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Release of hepatic cadmium by carbon tetrachloride treatment
ToYIC",oGY
AND APPLIED
Release KEIICHI
PHARMACOLOGY
of Hepatic TANAKA.
59,535-539(1981)
Cadmium
HIROKO
NOMURA,
by Carbon SATOMI
Tetrachloride
ONOSAKA,
AND
Treatment
KYONG-SON
MIN
Release of Hepatic Cadmium by Carbon Tetrachloride Treatment. TANAKA. K.. NOMURA. H.. ONOSAKA, S.. AND MIN, K. (1981). 7o.ricol. Appl. Plrctrmacol. 59, 535-539. To evaluate the possibility of metallothionein transfer from liver to kidney, an experimental hepatic disorder produced by carbon tetrachloride was examined to study the release of metallothionein from liver. ‘““Cd-exposed rats were treated with carbon tetrachloride and the kinetics of cadmium in the body was studied. Hepatic cadmium was significantly decreased in a dose-dependent manner. In contrast, cadmium in plasma. kidney. and urine was increased in proportion to the decrease in hepatic cadmium. No conspicuous changes in cadmium of other tissues and feces were observed. Cadmium in hepatic supernatant of carbon tetrachloride-treated rat was bound mostly to the metallothionein fraction in Sephadex G-75 gel filtration. and in kidney, plasma, or urine. Cd was also in the form of metallothionein. These results suggest that hepatic metallothionein can be released into blood and be transported to kidney and urine in some types of hepatic disorders.
Recent biochemical research on metallothionein (MT) has elucidated a possible physiological role for this protein, that is, the homeostatic control of essential trace elements (Cousins and Failla, 1980). These investigations, however, do not deny the role of MT in the defense mechanism against heavy metal intoxication, and MT is considered to have an important role in the fate of some heavy metals. especially cadmium (Cd), in the body (Webb, 1980). It has hitherto been unknown whether MT participates in the transport of Cd from liver to other tissues in Cd-exposed animals. The present authors (Tanaka et al., 1975) and Cherian and Shaikh (1975) first reported the distribution of MT in rats. There have been many reports which. however, were concerned mostly with the fate of injected MT. Still, very little is known about the fate of endogenous MT. It is commonly thought that in liver and kidney, the protein part of MT (thionein) is decomposed, probably
by some lysosomal proteinases (Feldman et trl., 1978). but Cd liberated by this turnover is not usually considered to leak from these hepatic and renal cells bound to MT (Kimura, 1980). However, a Cd-bound MTlike protein was found in the plasma of rats receiving Cd, at 0.5 mg/kg body wt, 6 days1 week, for 26 weeks (Suzuki, 1980), suggesting the possibility of MT leak from the cells, especially degenerated hepatic cells (Kawai et cl/., 1976). The present study was made to evaluate whether Cd bound to MT leaks from the liver into blood and further into kidney, using animals with liver injury caused experimentally with carbon tetrachloride. METHODS
E.\rlwrinzctrt I. Male Wistar-strain rats (average weight. 200 g) were divided into four groups. with six to eight in each group. ““CdCI, was injected sub-
536
TANAKA
ET AL.
TABLE EFFECTS
OF CARBON
TETRACHI
ORIDE
I
ON CADMIUM
IN TISSUES,
Duration I Day
I Week
PLASMA.
URINE.
AND
FECES
IN R,%rs”
of Cd injection 4 Weeks
8 Weeks
Liver Control CCI,
S.58 i 0.34 4.34 2 O.lP
61.78 it.06
-t 2.62 e 3.69”
184.65 90.76
1- 16.61 ‘- x.16’*
368.54 219.60
+ 14.20 2 16.40“
Kidney Control CCI,
1.85 i 0.19 4.50 t 0.49*r
19.12 f 0.68 34.82 i- O.‘4”x
95.53 120.38
i 6.23 t 3.07”
185.16 197.44
i 5.98 i 6.39
Small intestine Control ccl,
0.30 2 0.01 0.33 + 0.02
0.99 t 0.02 1.04 i- 0.02
2.91 -+ 0.16 3.48 i 0.04*
Pancreas Control CCI,
1.06 -t 0.08 1.58 i 0.04**.
