Camp. Biochem.fhysiol. Vol. 103C,No. I, pp. 3541, 1992
0306..4492/92 SO0 + 0.00 0 1992Pergamon Press Ltd
Printed in Great Britain
ON METALLOTHIONEIN, CADMIUM, COPPER AND ZINC RELATIONSHIPS IN THE LIVER AND KIDNEY OF ADULT RATS TADEUSZ WLOSTOWSKI Institute
of Biology, Biaiystok Branch of Warsaw University, Swierkowa 20B, 15-950 Bialystok, Poland (Received 24 January 1992; accepted for publication 26 February 1992)
Abstract-l. A short-term exposure of adult Wistar rats to Cu (50pg/ml) and Cd (10.0pg/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however. 2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis. 3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32-1.4 pg/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver. 4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (b-domain) in the liver and kidney was sufficient to bind 50-100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.
the cytoplasm and then transfer them into the rnitochondrial-lysosomal fraction, suggesting that under normal physiological conditions the protein may be involved in the hepatic metabolism of Cu. A question arises as to whether in other animal species a low concentration of Cd also stimulates synthesis of MT to be involved in cellular metabolism of Cu. Therefore, the present work was designed to examine the relationship between the concentration of MT in the post-mitochondrial fraction and the subcellular levels of Cd, Cu and Zn in the liver and kidney of rats exhibiting variable but normal concentrations of the metals in both organs.
lNTRODUCTION
Metallothionein (MT) is a cytoplasmic, heat stable, low-molecular weight, metal-binding protein, containing about 30% of cysteine residues (Cousins, 1985; Kagi and Schaffer, 1988; Bremner and Beattie, 1990). It was originally isolated from equine renal cortex by Margoshes and Vallee (1957). Later studies revealed that MT is an ubiquitous protein in the mammalian body, although the liver and the kidney are the most potent sites of MT induction @elazowski and Piotrowski, 1977; Piotrowski and Szymahska, 1978; Onosaka and Cherian, 1981). It has been shown that numerous metals, including cadmium (Cd), zinc (Zn), copper (Cu), mercury (Hg), silver (Ag) and bismuth (Bi) exhibit an ability to induce MT synthesis but the most powerful inducer of hepatic and renal MT is Cd (Piotrowski and Szymariska, 1978; Yagle and Palmiter, 1985; Kershaw et al., 1990; Yamada and Koizumi, 1991). Although MT was discovered 35 years ago the function specific to the protein still remains to be determined. As far the protein has been suggested to be involved in the detoxification of toxic metals (Cd, Hg), in the metabolism of Zn and Cu, as well as in the scavenging of free radicals (Cousins, 1985; Thornalley and Vasak, 1985; Bremner, 1987; Richards, 1989). A recent study from our laboratory revealed that in the liver of adult free-living bank voles (Clethrionomys glareolus), captured in a relatively unpolluted forest, MT is induced principally by small amounts of Cd (< 1.0 pg/g wet wt) (Wlostowski, 1992). The same study also demonstrated that MT, first induced by Cd, sequesters free Cu ions in
MATERIALS AND METHODS Forty-two male Wistar strain rats (250-300 g) were purchased from PAN Breeding Laboratory at Lomna, near Warsaw, and were individually housed in stainless steel cages in a room maintained at 22-25°C on a 12-hr light/dark cycle. The rats were fed ad libitum a standard laboratory diet (Murigran), containing (by AAS analysis) 60-65 fig Zn/g, 8-12 pg Cu/g and 0.2-0.4pg Cd/g dry wt. To obtain significantly variable but resembling, at least to some degree, realistic concentrations of Cd, Cu, Zn and MT in the liver and kidneys, the rats after arrival at the laboratory were divided into six groups (7 animals each) according to the drinking water: (1) C3 (control)-receiving for 3 days distilled water; (2) Cu3-receiving for 3 days distilled water containing 50.0 pg Cu/ml as CuCl,; (3) Cd3-receiving for 3 days distilled water containing 10.0 pg Cd/ml as CdCl,; (4) C9 (control+receiving for 9 days distilled water; (5) Cu6/Cd3-receiving for 6 days water containing 50.0 pg Cu/ml and subsequently (for 3 days) water containing lO.Oyg Cd/ml; (6) Cd6/Cu3-receiving for 6 days water containing lO.Opg Cd/ml and subsequently (for 3 days) water containing 50.0 pg Cu/ml. 35
36
TADEUSZ WLOSTOWSK~ Table 1. Total and subcellular
Grouo
concentrations
Nuclear fr.
Total f + f f f f
of cadmium in the liver and kidney of rats exposed to copper and cadmium
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6jCu3
0.62 0.64 0.97 0.67 0.93 0.97
0.18’ 0.12’ O.OXb 0.098 O.OXb 0.09b
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd61Cu3
0.64 + 0.20” 0.77 *0.12a 1.19 + 0.26b 0.66 f 0.098 1.04f0.16b 1.24 f O.lOb
0.20 0.15 0.26 0.20 0.27 0.27 0.20 0.23 0.34 0.23 0.27 0.35
Postmitochondrial
Liver * 0.04” *o.o5a + 0.04b +_0.03a + 0.03b f 0.04b Kidney + 0.01” + 0.03” * 0.05’ + 0.01’ + 0.03b t 0.03’
fr.
Mitochondriallysosomal fr.
0.41 0.47 0.58 0.43 0.58 0.62
f f f f f k
0.15’ 0.1 lab 0.04b 0.03s 0.04b 0.03b
0.01 0.02 0.13 0.03 0.08 0.09
f f * * f +
0.02” 0.020 0.04b 0.03’ 0.03b 0.03b
0.48 0.47 0.68 0.43 0.67 0.75
+ * * f + k
0.10” 0.07s 0.13b 0.09a 0.14b 0.06b
0.02 + 0.02” 0.07 f 0.06” 0.17 f O.OXb 0.05 + 0.021 0.13f0.11~ 0.14 + 0.03b
Values (pg/g wet wt) represent mean + SD. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 fig/ml) for 3 or 6 days. Mean values in columns with different superscript letters are significantlydifferent at P < 0.05.
After 3 or 9 days of the experiment the animals were anaesthetized with ether and the liver and the left kidney were removed. An aliquot (0.5 g) of the fresh liver or kidney was immediately transferred to 4.5 ml of 0.25 M sucrose solution (0°C) and homogenized with Teflon pestle in glass homogenizer. The homogenate was subjected to differential centrifugation at 4°C. The crude nuclear fraction (nuclei and cellular debris) was obtained by centrifuging the homogenate at 1300g for 5 min. The resulting supernatant was then centrifuged at 20,000 g for 20 min, yielding the pelleted mitochondrial-lysosomal fraction and supematant--the post-mitochondrial fraction (cytosol + microsomes). Aliquots (100 ~1) of the supernatants were removed for MT assay which was performed by using the thiomolybdateCd-saturation method (Klein et al., 1990). The remaining supernatants and the nuclear and mitochondrial-lysosomal fractions, as well as a portion (0.4 g) of the whole fresh tissue were digested with the mixed nitric and perchloric acids (Wlostowski ef al., 1988). The concentrations of Cu and Zn were determined by atomic absorption spectrophotometry in air-acetylene flame, whereas Cd analyses were carried out by electrothermal atomic absorption spectrophotometry using AAS 3 Carl Zeiss Jena instrument with an EA 3 furnace attachment. The one-way analysis of variance and Duncan’s multiple range test were used for the determination of the statistical significance of differences between means. The multiple regression analysis was used for the evaluation of the relationship between MT concentration in the post-mitoTable 2. Total and subcellular
Grouo Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3 Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6lCu3
concentrations
Total 3.53 4.68 4.20 4.38 4.87 7.05
* k + f f +
0.95s 0.65b 1.76ab 0.80b 0.66b I .8ff
6.33 f 0.95’ 11.12+2.24b 12.45 k 3.02b 10.76 + 3.45b 8.91 + 3.@ Il.97 + 3.37b
chondrial fraction and metal levels in the particular subcellular fractions of the liver and kidney.
