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Comparison of the cytotoxic effects of cadmium chloride and cadmium-metallothionein in LLC-PK1 cells
Life Sciences, Vol. 53, pp. PL 337-342 Printed in the USA
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
COMPARISON OF T H E CYTOTOXIC EFFECTS OF CADMIUM C H L O R I D E AND C A D M I U M - M E T A L L O T H I O N E I N IN LLC-PK 1 CELLS Walter C. Prozialeck, Dawn R. Wellington and Peter C. Lamar Department of Pharmacology Midwestern University Downers Grove, IL 60515 (Submitted July 19, 1993; accepted August 2, 1993; received in final form September 9, 1993)
Abstract. Recent studies have shown that ionic cadmium (Cd 2÷) can selectively damage the tight junctions between LLC-PK 1 cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd 2+ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd 2+ for 1-4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24-48 hours of exposure. Additional results showed that the cells accumulated Cd 2+ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK~ cells, but that it can kill the cells after prolonged exposure.
Cadmium is an important industrial and environmental pollutant that damages a variety of organs, including the liver, kidney, lung, testis, and placenta (for reviews see 1,2). Although the general toxic effects of cadmium have been well-characterized, the mechanisms underlying many of these effects have yet to be elucidated. The observation that some of the toxic effects of cadmium seem to involve an increase in the permeability of various endothelial and epithelial surfaces (3,4) led us to examine the effects of cadmium on cells of the established porcine renal epithelial cell line, LLC-PK 1. This cell line exhibits some of the properties of the proximal tubular epithelium and has been used as a model system for physiologic and toxicologic studies (5-7). The results of our studies showed that ionic cadmium (Cd 2÷) selectively damages the junctions between LLC-PK 1 cells (4,8,9). Exposure to micromolar concentrations of CdC12 for 1-4 hours causes the cells to separate from each other without killing them. This effect coincides with a drop in the transepithelial electrical resistance and changes in the structure adhering and occluding junctional complexes (4,8). The objective of the work described in this report was to determine whether or not cadmium that is bound to metallothionein, a low molecular weight metal-binding protein that plays a key role in cadmium metabolism in vivo (10), can disrupt the junctions between LLCPK1 cells in the same manner as free Cd 2÷. This is an important issue because much of the Send correspondence to Walter C. Prozialeck. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-338
Effects of Cd2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
circulating cadmium in vivo is bound to metallothionein (10,11), and there is evidence to suggest that Cd-metallothionein conjugates may be directly responsible for some of the nephrotoxic effects of cadmium (10-13).
Methods Preparation and characterization of the Cd-metallothionein complex. Rabbit metallothionein II was obtained from Sigma Chemical Co. (St. Louis, MO). Apometallothionein was prepared by treating the Sigma metallothionein with 0.01 M HC1 as described by Hunziker (14). The Cd-metallothionein complex (Cd-Mt) was formed by incubating the apometallothionein in the presence of a 20-fold molar excess of CdC1 z at pH 7.4. To form a radioactive Cd-Mt complex for use in uptake studies, 1°9Cd2+ (1 /~Ci/mg protein) was included in the reaction mixture. The Cd-Mt conjugate was separated from freeCd 2+ by chromatography on Sephadex G-25. The Cd-Mt complex formed in this manner appeared to be homogeneous when analyzed by polyacrylamide gel electrophoresis and contained 6-7 moles of Cd per mole of metallothionein. Cell culture and treatment procedures. LLC-PK~ cells were provided by Dr. James Mullin of the Lankenau Medical Research Center (Philadelphia, PA). The cells were grown in Falcon Cell Culture Inserts (Becton Dickinson and Co., Lincoln Park, N J) as described previously (8). For most studies, 1,500,000 cells in 4 ml medium were seeded into the inserts (apical compartment), which were maintained in standard 6-well culture flasks containing 4 ml medium/well (basolateral compartment). The cells were fed on the day after seeding and used on the second day. On the day of an experiment, the growing medium was drawn off and replaced with serum-free medium. The cells were exposed to Cd z+ or Cd-Mt by adding concentrated solutions of the agents to the medium in the apical or the