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In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney
Toxicology in Vitro 21 (2007) 734–740 www.elsevier.com/locate/toxinvit
In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney Lijuan Zhang, Xiaoqun Mu, Juanling Fu, Zongcan Zhou
¤
School of Public Health, Peking University, Beijing, 100083, PR China Received 5 August 2006; accepted 4 January 2007 Available online 20 January 2007
Abstract In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl2), phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in Bel-7402 cells and HK-2 cells (R2 D 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using speciWc cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity eVect after long-term exposure to a chemical. © 2007 Elsevier Ltd. All rights reserved. Keywords: In vitro cytotoxicity assay; Mitochondrial permeability transition; Target organ; Predict
1. Introduction Acute toxicity testing in animals is typically the initial step in the evaluation of the health eVects of a chemical, and its primary purpose is to provide information on potential health hazards that may result from a short-term exposure. Recent studies suggest that in vitro methods might be helpful in predicting acute toxicity and estimating toxic chemical concentrations in vivo. Some results (Spielmann et al., 1999) have illustrated that cytotoxicity data in vitro may be useful in identifying an appropriate starting dose for in vivo studies, and thus may potentially reduce the number of necessary animals for such determinations.
*
Corresponding author. Tel.: +86 10 82801531; fax: +86 10 62015583. E-mail address: [email protected] (Z. Zhou).
0887-2333/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.01.013
Other results (Ekwall et al., 2000) have indicated an association between the concentrations of chemicals leading to cytotoxicity in vitro and the concentrations of human lethal blood. A program utilizing in vitro methods to estimate toxicokinetic parameters and target organ toxicity has been proposed in order to enhance predictions of toxicity and reduce or replace animal use in some tests (Ekwall et al., 1999). Generally, there are many reasons for an organ to be as a target of toxic action of chemicals, such as toxicokinetics, biotransformation and toxicodynamics. Selective cytotoxicity may occur when some types of diVerentiated cells are more sensitive to the eVects of one particular toxicant than others. Numerous assays have been developed for assessing a chemical cytotoxicity in vitro. Standard operating procedure (SOP) for BALB/c 3T3 cells and normal human epidermal keratinocyte (NHK) cells using neutral red (NR) assay was recommended by the Interagency
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
Coordinating Committee on the Validation of Alternative Methods (ICCVAM, 2001). However, until recently, there has been little emphasis on how to apply the results of in vitro assay to predict target-organ toxicity in vivo. In the present paper, we treated liver and kidney cell lines with several known hepatotoxicants, acetaminophen (AAP), mitomycin (MMC) and cupric chloride (CuCl2), and nephrotoxicants, phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), then used NR and mitochondrial permeability transition (MPT) assays to elucidate the feasibility and limitations of using in vitro cytotoxicity assay to predict liver and kidney target-organ toxicity. 2. Materials and methods 2.1. Cell lines Human hepatoma cells (Bel-7402) obtained from institute of basic medical sciences, Peking University (Beijing, China) and human renal tubular epithelial cells (HK-2, ATCC CRL-2190™) provided by institute of basic medical sciences, Peking Union Medical College (Beijing, China), were maintained in RPMI-1640 (Gibco, USA) and in D/F12 (DMEM:F12 D 1:1, Gibco) medium respectively. Both the media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U penicillin G/100 g streptomycin sulfate per ml and incubated in an atmosphere containing 5% CO2 at 37 °C. 2.2. Materials The test chemicals AAP, MMC, CuCl2, phenacetin, CdCl2 and AA were obtained from Sigma (USA). MMC, CuCl2 and CdCl2 were solubilized in FBS free medium, AAP was solubilized in warm FBS free medium, and phenacetin in ethanol. Stock solutions of selected chemicals were prepared by 100£ and added to the cells by 1:100 dilution with culture medium. The Wnal concentration of ethanol was 0.1%. The stock solution of rhodamine 123 (Rh123, Sigma) was prepared in dimethyl sulfoxide (Sigma) (50 mg/ml) and the working concentration was 10 g/ml in phosphate-buVered saline (PBS). Other chemicals were of analytical grade or the best pharmaceutical grade. 2.3. Cytotoxicity of selected chemicals in Bel-7402 and HK-2 cells The NR assay was performed according to the recommended method (ICCVAM, 2001). Individual well of a tissue culture 96-well microtiter plates was inoculated with 0.2 ml of medium containing 2 £ 104 cells, and incubated for 24 h. Thereafter, growth medium was replaced with the exposure medium, consisting of 5%FBS-medium with diVerent concentrations of selected chemicals. Eight replicated wells were used per concentration of each chemical. After a 24 h exposure, cytotoxicity was assessed by NR
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assay, which was based on the uptaking and lysosomal accumulation of supravital dye. All experiments were performed at least three times. 2.4. Bioactivation assay Bel-7402 and HK-2 cells were seeded into 96-well microtiter plates. After 24 h, the cells were exposed to AAP and phenacetin in the presence of hepatic S9 mixture with FBS free medium for 4 h. Then with the mixture removed, fresh medium was added for additional 20 h. The S9 mixture of 1.0 ml consists of 0.5 ml 200 mM sodium phosphate buVer (pH 7.4), 0.02 ml 0.4 M MgCl2/1.65 M KCl, 0.01 ml 1 M glucose-6-phosphate, 0.1 ml NADP, 0.04 ml S9 preparation (35.5 mg protein/ml) and 0.435 ml H2O. For use in the NR assay, this mixture was diluted 1:10 (v/v) with the exposure medium with AAP and phenacetin. 2.5. Measurement of mitochondrial permeability transition (MPT) Bel-7402 (2 £ 105 cells/well) and HK-2 cells (3 £ 105 cells/well) were seeded into 6-well tissue culture plates with slides and incubated for 24 h, then treated with selected chemicals for another 24 h. The mitochondrial permeability transition was measured by the Xuorescent dye Rh123 (duplicated plates per treatment). At the end of treatment, the culture medium was removed, and the cells were washed twice in PBS and stained with 10 g/ml Rh123 for 30 min at 37 °C then washed twice in PBS. Confocal images were obtained by a laser scanning confocal microscope with excitation at 488 nm and emission at 530 nm. At least 30 image Welds with a similar degree of cell density in each experimental group were randomly captured with a laser scanning cofocal microscope (TCS SP2, Leica, Germany) and the Xuorescent intensities were analyzed using the confocal software (Leica, Germany). In each image Weld, the total number of pixels was quantiWed on gray scale (0–255 counts). The mean pixed value in each image Weld was obtained and expressed as mean § SD of the total number of mean pixel values in each experimental group. The Xuorescent intensities were expressed as percent Xuorescence change over control. The experiment was repeated three times. 2.6. Statistical analyses Results are expressed as means § SD. Linear regression analysis was used to compute the concentration of selected chemical needed to reduce absorbency of NR by 50%, in terms of IC50 values in NR uptake assay and the concentration that disrupts the MPT by 50%, in terms of EC50 values in MPT assay. Experimental data were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test for signiWcant diVerences. In all cases, the criterion for statistical signiWcance was P < 0.05.
