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Inhibitory effect of cadmium on collagenous peptide synthesis of embryonic chick bone in tissue culture
137
Toxicology Letters, 6 (1980) 137-139 o Elsevier/North-Holland Biomedical Press
INHIBITORY EFFECT OF CADMIUM ON COLLAGENOUS PEPTIDE SYNTHESIS OF EMBRYONIC CHICK BONE IN TISSUE CULTURE
TATSURO MIYAHARA,
TOSHI KOMURASAKI and HIROSHI KOZUKA
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical Sugitani, Toyama-Shi, Toyama 93041 (Japan)
University, 2630,
(Received March 6th, 1980) (Accepted March lOth, 1980)
SUMMARY
The effect of cadmium (Cd) on bone coll~en.s~thesis was assessed in organ cultures of embryonic femur by measuring the incorporation of [ 3HJ proline(Pro) into collagenous-digestible protein (CDP) using purified bacterial collagenase. Cd produced a decrease when C3H]Pro was incorporated in CDP. There was little alteration in the hydroxylation of [ 3H] Pro to [ 3H] hydroxyproline(Hyp) in CDP of Cd-treated bone. An accumulation of underhydroxylated collagen and a decrease in the activity of prolyl hydroxylase (EC 1.14.11.2) in Cd-treated bone was not observed. These results indicated that the inhibitory effect of Cd on collagen synthesis was largely due to inhibition of collagenous peptide synthesis without inhibition of its hydroxylation. INTRODUCTION
We have reported that Cd ~hibi~ the collagen synthesis of embryonic chick bone in tissue culture [I] . Protease-free bacterial collagenase is widely used to differentiate the labeling of CDP and NCP. The present study was undertaken to examine whether or not the ~co~oration of E3H]Pro into CDP and the hydroxylation of Pro to [ 3H] Hyp in CDP was affected by Cd.
Femurs from 9day chick embryo were cultured by the Roller-tube method
[l].Labeled CDP was determined by the method of Peterokofsky and Diegelman [Z] . Bacterial collagenase (Amano) was chromatographed on Sephadex G-200 and the fractions free of proteolytic activity were pooled. It was checked that this enzyme preparation did not hydrolyze [ 3H] tryptophanAbbreviations: CDP, collagenous-digestible protein; Hyp, hydroxyproline; NCP, non-collagen protein; Pro, proline; PTH, parathyroid hormone; TCA, trichloroacetic acid.
138
labeled g-day embryonic chick bone homogenates. [ 3H] Pro and [ 3H] Hyp were separated using a column of Dowex resin [ 31. Underhydroxylated collagen and the activity of prolyl hydroxylase in cultured bone were measured by the method of Chen and Raisz [4]. RESULTS
Table I shows the effect of Cd on the incorporation of [3H] Pro in CDP and NCP of bones cultivated with 2 ppm Cd for 2 days. Cd inhibited an incorporation of [ jH] Pro into CDP and NCP. Table II represents the effect of Cd on the hydroxylation of [‘HI Pro to [ 3H] Hyp in CDP. Cd produced little alteration in the ratio of [ 3H] Pro to [ 3H] Hyp. To confirm that Cd was without effect on the hydroxylation of Pro, the accumulation of underhydroxylated collagen in Cd-treated bone was examined. Bones were labeled with 3,4-[ 3Hz] Pro in the culture and assayed for hydroxylatable substrate with exogenous prolyl hydroxylase and cofactors. The radioTABLE I EFFECT OF Cd ON THE INCORPORATION OF [“HI PROLINE INTO COLLAGENASEDIGESTIBLE PROTEIN (CDP) AND NON-COLLAGEN PROTEIN (NCP) IN EMBRYONIC CHICK BONES CULTURED FOR 2 DAYS Cultured bones were homogenized with cold 5% TCA solution and TCA-insoluble fractions were treated with protease-free collagenase. Collagenase-digestible and -undigestible fractions were counted in a liquid scintillation counter. Values are means f S.E.M. of 4 cultures. Cd 2 ppm