7.87 -c 0.36 8.49 f 0.38
25.01 t 1.47 ‘26.53 2 0.99
54.86 60.39
Spleen Control ccl,
0.39 2 0.03 0.66 -t 0.01**
2.42 2.86
-+ 0.10 ? 0.09’
9.41 2 0.39 10.28 -t 0.61
19.07 2 0.57 20.01 2 1.37
Plasma Control ccl,
0.01 2 0.00 0.02 +- o.oo*
0.01 ?Z 0.00 0.12 5 0.03x
0.01 -+ 0.00 0.42 2 0.17*
0.03 e 0.00 0..55 + 0. Ii
0.1 i 0.0 98.8 -c 16.9**
0.3 -c 0.0 307.7 i 42.0**
3.0 i- 0.5 548.7 ‘r 165. I ‘.
7.1 t 0.3 6.3 i 3.2
5.0 2 2.1 14.8 f 4.3
Urine Control CCI,
0.3 2 0.1 2.7 t I.7
Feces Control ccl,
3.3 I? 0.4 I.8 -+ 0.4*
8.1 2 0.8 5.2 i- 0.9
6.78 t 0.57 5.89 t 0.57 -t 0.56 2 2.85
k
” Olive oil or Ccl, (0.25 ml/l00 g body weight) was administered to rats that were pretreated with Cd. and 24 hr later, rats were sacrificed. Each value represents the mean -t SE of three or four rats f&g for tissues and plasma, ~8124 hr for urine and feces). * Significantly different from respective control value, p < 0.05. ** Significantly different from respective control value, p < 0.01.
cutaneously to rats at a dose of 0.5 mg Cd/O.67 pCi/kg body wt one time or over a period of 1, 4, or 8 weeks (6 days per week). At I day after a single injection or the final injection of “Vd, half of the rats in each group were orally given CCI, in olive oil (0.25 ml CCl,/lOO g body wt) and the other half were administered olive oil alone. Rats were housed individually in metabolic cages, and urine and feces were collected for 24 hr. At 24 hr after Ccl, or olive oil administration. the rats were sacrificed by bleeding under light ether anesthesia, and the liver, kidney, spleen, pancreas, and small intestine were removed. Plasma was obtained by centrifugation of the heparinized blood at 3000 rpm for 10 min.
E.rprrimrnt 2. Twenty rats were previously injected with “‘%d for 8 weeks as in Experiment 1. At 1 day after the final administration, 0. l/12, t/6, or 113 ml/ 100 g body wt of CCL, was given to the rats (five in each group), and urine was collected for 24 hr in metabolic cages. Rats were sacrificed 24 hr after Ccl, or olive oil administration. and liver, kidney. and plasma were obtained. Eqerimenr 3. Ten rats that were given ““‘Cd for 4 weeks were administered olive oil or 0.25 ml/l00 g body wt of Ccl,. Rats were individually housed in metabolic cages for 5 days and urine was collected daily. At 5 days, rats were sacrificed and liver, kidney. and plasma were obtained.
RELEASE
OF HEPATIC
CADMIUM
537
The Cd content of all samples was calculated from the specific radioactivity of “‘“Cd. which was determined with an Aloka Auto Well scintillation counter (efficiency. 50%). For counting the tissue samples. a 0.25 M sucrose homogenate was used. The determination of zinc and copper in column eluates was made by a Hitachi 208 flame-type atomic absorption spectrometer.
The liver and kidney were homogenized in 5 vol of 0.25 r~f sucrose solution. Sediments were removed by centrifugation at lOS.000~ for 60 min at 4”C, and the supernatants were obtained. The plasma and urine were used without pretreatment. MT in each specimen was identified on a Sephadex G-75 column eluted with 50 rn!+t Tris-HCI buffer (pH 8.0).
