Interaction between cadmium and copper in relation to the collagen metabolism of embryonic chick bone in tissue culture

TOXICOLOGY

AND APPLIED

PHARMACOLOGY

75479-484

(1984)

Interaction between Cadmium and Copper in Relation to the Collagen Metabolism of Embryonic Chick Bone in Tissue Culture TATSURO

MIYAHARA, YOSHINORI Faculty

SACHIMI SUGIYAMA, TOSHIYUKI KAJI, RIEKO YAMASHITA, OH-E, TOSHIKO KURANO, AND HIROSHI KOZUKA

of Pharmaceutical Sciences, Toyama 2630 Sugitani, Toyama-shi,

Received

December

5, 1983;

Medical Toyama

and Pharmaceutical 930-01, Japan

accepted

March

University,

24, 1984

Interaction between Cadmium and Copper in Relation to the Collagen Metabolism of Embryonic Chick Bone in Tissue Culture. MIYAHARA, T., SUGIYAMA, S., KAJI, T., YAMASHITA, R., OH-E, Y., KURANO, T., AND KOZUKA, H. (1984). Toxicol. Appl. Pharmacol. 75, 479-484. To investigate the interaction between Cd and Cu in relation to the content of collagen in bone, femurs obtained from 9-day-old chick embryos were cultivated with a combination of Cd and Cu concentrations of 8.9 @M and below for 4 days. When 2.23 or 4.45 pM Cd was added to the medium containing 4.45 or 8.90 pM Cu, the presence of both Cd and Cu caused a remarkable decrease in collagen synthesis compared with a decrease in collagen synthesis caused by either Cd or Cu alone. The results show that Cd and Cu caused an interactive inhibition of collagen synthesis, which was not due to an increase in collagen degradation by Cd and Cu. At concentrations showing inhibition, Cd caused an increase in Cu content and Cu caused an increase in Cd content. The increase in Cd content was mainly caused by the accumulation of Cd in bone mineral. The increase in Cu content was due to binding to metallothionein-like protein in the cytosoi induced by Cd. The relationship between inhibition and the increase in both Cd and Cu was not confirmed after division of the bone into diaphysis and epiphysis. The present study showed that Cu aggravated the bone matrix damage caused by Cd.

Demineralization of bone and renal damage have been recognized in Japanese women as a disease entity called Itai-Itai disease, which is thought to be due, at least partly, to chronic Cd poisoning. Although the bone lesions of patients with Itai-Itai disease are osteomalatic (Webb, 1979), those of Cdtreated animals are osteoporotic (Yoshiki et al., 1975). A decrease in bone mineral has been observed in both humans and experimental animals; however, only the latter showed a decrease in bone matrix. The difference in bone lesions between humans and Cd-treated animals suggested that factors other than Cd participated in the etiology of Itai-Itai disease. In a previous study (Miyahara et al., 1983), we gave attention to Zn, Cu, and Pb as

factors other than Cd, since these metals were found with Cd in the bones of patients with Itai-Itai disease and in Cd-polluted rice (Kobashi et al., 1978). The interaction between Cd and Zn, Cu, or Pb in relation to the content of collagen and mineral in cultured embryonic bone was investigated. The results showed that Zn prevented a decrease in collagen content caused by Cd, although Cu and Pb showed no such protective effect. Neither Zn, Cu, nor Pb prevented the decrease in mineral content caused by Cd. On the other hand, as it has been reported that the interaction between Cd and Cu was recognized in the rat (Kojima and Tanaka, 1973; Sakamoto et al., 1981) and mouse (Kojima et al., 1974), the interaction between Cd and Cu in cultured bone was reexamined

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0041-008X/84

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Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

