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Sensitive response of cultured vascular smooth-muscle cells to cadmium cytotoxicity: comparison with cultured vascular endothelial cells and kidney epithelial LLC-PK1 cells
Toxicology Letters ELSEVIER
Toxicology Letters 89 (1996) 13 I - 137
Sensitive response of cultured vascular smooth-muscle cells to cadmium cytotoxicity: comparison with cultured vascular endothelial cells and kidney epithelial LLC-PKl cells Toshiyuki KajP,*, Megumi Suzuki”, Chika Yamamotoa, Yumiko Imaki”, Sawako Miyajima”, Yasuyuki Fujiwara”, Michiko Sakamotoa, Hiroshi Kozukab aDepartment of E,avironmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920- 11, Japan bDepartment of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
Received 20 March 1996; accepted 2 July 1996
Abstract Response to cadmium cytotoxicity of cultured bovine aortic smooth-muscle cells was compared with that of cultured bovine aortic endothelial cells and porcine kidney epithelial LLC-PKl cells. The cell damage was evaluated by morphology and the lactate dehydrogenase leakage assay. It was found that vascular smooth-muscle cells are markedly sensitive to cadmium cytotoxicity. The accumulation of intracellular cadmium was much higher but that of metallothionein was much less in vascular smooth-muscle cells than in LLC-PKl cells; vascular endothelial cells were in between vascular smooth-muscle cells and LLC-PKl cells. The content of reduced glutathione was slightly increased by cadmium in all three cell types. The present data suggest that a much lower inducibility of metallothionein with a high accumulation of intracellular cadmium in vascular smooth-muscle cells resulted in a marked sensitivity of the cells to cadmium cytotoxicity. Vascular smooth-muscle cells may be one of the critical target of cadmium toxicity. Keywords:
Cadmium;
* Corresponding 0378-4274/96/$15.00
Cytotoxicity;
Endothelial
cells; Metallothionein;
author. 0 1996 Elsevier Science Ireland Ltd. All rights reserved
PII SO378-4274(96)03797-6
Smooth
muscle cells; Vascular
132
T. Kaji et al. / Toxicology
1. Introduction
It has been suggested that cadmium in the environment may be a causal factor in the cardio -vascular diseases. An epidemiologic study showed that there is a relationship between environmental cadmium and vascular diseases [I]. In experimental animals, the metal induces atherosclerosis and hypertension [2]. Nolan and Shaikh [3] suggested that the initial effect of acute cadmium administration is on the integrity and permeability of the vascular endothelium; other necrotic changes of tissue occur secondarily. We also hypothesized that the first target of cadmium in the vascular tissue is vascular endothelial cells. It was found that cadmium destroys the monolayer of cultured endothelial cells [4]. The metal induces the synthesis of glycosaminoglycans [5] with an enhancement of heparin-like activity on the cell surface [6] and the release of plasminogen activator inhibitor type 1 from the cells [7]. These results clearly indicated that functional damage of endothelial cells may be an important component of cadmium-induced vascular lesions. On the other hand, cadmium is capable of acting on vascular smooth-muscle cells. The metal causes vasoconstriction in vitro [8,9] and hypertension in vivo [IO]. However, the exhibition of cadmium toxicity at the flvascular smooth-muscle cell level is not necessarily clear. The purpose of the present study is to clarify the sensitivity of vascular smooth-muscle cells to cadmium cytotoxicity using a cell culture system.
