Aristolochic Acid I Induces Ovarian Toxicity by Inhibition of Akt

Nov 18, 2014 - mouse ovary; western blot was used to assess apoptosis;. TUNEL assay was used to evaluate apoptotic cell death; and immunohistochemistr...
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Aristolochic Acid I Induces Ovarian Toxicity by Inhibition of Akt Phosphorylation Dong Hoon Kwak,† Ji-Hye Park,‡ Hak-Seung Lee,‡ Ji-Sook Moon,‡ and Seoul Lee*,‡ †

Institute for Glycoscience, Wonkwang University, Iksan 570-749, Republic of Korea Department of Pharmacology, Wonkwang University School of Medicine and Wonkwang Brain Research Institute, Iksan 570-749, Republic of Korea

‡

ABSTRACT: Aristolochic acids are natural products found in Chinese herbs of the Aristolochiaceae family. Aristolochic acid I (AAI) is a potent carcinogen and was found to be toxic in animal and clinical studies. Apoptosis is a rapid, selective process of physiological cell deletion that regulates the balance between cell proliferation and cell death and is induced by various kinds of damage. However, the toxicity of AAI during ovarian maturation in the mouse is unclear and is the subject of the present investigation. We used Chinese hamster ovaryK1 (CHO-K1) cells and an AAI injection mouse model: MTT assay was used to assess AA toxicity to cells; ovary size and weight were measured to determine the toxicity of AA to mouse ovary; western blot was used to assess apoptosis; TUNEL assay was used to evaluate apoptotic cell death; and immunohistochemistry was used to examine the local expression of apoptotic proteins in ovary tissue. We found that AAI significantly inhibits the viability of CHO-K1 cells and strongly induces apoptotic cell death in CHO-K1 cells and in mouse ovary. In addition, we observed that AAI markedly increases the expression of pro-apoptotic proteins, including Bax, caspase-3, caspase-9, and poly(ADP) ribose polymerase (PARP). In contrast, antiapoptotic proteins, such as Bcl-2 and survivin, were decreased by AAI treatment. Furthermore, we observed that ovary size and weight were significantly reduced and that the number of ovulated oocytes was markedly suppressed in AAI-treated mice. These results suggest that AAI strongly induces toxic damage during ovarian maturation by inhibiting Akt phosphorylation-mediated suppression of apoptosis.

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epithelium cells.7,8 Long-term exposure to high-dose AAI can cause severe organ toxicity, such as chronic renal failure, tubulointerstitial fibrosis, DNA damage and cell-cycle arrest, and urothelial cancer.4,5 AAI-mediated carcinogenesis and stomach tumor were induced by DNA adduct formation.9,10 Thus, AAI is a potent carcinogen that has been shown to be highly toxic in animal and clinical studies.11 Furthermore, several studies have demonstrated that renal tubular epithelial cells are very sensitive to apoptosis induced by AAI.4,12,13 Apoptosis is a selective process of physiological cell deletion that regulates the balance between cell proliferation and cell death and is induced by various types of cellular damage.14 The intrinsic apoptosis pathway regulates the activity of the survivin and B-cell lymphoma 2 (Bcl-2) family proteins.15 Another member of the Bcl-2 family, Bcl-2associated X protein (Bax), was the first protein to be shown to form a regulatory dimer with Bcl-2 and has been reported to block the ability of Bcl-2 to inhibit apoptosis, suggesting that Bax may promote apoptosis by functional antagonism through the formation of heterodimers with Bcl-2.16 Poly(ADP) ribose

INTRODUCTION Aristolochic acids (AAs) are a family of structurally related nitrophenanthrene carboxylic acids found in Chinese herbs; AAs are found exclusively in Aristolochiaceae.1 Earlier studies reported that AAs have therapeutic effects, including antifungal, antibacterial, and antiviral effects.2 Their toxicity received attention when a case study was published describing nephropathy, renal failure, and urinary tract cancer in more than 100 woman in Belgium who had taken herbal pills containing AAs for weight loss.3 Aristolochic acid-associated nephropathy (AAN) is one of the common causes of chronic kidney disease in China and is a worldwide problem.4 Pathologically, in both patients and animal models of AAN, chronic AAN is characterized by extensive tubulointerstitial fibrosis with atrophy and loss of the tubules.4,5 The major components of AAs are 8-methoxy-6-nitro-phenanthro-(3,4-d)1,3-dioxolo-5-carboxylic acid (AAI) and 6-nitro-phenanthro(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAII), and both AAI and AAII have been shown to cause direct damage to renal tubular cells.3 However, AAI is the main ingredient in herbal pills containing AAs, and it is more cytotoxic than AAII.6 Numerous researchers have reported AAI toxicity in multiple organs, including the stomach and intestine as well as in renal tubular © 2014 American Chemical Society