RESULTS
An addition of Cu (50.0 pg/ml) or Cd (10.0 pg/ml) to drinking water did not affect significantly the weights of animals (data not shown). Yet, the rats were found to drink significantly less (P < 0.01) water containing Cu (20-25 ml/rat/24 hr) than distilled water (control) or water containing Cd (25-35 ml/rat/24 hr). The addition of Cd to drinking water caused weak but statistically significant increase in the total concentration of this metal in the liver and kidney as early as after 3 days of the experiment (Table 1). This increase was accompanied by a significant increase in the Cd concentration in the nuclear, post-mitochondrial and mitochondrial-lysosomal fractions. Similar hepatic and renal levels of Cd to those in the group Cd3 were found in animals from the groups Cu6/Cd3 and Cd6/Cu3. The concentrations of Cu in the liver of animals from the group Cu3 and in the kidney of animals from the groups Cu3 and Cd3 were significantly higher than those from the control group C3 (Table 2). After 9 days of the experiment, a significant
of copper in the liver and kidney of rats exposed to copper and cadmium Nuclear fr.
1.28 I .25 1.68 1.50 1.78 2.31
f f + + k f
2.69 4.81 5.74 4.57 3.65 4.43
* f * f f +
Liver 0.46” 0.43* 0.96’b 0.71ab 0.37sb 0.70’ Kidney 1.06’ l.4Xb 0.73b I .29” 0.85” I .65k
Postmitochondrial
fr.
Mitochondriallysosomal fr.
I .52 + 2.78 + I .X7 f 2.04 + 2.45 f 3.65 f
0.66’ 0.34b 0.69’ 0.19’ 0.54be 0.77”
0.73 0.66 0.65 0.83 0.64 1.10
2.88 5.21 5.46 4.91 4.51 6.00
1.42’ 1.13b I .84b 1.62’ 1.97b I .43b
0.75 + 0.24’ I .09 f 0.5lUb 1.25 * 0.508b 1.29f 0.571b 0.75 + 0.38’ 1.63 f 0.6Xb
+ f + f * f
i 0.24’ f 0.21’ + 0.29’ +0.1X’ * 0.44* +0.76a
Values (fig/g wet wt) represent mean f S.D. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 figjml) for 3 or 6 days. Mean values in columns with different superscript letters are significantly different at P < 0.05.
Cd, Cu. Zn and metallothionein
37
Table 3. Total and subcellular concentrations of zinc in the liver and kidney of rats exposed to copper and cadmium Group
Nuclear fr.
Total
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
20.1 + 2.08 19.9 f 3.2” 21.4 k 0.4’ 19.0 *3.3a 20.2 + 2.7’ 25.2 f 3.9b
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
16.2 + 17.2 5 18.4 + 14.0 + 15.9 + 18.7 +
3.Sab 2.5b 2.5b I .5a 2.7ab l.5b
Postmitochondrial
Liver 8.1 f 1.5O 7.1 f I.72 8.8 * 0.78 7.7 + I .5* 7.2 f I .9a 9.0 _+2.0” Kidney 8.0 + I .Oa 8.1 f 3.0a 7.8 f 0.2” 6.2 _+0.4a 5.8 + I .6” 7.7 + 0.9a
fr.
Mitochondriallysosomal fr.
10.8 + 10.5 f 10.7 i 10.0 f II.1 f 12.7 +
1.4’ 1.4” 0.3” 2.4” 1.9a 1.8’
l.17~0.61p 2.29 f 1.30” 1.87 + 0.88” I .25 + 0.66a I .90 _+0.9v 3.80 k 0.80b
6.8 f 7.6 f 7.8 f 6.4 f 8.7 + 8.2 +
2.4’ 0.6” 1.7” 0.4” I .9’ 0.7”
I .43 + 1.53 f 2.74* 1.50 f I .46 + 2.82 f
0.87a 0.56’ I.OIb 0.89” 0.65ab l.16b
Values (pg/g wet wt) represent mean _+SD. of 7 rats. The rats received distilled water containing Cu (50.0 us/ml) or Cd (10.0 gniml) for 3 or 6 days. Mean values in columns with different superscript -. letters are significantly d&ent at P < 0.05.
increase in the Cu level was found only in the liver of animals from the group Cd6/Cu3. In general, an increase in the total hepatic Cu was accompanied by a significant increase of the Cu content in the postmitochondrial fraction, whereas in the kidney the increase was recorded in the post-mitochondrial and nuclear fractions. After three days of the experiment no significant differences in the hepatic and renal Zn concentrations were found to occur between the particular groups (Table 3). After 9 days of the exposure a significant increase in the total hepatic and renal Zn, as well as in the mitochondrial-lysosomal Zn of the organs in rats from the group Cd6/Cu3 was recorded. The addition of Cu or Cd to drinking water did not cause significant changes in the concentration of MT in the liver after 3 days of the exposure; however, it did bring about a significant increase in the MT level of the kidney (Table 4). After 9 days of the experiment a significant increase in the hepatic and renal MT levels was observed in the group Cd6/Cu3 as compared with the control groups C9 and the group Cu6/Cd3. To identify the relationship between the changes in the subcellular concentrations of Cd, Cu, Zn in the liver and kidney with the level of MT in the postmitochondrial fraction, the analytical data (N = 42) were subjected to the multiple regression analysis (Table 5). The analysis showed that a level of MT in the post-mitochondrial fraction of the liver depended primarily on the concentration of Cd in the range of
Table 4. Metallothionein content in the post-mitochondrial fraction of the liver and kidney of rats exposed to copper and cadmium Group Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
Liver 26.5 f 28. I f 29.6 f 23.0 + 22.7 + 51.9 f
6.3a 5.9” 5.6’ 13.g” 5.8” 5.5b
Kidney 35.8 _+6.3a 49.9 + 4.7M 49.0 + 4.gM 43.7 _+5.5’b 44.4 + 4.2ab 54.4 + 5.5Cd
Values @g/g wet wt) represent mean f S.D. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 pg/ml) for 3 or 6 days. Mean values in columns with different superscript letters are significantly different at P < 0.05.
0.32-1.4 pg/g wet wt (equations 1, 3, 5). The insignificant regression coefficients (in these equations) in the case of Cu and Zn indicated that the two metals did not affect an amount of MT in the cytoplasm. However, the positive linear correlation existed between the concentration of Cu in the post-mitochondrial fraction and the level of MT (equation 4) suggesting that a significant portion of the total post-mitochondrial Cu occupied metal-binding sites on MT, probably not influencing the number of its molecules. The idea was supported further by the fact that /?-domain of MT in the liver of experimental animals had the potential to bind 50-100% of Cu ions concentrated in the post-mitochondrial fraction (Table 6). In contrast to the liver, a level of MT in the post-mitochondrial fraction of the kidney related both to the concentration of Cd in the range of 0.34-l S9 pg/g wet wt and the concentration of Cu (equations 8-12). An amount of MT in the kidney also correlated positively with the concentration of this protein in the liver (equation 13) and with the level of Cd in the liver mitochondrial-lysosomal fraction (equation 14), suggesting that a substantial quantity of MT present in the kidney might be of hepatic origin. Equations 9 and 12 indicated that both Cd and Cu were associated with the protein in the cytoplasm. DISCUSSION
The present paper revealed that a short-term exposure of rats to low concentrations of Cu and Cd causes significant changes in the levels of Cd, Cu, Zn and MT in the liver and kidney, but the values obtained are most probably situated within the physiological range of concentrations since they approximate the control values obtained by other authors (Cousins et al., 1986; Leblondel et al., 1986; Nederbragt and van Zutphen, 1987; Blalock et al., 1988). It allows a suggestion that the relationships established in this study reflect the real relations occurring under normal conditions, especially in the case of slight and short-term changes in the concentrations of Cd, Cu and Zn in both organs. The multiple regression analysis between the concentration of MT in the post-mitochondrial fraction
TADEUSZ W~O~TOWSKI
38
Table 5. Multiple regression analysis between metallothionein (MT) level in the post-mitochondrial fraction and Cd, Zn and Co concentrations in the liver and kidney of rats under study* Eouation
No.
