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Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells
Toxicology in Vitro 24 (2010) 1092–1097
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells Ji Li a, Liang Zhang b,c, Zhenzhou Jiang a,d, Bin Shu a,e, Fu Li a, Qingli Bao a, Luyong Zhang a,* a
Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Pharmacology, Nanjing University of Chinese Medicine, Nanjing 210046, PR China c Jiangsu Province Key Lab. of Efficiency and Safety Evaluation of Chinese Medicine, PR China d Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, PR China e Jiangsu Center for Safety Evaluation of Drugs, Nanjing University of Technology, Nanjing 210009, PR China b
a r t i c l e
i n f o
Article history: Received 20 January 2010 Accepted 18 March 2010 Available online 23 March 2010 Keywords: Aristolochic acid I (AA-I) Aristololactam I (AL-I) Cytotoxicity Human proximal tubular epithelial cell lines (HK-2 cells)
a b s t r a c t Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-Imediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway. Ó 2010 Published by Elsevier Ltd.
1. Introduction Aristolochic acid I (AA-I) and aristololactam I (AL-I) are the main active components in plants of the Aristolochia species (Zhang et al., 2006). Aristolochia is a Chinese traditional herbal remedy with potent diuretic activity. In some European countries, it is utilized in weight-loss regimens. However, the clinical application of aristolochic acid (AA) has been limited by its severe nephrotoxicity. The nephrotoxic effect was first reported in 1964 (Jackson et al., 1964). It was again noted in 1993 in Belgium when a group of young female patients undergoing a slimming regimen that included Chinese herbs containing AA presented with rapidly progressing interstitial renal fibrosis (Vanherweghem et al., 1993, 1996). Debelle et al. reported that long-term exposure or overdose of AA may cause severe nephrotoxicity, which is characterized by chronic renal failure, tubulointerstitial fibrosis, and development of urothelial cancer (Debelle et al., 2002). Recent studies have revealed that AA-I can cause direct damage to renal tubular cells, and the toxicity was associated with the formation of promutagenic AA-DNA adducts (Li et al., 2006). However, the mechanism of the renal injury caused by AL-I remains unclear. In the present study, we investigated the role and
* Corresponding author. Tel.: +86 25 83271043; fax: +86 25 83271142. E-mail address: [email protected] (L. Zhang). 0887-2333/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.tiv.2010.03.012
in vitro mechanism of AL-I-mediated cytotoxicity. In our experimental conditions, AL-I induced apoptosis in the cells of the human renal tubular epithelial (HK-2) cell line. Caspase 3 activation was observed during the course of this apoptosis. We also observed that the cytotoxic potency of AL-I was stronger than that of AA-I. 2. Materials and methods 2.1. Cell culture and reagents HK-2 cells were obtained from American Type Culture Collection (ATCC, USA). Dulbecco modified Eagle medium/F12 (F12 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) fetal bovine serum (FBS), and trypsin/ethylene diamine tetraacetic acid (EDTA) were purchased from Gibco (Gibco Laboratories, NY, USA). The HK-2 cells were grown in DF12 supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. AA-I and AL-I (purity 98%, determined by high-performance liquid chromatography) were purchased from Zhengzhou Tianlin Pharm-tech Co., Ltd. (Zhengzhou, Henan, China). AA-I and AL-I were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions (15 mM), which were stored at 4 °C. Before use in the experiment, the stock solution was diluted to the indicated concentrations by using the culture medium. During the experiments, the DMSO content in the medium never exceeded 0.5% (v/v).
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2.6. Statistical analysis
Propidium iodide (PI), a colorigenic synthetic peptide substrate for caspase 3 proteases (Ac-DEVD-pNA), 3-(4,5-dimethyl-thiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and ribonuclease A (RNase A) were purchased from Sigma (St. Louis, MO, USA). Annexin V/PI apoptosis detection kit was obtained from Becton Dickinson (CA, USA).
Each experiment was repeated at least 3 times, and the data were expressed as mean ± SD values. Student’s t test was used for statistical analysis, and P values less than 0.05 were considered statistically significant.
2.2. MTT assay
3. Results
HK-2 cells (5 104 cells/ml) were treated with AA-I and AL-I. The culture was incubated with fresh medium containing different concentrations of AA-I and AL-I for 24 h, 48 h, or 72 h. Subsequently, MTT was added to each well to a final concentration of 0.5 mg/ml. After incubation for 4 h at 37 °C, the formazan crystals derived from MTT were dissolved in DMSO, and the absorbance at 570 nm was measured using a Model 680 Microplate Reader (BioRad Lab., UK).
