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Consecutive uptake of cadmium by KB cells in culture
TOXICOLOGY
AND
APPLIED
Consecutive
PHARMACOLOGY
Uptake
46,807-8 10 (1978)
of Cadmium
by KB Cells in Culture
Consecutive Uptake of Cadmium by KB Cells in Culture. MESHITSIJKA, S., AND ISHIZAWA, Toxicol. Appl. Pharmacol. 46,807-810. The uptake of cadmium ions by KB cells in culture has been analyzed by atomic absorption spectrophotometry in the course of the culture. The increase of the amount of Cd*+ taken up into the cells corresponded to the decrease of Cd*+ in the medium. The uptake of Cd*+ was initiated soon after the addition and ceased to reach a stationary state after approximately 10 hr. A consecutive uptake of Cd’+ was observed and Cd*+ accumulated; however, the cells continued to survive. M. (1978).
The influence of trace elements on cells in culture has been studied by several investigators. These studies can be divided into two groups: one is to reveal the specific roles of trace metals in the biological systems (Rubin, 1972, 1975; Waters et al., 1970; Thomas and Johnson, 1967); the other concerns the toxicity of foreign metals (Fischer, 1975, 1976; Van Giessen et al., 1973; Kono et al., 1976; Kajikawa et al., 1972; Waters et al., 1974; Brun and Brunk, 1974). Both groups of studies are concerned with the uptake of metals. The process of metal uptake, however, was not extensively studied toxicologically using the cells in culture (Kailis and Morgan, 1977; Schwarz and Matrone, 1975; Kalckar, 1976). Cadmium is one of the most important metals in the pollution of the environment (Friberg et al., 1974). In this communication, the consecutive uptake of Cd2+ and its accumulation by KB cells in culture are reported. METHODS
A human epidermoid carcinoma cell line, KB, was obtained from Dr. G. Kimura (Cancer Research Institute, Kyushu University). The culture medium was Eagle’s MEM (Daigoeiyokagaku Co., Ltd.) supplemented with 2% newborn calf serum (Microbiological Associates) and 100 units/ml of penicillin and 100 #g/ml of streptomycin. KB cells, 5 x 104, in 2 ml of culture medium were distributed in each tube (15 x 150 mm). Incubation was performed at 37OC by the stationary culture method. After an interval of several hours, further incubation of the replicate culture was undertaken. The growth of the cells was measured by the amount of protein produced, using Lowry’s method (Lowry et al., 1951). The culture medium was replaced with a fresh medium containing Cd2+ after an incubation period of 48 hr. Following that, the change in the amount of Cd*+ absorbed into the cells and the concentration of Cd*+ in the culture medium were analyzed for each harvested tube. Four replicate tubes were used for each harvest. Cd salts such as Cd(NO,),, CdSO,, and CdCl, of special grade (Wako Pure Chem. Ind. Ltd.) were used. The analysis procedure was as follows: The medium was filtrated into a clean tube, The residual cells attached to the wall of the tube 807
ooO1-008X/78/0463-0807%02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
808
SHORT
COMMUNICATIONS
were washed twice by phosphate-buffered saline having no Ca and Mg ions [PBS(-)] and then immersed in 1.5 ml of 1.0% NaOH solution. After the cells were lysed, 0.5 ml of 10% HNO, was added. The concentration of Cd*+ in each medium and the cell lysate was measured with a Hitachi 20’7 atomic absorption spectrophotometer (Friberg et al., 1974). RESULTS AND DISCUSSION The uptake of Cd’+ began immediately after the addition of Cd2+, without any induction period. For the first several hours, the concentration in the medium decreased linearly and reached a stationary state in about 10 hr. The decrease of Cd2+ concentration in the culture medium corresponded to an increase of Cd*+ in the cells, although there was a small loss of cells in the course of transferring the medium and washing cells with PBS(-), The uptakes of Cd2+ were similar in the concentration range of 0.2 to 0.6 ppm of Cdz+. Both the uptake of CdZf and the growth inhibition by CdZ+ were not influenced by counterions of Cd ‘+. Growth of the cells in the medium containing Cd2+ was inhibited and differed distinctively from that of control cells 1 day after adding Cd 2f. The higher the concentration of Cd2+ in the medium, the more severe the damage to the cells.