0.05Zn LiY&r 17.1 49.3Cd, P = 0.0000 -P = o.*b+ P = o.d9- P = 0.09
r
P-value
0.78
0.0000
I
MT=
2
MT = 3.9Cu, + 11.5
0.35
0.04
3
12.3 0.2Zn, 3.2Cu, MT= 64.1Cd, P = 0.0019 + P = 0.85+ P = 0.20 -P = 0.5
0.65
0.001 I
4
MT = 6.9Cu, + 13.2
0.40
0.02
5
4.8 0.6lZn, I .5cu l44Cd, MT= P = 0.0001 -P = 0.35 + P = 0.5 + P = 0.5
0.71
0.0004
6
MT=
3.1 l44Cd, I .5cu,, P = O.oooO-P = 0.83 + P = 0.7
0.71
0.0002
7
MT=
73Cd, O.O8Cu,, P = 0.004+P = 0.99 -P
6.9 = 0.54
0.64
0.0012
MT =
Kidney I .3cu, 9.4Cd, 0.5Zn, P=0.028+P=0.185+P=0.0024+P=0.019
0.75
0.0001
9
MT=
24.5 0.8Zn, 2.5Cu, 6.3Cd, P = 0.37 + P = 0.38 + P = 0.01 + P = 0.0002
0.66
0.0013
IO
MT=
3.201, 44Cd, 0.4Zn, P=0.01+P=0.51+P=0.002+P=0.013
0.73
0.0002
II
MT=
26.3 67Cd, l.lCU,, P = 0.0049+ P = 0.58 + P = 0.0004
0.50
0.015
12
MT=
28.5 27.6Cdr l.lCu,, P = 0.005+ P = 0.59+ P = 0.0001
0.50
0.015
I3
MT = 0.25MT, + 37.7
0.35
0.039
I4
MT = 72Cd,,, + 41
0.40
0.02
I5
MT = 24Cd,, + 33
0.28
0.09
8
15.9
16.1
*The data obtained from 42 animals were used for the analysis. t-Total metal concentration, n-nuclear fraction, p-post-mitochondrial drial-lysosomal fraction, L-liver.
fraction, ml-mitochon-
taken up by organelles composing the mitochondriallysosomal fraction. No such relationship was found in the rats under study. Although the concentration of MT in the rat liver did not change significantly as a result of the increase in hepatic Cu (equations 1, 3, 5), it is likely that essential amounts of Cu ions, being taken up by the liver, did associate with metal-binding sites on the pre-existing protein since there was a linear correlation between the Cu concentration in the post-mitochondrial fraction and the level of MT (equation 4). These data together with
and the levels of Cd, Cu and Zn in the particular subcellular fractions showed that MT synthesis in the rat liver is principally induced by small amounts of Cd; thus the data are consistent with our recent finding obtained for the free-living bank vole (Wiostowski, 1992). In the bank vole the same analysis revealed, however, that a level of MT in the post-mitochondrial fraction concurrently decreases as total hepatic Cu, as well as mitochondriallysosomal Cu increases, suggesting that MT, after being associated with Cu ions in the cytoplasm, is
Table 6. Metal-binding caoacitv of metallothionein in the liver and kidney of rats under study* a-domain cg Zn/g (%)
g-domain Pg Cnlg (%) Group Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
Liver I.53 I .62 I.71 1.33 I.31 3.00
(100) (58) (91) (65) (54) (82)
Kidney 2.07 2.88 2.83 2.53 2.56 3.14
(71) (55) (52) (52) (57) (52)
Liver
Kidney
I .05 (9)
I.41 (20) 1.98 (26) 1.94 (25) 1.73 (27) 1.76 (20) 2.16 (26)
I.11 1.17 0.91 0.90 2.06
(IO) (II) (9) (8) (16)
Percentage saturation of cc-domain by post-mitochondrial Cd Liver Kidney 22.6 24.6 28.8 27.4 37.5 17.5
19.7 13.8 20.4 14.4 22.2 20.2
*Calculated from the MT values of Table 4, assuming that b-domain preferentially binds 6 atoms of Cu (Nielson and Winge, 1984). whereas a-domain binds 4 atoms of Cd or Zn (Briggs and Armitage, 1982; Winge and Miklossy, 1982) (molecular weight of MT = 6600). Data shown are presented as Pg Me/g wet wt and as percent of the metal bound to MT in the post-mitochondrial fraction (in parentheses).
Cd, Cu, Zn and metallothionein
the fact that Cu was not able to induce MT synthesis (equation 5), suggest that an excess of Cu ions in the cytoplasm is bound by MT, first induced by small amounts of Cd. Binding of Cu by MT, induced by some other agent (e.g. Zn), has been also shown by Day et al. (1981) in the rat liver, by Yamamura and Suzuki (1984) in the liver of tortoise, and by Engel and Brouwer (1991) in digestive gland of the blue crab. Some recent studies (Porter, 1974; Mehra and Bremner, 1984; Verheesen et al., 1989; Engel and Brouwer, 1991; Wlostowski, 1992) have shown that Cu-MT complex formed in cytoplasm may be subsequently sequestered by lysosomal fraction, a finding not observed in this paper. It is likely that this discrepancy may have been due to the period of Cu exposure which in this study was probably too short to cause significant accumulation of the complex in the mitochondrial-lysosomal fraction, as it has been found in the bank vole (Wlostowski, 1992) or in the rats exposed for several weeks to large doses of dietary Cu (Verheesen et al., 1989). Alternatively, the strain used in this study might exhibit either a low rate of the Cu-MT accumulation in lysosomes or a high rate of its excretion from the organelles. Indeed, there are some differences between rat strains in the subcellular distribution of the complex. For example, in the liver of Brown Norway rats this complex accumulates mainly in lysosomes, while in Fisher rats-in the cytoplasm (Verheesen et nl., 1989). In contrast to the liver, the level of MT in the post-mitochondrial fraction of the kidney depended on the concentrations of both renal Cd and renal Cu (equations 8-12), as well as on the amount of this protein in the liver and the level of Cd in the liver mitochondrial-lysosomal fraction (equations 13 and 14). These data strongly suggest that apart from the induction of MT synthesis in the kidney by Cd and Cu, a substantial amount of the protein might be coming directly from the liver. Indeed, recent studies demonstrated that MT is released in small quantities from the liver and other tissues into the circulation (Tohyama and Shaikh, 1981; Hidalgo et al., 1988; Robertson et al., 1989) and subsequently the protein, due to its low molecular weight, is efficiently filtered and reabsorbed by the proximal tubules in the kidneys (Cherian and Shaikh, 1975; Suzuki et al., 1979). It is likely therefore that the rapid increase in MT concentration only in the kidney (Table 4) after 3 days of the exposure to Cu and Cd may have been due to this phenomenon, rather than to the de nouo synthesis of the protein under influence of Cd and Cu. Furthermore, equations 13, 14 and 15 (Table 5) describing the relations between the renal MT and the total hepatic MT (significant), the liver mitochondrial-lysosomal Cd (significant) and the liver postmitochondrial Cd (nonsignificant) respectively, suggest that a release of MT (probably in association with Cd) from the liver into the blood might follow the exocytosis of some granules composing the mitochondrial-lysosomal fraction. Although most of the filtered MT is reabsorbed by the proximal tubules, a small fraction is excreted in urine in concentration-dependent manner (Tohyama and Shaikh, 1981). Shaikh et al. (1990) showed that the quantity of MT in human urine correlated posi-
39