3.1. Cytotoxicities of AA-I and AL-I in HK-2 cells
2.3. Morphology of the HK-2 cells after treatment with AA-I and AL-I The morphologies of the HK-2 cells after exposure to 80 lM AAI or AL-I for 24 h were examined using a contrast microscope (1X71S8F-2; Olympus, Japan). After the treatments, the HK-2 cells were stained with Hoechst 33258 and visualized to investigate the incidence of apoptosis. To this end, the cells were seeded on coverslips, incubated with 80 lM AA-I or AL-I for 24 h, and then fixed with 4% paraformaldehyde for 10 min. The nuclear DNA was stained with 5 mg/ml Hoechst 33258 for another 5 min, and these cells were observed under a fluorescence microscope (1X71S8F-2; Olympus, Japan). 2.4. Detection of apoptosis and cell-cycle analysis 2.4.1. PI staining assay HK-2 cells were collected at the end of treatment, washed twice with ice-cold phosphate-buffered saline (PBS), and then fixed in 70% ethanol at 4 °C for 12 h. After fixation, the cells were washed twice with PBS and incubated in PBS containing PI, RNase A, Triton X-100 (0.5%) at 37 °C for 30 min. The fluorescence emitted from the propidium–DNA complex was measured using FACScan flow cytometry (Becton Dickinson, San Jones, CA, USA). Cells containing hypodiploid DNA were considered apoptotic. 2.4.2. Annexin V/PI staining assay The apoptosis ratio was measured using FACScan flow cytometer (Becton Dickenson, San Jose, CA, USA) according to the instructions provided with the annexin V/PI kit. Briefly, after treatment with 40 lM AA-I or AL-I for 24, 48, or 72 h, HK-2 cells were harvested and washed twice with pre-cooled PBS and resuspended in a binding buffer containing fluorescein isothiocyanate (FITC)conjugated annexin V antibody and PI. After incubation in the dark for 30 min, the cells were analyzed using flow cytometry. 2.5. Activation of caspase 3 The activity of caspase 3 was determined using a previously described method (Ito et al., 1999). Briefly, cells (106 ml 1) were harvested after treatment, washed thrice with PBS, and resuspended in ice-cold buffer containing 50 mM Tris–HCl, 1 mM EDTA, 10 mM ethylene glycol tetraacetic acid (EGTA), and 1 mM DTT. Cell lysates were centrifuged at 12,000 g for 5 min, and extracts containing 50 lg of the protein were incubated with 100 lM of the enzyme-specific colorimetric substrate Ac-DEVD-pNA at 37 °C for 2 h. The colorimetric release of p-nitroaniline from the Ac-DEVD-pNA substrate was measured by determining the absorbance at 405 nm.
The cytotoxicities of AA-I and AL-I in HK-2 cells were determined using the MTT assay. The cell growth inhibition after incubation with various concentrations of AA-I or AL-I for 24, 48, or 72 h are presented in Fig. 1A and B. As expected, AA-I and AL-I showed cytotoxic activity. In comparison with the control cells, HK-2 cells exposed to these agents for 24, 48, and 72 h showed a significant dose- and time-dependent inhibition of cell growth (P < 0.01 vs. control). DMSO alone did not show any inhibitory effect on the cell viability. As shown in Fig. 1C, the IC50 values of AA-I were higher than those of AL-I, which suggested that AL-I plays an important role in the AA-mediated cytotoxicity in HK-2 cells. 3.2. Morphological changes in HK-2 cells after exposure to AA-I and AL-I As shown in Fig. 2A, untreated HK-2 cells appeared to proliferate in spindle-like shapes in the wells, and HK-2 cells treated with AA-I (80 lM) contracted and became rounded. The morphology of the HK-2 cells exposed to AL-I (80 lM) for 24 h was similar to that of the cells treated with AA-I. After the treatments, the HK-2 cells were stained with Hoechst 33258 and visualized to investigate the incidence of apoptosis. The morphological images acquired using the microscope are shown in Fig. 2B. Apoptotic cells were characterized by nuclear condensation and chromatin margination. 3.3. Detection of apoptosis in the HK-2 cells treated with AA-I and AL-I 3.3.1. Effects of AA-I and AL-I on cell cycle The inhibitory effect of AA-I and AL-I on cell growth was further investigated using flow cytometric analysis of the cellular DNA content. As shown in Fig. 3A, apoptotic cells were identified by the sub-G1 apoptotic peaks, which could be attributed to their lower DNA content. Apoptotic peaks and increased percentage of apoptotic cells were observed after 24 h treatment with 40 lmol/ L AL-I or 80 lmol/L AA-I. The distributions of the cells in various phases of the cell cycle and the percentages of apoptotic cells after treatment with different concentrations of the test compounds are summarized in Fig. 3B. The increase in S-phase cell populations after treatment with AA-I or AL-I for 24 h were similar. This finding is consistent with the previous reports that AA caused S-phase arrest, accelerated the cell cycle, and caused abnormal proliferation in the epithelial cells of the urinary tract (Chang et al., 2006). After treatment with AA-I, the percentage of cells in the G2/M phase showed a marked increase, and the percentage of cells in the G1 phase showed a significant reduction; however, these effects were not as remarkable as those observed in the cells treated with AL-I. 3.3.2. Detection of apoptosis and necrosis Annexin V specifically binds to the negatively charged phosphatidylserine (PS) that is translocated from the inner surface of the cell membrane to the surface during early apoptosis (Fadok et al., 1992). PI is a non-specific DNA intercalating agent, which is ex-
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Fig. 1. Inhibition of cell viability by AA-I or AL-I in HK-2 cells. Concentration-dependent effect of AA-I (A), AL-I (B) on HK-2 cells inhibition, and the IC50 value compared with AA-I and AL-I (C). Data represent mean ± S.D. from three independent experiments.