Received: September 18, 2014 Published: November 18, 2014 2128

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PBS were diluted from a 10 mM AAI stock solution in PBS. At the end of the incubation period, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; 5 mg/mL in PBS) was added to each well, and the plate was incubated at 37 °C for 4 h. At the end of the incubation, the culture medium was discarded, and the wells were washed with PBS, after which 150 μL of DMSO was added to each well, and the plate was incubated with shaking at room temperature. Absorbance was measured at 540 nm using an ELX800 Universal Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA), and the percent (%) cell viability was calculated. In Situ Labeling of DNA in Apoptotic Cells. Apoptotic cells were identified in tissue sections (5 μm) and in CHO-K1 cells by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) using a TUNEL kit (Roche Ltd., Basel, Switzerland). Briefly, tissue sections and CHO-K1 cells were treated with proteinase K (20 mg/mL) for 15 min at room temperature before TdT was used to label the 3′-OH ends of DNA with digoxigeninlabeled nucleotides (1 h incubation at 37 °C). The sections and cells were then treated with an anti-digoxigenin antibody−peroxidase conjugate for 30 min at room temperature and stained with a fluorescence development solution for 3−6 min to produce the characteristic green color of positive cells. TUNEL-positive cells in seminiferous tubules in randomly chosen fields were counted under a light microscope. Apoptotic cell counting was carried out by two independent investigators, after which the average was calculated. AAI Treatment and Ovary Collection in Mice. Female ICR mice (4 weeks of age, Samtaco Co., Ltd., Gyungi-do, Korea) were raised under strict pathogen-free and microbiologically well-defined conditions, in accordance with the regulations of the ethics committee of the Korea Institute of Oriental Medicine (KIOM). The mice were treated with 10 mg/kg AAI (Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally for 4 weeks. Control mice were given PBS only via the same route. Animals were sacrificed and ovaries were collected after 4 weeks. Superovulation. Female ICR mice (8 weeks of age, Samtaco Co., Ltd., Gyungi-do, Korea) were superovulated via intraperitoneal administration of 5 IU equine chorionic gonadotropin (PMSG; Sigma-Aldrich, St. Louis, MO, USA). Forty six hours after PMSG treatment, mice were intraperitoneally treated with 5 IU of human chorionic gonadotropin (hCG; Sigma-Aldrich). The oocytes were collected form the oviducts 1 h after hCG administration. The oviducts were isolated and placed in a dish containing paraffin oil. The cumulus−oocyte complexes were dissected from the swollen ampulla and transferred to Toyota, Yokohama, and Hosi (TYH) medium under paraffin oil, followed by preincubation at 37 °C under a 5% CO2 atmosphere in a humidified incubator. Hematoxylin & Eosin (H&E) Staining and Immunohistochemistry. The ovaries were excised and weighed, after which all tissues were fixed in paraformaldehyde solution for 24 h. Ovary tissues were washed in 70% ethanol, soaked in a graded series of ethanol concentrations, and embedded in paraffin. Ovary tissue samples were cut into 5 μm thick sections that were stained with H&E. Alternative sections were prepared for immunohistochemistry (IHC). Next, sections were treated with 0.1% sodium citrate buffer (pH 6) at 100 °C for 10 min and washed with PBS. Sections were delineated by a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H2O2 for 15 min to inhibit endogenous peroxidase activity, after which they were washed with PBS and incubated for 18 h at 4 °C with primary antibodies: anti-Bax, anti-proliferating cell nuclear antigen (PCNA), anti-survivin, and anti-p53, all at a 1:200 dilution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After incubation with the primary antibodies, sections were washed three times for 5 min each with PBS, followed by incubation with biotinylated IgG and then with a streptavidin−peroxidase conjugate (ABC kit, Vector Laboratories, Burlingame, CA, USA). All incubation steps were separated by three washing steps. After washing three times for 5 min with PBS, sections were incubated with a DAB substrate containing diaminobenzidine (Vector Laboratories) for 5 min to stain the immunolabelled cells and were then counterstained with Mayer’s hematoxylin. Sections were covered with mounting medium and