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3. Results 3.1. EVects of selected chemicals on cytotoxicity Cytotoxicity of a 24 h-exposure to selected chemicals was evaluated in Bel-7402 cells and HK-2 cells using the NR assay (Table 1). The regression analysis of doseresponse curves and calculation of IC50 were done according to ICCVAM (2001). Based on the IC50 values, the hepatotoxicants, AAP, MMC and CuCl2 showed higher cytotoxicity in liver cells than that in kidney cells, nephrotoxicant, CdCl2 showed higher cytotoxiticy in kidney cells than that in liver cells. However, cytotoxicity of nephrotoxicants, phenacetin and AA was higher in liver cells than that in kidney cells. 3.2. Changes of mitochondrial permeability transition in Bel-7402 and HK-2 cells Bel-7402 and HK-2 cells were incubated with diVerent concentrations of selected chemicals for 24 h and stained with Rh123 then MPT was determined by a laser scanning confocal microscope. Concentrations of these chemicals were selected based on the IC50 values of cytotoxicity. All selected chemicals disrupted the MPT in both cell lines in a
dose-dependent manner. AAP and phenacetin increased MPT under their IC50 concentrations (Fig. 1 B1 and B4) in Bel-7402 cells. It showed that 1000 g/mL AAP and 600 g/ mL phenacetin increased the MPT in Bel-7402 cells, but decreased the MPT in HK-2 cells (Fig. 1a). 3.3. Comparison of IC50 and EC50 values Linear regression analysis was used to compute the concentrations of chemicals that reduce absorbency of NR by 50% and the concentrations that disrupt the MPT by 50%, in terms of the chemical IC50 values of cytotoxicity and EC50 values of MPT respectively (ICCVAM, 2001). The IC50 values of cytotoxicity and the EC50 values of MPT were Wt by the linear regression models of these selected chemicals in Bel-7402 cells and HK-2 cells (Fig. 2). The regression lines were y D 60.04 + 1.01x, R2 D 0.987 in Bel7402 cells and y D 174.61 + 1.02x, R2 D 0.823 in HK-2 cells, where y is EC50 of MPT and x is IC50 of cytotoxicity. 3.4. AAP and phenacetin cytotoxicity in Bel-7402 and HK-2 cells in the presence of S9 mixture Cytotoxicity of AAP and phenacetin was evaluated in the presence of a metabolic activating system. In the
Table 1 The IC50 (g/ml) values of AAP, MMC, CuCl2, phenacetin, CdCl2 and AA in Bel-7402 and HK-2 cellsa Cell type Bel-7402 HK-2
AAP 1070.00 § 25.09 1246.09 § 29.03
MMC 5.62 § 0.17 19.35 § 0.94
CuCl2 11.77 § 0.33 50.89 § 7.80
Phenacetin 289.80 § 7.91 408.19 § 6.32
CdCl2 3.73 § 0.20 1.35 § 0.21
AA 28.07 § 6.01 125.22 § 3.04
a Bel-7402 and HK-2 cells, seeded at 2 £ 104 cells/well, were incubated for 24 h in the presence of selected chemicals, then the neutral red (NR) assay was performed. Cell viability is based on incorporation of NR into viable cells. Linear regression analysis was used to compute the concentrations of selected chemicals needed to reduce absorbency of NR by 50%, termed IC50 values.
Fig. 1. Changes of mitochondrial permeability transition (MPT) in Bel-7402 and HK-2 cells after exposure to selected chemicals. Bel-7402 (2 £ 105 cell/ well) and HK-2 (3 £ 105 cells/well) cells were seeded into 6-well tissue culture plates and incubated for 24 h. The cells were stained with 10 g/ml Rh123 for 30 min at 37 °C after 24 h exposure to AAP, MMC, CuCl2, phenacetin, CdCl2, and AA. Confocal images were obtained with a laser scanning confocal microscope with excitation at 488 nm and emission at 530 nm. The changes of MPT were expressed as the ratios of test light density to the control respectively (means § SD) (b and c). Compared to the control respectively, *P < 0.05; **P < 0.01. 1000 g/ml AAP and 600 g/ml phenacetin increased the MPT in Bel-7402 cells, but decreased the MPT in HK-2 cells (a).