Fig. 2. (A) Morphology of HK-2 cells following various treatments for 24 h (phase contrast, 100). (B) Morphology of HK-2 cells stained with Hoechst 33258 following various treatments for 24 h (phase contrast, 200).
cluded by the plasma membrane of living cells, and thus can be used to distinguish necrotic cells from apoptotic and living cells by supravital staining without prior permeabilization (Zamai et al., 1996). This assay divides apoptotic cells into two stages: early (Annexin V+/PI ) and late apoptotic/necrotic (Annexin V+/ PI+).
In our experiment, we chose a range of doses of AA-I and AL-I (20–80 lM) to observe both early apoptosis and necrosis. HK-2 cells treated with different concentrations of AA-I and AL-I exhibited a significant progressive increase in annexin V+/PI staining; concurrently, the number of annexin V+/PI+ cells remained at low levels (Fig. 4). This finding indicated that AA-I and AL-I induced
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Fig. 3. Effects of various treatments on cell-cycle analysis of HK-2 cells. (A) Representative data of flow cytometry assay. (B) The summary (*P < 0.05, **P < 0.01, ***P < 0.0001 vs. control).
apoptosis in HK-2 cells in a dose-dependent manner without causing any obvious necrosis. 3.4. Effect of AA-I and AL-I on caspase 3 expression The caspase family can be divided into two major subgroups on the basis of the substrate specificity, sequence homology, and biochemical function of the proteases: caspase 1-like proteases (caspases 1, 4, and 5), and caspase 3-like proteases (caspases 2, 3, and 10) (Nicholson and Thornberry, 1997). Caspase 3 plays an especially pivotal role in the terminal phase of apoptosis (Wilson, 1998). To determine whether caspase 3 activity was involved in the apoptosis induced by AA-I and AL-I, we performed an assay using Ac-DEVD-pNA, which is a colorimetric substrate for caspase 3-like proteases. As shown in Fig. 5, AA-I and AL-I increased caspase 3like activity in a concentration-dependent manner. These data suggest that the activation of caspase 3-like proteases was involved in the apoptosis induced by AA-I and AL-I. 4. Discussion AL-I is one of the important metabolites of AA-I in vivo and it can enter and directly injure renal proximal tubule cells. Recent study reported that the concentration of AL-I increased rapidly during the first 12 h and reached near saturation after approxi-
mately 12 h after treatment with AA-I (20 lg/ml) in L-02 cells, and the maximal intracellular concentration of AL-I was reached at about 36 h (Yuan et al., 2009). In vivo, Ling et al. (2007) reported that the rat plasma profiles of AL-I could still be detected even after 20 h after a single oral dose of AA-I (20 mg/kg). These studies showed that there was a conversion of AA-I to AL-I in vitro, and AL-I is one of the important metabolites of AA-I in vivo. Furthermore, Shang et al. (2008) also reported that AL-I could enter renal proximal tubular epithelial cells in short time and accumulate in cytoplasm after treatment on HK-2 cells. This property may partially explain that AL-I can enter and directly injure renal proximal tubule cells. All of researches may contribute to explain the AL-I can lead to persistent renal toxicity in the development of aristolochic acid nephropathy. To elucidate the molecular mechanism of the AL-I induced cytotoxicity on HK-2 cells, we first compared the cellular toxicities of AA-I and AL-I by performing the MTT assay and found that under the experimental conditions, AL-I induced inhibition of cell growth at lower concentrations than those required for AA-I (Fig. 1). Moreover, the morphological changes in the HK-2 cells treated with AAI and AL-I indicated apoptosis (Figs. 2), which was further confirmed by flow cytometry (Figs. 3 and 4). We also found that AAI and AL-I were able to activate caspase 3 (Fig. 5), thus providing a reasonable explanation for their cytotoxic activities in HK-2 cells. Under the experimental conditions, AA-I and AL-I exert an antiproliferative effect on HK-2 cells by causing S-phase arrest. All the
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A
Early apoptotic cells(%) 60
**
AA-I AL-I
40
** **
20
µM 80
µM 40
20
co nt
µM
0 ro l
Percentage of apoptotic cells
B
Fig. 4. Apoptosis induced by AA-I or AL-I. HK-2 cells were treated with AA-I and AL-I 20, 40 and 80 lM for 24 h. (A) The percentage of early apoptotic cells treated with AA-I or AL-I of various concentrations. (B) The summary (*P < 0.05, **P < 0.01 vs. AA-I group).