2. Materials and methods 2.1. Materials Vascular smooth-muscle cells and endothelial cells derived from bovine aorta were gifts from Drs. Yutaka Nakashima and Katsuo Sueishi (First Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan). Epithelial LLC-PKl cells derived from porcine kidney were purchased from American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s
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89 (1996) 131-137
medium and ASF 301 medium were from Nissui Pharmaceutical (Tokyo, Japan) and Ajinomoto (Tokyo, Japan), respectively. Fetal bovine serum was obtained from Whittaker (Warkersville, MD). Tissue culture dishes and plates were from Corning (Corning, NY). Cadmium chloride, bovine hemoglobin, the lactate dehydrogenase kit and other reagents were purchased from Wako Purechemical Industries (Osaka, Japan). 2.2. Morphology Vascular endothelial cells, vascular smooth-muscle cells and LLC-PKI ceils were each cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in 6-well plates at 37°C in a humid atmosphere of 5% CO, in air until confluent (confluent cultures). In another experiment, the three cell types were each plated at 1 x lo5 cells/well into 6-well plates and cultured for 24 h in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (growing cultures). After culture, the cells were washed twice with serum-free ASF 301 medium and incubated at 37°C for 24 h in 1 ml of fresh ASF 301 medium in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 PM. The medium was then discarded and the cells were gently washed with Ca, Mg-free phosphate buffered saline (CMF-PBS). The cells were fixed with methanol and stained with Giemsa. 2.3. Biochemical evaluation of cell damage Confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were prepared in 24-well culture plates and treated with cadmium chloride at 0.5, 1, 2, 5 or 10 PM for 24 h in 0.25 ml of serum-free ASF 301 medium. The conditioned medium was collected and analyzed for the activity of lactate dehydrogenase as a marker of nonspecific cell damage. 2.4. Determination of intracellular cadmium Confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were each treated with cadmium chloride at 0.5, 1, 2, 5 or 10 PM for 24 h in serum-free ASF 301
T. Kaji et al. / Toxicology
medium in 6-well culture plates. After treatment, the medium was discarded and the cells were washed twice with CMF-PBS. The cells were next washed twice with ice-cold 2 mM EGTA solution to remove cadmium loosely bound to the cell surface [l l] and extracted with 1 ml of 0.1 M acetate buffer solution (pH 5.5) containing 10 mM EDTA at 4°C for 72 h. The extract was collected and the cells were gently washed with 1 ml of the buffer; the wash was combined with the collected extract. The extract was directly analyzed for cadmium content by flameless atomic absorption spectrophotometry (Nippon Jarrell Ash, AA8500). The cells after extraction were scraped off with a rubber policeman in the presence of 0.75 ml of CMF-PBS. After collection of the cell suspension, the well was washed with 0.75 ml of CMF-PBS; the wash was combined with the cell suspension. The cell homogenate was prepared by sonication and an aliquot was used for the determination of protein content by the method of Lowry et al. [ 121. The accumulation of intracellular cadmium was expressed as nmolimg protein. 2.5. Determination glutathione
of metallothionein
and reduced
Vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were cultured until confluent and treated with cadmium chloride at 0.5, 1, 2, 5 or 10 uM for 24 h in serum-free ASF 301 medium in 60-mm dishes. After treatment, the medium was discarded and the cells were washed twice with CMF-PBS. The cells were then scraped off with a rubber policeman in the presence of 1 ml of 0.25 M sucrose. The dish was washed with 1 ml of 0.25 M sucrose and the wash was combined with the cell suspension. The cell homogenate was prepared by sonication and used for the determination of metallothionein content by the cadmiumhemoglobin method [13] with some modifications as follows: A portion of the cell homogenate (0.5 ml) was transferred into a microtube and both 0.5 ml of 0.1 M Tris-H:Cl buffer solution (pH 8.0) and 0.05 ml of 10 ug/ml cadmium chloride solution were added. After -incubation for 10 min at room temperature, 0.1 ml of 2% bovine hemoglobin was added and the mixture was boiled for 2 min; the
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tube was centrifuged at 10 000 x g for 5 min. The addition of hemoglobin, boiling and the centrifugation were performed three times. The supernatant was analyzed for cadmium content by flameless atomic absorption spectrophotometry and the metallothionein content was calculated; another portion (0.3 ml) of the cell homogenate was used for the determination of protein content; the remainder was analyzed for the content of reduced glutathione by the fluorometric assay [14]. 2.6. Statistical
analysis
Data were analyzed for statistical significance by Student’s t-test. P values of less than 0.05 were considered to indicate statistically significant differences.
3. Results Fig. 1 shows the morphology of confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium at 10 uM or less for 24 h. In endothelial cells, the cell number was markedly decreased by cadmium at 5 uM or more; a necrotic change was extensively observed at 10 PM. In vascular smooth-muscle cells, a degenerative change was caused by cadmium at 1 uM or more; a severe damage of the cells was observed at 2 uM or more. In LLC-PKl cells, no destructive change was observed in the cell layer even after exposure to cadmium; damage of the cells was not severe. These observations were similar in growing cultures of the corresponding cell types (data not shown). Fig. 2 shows the leakage of lactate dehydrogenase into the medium from vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after a 24-h incubation. The highest and marked leakage of the enzyme was observed in vascular smooth-muscle cells after exposure to cadmium at 0.5 uM or more, supporting the morphological observation. In endothelial cells, the leakage was significantly increased by cadmium at 5 uM or more; this change was also consistent with the morphology. In LLC-PKl
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T. Kaji et al. / Toxicology Letters 89 (1996) 131-137
Fig. 1. Morphology of confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium chloride at 0.5, 1, 2, 5 or 10 uM for 24 h. Al, endothelial cells (control); A2, endothelial cells treated with 0.5 nM cadmium; A3, endothelial cells treated with 1 nM cadmium; A4, endothelial cells treated with 2 uM cadmium; A5, endothelial cells treated with 5 )IM cadmium; A6, endothelial cells treated with 10 nM cadmium; Bl, vascular smooth-muscle cells (control); B2, vascular smooth-muscle cells treated with 0.5 nM cadmium; B3, vascular smooth-muscle cells treated with 1 uM cadmium; B4, vascular smooth-muscle cells treated with 2 nM cadmium; B5, vascular smooth-muscle cells treated with 5 uM cadmium; B6, vascular smooth-muscle cells treated with 10 uM cadmium; Cl, LLC-PKl cells (control); C2, LLC-PKl cells treated with 0.5 nM cadmium; C3, LLC-PKl cells treated with 1 uM cadmium; C4, LLC-PKl cells treated with 2 uM cadmium; C5, LLC-PKl cells treated with 5 nM cadmium; C6, LLC-PKl cells treated with 10 nM cadmium.