MIYAHARA

480

in the present study. Consequently, it was found that Cu enhanced the decrease in collagen content caused by Cd. METHODS Culture technique. Fertilized eggs were incubated for 9 days in an electric incubator, and whole femurs were dissected from the embryonic chicken under sterile conditions. A femur was added to 1.5 ml of the culture medium in a Leighton-type culture tube. The tube was incubated at 38’C for 4 days in a rotating culture drum apparatus at an angle of 5” and at l/5 rpm; the medium was changed every 2 days. The medium was BGJbHW* medium (Endo, l974), except that the concentration of NaHCOr was 114 mg/dl. An aqueous solution of CdClz or CuSO, was added to the culture medium. An aqueous solution of L[U-‘4C)cystine (3 19.0 &i/nmol, NEN) was added to the medium to give a concentration of 0.06 rCi/ml. Analytical method. Whole bone was extracted for 2 days with cold 0.1 M acetate buffer containing 0.01 M EDTA (pH 5.5). Under certain circumstances, the epiphysis was cut from the diaphysis, and each part was extracted. The extracts were analyzed for Cd and Cu content by flameless atomic absorption spectrophotometry (Nippon Jarell Ash A-855, FCA-100). Demineralized bone was immersed in 0.1 ml 4 N NaOH and autoclaved at 120°C for 30 min. One milliliter of the growth medium was evaporated to dryness in the test tube, dissolved in 2.0 ml 4 N NaOH, and autoclaved at 120°C for IO min. The hydrolyzates were analyzed for hydroxyproline (Hyp) (Huszar et al., 1980). Since one femur of the same embryo was cultured and the other was uncultured, the amount of Hyp synthesized was calculated by subtracting the Hyp content of uncultured bone from the total Hyp content recovered in the bone and medium after culture. Differences in group means were assessed by Student’s t test. Preparation of subcellular fractions. The bone shafts of 45 hones cultured for 4 days were homogenized in 1.5 ml 0.25 M sucrose at O’C to avoid mixing of bone mineral with other fractions. The homogenate was centrifuged at 50g for 7 min. The pellet was considered the bone mineral. The supernatant fraction was mixed with cartilage ends and homogenized. The homogenate was centrifuged at 85OOg for 10 min. The precipitate was again homogenized in 4 ml 0.25 M sucrose. This homogenate was mixed with the supematant fraction obtained in the second step. This mixture was centrifuged at 50g for 7 min to obtain the unbroken cells. The supematant fraction was centrifuged at 85Og for IO min to obtain the nuclear fraction. The supematant fraction was then centrifuged at 20,OOOg for I8 min to obtain the mitochondrial fraction. The remaining supematant fraction was centrifuged at 100,OOOgfor 60 min. The supematant

E-f AL. fraction and precipitate obtained were considered the cytosol and microsomal fractions, respectively. Gel filtration of the 100,OOOgsupernatant fraction. The cytosol fraction was chromatographed on a Sephadex G75 column (2.8 x 96 cm, 24 ml/hr) with 0.01 M TrisHCI buffer (pH 8.6) containing 0.05 M NaCl at 0°C. The amount of Cd and Cu in each fraction was measured by flameless atomic absorption spectrometry. The 14C radioactivity in each fraction was measured in a liquid scintillation spectrometer (LSC 903, Aloka).

RESULTS Efect of Cu on the Hydroxyproline Content in the Presence or Absence of Cd Femurs were cultured in the combination of Cd and Cu concentrations of 8.9 I.IM and below. As shown in Fig. 1, when 2.23 or 4.45 I.IM Cd was added to the medium with 4.45 or 8.90 PM Cu, the presence of both Cd and Cu caused a remarkable decrease in Hyp synthesis compared with a decrease in Hyp synthesis caused by Cd or Cu alone. The results show that Cd and Cu caused a synergistic inhibition on the collagen synthesis. The percentage of Hyp released into the medium (relative to the total Hyp found in the bone and medium) was not influenced by Cd alone, but it was decreased by the presence of both Cd and Cu (Fig. 2). The presence of both Cd and Cu also may cause a weaker synergistic inhibition on the collagen degradation than that on the collagen synthesis.

** Cu 0 **cu

21:

\**Cu 01

2.23

4.45 Cd (PM)

,,M

4.45

NM

8.90

UM

8.90

FIG. 1. Interaction between Cd and Cu in relation to the collagen synthesis of the bone cultured for 4 days. Values are X f SE for five bones. *, Significantly different from control, p < 0.05; **, significantly different from control, p < 0.01.

INTERACTION

BETWEEN

Cd AND Cu IN EMBRYONIC

BONE

481

u 4.45 C" 8.90

OL

2.23

4.45 Cd(uM)

OL

8.90

2.23

!JM PM

8.9U

4.45 Cd(W)

FIG. 2. Interaction between Cd and Cu in relation to the collagen degradation of the bone cultured for 4 days. Values are X f SE for five bones. *, Significantly different from control, p < 0.05; **, significantly different from control, p i 0.0 1; ***, significantly different from control, p < 0.001.

FIG. 4. Effect of Cd on the Cu content in bone cultured for 4 days. Values are X + SE for five bones. *, Significantly different from Cd untreatment in each Cu level, p < 0.05; **, significantly different from Cd untreatment in each Cu level, p K 0.01; ***, significantly different from Cd untreatment in each Cu level, p < 0.001.

