifuging at 2300 g for 10 min after allowing blood to stand for 30 min. Five rats were used per data point and each datum before the injection was used as the respective control because activity of CHE in serum was shown n o t to be dependent on routes of blood collecting (tail vein or carotid artery) in preliminary experiments. For the recovery experiment, blood samples were obtained from 25 rats of each sex (15 weeks old) b y cutting tails before the injection of cadmium (1.5 mg Cd/kg b o d y wt) and these data served as the control. Groups of 5 animals were then killed by exsanguination at 12 h, 2, 3, 5 and 7 days after the injection. Serum was separated as mentioned above. Determinations o f enzyme activities in serum Serum was separated by centrifuging at 2300 g for 10 min. The activities of CHE, GOT and GPT in serum were determined on a GEMSAEC Fast Analyzer using Cholinesterase-Colour-Test [11], GOT Monostest [12] and GPT opt. [13] (Boehringer-Mannheim Co., Mannheim) according to the manufacturer's monographs. Control serum (Precinorm EA} was used as the reference sample. Statistical analysis Statistical evaluation of alterations from the controls were made by Welch's t-test. A value of P < 0.05 was accepted as significant and marked with a star.
RESULTS Four-week-old male and female rats were purchased at the same time and then maintained until 8, 15 and 22 weeks of age. The b o d y weights of females and males at these ages were 147.1 + 5.2 and 219.1 + 8.8, 221.6 +- 10.3 and 351.6 + 18.4, and 219.9 -+ 10.3 and 388.5 + 42.0 g, respectively. The CHE activity in the control serum of the male rats was low and remained at a relatively constant level throughout (Figs. 1A--C). Control CHE activity for female rats was a b o u t 3--7 times higher than that o f the males and increased with age {Figs. 1D--F). A single injection of cadmium caused a time
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Fig. 1. Changes in serum CHE activity with dose, age and sex after a single s.c. injection o f cadmium. Male (Panels A, B, and C) and female (Panels D, E, and F) rats were injected s.c. with cadmium chloride at doses o f 1.0 (a), 1.5 (o) and 2.0 (=) mg Cd/kg b o d y weight. Blood samples were obtained by cutting the tail of each rat twice; once before the injection (the day 0) and a second time 1 day after the injection. All rats were killed by exsanguination on day 2 after the injection. Ages of the rats were as follows: 8 (Panels A and D), 15 (Panels B and E) and 22 weeks old (Panels C and F). The data have been expressed as means _+ S.D. o f 5 samples.
one post-injection and attained the lowest level (60% less than the control value) on days 2 and 3 after the injection. CHE activity recovered almost to control levels on day 5 after the injection (Fig. 2A). The time-course of CHE activity for the female rat (Fig. 2B) was similar to that for the male. Again, enzymic activity started to decrease on day 1, was the lowest level on days 2 and 3 after the injection (60% less than the control value), and returned to the control level on day 5 after the injection (Fig. 2B). In contrast to the marked effect of cadmium on CHE activity, the metal did not cause an increase in GPT activity except for in the oldest group of males (Fig. 3). Some of the data in Fig. 3 showed rather unexplainable decreases in view of the fact that GPT activity in serum is known to increase
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Fig. 2. Changes in serum CHE activity with time after a single s.c. injection o f cadmium (1.5 mg Cd2*/kg body wt). Tail-vein blood samples obtained from male (Panel A) and female (Panel B) rats (15 weeks old) prior to the injection of cadmium (data are for 25 sera (mean ~ S.D.)) served as controls. The data for 12 h, 3, 3, 5 and 7 days correspond to those of sera obtained by killing 5 rats at the respective times. The data points for day 1 are the same as those used in Figs. 1B and 1E.