basolateral compartments. Evaluation of cell-cell junctions. The integrity of intercellular junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The effects on cell morphology were evaluated by trained observers who were not aware of the treatments the cells received. Transepithelial resistance was measured at 37°C using an EVOM Epithelial Volt-Ohm Meter and STX-2 electrodes (World Precision Instruments, New Haven, CT) as described previously (8). Accumulation of C d 2+ and Cd-Mt. Cells on Falcon inserts were incubated at 37°C in the presence of 20 ~M 1°9CDC12 (0.1 ~Ci) or 100 ~zM l°gCd-Mt (0.1 ~Ci) added to either the apical or the basolateral compartment. After incubation, the solution was aspirated off, and the cells were washed by rapidly immersing the inserts into MOPS-buffered saline for 5 seconds. The wash solution was aspirated from the apical cell surface and gently blotted from the basolateral surface of the cell culture insert. The cells were then solubilized, assayed for protein content and counted for radioactivity as described previously (9). Cell viability and protein assays. Cell viability was assessed by monitoring the release of lactate dehydrogenase into the medium (4). Cellular protein content was determined using BCA Protein Assay Kits (Pierce, Inc., Rockford, IL). Results Effects of Cd> and Cd-Mt on cell morphology. The photo on the left in Figure 1 shows the typical appearance of normal LLC-PK 1 cells. Note that the cells are packed tightly with
Vol. 53, No. 20, 1993
Effects of Cd2+ and Cd-metallothionein
PL-339
little light being transmitted between them. The middle photo shows cells that had been treated for 4 hours with 20 t~M Cd 2÷. Note that the CdZ+-treated cells appear to be separating from each other, as evidenced by the bright borders or "halos" around the individual cells. Also note that the Cd2+-treated cells have not detached from the growing surface. The bottom photo shows cells that had been treated with Cd-Mt (100 ~M) for 48 hours. Note that dead cells and cellular debris are present, although most of the cell monolayer remains intact. In contrast to the Cd2+-treated cells, the Cd-Mt treated cells do not appear to be separating from each other. Cells that had been exposed to lower concentrations of the Cd-Mt complex showed normal morphology even after 48 hours (not shown). Table 1 summarizes the morphologic effects of Cd z+ and Cd-Mt. Note that the cells that were exposed to Cd z+ showed significant changes in cell morphology within 2 hours. These early morphologic changes were similar to those described in Figure 1 and consisted of focal areas of increased light transmission between cells and the appearance of "halos" around the cells. Few of the cells detached from the growing surface until 24-48 hours of exposure. By contrast, the cells that were exposed to the Cd-Mt complex showed little evidence of cellcell separation but did begin to detach from the growing surface after 48-72 hours of exposure. Effects on transepithelial resistance and cell viability. Results presented in Figure 2 show that Cd 2+ caused a rapid decrease in the electrical resistance that coincided with the separation of the cells (compare with data in Table 1). The resistance was reduced significantly by 2 hours and continued to fall over the course of the experiment. During the time in which the resistance was dropping most rapidly (2-8 hours), there was no change in cell viability, indicating that Cd z+ selectively damaged the junctions between the cells. By contrast, exposure to the Cd-Mt complex had little effect on the transepithelial electrical resistance until the cells began to die and detach from the growing surface which did not occur until 48-72 hours of exposure. These results indicate the Cd-Mt complex does not specifically damage the junctions between cells, but that it can kill the cells after prolonged exposure. Accumulation of Cd 2+ and Cd-Mt. Figure 3 shows the accumulation of Cd 2+ and Cd-Mt from the apical and basolateral compartments of LLC-PK 1 cells on Falcon Cell Inserts. Note that the cells accumulated more Cd z+ from the basolateral compartment than from the apical compartment. Also note that the accumulation of Cd-Mt was significantly less than that of Cd z+. Moreover, there was no difference in the accumulation of the Cd-Mt complex from the apical and the basolateral compartments.
)
Control
201zM Cd 2+ (4 hrs)
1001zM C d - M t (48 hrs)
Fig. 1
Effects of Cd z+ and C d - M t on the General Morphology of LLC-PK~ cells. LLC-PK~cells in plastic culture flasks were exposed to 20 #M Cdz+ or 100/~M Cd-Mt as described in the Methods.