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
Fig. 1 (continued)
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Fig. 2. Regression between cytotoxicity (IC50) and mitochondrial permeability transition (MPT) (EC50) values for selected chemicals in each cell line. Cytotoxicity curves were generated and linear regression analysis was used to compute the concentrations of selected chemicals needed to reduce absorbency of NR by 50%, termed the IC50 values. Mitochondrial permeability transition curves were generated and linear regression analysis was used to compute the concentrations that disrupt the MPT by 50%, termed the EC50 values. The IC50 values of cytotoxicity and the EC50 values of MPT were Wt by the linear regression models of selected chemicals in Bel-7402 cells and HK-2 cells. The regression lines were Ò D 60.04 + 1.01x, R2 D 0.987 in Bel-7402 cells (a) and Ò D 174.61 + 1.02x, R2 D 0.823 in HK-2 cells (b). 0.4
## #
A540nm(NR assay)
0.35
#
*
*
0.3
##
##
##
*
##
##
**
0.25 0.2 0.15 0.1 0.05 0 Con1 15
47.4 150 474 1500
AAP ( g/ml)
5
15.8
50 158 500
Phenacetin ( g/ml) Bel-7402 cells
Con2 15
47.4 150 474 1500
5
AAP ( g/ml)
15.8
50
158 500
Phenacetin ( g/ml) HK-2 cells
Fig. 3. AAP and phenacetin cytotoxicity in Bel-7402 and HK-2 cells was determined with the neutral red (NR) assay (n D 4). After a 4 h exposure to selected chemicals with or without S9 mixture, the cells were continued cultivating in fresh medium for another 20 h. Results were expressed as the means § SD of the absorbency of NR. Compared to the control in each cell line respectively, *P < 0.05; **P < 0.01. Compared the absorbency of NR without S9 mixture with that with S9 mixture at the same dose of each chemical, #P < 0.05; ##P < 0.01.
presence of S9 mixture, AAP and phenacetin showed cytotoxicity in Bel-7402 cells. However, cytotoxicity of these two chemicals showed no signiWcant diVerences at all detected concentrations in the absence of S9 mixture, compared to the negative control group. Furthermore, in HK-2 cells, cytotoxicity of AAP and phenacetin with S9 mixture was higher than that without S9 mixture at each dose tested (P < 0.01) (Fig. 3). 4. Discussion Research over the last 50 years has been conducted to evaluate the potential use of in vitro cell systems for predicting acute toxic eVects in vivo. SigniWcant correlations between cytotoxicity in vitro and animal lethality have been demonstrated on numerous papers (Garle et al., 1994). The in vitro cytotoxicity assays can reduce and replace the use of laboratory animals for acute toxicity testing. Recently,
in vitro cytotoxicity assays have been recommended to predict target-organ toxicity of a chemical in vivo (ICCVAM, 2001). Liver and kidney are important target organs of toxic eVects of chemicals, since they are primary involved in metabolism and excretion of chemicals. Therefore, we selected hepatoxicants and nephrotoxicants using human hepatoma cells Bel-7402 (Chen et al., 1978) and human renal tubular epithelial cells (HK-2, ATCC CRL-2190™) to elucidate the feasibility of in vitro cytotoxicity assay to predict liver and kidney target-organ toxicity. In this paper hepatoxicants, AAP, CuCl2 and MMC, and nephrotoxicant, CdCl2, phenacetin and AA, were selected to study the compatibility and limitations of predicting target-organ toxicity using cytotoxicity assay on human cells. Acetaminophen can produce a centrilobular hepatic necrosis in man and experimental animal in acute toxic exposure (Buckley and Eddleston, 2005). Acetaminophen is metabolized via the CYP450-pathway to a highly reactive
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
N-acetyl-p-benzoquinoneimine (NAPQI). The toxic intermediate NAPQI is normally detoxiWed by endogenous glutathione (GSH). Toxicity occurs when the amount of NAPQI increases and depletes endogenous GSH by 70% or more of normal stores. Mitomycin is an anticancer drug and acts as an alkylating agent, primarily inhibits DNA synthesis and can cause DNA crosslinks (Celli and Jaiswal, 2003). Mitomycin is mainly metabolized in liver, involving in CYP450 reductase (Tomasz and Lipman, 1981) and DT-diaphorase (DTD) (Mikami et al., 1996). After acute exposure, MMC can cause gastrointestinal toxicity and bone marrow toxicity. The liver toxicity and hemolytic-uremic syndrome are also observed. The chronic toxicity of MMC is mainly kidney toxicity. Hepatic toxicity resulting from Cu2+ overload was the result of the redox cycling Haber-Weiss reaction in which cuprous ions react with H2O2 to form highly reactive oxygen species (ROS), which in turn cause membrane lipid peroxidation and membrane disruption (Luza and Speisky, 1996). Copper is stored by the liver in storage protein metallothionien (MT) and excreted by the transport protein ceruloplasmin into the bile (Papadimitriou and Loumbourdis, 2003). The nephrototoxicity produced by Cd exposure is probably due to the formation of Cd-Metallothionein complex (CdMT) (Liu et al., 1999). Cadmiummetallothionein is toxic when taken up by proximal tubular cells (O`Brien and Salacinski, 1998). Based on the NR assay, the IC50 values of AAP, MMC and CuCl2 showed higher cytotoxicity in liver cells than that in kidney cells (Table 1). The nephrotoxicant, CdCl2 showed higher cytotoxicity in kidney cells than that in liver cells. All these indicated that cytotoxicity of these chemicals in liver and kidney cells in vitro showed good compatibility with their acute target-organ toxicity in vivo. Phenacetin is a typical kidney toxicant and carcinogen (Murray and Goldberg, 1975). Phenacetin can mainly be metabolized to 4 diVerent compounds, AAP, phenacetin3,4-epoxide, N-hydroxyphenacetin and 2-hydroxyphenetidine in liver. These metabolites are ultimately excreted into kidney and damage renal and urinary tract. The contribution of AAP and NAPQI to phenacetin-induced renal toxicity is less than that caused by other reactive metabolites (Veronese et al., 1985). Aristolochic acid can induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called aristolochic acid nephropathy (AAN) (Liu et al., 2003). In studies on pharmacodynamic characteristics of aristolochic acid I (AA-I) in rats, oral administration AA-I is rapidly distributed to all theviscera or tissue, whose peak appeared in 5 min and the vallecula was from 24 to 48 h. The highest distribution Wrstly is the liver by the 2nd day, then in the kidney and in the liver by the 4th day, Wnally in the kidney after the 30th and the 40th day (Su et al., 2004). Based on the IC50 values obtained from NR assay (Table 1), the reason why kidney cytotoxicity of phenacetin and AA in vitro was lower than that in liver cells might be due to the absence of toxicokinetic processes of the whole organism in cell culture system in vitro.