folds of caspase 3 activity
10
AA-I AL-I
8
**
6
* *
4 2
µM 80
µM
µM 40
ro l
20
co nt
µM 80 µM
40
co nt
ro l 20 µM
0
Fig. 5. Activation of caspase 3 induced by AA-I or AL-I. HK-2 cells were treated without (control) or with AA-I or AL-I (20, 40 and 80 lM) for 24 h. Enzymatic activity of caspase 3 was detected by incubation with specific colorigenic substrates, Ac-DEVD-pNA (*P < 0.05, **P < 0.01 vs. control).
sation, and nuclear fragmentation, support the notion of apoptosis. The morphological changes as well as the cytochemical evidence in the present study clearly proved that AA-I and AL-I primarily caused caspase 3-dependent apoptosis rather than necrosis in HK-2 cells. The apoptotic activity of AL-I was more potent and obvious than that of AA-I. In conclusion, the experimental data showed the cytotoxic effects of various concentrations of AA-I and AL-I on HK-2 cells, including the inhibition of cell growth, the changes in the morphology and cell cycle, and the apoptosis-inducing activity. Furthermore, the caspase 3 assay clearly proved that the effects of AA-I and AL-I in HK-2 cells were primarily mediated by caspase 3dependent apoptosis. To our knowledge, this is the first study to reveal that the cytotoxic effect of AL-I on HK-2 cells is stronger than that of AA-I and that the cytotoxic mechanism is associated with caspase 3-dependent apoptosis.
Conflict of interest statement morphological characteristics observed in the HK-2 cells treated with AA-I or AL-I, including cellular shrinkage, chromatin conden-
None declared.
J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097
Acknowledgements The authors thank Dr. Liu Jun (Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing, China) for revision on the manuscript. This project was supported by National Natural Science Foundation of China: Project for Young Scientists Fund (No. 3070116), and Mega-projects of Science Research for the 11th Five-Year Plan: Standardized platform construction and scientific application in new technologies for new drug screening (No. 2009ZX09302002), and Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of finance (No. 200707008).
References Chang, H.R., Lian, J.D., Lo, C.W., Chang, Y.C., Yang, M.Y., Wang, C.J., 2006. Induction of urothelial proliferation in rats by aristolochic acid through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2. Food Chem. Toxicol. 44, 28–35. Debelle, F.D., Nortier, J.L., De Prez, E.G., Garbar, C.H., Vienne, A.R., Salmon, I.J., Deschodt-Lanckman, M.M., Vanherweghem, J.L., 2002. Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats. J. Am. Soc. Nephrol. 13, 431–436. Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., Henson, P.M., 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216. Ito, A., Uehara, T., Tokumitsu, A., Okuma, Y., Nomura, Y., 1999. Possible involvement of cytochrome c release and sequential activation of caspases in ceramideinduced apoptosis in SK-N-MC cells. Biochim. Biophys. Acta 1452, 263–274.