PL-340
Effects of Cd 2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
TABLE I Summary of the Effects of Cd z÷ and Cd-Mt on Cell Morphology TREATMENT
TIME OF EXPOSURE (hours)
Cd 2+ (20 uM) ~,
1. ,L Cd-Mt (100 #M) 1,
MORPHOLOGY (separation) (detachment)
0 2 4
0 1 1
0 0 0
8
2
o
24 48
4 4
3 4
0 2 4
0 0 0
0 0 0
8
0
0
24
0
0
.1.
48
0
1
~,
72
4
4
LLC-PK 1 cells on Falcon Cell Culture Inserts were exposed to 20 #M Cd 2+ or 100 # M Cd-Mt from both the apical and basolateral compartments. Morphologic changes, particularly cell separation and cell detachment were evaluated by trained observers who were not aware of the treatments the cells had received. Cell separation and cell detachment were rated on scales of 0-4, with 0 representing no change and 4 representing complete separation or detachment.
._1
12o~
Cd2÷(20/J,M)
o--o ~--t~
o ,ooi
+~÷
Resietonee Viobility
C d - M t (100/J.M)
o--o
Ruietonce
'0'~..~.~
0 80U 0
\
U.I ~ 20. b.I Q. 0
8
18
24
32
40
48
~
64
72 ""
80
o!
0
+,
8
•
o
•
10
24
32
,
40
,
48
,
N
' ,
64
4
72
BO
TIME (hours)
TIME(hours) Fig. 2
Effects o / C d z+ and C d - M t on Transepithelial Resistance and Cell Viability. Cells on Falcon Inserts were exposed to 20 #M Cd 2+ or 100 # M Cd-Mt from both the apical and basolateral compartments. The transepithelial electrical resistance and cell viability were determined as described in the Methods. Each point represents the mean +- standard deviation of 3-6 replicate samples. Control samples that were incubated in the absence of Cd 2+ or Cd-Mt (not displayed) showed no change in resistance or viability over the course of the experiment.
Discussion These results show that the effects of Cd-Mt on LLC-PK 1 cells are quite different from those produced by Cd z÷. One of the earliest and most striking effects of Cd 2÷ was the loosening of the junctions between the cells. When the cells were exposed to micromolar concentrations of Cd 2÷, the junctional changes were evident after 1-4 hours, whereas more severe cytotoxic effects and cell death did not occur until 8-24 hours. By contrast, even at concentrations as high as 100/~M, the Cd-Mt complex had no apparent effect on the junctions between LLC-PK t cells, even though it did kill the cells after 24-48 hours of exposure.
Vol. 53, No. 20, 1993
Effects of Cd 2+ and Cd-metallothionein
PL-341
18!
111. Apicol Cd I+
O--O ~--/'
16-
[
IIIJ¢ Z ,~141"i O
Basolateral Cd 1+
Apicol Cd-Mt Bollolotero Cd-Mt
o--o A--A
[
O ' ~ _ 64
o#4 0 2
4.
II
I!
10 12 14 ?11 18 20 22 24 26
TIME (hours)
oA--'~--I"-T 0
2
4
6
,
II
,
,
*
,
:
:
:
!
10 12 14 16 16 20 22 24 26
TIME (hours) Fig. 3
Acczmt~=ion o f Cd 2* and C d - M t L/.C-PKI. The cells were exposed to 20 #M ~°9Cd2+or 100/~M
l°gCd-]Vlt from either the apical or the basolateral compartment and accumulation of ]°gCd was determined by the procedure described in the Methods. Each point represents the mean -+ standard deviation of 3 replicate samples.