Bones after culture were analyzed for content of Cd or Cu. Figure 3 shows the effect of Cu on the Cd content in bone. The Cd content in the presence of Cu was clearly larger than that in the absence of Cu. The Cu content in the presence of 2.23 and 4.45 pM Cd was larger than that in the absence of Cd, but the content in the presence of 8.90 PM Cd was equal to that in the absence of Cd (Fig. 4). At the concentrations of Cd and Cu which caused a synergistic inhibition on collagen synthesis, both Cd and Cu contents in the presence of both elements were recognized to be larger than that in the presence of Cd or Cu alone.

Content of Hydroxyproline, Diaphysis and Epiphysis

*** 6

***

Cd 8.90

u"

Cd 4.45

II"

Cd 2.23

UM

*** 4

Cu

IuM)

FIG. 3. Effect of Cu on the Cd content in bone cultured for 4 days. Values are X + SE for five bones. **, Significantly different from Cu untreatment in each Cd level, p < 0.01; ***, significantly different from Cu untreatment in each Cd level, p < 0.001.

Cd, or CM in

Femurs cultured with Cd or Cu or both were separated into diaphysis and epiphysis, and each part was analyzed for Hyp, Cd, and Cu. An increase in Hyp content after cultivation was expressed as a ratio of Hyp content after culture to Hyp content before culture. A synergistic effect of Cd and Cu in decreasing Hyp content was recognized in the epiphysis but it was not shown in the diaphysis (Fig. 5). Therefore, a synergistic inhibition of collagen synthesis recognized in whole bone may be due mainly to that in the epiphysis. An increase in Cu content in the presence of both Cd and Cu observed in whole bone was also recognized in both the epiphysis and the diaphysis. Although a large increase in Cd content in the presence of both Cd and Cu was recognized in diaphysis, the Cd content of epiphysis in the presence of both Cd and Cu was slightly smaller than that in the presence of Cd alone. Subcellular Distribution

of Cd and Cu

The Cu content of each subcellular fraction in the presence of both Cd and Cu was larger than that in the presence of Cu alone, except

MIYAHARA

482 lu

Epiphysis

Whole

bone

0

CdCu+Cd

h 2.0 2 2 2 2 m ;c 2 1.0

* I

0

0

cu CdCu+Cd

0

vA Cu Cd Cu+Cd

Cu

CdCu+Cd

Cu Cd

Cu+Cd

Cu

Cu Cd

Cu+Cd

FIG. 5. Interaction between Cd and Cu in relation to the hydroxyproline content in the diaphysis and epiphysis of bone cultured with 4.45 /rM Cd and 4.45 pM Cu for 4 days. Values are X k SE for five bones. *, Significantly different from control, p < 0.05; ***, significantly different from control, p < 0.001.

in the microsomal and mineral fractions (Table 1). An increase in Cu content was especially remarkable in the cytosol. Although the Cd content of each particular fraction in the presence of both Cd and Cu was larger than that in the presence of Cd alone, a large decrease in the Cd content was recognized in the cytosol fraction. A large increase in the Cd content of bone mineral fraction was found in the presence of both Cd and Cu.

Distribution of Cd and Cu in the Cytosol When 4.45 ~.LM Cd, 4.45 I.IM Cu, or both were cultured with [‘4C]cystine, and the cyTABLE SUBCELLULAR

DISTRIBUTIONOF

Cd

ANDCUIN

ET AL,

tosol obtained from bone homogenate was fractionated by gel filtration, the first fraction was eluted near the void volume of about 170 ml, the second fraction was eluted at about 340 ml, which corresponded to about M, 10,000 (metallothionein (MT) fraction), and the third fraction was eluted at about 470 ml, which corresponded to about 2,500 molecular weight (Fig. 6). The cytosol obtained from Cu-treated bones showed a very small peak in the MT fraction, but that from Cd-treated ones showed a large peak in the MT fraction. The Cd content of the MT fraction in the presence of both Cd and Cu decreased compared with that in the presence of Cd alone. The Cu content in the presence of both Cd and Cu was about four times that in the presence of Cu alone. The amount of Cd in the MT fraction induced by Cd alone was equal to the sum of Cd and Cu in the MT fraction obtained from bones in the presence of both Cd and Cu. DISCUSSION The bone damage caused by Cd in both humans and animals is thought to be due either to a direct action on the bone (Yoshiki et al., 1975) or to an indirect action accompanying the damage caused in the kidney or intestine (Murata, 197 1). To clarify the direct action of Cd on bone, we have studied biochemically and histologically the effect of Cd I