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Jackson, L., Kofman, S., Weiss, A., Brodovsky, H., 1964. Aristolochic acid (Nsc50413): phase I clinical study. Cancer Chemoth. Rep. 42, 35–37. Li, Y., Liu, Z., Guo, X., Shu, J., Chen, Z., Li, L., 2006. Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro. Arch. Toxicol. 80, 524–532. Ling, Z., Jiang, Z.Z., Huang, X., Zhang, L.Y., Xu, X.Y., 2007. A liquid chromatography– mass spectrometry method for the quantitation of aristololactam-I in rat plasma. Biomed. Chromatogr. 21, 10–14. Nicholson, D.W., Thornberry, N.A., 1997. Caspases: killer proteases. Trend. Biochem. Sci. 22, 299–306. Shang, P., Wang, X., Li, X.M., Tang, J.W., Cai, S.Q., 2008. Observation of penetration, distribution and accumulation in human renal proximal tubular epithelial cells by aristololactam-I. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China J. Chinese Mater. Med. 33, 793–797. Vanherweghem, J.L., Abramowicz, D., Tielemans, C., Depierreux, M., 1996. Effects of steroids on the progression of renal failure in chronic interstitial renal fibrosis: a pilot study in Chinese herbs nephropathy. Am. J. Kidney Dis. 27, 209–215. Vanherweghem, J.L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R., et al., 1993. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387– 391. Wilson, M.R., 1998. Apoptotic signal transduction: emerging pathways. Biochemistry and Cell Biology = Biochim. Biol. Cell. 76, 573–582. Yuan, J., Luo, X., Guo, M., Wu, J., Yang, W., Yu, R., Yao, S., 2009. Determination of aristolochic acid I and its metabolites in cell culture with a hyphenated highperformance liquid chromatographic technique for cell toxicology. Talanta 78, 1141–1147. Zamai, L., Falcieri, E., Marhefka, G., Vitale, M., 1996. Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation. Cytometry 23, 303–311. Zhang, C., Wang, X., Shang, M., Yu, J., Xu, Y., Li, Z., Lei, L., Li, X., Cai, S., Namba, T., 2006. Simultaneous determination of five aristolochic acids and two aristololactams in Aristolochia plants by high-performance liquid chromatography. Biomed. Chromatogr. 20, 309–318.
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells Ji Li a, Liang Zhang b,c, Zhenzhou Jiang a,d, Bin Shu a,e, Fu Li a, Qingli Bao a, Luyong Zhang a,* a
Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Pharmacology, Nanjing University of Chinese Medicine, Nanjing 210046, PR China c Jiangsu Province Key Lab. of Efficiency and Safety Evaluation of Chinese Medicine, PR China d Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, PR China e Jiangsu Center for Safety Evaluation of Drugs, Nanjing University of Technology, Nanjing 210009, PR China b
a r t i c l e
i n f o
Article history: Received 20 January 2010 Accepted 18 March 2010 Available online 23 March 2010 Keywords: Aristolochic acid I (AA-I) Aristololactam I (AL-I) Cytotoxicity Human proximal tubular epithelial cell lines (HK-2 cells)
a b s t r a c t Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-Imediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway. Ó 2010 Published by Elsevier Ltd.
1. Introduction Aristolochic acid I (AA-I) and aristololactam I (AL-I) are the main active components in plants of the Aristolochia species (Zhang et al., 2006). Aristolochia is a Chinese traditional herbal remedy with potent diuretic activity. In some European countries, it is utilized in weight-loss regimens. However, the clinical application of aristolochic acid (AA) has been limited by its severe nephrotoxicity. The nephrotoxic effect was first reported in 1964 (Jackson et al., 1964). It was again noted in 1993 in Belgium when a group of young female patients undergoing a slimming regimen that included Chinese herbs containing AA presented with rapidly progressing interstitial renal fibrosis (Vanherweghem et al., 1993, 1996). Debelle et al. reported that long-term exposure or overdose of AA may cause severe nephrotoxicity, which is characterized by chronic renal failure, tubulointerstitial fibrosis, and development of urothelial cancer (Debelle et al., 2002). Recent studies have revealed that AA-I can cause direct damage to renal tubular cells, and the toxicity was associated with the formation of promutagenic AA-DNA adducts (Li et al., 2006). However, the mechanism of the renal injury caused by AL-I remains unclear. In the present study, we investigated the role and
* Corresponding author. Tel.: +86 25 83271043; fax: +86 25 83271142. E-mail address: [email protected] (L. Zhang). 0887-2333/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.tiv.2010.03.012
in vitro mechanism of AL-I-mediated cytotoxicity. In our experimental conditions, AL-I induced apoptosis in the cells of the human renal tubular epithelial (HK-2) cell line. Caspase 3 activation was observed during the course of this apoptosis. We also observed that the cytotoxic potency of AL-I was stronger than that of AA-I. 2. Materials and methods 2.1. Cell culture and reagents HK-2 cells were obtained from American Type Culture Collection (ATCC, USA). Dulbecco modified Eagle medium/F12 (F12 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) fetal bovine serum (FBS), and trypsin/ethylene diamine tetraacetic acid (EDTA) were purchased from Gibco (Gibco Laboratories, NY, USA). The HK-2 cells were grown in DF12 supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. AA-I and AL-I (purity 98%, determined by high-performance liquid chromatography) were purchased from Zhengzhou Tianlin Pharm-tech Co., Ltd. (Zhengzhou, Henan, China). AA-I and AL-I were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions (15 mM), which were stored at 4 °C. Before use in the experiment, the stock solution was diluted to the indicated concentrations by using the culture medium. During the experiments, the DMSO content in the medium never exceeded 0.5% (v/v).