Besides not showing any junctional changes in response to Cd-Mt, the LLC-PK~ cells were generally less sensitive to the lethal effects of Cd-Mt than to those of Cd 2÷. This was somewhat surprising since previous studies had shown that primary cultures of rat renal epithelial cells were more sensitive to Cd-Mt than to Cd z+ (15). Furthermore, a variety of in vivo studies have shown that Cd-Mt conjugates are generally more potent nephrotoxic agents than Cd 2÷ (10,11). However, other studies have shown that cells in culture are relatively resistant to the cytotoxic actions of Cd-Mt (16,17). While our studies were in progress, Chin and Templeton (17) also published data showing that LLC-PK~ cells were relatively resistant to the lethal effects of Cd-Mt. They suggested that one possible explanation for the differences between the in vivo and in vitro results may be that renal epithelial cells in vivo contain apical or basolateral transport systems for Cd-Mt that are lacking in cultured cells. While the results of our studies showed that there is no difference in the accumulation of Cd-Mt from the apical and the basolateral compartments of cultured LLC-PK 1 cells, they do not rule out the possibility that different transport systems may exist in vivo. The specific molecular mechanisms by which Cd 2÷ and Cd-Mt produce their cytotoxic effects are not well understood. Most studies in this area have focused on identifying the mechanisms by which these agents kill cells. Results of those studies have shown that both Cd 2÷ and Ct-Mt can disrupt a variety of intracellular processes that could lead to the metabolic derangement and death of cells (2,16,17). Recent findings from our laboratory suggest that the junctional effects of Cd 2÷ in LLC-PK~ cells may result from the interaction of Cd 2÷ with Ca z÷ binding sites on the cell adhesion molecule, E-cadherin, or similar proteins on the basolateral cell surface, whereas the lethal effects of Cd 2÷ probably involve actions at intracellular sites (8,9). In light of these findings, it is not surprising that the Cd-Mt complex does not damage the junctions between LLC-PK 1 cells. Because of the high affinity of the interaction between Cd 2+ and metallothionein, Cd 2÷ that is bound to the protein would not be able to interact with the Ca 2+ binding sites on the cell surface. It should be noted that once the Cd-Mt complex is taken up by the cells, Cd 2÷ can be released intracellularly through the actions of lysosomal enzymes (for review see 11). Thus, while the actions of Cd 2÷ and Cd-Mt on the cell surface (i.e., the junctional effects) would be quite different, their intracellular toxic effects might be similar. Although most of our studies have focused on characterizing the junction-perturbing effects of Cd 2+ in LLC-PK 1 cells, we have recently obtained preliminary evidence that Cd a÷ can
PL-342
Effects of Cd2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
selectively damage intercellular junctions in several other types of epithelial cells. In view of the circumstantial evidence that many of the effects of Cd 2+ may result from an increase in the permeability of various epithelial and endothelial surfaces, the finding that ionic Cd 2÷, but not Cd-Mt, can selectively damage epithelial cell-cell junctions could have important implications concerning the mechanisms of Cd 2÷ toxicity in vivo. This mechanism may be most relevant to situations in which significant amounts of free Cd 2÷ would be available to act on epithelial cells. Some situations in which this might be the case include high level respiratory and oral exposure, where large amounts of free Cd z+ could act on the pulmonary or gastrointestinal epithelia (18). On the other hand, it seems less likely that such a mechanism would be applicable to situations in which most of the circulating Cd 2+ is bound to metallothionein, a condition that exists with chronic, low-level Cd 2+ exposure (18). Acknowledgements
This work was supported by NIH Grant #ES05656 to W.C.P. The authors gratefully acknowledge the excellent technical assistance of Paul Tomcykoskiand thank Barbara Le Breton for typing the manuscript. References
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