cells, a significant increase in the leakage was caused by cadmium at 5 uM or more. At 10 PM, lactate dehydrogenase was leaked more from LLC-PKl cells than from vascular endothelial cells. The rH]adenine release assay, which sensitively detects the damage of endothelial cells, also indicated that cadmium at 10 uM exhibited cytotoxicity more severely in LLC-PKl cells than in endothelial cells (data not shown).
The intracellular accumulation of cadmium is an important factor of exhibiting cytotoxicity of the metal. As shown in Fig. 3, cadmium markedly accumulated in vascular smooth-muscle cells in comparison with in LLC-PKl cells. The accumulation of the metal within endothelial cells was in between vascular smooth-muscle cells and LLCPKl cells. Fig. 4 shows the intracellular accumulation of
T. Kaji et al. / Toxicology
metallothionein in vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium for 24 h. Endothelial cells greatly responded to cadmium at 2 uM or less; however, at .5 PM, metallothionein content was not increased further. In vascular smoothmuscle cells, cadmium at 2 uM or less induced metallothionein only slightly. Since a severe damage was seen in thl: cells treated with cadmium at 5 uM or more, metallothionein induction was not examined at these concentrations. In contrast, cadmium at 10 uM. or less strongly induced metallothionein in a concentration-dependent manner. Finally, the effect of cadmium on the content of cellular reduced glutathione was examined since this substance is capable of protection against the cytotoxicity of the metal [15]. As shown in Fig. 5, cadmium treatment resulted in a slight but significant increase in the glutathione content in all tested cell types.
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89 (1996) 131-137
0
135
25
5.0
7.5
IO
Cadmium Gmcentrabon t,,M) Fig. 3. Intracellular accumulation of cadmium in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples. All S.E. values are smaller than the symbols.
4. Discussion
0
1, 0
23
5.0
7.5
IO
CadmiLcn Concentration (,,M) Fig. 2. Leakage of lactate dehydrogenase from vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are mean f S.E. of four samples. Significantly different from the corresponding control, **P < 0.01; ***p < 0.001.
It has been suggested that one of the targets of cadmium toxicity is the vascular tissue [1,2], however, toxicity of the metal at the cell level has not been clear. In the present study, we morphologically demonstrated that vascular smooth-muscle cells sensitively respond to the cytotoxicity of cadmium in a culture system. The sensitive response was supported by a marked leakage of lactate dehydrogenase from the cells. These results clearly indicated that vascular smooth-muscle cells may be a principal target of cadmium in the vascular tissue as well as vascular endothelial cells [4-71. The monolayer of endothelial cells was destroyed by cadmium without a marked leakage of lactate dehydrogenase. In contrast, cadmium at 10 uM induced a marked leakage of the enzyme from LLC-PKl cells but their monolayer was maintained. This apparent discrepancy may indicate
T. Kaji rt al. i Toxicology Letters 89 (1996) 131-137
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the possibility that LLC-PKl cells repair the monolayer after the cell damage caused by cadmium much more actively than endothelial cells do. In other words, a sensitivity of endothelial cell monolayer to cadmium [4] may be due to a lower ability of the repair of damaged monolayer. The most sensitive cell type to the cytotoxicity of cadmium was vascular smooth-muscle cells in tested three cell types. It was shown that intracellular accumulation of cadmium was higher in vascular smooth-muscle cells and endothelial cells than in LLC-PKl cells; in contrast, metallothionein induction was very slight in vascular smooth-muscle cells. It is thus suggested that the considerable part of intracellular cadmium would not be sequestered by metallothionein and would exist as the toxic form in both the particulate fraction [16] and the high molecular weight fraction of the cytosol fraction [17] in vascular smooth-muscle cells. In LLC-PKl cells, an active induction of metallothionein by lower accumulation of intracellular cadmium would help the maintenance of their monolayer even if the concentration of the metal in the medium was high. Severe damage of endothelial cell layer may be due to a low inducibility of metallothionein with a high accumulation of intracellular cadmium when
3
t
0
1
t
1
0
5
lo
0
5
m
0
5
10
cadmiunConcamalion(rM)
Fig. 4 1ntracellular accumulation of metallothionein in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples. All S.E. values are smaller than the symbols.