tively with Cd levels both in liver and kidneys. Moreover, Mitane et al. (1986) demonstrated that urinary MT had a significant correlation not only with urinary Cd but also with urinary Cu in people environmentally exposed to Cd, suggesting that both metals were excreted as a complex with the protein. These authors assumed that an elevated excretion of Cu-MTCd complex was a result of renal tubular dysfunction due to Cd exposure. However, essential amounts of MT in urine were also found in normal rats (Robertson et al., 1989) which would indicate that excretion of MT in urine is a physiological process. Thus, it cannot be excluded that MT under normal physiological conditions, alike MT under Cd exposure (Suzuki, 1981), liberated from the liver, binds free Cu ions in plasma and transports them to the kidneys where a part of the complex is reabsorbed, increasmg the concentration of Cu-MT (Szymanska and Zelazowski, 1979) and the remnant excreted in urine. It is possible that the circulating MT may participate, at least to some extent, in the control of free Cu ions or in their detoxification. High cytotoxicity of Cu to both erythrocytes (Hochstein et al., 1978) and hepatocytes (Stacey and Klaassen, 1981) might account for this function of MT in the circulation. The assumption requires, however, further studies since it is based mainly on indirect inference. The results obtained from this study indicated also that under normal conditions Zn was not able to induce MT synthesis in the rat liver and kidney, which confirms previous observations (Blalock et al., 1988; Bremner and Beattie, 1990; Wlostowski, 1992). The multiple regression analysis did not show either a significant relationship between the level of MT and the concentration of Zn in the post-mitochondrial fraction of the liver and kidney, suggesting that MT had not bound significant amounts of Zn in both organs. But taking into account the fact that c(domain of MT binds preferentially 4 atoms of Cd or Zn (Briggs and Armitage, 1982; Winge and Miklossy, 1982), whereas B-domain has the potential to bind preferentially 6 atoms of Cu (Nielson and Winge, 1984), the total concentration of Cd in the post-mitochondrial fraction of the liver and kidney would be sufficient to saturate maximally 17-37 and 13-22% of metal-binding sites in cc-domain, respectively (Table 6); thus, it cannot be excluded that Zn ions were bound to the remaining sites in a-domain, although they occurred in insufficient amounts (Table 6) to give a significant correlation between their concentration and MT level. It is also noteworthy that a-domain of MT in the liver and kidney was able to bind potentially 0.9-2.16 pg Zn/g wet wt (Table 6), which approximated the values (0.5-2.5 pg Zn in MT/g wet wt) obtained by analysis for control rats (Brady and Helvig, 1984). Thus, it is reasonable to assume that under normal physiological conditions a-domain of MT binds both nonsignificant amounts of Zn and small but statistically significant quantities of Cd (Table 5). In contrast, B-domain of MT was able to bind 50-100% of Cu ions concentrated in the post-mitochondrial fraction of the liver and kidney (Table 6). Such a great Cu-binding capacity of MT under normal conditions supports the assumption that MT may function as an intracellular buffer of
TADEUSZWLOSTOWSKI
40
Cu, ensuring the maintenance of Cu concentration in the particular compartments of the cell at level compatible with normal cellular requirements (Richards, 1989; Engel and Brouwer, 1991; Wlostowski, 1992). It is an open question, however, whether MT plays a similar role in the Zn and Cd metabolism under physiological conditions or whether the metals determine rather the metabolism of the protein only. There is some evidence in literature supporting the latter possibility. Apart from the stimulation of MT production by small amounts of Cd (Table 5, Wlostowski, 1992), this metal together with Zn may be involved in the stabilization of thionein molecules in cytoplasm. A convincing evidence of this was provided by Fleet et al. (1990) who showed in chicken that under Zn deficiency state, although an induction of MT mRNA synthesis occurred in the liver, the protein was not detected in the cytoplasm until adequate amounts of Cd or Zn were added. So it is reasonable to conclude that when synthesis of MT in the liver is overexpressed e.g. upon an injection of Fe, endotoxin or interleukin-1 (DiSilvestro and Cousins, 1984; McCormick, 1987; Fleet et al., 1990) the amount of Cd ions and even Zn ions in the cytoplasm is not sufficient to produce a stable complex with newly synthesized thionein molecules and that is why the protein either forms a complex with Zn at the expense of plasma Zn, which results in a simultaneous decrease in plasma Zn and an increase in hepatic Zn (DiSilvestro and Cousins, 1984; Bremner and Beattie, 1990), or in the case of plasma Zn deficiency it undergoes immediate degradation (Fleet et al., 1990). Obviously, further studies are needed to establish the exact role of Cd and Zn in the metabolism of MT, especially under physiological conditions. Collectively, this investigation confirms our recent finding (Wiostowski, 1992), indicating that under normal conditions a level of MT in the liver of adult rodents is related mainly to small amounts of Cd. In comparison, renal MT depends not only on the low level of Cd but also on the hepatic MT which is probably secreted into the circulation and then filtered and reabsorbed in the kidneys. Additionally, the fact that MT, first induced by Cd, binds significant amounts of Cu gives further support to the assumption that the function of MT in normal organism is in the metabolism (regulation, detoxification) of Cu.
Cousins R. J. (1985) Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65, 238-309.
Cousins R. J., Dunn M. A., Leinart A., Yedinak K. C. and DiSilvestro R. A. (1986) Coordinate regulation of zinc metabolism and metallothionein gene expression in rats. Am. J. Physiol. 251, E688-E694. Day F. A., Panemangalore M. and Brady F. 0. (1981) In uiuo and ex uivo effects of copper on rat liver metallothionein. Proc. Sot. exp. Biol. Med. 168, 306-310. DiSilvestro R. A. and Cousins R. J. (1984) Mediation of endotoxin-induced changes in zinc metabolism in rats. Am. J. Physiol. 247, E43&E441.
Engel D. W. and Brouwer M. (1991) Short-term metallothionein and copper changes in blue crabs at ecdysis. Biol. Bull. 180, 447452.
Fleet J. C., Andrews G. K. and McCormick C. C. (1990) Iron-induced metallothionein in chick liver: a rapid, route-dependent effect independent of zinc status. J. Nutr. 120, 121-4-1222. Hidaleo J.. Giralt M.. Garvev J. S. and Armario A. (1988) Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions. Am. J. Physiol. 254, E71-E78. Hochstein P., Kumar K. S. and Forman S. J. (1978) Mechanisms of copper toxicity in red cells. Prog. C/in. Biol. Res. 21, 669686.
Kagi J. H. R. and SchatTer A. (1988) Biochemistry of metallothionein. Biochemistry 27, 8509-8515. Kershaw W. C., Lehman-McKeeman L. D. and Klaassen C. D. (1990) Hepatic isometallothioneins in mice: induction in adults and postnatal ontogeny. Toxicol. appl. Pharmacol. 104, 267-275. Klein D., Bartsch R. and Summer K. H. (1990) Quantitation of Cu-containing metallothionein by a Cd-saturation method. Anal. Biochem. 189, 35-39. Leblondel G., Mauras Y. and Allain P. (1986) Tissue distribution of some elements in rats. Biol. Trace Elem. Res. 10, 327-333. Margoshes M. and Vallee B. L. (1957) A cadmium protein from equine kidney cortex. J. Am. them. Sot. 79, 48134814. Mehra R. K. and Bremner I. (1984) Species differences in the occurrence of copper-metallothionein in the particulate fractions of the liver of copper-loaded animals. Biochem. J. 219, 539-546.
McCormick C. C. (1987) Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement. Proc. Sot. exp. Biol. Med. 185, 413419. Mitane Y., Tohyama C. and Saito H. (1986) The role of metallothionein in the elevated excretion of copper in urine from people living in a cadmium-polluted area. Fund. appl. k’oxkol. 6, 285-29
REFERENCES Blalock T. L., Dunn M. A. and Cousins R. J. (1988) Metallothionein gene expression in rats: tissue-specific regulation by dietary copper and zinc. J. Nutr. 118, 222-228. Brady F. 0. and Helvig B. (1984) Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver. Am. J. Phvsiol. 247, E318-E322. Bremner I. (1987) Involvement of metallothionein in the henatic metabolism of copper. J. Nutr. 117, 19-29. Bremner I. and Beattie J. H.