L. FRIBERG, C.-G. ELINDER, T. KJELLSTROM and G. N O R D B E R G (Eds), Cadmium and Health: a Toxicological and Epidemiological Appraisal, Vols. 1-2, CRC Press, Boca Raton, FL. (1986). A.W.F. MORSELT, Toxicology 70 1-132 (1991). C.V. NOLAN and Z.A. SHAIKH, Life Sci. 39 1403-1409 (1986). W.C. P R O Z I A L E C K and R.J.NIEWENHUIS, Toxicol. Appl. Pharmacol. 10__].781-97 (1991). G. G S T R A U N T H A L E R , D. STEINMASSL and W. PFALLER, Toxicol. Lett. 53 1-7 (1990). J.M. MULLIN and T.G. O'BRIEN, Am. J. Physiol. 251 C597-602 (1986). I.M. B R U G G E M A N , J.H.M. TEMMINK and P.J. VAN BLADEREN, Toxicol. In Vitro 6_ 195-200 (1992). W.C. P R O Z I A L E C K and R.J. NIEWENHUIS, Biochem. Biophys. Res. Comm. 181 1118-1123 (1991). W.C. P R O Z I A L E C K and P.C. LAMAR, Arch. Toxicol. 67 113-119 (1993). M. WEBB, Cadmium, Handbook Exp. Pharmacol. 80 E.C. Foulkes (Ed) 281-337, Springer-Verlag, New York (1986). C.G. E L I N D E R and M. N O R D B E R G , Cadmium and Health: a Toxicolological and Epidemiological Appraisal, Vol. 1 (L. Friberg, C.-G. Elinder, T. Kjellstr6m and G.F. Nordberg (Eds) 65-79, CRC Press, Boca Raton, FL (1986). M.G. CHERIAN and Z.A. SHAIKH, Biochem. Biophys. Res. Commun. 65 863-869 (1975). R.E. DUDLEY, L.M. G A M M A L and C.D. KLAASSEN, Toxicol. Appl. Pharmacol. 77 414-426 (1985). P.E. H U N Z I K E R , Meth. Enzymol. 205 451-452 (1991). M.G. CHERIAN, In Vitro Cell. Dev. Biol. 21 505-508 (1983). L.E. SENDELBACH, W.M. BRACKEN and C.D. KLAASSEN, Toxicology 55 83-91, 1989. T.A. CHIN and D.M. TEMPLETON, Toxicol. Appl. Pharmacol. 116 133-141 (1992). G.F. N O R D B E R G , T. KJELLSTROM and M. N O R D B E R G , Cadmium and Health: a Toxicologic and Epidemiologic Appraisal, Vol. 1 L. Friberg, C.-G. Elinder, T. Kjellstr6m and G.F. Nordberg (Eds) 103-178, CRC Press, Boca Raton, FL (1986).
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
COMPARISON OF T H E CYTOTOXIC EFFECTS OF CADMIUM C H L O R I D E AND C A D M I U M - M E T A L L O T H I O N E I N IN LLC-PK 1 CELLS Walter C. Prozialeck, Dawn R. Wellington and Peter C. Lamar Department of Pharmacology Midwestern University Downers Grove, IL 60515 (Submitted July 19, 1993; accepted August 2, 1993; received in final form September 9, 1993)
Abstract. Recent studies have shown that ionic cadmium (Cd 2÷) can selectively damage the tight junctions between LLC-PK 1 cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd 2+ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd 2+ for 1-4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24-48 hours of exposure. Additional results showed that the cells accumulated Cd 2+ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK~ cells, but that it can kill the cells after prolonged exposure.
Cadmium is an important industrial and environmental pollutant that damages a variety of organs, including the liver, kidney, lung, testis, and placenta (for reviews see 1,2). Although the general toxic effects of cadmium have been well-characterized, the mechanisms underlying many of these effects have yet to be elucidated. The observation that some of the toxic effects of cadmium seem to involve an increase in the permeability of various endothelial and epithelial surfaces (3,4) led us to examine the effects of cadmium on cells of the established porcine renal epithelial cell line, LLC-PK 1. This cell line exhibits some of the properties of the proximal tubular epithelium and has been used as a model system for physiologic and toxicologic studies (5-7). The results of our studies showed that ionic cadmium (Cd 2÷) selectively damages the junctions between LLC-PK 1 cells (4,8,9). Exposure to micromolar concentrations of CdC12 for 1-4 hours causes the cells to separate from each other without killing them. This effect coincides with a drop in the transepithelial electrical resistance and changes in the structure adhering and occluding junctional complexes (4,8). The objective of the work described in this report was to determine whether or not cadmium that is bound to metallothionein, a low molecular weight metal-binding protein that plays a key role in cadmium metabolism in vivo (10), can disrupt the junctions between LLCPK1 cells in the same manner as free Cd 2÷. This is an important issue because much of the Send correspondence to Walter C. Prozialeck. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-338
Effects of Cd2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
circulating cadmium in vivo is bound to metallothionein (10,11), and there is evidence to suggest that Cd-metallothionein conjugates may be directly responsible for some of the nephrotoxic effects of cadmium (10-13).
