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Use of flow cytometry and confocal microscopy techniques to investigate early CdCl2-induced nephrotoxicity in vitro
Toxicology in Vitro 15 (2001) 407–412 www.elsevier.com/locate/toxinvit
Use of flow cytometry and confocal microscopy techniques to investigate early CdCl2-induced nephrotoxicity in vitro A. Alvarez-Barrientosa, J.E. O’Connorb, R. Nieto Castilloa, A.B. Moreno Morenoa, P. Prietoc,* a Flow Cytometry and Confocal Microscopy Unit, Complutense University 28040 Madrid, Spain Department of Biochemistry and Molecular Biology, University of Valencia, 46010 Valencia, Spain c ECVAM, Institute for Health & Consumer Protection, European Commission, Joint Research Centre, 21020 Ispra (Va), Italy b
Abstract CdCl2 is a well-known toxic compound for the kidney in vivo and in vitro. We report here part of the results of an ECVAM (European Centre for the Validation of Alternative Methods) contract study, aimed at establishing and assessing several flow cytometric and confocal microscopic endpoints for use in an in vitro nephrotoxicity model. Three renal tubule cell lines, OK (opossum, proximal tubule origin), LLC-PK1 (pig, proximal tubule origin) and MDCK (dog, distal tubule origin) were exposed for 1, 5 and 24 h to 25 mm and 100 mm CdCl2. The results obtained for mitochondrial membrane potential showed a decrease in all the cell lines after 5 h of treatment with both CdCl2 concentrations. In some cases, this decrease was detected by flow cytometry after a 1-h exposure. On the contrary, intracellular Ca2+ increased in a time-dependent and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 mm CdCl2. However, cell viability was not affected by 25 mm CdCl2. Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca2+ levels in renal tubular epithelial cell lines treated with CdCl2. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Nephrotoxicity; Flow cytometry; Confocal microscopy; CdCl2; In vitro
1. Introduction Nephrotoxicity is one of the major side-effects of exposure to xenobiotics, of which CdCl2 is a particularly potent toxic compound that causes damage to the proximal tubular epithelium in vivo and in vitro (Prozialeck and Lamar, 1995; Bandyopadhyay et al., 1997). Although CdCl2-induced toxicity has been widely studied, the precise mechanism of action is still unclear. It is known that cadmium interferes with calcium homeostasis, and this could play an important role in cadmium toxicity (Conner and Fowler, 1993; Faurskov and Bjerregaard, 1997; Zimmerhackl et al., 1998). In addition, proximal renal tubule epithelial cells are rich in mitochondria, and mitochondrial dysfunction seems Abbreviations: ECVAM, European Centre for the Validation of Alternative Methods; EU, European Union; ATCC, American Type Culture Collection; ECACC, European Collection of Cell Cultures; CdCl2, cadmium chloride; DMEM, Dulbecco’s modified Eagle’s medium; PI, propidium iodide; Rh123, Rhodamine 123. * Corresponding author. Tel.: +39-332-785534; fax: +39-332785336. E-mail address: [email protected] (P. Prietoo).
to be involved in several types of xenobiotic-induced nephrotoxicity (Aleo et al., 1991; Campbell and AlNasser, 1996). Flow cytometry and confocal microscopy technologies are complementary, and their combination provides new opportunities for the development of relevant and reliable endpoints in quantitative cell toxicology (O’Connor, 1996). On this basis, flow cytometry and confocal microscopy will be used as complementary approaches to measure intracellular calcium levels and mitochondrial membrane potential, as early biochemical indicators of early CdCl2-induced nephrotoxicity in vitro. All the results shown here are part of an ECVAM contract study.
