Toxicology in Vitro 24 (2010) 1092–1097

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Toxicology in Vitro

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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 Chinab Department of Pharmacology, Nanjing University of Chinese Medicine, Nanjing 210046, PR Chinac Jiangsu Province Key Lab. of Efficiency and Safety Evaluation of Chinese Medicine, PR Chinad Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, PR Chinae Jiangsu Center for Safety Evaluation of Drugs, Nanjing University of Technology, Nanjing 210009, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 January 2010Accepted 18 March 2010Available online 23 March 2010

Keywords:Aristolochic acid I (AA-I)Aristololactam I (AL-I)CytotoxicityHuman proximal tubular epithelial cell lines(HK-2 cells)

0887-2333/$ - see front matter � 2010 Published bydoi:10.1016/j.tiv.2010.03.012

* Corresponding author. Tel.: +86 25 83271043; faxE-mail address: lyzhang@cpu.edu.cn (L. Zhang).

Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aris-tolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also partici-pate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-I-mediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells ofthe human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-Iand AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of cas-pase 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 bythe 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 alsosuggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects ofthese molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway.

� 2010 Published by Elsevier Ltd.

1. Introduction in vitro mechanism of AL-I-mediated cytotoxicity. In our experi-

Aristolochic acid I (AA-I) and aristololactam I (AL-I) are the mainactive components in plants of the Aristolochia species (Zhang et al.,2006). Aristolochia is a Chinese traditional herbal remedy with po-tent diuretic activity. In some European countries, it is utilized inweight-loss regimens. However, the clinical application of aristol-ochic acid (AA) has been limited by its severe nephrotoxicity. Thenephrotoxic effect was first reported in 1964 (Jackson et al.,1964). It was again noted in 1993 in Belgium when a group ofyoung female patients undergoing a slimming regimen that in-cluded Chinese herbs containing AA presented with rapidly pro-gressing interstitial renal fibrosis (Vanherweghem et al., 1993,1996). Debelle et al. reported that long-term exposure or overdoseof AA may cause severe nephrotoxicity, which is characterized bychronic renal failure, tubulointerstitial fibrosis, and developmentof urothelial cancer (Debelle et al., 2002). Recent studies have re-vealed that AA-I can cause direct damage to renal tubular cells,and the toxicity was associated with the formation of promutagen-ic AA-DNA adducts (Li et al., 2006).

However, the mechanism of the renal injury caused by AL-I re-mains unclear. In the present study, we investigated the role and

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mental conditions, AL-I induced apoptosis in the cells of the humanrenal tubular epithelial (HK-2) cell line. Caspase 3 activation wasobserved during the course of this apoptosis. We also observedthat 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 Collec-tion (ATCC, USA). Dulbecco modified Eagle medium/F12 (F12 with4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) fetalbovine 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 at37 �C in a 5% CO2 atmosphere.

AA-I and AL-I (purity 98%, determined by high-performanceliquid chromatography) were purchased from Zhengzhou TianlinPharm-tech Co., Ltd. (Zhengzhou, Henan, China). AA-I and AL-Iwere dissolved in dimethyl sulfoxide (DMSO) to obtain stocksolutions (15 mM), which were stored at 4 �C. Before use in theexperiment, the stock solution was diluted to the indicated con-centrations by using the culture medium. During the experiments,the DMSO content in the medium never exceeded 0.5% (v/v).

J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097 1093

Propidium iodide (PI), a colorigenic synthetic peptide substratefor caspase 3 proteases (Ac-DEVD-pNA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and ribonucleaseA (RNase A) were purchased from Sigma (St. Louis, MO, USA). An-nexin V/PI apoptosis detection kit was obtained from Becton Dick-inson (CA, USA).

2.2. MTT assay

HK-2 cells (5 � 104 cells/ml) were treated with AA-I and AL-I.The culture was incubated with fresh medium containing differentconcentrations of AA-I and AL-I for 24 h, 48 h, or 72 h. Subse-quently, MTT was added to each well to a final concentration of0.5 mg/ml. After incubation for 4 h at 37 �C, the formazan crystalsderived from MTT were dissolved in DMSO, and the absorbance at570 nm was measured using a Model 680 Microplate Reader (Bio-Rad Lab., UK).

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 AA-I 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 Hoechst33258 and visualized to investigate the incidence of apoptosis. Tothis end, the cells were seeded on coverslips, incubated with 80 lMAA-I or AL-I for 24 h, and then fixed with 4% paraformaldehyde for10 min. The nuclear DNA was stained with 5 mg/ml Hoechst 33258for another 5 min, and these cells were observed under a fluores-cence microscope (1X71S8F-2; Olympus, Japan).