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2.6. Statistical analysis
Propidium iodide (PI), a colorigenic synthetic peptide substrate for caspase 3 proteases (Ac-DEVD-pNA), 3-(4,5-dimethyl-thiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and ribonuclease A (RNase A) were purchased from Sigma (St. Louis, MO, USA). Annexin V/PI apoptosis detection kit was obtained from Becton Dickinson (CA, USA).
Each experiment was repeated at least 3 times, and the data were expressed as mean ± SD values. Student’s t test was used for statistical analysis, and P values less than 0.05 were considered statistically significant.
2.2. MTT assay
3. Results
HK-2 cells (5 104 cells/ml) were treated with AA-I and AL-I. The culture was incubated with fresh medium containing different concentrations of AA-I and AL-I for 24 h, 48 h, or 72 h. Subsequently, MTT was added to each well to a final concentration of 0.5 mg/ml. After incubation for 4 h at 37 °C, the formazan crystals derived from MTT were dissolved in DMSO, and the absorbance at 570 nm was measured using a Model 680 Microplate Reader (BioRad Lab., UK).
3.1. Cytotoxicities of AA-I and AL-I in HK-2 cells
2.3. Morphology of the HK-2 cells after treatment with AA-I and AL-I The morphologies of the HK-2 cells after exposure to 80 lM AAI or AL-I for 24 h were examined using a contrast microscope (1X71S8F-2; Olympus, Japan). After the treatments, the HK-2 cells were stained with Hoechst 33258 and visualized to investigate the incidence of apoptosis. To this end, the cells were seeded on coverslips, incubated with 80 lM AA-I or AL-I for 24 h, and then fixed with 4% paraformaldehyde for 10 min. The nuclear DNA was stained with 5 mg/ml Hoechst 33258 for another 5 min, and these cells were observed under a fluorescence microscope (1X71S8F-2; Olympus, Japan). 2.4. Detection of apoptosis and cell-cycle analysis 2.4.1. PI staining assay HK-2 cells were collected at the end of treatment, washed twice with ice-cold phosphate-buffered saline (PBS), and then fixed in 70% ethanol at 4 °C for 12 h. After fixation, the cells were washed twice with PBS and incubated in PBS containing PI, RNase A, Triton X-100 (0.5%) at 37 °C for 30 min. The fluorescence emitted from the propidium–DNA complex was measured using FACScan flow cytometry (Becton Dickinson, San Jones, CA, USA). Cells containing hypodiploid DNA were considered apoptotic. 2.4.2. Annexin V/PI staining assay The apoptosis ratio was measured using FACScan flow cytometer (Becton Dickenson, San Jose, CA, USA) according to the instructions provided with the annexin V/PI kit. Briefly, after treatment with 40 lM AA-I or AL-I for 24, 48, or 72 h, HK-2 cells were harvested and washed twice with pre-cooled PBS and resuspended in a binding buffer containing fluorescein isothiocyanate (FITC)conjugated annexin V antibody and PI. After incubation in the dark for 30 min, the cells were analyzed using flow cytometry. 2.5. Activation of caspase 3 The activity of caspase 3 was determined using a previously described method (Ito et al., 1999). Briefly, cells (106 ml 1) were harvested after treatment, washed thrice with PBS, and resuspended in ice-cold buffer containing 50 mM Tris–HCl, 1 mM EDTA, 10 mM ethylene glycol tetraacetic acid (EGTA), and 1 mM DTT. Cell lysates were centrifuged at 12,000 g for 5 min, and extracts containing 50 lg of the protein were incubated with 100 lM of the enzyme-specific colorimetric substrate Ac-DEVD-pNA at 37 °C for 2 h. The colorimetric release of p-nitroaniline from the Ac-DEVD-pNA substrate was measured by determining the absorbance at 405 nm.