Methods Preparation and characterization of the Cd-metallothionein complex. Rabbit metallothionein II was obtained from Sigma Chemical Co. (St. Louis, MO). Apometallothionein was prepared by treating the Sigma metallothionein with 0.01 M HC1 as described by Hunziker (14). The Cd-metallothionein complex (Cd-Mt) was formed by incubating the apometallothionein in the presence of a 20-fold molar excess of CdC1 z at pH 7.4. To form a radioactive Cd-Mt complex for use in uptake studies, 1°9Cd2+ (1 /~Ci/mg protein) was included in the reaction mixture. The Cd-Mt conjugate was separated from freeCd 2+ by chromatography on Sephadex G-25. The Cd-Mt complex formed in this manner appeared to be homogeneous when analyzed by polyacrylamide gel electrophoresis and contained 6-7 moles of Cd per mole of metallothionein. Cell culture and treatment procedures. LLC-PK~ cells were provided by Dr. James Mullin of the Lankenau Medical Research Center (Philadelphia, PA). The cells were grown in Falcon Cell Culture Inserts (Becton Dickinson and Co., Lincoln Park, N J) as described previously (8). For most studies, 1,500,000 cells in 4 ml medium were seeded into the inserts (apical compartment), which were maintained in standard 6-well culture flasks containing 4 ml medium/well (basolateral compartment). The cells were fed on the day after seeding and used on the second day. On the day of an experiment, the growing medium was drawn off and replaced with serum-free medium. The cells were exposed to Cd z+ or Cd-Mt by adding concentrated solutions of the agents to the medium in the apical or the basolateral compartments. Evaluation of cell-cell junctions. The integrity of intercellular junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The effects on cell morphology were evaluated by trained observers who were not aware of the treatments the cells received. Transepithelial resistance was measured at 37°C using an EVOM Epithelial Volt-Ohm Meter and STX-2 electrodes (World Precision Instruments, New Haven, CT) as described previously (8). Accumulation of C d 2+ and Cd-Mt. Cells on Falcon inserts were incubated at 37°C in the presence of 20 ~M 1°9CDC12 (0.1 ~Ci) or 100 ~zM l°gCd-Mt (0.1 ~Ci) added to either the apical or the basolateral compartment. After incubation, the solution was aspirated off, and the cells were washed by rapidly immersing the inserts into MOPS-buffered saline for 5 seconds. The wash solution was aspirated from the apical cell surface and gently blotted from the basolateral surface of the cell culture insert. The cells were then solubilized, assayed for protein content and counted for radioactivity as described previously (9). Cell viability and protein assays. Cell viability was assessed by monitoring the release of lactate dehydrogenase into the medium (4). Cellular protein content was determined using BCA Protein Assay Kits (Pierce, Inc., Rockford, IL). Results Effects of Cd> and Cd-Mt on cell morphology. The photo on the left in Figure 1 shows the typical appearance of normal LLC-PK 1 cells. Note that the cells are packed tightly with
Vol. 53, No. 20, 1993
Effects of Cd2+ and Cd-metallothionein
PL-339
little light being transmitted between them. The middle photo shows cells that had been treated for 4 hours with 20 t~M Cd 2÷. Note that the CdZ+-treated cells appear to be separating from each other, as evidenced by the bright borders or "halos" around the individual cells. Also note that the Cd2+-treated cells have not detached from the growing surface. The bottom photo shows cells that had been treated with Cd-Mt (100 ~M) for 48 hours. Note that dead cells and cellular debris are present, although most of the cell monolayer remains intact. In contrast to the Cd2+-treated cells, the Cd-Mt treated cells do not appear to be separating from each other. Cells that had been exposed to lower concentrations of the Cd-Mt complex showed normal morphology even after 48 hours (not shown). Table 1 summarizes the morphologic effects of Cd z+ and Cd-Mt. Note that the cells that were exposed to Cd z+ showed significant changes in cell morphology within 2 hours. These early morphologic changes were similar to those described in Figure 1 and consisted of focal areas of increased light transmission between cells and the appearance of "halos" around the cells. Few of the cells detached from the growing surface until 24-48 hours of exposure. By contrast, the cells that were exposed to the Cd-Mt complex showed little evidence of cellcell separation but did begin to detach from the growing surface after 48-72 hours of exposure. Effects on transepithelial resistance and cell viability. Results presented in Figure 2 show that Cd 2+ caused a rapid decrease in the electrical resistance that coincided with the separation of the cells (compare with data in Table 1). The resistance was reduced significantly by 2 hours and continued to fall over the course of the experiment. During the time in which the resistance was dropping most rapidly (2-8 hours), there was no change in cell viability, indicating that Cd z+ selectively damaged the junctions between the cells. By contrast, exposure to the Cd-Mt complex had little effect on the transepithelial electrical resistance until the cells began to die and detach from the growing surface which did not occur until 48-72 hours of exposure. These results indicate the Cd-Mt complex does not specifically damage the junctions between cells, but that it can kill the cells after prolonged exposure. Accumulation of Cd 2+ and Cd-Mt. Figure 3 shows the accumulation of Cd 2+ and Cd-Mt from the apical and basolateral compartments of LLC-PK 1 cells on Falcon Cell Inserts. Note that the cells accumulated more Cd z+ from the basolateral compartment than from the apical compartment. Also note that the accumulation of Cd-Mt was significantly less than that of Cd z+. Moreover, there was no difference in the accumulation of the Cd-Mt complex from the apical and the basolateral compartments.