2. Materials and methods 2.1. Culture of renal cell lines Two proximal renal tubule cell lines, LLC-PK1 (ATCC, CRL-1392, derived from pig) and OK (ATCC, CRL-1840, derived from opossum) and the distal renal
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(01)00044-3
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tubular cell line MDCK (ECACC, CB2270, derived from dog), were obtained from ECVAM at passages 198, 38 and 75, respectively. Cells were grown at 37 C in 95% air and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 5.5 mm glucose. The optimal culture conditions were defined, for example, according to the requirements of trypsin treatment (0.25% trypsin — 0.03% EDTA) for efficient detachment of cell monolayers. MDCK cells required a more sustained action of trypsin than OK and LLC-PK1 cells for obtaining cell suspensions adequate for subculturing, plating and flow cytometric analysis. The cultures were routinely examined for their morphological features by standard and confocal fluorescence microscopy. All the tissue culture plasticware was from Falcon (Becton-Dickinson); culture media and media components were from Gibco (Life Technologies), fluorescent probes and kits were from Molecular Probes (Eugene, USA). 2.2. Treatment The toxicity range of CdCl2 (Sigma Chemical Co., C5081) was established on the basis of lethality parameters, as defined by the assay with propidium iodide (PI). Based on an initial screening, cell cultures were incubated in the presence of 0, 25 and 50 mm CdCl2 for a time range of 1, 5 and 24 h. At the destined time points, the cell cultures were examined by confocal microscopy and cell suspensions by flow cytometry. All parameters described below were determined before and after treatment with CdCl2.
membrane potential with the general changes in cell morphology and cell death (as revealed by PI staining). 2.6. Cytoplasmic Ca2+ levels Intracellular Ca2+ was determined by using Fluo-3 AM (Monteiro et al., 1999). In order to exclude dead or dying cells from the analysis, PI counterstaining was used. Fluo-3 staining was also visualized by confocal microscopy, in order to correlate the changes in intracellular calcium content and compartimentation with the general changes in cell morphology and cell death (as revealed by PI staining).
3. Results 3.1. Cell viability The results of the flow cytometric analysis of cell viability are shown in Fig. 1. There was a concentrationdependent decrease in cell viability, as revealed by a reduced percentage of cells excluding PI, in all the cell lines. For each concentration tested, there was a timedependent increase in the relative number of cells taking up PI, that is, non-viable cells. This decrease in cell viability was especially evident at the highest concentration tested.
2.3. Cell viability Cell viability was estimated from the degree of integrity of plasma membranes, by using PI staining (Darzynkiewicz et al., 1997). 2.4. Apoptosis Apoptosis was analyzed initially by the quantitation of residual DNA content by flow cytometric analysis (Darzynkiewicz et al., 1997), and by the detection of well-defined morphological changes by confocal microscopy. 2.5. Mitochondrial membrane potential Mitochondrial membrane potential was determined by using Rhodamine 123 (Rh123) (O’Connor et al., 1998). In order to exclude dead or dying cells from the analysis, PI counterstaining was used. Additionally, Rh123 staining was visualized by confocal microscopy, in order to correlate the changes in mitochondrial
Fig. 1. Effects of CdCl2 on cell viability determined by flow cytometric analysis of propidium iodide (PI) uptake. Control PI staining is assumed as 100% and the variations are represented as percentage. (*) MDCK cells, (&) LLC-PK1 cells, (~) OK cells. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P <0.05; **P <0.005; Student’s t-test).
A. Alvarez-Barrientos et al. / Toxicology in Vitro 15 (2001) 407–412
Fig. 2. Effects of CdCl2 on apoptosis in (A) MDCK cells, (B) OK cells, (C) LL-CPK1 cells, determined by flow cytometric quantification of cells with sub-diploid DNA content. (*) Control, (&) 25 mm, (~) 100 mm. The data show the meanS.E.M. of the percentage of cells with sub-diploid DNA content. Each point represents at least the meanS.E.M. of five experiments. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P<0.05; **P<0.005; Student’s t-test).
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Fig. 3. Effects of CdCl2 on intracellular calcium, determined by flow cytometric quantification of Fluo-3 fluorescence per cell, and on mitochondrial membrane potential, determined flow cytometric quantification of Rh123 fluorescence per cell, in (A) MDCK cells, (B) OK cells, (C) LLC-PK1 cells. (*) Rh123 and 25 mm CdCl2, (~) Rh123 and 100 mm CdCl2 (&) Fluo-3 and 25 mm CdCl2, (&) Fluo-3 100 mm CdCl2, The data show the meanSEM of the percentage of variation vs control. Each point represents at least five experiments. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P<0.05; **P<0.005; Student’s t-test).