FIG. 1. Consecutive uptake of Cd 2+by KB cells. To each culture tube 0.5 ppm of Cd2+ was added (arrow). The change of Cd2+ concentration in culture medium (+-) and the change of Cd2+ content in KB cells in a tube (A-) were observed. Soon after the addition of Cd*+ there was a rapid decrease in Cd’+ concentration in the medium. The decrease of Cdr+ concentration in medium corresponded to the increase of Cd”+ content in cells.
SHORT
COMMUNICATIONS
809
The uptake of Cd*+ by KB cells was observed several times for cells in the same tubes, as is shown in Fig. 1. Ten or 20 hr after the initial addition of Cd2+ (0.5 ppm), the concentration of Cd*+ became constant. On adding Cd2+ the uptake of Cd2+ started again. This process took place repeatedly, each time Cd2+ was added to fresh growth medium. When the medium was not changed at the time of a further addition of Cd2+, the uptake again was observed to take place repeatedly. The amount of Cd2+ in the cells was analyzed in lysate of cells for each set of replicates. Since the growth of cells was inhibited by the addition of Cd2+, the amount of protein in the cells in the tubes changed slightly. Therefore, Cd2+ accumulated in a stepwise manner in the cells. When KB cells, cultured with Cd2+, were incubated in clean growth medium without Cd2+, the cells beg an to multiply again, but no release of Cd2+ from the cells was observed. It seems likely that Cd2+ was taken up into the cells and fixed somewhere within the cells. No zinc ion was taken up by KB cells from the medium under the same experimental conditions; the concentration of Zn2+ was measured by atomic absorption spectrophotometry. The absorption of 65Zn ion in mouse 3T3 cells was reported by Schwarz and Matrone (1975); however, the amount of Zn2+ in cells was negligible compared with that of Cd2+ in our experiments. Although Zn2+ and Cd2+ are very similar in the chemical behavior, KB cells clearly distinguish Zn2+ from Cd2+. REFERENCES H., AND BRIJNK, LJ. (1974). Leadinducedinjury of in vitro cultured rat fibroblasts.Acta Pathol. Microbial. Stand. 82, 3 11-3 18. FISCHER,A. B. (1975). The effect of leadon cellscultivated in vitro. I. Acute effect. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Abt. 1 Orig. B 161, 26-37. FISCHER,A. B. (1976). The effect of lead on cellscultivated in vitro. II. Chronic exposureand developmentof resistance.Zentralbl. Bakteriol. Parasitenk. Infektionskr. Abt. I Orig. B 161, 3 17-330. FRIBERG,L., PISCATOR, M., NORDBERG, G. F., ANDKJELLSTROEM, T. (1974). Cadmium in the Environment, 2nd ed.CRC Press,Cleveland. BRUN,
KAILIS, S. G., ANDMORGAN,E. H. (1977).Iron uptakeby immatureerythroid cellsmechanism of dependence on metabolicenergy.Biochim. Biophys. Acta 464,389-398. KAJIKAWA, M., SUZUKI,N., SUZUE,R., ANDKAWADA, S. (1972). Detection of heavy metal toxicity by cell culturemethod(1). Japan. J. Nutr. 30, 159- 162. KALCKAR,H. M. (1976).Cellularregulationof transportanduptakeof nutrient: An overview.1. Ceil. Comp. Physiol. 89, 503-5 16.
KONO, E., IWATA, S., IKEBUCHI,M., AND HORIGUCHI,S. (1976). Modifying effects of inactivated bovine serumof the acutetoxicity of HgClCH,. Japan. J. Ind. Health 18, 502506.