polymerase (PARP) is a nuclear enzyme responsible for the poly(ADP) ribosylation of chromosomal proteins, and PARP activation is induced by formation of DNA strand breaks during apoptosis.17 PARP is a substrate for caspase (cysteinedependent aspartate-directed protease) family members such as caspase-3.18,19 During the apoptotic process, caspases are central regulators that are involved in both the death receptordependent and mitochondria-dependent apoptosis pathways.20 Another anti-apoptotic factor, survivin, is a caspase inhibitor that represses apoptosis by inhibiting the activation of caspase3.21 AAI-mediated apoptosis induced the inhibition of aquaporin-1 expression in HK-2 cells,22 and AAI increased apoptosis by extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity.23 AAI triggered the caspase/mitochondrial apoptotic pathway, which was induced by a change in the mitochondrial membrane potential, an enhanced Bax/Bcl-2 ratio, caspase-3 activation, and cytochrome c release in LLCPK1 cells.24 AAI evokes a rapid influx of extracellular Ca2+ and an increase in intracellular endoplasmic reticulum Ca2+ stores, which, in turn, causes mitochondria stress, resulting in apoptosis by activation of caspases.25 The effects of several apoptotic stimuli were shown to be induced by downregulation of the Akt signaling pathway.26 Akt is a serine−threonine protein kinase that is implicated in survival signaling in a wide a variety of cells, such as fibroblasts and neuronal cells, and inhibition of Akt signaling contributes to apoptosis.27 Bcl-2-associated death promoter (BAD) is a pro-apoptotic protein of the Bcl-2 family. Akt phosphorylates BAD on Ser136, which results in BAD dissociating from the Bcl-2/Bcl-X complex and losing its pro-apoptotic function. Akt can also activate NF-κB by regulating IκB kinase (IKK), resulting in transcription of pro-survival genes.28 The ovary is an organ that undergoes functional and extensive morphological changes during each reproductive cycle. Follicles develop into 1 of 3 cell types: oocytes, granulosa cells, or thecal cells. The development of follicles within the ovary is under the tight control of both hormones and growth factors.29 The number of follicles provides important information about the extensive morphological changes that take place in the ovary, for example, in determining the relative roles of gonadotrophic and steroid hormones in regulating the survival and maturation of follicles during the female reproductive cycle.30 The development of ovarian follicles is highly dependent on changes induced by hormones in oocytes and granulosa cells.31 Follicle maturation continues until ovulation, when eggs are extruded from the ovary for fertilization and the remaining follicular cells are luteinized.32 However, the toxicity of AA to reproductive organs such as the ovary is unknown; thus, the toxicity of AAs such as AAI should be further studied. Therefore, we investigated the toxicity of AAI in Chinese hamster ovary-K1 (CHO-K1) cells as well as in murine ovaries in vivo. We suggest that AAI suppresses maturation of the mouse ovary by inducing apoptosis.

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MATERIALS AND METHODS

Cell Culture and in Vitro Cytotoxicity Assay. CHO-K1 cells (ATCC, Manassas, VA, USA) were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) at 37 °C in a humidified atmosphere of 5% CO2. CHO-K1 cells (1 × 104 cells/well) were maintained in a 96-well culture plate for 72 h with AAI (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM) prepared in phosphate-buffered saline (PBS) vehicle; working solutions of AAI in 2129

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Figure 1. Dose-dependent apoptotic cell death induced by AAI treatment in CHO-K1 cells. (a) CHO-K1 cells were treated with AAI (0−1000 μM), and cell viability was determined by MTT assay (b) Apoptotic death-positive cells (green color) among CHO-K1 cells treated with AAI (30 μM) were determined by TUNEL staining. Data show the mean ± SD of three experiments. $$P < 0.01 vs control; **P < 0.01 vs control.

Figure 2. Effects of treatment with AAI on apoptotic proteins in CHO-K1 cells. CHO-K1 cells were treated with AAI (30 μM) and harvested at 15 and 30 min as well as 3, 6, and 24 h. Panels indicate the expression of pro- and anti-apoptotic proteins Bcl-2, survivin, PCNA, caspase-9, caspase-3, Bax, and PARP. 2130

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Figure 3. Effects of treatment with AAI on ovarian maturation in mice. The ovaries were treated with 10 mg/kg AAI intraperitoneally for 4 weeks. (A) Body weight changes in AAI-treated mice. (B) Changes in the ovary size of AAI-treated mice. (C) Changes in ovary weight and (D) number of ovulated oocytes in AAI-treated and normal mice. *P < 0.05 and **P < 0.01 vs control. Scale bar is 200 μm.