RESULTS
Table 1 shows the Cd concentration in several organs and plasma, the amount of Cd in urine and feces excreted within 24 hr of the rats that were given 0.5 mg/kg of Cd for different periods, and also the effects of the Ccl, treatment (Experiment 1). The Cd concentration in each organ increased according to the duration of its administration, while that in the plasma did not increase as much even in the rats that were given Cd for a 8-week period. Ccl, given at a dose of 0.25 ml/100 g body wt to the Cdpretreated rats induced a significant decrease in the Cd concentration of liver in all the groups at 24 hr after administration, showing a decrease to approximately half of the control, especially in the rats that received Cd treatment for 1- and 4-week periods. In contrast, Ccl, increased the Cd concentration in the kidney. The increase was conspicuous in those that were given Cd for shorter durations, but it was almost unrecognizable in those receiving Cd for an 8week period. An increase in the concentration of Cd following Ccl, treatment in the
FIG. I. Dose-response of Cd in liver, plasma, and urine to Ccl,. Rats pretreated with Cd for an S-week period (0.5 mgikg body wt. 6 days/week) were administered olive oil or l/l?. 116, or 113 ml/100 g body wt of Ccl,. Urine was collected for 24 hr prior to sacrifice. Each value shows the mean 3 SE offive rats.
small intestine, pancreas, and spleen was not so remarkable as was observed in the kidney. The Cd concentration in the plasma increased conspicuously after Ccl, treatment even in those that were given Cd for an g-week period. The excretion of Cd in urine was only 3 pglday in the rats that were given Cd for an S-week period. After Ccl, treatment, it increased drastically, up to approximately 550 @g/day. In the feces, however. the change was not drastic. Dose -Response to Ccl, CCII, 0- l/3 ml/l00 g body wt, was given to the rats that had been given Cd for an S-week period (Experiment 2). The total amount of Cd in the liver, the concentration of Cd in the plasma, and the excretion in the urine at 24 hr are shown in Fig. 1. The total amount of Cd in the liver decreased according to the increase in the dose of CC&, and correspondingly, the amount of Cd in the urine increased. The Cd concentration in the plasma increased linearly according to the dose. The total amount of Cd in the kidney increased by approximately 100 Fg in all the rats of different groups that received Ccl, at different doses. Time Course qf Urinay after Ccl, Treatment
Excretion
of Cd
An increase in urinary Cd excretion was studied following the oral administration of
538
TANAKA 4GC
0
ET AL
DISCUSSION
1:3.+5 D&Y
FIG. 2. Effect of CCI, on urinary excretion of Cd. Rats that were given Cd for 4 weeks (0.5 mg/kg body wt. 6 days/week) were administered olive oil or 0.35 ml/l00 g body wt of Ccl, and 24-hr urine was collected for 5 days. Each value shows the mean of five rats.
0.25 ml/100 g body wt of Ccl, to the rats that previously were given Cd for a 4-week period (Experiment 3). The daily amount of Cd excretion in the urine examined for 5 days following Ccl, treatment is shown in Fig. 2. A conspicuous increase occurred within 48 hr following Ccl, treatment, followed by a decrease to the control level. At 5 days, the total amount of Cd in the liver decreased to approximately one-half that of the control, and almost 70% of the loss was recovered from the urine. The Cd level in the plasma at 5 days was not different from that of the control.
Chemical forms of Cd in the rats following Ccl, administration were studied by means of gel filtration using Sephadex G-75 column. Figure 3 shows elution profiles of the supernatant of the liver and kidney homogenates of the rats that received 0.25 ml/100 g body wt of CCL, after Cd administration for an g-week period (Experiment 1). In each organ, Cd of the supernatant was eluted in the MT fraction. Figure 4 shows elution profiles of the plasma and urine from the same rat. In each specimen, the main component of Cd was eluted in the fraction corresponding to the rat hepatic MT, suggesting that Cd in the plasma and urine after Ccl., treatment exists in the form of MT.
These results show that the administration of CCI, to rats pretreated with Cd caused a decrease in hepatic Cd, an increase in plasma Cd, an increase in renal Cd, and a remarkable increase in urinary excretion of Cd. This indicates that Ccl, administration accelerates Cd release from the liver to the blood, and the released Cd is redistributed in the kidney or excreted in the urine. More interesting is that Cd seems to be transferred in the form of MT in this process. This is supported by the following facts: (11 Cd exists mostly in the MT form in the liver and kidney at 24 hr following the administration of Ccl, when release of Cd from the liver occurs: (2) the MT-form Cd increases in the plasma and urine according to the elevation of the Cd level in them; and (3) the change in Cd following Ccl, treatment is similar to the behavior of exogenous MT following its injection, i.e., (a) Cd was redistributed mostly to the kidney and urine, the same as the distribution of injected MT (Tanaka t)t al.. 19751, and (b) the urinary excretion of Cd was accelerated by an increase in plasma Cd as by an increased amount of plasma MT (Foulkes, 1978).