)
Control
201zM Cd 2+ (4 hrs)
1001zM C d - M t (48 hrs)
Fig. 1
Effects of Cd z+ and C d - M t on the General Morphology of LLC-PK~ cells. LLC-PK~cells in plastic culture flasks were exposed to 20 #M Cdz+ or 100/~M Cd-Mt as described in the Methods.
PL-340
Effects of Cd 2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
TABLE I Summary of the Effects of Cd z÷ and Cd-Mt on Cell Morphology TREATMENT
TIME OF EXPOSURE (hours)
Cd 2+ (20 uM) ~,
1. ,L Cd-Mt (100 #M) 1,
MORPHOLOGY (separation) (detachment)
0 2 4
0 1 1
0 0 0
8
2
o
24 48
4 4
3 4
0 2 4
0 0 0
0 0 0
8
0
0
24
0
0
.1.
48
0
1
~,
72
4
4
LLC-PK 1 cells on Falcon Cell Culture Inserts were exposed to 20 #M Cd 2+ or 100 # M Cd-Mt from both the apical and basolateral compartments. Morphologic changes, particularly cell separation and cell detachment were evaluated by trained observers who were not aware of the treatments the cells had received. Cell separation and cell detachment were rated on scales of 0-4, with 0 representing no change and 4 representing complete separation or detachment.
._1
12o~
Cd2÷(20/J,M)
o--o ~--t~
o ,ooi
+~÷
Resietonee Viobility
C d - M t (100/J.M)
o--o
Ruietonce
'0'~..~.~
0 80U 0
\
U.I ~ 20. b.I Q. 0
8
18
24
32
40
48
~
64
72 ""
80
o!
0
+,
8
•
o
•
10
24
32
,
40
,
48
,
N
' ,
64
4
72
BO
TIME (hours)
TIME(hours) Fig. 2
Effects o / C d z+ and C d - M t on Transepithelial Resistance and Cell Viability. Cells on Falcon Inserts were exposed to 20 #M Cd 2+ or 100 # M Cd-Mt from both the apical and basolateral compartments. The transepithelial electrical resistance and cell viability were determined as described in the Methods. Each point represents the mean +- standard deviation of 3-6 replicate samples. Control samples that were incubated in the absence of Cd 2+ or Cd-Mt (not displayed) showed no change in resistance or viability over the course of the experiment.
Discussion These results show that the effects of Cd-Mt on LLC-PK 1 cells are quite different from those produced by Cd z÷. One of the earliest and most striking effects of Cd 2÷ was the loosening of the junctions between the cells. When the cells were exposed to micromolar concentrations of Cd 2÷, the junctional changes were evident after 1-4 hours, whereas more severe cytotoxic effects and cell death did not occur until 8-24 hours. By contrast, even at concentrations as high as 100/~M, the Cd-Mt complex had no apparent effect on the junctions between LLC-PK t cells, even though it did kill the cells after 24-48 hours of exposure.
Vol. 53, No. 20, 1993
Effects of Cd 2+ and Cd-metallothionein
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18!