l Wsc&
Smooth-Mmck
*** ”
*** LLC-PKl
0
Cedk
*** cl
c&k
Lb 0
8 25
5.0
75
10
Cadmiim Concentration @4) Fig. 5. Intracellular accumulation of reduced glutathione in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples, All S.E. values are smaller than the symbols. Significantly different from the corresponding control, *P < 0.05; **P < 0.01; ***p < 0.001.
the cadmium concentration in the medium was 5 uM or more. Furthermore, although the possibility that cadmium cytotoxicity may be at least partly due to lipid peroxidation [18] cannot be excluded, it is unlikely that the sensitive response of vascular smooth-muscle cells to cadmium may result from a decrease in cellular reduced glutathione which is a potent scavenger of free radicals. We previously reported that zinc, a potent and physiological inducer of metallothionein, is not effective for cultured vascular endothelial cells [19]. Also in vascular smooth-muscle cells, zinc as well as cadmium induced metallothionein only slightly at 200 uM or less (data not shown). The physiological significance of the lower inducibility of metallothionein in vascular-composing cell types is not evident, however, an assumption can be made that metallothionein may play a unique role in vascular smooth-muscle cells as well as endothelial cells [20] such as scavenging vasodila-
T. Kaji et al. / Toxicology Letters 89 (1996) 131-137
tor nitric oxide [21]. However, this remains to be elucidated. The present data suggested that vascular smooth-muscle cells are sensitive to cadmium cytotoxicity, mainly due to a low inducibility of metallothionein. Induction of the protein in vascular-composing cells should be investigated further. Consequently, it was suggested that vascular smooth-muscle cells as well as endothelial cells may be a critical target of cadmium toxicity. Cadmium may cause a functional damage of vascular smooth-muscle cells at much lower levels.
References 111Carroll, R.E. (1966) The relationship of cadmium in the air to cardiovascular disease death rates. J. Am. Med. Assoc. 198, 267-269. 121Revis, N.W., Zinsmeister, A.R. and Bull, R. (1981) Atherosclerosis and hypertension induction by lead and cadmium ions: an effect prevented by calcium ion. Proc. Natl. Acad. Sci. L’SA 78, 649446498. [31 Nolan, C.V. and Shaikh, Z.A. (1986) The vascular endothelium as a target tissue in acute cadmium toxicity. Life Sci. 39, 1403--1409. [41 Kaji, T., Mishima, A., Yamamoto, C., Sakamoto, M. and Koizumi, F. (19921 Effect of cadmium on the monolayer maintenance of vascular endothelial cells in culture. Toxicology 11, 261-276. 151Kaji, T., Ohkawara, S., Inada, M., Yamamoto, C., Sakamoto, M. and Kozuka, H. (1994) Cadmium stimulation of glycosaminoglycan synthesis by cultured vascular endothelial cells: comparison of various cell types. Biol. Pharm. Bull. 17, 454-451. PI Kaji, T., Ohkawara, S., Yamamoto, C., Sakamoto, M. and Kozuka, H. (1994) Cadmium-induced alteration of glycosaminoglycam with an enhancement of heparin-like activity in cultured vascular endothelial cells. Toxicology 94, 161-171. 171Yamamoto, C., Kaji, T., Sakamoto, M. and Kozuka, H. (1993) Cadmium ,stimulation of plasminogen activator inhibitor-l release from human vascular endothelial cells in culture. Toxicology 83, 215-223. PI Niwa, A. and Suzuki, A. (1982) Effect of cadmium on the tension of isolated rat aorta (a possible mechanism for
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cadmium-induced hypertension). J. Toxicol. Sci. 7, 51-60. [91 Evans, D.H. and Weingarten, K. (1990) The effect of cadmium and other metals on vascular smooth muscle of the dog shark, Saualus acanthias. Toxicology 61, 275281. UOI Schroeder, H.A. and Vinton, W.H. (1962) Hypertension induced in rats by small dose of cadmium. Am. J. Physiol. 202, 515-518. [ill Blazka, M.E. and Shaikh, Z.A. (1991) Differences in cadmium and mercury uptakes by hepatocytes: role of calcium channels. Toxicol. Appl. Pharmacol. 110, 355363. WI Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 1131Onosaka, S., Tanaka, K., Doi, M. and Okahara, K. (1978) A simplified procedure for determination of metallothionein in animal tissue. Eisei Kagaku 24, 128- 131. v41 Cohn, V.H. and Lyle, J. (1966) A fluorometric assay for glutathione. Anal. Biochem. 14, 4344440. P51 Singhal, R.K., Anderson, M.E. and Melster, A. (1987) Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1, 220-223. 