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An event now recognized mitochondria as a central controller of cytotoxicity and the MPT play an important role in maintaining mitochondrial structure and function. In this paper, the mitochondrial membrane potential was determined to reXect mitochondrial function. Cell death is most commonly associated with the collapse of MPT (Green and Kroemer, 2004). However, in some instances, cell death is associated with increased MPT. Increased mitochondrial permeability transition can result from reversed activity of the mitochondrial F0F1ATPase caused by reduced ADP transport into the mitochondria (Khaled et al., 2001). All selected chemicals disrupted the MPT of both cells in a dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in each cell line (Fig. 2). But the reason why 80 g/ml aristolochic acid and the concentration causing Bel-7402 cells growth inhibition less than 50% have no eVect on the MPT needs future research. Bioactivation is an important consideration in in vitro assay. As most cell lines lose their P-450 enzymatic activity when maintained in culture, then a common protocol for an in vitro assay is to incorporate with hepatic S9 mixtrue. In our conditions, the cytotoxicity of AAP and phenacetin were strengthened in the presence of S9 mixture. The results in this paper indicated that in vitro cytotoxicity assay using Bel-7402 cells and HK-2 cells can predict liver and kidney target-organ toxicity in vivo. When cytotoxicity assay in speciWc cells show no compatibility with target-organ toxicity in vivo, the ADME of the chemical should be further studied, since in vitro system is absence the intact toxicokinetic process. Acknowledgements This work is supported by 211 project of Peking University, China. References Buckley, N., Eddleston, M., 2005. Paracetamol (acetaminophen) poisoning. Clinical Evidence, 1738–1744. Celli, C.M., Jaiswal, A.K., 2003. Role of GRP58 in mitomycin C-induced DNA cross-linking. Cancer Research 63, 6016–6025. Chen, R.M., Zhau, D.H., Ye, J.Z., Shen, D.W., 1978. The establishment and characterization of human liver cancer cell line Bel-7402. Shi Yan Sheng Wu Xue Bao 11, 37–50. Ekwall, B., Clemedson, C., Ekwall, B., Ring, P., Romert, L., 1999. EDIT: A new international multicentre programme to develop and evaluate batteries on in vitro tests for acute and chronic systemic toxicity. Alternatives to Laboratory Animals 27, 339–349. Ekwall, B., Ekwall, B., SjÖstrÖm, M., 2000. MEIC evaluation of acute systemic toxicity: Part VIII. Multivariate partial least squares evaluation, including the selection of a battery cell line tests with a good prediction of human acute lethal peak blood concentrations for 50 chemicals. Alternatives to Laboratory Animals 28, 201–234. Garle, M.J., Fentem, J.M., Fry, J.R., 1994. In vitro cytotoxicity tests for the prediction of acute toxicity in vivo. Toxicology in Vitro 8, 1303–1312. Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629.