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analyzed under a BX 40 light microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner except that the primary antibody incubation step was omitted. Protein Extraction and Western Blotting. Cultured cells and ovary tissues were lysed with ice-cold Pro-PREPTM buffer (INtRON Biotechnology, Gyeonggido, Korea). The cell and tissue lysates were centrifuged at 14 000 rpm for 20 min at 4 °C to remove insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Protein extracts were separated in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes at 150 mA for 1 h. The membranes were then blocked for 1 h at room temperature with PBS containing 5% bovine serum albumin (BSA) and incubated with primary antibodies at 1:1000 dilutions (anti-Akt, anti-pAkt, anti-phosphorylation-phosphoinositide-dependent kinase 1 (p-PDK1), anti-Bax, anti-Bcl2, anticaspase-3, anti-caspase-9, anti-survivin, anti-PCNA, anti-β-actin, and anti-PARP; Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C and subsequently with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000 dilution, Cell Signaling Technology) for 1 h at room temperature. Peroxidase activity was visualized using an ECL kit (Thermo Fisher Scientific, Waltham, MA, USA). Statistical Analysis. All data is presented as the mean ± standard deviation (SD), and all determinations were repeated three times. One-way analysis of variance (ANOVA) was used to evaluate differences among multiple groups, and an independent t-test was used to evaluate differences between two treatment groups. The data were analyzed using GraphPad Prism software (GraphPad Software Inc., Chicago, IL, USA), and P < 0.05 was considered to be statistically significant.

RESULTS

AAI-Induced Apoptosis in CHO-K1 Cells. CHO-K1 cell survival was determined by MTT assay. The viability of CHOK1 cells decreased with exposure to increasing concentrations of AAI, and the IC50 of AAI in CHO-K1 cells was 30 μM (Figure 1). To determine whether AAI-induced cell death was due to apoptosis, we analyzed apoptotic cell death in CHO-K1 cells by double staining with DAPI and TUNEL. The frequency and fluorescence intensity of TUNEL-positive cells increased in AAI-treated CHO-K1 cells. In addition, the number of apoptotic cells was markedly increased in CHO-K1 cells 24 h after AAI treatment as compared to that with in control CHOK1 cells (Figure 1). Apoptotic Regulatory Protein Expression in AAITreated CHO-K1 Cells. The relationship between apoptosis and AAI treatment was evaluated by evaluating the changes in the expression of apoptotic regulatory proteins after a single treatment with 30 μM of AAI over a time course. The expression of Bax, caspase-3, caspase-8, caspase-9, and PARP was increased in AAI-treated CHO-K1 cells. Moreover, expression of the anti-apoptotic regulatory protein Bcl-2 was decreased in AAI-treated cells. In addition, AAI treatment also inhibited expression of proliferative proteins such as survivin and PCNA in CHO-K1 cells (Figure 2). Ovary and Body Weight Changes in AAI-Treated Mice. Animals treated with AAI showed significantly decreased body weight after 3 and 4 weeks of treatment (P < 0.001) as compared to that in control mice treated with PBS (Figure 3A). The ovary weight in AAI-treated mice was 50% less than that of the control mice (Figure 3B). After animals were superovulated 2131

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Figure 4. AAI induces apoptosis during ovarian maturation. Mice were treated with AAI (10 mg/kg) for 4 weeks and were superovulated using PMSG and hCG. (a) TUNEL assay results for apoptosis-positive cells after AAI treatment. (b) Western blot analysis of pro- and anti-apoptotic proteins in the ovary. (c) Immunohistochemistry for pro- and anti-apoptotic factors in ovary tissue.