2.4. Detection of apoptosis and cell-cycle analysis

2.4.1. PI staining assayHK-2 cells were collected at the end of treatment, washed twice

with ice-cold phosphate-buffered saline (PBS), and then fixed in70% ethanol at 4 �C for 12 h. After fixation, the cells were washedtwice with PBS and incubated in PBS containing PI, RNase A, TritonX-100 (0.5%) at 37 �C for 30 min. The fluorescence emitted from thepropidium–DNA complex was measured using FACScan flowcytometry (Becton Dickinson, San Jones, CA, USA). Cells containinghypodiploid DNA were considered apoptotic.

2.4.2. Annexin V/PI staining assayThe apoptosis ratio was measured using FACScan flow cytome-

ter (Becton Dickenson, San Jose, CA, USA) according to the instruc-tions provided with the annexin V/PI kit. Briefly, after treatmentwith 40 lM AA-I or AL-I for 24, 48, or 72 h, HK-2 cells were har-vested and washed twice with pre-cooled PBS and resuspendedin a binding buffer containing fluorescein isothiocyanate (FITC)-conjugated annexin V antibody and PI. After incubation in the darkfor 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 de-scribed method (Ito et al., 1999). Briefly, cells (106 ml�1) were har-vested after treatment, washed thrice with PBS, and resuspendedin ice-cold buffer containing 50 mM Tris–HCl, 1 mM EDTA,10 mM ethylene glycol tetraacetic acid (EGTA), and 1 mM DTT. Celllysates were centrifuged at 12,000 g for 5 min, and extracts con-taining 50 lg of the protein were incubated with 100 lM of the en-zyme-specific colorimetric substrate Ac-DEVD-pNA at 37 �C for 2 h.The colorimetric release of p-nitroaniline from the Ac-DEVD-pNAsubstrate was measured by determining the absorbance at 405 nm.

2.6. Statistical analysis

Each experiment was repeated at least 3 times, and the datawere expressed as mean ± SD values. Student’s t test was usedfor statistical analysis, and P values less than 0.05 were consideredstatistically significant.

3. Results

3.1. Cytotoxicities of AA-I and AL-I in HK-2 cells

The cytotoxicities of AA-I and AL-I in HK-2 cells were deter-mined using the MTT assay. The cell growth inhibition after incu-bation with various concentrations of AA-I or AL-I for 24, 48, or72 h are presented in Fig. 1A and B.

As expected, AA-I and AL-I showed cytotoxic activity. In com-parison with the control cells, HK-2 cells exposed to these agentsfor 24, 48, and 72 h showed a significant dose- and time-dependentinhibition of cell growth (P < 0.01 vs. control). DMSO alone did notshow 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 sug-gested that AL-I plays an important role in the AA-mediated cyto-toxicity in HK-2 cells.

3.2. Morphological changes in HK-2 cells after exposure to AA-I andAL-I

As shown in Fig. 2A, untreated HK-2 cells appeared to prolifer-ate in spindle-like shapes in the wells, and HK-2 cells treated withAA-I (80 lM) contracted and became rounded. The morphology ofthe HK-2 cells exposed to AL-I (80 lM) for 24 h was similar to thatof the cells treated with AA-I.

After the treatments, the HK-2 cells were stained with Hoechst33258 and visualized to investigate the incidence of apoptosis. Themorphological images acquired using the microscope are shown inFig. 2B. Apoptotic cells were characterized by nuclear condensationand 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 cycleThe inhibitory effect of AA-I and AL-I on cell growth was further

investigated using flow cytometric analysis of the cellular DNAcontent. As shown in Fig. 3A, apoptotic cells were identified bythe sub-G1 apoptotic peaks, which could be attributed to theirlower DNA content. Apoptotic peaks and increased percentage ofapoptotic 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 variousphases of the cell cycle and the percentages of apoptotic cells aftertreatment with different concentrations of the test compounds aresummarized in Fig. 3B. The increase in S-phase cell populationsafter treatment with AA-I or AL-I for 24 h were similar. This findingis consistent with the previous reports that AA caused S-phasearrest, accelerated the cell cycle, and caused abnormal proliferationin the epithelial cells of the urinary tract (Chang et al., 2006). Aftertreatment with AA-I, the percentage of cells in the G2/M phaseshowed a marked increase, and the percentage of cells in the G1phase showed a significant reduction; however, these effects werenot as remarkable as those observed in the cells treated with AL-I.

3.3.2. Detection of apoptosis and necrosisAnnexin V specifically binds to the negatively charged phospha-

tidylserine (PS) that is translocated from the inner surface of thecell membrane to the surface during early apoptosis (Fadok et al.,1992). PI is a non-specific DNA intercalating agent, which is ex-

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 withAA-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 varioustreatments for 24 h (phase contrast, 200�).

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cluded by the plasma membrane of living cells, and thus can beused to distinguish necrotic cells from apoptotic and living cellsby supravital staining without prior permeabilization (Zamaiet 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-2cells treated with different concentrations of AA-I and AL-I exhib-ited a significant progressive increase in annexin V+/PI� staining;concurrently, the number of annexin V+/PI+ cells remained at lowlevels (Fig. 4). This finding indicated that AA-I and AL-I induced

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).