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MDCK cells showed the highest sensitivity to CdCl2. OK, and LLC-PK1 showed a similar behaviour, and both cell lines were affected only at higher CdCl2 concentrations and longer times of exposure than MDCK cells. LLC-PK1 was the least sensitive of all the cell types tested, and cell death induced by CdCl2 in this line was only significant at the highest concentration and time of exposure.
3.2. Apoptosis The results of the flow cytometric analysis of apoptosis are shown in Fig. 2. Apoptosis was only evident after 24 h of exposure. The observation by confocal microscopy of cells with morphological signs of apoptosis (including chromatin condensation and fragmentation,
Plate 1. Correlation of flow cytometric and confocal microscopic analysis of mitochondrial membrane potential after using Rh123/PI staining in MDCK cells treated with 100 mm CdCl2 (A: control; B: 1-h exposure; C: 24-h exposure). Green cells are live cells with mitochondria-associated Rh123 fluorescence. Red cells are dead cells (PI positive).
Plate 2. Changes in intracellular Ca2+ distribution in OK cells treated with 100 mm CdCl2, as detected by confocal microscopic analysis by using Fluo-3/PI staining. Green cells are live cells with increased FLUO-3 fluorescence. Red cells are dead cells (PI positive).
A. Alvarez-Barrientos et al. / Toxicology in Vitro 15 (2001) 407–412
cytoplasm disorganization) was generally consistent with the data generated by flow cytometry. MDCK was the most sensitive cell line, since it showed apoptosis with both CdCl2 concentrations tested. The increase observed in control cultures was not statistically significant. LLC-PK1 showed apoptosis only at the highest concentration. 25 mm showed a timedependent decrease on apoptosis. The results obtained with OK cells require further evaluation, because of their inconsistency with parallel observations by confocal microscopy. 3.3. Mitochondrial membrane potential The results of the analysis of the effects of CdCl2 on the mitochondrial membrane potential are shown in Fig. 3. There was a concentration- and time-dependent decrease of Rh123 fluorescence in all three cell lines, indicating a reduced mitochondrial membrane potential. MDCK cells showed the clearest concentration and time dependence in the reduction of mitochondrial membrane potential induced by CdCl2. OK was the least sensitive cell line. LLC-PK1 showed no evident changes at the lowest concentrations. However, there was a time-dependent decrease of Rh123 fluorescence with the highest concentration tested. A correlation between flow cytometric and confocal microscopic analysis for MDCK cells treated with 100 mm CdCl2, is shown in Plate 1. 3.4. Cytoplasmic Ca2+ levels The results of the flow cytometric analysis of intracellular Ca2+ are shown in Fig. 3. There was a concentration- and time-dependent increase of Fluo-3 fluorescence in all cell lines, indicating an increased content of intracellular calcium, as detected by flow cytometry. As the flow cytometric measurements were restricted to live cells, these data reveal toxic effects preceding cell death. The increase in intracellular calcium was evident even at the lowest concentration and exposure, in the absence of cell death. MDCK cells showed the clearest dependence on concentration and time for CdCl2 toxicity. The LLC-PK1 cell line showed differences between CdCl2 concentrations only after 24 h of exposure. The LLC-PK1 and OK cell lines were both less sensitive than MDCK cells and were only affected at higher concentrations and longer CdCl2 exposures. Plate 2 shows the intracellular Ca2+ distribution in OK cells treated with 100 mm CdCl2 by using confocal microscopy.