Figure 5. AAI inhibits Akt phosphorylation in CHO-K1 cells and ICR mouse ovaries. CHO-K1 cells were treated with AA-I (30 μM) for 24 h, and mice were treated with AAI (10 mg/kg) for 4 weeks. Akt and phospho-Akt were detected by western blot in CHO-K1 cells (a) and ovary tissue (b). The results are normalized to total Akt protein. ** P < 0.01 vs control.

by PMSG and hCG, the number of superovulated follicles in the ovaries of AAI-treated mice was significantly decreased compared with that in control mice (Figure 3C). In addition, AAI treatment significantly decreased ovary size after 4 weeks (Figure 3) compared with that of control mice (Figure 3D). AAI-Induced Apoptotic Changes in Ovary Tissue. TUNEL-positive cells were detected in ovary tissue sections (Figure 4A). Apoptotic cell death was quantitated by counting TUNEL-positive cells. After treatment with a single dose of 10 mg/kg/day AAI for 4 weeks, the number of TUNEL-positive cells was 149 ± 7, whereas the corresponding number after PBS control treatment was 3 (Figure 4A). This result indicates that AAI induced apoptotic cell death during ovary maturation. Furthermore, AAI treatment induced the expression of apoptotic regulatory proteins (Figure 4B). An investigation of the relationship between AAI treatment and the expression of

apoptotic regulatory proteins showed that Bcl-2 expression was decreased by AAI-treatment, whereas levels of pro-apoptotic proteins, including Bax, caspase-8, caspase-9, caspase-3, and PARP, were significantly increased by treatment with AAI (Figure 4B). We detected the localization of pro- and antiapoptotic protein expression in ovary tissue by immunohistochemistry. In AAI-treated ovary tissue, expression of proapoptotic proteins, including Bax, p53, caspase-3, and caspase-9, was significantly increased compared to that in control tissue, and the expression of anti-apoptotic proteins, such as Bcl-2, PCNA, and survivin, was markedly decreased compared with that in control tissue (Figure 4C). AAI-Regulated Akt Phosphorylation. The ERK1/2 and Akt signal transduction cascades represent key pathways that regulate cellular proliferation.26 Therefore, we examined the effect of AAI on Akt phosphorylation (Figure 5). Treatment 2132

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that AAI markedly increased the expression of caspase-3, caspase-9, and PARP in a manner that might be relevant to AAI-induced apoptosis in CHO-K1 cells and ICR mouse ovaries. These result indicated that initiator caspase-9 and PARP are activated by AAI, after which they trigger a cascade that activates downstream executioner caspase-3. Thus, our data suggest that AAI induces apoptosis through a caspase-3dependent mechanism. Bcl-2 family members regulate apoptosis by controlling mitochondrial membrane potential.39 The Bcl-2 family consists of both pro- and anti-apoptotic members, including the antiapoptotic protein Bcl-2 and the pro-apoptotic protein Bax. Bcl2 acts as an anti-apoptotic factor,40 whereas Bax acts as a proapoptotic factor by activating caspase-9, which subsequently activates caspase-3.41 In this study, we found that AAI induced prolonged expression of Bax and decreased Bcl-2 expression. These results suggest that AAI induced apoptosis-mediated damage in the ovary. Survivin is anti-apoptotic protein that can effectively inhibit apoptotic processes by specifically binding caspase-3 and caspase-9.42 Others have reported that survivin protects ovaries from apoptosis and might support luteal function.43 Inhibition of apoptosis increases cell proliferation and proliferation markers such as PCNA, which is an acidic nucleoprotein that is expressed only in proliferating cells and is essential for DNA replication.44 Others have reported that expression of PCNA increases during oocyte growth and follicle development.45 In this study, we found that AAI significantly decreased survivin and PCNA expression in ICR mouse ovaries and in CHO-K1 cells as compared with that in their respective control groups. These results suggest that AAI inhibits cell proliferation by inducing apoptosis during the maturation of the ovary. Akt is an important player in signal transduction involved in cell survival and is thought to regulate apoptosis.27 Some studies have reported that activation of the Akt pathway inhibits apoptosis.33,46 Phosphorylation of Akt inhibits apoptosis by causing it to phosphorylate a number of downstream targets, including anti-apoptotic Bcl-2 family members, which regulate apoptosis upstream of mitochondrial-mediated caspase-9 activation.28 In addition, Akt activation inhibits PARP activation. Wang et al. reported that Akt activation inhibits the activation of caspase-9 and caspase-3, which suppresses apoptosis. 47 We found a significant inhibition of Akt phosphorylation in AAI-treated CHO-K1 cells and ICR mouse ovary tissue. These results suggest that AAI induces apoptosis by inhibiting Akt phosphorylation-mediated suppression of anti-apoptotic proteins, including Bcl-2 and PARP. In Belgium, renal failure and urinary tract cancer were reported in more than 100 woman who had taken herbal pills containing AAs for weight loss.3 Recently, it was reported that, when used at an appropriate concentration and treatment period, herbal extracts containing AAs inhibited lipid accumulation by downregulating PPAR-γ and C/EBP-a through regulation of the Akt and ERK 1/2 pathways in 3T3-L1 adipocytes and HFD-induced obese mice without side effects, but high-dose and long-term treatment with herbal extracts containing AAs very significantly reduced body weight due to toxicity.48 This result indicated that weight loss from herbal pills containing AAs was induced by the toxicity of AAs, including direct damage to the kidney, DNA adduct formation, and apoptosis resulting from high-dose and long-term treatment.