TCA-soluble fraction (dpm)
CDP (dpm)
NCP (dpm)
-
23 335 * 3925 (199) 7005 * 1400 (39)
18 912 k 5086
28 083 + 5565 (199) 12 599 + 2391 (59)
+
(199) 3776 r 1148 (23)
TABLE II EFFECT OF Cd ON THE HYDROXYLATION OF [“HIPROLINE PROLINE IN COLLAGENASE-DIGESTIBLE PROTEIN
TO [“HIHYDROXY-
CDP was hydrolyzed in 6 N HCl and the hydrolysate was chromatographed on Dowex (50W X 8) to separate [3H] Hyp from [“HI Pro. Values were means * S.E.M. of 4 cultures. Cd 2 ppm
[“HIHYP (dpm)
[3H]Pro (dpm)
[3HlHyp/[3HlPro
+
8486 + 2546 1699 f 472
9735 + 2943 2157 i 748
0.88 f 0.04 0.86 r 0.07
139
activity of tritiated water formed in Cd-treated bone was approximately similar to that in control bone. As it has been reported that Cd inhibits prolyl hydroxylase [ 51, the activity of prolyl hydroxylase in Cd-treated bone was investigated. When bone enzyme extracts were assayed for available prolyl hydroxylase, the enzyme activity of Cd-treated bone was similar to that of control bone. DISCUSSION
It has been reported briefly that Cd inhibits prolyl hydroxylase [ 51. We noted that Cd inhibited a partially purified prolyl hydroxylase at levels above 0.5 ppm. Even if Cd inhibited the enzyme, it would not always inhibit the hydroxylation of Pro in CDP of embryonic bone in tissue culture. Hydroxylation of [ 3H] Pro to E3H]Hyp was unaffected by Cd (Table II), and this finding was supported by the slight difference in the amount of underhydroxylated collagen and the activity of prolyl hydroxylase between Cd-treated bone and control bone. On the other hand, Cd was found to inhibit the incorporation of labeled Pro into CDP. From these results it is postulated that an inhibitory effect of Cd on collagen synthesis in cultured bone is caused mainly by the i~ibition of collagenous peptide formation without the inhibition of its hydroxylation. The inhibitory effect of Cd on CDP formation was.slightly stronger than that on NCP formation, but was not specific compared with the inhibitor effect of PTH on the labeling of CDP, since PTH did not produce any alteration in the labeling of NCP or in amino acid uptake [6]. As shown in Table I, the incorporation of [ 3H] Pro into TCA-soluble fraction was inhibited by Cd. This result indicates that the inhibition of CDP formation by Cd may be ascribed partly to a decrease in amino acid uptake or‘in precursor pool size.
REFERENCES 1 T. Miyahara, T. Kato, S. Nakagawa, H. Kozuka, T. Sakai, N. Nomura and N. Takayanagi, Influence of poisonous metals on the bone metabolism, I. The effect of cadmium on the ossification of chick-embryo tibia in tissue culture, J. Hyg. Chem. (Japan), 24 (1968) 36-42. 2 B. Peterkofsky and RF. Diegelman, Use of a mixture of protease-free collagenase for the specific assay of radioactive collagen in the presence of other protein, Biochemistry, 10 (1971) 988-994. 3 S. Moore and W.H. Stein, Procedures for the chromatographic determination of amino acid on four percent cross-linked sulfonated polystyrene resins, J. Biol. Chem., 211 (1954) 893-906. 4 T.L. Chen and L.G. Raisz, The effect of ascorbic acid deficiency on tialcium and collagen metabolism in cultured fetal rat bones, Calcif. Tiss. Res., 17 (1975) 113-127. 5 R.S. Rapaka, K.R. Sorensen, S.D. Lee and R.S. Bhatnagen, Inhibition of hydroxyproline synthesis by palladium ions, Biochim. Biophys. Acta, 429 (1976) 63-71. 6 J.W. Dietrich, E.M. Canalis, D.M. Maina and L.G. Raisz, Hormonal control of bone collagen synthesis in vitro: Effects of parathyroid hormone and calcitonin, Endocrinology, 98 (1976) 943-949.