0306..4492/92 SO0 + 0.00 0 1992Pergamon Press Ltd
Printed in Great Britain
ON METALLOTHIONEIN, CADMIUM, COPPER AND ZINC RELATIONSHIPS IN THE LIVER AND KIDNEY OF ADULT RATS TADEUSZ WLOSTOWSKI Institute
of Biology, Biaiystok Branch of Warsaw University, Swierkowa 20B, 15-950 Bialystok, Poland (Received 24 January 1992; accepted for publication 26 February 1992)
Abstract-l. A short-term exposure of adult Wistar rats to Cu (50pg/ml) and Cd (10.0pg/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however. 2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis. 3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32-1.4 pg/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver. 4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (b-domain) in the liver and kidney was sufficient to bind 50-100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.
the cytoplasm and then transfer them into the rnitochondrial-lysosomal fraction, suggesting that under normal physiological conditions the protein may be involved in the hepatic metabolism of Cu. A question arises as to whether in other animal species a low concentration of Cd also stimulates synthesis of MT to be involved in cellular metabolism of Cu. Therefore, the present work was designed to examine the relationship between the concentration of MT in the post-mitochondrial fraction and the subcellular levels of Cd, Cu and Zn in the liver and kidney of rats exhibiting variable but normal concentrations of the metals in both organs.
lNTRODUCTION
Metallothionein (MT) is a cytoplasmic, heat stable, low-molecular weight, metal-binding protein, containing about 30% of cysteine residues (Cousins, 1985; Kagi and Schaffer, 1988; Bremner and Beattie, 1990). It was originally isolated from equine renal cortex by Margoshes and Vallee (1957). Later studies revealed that MT is an ubiquitous protein in the mammalian body, although the liver and the kidney are the most potent sites of MT induction @elazowski and Piotrowski, 1977; Piotrowski and Szymahska, 1978; Onosaka and Cherian, 1981). It has been shown that numerous metals, including cadmium (Cd), zinc (Zn), copper (Cu), mercury (Hg), silver (Ag) and bismuth (Bi) exhibit an ability to induce MT synthesis but the most powerful inducer of hepatic and renal MT is Cd (Piotrowski and Szymariska, 1978; Yagle and Palmiter, 1985; Kershaw et al., 1990; Yamada and Koizumi, 1991). Although MT was discovered 35 years ago the function specific to the protein still remains to be determined. As far the protein has been suggested to be involved in the detoxification of toxic metals (Cd, Hg), in the metabolism of Zn and Cu, as well as in the scavenging of free radicals (Cousins, 1985; Thornalley and Vasak, 1985; Bremner, 1987; Richards, 1989). A recent study from our laboratory revealed that in the liver of adult free-living bank voles (Clethrionomys glareolus), captured in a relatively unpolluted forest, MT is induced principally by small amounts of Cd (< 1.0 pg/g wet wt) (Wlostowski, 1992). The same study also demonstrated that MT, first induced by Cd, sequesters free Cu ions in
MATERIALS AND METHODS Forty-two male Wistar strain rats (250-300 g) were purchased from PAN Breeding Laboratory at Lomna, near Warsaw, and were individually housed in stainless steel cages in a room maintained at 22-25°C on a 12-hr light/dark cycle. The rats were fed ad libitum a standard laboratory diet (Murigran), containing (by AAS analysis) 60-65 fig Zn/g, 8-12 pg Cu/g and 0.2-0.4pg Cd/g dry wt. To obtain significantly variable but resembling, at least to some degree, realistic concentrations of Cd, Cu, Zn and MT in the liver and kidneys, the rats after arrival at the laboratory were divided into six groups (7 animals each) according to the drinking water: (1) C3 (control)-receiving for 3 days distilled water; (2) Cu3-receiving for 3 days distilled water containing 50.0 pg Cu/ml as CuCl,; (3) Cd3-receiving for 3 days distilled water containing 10.0 pg Cd/ml as CdCl,; (4) C9 (control+receiving for 9 days distilled water; (5) Cu6/Cd3-receiving for 6 days water containing 50.0 pg Cu/ml and subsequently (for 3 days) water containing lO.Oyg Cd/ml; (6) Cd6/Cu3-receiving for 6 days water containing lO.Opg Cd/ml and subsequently (for 3 days) water containing 50.0 pg Cu/ml. 35
36
TADEUSZ WLOSTOWSK~ Table 1. Total and subcellular
Grouo
concentrations
Nuclear fr.
Total f + f f f f
of cadmium in the liver and kidney of rats exposed to copper and cadmium
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6jCu3
0.62 0.64 0.97 0.67 0.93 0.97
0.18’ 0.12’ O.OXb 0.098 O.OXb 0.09b
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd61Cu3
0.64 + 0.20” 0.77 *0.12a 1.19 + 0.26b 0.66 f 0.098 1.04f0.16b 1.24 f O.lOb
0.20 0.15 0.26 0.20 0.27 0.27 0.20 0.23 0.34 0.23 0.27 0.35
Postmitochondrial
Liver * 0.04” *o.o5a + 0.04b +_0.03a + 0.03b f 0.04b Kidney + 0.01” + 0.03” * 0.05’ + 0.01’ + 0.03b t 0.03’
fr.
Mitochondriallysosomal fr.
0.41 0.47 0.58 0.43 0.58 0.62
f f f f f k
0.15’ 0.1 lab 0.04b 0.03s 0.04b 0.03b
0.01 0.02 0.13 0.03 0.08 0.09
f f * * f +
0.02” 0.020 0.04b 0.03’ 0.03b 0.03b
0.48 0.47 0.68 0.43 0.67 0.75
+ * * f + k
0.10” 0.07s 0.13b 0.09a 0.14b 0.06b
0.02 + 0.02” 0.07 f 0.06” 0.17 f O.OXb 0.05 + 0.021 0.13f0.11~ 0.14 + 0.03b
Values (pg/g wet wt) represent mean + SD. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 fig/ml) for 3 or 6 days. Mean values in columns with different superscript letters are significantlydifferent at P < 0.05.
After 3 or 9 days of the experiment the animals were anaesthetized with ether and the liver and the left kidney were removed. An aliquot (0.5 g) of the fresh liver or kidney was immediately transferred to 4.5 ml of 0.25 M sucrose solution (0°C) and homogenized with Teflon pestle in glass homogenizer. The homogenate was subjected to differential centrifugation at 4°C. The crude nuclear fraction (nuclei and cellular debris) was obtained by centrifuging the homogenate at 1300g for 5 min. The resulting supernatant was then centrifuged at 20,000 g for 20 min, yielding the pelleted mitochondrial-lysosomal fraction and supematant--the post-mitochondrial fraction (cytosol + microsomes). Aliquots (100 ~1) of the supernatants were removed for MT assay which was performed by using the thiomolybdateCd-saturation method (Klein et al., 1990). The remaining supernatants and the nuclear and mitochondrial-lysosomal fractions, as well as a portion (0.4 g) of the whole fresh tissue were digested with the mixed nitric and perchloric acids (Wlostowski ef al., 1988). The concentrations of Cu and Zn were determined by atomic absorption spectrophotometry in air-acetylene flame, whereas Cd analyses were carried out by electrothermal atomic absorption spectrophotometry using AAS 3 Carl Zeiss Jena instrument with an EA 3 furnace attachment. The one-way analysis of variance and Duncan’s multiple range test were used for the determination of the statistical significance of differences between means. The multiple regression analysis was used for the evaluation of the relationship between MT concentration in the post-mitoTable 2. Total and subcellular
Grouo Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3 Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6lCu3
concentrations
Total 3.53 4.68 4.20 4.38 4.87 7.05
* k + f f +
0.95s 0.65b 1.76ab 0.80b 0.66b I .8ff
6.33 f 0.95’ 11.12+2.24b 12.45 k 3.02b 10.76 + 3.45b 8.91 + 3.@ Il.97 + 3.37b
chondrial fraction and metal levels in the particular subcellular fractions of the liver and kidney.