4. Discussion CdCl2-induced nephrotoxicity was studied in renal tubular cell lines, focusing on the relationship between
411
mitochondrial damage, cytoplasmic C2+ levels, apoptosis and cell death. The results obtained after exposure of LLC-PK1, OK and MDCK for 1, 5 and 24 h to 25 and 100 mm CdCl2, showed a decrease in mitochondrial membrane potential in all the cell lines after 5 h of treatment with both CdCl2 concentrations. This decrease was detected in LLC-PK1 cells by flow cytometry after a 1-h exposure to 100 mm CdCl2. Intracellular Ca2+, however, increased in a time- and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 mm CdCl2. Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca2+ levels in renal tubular epithelial cell lines treated with CdCl2. This decrease of mitochondrial membrane potential and the increase of intracellular Ca2+ indicate a clear mitochondrial dysfunction together with an alteration of Ca2+ homeostasis. These results are in agreement with other investigations that indicate that nephrotoxicity is associated with many Ca2+-dependent processes (Prozialeck and Niewenhuis, 1991), and that mitochondrial dysfunction seems to play also an important role in several types of xenobiotics-induced nephrotoxicity (Aleo et al., 1991; Campbell and Al-Nasser, 1996). In addition, these early alterations could result in a further loss of barrier function, as measured by transepithelial resistance changes, and the decrease of ATP/ADP ratio observed when these cell lines are exposed to CdCl2 (data not shown), and also could explain later CdCl2 cellular toxicity in all three cell lines studied.
Acknowledgements This work was supported by the EU contract 153481999-10 F1ED ISP ES, entitled ‘‘An evaluation of the reproducibility and transferability of flow cytometry and confocal microscopic endpoints in an in vitro nephrotoxicity and in vitro metabolism models’’.
References Aleo, M.D., Wyatt, R.D., Schnellmann, R.G., 1991. Mitochondrial dysfunction is an early event in ochratoxin a but not osoporein toxicity to rat renal proximal tubules. Toxicology and Applied Pharmacology 107, 73–80. Bandyopadhyay, D., Chatterjee, A.K., Datta, A., 1997. Effect of cadmium on purified hepatic flavokinase: involvement of reactive–SH group(s) in the inactivation of flavokinase by cadmium. Life Sciences 60, 1891–1903. Campbell, P.I., Al-Nasser, I.A., 1996. Renal insufficiency induced by cisplatin in rats is ameliorated by cyclosporin A. Toxicology 114, 11–17. Conner, E.A., Fowler, B.A., 1993. Mechanisms of metal-induced nephrotoxicity. In: Toxicology of the Kidney, 2nd Edition. Raven Press, New York, pp. 437–457.
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Darzynkiewicz, D., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1–20. Faurskov, B., Bjerregaard, H.F., 1997. Effect of cadmium on active ion transport and cytotoxicity in culture renal epithelial cells (A6). Toxicology in Vitro 11, 717–722. Monteiro, M.C., Sansonetty, F., 1999. Gonc¸alves, M. J. and O’Connor, J.E. A flow cytometric kinetic assay of platelet activation in whole blood using Fluo-3 and CD41. Cytometry 35, 302–310. O’Connor, J.E., 1996. Flow cytometry versus fluorescence microscopy. In: Slavik, J. (Ed.), Fluorescence Microscopy and Fluorescence Probes. Plenum Press, New York, pp. 61–66. O’Connor, J.E., Vargas, J.L., Kimler, B.F., Herna´ndez-Yago, J.,
Grisolı´a, S., 1998. Use of rhodamine-123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria. Biochemical and Biophysical Research Communications 151, 568–573. Prozialeck, W.C., Lamar, P.C., 1995. Effects of glutathione depletion on the cytotoxic actions of cadmium in llc-pk1 cells. Toxicology and Applied Pharmacology 134, 285–295. Prozialeck, W.C., Niewenhuis, R.J., 1991. Cadmium (Cd2+) Disrupts intracellular junctions and actin filaments in LLC-PK1 cells. Toxicology and Applied Pharmacology 107, 81–97. Zimmerhackl, L.B., Momm, F., Wiegele, G., Brandis, M., 1998. Cadmium is more toxic to LLC-PK1 cells than MDCK cells acting on the cadherin–catenin complex. American Journal of Physiology 275 (Renal Physiology 44), F143–153.