1161Goering, P.L. and Klaassen, C.D. (1984) Zinc-induced tolerance to cadmium hepatotoxicity. Toxicol. Appl. Pharmacol. 74, 299-307. u71 Kudo, N., Yamashita, S. and Waku, K. (1986) Protection against cadmium toxicity by zinc: decrease in the Cd-high molecular weight protein fraction in rats liver and kidney on Zn pretreatment. Toxicology 40, 267-277. 1181Jamall, IS. and Smith, J.C. (1985) Effects of cadmium on glutathione peroxidase, superoxide dismutase, and lipid peroxidation in rat heart: a possible mechanism of cadmium cardiotoxicity. Toxicol. Appl. Pharmacol. 80, 333 42. [I91 Kaji, T., Mishima, A., Koyanagi, E., Yamamoto, C., Sakamoto, M. and Kozuka, H. (1992) Possible mechanism for zinc protection against cadmium cytotoxicity in cultured vascular endothelial cells. Toxicology 76, 257270. PO1 Kaji, T., Yamamoto, C., Tsubaki, S., Ohkawara, S., Sakamoto, M., Sato, M. and Kozuka, H. (1993) Metallothionein induction by cadmium, cytokines, thrombin and endothelin-1 in cultured vascular endothelial cells. Life Sci. 53, 1185-1191. [21] Palmer, R.M., Ferrige, A.G. and Moncada, S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524526.
Toxicology Letters 89 (1996) 13 I - 137
Sensitive response of cultured vascular smooth-muscle cells to cadmium cytotoxicity: comparison with cultured vascular endothelial cells and kidney epithelial LLC-PKl cells Toshiyuki KajP,*, Megumi Suzuki”, Chika Yamamotoa, Yumiko Imaki”, Sawako Miyajima”, Yasuyuki Fujiwara”, Michiko Sakamotoa, Hiroshi Kozukab aDepartment of E,avironmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920- 11, Japan bDepartment of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
Received 20 March 1996; accepted 2 July 1996
Abstract Response to cadmium cytotoxicity of cultured bovine aortic smooth-muscle cells was compared with that of cultured bovine aortic endothelial cells and porcine kidney epithelial LLC-PKl cells. The cell damage was evaluated by morphology and the lactate dehydrogenase leakage assay. It was found that vascular smooth-muscle cells are markedly sensitive to cadmium cytotoxicity. The accumulation of intracellular cadmium was much higher but that of metallothionein was much less in vascular smooth-muscle cells than in LLC-PKl cells; vascular endothelial cells were in between vascular smooth-muscle cells and LLC-PKl cells. The content of reduced glutathione was slightly increased by cadmium in all three cell types. The present data suggest that a much lower inducibility of metallothionein with a high accumulation of intracellular cadmium in vascular smooth-muscle cells resulted in a marked sensitivity of the cells to cadmium cytotoxicity. Vascular smooth-muscle cells may be one of the critical target of cadmium toxicity. Keywords:
Cadmium;
* Corresponding 0378-4274/96/$15.00
Cytotoxicity;
Endothelial
cells; Metallothionein;
author. 0 1996 Elsevier Science Ireland Ltd. All rights reserved
PII SO378-4274(96)03797-6
Smooth
muscle cells; Vascular
132
T. Kaji et al. / Toxicology
1. Introduction
It has been suggested that cadmium in the environment may be a causal factor in the cardio -vascular diseases. An epidemiologic study showed that there is a relationship between environmental cadmium and vascular diseases [I]. In experimental animals, the metal induces atherosclerosis and hypertension [2]. Nolan and Shaikh [3] suggested that the initial effect of acute cadmium administration is on the integrity and permeability of the vascular endothelium; other necrotic changes of tissue occur secondarily. We also hypothesized that the first target of cadmium in the vascular tissue is vascular endothelial cells. It was found that cadmium destroys the monolayer of cultured endothelial cells [4]. The metal induces the synthesis of glycosaminoglycans [5] with an enhancement of heparin-like activity on the cell surface [6] and the release of plasminogen activator inhibitor type 1 from the cells [7]. These results clearly indicated that functional damage of endothelial cells may be an important component of cadmium-induced vascular lesions. On the other hand, cadmium is capable of acting on vascular smooth-muscle cells. The metal causes vasoconstriction in vitro [8,9] and hypertension in vivo [IO]. However, the exhibition of cadmium toxicity at the flvascular smooth-muscle cell level is not necessarily clear. The purpose of the present study is to clarify the sensitivity of vascular smooth-muscle cells to cadmium cytotoxicity using a cell culture system.