The cytotoxicities of AA-I and AL-I in HK-2 cells were determined using the MTT assay. The cell growth inhibition after incubation with various concentrations of AA-I or AL-I for 24, 48, or 72 h are presented in Fig. 1A and B. As expected, AA-I and AL-I showed cytotoxic activity. In comparison with the control cells, HK-2 cells exposed to these agents for 24, 48, and 72 h showed a significant dose- and time-dependent inhibition of cell growth (P < 0.01 vs. control). DMSO alone did not show any inhibitory effect on the cell viability. As shown in Fig. 1C, the IC50 values of AA-I were higher than those of AL-I, which suggested that AL-I plays an important role in the AA-mediated cytotoxicity in HK-2 cells. 3.2. Morphological changes in HK-2 cells after exposure to AA-I and AL-I As shown in Fig. 2A, untreated HK-2 cells appeared to proliferate in spindle-like shapes in the wells, and HK-2 cells treated with AA-I (80 lM) contracted and became rounded. The morphology of the HK-2 cells exposed to AL-I (80 lM) for 24 h was similar to that of the cells treated with AA-I. After the treatments, the HK-2 cells were stained with Hoechst 33258 and visualized to investigate the incidence of apoptosis. The morphological images acquired using the microscope are shown in Fig. 2B. Apoptotic cells were characterized by nuclear condensation and chromatin margination. 3.3. Detection of apoptosis in the HK-2 cells treated with AA-I and AL-I 3.3.1. Effects of AA-I and AL-I on cell cycle The inhibitory effect of AA-I and AL-I on cell growth was further investigated using flow cytometric analysis of the cellular DNA content. As shown in Fig. 3A, apoptotic cells were identified by the sub-G1 apoptotic peaks, which could be attributed to their lower DNA content. Apoptotic peaks and increased percentage of apoptotic cells were observed after 24 h treatment with 40 lmol/ L AL-I or 80 lmol/L AA-I. The distributions of the cells in various phases of the cell cycle and the percentages of apoptotic cells after treatment with different concentrations of the test compounds are summarized in Fig. 3B. The increase in S-phase cell populations after treatment with AA-I or AL-I for 24 h were similar. This finding is consistent with the previous reports that AA caused S-phase arrest, accelerated the cell cycle, and caused abnormal proliferation in the epithelial cells of the urinary tract (Chang et al., 2006). After treatment with AA-I, the percentage of cells in the G2/M phase showed a marked increase, and the percentage of cells in the G1 phase showed a significant reduction; however, these effects were not as remarkable as those observed in the cells treated with AL-I. 3.3.2. Detection of apoptosis and necrosis Annexin V specifically binds to the negatively charged phosphatidylserine (PS) that is translocated from the inner surface of the cell membrane to the surface during early apoptosis (Fadok et al., 1992). PI is a non-specific DNA intercalating agent, which is ex-
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Fig. 1. Inhibition of cell viability by AA-I or AL-I in HK-2 cells. Concentration-dependent effect of AA-I (A), AL-I (B) on HK-2 cells inhibition, and the IC50 value compared with AA-I and AL-I (C). Data represent mean ± S.D. from three independent experiments.
Fig. 2. (A) Morphology of HK-2 cells following various treatments for 24 h (phase contrast, 100). (B) Morphology of HK-2 cells stained with Hoechst 33258 following various treatments for 24 h (phase contrast, 200).
cluded by the plasma membrane of living cells, and thus can be used to distinguish necrotic cells from apoptotic and living cells by supravital staining without prior permeabilization (Zamai et al., 1996). This assay divides apoptotic cells into two stages: early (Annexin V+/PI ) and late apoptotic/necrotic (Annexin V+/ PI+).
In our experiment, we chose a range of doses of AA-I and AL-I (20–80 lM) to observe both early apoptosis and necrosis. HK-2 cells treated with different concentrations of AA-I and AL-I exhibited a significant progressive increase in annexin V+/PI staining; concurrently, the number of annexin V+/PI+ cells remained at low levels (Fig. 4). This finding indicated that AA-I and AL-I induced
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Fig. 3. Effects of various treatments on cell-cycle analysis of HK-2 cells. (A) Representative data of flow cytometry assay. (B) The summary (*P < 0.05, **P < 0.01, ***P < 0.0001 vs. control).