RESULTS
An addition of Cu (50.0 pg/ml) or Cd (10.0 pg/ml) to drinking water did not affect significantly the weights of animals (data not shown). Yet, the rats were found to drink significantly less (P < 0.01) water containing Cu (20-25 ml/rat/24 hr) than distilled water (control) or water containing Cd (25-35 ml/rat/24 hr). The addition of Cd to drinking water caused weak but statistically significant increase in the total concentration of this metal in the liver and kidney as early as after 3 days of the experiment (Table 1). This increase was accompanied by a significant increase in the Cd concentration in the nuclear, post-mitochondrial and mitochondrial-lysosomal fractions. Similar hepatic and renal levels of Cd to those in the group Cd3 were found in animals from the groups Cu6/Cd3 and Cd6/Cu3. The concentrations of Cu in the liver of animals from the group Cu3 and in the kidney of animals from the groups Cu3 and Cd3 were significantly higher than those from the control group C3 (Table 2). After 9 days of the experiment, a significant
of copper in the liver and kidney of rats exposed to copper and cadmium Nuclear fr.
1.28 I .25 1.68 1.50 1.78 2.31
f f + + k f
2.69 4.81 5.74 4.57 3.65 4.43
* f * f f +
Liver 0.46” 0.43* 0.96’b 0.71ab 0.37sb 0.70’ Kidney 1.06’ l.4Xb 0.73b I .29” 0.85” I .65k
Postmitochondrial
fr.
Mitochondriallysosomal fr.
I .52 + 2.78 + I .X7 f 2.04 + 2.45 f 3.65 f
0.66’ 0.34b 0.69’ 0.19’ 0.54be 0.77”
0.73 0.66 0.65 0.83 0.64 1.10
2.88 5.21 5.46 4.91 4.51 6.00
1.42’ 1.13b I .84b 1.62’ 1.97b I .43b
0.75 + 0.24’ I .09 f 0.5lUb 1.25 * 0.508b 1.29f 0.571b 0.75 + 0.38’ 1.63 f 0.6Xb
+ f + f * f
i 0.24’ f 0.21’ + 0.29’ +0.1X’ * 0.44* +0.76a
Values (fig/g wet wt) represent mean f S.D. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 figjml) for 3 or 6 days. Mean values in columns with different superscript letters are significantly different at P < 0.05.
Cd, Cu. Zn and metallothionein
37
Table 3. Total and subcellular concentrations of zinc in the liver and kidney of rats exposed to copper and cadmium Group
Nuclear fr.
Total
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
20.1 + 2.08 19.9 f 3.2” 21.4 k 0.4’ 19.0 *3.3a 20.2 + 2.7’ 25.2 f 3.9b
Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
16.2 + 17.2 5 18.4 + 14.0 + 15.9 + 18.7 +
3.Sab 2.5b 2.5b I .5a 2.7ab l.5b
Postmitochondrial
Liver 8.1 f 1.5O 7.1 f I.72 8.8 * 0.78 7.7 + I .5* 7.2 f I .9a 9.0 _+2.0” Kidney 8.0 + I .Oa 8.1 f 3.0a 7.8 f 0.2” 6.2 _+0.4a 5.8 + I .6” 7.7 + 0.9a
fr.
Mitochondriallysosomal fr.
10.8 + 10.5 f 10.7 i 10.0 f II.1 f 12.7 +
1.4’ 1.4” 0.3” 2.4” 1.9a 1.8’
l.17~0.61p 2.29 f 1.30” 1.87 + 0.88” I .25 + 0.66a I .90 _+0.9v 3.80 k 0.80b
6.8 f 7.6 f 7.8 f 6.4 f 8.7 + 8.2 +
2.4’ 0.6” 1.7” 0.4” I .9’ 0.7”
I .43 + 1.53 f 2.74* 1.50 f I .46 + 2.82 f
0.87a 0.56’ I.OIb 0.89” 0.65ab l.16b
Values (pg/g wet wt) represent mean _+SD. of 7 rats. The rats received distilled water containing Cu (50.0 us/ml) or Cd (10.0 gniml) for 3 or 6 days. Mean values in columns with different superscript -. letters are significantly d&ent at P < 0.05.
increase in the Cu level was found only in the liver of animals from the group Cd6/Cu3. In general, an increase in the total hepatic Cu was accompanied by a significant increase of the Cu content in the postmitochondrial fraction, whereas in the kidney the increase was recorded in the post-mitochondrial and nuclear fractions. After three days of the experiment no significant differences in the hepatic and renal Zn concentrations were found to occur between the particular groups (Table 3). After 9 days of the exposure a significant increase in the total hepatic and renal Zn, as well as in the mitochondrial-lysosomal Zn of the organs in rats from the group Cd6/Cu3 was recorded. The addition of Cu or Cd to drinking water did not cause significant changes in the concentration of MT in the liver after 3 days of the exposure; however, it did bring about a significant increase in the MT level of the kidney (Table 4). After 9 days of the experiment a significant increase in the hepatic and renal MT levels was observed in the group Cd6/Cu3 as compared with the control groups C9 and the group Cu6/Cd3. To identify the relationship between the changes in the subcellular concentrations of Cd, Cu, Zn in the liver and kidney with the level of MT in the postmitochondrial fraction, the analytical data (N = 42) were subjected to the multiple regression analysis (Table 5). The analysis showed that a level of MT in the post-mitochondrial fraction of the liver depended primarily on the concentration of Cd in the range of
Table 4. Metallothionein content in the post-mitochondrial fraction of the liver and kidney of rats exposed to copper and cadmium Group Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
Liver 26.5 f 28. I f 29.6 f 23.0 + 22.7 + 51.9 f
6.3a 5.9” 5.6’ 13.g” 5.8” 5.5b
Kidney 35.8 _+6.3a 49.9 + 4.7M 49.0 + 4.gM 43.7 _+5.5’b 44.4 + 4.2ab 54.4 + 5.5Cd
Values @g/g wet wt) represent mean f S.D. of 7 rats. The rats received distilled water containing Cu (50.0 pg/ml) or Cd (10.0 pg/ml) for 3 or 6 days. Mean values in columns with different superscript letters are significantly different at P < 0.05.
0.32-1.4 pg/g wet wt (equations 1, 3, 5). The insignificant regression coefficients (in these equations) in the case of Cu and Zn indicated that the two metals did not affect an amount of MT in the cytoplasm. However, the positive linear correlation existed between the concentration of Cu in the post-mitochondrial fraction and the level of MT (equation 4) suggesting that a significant portion of the total post-mitochondrial Cu occupied metal-binding sites on MT, probably not influencing the number of its molecules. The idea was supported further by the fact that /?-domain of MT in the liver of experimental animals had the potential to bind 50-100% of Cu ions concentrated in the post-mitochondrial fraction (Table 6). In contrast to the liver, a level of MT in the post-mitochondrial fraction of the kidney related both to the concentration of Cd in the range of 0.34-l S9 pg/g wet wt and the concentration of Cu (equations 8-12). An amount of MT in the kidney also correlated positively with the concentration of this protein in the liver (equation 13) and with the level of Cd in the liver mitochondrial-lysosomal fraction (equation 14), suggesting that a substantial quantity of MT present in the kidney might be of hepatic origin. Equations 9 and 12 indicated that both Cd and Cu were associated with the protein in the cytoplasm. DISCUSSION
The present paper revealed that a short-term exposure of rats to low concentrations of Cu and Cd causes significant changes in the levels of Cd, Cu, Zn and MT in the liver and kidney, but the values obtained are most probably situated within the physiological range of concentrations since they approximate the control values obtained by other authors (Cousins et al., 1986; Leblondel et al., 1986; Nederbragt and van Zutphen, 1987; Blalock et al., 1988). It allows a suggestion that the relationships established in this study reflect the real relations occurring under normal conditions, especially in the case of slight and short-term changes in the concentrations of Cd, Cu and Zn in both organs. The multiple regression analysis between the concentration of MT in the post-mitochondrial fraction
TADEUSZ W~O~TOWSKI
38
Table 5. Multiple regression analysis between metallothionein (MT) level in the post-mitochondrial fraction and Cd, Zn and Co concentrations in the liver and kidney of rats under study* Eouation
No.