45 BONES CULTURED

WITH

4.45

PM Cd or4.45

PM CU OR BETH

Cd plus Cu treatment (nmol) Subcellular fraction

Cd treatment (Cd content nmol)

Mineral Cell debris Nuclei Mitochondria Microsome Cytosol

8.0 0.7 1.7 4.4 3.8 41.5

Total

60.1

Cu treatment (Cu content nmol)

Cd content

Cu content

2.4 2.0 2.1 3.0 7.4 4.6

39.1 1.3 2.6 5.2 3.8 29.2

1.9 5.5 6.9 5.6 4.2 19.4

21.5

81.2

43.5

INTERACTION (a)

200

(b)

BETWEEN

Cd AND Cu IN EMBRYONIC

Cc) (d)

300 Elution

400 volume(ml)

500

FIG. 6. Sephadex G-75 elution profile of Cd (----), Cu (- - -) and l/2 cystine (- . -) in 100,OOOg supernatant fraction from 45 bones cultured with 4.45 PM Cu (A), 4.45 PM Cd (B), or both (C) in the presence of 0.06 pCi/ml L[U-‘%Z]cystine for 4 days. Molecular weight was estimated by Blue Dextran (a), egg albumin (b), chymotrypsinogen (c), cytochrome c (d), and DPN-alanine.

on the ossification of cultured chick embryo bone (Sakai et al., 1975; Miyahara et al., 1978, 1980a,b,c). The results show that Cd inhibited the bone matrix formation and brought about a malfunction in the ossification process. Since Cd-treated bone in the culture system showed a decrease in both collagen and mineral, the change in cultured bone was similar to osteoporosis. In a previous study (Miyahara et al., 1983), Zn was shown to prevent a decrease in collagen content caused by Cd and not to prevent a decrease in mineral content caused by Cd. The change in Cd plus Zn-treated bone was similar to osteomalacia. When 8.9 I.~M (1 ppm) Cd was added to the medium with Cu at 8.9 PM or above, no interaction between Cd and Cu was recognized. However, Kojima et al. (1974) have reported that the presence of both Cd and Cu caused a large decrease in rat bone weight compared with the Cd or Cu treatment. Therefore, in the present study the interaction between Cd and Cu was reexamined at combined concentrations lower

BONE

483

than those used in the previous study. The results showed that Cd and Cu have an interactive effect on the inhibition of collagen synthesis in cultured bone. Although antagonism between Cd and Cu is common (Wada, 1975), an interactive inhibition by Cd and Cu found in our system and in rat is uncommon in the interaction between Cd and Cu. Since a negative interaction between both Cd and Cu content and Hyp synthesis occurred, it was suggested at first that the inhibition by Cd and Cu was due to an increase in both Cd and Cu content in the coexistence of Cd and Cu. However, the inhibition in whole bone was mainly reflected by an inhibition in the epiphysis where the Cu content was increased in the presence of both Cd and Cu, but the Cd content was not increased. This result suggested that a synergistic effect of Cd and Cu in decreasing collagen synthesis may be due to an increase in Cu caused by the concomitant presence of increased amounts of Cd. The increase in Cu in the presence of Cd occurred in the nuclear, mitochondrial, and cytosol fractions (Table 1). Although the chemical form of Cu in the nuclear and mitochondrial fractions was not determined, about 80% of the Cu in the cytosol was bound to MT-like protein (Fig. 6). Therefore, the effect of Cd and Cu in decreasing collagen synthesis may be due partly to the increase in Cu content. Recently, 50 PM Cd was reported to inhibit Cu uptake by isolated hepatocytes (Schmitt et al., 1983). In our system, 2.23 and 4.45 PM Cd stimulated Cu accumulation, but 8.90 FM Cd had no effect on Cu accumulation. If a higher concentration of Cd was used, accumulation of Cu in bone may also be inhibited in our system. An increase in Cu accumulation by Cd may be due to the increased content of Cu in the cytosol, which may be part of the reason that some Cd in MT-like protein induced by Cd was replaced by Cu. Cu also has a higher affinity for MT than does Cd (Susuki, 198 1). On the other hand, it has been reported that 0.51 and 0.89 ppm Cu inhibited Cd