FIG. 3. Elution protile of hepatic and renal aupernatant from a CCI,-treated rat on a Sephadex G-75 column. A rat pretreated with Cd for a R-week period 10,s mg/kg body wt. 6 days/week) was administered 0.25 ml/100 g body wt of Ccl,. Twenty-four hours after administration, liver and kidney were re moved and homogenized with 5 vol ot‘0.75 M sucrose. Four milliliters of 105,0001: supernatant fraction of homogenate was charged on a Sephadex G-75 column (1.8 * 47.5 cm) and eluted with 50 mM Tris-HCI buffer, nH 8.0.
RELEASE
OF
HEPATIC
539
CADMIUM
injury may lead to membrane damage in which the cellular MT. together with some enzymes, leaks out into the blood. and the MT may be reabsorbed in the kidney to some degree, but most is excreted in the urine.
FIN,. 4. Elution profile treated rat on a Sephadex of plasma or urine from
of plasma and urine of a CCI,G-75 column. Two milliliters the same rat as in Fig. 3 was
charged on a Sephadex G-75 column (2.0 * 16.0 cm) and eluted with 50 m.v Tris-HCI buffer. pH X.0. The bracket shows the fraction of rat hepatic MT eluted on the same column.
The mechanism of MT release from the liver owing to the effect of Ccl, is still under investigation. However, as one possibility, we believe that MT behaves in the same way as the released enzymes in the case of hepatic injury. Changes in the MT level in the plasma after Ccl, administration were not followed, but it appears that the MT level in the plasma has a peak at 24-48 hr corresponding to the urinary Cd peak following Ccl, administration (Fig. 2). These changes with time of MT coincide with those of the released enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). in the plasma following the administration of Ccl, (Zimmerman, 1973). Since the severity of hepatic injury caused experimentally by Ccl, is usually proportional to the dose of the drug, the activity of the enzymes such as GOT. GPT, and lactate dehydrogenase (LDH) in the plasma increased in proportion to the dose of Ccl, administered. There was also a correlation between the plasma Cd level and the dose of Ccl, (Fig. 1). Moreover, in our preliminary data (Tanaka and Min, 1980). there was a significant correlation between the plasma Cd level and the activity of GOT, GPT, or LDH in the plasma. These facts are suggestive that MT is released into the blood in the same way as certain enzymes in the liver cells. Liver
REFERENCES M.
CHERIAN.
lism of protein.
G..
END
molecular aspects and homeostasis.
(J. 0. Nriagu, Wiley and Sons,
F~I.DM.XN.
ed.). New
Degradation
vitro.
Part York.
.Qi~>plr.v.\.
hts. E. C. cadmium-metallothionein.
(1978).
FOLJI
II.
pp.
121-135.
John
M. L., AND COVSI~S.
of rat
Riochi,rl.
65,
f-.411 t h. M. L. (1980). Cellular of mammalian zmc metaboIn %i,lc, if? rllc t‘,r~.irr~/lrtl<~/r/
S. L.. F+ILL.~,
( 1978).
Metabo-
injected cadmium-binding Bio/)lt?-~. Rr.,. C‘OVI?)II~/I.
X63-X69. COUSINS. R. J.. &ND and lism
Z. A. (1975).
SHAihfI.
intravenously Bi
liver AC,/II
R. J.
metallothioneins 544.
in
63X-646.
Renal tubular transport 7~~ti[~r,/. c\p[~/. f/ltrr,n+
of
(~01. 45, 505-511. li~w.41.
K.,
FLlhl:D4.
h;..
.AND
M.
KIMLIRA.
(1976).
Morphological alterations in experimental cadmium exposure with special reference to the onset of renal lesion. In E(/cc.r.\ IIII~ D,~.\~,-~~,.~l’otr,\l, Hcll~ri~~r~.\hil).\
in
Amsterdam. M. (19801. Biosynthehis mammahan cultured cells.
of metallothionein ./(li>tr,~. J. Hxr.