0.05Zn LiY&r 17.1 49.3Cd, P = 0.0000 -P = o.*b+ P = o.d9- P = 0.09
r
P-value
0.78
0.0000
I
MT=
2
MT = 3.9Cu, + 11.5
0.35
0.04
3
12.3 0.2Zn, 3.2Cu, MT= 64.1Cd, P = 0.0019 + P = 0.85+ P = 0.20 -P = 0.5
0.65
0.001 I
4
MT = 6.9Cu, + 13.2
0.40
0.02
5
4.8 0.6lZn, I .5cu l44Cd, MT= P = 0.0001 -P = 0.35 + P = 0.5 + P = 0.5
0.71
0.0004
6
MT=
3.1 l44Cd, I .5cu,, P = O.oooO-P = 0.83 + P = 0.7
0.71
0.0002
7
MT=
73Cd, O.O8Cu,, P = 0.004+P = 0.99 -P
6.9 = 0.54
0.64
0.0012
MT =
Kidney I .3cu, 9.4Cd, 0.5Zn, P=0.028+P=0.185+P=0.0024+P=0.019
0.75
0.0001
9
MT=
24.5 0.8Zn, 2.5Cu, 6.3Cd, P = 0.37 + P = 0.38 + P = 0.01 + P = 0.0002
0.66
0.0013
IO
MT=
3.201, 44Cd, 0.4Zn, P=0.01+P=0.51+P=0.002+P=0.013
0.73
0.0002
II
MT=
26.3 67Cd, l.lCU,, P = 0.0049+ P = 0.58 + P = 0.0004
0.50
0.015
12
MT=
28.5 27.6Cdr l.lCu,, P = 0.005+ P = 0.59+ P = 0.0001
0.50
0.015
I3
MT = 0.25MT, + 37.7
0.35
0.039
I4
MT = 72Cd,,, + 41
0.40
0.02
I5
MT = 24Cd,, + 33
0.28
0.09
8
15.9
16.1
*The data obtained from 42 animals were used for the analysis. t-Total metal concentration, n-nuclear fraction, p-post-mitochondrial drial-lysosomal fraction, L-liver.
fraction, ml-mitochon-
taken up by organelles composing the mitochondriallysosomal fraction. No such relationship was found in the rats under study. Although the concentration of MT in the rat liver did not change significantly as a result of the increase in hepatic Cu (equations 1, 3, 5), it is likely that essential amounts of Cu ions, being taken up by the liver, did associate with metal-binding sites on the pre-existing protein since there was a linear correlation between the Cu concentration in the post-mitochondrial fraction and the level of MT (equation 4). These data together with
and the levels of Cd, Cu and Zn in the particular subcellular fractions showed that MT synthesis in the rat liver is principally induced by small amounts of Cd; thus the data are consistent with our recent finding obtained for the free-living bank vole (Wiostowski, 1992). In the bank vole the same analysis revealed, however, that a level of MT in the post-mitochondrial fraction concurrently decreases as total hepatic Cu, as well as mitochondriallysosomal Cu increases, suggesting that MT, after being associated with Cu ions in the cytoplasm, is
Table 6. Metal-binding caoacitv of metallothionein in the liver and kidney of rats under study* a-domain cg Zn/g (%)
g-domain Pg Cnlg (%) Group Control-C3 cu3 Cd3 Control-C9 Cu6/Cd3 Cd6/Cu3
Liver I.53 I .62 I.71 1.33 I.31 3.00
(100) (58) (91) (65) (54) (82)
Kidney 2.07 2.88 2.83 2.53 2.56 3.14
(71) (55) (52) (52) (57) (52)
Liver
Kidney
I .05 (9)
I.41 (20) 1.98 (26) 1.94 (25) 1.73 (27) 1.76 (20) 2.16 (26)
I.11 1.17 0.91 0.90 2.06
(IO) (II) (9) (8) (16)
Percentage saturation of cc-domain by post-mitochondrial Cd Liver Kidney 22.6 24.6 28.8 27.4 37.5 17.5
19.7 13.8 20.4 14.4 22.2 20.2
*Calculated from the MT values of Table 4, assuming that b-domain preferentially binds 6 atoms of Cu (Nielson and Winge, 1984). whereas a-domain binds 4 atoms of Cd or Zn (Briggs and Armitage, 1982; Winge and Miklossy, 1982) (molecular weight of MT = 6600). Data shown are presented as Pg Me/g wet wt and as percent of the metal bound to MT in the post-mitochondrial fraction (in parentheses).
Cd, Cu, Zn and metallothionein
the fact that Cu was not able to induce MT synthesis (equation 5), suggest that an excess of Cu ions in the cytoplasm is bound by MT, first induced by small amounts of Cd. Binding of Cu by MT, induced by some other agent (e.g. Zn), has been also shown by Day et al. (1981) in the rat liver, by Yamamura and Suzuki (1984) in the liver of tortoise, and by Engel and Brouwer (1991) in digestive gland of the blue crab. Some recent studies (Porter, 1974; Mehra and Bremner, 1984; Verheesen et al., 1989; Engel and Brouwer, 1991; Wlostowski, 1992) have shown that Cu-MT complex formed in cytoplasm may be subsequently sequestered by lysosomal fraction, a finding not observed in this paper. It is likely that this discrepancy may have been due to the period of Cu exposure which in this study was probably too short to cause significant accumulation of the complex in the mitochondrial-lysosomal fraction, as it has been found in the bank vole (Wlostowski, 1992) or in the rats exposed for several weeks to large doses of dietary Cu (Verheesen et al., 1989). Alternatively, the strain used in this study might exhibit either a low rate of the Cu-MT accumulation in lysosomes or a high rate of its excretion from the organelles. Indeed, there are some differences between rat strains in the subcellular distribution of the complex. For example, in the liver of Brown Norway rats this complex accumulates mainly in lysosomes, while in Fisher rats-in the cytoplasm (Verheesen et nl., 1989). In contrast to the liver, the level of MT in the post-mitochondrial fraction of the kidney depended on the concentrations of both renal Cd and renal Cu (equations 8-12), as well as on the amount of this protein in the liver and the level of Cd in the liver mitochondrial-lysosomal fraction (equations 13 and 14). These data strongly suggest that apart from the induction of MT synthesis in the kidney by Cd and Cu, a substantial amount of the protein might be coming directly from the liver. Indeed, recent studies demonstrated that MT is released in small quantities from the liver and other tissues into the circulation (Tohyama and Shaikh, 1981; Hidalgo et al., 1988; Robertson et al., 1989) and subsequently the protein, due to its low molecular weight, is efficiently filtered and reabsorbed by the proximal tubules in the kidneys (Cherian and Shaikh, 1975; Suzuki et al., 1979). It is likely therefore that the rapid increase in MT concentration only in the kidney (Table 4) after 3 days of the exposure to Cu and Cd may have been due to this phenomenon, rather than to the de nouo synthesis of the protein under influence of Cd and Cu. Furthermore, equations 13, 14 and 15 (Table 5) describing the relations between the renal MT and the total hepatic MT (significant), the liver mitochondrial-lysosomal Cd (significant) and the liver postmitochondrial Cd (nonsignificant) respectively, suggest that a release of MT (probably in association with Cd) from the liver into the blood might follow the exocytosis of some granules composing the mitochondrial-lysosomal fraction. Although most of the filtered MT is reabsorbed by the proximal tubules, a small fraction is excreted in urine in concentration-dependent manner (Tohyama and Shaikh, 1981). Shaikh et al. (1990) showed that the quantity of MT in human urine correlated posi-
39