Use of flow cytometry and confocal microscopy techniques to investigate early CdCl2-induced nephrotoxicity in vitro A. Alvarez-Barrientosa, J.E. O’Connorb, R. Nieto Castilloa, A.B. Moreno Morenoa, P. Prietoc,* a Flow Cytometry and Confocal Microscopy Unit, Complutense University 28040 Madrid, Spain Department of Biochemistry and Molecular Biology, University of Valencia, 46010 Valencia, Spain c ECVAM, Institute for Health & Consumer Protection, European Commission, Joint Research Centre, 21020 Ispra (Va), Italy b
Abstract CdCl2 is a well-known toxic compound for the kidney in vivo and in vitro. We report here part of the results of an ECVAM (European Centre for the Validation of Alternative Methods) contract study, aimed at establishing and assessing several flow cytometric and confocal microscopic endpoints for use in an in vitro nephrotoxicity model. Three renal tubule cell lines, OK (opossum, proximal tubule origin), LLC-PK1 (pig, proximal tubule origin) and MDCK (dog, distal tubule origin) were exposed for 1, 5 and 24 h to 25 mm and 100 mm CdCl2. The results obtained for mitochondrial membrane potential showed a decrease in all the cell lines after 5 h of treatment with both CdCl2 concentrations. In some cases, this decrease was detected by flow cytometry after a 1-h exposure. On the contrary, intracellular Ca2+ increased in a time-dependent and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 mm CdCl2. However, cell viability was not affected by 25 mm CdCl2. Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca2+ levels in renal tubular epithelial cell lines treated with CdCl2. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Nephrotoxicity; Flow cytometry; Confocal microscopy; CdCl2; In vitro
1. Introduction Nephrotoxicity is one of the major side-effects of exposure to xenobiotics, of which CdCl2 is a particularly potent toxic compound that causes damage to the proximal tubular epithelium in vivo and in vitro (Prozialeck and Lamar, 1995; Bandyopadhyay et al., 1997). Although CdCl2-induced toxicity has been widely studied, the precise mechanism of action is still unclear. It is known that cadmium interferes with calcium homeostasis, and this could play an important role in cadmium toxicity (Conner and Fowler, 1993; Faurskov and Bjerregaard, 1997; Zimmerhackl et al., 1998). In addition, proximal renal tubule epithelial cells are rich in mitochondria, and mitochondrial dysfunction seems Abbreviations: ECVAM, European Centre for the Validation of Alternative Methods; EU, European Union; ATCC, American Type Culture Collection; ECACC, European Collection of Cell Cultures; CdCl2, cadmium chloride; DMEM, Dulbecco’s modified Eagle’s medium; PI, propidium iodide; Rh123, Rhodamine 123. * Corresponding author. Tel.: +39-332-785534; fax: +39-332785336. E-mail address: [email protected] (P. Prietoo).
to be involved in several types of xenobiotic-induced nephrotoxicity (Aleo et al., 1991; Campbell and AlNasser, 1996). Flow cytometry and confocal microscopy technologies are complementary, and their combination provides new opportunities for the development of relevant and reliable endpoints in quantitative cell toxicology (O’Connor, 1996). On this basis, flow cytometry and confocal microscopy will be used as complementary approaches to measure intracellular calcium levels and mitochondrial membrane potential, as early biochemical indicators of early CdCl2-induced nephrotoxicity in vitro. All the results shown here are part of an ECVAM contract study.