35,
22x-129. SC,ILIKI. Y. (1980). zinc and copper flelllth
18.
T.4N.4K.4.
lothionein
and in blood
of
cadmium, rats. I/r
19-19.
h;..
Oh4timA.
Metallothlonein distribution
StiED.4.
li..
K. (1975). Fate in rats. 7o.\ir.r~/.
OluOS.ihh.
s..
.41\D
of “‘!‘Cd-labeled metal,q/~/‘/, /‘/~~~rr,r~rc~<~/. 33,
158-266. K., AUD MIN. K. t 1980). Release lothionein from liver by hepatic disorder. S~t~fpo.\irrttl ot, Etll~irlJrlnlctlrcll Po//!rtllrlrs ~.o/o~?.. Kobe. Japan.
TANAh.4.
WFBB. i\rr~..
M. (1980). BLdrcmi.vrr~
Webb. ed.). Amsterdam.
pp.
The
metallothioneins. or~d Bio/,,gy
195-266.
H. J. (1973). totoxicity. In Lllh0rrlrorx LXsc,~r.sr.\ (F. W. Sundermann man”. Jr.. eds.). Japan ed.. Shoten, Tokyo.
ZIMMEKMAN.
o/
of metai7/l<, 7rlr linti 70 ri-
In 71r(, C‘l?czrtrC‘t~dmi/oc (M.
ElsevierINorth-Holland. Manifestation of hepaI>iu,qno.vi.\ c!/ Li~,cr and F. W. Sunderpp. 329-339. Hirokawa-
AND APPLIED
Release KEIICHI
PHARMACOLOGY
of Hepatic TANAKA.
59,535-539(1981)
Cadmium
HIROKO
NOMURA,
by Carbon SATOMI
Tetrachloride
ONOSAKA,
AND
Treatment
KYONG-SON
MIN
Release of Hepatic Cadmium by Carbon Tetrachloride Treatment. TANAKA. K.. NOMURA. H.. ONOSAKA, S.. AND MIN, K. (1981). 7o.ricol. Appl. Plrctrmacol. 59, 535-539. To evaluate the possibility of metallothionein transfer from liver to kidney, an experimental hepatic disorder produced by carbon tetrachloride was examined to study the release of metallothionein from liver. ‘““Cd-exposed rats were treated with carbon tetrachloride and the kinetics of cadmium in the body was studied. Hepatic cadmium was significantly decreased in a dose-dependent manner. In contrast, cadmium in plasma. kidney. and urine was increased in proportion to the decrease in hepatic cadmium. No conspicuous changes in cadmium of other tissues and feces were observed. Cadmium in hepatic supernatant of carbon tetrachloride-treated rat was bound mostly to the metallothionein fraction in Sephadex G-75 gel filtration. and in kidney, plasma, or urine. Cd was also in the form of metallothionein. These results suggest that hepatic metallothionein can be released into blood and be transported to kidney and urine in some types of hepatic disorders.
Recent biochemical research on metallothionein (MT) has elucidated a possible physiological role for this protein, that is, the homeostatic control of essential trace elements (Cousins and Failla, 1980). These investigations, however, do not deny the role of MT in the defense mechanism against heavy metal intoxication, and MT is considered to have an important role in the fate of some heavy metals. especially cadmium (Cd), in the body (Webb, 1980). It has hitherto been unknown whether MT participates in the transport of Cd from liver to other tissues in Cd-exposed animals. The present authors (Tanaka et al., 1975) and Cherian and Shaikh (1975) first reported the distribution of MT in rats. There have been many reports which. however, were concerned mostly with the fate of injected MT. Still, very little is known about the fate of endogenous MT. It is commonly thought that in liver and kidney, the protein part of MT (thionein) is decomposed, probably
by some lysosomal proteinases (Feldman et trl., 1978). but Cd liberated by this turnover is not usually considered to leak from these hepatic and renal cells bound to MT (Kimura, 1980). However, a Cd-bound MTlike protein was found in the plasma of rats receiving Cd, at 0.5 mg/kg body wt, 6 days1 week, for 26 weeks (Suzuki, 1980), suggesting the possibility of MT leak from the cells, especially degenerated hepatic cells (Kawai et cl/., 1976). The present study was made to evaluate whether Cd bound to MT leaks from the liver into blood and further into kidney, using animals with liver injury caused experimentally with carbon tetrachloride. METHODS
E.\rlwrinzctrt I. Male Wistar-strain rats (average weight. 200 g) were divided into four groups. with six to eight in each group. ““CdCI, was injected sub-
536
TANAKA
ET AL.