2. Materials and methods 2.1. Materials Vascular smooth-muscle cells and endothelial cells derived from bovine aorta were gifts from Drs. Yutaka Nakashima and Katsuo Sueishi (First Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan). Epithelial LLC-PKl cells derived from porcine kidney were purchased from American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s
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89 (1996) 131-137
medium and ASF 301 medium were from Nissui Pharmaceutical (Tokyo, Japan) and Ajinomoto (Tokyo, Japan), respectively. Fetal bovine serum was obtained from Whittaker (Warkersville, MD). Tissue culture dishes and plates were from Corning (Corning, NY). Cadmium chloride, bovine hemoglobin, the lactate dehydrogenase kit and other reagents were purchased from Wako Purechemical Industries (Osaka, Japan). 2.2. Morphology Vascular endothelial cells, vascular smooth-muscle cells and LLC-PKI ceils were each cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in 6-well plates at 37°C in a humid atmosphere of 5% CO, in air until confluent (confluent cultures). In another experiment, the three cell types were each plated at 1 x lo5 cells/well into 6-well plates and cultured for 24 h in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (growing cultures). After culture, the cells were washed twice with serum-free ASF 301 medium and incubated at 37°C for 24 h in 1 ml of fresh ASF 301 medium in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 PM. The medium was then discarded and the cells were gently washed with Ca, Mg-free phosphate buffered saline (CMF-PBS). The cells were fixed with methanol and stained with Giemsa. 2.3. Biochemical evaluation of cell damage Confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were prepared in 24-well culture plates and treated with cadmium chloride at 0.5, 1, 2, 5 or 10 PM for 24 h in 0.25 ml of serum-free ASF 301 medium. The conditioned medium was collected and analyzed for the activity of lactate dehydrogenase as a marker of nonspecific cell damage. 2.4. Determination of intracellular cadmium Confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were each treated with cadmium chloride at 0.5, 1, 2, 5 or 10 PM for 24 h in serum-free ASF 301
T. Kaji et al. / Toxicology
medium in 6-well culture plates. After treatment, the medium was discarded and the cells were washed twice with CMF-PBS. The cells were next washed twice with ice-cold 2 mM EGTA solution to remove cadmium loosely bound to the cell surface [l l] and extracted with 1 ml of 0.1 M acetate buffer solution (pH 5.5) containing 10 mM EDTA at 4°C for 72 h. The extract was collected and the cells were gently washed with 1 ml of the buffer; the wash was combined with the collected extract. The extract was directly analyzed for cadmium content by flameless atomic absorption spectrophotometry (Nippon Jarrell Ash, AA8500). The cells after extraction were scraped off with a rubber policeman in the presence of 0.75 ml of CMF-PBS. After collection of the cell suspension, the well was washed with 0.75 ml of CMF-PBS; the wash was combined with the cell suspension. The cell homogenate was prepared by sonication and an aliquot was used for the determination of protein content by the method of Lowry et al. [ 121. The accumulation of intracellular cadmium was expressed as nmolimg protein. 2.5. Determination glutathione
of metallothionein
and reduced
Vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells were cultured until confluent and treated with cadmium chloride at 0.5, 1, 2, 5 or 10 uM for 24 h in serum-free ASF 301 medium in 60-mm dishes. After treatment, the medium was discarded and the cells were washed twice with CMF-PBS. The cells were then scraped off with a rubber policeman in the presence of 1 ml of 0.25 M sucrose. The dish was washed with 1 ml of 0.25 M sucrose and the wash was combined with the cell suspension. The cell homogenate was prepared by sonication and used for the determination of metallothionein content by the cadmiumhemoglobin method [13] with some modifications as follows: A portion of the cell homogenate (0.5 ml) was transferred into a microtube and both 0.5 ml of 0.1 M Tris-H:Cl buffer solution (pH 8.0) and 0.05 ml of 10 ug/ml cadmium chloride solution were added. After -incubation for 10 min at room temperature, 0.1 ml of 2% bovine hemoglobin was added and the mixture was boiled for 2 min; the
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tube was centrifuged at 10 000 x g for 5 min. The addition of hemoglobin, boiling and the centrifugation were performed three times. The supernatant was analyzed for cadmium content by flameless atomic absorption spectrophotometry and the metallothionein content was calculated; another portion (0.3 ml) of the cell homogenate was used for the determination of protein content; the remainder was analyzed for the content of reduced glutathione by the fluorometric assay [14]. 2.6. Statistical
analysis
Data were analyzed for statistical significance by Student’s t-test. P values of less than 0.05 were considered to indicate statistically significant differences.