2. Materials and methods 2.1. Culture of renal cell lines Two proximal renal tubule cell lines, LLC-PK1 (ATCC, CRL-1392, derived from pig) and OK (ATCC, CRL-1840, derived from opossum) and the distal renal
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(01)00044-3
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A. Alvarez-Barrientos et al. / Toxicology in Vitro 15 (2001) 407–412
tubular cell line MDCK (ECACC, CB2270, derived from dog), were obtained from ECVAM at passages 198, 38 and 75, respectively. Cells were grown at 37 C in 95% air and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 5.5 mm glucose. The optimal culture conditions were defined, for example, according to the requirements of trypsin treatment (0.25% trypsin — 0.03% EDTA) for efficient detachment of cell monolayers. MDCK cells required a more sustained action of trypsin than OK and LLC-PK1 cells for obtaining cell suspensions adequate for subculturing, plating and flow cytometric analysis. The cultures were routinely examined for their morphological features by standard and confocal fluorescence microscopy. All the tissue culture plasticware was from Falcon (Becton-Dickinson); culture media and media components were from Gibco (Life Technologies), fluorescent probes and kits were from Molecular Probes (Eugene, USA). 2.2. Treatment The toxicity range of CdCl2 (Sigma Chemical Co., C5081) was established on the basis of lethality parameters, as defined by the assay with propidium iodide (PI). Based on an initial screening, cell cultures were incubated in the presence of 0, 25 and 50 mm CdCl2 for a time range of 1, 5 and 24 h. At the destined time points, the cell cultures were examined by confocal microscopy and cell suspensions by flow cytometry. All parameters described below were determined before and after treatment with CdCl2.
membrane potential with the general changes in cell morphology and cell death (as revealed by PI staining). 2.6. Cytoplasmic Ca2+ levels Intracellular Ca2+ was determined by using Fluo-3 AM (Monteiro et al., 1999). In order to exclude dead or dying cells from the analysis, PI counterstaining was used. Fluo-3 staining was also visualized by confocal microscopy, in order to correlate the changes in intracellular calcium content and compartimentation with the general changes in cell morphology and cell death (as revealed by PI staining).
3. Results 3.1. Cell viability The results of the flow cytometric analysis of cell viability are shown in Fig. 1. There was a concentrationdependent decrease in cell viability, as revealed by a reduced percentage of cells excluding PI, in all the cell lines. For each concentration tested, there was a timedependent increase in the relative number of cells taking up PI, that is, non-viable cells. This decrease in cell viability was especially evident at the highest concentration tested.
2.3. Cell viability Cell viability was estimated from the degree of integrity of plasma membranes, by using PI staining (Darzynkiewicz et al., 1997). 2.4. Apoptosis Apoptosis was analyzed initially by the quantitation of residual DNA content by flow cytometric analysis (Darzynkiewicz et al., 1997), and by the detection of well-defined morphological changes by confocal microscopy. 2.5. Mitochondrial membrane potential Mitochondrial membrane potential was determined by using Rhodamine 123 (Rh123) (O’Connor et al., 1998). In order to exclude dead or dying cells from the analysis, PI counterstaining was used. Additionally, Rh123 staining was visualized by confocal microscopy, in order to correlate the changes in mitochondrial
Fig. 1. Effects of CdCl2 on cell viability determined by flow cytometric analysis of propidium iodide (PI) uptake. Control PI staining is assumed as 100% and the variations are represented as percentage. (*) MDCK cells, (&) LLC-PK1 cells, (~) OK cells. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P <0.05; **P <0.005; Student’s t-test).
A. Alvarez-Barrientos et al. / Toxicology in Vitro 15 (2001) 407–412
Fig. 2. Effects of CdCl2 on apoptosis in (A) MDCK cells, (B) OK cells, (C) LL-CPK1 cells, determined by flow cytometric quantification of cells with sub-diploid DNA content. (*) Control, (&) 25 mm, (~) 100 mm. The data show the meanS.E.M. of the percentage of cells with sub-diploid DNA content. Each point represents at least the meanS.E.M. of five experiments. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P<0.05; **P<0.005; Student’s t-test).
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Fig. 3. Effects of CdCl2 on intracellular calcium, determined by flow cytometric quantification of Fluo-3 fluorescence per cell, and on mitochondrial membrane potential, determined flow cytometric quantification of Rh123 fluorescence per cell, in (A) MDCK cells, (B) OK cells, (C) LLC-PK1 cells. (*) Rh123 and 25 mm CdCl2, (~) Rh123 and 100 mm CdCl2 (&) Fluo-3 and 25 mm CdCl2, (&) Fluo-3 100 mm CdCl2, The data show the meanSEM of the percentage of variation vs control. Each point represents at least five experiments. Asterisks indicate significant differences between CdCl2-exposed cultures and their respective controls (*P<0.05; **P<0.005; Student’s t-test).