TABLE EFFECTS
OF CARBON
TETRACHI
ORIDE
I
ON CADMIUM
IN TISSUES,
Duration I Day
I Week
PLASMA.
URINE.
AND
FECES
IN R,%rs”
of Cd injection 4 Weeks
8 Weeks
Liver Control CCI,
S.58 i 0.34 4.34 2 O.lP
61.78 it.06
-t 2.62 e 3.69”
184.65 90.76
1- 16.61 ‘- x.16’*
368.54 219.60
+ 14.20 2 16.40“
Kidney Control CCI,
1.85 i 0.19 4.50 t 0.49*r
19.12 f 0.68 34.82 i- O.‘4”x
95.53 120.38
i 6.23 t 3.07”
185.16 197.44
i 5.98 i 6.39
Small intestine Control ccl,
0.30 2 0.01 0.33 + 0.02
0.99 t 0.02 1.04 i- 0.02
2.91 -+ 0.16 3.48 i 0.04*
Pancreas Control CCI,
1.06 -t 0.08 1.58 i 0.04**.
7.87 -c 0.36 8.49 f 0.38
25.01 t 1.47 ‘26.53 2 0.99
54.86 60.39
Spleen Control ccl,
0.39 2 0.03 0.66 -t 0.01**
2.42 2.86
-+ 0.10 ? 0.09’
9.41 2 0.39 10.28 -t 0.61
19.07 2 0.57 20.01 2 1.37
Plasma Control ccl,
0.01 2 0.00 0.02 +- o.oo*
0.01 ?Z 0.00 0.12 5 0.03x
0.01 -+ 0.00 0.42 2 0.17*
0.03 e 0.00 0..55 + 0. Ii
0.1 i 0.0 98.8 -c 16.9**
0.3 -c 0.0 307.7 i 42.0**
3.0 i- 0.5 548.7 ‘r 165. I ‘.
7.1 t 0.3 6.3 i 3.2
5.0 2 2.1 14.8 f 4.3
Urine Control CCI,
0.3 2 0.1 2.7 t I.7
Feces Control ccl,
3.3 I? 0.4 I.8 -+ 0.4*
8.1 2 0.8 5.2 i- 0.9
6.78 t 0.57 5.89 t 0.57 -t 0.56 2 2.85
k
” Olive oil or Ccl, (0.25 ml/l00 g body weight) was administered to rats that were pretreated with Cd. and 24 hr later, rats were sacrificed. Each value represents the mean -t SE of three or four rats f&g for tissues and plasma, ~8124 hr for urine and feces). * Significantly different from respective control value, p < 0.05. ** Significantly different from respective control value, p < 0.01.
cutaneously to rats at a dose of 0.5 mg Cd/O.67 pCi/kg body wt one time or over a period of 1, 4, or 8 weeks (6 days per week). At I day after a single injection or the final injection of “Vd, half of the rats in each group were orally given CCI, in olive oil (0.25 ml CCl,/lOO g body wt) and the other half were administered olive oil alone. Rats were housed individually in metabolic cages, and urine and feces were collected for 24 hr. At 24 hr after Ccl, or olive oil administration. the rats were sacrificed by bleeding under light ether anesthesia, and the liver, kidney, spleen, pancreas, and small intestine were removed. Plasma was obtained by centrifugation of the heparinized blood at 3000 rpm for 10 min.
E.rprrimrnt 2. Twenty rats were previously injected with “‘%d for 8 weeks as in Experiment 1. At 1 day after the final administration, 0. l/12, t/6, or 113 ml/ 100 g body wt of CCL, was given to the rats (five in each group), and urine was collected for 24 hr in metabolic cages. Rats were sacrificed 24 hr after Ccl, or olive oil administration. and liver, kidney. and plasma were obtained. Eqerimenr 3. Ten rats that were given ““‘Cd for 4 weeks were administered olive oil or 0.25 ml/l00 g body wt of Ccl,. Rats were individually housed in metabolic cages for 5 days and urine was collected daily. At 5 days, rats were sacrificed and liver, kidney. and plasma were obtained.