3. Results Fig. 1 shows the morphology of confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium at 10 uM or less for 24 h. In endothelial cells, the cell number was markedly decreased by cadmium at 5 uM or more; a necrotic change was extensively observed at 10 PM. In vascular smooth-muscle cells, a degenerative change was caused by cadmium at 1 uM or more; a severe damage of the cells was observed at 2 uM or more. In LLC-PKl cells, no destructive change was observed in the cell layer even after exposure to cadmium; damage of the cells was not severe. These observations were similar in growing cultures of the corresponding cell types (data not shown). Fig. 2 shows the leakage of lactate dehydrogenase into the medium from vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after a 24-h incubation. The highest and marked leakage of the enzyme was observed in vascular smooth-muscle cells after exposure to cadmium at 0.5 uM or more, supporting the morphological observation. In endothelial cells, the leakage was significantly increased by cadmium at 5 uM or more; this change was also consistent with the morphology. In LLC-PKl
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T. Kaji et al. / Toxicology Letters 89 (1996) 131-137
Fig. 1. Morphology of confluent cultures of vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium chloride at 0.5, 1, 2, 5 or 10 uM for 24 h. Al, endothelial cells (control); A2, endothelial cells treated with 0.5 nM cadmium; A3, endothelial cells treated with 1 nM cadmium; A4, endothelial cells treated with 2 uM cadmium; A5, endothelial cells treated with 5 )IM cadmium; A6, endothelial cells treated with 10 nM cadmium; Bl, vascular smooth-muscle cells (control); B2, vascular smooth-muscle cells treated with 0.5 nM cadmium; B3, vascular smooth-muscle cells treated with 1 uM cadmium; B4, vascular smooth-muscle cells treated with 2 nM cadmium; B5, vascular smooth-muscle cells treated with 5 uM cadmium; B6, vascular smooth-muscle cells treated with 10 uM cadmium; Cl, LLC-PKl cells (control); C2, LLC-PKl cells treated with 0.5 nM cadmium; C3, LLC-PKl cells treated with 1 uM cadmium; C4, LLC-PKl cells treated with 2 uM cadmium; C5, LLC-PKl cells treated with 5 nM cadmium; C6, LLC-PKl cells treated with 10 nM cadmium.
cells, a significant increase in the leakage was caused by cadmium at 5 uM or more. At 10 PM, lactate dehydrogenase was leaked more from LLC-PKl cells than from vascular endothelial cells. The rH]adenine release assay, which sensitively detects the damage of endothelial cells, also indicated that cadmium at 10 uM exhibited cytotoxicity more severely in LLC-PKl cells than in endothelial cells (data not shown).
The intracellular accumulation of cadmium is an important factor of exhibiting cytotoxicity of the metal. As shown in Fig. 3, cadmium markedly accumulated in vascular smooth-muscle cells in comparison with in LLC-PKl cells. The accumulation of the metal within endothelial cells was in between vascular smooth-muscle cells and LLCPKl cells. Fig. 4 shows the intracellular accumulation of
T. Kaji et al. / Toxicology
metallothionein in vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium for 24 h. Endothelial cells greatly responded to cadmium at 2 uM or less; however, at .5 PM, metallothionein content was not increased further. In vascular smoothmuscle cells, cadmium at 2 uM or less induced metallothionein only slightly. Since a severe damage was seen in thl: cells treated with cadmium at 5 uM or more, metallothionein induction was not examined at these concentrations. In contrast, cadmium at 10 uM. or less strongly induced metallothionein in a concentration-dependent manner. Finally, the effect of cadmium on the content of cellular reduced glutathione was examined since this substance is capable of protection against the cytotoxicity of the metal [15]. As shown in Fig. 5, cadmium treatment resulted in a slight but significant increase in the glutathione content in all tested cell types.