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MDCK cells showed the highest sensitivity to CdCl2. OK, and LLC-PK1 showed a similar behaviour, and both cell lines were affected only at higher CdCl2 concentrations and longer times of exposure than MDCK cells. LLC-PK1 was the least sensitive of all the cell types tested, and cell death induced by CdCl2 in this line was only significant at the highest concentration and time of exposure.
3.2. Apoptosis The results of the flow cytometric analysis of apoptosis are shown in Fig. 2. Apoptosis was only evident after 24 h of exposure. The observation by confocal microscopy of cells with morphological signs of apoptosis (including chromatin condensation and fragmentation,
Plate 1. Correlation of flow cytometric and confocal microscopic analysis of mitochondrial membrane potential after using Rh123/PI staining in MDCK cells treated with 100 mm CdCl2 (A: control; B: 1-h exposure; C: 24-h exposure). Green cells are live cells with mitochondria-associated Rh123 fluorescence. Red cells are dead cells (PI positive).
Plate 2. Changes in intracellular Ca2+ distribution in OK cells treated with 100 mm CdCl2, as detected by confocal microscopic analysis by using Fluo-3/PI staining. Green cells are live cells with increased FLUO-3 fluorescence. Red cells are dead cells (PI positive).
A. Alvarez-Barrientos et al. / Toxicology in Vitro 15 (2001) 407–412
cytoplasm disorganization) was generally consistent with the data generated by flow cytometry. MDCK was the most sensitive cell line, since it showed apoptosis with both CdCl2 concentrations tested. The increase observed in control cultures was not statistically significant. LLC-PK1 showed apoptosis only at the highest concentration. 25 mm showed a timedependent decrease on apoptosis. The results obtained with OK cells require further evaluation, because of their inconsistency with parallel observations by confocal microscopy. 3.3. Mitochondrial membrane potential The results of the analysis of the effects of CdCl2 on the mitochondrial membrane potential are shown in Fig. 3. There was a concentration- and time-dependent decrease of Rh123 fluorescence in all three cell lines, indicating a reduced mitochondrial membrane potential. MDCK cells showed the clearest concentration and time dependence in the reduction of mitochondrial membrane potential induced by CdCl2. OK was the least sensitive cell line. LLC-PK1 showed no evident changes at the lowest concentrations. However, there was a time-dependent decrease of Rh123 fluorescence with the highest concentration tested. A correlation between flow cytometric and confocal microscopic analysis for MDCK cells treated with 100 mm CdCl2, is shown in Plate 1. 3.4. Cytoplasmic Ca2+ levels The results of the flow cytometric analysis of intracellular Ca2+ are shown in Fig. 3. There was a concentration- and time-dependent increase of Fluo-3 fluorescence in all cell lines, indicating an increased content of intracellular calcium, as detected by flow cytometry. As the flow cytometric measurements were restricted to live cells, these data reveal toxic effects preceding cell death. The increase in intracellular calcium was evident even at the lowest concentration and exposure, in the absence of cell death. MDCK cells showed the clearest dependence on concentration and time for CdCl2 toxicity. The LLC-PK1 cell line showed differences between CdCl2 concentrations only after 24 h of exposure. The LLC-PK1 and OK cell lines were both less sensitive than MDCK cells and were only affected at higher concentrations and longer CdCl2 exposures. Plate 2 shows the intracellular Ca2+ distribution in OK cells treated with 100 mm CdCl2 by using confocal microscopy.