Toxicology Letters, 6 (1980) 137-139 o Elsevier/North-Holland Biomedical Press
INHIBITORY EFFECT OF CADMIUM ON COLLAGENOUS PEPTIDE SYNTHESIS OF EMBRYONIC CHICK BONE IN TISSUE CULTURE
TATSURO MIYAHARA,
TOSHI KOMURASAKI and HIROSHI KOZUKA
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical Sugitani, Toyama-Shi, Toyama 93041 (Japan)
University, 2630,
(Received March 6th, 1980) (Accepted March lOth, 1980)
SUMMARY
The effect of cadmium (Cd) on bone coll~en.s~thesis was assessed in organ cultures of embryonic femur by measuring the incorporation of [ 3HJ proline(Pro) into collagenous-digestible protein (CDP) using purified bacterial collagenase. Cd produced a decrease when C3H]Pro was incorporated in CDP. There was little alteration in the hydroxylation of [ 3H] Pro to [ 3H] hydroxyproline(Hyp) in CDP of Cd-treated bone. An accumulation of underhydroxylated collagen and a decrease in the activity of prolyl hydroxylase (EC 1.14.11.2) in Cd-treated bone was not observed. These results indicated that the inhibitory effect of Cd on collagen synthesis was largely due to inhibition of collagenous peptide synthesis without inhibition of its hydroxylation. INTRODUCTION
We have reported that Cd ~hibi~ the collagen synthesis of embryonic chick bone in tissue culture [I] . Protease-free bacterial collagenase is widely used to differentiate the labeling of CDP and NCP. The present study was undertaken to examine whether or not the ~co~oration of E3H]Pro into CDP and the hydroxylation of Pro to [ 3H] Hyp in CDP was affected by Cd.
Femurs from 9day chick embryo were cultured by the Roller-tube method
[l].Labeled CDP was determined by the method of Peterokofsky and Diegelman [Z] . Bacterial collagenase (Amano) was chromatographed on Sephadex G-200 and the fractions free of proteolytic activity were pooled. It was checked that this enzyme preparation did not hydrolyze [ 3H] tryptophanAbbreviations: CDP, collagenous-digestible protein; Hyp, hydroxyproline; NCP, non-collagen protein; Pro, proline; PTH, parathyroid hormone; TCA, trichloroacetic acid.
138
labeled g-day embryonic chick bone homogenates. [ 3H] Pro and [ 3H] Hyp were separated using a column of Dowex resin [ 31. Underhydroxylated collagen and the activity of prolyl hydroxylase in cultured bone were measured by the method of Chen and Raisz [4]. RESULTS
Table I shows the effect of Cd on the incorporation of [3H] Pro in CDP and NCP of bones cultivated with 2 ppm Cd for 2 days. Cd inhibited an incorporation of [ jH] Pro into CDP and NCP. Table II represents the effect of Cd on the hydroxylation of [‘HI Pro to [ 3H] Hyp in CDP. Cd produced little alteration in the ratio of [ 3H] Pro to [ 3H] Hyp. To confirm that Cd was without effect on the hydroxylation of Pro, the accumulation of underhydroxylated collagen in Cd-treated bone was examined. Bones were labeled with 3,4-[ 3Hz] Pro in the culture and assayed for hydroxylatable substrate with exogenous prolyl hydroxylase and cofactors. The radioTABLE I EFFECT OF Cd ON THE INCORPORATION OF [“HI PROLINE INTO COLLAGENASEDIGESTIBLE PROTEIN (CDP) AND NON-COLLAGEN PROTEIN (NCP) IN EMBRYONIC CHICK BONES CULTURED FOR 2 DAYS Cultured bones were homogenized with cold 5% TCA solution and TCA-insoluble fractions were treated with protease-free collagenase. Collagenase-digestible and -undigestible fractions were counted in a liquid scintillation counter. Values are means f S.E.M. of 4 cultures. Cd 2 ppm
TCA-soluble fraction (dpm)
CDP (dpm)
NCP (dpm)