RELEASE
OF HEPATIC
CADMIUM
537
The Cd content of all samples was calculated from the specific radioactivity of “‘“Cd. which was determined with an Aloka Auto Well scintillation counter (efficiency. 50%). For counting the tissue samples. a 0.25 M sucrose homogenate was used. The determination of zinc and copper in column eluates was made by a Hitachi 208 flame-type atomic absorption spectrometer.
The liver and kidney were homogenized in 5 vol of 0.25 r~f sucrose solution. Sediments were removed by centrifugation at lOS.000~ for 60 min at 4”C, and the supernatants were obtained. The plasma and urine were used without pretreatment. MT in each specimen was identified on a Sephadex G-75 column eluted with 50 rn!+t Tris-HCI buffer (pH 8.0).
RESULTS
Table 1 shows the Cd concentration in several organs and plasma, the amount of Cd in urine and feces excreted within 24 hr of the rats that were given 0.5 mg/kg of Cd for different periods, and also the effects of the Ccl, treatment (Experiment 1). The Cd concentration in each organ increased according to the duration of its administration, while that in the plasma did not increase as much even in the rats that were given Cd for a 8-week period. Ccl, given at a dose of 0.25 ml/100 g body wt to the Cdpretreated rats induced a significant decrease in the Cd concentration of liver in all the groups at 24 hr after administration, showing a decrease to approximately half of the control, especially in the rats that received Cd treatment for 1- and 4-week periods. In contrast, Ccl, increased the Cd concentration in the kidney. The increase was conspicuous in those that were given Cd for shorter durations, but it was almost unrecognizable in those receiving Cd for an 8week period. An increase in the concentration of Cd following Ccl, treatment in the
FIG. I. Dose-response of Cd in liver, plasma, and urine to Ccl,. Rats pretreated with Cd for an S-week period (0.5 mgikg body wt. 6 days/week) were administered olive oil or l/l?. 116, or 113 ml/100 g body wt of Ccl,. Urine was collected for 24 hr prior to sacrifice. Each value shows the mean 3 SE offive rats.
small intestine, pancreas, and spleen was not so remarkable as was observed in the kidney. The Cd concentration in the plasma increased conspicuously after Ccl, treatment even in those that were given Cd for an g-week period. The excretion of Cd in urine was only 3 pglday in the rats that were given Cd for an S-week period. After Ccl, treatment, it increased drastically, up to approximately 550 @g/day. In the feces, however. the change was not drastic. Dose -Response to Ccl, CCII, 0- l/3 ml/l00 g body wt, was given to the rats that had been given Cd for an S-week period (Experiment 2). The total amount of Cd in the liver, the concentration of Cd in the plasma, and the excretion in the urine at 24 hr are shown in Fig. 1. The total amount of Cd in the liver decreased according to the increase in the dose of CC&, and correspondingly, the amount of Cd in the urine increased. The Cd concentration in the plasma increased linearly according to the dose. The total amount of Cd in the kidney increased by approximately 100 Fg in all the rats of different groups that received Ccl, at different doses. Time Course qf Urinay after Ccl, Treatment
Excretion
of Cd
An increase in urinary Cd excretion was studied following the oral administration of
538
TANAKA 4GC
0
ET AL
DISCUSSION
1:3.+5 D&Y
FIG. 2. Effect of CCI, on urinary excretion of Cd. Rats that were given Cd for 4 weeks (0.5 mg/kg body wt. 6 days/week) were administered olive oil or 0.35 ml/l00 g body wt of Ccl, and 24-hr urine was collected for 5 days. Each value shows the mean of five rats.
0.25 ml/100 g body wt of Ccl, to the rats that previously were given Cd for a 4-week period (Experiment 3). The daily amount of Cd excretion in the urine examined for 5 days following Ccl, treatment is shown in Fig. 2. A conspicuous increase occurred within 48 hr following Ccl, treatment, followed by a decrease to the control level. At 5 days, the total amount of Cd in the liver decreased to approximately one-half that of the control, and almost 70% of the loss was recovered from the urine. The Cd level in the plasma at 5 days was not different from that of the control.