when liver damage occurs and that is increased by repeated injections of cadmium [10]. Time-dependent changes in GPT activity were also followed subsequent to the administration of 1.5 mg Cd/kg body weight to 15-week-old male and female rats (Fig. 4). Most of the changes were not significant compared to the control and some values lower than the controls were again observed. Direct effect of cadmium on CHE activity in serum was examined in vitro by the following experiment. Cadmium chloride was added in vitro to serum o f 10-week-old male rats to a concentration of up to 15 #g/ml serum. CHE activity in serum was measured right after the addition or after incubation for 60 min at 37°C. The activity was n o t depressed in any experiments compared to a control value before addition of cadmium (around 600 mU/ml serum). DISCUSSION Enzyme activities in serum or plasma are used clinically to diagnose abnormal functioning of a wide variety of organs. Enzymes that exist in the soluble fraction of organs leak into the body fluid when these organs are injured, thus, their serum or plasma activities increase with the extent of injury. On the other hand, the serum or plasma activity of enzymes that are secreted into the body fluid by normal organs decreases when these organs are injured. GPT and GOT belong to the former group of enzymes and are
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( doy )
Fig. 3. Changes in serum GPT activity with dose, age and sex after a single s.c. injection o f c a d m i u m . The same sera used for Fig. 1 were used in this experiment. Male (Panels A, B, and C) and female (Panels D, E, and F) rats o f 8 (Panels A and D), 15 (Panels B and E) and 22 weeks old (Panels C and F) were injected with c a d m i u m at ~oses of 1.0 (A), 1.5 0 ) and 2.0 ( - ) mg Cd/kg b o d y weight. The data for day 1 in the panels C and F were not obtained due to a lack o f serum. The data for the highest dose in the panel C were 31.4 ± 4.8 (day 0) and 301 *- 233 m U / m l serum (day 2), and were not s h o w n because t h e y were off-scale. The data were expressed as means ± S.D. o f 5 samples.
generally used as markers diagnostic of hepatic function. GPT is known clinically to be a more specific marker to liver function than is GOT [14]. The target organ for the toxicity of cadmium is widely recognized to be the kidneys, especially in the case of chronic cadmium toxicity. Therefore, various kinds of marker substances including urinary enzymes have been used to diagnose renal dysfunction and injury. On the other hand, marker substances for the toxicological effect of cadmium on the liver are mostly limited to those that are leaked from the damaged liver. Although CHE activity in serum or plasma has previously been determined in an epidemiological survey
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A ~3
40 B 30
3O
2O
i
10
10 I
!
2 ~4 Time after injection ( day ) Fig. 4. Changes in serum GPT activity with time after a single s.c. injection of cadmium. Panels A (male) and B (female) for GPT activity correspond to those in Fig. 2.
of workers exposed to cadmium [8] and in 2 experimental studies of cadmium toxicity [7,9], those studies made no a t t e m p t to explore differences between these 2 types of enzymes in sensitivity to the effects of cadmium on the liver. The present study revealed that the CHE activity in serum is markedly reduced by a single injection of a relatively low dose of cadmium. Although cadmium concentration in serum is k n o w n to increase right after the injection of cadmium, the metal is rapidly transferred mainly to the liver and the serum concentration was decreased: to a marginally detectable level within several hours [15]. Therefore, result of the time-course experiment shown in Fig. 2 suggests that the depression of CHE activity in serum is not due to a direct effect of cadmium on CHE in serum. The in vitro experiment further confirmed that the depression of CHE activity in serum was n o t due to a direct effect on CHE but was due to a decrease in a m o u n t of CHE. A signififcant dose-response relationship was not observed for the CHE activity by the doses used in the present study (Fig. 1). This m a y indicate that the doses were too high to observe a dose-response relationship irrespective of too low doses to observe significant alterations in the conventional indicators, leaked enzymes from liver. As the activity of CHE in serum is t h a t of a pseudo-cholinesterase and because it originates from the liver, reductions in CHE activity indicate that at least a part of the secretory functions of the liver and/or the biosynthesis of the secretory enzyme are damaged by cadmium. On the other hand, the GPT activity in serum was not increased by the cadmium doses used in the present study suggesting that the liver was n o t injured to the extent that intracellular soluble enzymes were leaked into the body fluid. The activity of serum GOT was also determined in this study (data not shown}. However, no significant increase in the activity of this enzyme was observed. These results suggest that both GPT and GOT activities in serum are less sensitive markers of cadmium toxicity than is serum CHE activity.