LOWRY,0. H., RO~EBROUGH, N. .I., FARR, A. L., AND RANDALL,R. J. (1951). Protein measurement with the Folin phenolreagent.J. Biol. Chem. 193,265-275. RUBIN,H. (1972).Inhibition of DNA synthesisin animalcellsby ethylenediaminetetraacetate andits reversalby zinc. Proc. Nat. Acad. Sci. USA 69, 7 12-716. RUBIN, H. (1975). Nonspecificnature of the stimulusto DNA synthesesin culturesof chick embryocells.Proc. Nat. Acad. Sci. USA 72, 1676-1680. SCHWARZ, F. J., AND MATRONE, G. (1975). Methodological studieson the uptake of Zn by 3T3 cells.Proc. Sot. Exp. Biol. Med. 149,888-892. THOMAS,J. A., ANDJOHNSON, M. J. (1967). Trace-metalrequirementsof NCTC clone 929 strainL cells.J. Nat. Cancer Inst. 39, 337-345.
810
SHORT
COMMUNICATIONS
GIESSEN, G. J., GRIM, J. A., PETERING, D. H., AND PETERING, H. G. (1973). Effect of heavy metals on the in vitro cytotoxicity of 3-ethoxy-2-oxobuthylaldehyde.J. Nat. Cancer Inst. 51, 139-146. WATERS, M. D., MOORE,R. D., AMATO, J. J., AND HOUK, J. C. (1970). Zinc sulfate-failureas an accelerationof collagenbiosynthesisand iibroblast proliferation. Proc. Sot. Exp. BioE. VAN
Med. 138,313-311. M. D., GARDNER, D. E., AND COFFIN, D. L. (1974). Cytotoxic effect of vanadiumon rabbit alveolarmacrophages in vitro. Toxicol. Appl. Pharmacol. 28,253-263.
WATERS,
SHUNSUKE MESHITSUKA MASAICHI ISHIZAWA
Department of Public Health Tottori University School of Medicine Yonago 683, Japan Received February
16,1978;
accepted August I7,1978
AND
APPLIED
Consecutive
PHARMACOLOGY
Uptake
46,807-8 10 (1978)
of Cadmium
by KB Cells in Culture
Consecutive Uptake of Cadmium by KB Cells in Culture. MESHITSIJKA, S., AND ISHIZAWA, Toxicol. Appl. Pharmacol. 46,807-810. The uptake of cadmium ions by KB cells in culture has been analyzed by atomic absorption spectrophotometry in the course of the culture. The increase of the amount of Cd*+ taken up into the cells corresponded to the decrease of Cd*+ in the medium. The uptake of Cd*+ was initiated soon after the addition and ceased to reach a stationary state after approximately 10 hr. A consecutive uptake of Cd’+ was observed and Cd*+ accumulated; however, the cells continued to survive. M. (1978).