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ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)., 2001. Guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity. NIH Publication 01-4500. Research Triangle Park, NC: National Institute for Environmental Health Sciences. Available on the Internet at http:// iccvam.niehs.nih.gov/. Khaled, A.R., Reynolds, D.A., Young, H.A., Thompson, C.B., Muegge, K., Durum, S.K., 2001. Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F0F1-ATPase. The Journal of Biological Chemistry 276, 6453–6462. Liu, M.C., Maruyama, S., Mizuno, M., Morita, Y., Hanaki, S., Yuzawa, Y., Matsuo, S., 2003. The nephrotoxicity of arstolochia manshuriensis in rats is attributable to its aristolochic acids. Clinical and Experimental Nephrology 7, 186–194. Liu, Y., Liu, J., Habeebu, S.S., Klaassen, C.D., 1999. Metallothionein protects against the nephrotoxicity produced by chronic CdMT exposure. Toxicological Sciences 50, 221–227. Luza, S.C., Speisky, H.C., 1996. Liver copper storage and transport during development: implications for cytotoxicity. The American of Clinical Nutrition 63, 812S–820S. Mikami, K., Naito, M., Tomida, A., Yamada, M., Sirakusa, T., Tsuruo, T., 1996. DT-diaphorase as a critical determinant of sensitivity to mito-
mycin C in human colon and gastric carcinoma cell lines. Cancer Research 56, 2823–2826. Murray, T., Goldberg, M., 1975. Analgesic abuse and renal disease. Annual Review of Medicine 26, 537–550. O`Brien, P., Salacinski, H.J., 1998. Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals. Archives of Toxicology 72, 690–700. Papadimitriou, E.A., Loumbourdis, N.S., 2003. Copper kinetics and hepatic metallothionein levels in the frog Rana ridibunda, after exposure to CuCl2. Biometals 16, 271–277. Spielmann, H., Genschow, E., Leibsch, M., Halle, W., 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. Alternatives to Laboratory Animals 27, 957–966. Su, T., Qu, L., Zhang, C.L., Cai, S.Q., Li, X.M., 2004. Studies on pharmacodynamic characteristics of aristolochic acid I in rats. Journal of Chinese Materia Medica 29, 676–681. Tomasz, M., Lipman, R., 1981. Reductive mechanism and alkylating activity of mitomycin C induced by rat liver microsomes. Biochemistry 20, 5056–5061. Veronese, M.E., McLean, S., D`Souza, C.A., Davies, N.W., 1985. Formation of reactive metabolites of phenacetin in humans and rats. Xenobiotica 15, 929–940.
In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney Lijuan Zhang, Xiaoqun Mu, Juanling Fu, Zongcan Zhou
¤
School of Public Health, Peking University, Beijing, 100083, PR China Received 5 August 2006; accepted 4 January 2007 Available online 20 January 2007
Abstract In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl2), phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in Bel-7402 cells and HK-2 cells (R2 D 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using speciWc cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity eVect after long-term exposure to a chemical. © 2007 Elsevier Ltd. All rights reserved. Keywords: In vitro cytotoxicity assay; Mitochondrial permeability transition; Target organ; Predict
1. Introduction Acute toxicity testing in animals is typically the initial step in the evaluation of the health eVects of a chemical, and its primary purpose is to provide information on potential health hazards that may result from a short-term exposure. Recent studies suggest that in vitro methods might be helpful in predicting acute toxicity and estimating toxic chemical concentrations in vivo. Some results (Spielmann et al., 1999) have illustrated that cytotoxicity data in vitro may be useful in identifying an appropriate starting dose for in vivo studies, and thus may potentially reduce the number of necessary animals for such determinations.
*
Corresponding author. Tel.: +86 10 82801531; fax: +86 10 62015583. E-mail address: [email protected] (Z. Zhou).
0887-2333/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.01.013
Other results (Ekwall et al., 2000) have indicated an association between the concentrations of chemicals leading to cytotoxicity in vitro and the concentrations of human lethal blood. A program utilizing in vitro methods to estimate toxicokinetic parameters and target organ toxicity has been proposed in order to enhance predictions of toxicity and reduce or replace animal use in some tests (Ekwall et al., 1999). Generally, there are many reasons for an organ to be as a target of toxic action of chemicals, such as toxicokinetics, biotransformation and toxicodynamics. Selective cytotoxicity may occur when some types of diVerentiated cells are more sensitive to the eVects of one particular toxicant than others. Numerous assays have been developed for assessing a chemical cytotoxicity in vitro. Standard operating procedure (SOP) for BALB/c 3T3 cells and normal human epidermal keratinocyte (NHK) cells using neutral red (NR) assay was recommended by the Interagency
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
Coordinating Committee on the Validation of Alternative Methods (ICCVAM, 2001). However, until recently, there has been little emphasis on how to apply the results of in vitro assay to predict target-organ toxicity in vivo. In the present paper, we treated liver and kidney cell lines with several known hepatotoxicants, acetaminophen (AAP), mitomycin (MMC) and cupric chloride (CuCl2), and nephrotoxicants, phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), then used NR and mitochondrial permeability transition (MPT) assays to elucidate the feasibility and limitations of using in vitro cytotoxicity assay to predict liver and kidney target-organ toxicity. 2. Materials and methods 2.1. Cell lines Human hepatoma cells (Bel-7402) obtained from institute of basic medical sciences, Peking University (Beijing, China) and human renal tubular epithelial cells (HK-2, ATCC CRL-2190™) provided by institute of basic medical sciences, Peking Union Medical College (Beijing, China), were maintained in RPMI-1640 (Gibco, USA) and in D/F12 (DMEM:F12 D 1:1, Gibco) medium respectively. Both the media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U penicillin G/100 g streptomycin sulfate per ml and incubated in an atmosphere containing 5% CO2 at 37 °C. 2.2. Materials The test chemicals AAP, MMC, CuCl2, phenacetin, CdCl2 and AA were obtained from Sigma (USA). MMC, CuCl2 and CdCl2 were solubilized in FBS free medium, AAP was solubilized in warm FBS free medium, and phenacetin in ethanol. Stock solutions of selected chemicals were prepared by 100£ and added to the cells by 1:100 dilution with culture medium. The Wnal concentration of ethanol was 0.1%. The stock solution of rhodamine 123 (Rh123, Sigma) was prepared in dimethyl sulfoxide (Sigma) (50 mg/ml) and the working concentration was 10 g/ml in phosphate-buVered saline (PBS). Other chemicals were of analytical grade or the best pharmaceutical grade. 2.3. Cytotoxicity of selected chemicals in Bel-7402 and HK-2 cells The NR assay was performed according to the recommended method (ICCVAM, 2001). Individual well of a tissue culture 96-well microtiter plates was inoculated with 0.2 ml of medium containing 2 £ 104 cells, and incubated for 24 h. Thereafter, growth medium was replaced with the exposure medium, consisting of 5%FBS-medium with diVerent concentrations of selected chemicals. Eight replicated wells were used per concentration of each chemical. After a 24 h exposure, cytotoxicity was assessed by NR