-
23 335 * 3925 (199) 7005 * 1400 (39)
18 912 k 5086
28 083 + 5565 (199) 12 599 + 2391 (59)
+
(199) 3776 r 1148 (23)
TABLE II EFFECT OF Cd ON THE HYDROXYLATION OF [“HIPROLINE PROLINE IN COLLAGENASE-DIGESTIBLE PROTEIN
TO [“HIHYDROXY-
CDP was hydrolyzed in 6 N HCl and the hydrolysate was chromatographed on Dowex (50W X 8) to separate [3H] Hyp from [“HI Pro. Values were means * S.E.M. of 4 cultures. Cd 2 ppm
[“HIHYP (dpm)
[3H]Pro (dpm)
[3HlHyp/[3HlPro
+
8486 + 2546 1699 f 472
9735 + 2943 2157 i 748
0.88 f 0.04 0.86 r 0.07
139
activity of tritiated water formed in Cd-treated bone was approximately similar to that in control bone. As it has been reported that Cd inhibits prolyl hydroxylase [ 51, the activity of prolyl hydroxylase in Cd-treated bone was investigated. When bone enzyme extracts were assayed for available prolyl hydroxylase, the enzyme activity of Cd-treated bone was similar to that of control bone. DISCUSSION
It has been reported briefly that Cd inhibits prolyl hydroxylase [ 51. We noted that Cd inhibited a partially purified prolyl hydroxylase at levels above 0.5 ppm. Even if Cd inhibited the enzyme, it would not always inhibit the hydroxylation of Pro in CDP of embryonic bone in tissue culture. Hydroxylation of [ 3H] Pro to E3H]Hyp was unaffected by Cd (Table II), and this finding was supported by the slight difference in the amount of underhydroxylated collagen and the activity of prolyl hydroxylase between Cd-treated bone and control bone. On the other hand, Cd was found to inhibit the incorporation of labeled Pro into CDP. From these results it is postulated that an inhibitory effect of Cd on collagen synthesis in cultured bone is caused mainly by the i~ibition of collagenous peptide formation without the inhibition of its hydroxylation. The inhibitory effect of Cd on CDP formation was.slightly stronger than that on NCP formation, but was not specific compared with the inhibitor effect of PTH on the labeling of CDP, since PTH did not produce any alteration in the labeling of NCP or in amino acid uptake [6]. As shown in Table I, the incorporation of [ 3H] Pro into TCA-soluble fraction was inhibited by Cd. This result indicates that the inhibition of CDP formation by Cd may be ascribed partly to a decrease in amino acid uptake or‘in precursor pool size.
REFERENCES 1 T. Miyahara, T. Kato, S. Nakagawa, H. Kozuka, T. Sakai, N. Nomura and N. Takayanagi, Influence of poisonous metals on the bone metabolism, I. The effect of cadmium on the ossification of chick-embryo tibia in tissue culture, J. Hyg. Chem. (Japan), 24 (1968) 36-42. 2 B. Peterkofsky and RF. Diegelman, Use of a mixture of protease-free collagenase for the specific assay of radioactive collagen in the presence of other protein, Biochemistry, 10 (1971) 988-994. 3 S. Moore and W.H. Stein, Procedures for the chromatographic determination of amino acid on four percent cross-linked sulfonated polystyrene resins, J. Biol. Chem., 211 (1954) 893-906. 4 T.L. Chen and L.G. Raisz, The effect of ascorbic acid deficiency on tialcium and collagen metabolism in cultured fetal rat bones, Calcif. Tiss. Res., 17 (1975) 113-127. 5 R.S. Rapaka, K.R. Sorensen, S.D. Lee and R.S. Bhatnagen, Inhibition of hydroxyproline synthesis by palladium ions, Biochim. Biophys. Acta, 429 (1976) 63-71. 6 J.W. Dietrich, E.M. Canalis, D.M. Maina and L.G. Raisz, Hormonal control of bone collagen synthesis in vitro: Effects of parathyroid hormone and calcitonin, Endocrinology, 98 (1976) 943-949.