with AAI significantly suppressed Akt phosphorylation compared with that in control CHO-K1 cells (Figure 5A). In ovary tissue, AAI also significantly decreased Akt phosphorylation (Figure 5B). We also measured PDK1 expression, but PDK1 expression was similar between the control and AAItreated groups (data not shown). These results indicate that AAI directly inhibits Akt phosphorylation.

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DISCUSSION AA-containing Chinese herbs have been a worldwide problem because of their association with toxicity to multiple organs, including the stomach and intestine, as well as to renal tubular epithelium cells and cancerous cells.7,8,34 Toxicity of AA is a worldwide health concern, but the toxic effects of AAI during ovarian maturation have not been reported. Therefore, we investigated the toxicity of AAI in CHO-K1 cells and in the ovaries of ICR mice. We found that AAI toxicity in CHO-K1 cells was mediated by apoptosis induced via upregulation of pro-apoptotic proteins. In vivo studies showed that AAI-induced reductions in ovary weight and size were accompanied by apoptotic cell death along with increased expression of proapoptotic proteins. AA-containing natural products are often sold as traditional Chinese medicines meant to effect weight loss and improve the immune system.35 Preclinical evaluation has shown that AA produces severe toxicity associated with apoptosis.3 Apoptosis is a programmed cell death process that occurs in many multicellular organisms as a part of normal physiological function. However, excessive apoptosis causes tissue damage that leads to a loss of function and structure. In this study, we found that apoptotic cell death was induced in CHO-K1 cells and ICR mouse ovaries by AAI treatment. In a previous report, it was found that AAI-mediated tubular cell apoptosis induced tubular injury that played an important role in the development of kidney damage. Another study showed that AAI induced apoptosis in HK-2 cells.7 Our finding that the AAI-induced toxicity in CHO-K1 cells and ICR mouse ovaries was associated with apoptosis corroborates these previous reports. We investigated the toxicity of AAI in ICR mouse ovaries. We found that body weight, ovary weight, and ovary size were significantly decreased by 4 weeks of AAI treatment as compared with that from control treatment. In addition, when superovulation was induced by PMSG and hCG, we found markedly decreased numbers of superovulated oocytes in the AAI-treated mice as compared with that in the control mice. Several studies have reported that AA is toxic to the kidney and that it induces carcinogenesis10 and DNA damage.36 These studies support our hypothesis that AAI-induced damage during ovarian maturation involves apoptosis. Apoptosis is regulated by the dynamic balance between proand anti-apoptotic factors. Disrupting this balance by reducing the activity of pro-apoptotic factors or increasing the activity of anti-apoptotic factors interferes with the regulation of apoptosis. Using a TUNEL assay, we found that AAI induced apoptotic cell death in CHO-K1 cells and ovary tissue. These results indicate that AAI activated caspases, which are intracellular cysteine proteases that act as pro-apoptotic factors. Among the caspases, caspase-3 is a central effector caspase that is implicated in several modes of cell death.8 AAI induces apoptosis through a caspase-3-dependent mechanism.37 PARP is a substrate of caspases, particularly of caspase-3.38 In addition, caspase-9 is also important for drug-induced apoptosis. On the basis of the results of this study, we found 2133