J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097 1095

apoptosis in HK-2 cells in a dose-dependent manner without caus-ing 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 onthe basis of the substrate specificity, sequence homology, and bio-chemical function of the proteases: caspase 1-like proteases (casp-ases 1, 4, and 5), and caspase 3-like proteases (caspases 2, 3, and10) (Nicholson and Thornberry, 1997). Caspase 3 plays an espe-cially pivotal role in the terminal phase of apoptosis (Wilson,1998).

To determine whether caspase 3 activity was involved in theapoptosis induced by AA-I and AL-I, we performed an assay usingAc-DEVD-pNA, which is a colorimetric substrate for caspase 3-likeproteases. As shown in Fig. 5, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These data sug-gest that the activation of caspase 3-like proteases was involved inthe apoptosis induced by AA-I and AL-I.

4. Discussion

AL-I is one of the important metabolites of AA-I in vivo and itcan enter and directly injure renal proximal tubule cells. Recentstudy reported that the concentration of AL-I increased rapidlyduring 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 reachedat about 36 h (Yuan et al., 2009). In vivo, Ling et al. (2007) reportedthat the rat plasma profiles of AL-I could still be detected even after20 h after a single oral dose of AA-I (20 mg/kg). These studiesshowed that there was a conversion of AA-I to AL-I in vitro, andAL-I is one of the important metabolites of AA-I in vivo. Further-more, Shang et al. (2008) also reported that AL-I could enter renalproximal tubular epithelial cells in short time and accumulate incytoplasm after treatment on HK-2 cells. This property may par-tially explain that AL-I can enter and directly injure renal proximaltubule cells. All of researches may contribute to explain the AL-Ican lead to persistent renal toxicity in the development of aristol-ochic acid nephropathy.

To elucidate the molecular mechanism of the AL-I induced cyto-toxicity on HK-2 cells, we first compared the cellular toxicities ofAA-I and AL-I by performing the MTT assay and found that underthe experimental conditions, AL-I induced inhibition of cell growthat lower concentrations than those required for AA-I (Fig. 1). More-over, the morphological changes in the HK-2 cells treated with AA-I and AL-I indicated apoptosis (Figs. 2), which was further con-firmed by flow cytometry (Figs. 3 and 4). We also found that AA-I and AL-I were able to activate caspase 3 (Fig. 5), thus providinga reasonable explanation for their cytotoxic activities in HK-2 cells.

Under the experimental conditions, AA-I and AL-I exert an anti-proliferative effect on HK-2 cells by causing S-phase arrest. All the

Early apoptotic cells(%)

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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 orAL-I of various concentrations. (B) The summary (*P < 0.05, **P < 0.01 vs. AA-I group).

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Fig. 5. Activation of caspase 3 induced by AA-I or AL-I. HK-2 cells were treatedwithout (control) or with AA-I or AL-I (20, 40 and 80 lM) for 24 h. Enzymaticactivity of caspase 3 was detected by incubation with specific colorigenicsubstrates, Ac-DEVD-pNA (*P < 0.05, **P < 0.01 vs. control).

1096 J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097

morphological characteristics observed in the HK-2 cells treatedwith AA-I or AL-I, including cellular shrinkage, chromatin conden-

sation, and nuclear fragmentation, support the notion of apoptosis.The morphological changes as well as the cytochemical evidence inthe present study clearly proved that AA-I and AL-I primarilycaused caspase 3-dependent apoptosis rather than necrosis inHK-2 cells. The apoptotic activity of AL-I was more potent andobvious than that of AA-I.

In conclusion, the experimental data showed the cytotoxic ef-fects of various concentrations of AA-I and AL-I on HK-2 cells,including the inhibition of cell growth, the changes in the morphol-ogy and cell cycle, and the apoptosis-inducing activity. Further-more, the caspase 3 assay clearly proved that the effects of AA-Iand AL-I in HK-2 cells were primarily mediated by caspase 3-dependent apoptosis. To our knowledge, this is the first study to re-veal that the cytotoxic effect of AL-I on HK-2 cells is stronger thanthat of AA-I and that the cytotoxic mechanism is associated withcaspase 3-dependent apoptosis.

Conflict of interest statement

None declared.

J. Li et al. / Toxicology in Vitro 24 (2010) 1092–1097 1097

Acknowledgements

The authors thank Dr. Liu Jun (Jiangsu Center for Drug Screen-ing, China Pharmaceutical University, Nanjing, China) for revisionon the manuscript.

This project was supported by National Natural Science Founda-tion of China: Project for Young Scientists Fund (No. 3070116), andMega-projects of Science Research for the 11th Five-Year Plan:Standardized platform construction and scientific application innew technologies for new drug screening (No. 2009ZX09302-002), and Specific Fund for Public Interest Research of TraditionalChinese Medicine, Ministry of finance (No. 200707008).

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