The influence of trace elements on cells in culture has been studied by several investigators. These studies can be divided into two groups: one is to reveal the specific roles of trace metals in the biological systems (Rubin, 1972, 1975; Waters et al., 1970; Thomas and Johnson, 1967); the other concerns the toxicity of foreign metals (Fischer, 1975, 1976; Van Giessen et al., 1973; Kono et al., 1976; Kajikawa et al., 1972; Waters et al., 1974; Brun and Brunk, 1974). Both groups of studies are concerned with the uptake of metals. The process of metal uptake, however, was not extensively studied toxicologically using the cells in culture (Kailis and Morgan, 1977; Schwarz and Matrone, 1975; Kalckar, 1976). Cadmium is one of the most important metals in the pollution of the environment (Friberg et al., 1974). In this communication, the consecutive uptake of Cd2+ and its accumulation by KB cells in culture are reported. METHODS
A human epidermoid carcinoma cell line, KB, was obtained from Dr. G. Kimura (Cancer Research Institute, Kyushu University). The culture medium was Eagle’s MEM (Daigoeiyokagaku Co., Ltd.) supplemented with 2% newborn calf serum (Microbiological Associates) and 100 units/ml of penicillin and 100 #g/ml of streptomycin. KB cells, 5 x 104, in 2 ml of culture medium were distributed in each tube (15 x 150 mm). Incubation was performed at 37OC by the stationary culture method. After an interval of several hours, further incubation of the replicate culture was undertaken. The growth of the cells was measured by the amount of protein produced, using Lowry’s method (Lowry et al., 1951). The culture medium was replaced with a fresh medium containing Cd2+ after an incubation period of 48 hr. Following that, the change in the amount of Cd*+ absorbed into the cells and the concentration of Cd*+ in the culture medium were analyzed for each harvested tube. Four replicate tubes were used for each harvest. Cd salts such as Cd(NO,),, CdSO,, and CdCl, of special grade (Wako Pure Chem. Ind. Ltd.) were used. The analysis procedure was as follows: The medium was filtrated into a clean tube, The residual cells attached to the wall of the tube 807
ooO1-008X/78/0463-0807%02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
808
SHORT
COMMUNICATIONS
were washed twice by phosphate-buffered saline having no Ca and Mg ions [PBS(-)] and then immersed in 1.5 ml of 1.0% NaOH solution. After the cells were lysed, 0.5 ml of 10% HNO, was added. The concentration of Cd*+ in each medium and the cell lysate was measured with a Hitachi 20’7 atomic absorption spectrophotometer (Friberg et al., 1974). RESULTS AND DISCUSSION The uptake of Cd’+ began immediately after the addition of Cd2+, without any induction period. For the first several hours, the concentration in the medium decreased linearly and reached a stationary state in about 10 hr. The decrease of Cd2+ concentration in the culture medium corresponded to an increase of Cd*+ in the cells, although there was a small loss of cells in the course of transferring the medium and washing cells with PBS(-), The uptakes of Cd2+ were similar in the concentration range of 0.2 to 0.6 ppm of Cdz+. Both the uptake of CdZf and the growth inhibition by CdZ+ were not influenced by counterions of Cd ‘+. Growth of the cells in the medium containing Cd2+ was inhibited and differed distinctively from that of control cells 1 day after adding Cd 2f. The higher the concentration of Cd2+ in the medium, the more severe the damage to the cells.
FIG. 1. Consecutive uptake of Cd 2+by KB cells. To each culture tube 0.5 ppm of Cd2+ was added (arrow). The change of Cd2+ concentration in culture medium (+-) and the change of Cd2+ content in KB cells in a tube (A-) were observed. Soon after the addition of Cd*+ there was a rapid decrease in Cd’+ concentration in the medium. The decrease of Cdr+ concentration in medium corresponded to the increase of Cd”+ content in cells.
SHORT
COMMUNICATIONS
809