Chemical forms of Cd in the rats following Ccl, administration were studied by means of gel filtration using Sephadex G-75 column. Figure 3 shows elution profiles of the supernatant of the liver and kidney homogenates of the rats that received 0.25 ml/100 g body wt of CCL, after Cd administration for an g-week period (Experiment 1). In each organ, Cd of the supernatant was eluted in the MT fraction. Figure 4 shows elution profiles of the plasma and urine from the same rat. In each specimen, the main component of Cd was eluted in the fraction corresponding to the rat hepatic MT, suggesting that Cd in the plasma and urine after Ccl., treatment exists in the form of MT.
These results show that the administration of CCI, to rats pretreated with Cd caused a decrease in hepatic Cd, an increase in plasma Cd, an increase in renal Cd, and a remarkable increase in urinary excretion of Cd. This indicates that Ccl, administration accelerates Cd release from the liver to the blood, and the released Cd is redistributed in the kidney or excreted in the urine. More interesting is that Cd seems to be transferred in the form of MT in this process. This is supported by the following facts: (11 Cd exists mostly in the MT form in the liver and kidney at 24 hr following the administration of Ccl, when release of Cd from the liver occurs: (2) the MT-form Cd increases in the plasma and urine according to the elevation of the Cd level in them; and (3) the change in Cd following Ccl, treatment is similar to the behavior of exogenous MT following its injection, i.e., (a) Cd was redistributed mostly to the kidney and urine, the same as the distribution of injected MT (Tanaka t)t al.. 19751, and (b) the urinary excretion of Cd was accelerated by an increase in plasma Cd as by an increased amount of plasma MT (Foulkes, 1978).
FIG. 3. Elution protile of hepatic and renal aupernatant from a CCI,-treated rat on a Sephadex G-75 column. A rat pretreated with Cd for a R-week period 10,s mg/kg body wt. 6 days/week) was administered 0.25 ml/100 g body wt of Ccl,. Twenty-four hours after administration, liver and kidney were re moved and homogenized with 5 vol ot‘0.75 M sucrose. Four milliliters of 105,0001: supernatant fraction of homogenate was charged on a Sephadex G-75 column (1.8 * 47.5 cm) and eluted with 50 mM Tris-HCI buffer, nH 8.0.
RELEASE
OF
HEPATIC
539
CADMIUM
injury may lead to membrane damage in which the cellular MT. together with some enzymes, leaks out into the blood. and the MT may be reabsorbed in the kidney to some degree, but most is excreted in the urine.
FIN,. 4. Elution profile treated rat on a Sephadex of plasma or urine from
of plasma and urine of a CCI,G-75 column. Two milliliters the same rat as in Fig. 3 was
charged on a Sephadex G-75 column (2.0 * 16.0 cm) and eluted with 50 m.v Tris-HCI buffer. pH X.0. The bracket shows the fraction of rat hepatic MT eluted on the same column.
The mechanism of MT release from the liver owing to the effect of Ccl, is still under investigation. However, as one possibility, we believe that MT behaves in the same way as the released enzymes in the case of hepatic injury. Changes in the MT level in the plasma after Ccl, administration were not followed, but it appears that the MT level in the plasma has a peak at 24-48 hr corresponding to the urinary Cd peak following Ccl, administration (Fig. 2). These changes with time of MT coincide with those of the released enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). in the plasma following the administration of Ccl, (Zimmerman, 1973). Since the severity of hepatic injury caused experimentally by Ccl, is usually proportional to the dose of the drug, the activity of the enzymes such as GOT. GPT, and lactate dehydrogenase (LDH) in the plasma increased in proportion to the dose of Ccl, administered. There was also a correlation between the plasma Cd level and the dose of Ccl, (Fig. 1). Moreover, in our preliminary data (Tanaka and Min, 1980). there was a significant correlation between the plasma Cd level and the activity of GOT, GPT, or LDH in the plasma. These facts are suggestive that MT is released into the blood in the same way as certain enzymes in the liver cells. Liver
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