111. Apicol Cd I+
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TIME (hours)
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,
,
*
,
:
:
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10 12 14 16 16 20 22 24 26
TIME (hours) Fig. 3
Acczmt~=ion o f Cd 2* and C d - M t L/.C-PKI. The cells were exposed to 20 #M ~°9Cd2+or 100/~M
l°gCd-]Vlt from either the apical or the basolateral compartment and accumulation of ]°gCd was determined by the procedure described in the Methods. Each point represents the mean -+ standard deviation of 3 replicate samples.
Besides not showing any junctional changes in response to Cd-Mt, the LLC-PK~ cells were generally less sensitive to the lethal effects of Cd-Mt than to those of Cd 2÷. This was somewhat surprising since previous studies had shown that primary cultures of rat renal epithelial cells were more sensitive to Cd-Mt than to Cd z+ (15). Furthermore, a variety of in vivo studies have shown that Cd-Mt conjugates are generally more potent nephrotoxic agents than Cd 2÷ (10,11). However, other studies have shown that cells in culture are relatively resistant to the cytotoxic actions of Cd-Mt (16,17). While our studies were in progress, Chin and Templeton (17) also published data showing that LLC-PK~ cells were relatively resistant to the lethal effects of Cd-Mt. They suggested that one possible explanation for the differences between the in vivo and in vitro results may be that renal epithelial cells in vivo contain apical or basolateral transport systems for Cd-Mt that are lacking in cultured cells. While the results of our studies showed that there is no difference in the accumulation of Cd-Mt from the apical and the basolateral compartments of cultured LLC-PK 1 cells, they do not rule out the possibility that different transport systems may exist in vivo. The specific molecular mechanisms by which Cd 2÷ and Cd-Mt produce their cytotoxic effects are not well understood. Most studies in this area have focused on identifying the mechanisms by which these agents kill cells. Results of those studies have shown that both Cd 2÷ and Ct-Mt can disrupt a variety of intracellular processes that could lead to the metabolic derangement and death of cells (2,16,17). Recent findings from our laboratory suggest that the junctional effects of Cd 2÷ in LLC-PK~ cells may result from the interaction of Cd 2÷ with Ca z÷ binding sites on the cell adhesion molecule, E-cadherin, or similar proteins on the basolateral cell surface, whereas the lethal effects of Cd 2÷ probably involve actions at intracellular sites (8,9). In light of these findings, it is not surprising that the Cd-Mt complex does not damage the junctions between LLC-PK 1 cells. Because of the high affinity of the interaction between Cd 2+ and metallothionein, Cd 2÷ that is bound to the protein would not be able to interact with the Ca 2+ binding sites on the cell surface. It should be noted that once the Cd-Mt complex is taken up by the cells, Cd 2÷ can be released intracellularly through the actions of lysosomal enzymes (for review see 11). Thus, while the actions of Cd 2÷ and Cd-Mt on the cell surface (i.e., the junctional effects) would be quite different, their intracellular toxic effects might be similar. Although most of our studies have focused on characterizing the junction-perturbing effects of Cd 2+ in LLC-PK 1 cells, we have recently obtained preliminary evidence that Cd a÷ can
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Effects of Cd2+ and Cd-metallothionein
Vol. 53, No. 20, 1993
selectively damage intercellular junctions in several other types of epithelial cells. In view of the circumstantial evidence that many of the effects of Cd 2+ may result from an increase in the permeability of various epithelial and endothelial surfaces, the finding that ionic Cd 2÷, but not Cd-Mt, can selectively damage epithelial cell-cell junctions could have important implications concerning the mechanisms of Cd 2÷ toxicity in vivo. This mechanism may be most relevant to situations in which significant amounts of free Cd 2÷ would be available to act on epithelial cells. Some situations in which this might be the case include high level respiratory and oral exposure, where large amounts of free Cd z+ could act on the pulmonary or gastrointestinal epithelia (18). On the other hand, it seems less likely that such a mechanism would be applicable to situations in which most of the circulating Cd 2+ is bound to metallothionein, a condition that exists with chronic, low-level Cd 2+ exposure (18). Acknowledgements
This work was supported by NIH Grant #ES05656 to W.C.P. The authors gratefully acknowledge the excellent technical assistance of Paul Tomcykoskiand thank Barbara Le Breton for typing the manuscript. References
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
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