Letters
89 (1996) 131-137
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Cadmium Gmcentrabon t,,M) Fig. 3. Intracellular accumulation of cadmium in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples. All S.E. values are smaller than the symbols.
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CadmiLcn Concentration (,,M) Fig. 2. Leakage of lactate dehydrogenase from vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are mean f S.E. of four samples. Significantly different from the corresponding control, **P < 0.01; ***p < 0.001.
It has been suggested that one of the targets of cadmium toxicity is the vascular tissue [1,2], however, toxicity of the metal at the cell level has not been clear. In the present study, we morphologically demonstrated that vascular smooth-muscle cells sensitively respond to the cytotoxicity of cadmium in a culture system. The sensitive response was supported by a marked leakage of lactate dehydrogenase from the cells. These results clearly indicated that vascular smooth-muscle cells may be a principal target of cadmium in the vascular tissue as well as vascular endothelial cells [4-71. The monolayer of endothelial cells was destroyed by cadmium without a marked leakage of lactate dehydrogenase. In contrast, cadmium at 10 uM induced a marked leakage of the enzyme from LLC-PKl cells but their monolayer was maintained. This apparent discrepancy may indicate
T. Kaji rt al. i Toxicology Letters 89 (1996) 131-137
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the possibility that LLC-PKl cells repair the monolayer after the cell damage caused by cadmium much more actively than endothelial cells do. In other words, a sensitivity of endothelial cell monolayer to cadmium [4] may be due to a lower ability of the repair of damaged monolayer. The most sensitive cell type to the cytotoxicity of cadmium was vascular smooth-muscle cells in tested three cell types. It was shown that intracellular accumulation of cadmium was higher in vascular smooth-muscle cells and endothelial cells than in LLC-PKl cells; in contrast, metallothionein induction was very slight in vascular smooth-muscle cells. It is thus suggested that the considerable part of intracellular cadmium would not be sequestered by metallothionein and would exist as the toxic form in both the particulate fraction [16] and the high molecular weight fraction of the cytosol fraction [17] in vascular smooth-muscle cells. In LLC-PKl cells, an active induction of metallothionein by lower accumulation of intracellular cadmium would help the maintenance of their monolayer even if the concentration of the metal in the medium was high. Severe damage of endothelial cell layer may be due to a low inducibility of metallothionein with a high accumulation of intracellular cadmium when
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Fig. 4 1ntracellular accumulation of metallothionein in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples. All S.E. values are smaller than the symbols.
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Cadmiim Concentration @4) Fig. 5. Intracellular accumulation of reduced glutathione in cultured vascular endothelial cells, vascular smooth-muscle cells and LLC-PKl cells after exposure to cadmium. Confluent cultures of bovine aortic endothelial cells, bovine aortic smooth-muscle cells and porcine kidney epithelial LLC-PKl cells were each incubated at 37°C for 24 h in the presence of cadmium chloride at 0.5, 1, 2, 5 or 10 uM. Values are means of five samples, All S.E. values are smaller than the symbols. Significantly different from the corresponding control, *P < 0.05; **P < 0.01; ***p < 0.001.
the cadmium concentration in the medium was 5 uM or more. Furthermore, although the possibility that cadmium cytotoxicity may be at least partly due to lipid peroxidation [18] cannot be excluded, it is unlikely that the sensitive response of vascular smooth-muscle cells to cadmium may result from a decrease in cellular reduced glutathione which is a potent scavenger of free radicals. We previously reported that zinc, a potent and physiological inducer of metallothionein, is not effective for cultured vascular endothelial cells [19]. Also in vascular smooth-muscle cells, zinc as well as cadmium induced metallothionein only slightly at 200 uM or less (data not shown). The physiological significance of the lower inducibility of metallothionein in vascular-composing cell types is not evident, however, an assumption can be made that metallothionein may play a unique role in vascular smooth-muscle cells as well as endothelial cells [20] such as scavenging vasodila-
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tor nitric oxide [21]. However, this remains to be elucidated. The present data suggested that vascular smooth-muscle cells are sensitive to cadmium cytotoxicity, mainly due to a low inducibility of metallothionein. Induction of the protein in vascular-composing cells should be investigated further. Consequently, it was suggested that vascular smooth-muscle cells as well as endothelial cells may be a critical target of cadmium toxicity. Cadmium may cause a functional damage of vascular smooth-muscle cells at much lower levels.
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