484

MIYAHARA

uptake by cultured cells (Meshitsuka et al., 1982). In our system, Cd accumulation in the diaphysis was stimulated by 4.45 @M Cu. Cd accumulation in epiphysis was slightly inhibited by 4.45 I.IM Cu. Since the epiphysis contains no mineral, it is closer to cultured cells than is the diaphysis. If Cu were increased, Cd accumulation in the epiphysis may be inhibited by Cu. Based on these and earlier studies, Cu aggravates Cd-induced bone matrix damage. Zn appears to protect Cd-induced injury. Therefore, it is suggested by these data that the ratio of the metals as well as each dose of three metals influences whether bone damage in vivo is osteoporotic or osteomalatic. REFERENCES ENDO, H. (1974). Cartilage matrix metabolism of chondrocytes changing through their life span and its possible hormonal control, as demonstrated by tissue culture studies. Connect. Tissue 6, 139-148. HUSZAR, G., MAIOCCO, J., AND NAFTOLIN, F. (1980). Monitoring of collagen and collagen fragments in chromatography of protein mixtures. Anal. B&hem. 105,424-429. KOEIASHI, K., NAKAI, N., HASE, J., MIYAHARA, T., KOZUKA, H., AND FLIJII, M. (1978). Chemical forms of cadmium in cadmium-polluted rice. 1. Binding properties of glutelin-cadmium complex. J. Hyg. Chem. (Japan) 24, 3 14-32 1. KOJIMA, R., AND TANAKA, E. (1973). Additive effects of copper on cadmium toxicity. Med. Biol. 86, 173176. KOJIMA, R., ISHIGURO, E., AND TANII, M. (1974). Effect of simultaneous administration of cadmium and copper in mice. Japan, J. Pub. Health 21, 281-289. MESHITSUKA, S., OHSHIRO, H., AND NOSE, T. (1982). Release of preabsorbed Cd from cultured cells by the addition of Cu ion. Experientia 38, 1473- 1474. MIYAHARA, T., KATO, T., NAKAGAWA, S., KOZUKA, H., SAKAI, T., NOMURA, N., AND TAKAYANAGI, N. (1978). Influence of poisonous metals on the bone

ET AL. metabolism. I. The effect of cadmium on the ossification of chick-embryo tibia in tissue culture. J. Hyg. Chem. (Japan) 24, 36-42. MIYAHARA, T., MIYAKOSHI, M., SAITO, Y., AND KoZUKA, H. (1980a). Influence of poisonous metals on bone metabolism. III. The effect of cadmium on bone resorption in tissue culture. Toxicol. Appl. Pharmacol. 55, 477-483. MIYAHARA, T., KOMURASAKI, T., OH-E, Y., AND KoZUKA, H. (1980b). Inhibition of hydroxyproline synthesis by cadmium ion in cultured embryonic chick bone. Japan. J. Pub. Health 27, 361-366. MIYAHARA, T., MIYAKOSHI, M., AND KOZUKA, H. (1980~). Inhibitory effect of cadmium on the mineralization of embryonic chick femurs in tissue culture. Japan. J. Pub. Health 21, 323-328. MIYAHARA, T., OH-E, Y., TAKAINE, E., AND KOZUKA, H. (1983). Interaction between cadmium and zinc, copper, or lead in relation to the collagen and mineral content of embryonic chick bone in tissue culture. Toxicol. Appl. Pharmacol. 67, 4 l-48, MURATA, I. (197 1). Chronic entero-osteo-nephropathy cadmium. J. Japan. Med. Assoc. 65, 15-42. SAKAI, T., MIYAHARA, T., SANEI, K., NOMURA, N., AND TAKAYANAGI, N. (1975). Hygienic chemical studies on environmental pollution. I. Effect of cadmium on chick-embryo tibia in tissue culture. J. Hyg. Chem. (Japan) 21, 35-41. SAKAMOTO, M., KAWAHARA, K., AND KUSHIHATA, T. (198 1). Effect of copper-cadmium interaction on copper metabolism. I. Distribution of copper and cadmium in tissue of copper- and/or cadmium-administered rats for three months. Japan. J. Pub. Health 28, 167-170. SCHMITT, R. C., DARWISH, H. M., CHENEY, J. C., AND ETINGEN, M. J. (1983). Copper transport kinetics by isolated rat hepatocytes. Amer. J. Physiol. 244, G 183Gl91. SUZUKI, K. T. (198 1). Heavy metals and mctallothionein. J. Syn. Org. Chem. Japan 39, 1073-1082. WADA, 0. (1975). Interaction among metals. Metab. Dis. 12, 219-227. WEBB, M. (1979). The Chemistry, Biochemistry, and Biology of Cadmium, pp. 446-447. EIsevier/NorthHolland, Amsterdam/New York. YOSHIKI, S., YANAGISAWA, T., KIMURA, M., OTAKI, N., SUZUKI, M., AND SUDA, T. (1975). Bone and kidney lesions in experimental cadmium intoxication. Arch. Environ. Health 30, 559-562.