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assay, which was based on the uptaking and lysosomal accumulation of supravital dye. All experiments were performed at least three times. 2.4. Bioactivation assay Bel-7402 and HK-2 cells were seeded into 96-well microtiter plates. After 24 h, the cells were exposed to AAP and phenacetin in the presence of hepatic S9 mixture with FBS free medium for 4 h. Then with the mixture removed, fresh medium was added for additional 20 h. The S9 mixture of 1.0 ml consists of 0.5 ml 200 mM sodium phosphate buVer (pH 7.4), 0.02 ml 0.4 M MgCl2/1.65 M KCl, 0.01 ml 1 M glucose-6-phosphate, 0.1 ml NADP, 0.04 ml S9 preparation (35.5 mg protein/ml) and 0.435 ml H2O. For use in the NR assay, this mixture was diluted 1:10 (v/v) with the exposure medium with AAP and phenacetin. 2.5. Measurement of mitochondrial permeability transition (MPT) Bel-7402 (2 £ 105 cells/well) and HK-2 cells (3 £ 105 cells/well) were seeded into 6-well tissue culture plates with slides and incubated for 24 h, then treated with selected chemicals for another 24 h. The mitochondrial permeability transition was measured by the Xuorescent dye Rh123 (duplicated plates per treatment). At the end of treatment, the culture medium was removed, and the cells were washed twice in PBS and stained with 10 g/ml Rh123 for 30 min at 37 °C then washed twice in PBS. Confocal images were obtained by a laser scanning confocal microscope with excitation at 488 nm and emission at 530 nm. At least 30 image Welds with a similar degree of cell density in each experimental group were randomly captured with a laser scanning cofocal microscope (TCS SP2, Leica, Germany) and the Xuorescent intensities were analyzed using the confocal software (Leica, Germany). In each image Weld, the total number of pixels was quantiWed on gray scale (0–255 counts). The mean pixed value in each image Weld was obtained and expressed as mean § SD of the total number of mean pixel values in each experimental group. The Xuorescent intensities were expressed as percent Xuorescence change over control. The experiment was repeated three times. 2.6. Statistical analyses Results are expressed as means § SD. Linear regression analysis was used to compute the concentration of selected chemical needed to reduce absorbency of NR by 50%, in terms of IC50 values in NR uptake assay and the concentration that disrupts the MPT by 50%, in terms of EC50 values in MPT assay. Experimental data were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test for signiWcant diVerences. In all cases, the criterion for statistical signiWcance was P < 0.05.
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3. Results 3.1. EVects of selected chemicals on cytotoxicity Cytotoxicity of a 24 h-exposure to selected chemicals was evaluated in Bel-7402 cells and HK-2 cells using the NR assay (Table 1). The regression analysis of doseresponse curves and calculation of IC50 were done according to ICCVAM (2001). Based on the IC50 values, the hepatotoxicants, AAP, MMC and CuCl2 showed higher cytotoxicity in liver cells than that in kidney cells, nephrotoxicant, CdCl2 showed higher cytotoxiticy in kidney cells than that in liver cells. However, cytotoxicity of nephrotoxicants, phenacetin and AA was higher in liver cells than that in kidney cells. 3.2. Changes of mitochondrial permeability transition in Bel-7402 and HK-2 cells Bel-7402 and HK-2 cells were incubated with diVerent concentrations of selected chemicals for 24 h and stained with Rh123 then MPT was determined by a laser scanning confocal microscope. Concentrations of these chemicals were selected based on the IC50 values of cytotoxicity. All selected chemicals disrupted the MPT in both cell lines in a
dose-dependent manner. AAP and phenacetin increased MPT under their IC50 concentrations (Fig. 1 B1 and B4) in Bel-7402 cells. It showed that 1000 g/mL AAP and 600 g/ mL phenacetin increased the MPT in Bel-7402 cells, but decreased the MPT in HK-2 cells (Fig. 1a). 3.3. Comparison of IC50 and EC50 values Linear regression analysis was used to compute the concentrations of chemicals that reduce absorbency of NR by 50% and the concentrations that disrupt the MPT by 50%, in terms of the chemical IC50 values of cytotoxicity and EC50 values of MPT respectively (ICCVAM, 2001). The IC50 values of cytotoxicity and the EC50 values of MPT were Wt by the linear regression models of these selected chemicals in Bel-7402 cells and HK-2 cells (Fig. 2). The regression lines were y D 60.04 + 1.01x, R2 D 0.987 in Bel7402 cells and y D 174.61 + 1.02x, R2 D 0.823 in HK-2 cells, where y is EC50 of MPT and x is IC50 of cytotoxicity. 3.4. AAP and phenacetin cytotoxicity in Bel-7402 and HK-2 cells in the presence of S9 mixture Cytotoxicity of AAP and phenacetin was evaluated in the presence of a metabolic activating system. In the
Table 1 The IC50 (g/ml) values of AAP, MMC, CuCl2, phenacetin, CdCl2 and AA in Bel-7402 and HK-2 cellsa Cell type Bel-7402 HK-2
AAP 1070.00 § 25.09 1246.09 § 29.03
MMC 5.62 § 0.17 19.35 § 0.94
CuCl2 11.77 § 0.33 50.89 § 7.80
Phenacetin 289.80 § 7.91 408.19 § 6.32
CdCl2 3.73 § 0.20 1.35 § 0.21
AA 28.07 § 6.01 125.22 § 3.04
a Bel-7402 and HK-2 cells, seeded at 2 £ 104 cells/well, were incubated for 24 h in the presence of selected chemicals, then the neutral red (NR) assay was performed. Cell viability is based on incorporation of NR into viable cells. Linear regression analysis was used to compute the concentrations of selected chemicals needed to reduce absorbency of NR by 50%, termed IC50 values.