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A l t h o u g h m o r e e x a m p l e s are n e c e s s a r y b e f o r e a d e f i n i t i v e c o n c l u s i o n c a n be m a d e , t h e p r e s e n t r e s u l t s s u g g e s t t h a t s e r u m e n z y m e s w h i c h are s e c r e t e d f r o m t h e n o r m a l f u n c t i o n i n g l i v e r m a y b e m o r e s e n s i t i v e i n d i c a t o r s o f cadm i u m t o x i c i t y t h a n t h o s e e n z y m e s w h i c h are r e l e a s e d f r o m s e v e r e l y d a m a g e d liver cells. ACKNOWLEDGEMENT K . T . S . a n d Y . A . e x p r e s s t h e i r t h a n k s t o Dr. I c h i r o W a k i s a k a f o r his e n couragement. REFERENCES 1 E. Aughey, G.S. Fell, R. Scott and M. Black, Histopathology of early effects of oral cadmium in rat kidney. Environ. Health Perspect., 54 (1984) 153. 2 L. Friberg, Cadmium and the kidney. Environ. Health Perspect., 54 (1984) 1. 3 R.R. Lauwerys, A. Bernard, H.A. Roels, J.-P. Buchet and C. Viau, Characterization of cadmium proteinuria in man and rat. Environ. Health Perspect., 54 (1984) 147. 4 K. Nogawa, Biological indicators of cadmium nephrotoxicity in persons with low-level cadmium exposure. Environ. Health Perspect., 54 (1984) 163. 5 Z.A. Shaikb and C. Tobyama, Urinary metallothionein as an indicator of cadmium body burden and of cadmium-induced nephrotoxicity. Environ. Health Perspect., 54 (1984) 171. 6 R.E. Dudley, D.J. Svoboda and C.D. Klaassen, Acute exposure to cadmium causes severe liver injury in rats. Toxicol. Appl. Pbarmacol., 65 {1982) 302. 7 M.S. Dubale and P. Shah, Biochemical alterations induced by cadmium in the liver of Channa punctatus. Environ. Res., 26 (1981) 110. 8 R.R. Lauwerys, J.P. Bucbet, H.A. Roles, J. Brouwers and D. Stanescu, Epidemiological survey of workers exposed to cadmium. Arch. Environ. Health, 28 (1974) 145. 9 S.V.S. Rana, V.P. Agrawal and N.G. Bhardwaj, A qualitative study of few enzymes in the liver of cadmium fed rats. Ind. H~alth, 21 (1983) 137. 10 H. Uehara, Y, Aoki, R. Kawamura, H. Sunaga, M. Yamamura, M. Nishikawa, N. Shimojo and K.T. Suzuki, E[fects oi diet on tissue accumulation of cadmium and its toxicological indicators. Eisei Kagaku, 31 (1985) P-24. 11 G.L. Ellman, K.D. Courtney, V. Andres Jr. and R.M. Featherstone, A new and rapid colorimetric determination of acetbylcbolinesterase activity. Biochem. Pbarmacol., 7 (1961) 88. 12 A. Karmen, A note on the spectropbotometric assay of glutamic oxaloacetic transaminase in b u m a n blood serum. J. Clin. Invest., 34 (1955) 131. 13 F. Wroblewski and J.S. LaDue, Serum glutamic-pyruvic transaminase in cardiac and hepatic disease. Proc. Soc. Exp. Biol. Med., 91 {1956) 569. 14 H. Suzuki, Selection of liver function tests. Saishin Igaku, 35 (1980) 2471. 15 K.T. Suzuki and M. Yamamura, Changes of metal contents and isometallothionein levels in rat tissues after cadmium loading. Biochem. Pharmacol., 29 (1980) 2407.
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