apoptosis in HK-2 cells in a dose-dependent manner without causing any obvious necrosis. 3.4. Effect of AA-I and AL-I on caspase 3 expression The caspase family can be divided into two major subgroups on the basis of the substrate specificity, sequence homology, and biochemical function of the proteases: caspase 1-like proteases (caspases 1, 4, and 5), and caspase 3-like proteases (caspases 2, 3, and 10) (Nicholson and Thornberry, 1997). Caspase 3 plays an especially pivotal role in the terminal phase of apoptosis (Wilson, 1998). To determine whether caspase 3 activity was involved in the apoptosis induced by AA-I and AL-I, we performed an assay using Ac-DEVD-pNA, which is a colorimetric substrate for caspase 3-like proteases. As shown in Fig. 5, AA-I and AL-I increased caspase 3like activity in a concentration-dependent manner. These data suggest that the activation of caspase 3-like proteases was involved in the apoptosis induced by AA-I and AL-I. 4. Discussion AL-I is one of the important metabolites of AA-I in vivo and it can enter and directly injure renal proximal tubule cells. Recent study reported that the concentration of AL-I increased rapidly during the first 12 h and reached near saturation after approxi-
mately 12 h after treatment with AA-I (20 lg/ml) in L-02 cells, and the maximal intracellular concentration of AL-I was reached at about 36 h (Yuan et al., 2009). In vivo, Ling et al. (2007) reported that the rat plasma profiles of AL-I could still be detected even after 20 h after a single oral dose of AA-I (20 mg/kg). These studies showed that there was a conversion of AA-I to AL-I in vitro, and AL-I is one of the important metabolites of AA-I in vivo. Furthermore, Shang et al. (2008) also reported that AL-I could enter renal proximal tubular epithelial cells in short time and accumulate in cytoplasm after treatment on HK-2 cells. This property may partially explain that AL-I can enter and directly injure renal proximal tubule cells. All of researches may contribute to explain the AL-I can lead to persistent renal toxicity in the development of aristolochic acid nephropathy. To elucidate the molecular mechanism of the AL-I induced cytotoxicity on HK-2 cells, we first compared the cellular toxicities of AA-I and AL-I by performing the MTT assay and found that under the experimental conditions, AL-I induced inhibition of cell growth at lower concentrations than those required for AA-I (Fig. 1). Moreover, the morphological changes in the HK-2 cells treated with AAI and AL-I indicated apoptosis (Figs. 2), which was further confirmed by flow cytometry (Figs. 3 and 4). We also found that AAI and AL-I were able to activate caspase 3 (Fig. 5), thus providing a reasonable explanation for their cytotoxic activities in HK-2 cells. Under the experimental conditions, AA-I and AL-I exert an antiproliferative effect on HK-2 cells by causing S-phase arrest. All the
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A
Early apoptotic cells(%) 60
**
AA-I AL-I
40
** **
20
µM 80
µM 40
20
co nt
µM
0 ro l
Percentage of apoptotic cells
B
Fig. 4. Apoptosis induced by AA-I or AL-I. HK-2 cells were treated with AA-I and AL-I 20, 40 and 80 lM for 24 h. (A) The percentage of early apoptotic cells treated with AA-I or AL-I of various concentrations. (B) The summary (*P < 0.05, **P < 0.01 vs. AA-I group).
folds of caspase 3 activity
10
AA-I AL-I
8
**
6
* *
4 2
µM 80
µM
µM 40
ro l
20
co nt
µM 80 µM
40
co nt
ro l 20 µM
0
Fig. 5. Activation of caspase 3 induced by AA-I or AL-I. HK-2 cells were treated without (control) or with AA-I or AL-I (20, 40 and 80 lM) for 24 h. Enzymatic activity of caspase 3 was detected by incubation with specific colorigenic substrates, Ac-DEVD-pNA (*P < 0.05, **P < 0.01 vs. control).
sation, and nuclear fragmentation, support the notion of apoptosis. The morphological changes as well as the cytochemical evidence in the present study clearly proved that AA-I and AL-I primarily caused caspase 3-dependent apoptosis rather than necrosis in HK-2 cells. The apoptotic activity of AL-I was more potent and obvious than that of AA-I. In conclusion, the experimental data showed the cytotoxic effects of various concentrations of AA-I and AL-I on HK-2 cells, including the inhibition of cell growth, the changes in the morphology and cell cycle, and the apoptosis-inducing activity. Furthermore, the caspase 3 assay clearly proved that the effects of AA-I and AL-I in HK-2 cells were primarily mediated by caspase 3dependent apoptosis. To our knowledge, this is the first study to reveal that the cytotoxic effect of AL-I on HK-2 cells is stronger than that of AA-I and that the cytotoxic mechanism is associated with caspase 3-dependent apoptosis.
Conflict of interest statement morphological characteristics observed in the HK-2 cells treated with AA-I or AL-I, including cellular shrinkage, chromatin conden-
None declared.
J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097
Acknowledgements The authors thank Dr. Liu Jun (Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing, China) for revision on the manuscript. This project was supported by National Natural Science Foundation of China: Project for Young Scientists Fund (No. 3070116), and Mega-projects of Science Research for the 11th Five-Year Plan: Standardized platform construction and scientific application in new technologies for new drug screening (No. 2009ZX09302002), and Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of finance (No. 200707008).
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