The uptake of Cd*+ by KB cells was observed several times for cells in the same tubes, as is shown in Fig. 1. Ten or 20 hr after the initial addition of Cd2+ (0.5 ppm), the concentration of Cd*+ became constant. On adding Cd2+ the uptake of Cd2+ started again. This process took place repeatedly, each time Cd2+ was added to fresh growth medium. When the medium was not changed at the time of a further addition of Cd2+, the uptake again was observed to take place repeatedly. The amount of Cd2+ in the cells was analyzed in lysate of cells for each set of replicates. Since the growth of cells was inhibited by the addition of Cd2+, the amount of protein in the cells in the tubes changed slightly. Therefore, Cd2+ accumulated in a stepwise manner in the cells. When KB cells, cultured with Cd2+, were incubated in clean growth medium without Cd2+, the cells beg an to multiply again, but no release of Cd2+ from the cells was observed. It seems likely that Cd2+ was taken up into the cells and fixed somewhere within the cells. No zinc ion was taken up by KB cells from the medium under the same experimental conditions; the concentration of Zn2+ was measured by atomic absorption spectrophotometry. The absorption of 65Zn ion in mouse 3T3 cells was reported by Schwarz and Matrone (1975); however, the amount of Zn2+ in cells was negligible compared with that of Cd2+ in our experiments. Although Zn2+ and Cd2+ are very similar in the chemical behavior, KB cells clearly distinguish Zn2+ from Cd2+. REFERENCES H., AND BRIJNK, LJ. (1974). Leadinducedinjury of in vitro cultured rat fibroblasts.Acta Pathol. Microbial. Stand. 82, 3 11-3 18. FISCHER,A. B. (1975). The effect of leadon cellscultivated in vitro. I. Acute effect. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Abt. 1 Orig. B 161, 26-37. FISCHER,A. B. (1976). The effect of lead on cellscultivated in vitro. II. Chronic exposureand developmentof resistance.Zentralbl. Bakteriol. Parasitenk. Infektionskr. Abt. I Orig. B 161, 3 17-330. FRIBERG,L., PISCATOR, M., NORDBERG, G. F., ANDKJELLSTROEM, T. (1974). Cadmium in the Environment, 2nd ed.CRC Press,Cleveland. BRUN,
KAILIS, S. G., ANDMORGAN,E. H. (1977).Iron uptakeby immatureerythroid cellsmechanism of dependence on metabolicenergy.Biochim. Biophys. Acta 464,389-398. KAJIKAWA, M., SUZUKI,N., SUZUE,R., ANDKAWADA, S. (1972). Detection of heavy metal toxicity by cell culturemethod(1). Japan. J. Nutr. 30, 159- 162. KALCKAR,H. M. (1976).Cellularregulationof transportanduptakeof nutrient: An overview.1. Ceil. Comp. Physiol. 89, 503-5 16.
KONO, E., IWATA, S., IKEBUCHI,M., AND HORIGUCHI,S. (1976). Modifying effects of inactivated bovine serumof the acutetoxicity of HgClCH,. Japan. J. Ind. Health 18, 502506.
LOWRY,0. H., RO~EBROUGH, N. .I., FARR, A. L., AND RANDALL,R. J. (1951). Protein measurement with the Folin phenolreagent.J. Biol. Chem. 193,265-275. RUBIN,H. (1972).Inhibition of DNA synthesisin animalcellsby ethylenediaminetetraacetate andits reversalby zinc. Proc. Nat. Acad. Sci. USA 69, 7 12-716. RUBIN, H. (1975). Nonspecificnature of the stimulusto DNA synthesesin culturesof chick embryocells.Proc. Nat. Acad. Sci. USA 72, 1676-1680. SCHWARZ, F. J., AND MATRONE, G. (1975). Methodological studieson the uptake of Zn by 3T3 cells.Proc. Sot. Exp. Biol. Med. 149,888-892. THOMAS,J. A., ANDJOHNSON, M. J. (1967). Trace-metalrequirementsof NCTC clone 929 strainL cells.J. Nat. Cancer Inst. 39, 337-345.
810
SHORT
COMMUNICATIONS
GIESSEN, G. J., GRIM, J. A., PETERING, D. H., AND PETERING, H. G. (1973). Effect of heavy metals on the in vitro cytotoxicity of 3-ethoxy-2-oxobuthylaldehyde.J. Nat. Cancer Inst. 51, 139-146. WATERS, M. D., MOORE,R. D., AMATO, J. J., AND HOUK, J. C. (1970). Zinc sulfate-failureas an accelerationof collagenbiosynthesisand iibroblast proliferation. Proc. Sot. Exp. BioE. VAN
Med. 138,313-311. M. D., GARDNER, D. E., AND COFFIN, D. L. (1974). Cytotoxic effect of vanadiumon rabbit alveolarmacrophages in vitro. Toxicol. Appl. Pharmacol. 28,253-263.
WATERS,
SHUNSUKE MESHITSUKA MASAICHI ISHIZAWA
Department of Public Health Tottori University School of Medicine Yonago 683, Japan Received February
16,1978;
accepted August I7,1978