Fig. 1. Changes of mitochondrial permeability transition (MPT) in Bel-7402 and HK-2 cells after exposure to selected chemicals. Bel-7402 (2 £ 105 cell/ well) and HK-2 (3 £ 105 cells/well) cells were seeded into 6-well tissue culture plates and incubated for 24 h. The cells were stained with 10 g/ml Rh123 for 30 min at 37 °C after 24 h exposure to AAP, MMC, CuCl2, phenacetin, CdCl2, and AA. Confocal images were obtained with a laser scanning confocal microscope with excitation at 488 nm and emission at 530 nm. The changes of MPT were expressed as the ratios of test light density to the control respectively (means § SD) (b and c). Compared to the control respectively, *P < 0.05; **P < 0.01. 1000 g/ml AAP and 600 g/ml phenacetin increased the MPT in Bel-7402 cells, but decreased the MPT in HK-2 cells (a).
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
Fig. 1 (continued)
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Fig. 2. Regression between cytotoxicity (IC50) and mitochondrial permeability transition (MPT) (EC50) values for selected chemicals in each cell line. Cytotoxicity curves were generated and linear regression analysis was used to compute the concentrations of selected chemicals needed to reduce absorbency of NR by 50%, termed the IC50 values. Mitochondrial permeability transition curves were generated and linear regression analysis was used to compute the concentrations that disrupt the MPT by 50%, termed the EC50 values. The IC50 values of cytotoxicity and the EC50 values of MPT were Wt by the linear regression models of selected chemicals in Bel-7402 cells and HK-2 cells. The regression lines were Ò D 60.04 + 1.01x, R2 D 0.987 in Bel-7402 cells (a) and Ò D 174.61 + 1.02x, R2 D 0.823 in HK-2 cells (b). 0.4
## #
A540nm(NR assay)
0.35
#
*
*
0.3
##
##
##
*
##
##
**
0.25 0.2 0.15 0.1 0.05 0 Con1 15
47.4 150 474 1500
AAP ( g/ml)
5
15.8
50 158 500
Phenacetin ( g/ml) Bel-7402 cells
Con2 15
47.4 150 474 1500
5
AAP ( g/ml)
15.8
50
158 500
Phenacetin ( g/ml) HK-2 cells
Fig. 3. AAP and phenacetin cytotoxicity in Bel-7402 and HK-2 cells was determined with the neutral red (NR) assay (n D 4). After a 4 h exposure to selected chemicals with or without S9 mixture, the cells were continued cultivating in fresh medium for another 20 h. Results were expressed as the means § SD of the absorbency of NR. Compared to the control in each cell line respectively, *P < 0.05; **P < 0.01. Compared the absorbency of NR without S9 mixture with that with S9 mixture at the same dose of each chemical, #P < 0.05; ##P < 0.01.
presence of S9 mixture, AAP and phenacetin showed cytotoxicity in Bel-7402 cells. However, cytotoxicity of these two chemicals showed no signiWcant diVerences at all detected concentrations in the absence of S9 mixture, compared to the negative control group. Furthermore, in HK-2 cells, cytotoxicity of AAP and phenacetin with S9 mixture was higher than that without S9 mixture at each dose tested (P < 0.01) (Fig. 3). 4. Discussion Research over the last 50 years has been conducted to evaluate the potential use of in vitro cell systems for predicting acute toxic eVects in vivo. SigniWcant correlations between cytotoxicity in vitro and animal lethality have been demonstrated on numerous papers (Garle et al., 1994). The in vitro cytotoxicity assays can reduce and replace the use of laboratory animals for acute toxicity testing. Recently,
in vitro cytotoxicity assays have been recommended to predict target-organ toxicity of a chemical in vivo (ICCVAM, 2001). Liver and kidney are important target organs of toxic eVects of chemicals, since they are primary involved in metabolism and excretion of chemicals. Therefore, we selected hepatoxicants and nephrotoxicants using human hepatoma cells Bel-7402 (Chen et al., 1978) and human renal tubular epithelial cells (HK-2, ATCC CRL-2190™) to elucidate the feasibility of in vitro cytotoxicity assay to predict liver and kidney target-organ toxicity. In this paper hepatoxicants, AAP, CuCl2 and MMC, and nephrotoxicant, CdCl2, phenacetin and AA, were selected to study the compatibility and limitations of predicting target-organ toxicity using cytotoxicity assay on human cells. Acetaminophen can produce a centrilobular hepatic necrosis in man and experimental animal in acute toxic exposure (Buckley and Eddleston, 2005). Acetaminophen is metabolized via the CYP450-pathway to a highly reactive
L. Zhang et al. / Toxicology in Vitro 21 (2007) 734–740
N-acetyl-p-benzoquinoneimine (NAPQI). The toxic intermediate NAPQI is normally detoxiWed by endogenous glutathione (GSH). Toxicity occurs when the amount of NAPQI increases and depletes endogenous GSH by 70% or more of normal stores. Mitomycin is an anticancer drug and acts as an alkylating agent, primarily inhibits DNA synthesis and can cause DNA crosslinks (Celli and Jaiswal, 2003). Mitomycin is mainly metabolized in liver, involving in CYP450 reductase (Tomasz and Lipman, 1981) and DT-diaphorase (DTD) (Mikami et al., 1996). After acute exposure, MMC can cause gastrointestinal toxicity and bone marrow toxicity. The liver toxicity and hemolytic-uremic syndrome are also observed. The chronic toxicity of MMC is mainly kidney toxicity. Hepatic toxicity resulting from Cu2+ overload was the result of the redox cycling Haber-Weiss reaction in which cuprous