4. Discussion CdCl2-induced nephrotoxicity was studied in renal tubular cell lines, focusing on the relationship between
411
mitochondrial damage, cytoplasmic C2+ levels, apoptosis and cell death. The results obtained after exposure of LLC-PK1, OK and MDCK for 1, 5 and 24 h to 25 and 100 mm CdCl2, showed a decrease in mitochondrial membrane potential in all the cell lines after 5 h of treatment with both CdCl2 concentrations. This decrease was detected in LLC-PK1 cells by flow cytometry after a 1-h exposure to 100 mm CdCl2. Intracellular Ca2+, however, increased in a time- and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 mm CdCl2. Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca2+ levels in renal tubular epithelial cell lines treated with CdCl2. This decrease of mitochondrial membrane potential and the increase of intracellular Ca2+ indicate a clear mitochondrial dysfunction together with an alteration of Ca2+ homeostasis. These results are in agreement with other investigations that indicate that nephrotoxicity is associated with many Ca2+-dependent processes (Prozialeck and Niewenhuis, 1991), and that mitochondrial dysfunction seems to play also an important role in several types of xenobiotics-induced nephrotoxicity (Aleo et al., 1991; Campbell and Al-Nasser, 1996). In addition, these early alterations could result in a further loss of barrier function, as measured by transepithelial resistance changes, and the decrease of ATP/ADP ratio observed when these cell lines are exposed to CdCl2 (data not shown), and also could explain later CdCl2 cellular toxicity in all three cell lines studied.
Acknowledgements This work was supported by the EU contract 153481999-10 F1ED ISP ES, entitled ‘‘An evaluation of the reproducibility and transferability of flow cytometry and confocal microscopic endpoints in an in vitro nephrotoxicity and in vitro metabolism models’’.
References Aleo, M.D., Wyatt, R.D., Schnellmann, R.G., 1991. Mitochondrial dysfunction is an early event in ochratoxin a but not osoporein toxicity to rat renal proximal tubules. Toxicology and Applied Pharmacology 107, 73–80. Bandyopadhyay, D., Chatterjee, A.K., Datta, A., 1997. Effect of cadmium on purified hepatic flavokinase: involvement of reactive–SH group(s) in the inactivation of flavokinase by cadmium. Life Sciences 60, 1891–1903. Campbell, P.I., Al-Nasser, I.A., 1996. Renal insufficiency induced by cisplatin in rats is ameliorated by cyclosporin A. Toxicology 114, 11–17. Conner, E.A., Fowler, B.A., 1993. Mechanisms of metal-induced nephrotoxicity. In: Toxicology of the Kidney, 2nd Edition. Raven Press, New York, pp. 437–457.
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Darzynkiewicz, D., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1–20. Faurskov, B., Bjerregaard, H.F., 1997. Effect of cadmium on active ion transport and cytotoxicity in culture renal epithelial cells (A6). Toxicology in Vitro 11, 717–722. Monteiro, M.C., Sansonetty, F., 1999. Gonc¸alves, M. J. and O’Connor, J.E. A flow cytometric kinetic assay of platelet activation in whole blood using Fluo-3 and CD41. Cytometry 35, 302–310. O’Connor, J.E., 1996. Flow cytometry versus fluorescence microscopy. In: Slavik, J. (Ed.), Fluorescence Microscopy and Fluorescence Probes. Plenum Press, New York, pp. 61–66. O’Connor, J.E., Vargas, J.L., Kimler, B.F., Herna´ndez-Yago, J.,
Grisolı´a, S., 1998. Use of rhodamine-123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria. Biochemical and Biophysical Research Communications 151, 568–573. Prozialeck, W.C., Lamar, P.C., 1995. Effects of glutathione depletion on the cytotoxic actions of cadmium in llc-pk1 cells. Toxicology and Applied Pharmacology 134, 285–295. Prozialeck, W.C., Niewenhuis, R.J., 1991. Cadmium (Cd2+) Disrupts intracellular junctions and actin filaments in LLC-PK1 cells. Toxicology and Applied Pharmacology 107, 81–97. Zimmerhackl, L.B., Momm, F., Wiegele, G., Brandis, M., 1998. Cadmium is more toxic to LLC-PK1 cells than MDCK cells acting on the cadherin–catenin complex. American Journal of Physiology 275 (Renal Physiology 44), F143–153.