tively with Cd levels both in liver and kidneys. Moreover, Mitane et al. (1986) demonstrated that urinary MT had a significant correlation not only with urinary Cd but also with urinary Cu in people environmentally exposed to Cd, suggesting that both metals were excreted as a complex with the protein. These authors assumed that an elevated excretion of Cu-MTCd complex was a result of renal tubular dysfunction due to Cd exposure. However, essential amounts of MT in urine were also found in normal rats (Robertson et al., 1989) which would indicate that excretion of MT in urine is a physiological process. Thus, it cannot be excluded that MT under normal physiological conditions, alike MT under Cd exposure (Suzuki, 1981), liberated from the liver, binds free Cu ions in plasma and transports them to the kidneys where a part of the complex is reabsorbed, increasmg the concentration of Cu-MT (Szymanska and Zelazowski, 1979) and the remnant excreted in urine. It is possible that the circulating MT may participate, at least to some extent, in the control of free Cu ions or in their detoxification. High cytotoxicity of Cu to both erythrocytes (Hochstein et al., 1978) and hepatocytes (Stacey and Klaassen, 1981) might account for this function of MT in the circulation. The assumption requires, however, further studies since it is based mainly on indirect inference. The results obtained from this study indicated also that under normal conditions Zn was not able to induce MT synthesis in the rat liver and kidney, which confirms previous observations (Blalock et al., 1988; Bremner and Beattie, 1990; Wlostowski, 1992). The multiple regression analysis did not show either a significant relationship between the level of MT and the concentration of Zn in the post-mitochondrial fraction of the liver and kidney, suggesting that MT had not bound significant amounts of Zn in both organs. But taking into account the fact that c(domain of MT binds preferentially 4 atoms of Cd or Zn (Briggs and Armitage, 1982; Winge and Miklossy, 1982), whereas B-domain has the potential to bind preferentially 6 atoms of Cu (Nielson and Winge, 1984), the total concentration of Cd in the post-mitochondrial fraction of the liver and kidney would be sufficient to saturate maximally 17-37 and 13-22% of metal-binding sites in cc-domain, respectively (Table 6); thus, it cannot be excluded that Zn ions were bound to the remaining sites in a-domain, although they occurred in insufficient amounts (Table 6) to give a significant correlation between their concentration and MT level. It is also noteworthy that a-domain of MT in the liver and kidney was able to bind potentially 0.9-2.16 pg Zn/g wet wt (Table 6), which approximated the values (0.5-2.5 pg Zn in MT/g wet wt) obtained by analysis for control rats (Brady and Helvig, 1984). Thus, it is reasonable to assume that under normal physiological conditions a-domain of MT binds both nonsignificant amounts of Zn and small but statistically significant quantities of Cd (Table 5). In contrast, B-domain of MT was able to bind 50-100% of Cu ions concentrated in the post-mitochondrial fraction of the liver and kidney (Table 6). Such a great Cu-binding capacity of MT under normal conditions supports the assumption that MT may function as an intracellular buffer of
TADEUSZWLOSTOWSKI
40
Cu, ensuring the maintenance of Cu concentration in the particular compartments of the cell at level compatible with normal cellular requirements (Richards, 1989; Engel and Brouwer, 1991; Wlostowski, 1992). It is an open question, however, whether MT plays a similar role in the Zn and Cd metabolism under physiological conditions or whether the metals determine rather the metabolism of the protein only. There is some evidence in literature supporting the latter possibility. Apart from the stimulation of MT production by small amounts of Cd (Table 5, Wlostowski, 1992), this metal together with Zn may be involved in the stabilization of thionein molecules in cytoplasm. A convincing evidence of this was provided by Fleet et al. (1990) who showed in chicken that under Zn deficiency state, although an induction of MT mRNA synthesis occurred in the liver, the protein was not detected in the cytoplasm until adequate amounts of Cd or Zn were added. So it is reasonable to conclude that when synthesis of MT in the liver is overexpressed e.g. upon an injection of Fe, endotoxin or interleukin-1 (DiSilvestro and Cousins, 1984; McCormick, 1987; Fleet et al., 1990) the amount of Cd ions and even Zn ions in the cytoplasm is not sufficient to produce a stable complex with newly synthesized thionein molecules and that is why the protein either forms a complex with Zn at the expense of plasma Zn, which results in a simultaneous decrease in plasma Zn and an increase in hepatic Zn (DiSilvestro and Cousins, 1984; Bremner and Beattie, 1990), or in the case of plasma Zn deficiency it undergoes immediate degradation (Fleet et al., 1990). Obviously, further studies are needed to establish the exact role of Cd and Zn in the metabolism of MT, especially under physiological conditions. Collectively, this investigation confirms our recent finding (Wiostowski, 1992), indicating that under normal conditions a level of MT in the liver of adult rodents is related mainly to small amounts of Cd. In comparison, renal MT depends not only on the low level of Cd but also on the hepatic MT which is probably secreted into the circulation and then filtered and reabsorbed in the kidneys. Additionally, the fact that MT, first induced by Cd, binds significant amounts of Cu gives further support to the assumption that the function of MT in normal organism is in the metabolism (regulation, detoxification) of Cu.
Cousins R. J. (1985) Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65, 238-309.
Cousins R. J., Dunn M. A., Leinart A., Yedinak K. C. and DiSilvestro R. A. (1986) Coordinate regulation of zinc metabolism and metallothionein gene expression in rats. Am. J. Physiol. 251, E688-E694. Day F. A., Panemangalore M. and Brady F. 0. (1981) In uiuo and ex uivo effects of copper on rat liver metallothionein. Proc. Sot. exp. Biol. Med. 168, 306-310. DiSilvestro R. A. and Cousins R. J. (1984) Mediation of endotoxin-induced changes in zinc metabolism in rats. Am. J. Physiol. 247, E43&E441.
Engel D. W. and Brouwer M. (1991) Short-term metallothionein and copper changes in blue crabs at ecdysis. Biol. Bull. 180, 447452.
Fleet J. C., Andrews G. K. and McCormick C. C. (1990) Iron-induced metallothionein in chick liver: a rapid, route-dependent effect independent of zinc status. J. Nutr. 120, 121-4-1222. Hidaleo J.. Giralt M.. Garvev J. S. and Armario A. (1988) Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions. Am. J. Physiol. 254, E71-E78. Hochstein P., Kumar K. S. and Forman S. J. (1978) Mechanisms of copper toxicity in red cells. Prog. C/in. Biol. Res. 21, 669686.
Kagi J. H. R. and SchatTer A. (1988) Biochemistry of metallothionein. Biochemistry 27, 8509-8515. Kershaw W. C., Lehman-McKeeman L. D. and Klaassen C. D. (1990) Hepatic isometallothioneins in mice: induction in adults and postnatal ontogeny. Toxicol. appl. Pharmacol. 104, 267-275. Klein D., Bartsch R. and Summer K. H. (1990) Quantitation of Cu-containing metallothionein by a Cd-saturation method. Anal. Biochem. 189, 35-39. Leblondel G., Mauras Y. and Allain P. (1986) Tissue distribution of some elements in rats. Biol. Trace Elem. Res. 10, 327-333. Margoshes M. and Vallee B. L. (1957) A cadmium protein from equine kidney cortex. J. Am. them. Sot. 79, 48134814. Mehra R. K. and Bremner I. (1984) Species differences in the occurrence of copper-metallothionein in the particulate fractions of the liver of copper-loaded animals. Biochem. J. 219, 539-546.
McCormick C. C. (1987) Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement. Proc. Sot. exp. Biol. Med. 185, 413419. Mitane Y., Tohyama C. and Saito H. (1986) The role of metallothionein in the elevated excretion of copper in urine from people living in a cadmium-polluted area. Fund. appl. k’oxkol. 6, 285-29
REFERENCES Blalock T. L., Dunn M. A. and Cousins R. J. (1988) Metallothionein gene expression in rats: tissue-specific regulation by dietary copper and zinc. J. Nutr. 118, 222-228. Brady F. 0. and Helvig B. (1984) Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver. Am. J. Phvsiol. 247, E318-E322. Bremner I. (1987) Involvement of metallothionein in the henatic metabolism of copper. J. Nutr. 117, 19-29. Bremner I. and Beattie J. H.