ions react with H2O2 to form highly reactive oxygen species (ROS), which in turn cause membrane lipid peroxidation and membrane disruption (Luza and Speisky, 1996). Copper is stored by the liver in storage protein metallothionien (MT) and excreted by the transport protein ceruloplasmin into the bile (Papadimitriou and Loumbourdis, 2003). The nephrototoxicity produced by Cd exposure is probably due to the formation of Cd-Metallothionein complex (CdMT) (Liu et al., 1999). Cadmiummetallothionein is toxic when taken up by proximal tubular cells (O`Brien and Salacinski, 1998). Based on the NR assay, the IC50 values of AAP, MMC and CuCl2 showed higher cytotoxicity in liver cells than that in kidney cells (Table 1). The nephrotoxicant, CdCl2 showed higher cytotoxicity in kidney cells than that in liver cells. All these indicated that cytotoxicity of these chemicals in liver and kidney cells in vitro showed good compatibility with their acute target-organ toxicity in vivo. Phenacetin is a typical kidney toxicant and carcinogen (Murray and Goldberg, 1975). Phenacetin can mainly be metabolized to 4 diVerent compounds, AAP, phenacetin3,4-epoxide, N-hydroxyphenacetin and 2-hydroxyphenetidine in liver. These metabolites are ultimately excreted into kidney and damage renal and urinary tract. The contribution of AAP and NAPQI to phenacetin-induced renal toxicity is less than that caused by other reactive metabolites (Veronese et al., 1985). Aristolochic acid can induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called aristolochic acid nephropathy (AAN) (Liu et al., 2003). In studies on pharmacodynamic characteristics of aristolochic acid I (AA-I) in rats, oral administration AA-I is rapidly distributed to all theviscera or tissue, whose peak appeared in 5 min and the vallecula was from 24 to 48 h. The highest distribution Wrstly is the liver by the 2nd day, then in the kidney and in the liver by the 4th day, Wnally in the kidney after the 30th and the 40th day (Su et al., 2004). Based on the IC50 values obtained from NR assay (Table 1), the reason why kidney cytotoxicity of phenacetin and AA in vitro was lower than that in liver cells might be due to the absence of toxicokinetic processes of the whole organism in cell culture system in vitro.
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An event now recognized mitochondria as a central controller of cytotoxicity and the MPT play an important role in maintaining mitochondrial structure and function. In this paper, the mitochondrial membrane potential was determined to reXect mitochondrial function. Cell death is most commonly associated with the collapse of MPT (Green and Kroemer, 2004). However, in some instances, cell death is associated with increased MPT. Increased mitochondrial permeability transition can result from reversed activity of the mitochondrial F0F1ATPase caused by reduced ADP transport into the mitochondria (Khaled et al., 2001). All selected chemicals disrupted the MPT of both cells in a dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in each cell line (Fig. 2). But the reason why 80 g/ml aristolochic acid and the concentration causing Bel-7402 cells growth inhibition less than 50% have no eVect on the MPT needs future research. Bioactivation is an important consideration in in vitro assay. As most cell lines lose their P-450 enzymatic activity when maintained in culture, then a common protocol for an in vitro assay is to incorporate with hepatic S9 mixtrue. In our conditions, the cytotoxicity of AAP and phenacetin were strengthened in the presence of S9 mixture. The results in this paper indicated that in vitro cytotoxicity assay using Bel-7402 cells and HK-2 cells can predict liver and kidney target-organ toxicity in vivo. When cytotoxicity assay in speciWc cells show no compatibility with target-organ toxicity in vivo, the ADME of the chemical should be further studied, since in vitro system is absence the intact toxicokinetic process. Acknowledgements This work is supported by 211 project of Peking University, China. References Buckley, N., Eddleston, M., 2005. Paracetamol (acetaminophen) poisoning. Clinical Evidence, 1738–1744. Celli, C.M., Jaiswal, A.K., 2003. Role of GRP58 in mitomycin C-induced DNA cross-linking. Cancer Research 63, 6016–6025. Chen, R.M., Zhau, D.H., Ye, J.Z., Shen, D.W., 1978. The establishment and characterization of human liver cancer cell line Bel-7402. Shi Yan Sheng Wu Xue Bao 11, 37–50. Ekwall, B., Clemedson, C., Ekwall, B., Ring, P., Romert, L., 1999. EDIT: A new international multicentre programme to develop and evaluate batteries on in vitro tests for acute and chronic systemic toxicity. Alternatives to Laboratory Animals 27, 339–349. Ekwall, B., Ekwall, B., SjÖstrÖm, M., 2000. MEIC evaluation of acute systemic toxicity: Part VIII. Multivariate partial least squares evaluation, including the selection of a battery cell line tests with a good prediction of human acute lethal peak blood concentrations for 50 chemicals. Alternatives to Laboratory Animals 28, 201–234. Garle, M.J., Fentem, J.M., Fry, J.R., 1994. In vitro cytotoxicity tests for the prediction of acute toxicity in vivo. Toxicology in Vitro 8, 1303–1312. Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629.
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