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(8) Pozdzik, A. A., Salmon, I. J., Husson, C. P., Decaestecker, C., Rogier, E., Bourgeade, M. F., Deschodt-Lanckman, M. M., Vanherweghem, J. L., and Nortier, J. L. (2008) Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy. Nephrol., Dial., Transplant. 23, 2480− 2491. (9) Wang, Y., Meng, F., Arlt, V. M., Mei, N., Chen, T., and Parsons, B. L. (2011) Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction. Mutagenesis 26, 619−628. (10) Hadjiolov, D., Fernando, R. C., Schmeiser, H. H., Wiessler, M., Hadjiolov, N., and Pirajnov, G. (1993) Effect of diallyl sulfide on aristolochic acid-induced forestomach carcinogenesis in rats. Carcinogenesis 14, 407−410. (11) Cosyns, J. P., Jadoul, M., Squifflet, J. P., Wese, F. X., and van Ypersele de Strihou, C. (1999) Urothelial lesions in Chinese-herb nephropathy. Am. J. Kidney Dis. 33, 1011−1017. (12) Pozdzik, A. A., Salmon, I. J., Debelle, F. D., Decaestecker, C., Van den Branden, C., Verbeelen, D., Deschodt-Lanckman, M. M., Vanherweghem, J. L., and Nortier, J. L. (2008) Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation. Kidney Int. 73, 595−607. (13) Gao, R., Zheng, F., Liu, Y., Zheng, D., Li, X., Bo, Y., and Liu, Y. (2000) Aristolochic acid I-induced apoptosis in LLC-PK1 cells and amelioration of the apoptotic damage by calcium antagonist. Chin. Med. J. 113, 418−424. (14) Mori, C., Nakamura, N., Dix, D. J., Fujioka, M., Nakagawa, S., Shiota, K., and Eddy, E. M. (1997) Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70-2 knockout mice. Dev. Dyn. 208, 125−136. (15) Taylor, R. C., Cullen, S. P., and Martin, S. J. (2008) Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231−241. (16) Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609−619. (17) Ferro, A. M., and Olivera, B. M. (1982) Poly(ADP-ribosylation) in vitro. Reaction parameters and enzyme mechanism. J. Biol. Chem. 257, 7808−7813. (18) Ohashi, Y., Ueda, K., Kawaichi, M., and Hayaishi, O. (1983) Activation of DNA ligase by poly(ADP-ribose) in chromatin. Proc. Natl. Acad. Sci. U.S.A. 80, 3604−3607. (19) Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmAinhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81, 801−809. (20) Debatin, K. M. (2004) Apoptosis pathways in cancer and cancer therapy. Cancer Immunol. Immunother. 53, 153−159. (21) Wang, W., Luo, H., and Wang, A. (2006) Expression of survivin and correlation with PCNA in osteosarcoma. J. Surg. Oncol. 93, 578− 584. (22) Zhang, L., Li, J., Jiang, Z., Sun, L., Mei, X., Yong, B., and Zhang, L. (2011) Inhibition of aquaporin-1 expression by RNAi protects against aristolochic acid I-induced apoptosis in human proximal tubular epithelial (HK-2) cells. Biochem. Biophys. Res. Commun. 405, 68−73. (23) Zeng, Y., Yang, X., Wang, J., Fan, J., Kong, Q., and Yu, X. (2012) Aristolochic acid I induced autophagy extenuates cell apoptosis via ERK 1/2 pathway in renal tubular epithelial cells. PLoS One 7, e30312. (24) Yang, H., Dou, Y., Zheng, X., Tan, Y., Cheng, J., Li, L., Du, Y., Zhu, D., and Lou, Y. (2011) Cysteinyl leukotrienes synthesis is involved in aristolochic acid I-induced apoptosis in renal proximal tubular epithelial cells. Toxicology 287, 38−45. (25) Hsin, Y. H., Cheng, C. H., Tzen, J. T., Wu, M. J., Shu, K. H., and Chen, H. C. (2006) Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells. Apoptosis 11, 2167−2177.

The results of this study indicate that AAI produces strong toxic effects during ovarian maturation by inhibiting Akt, which induces apoptosis, and suggest that high-dose and long-term AAI treatment increases the likelihood of reproductive toxicity in woman. Therefore, herbal pills containing AAs should be prohibited from use in order to maintain reproductive health.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +82-63-850-6780. Funding

This study was supported by a grant from Wonkwang University, 2014. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jin Yeul Ma of the Korea Institute of Oriental Medicine for critical review of the manuscript. We also thank Young-Kug Choo of the Department of Biological Science, College of Natural Sciences, Wonkwang University for critical review of the manuscript.

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ABBREVIATIONS Bcl-2, lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly(ADP) ribose polymerase; AAs, aristolochic acids; IKK, IκB kinase; AAI, aristolochic acid I; CHO-K1, Chinese hamster ovary-K1; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; KIOM, Korea Institute of Oriental Medicine; H&E, hematoxylin and eosin; AAII, aristolochic acid II; FBS, fetal bovine serum; PBS, phosphatebuffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; hCG, human chorionic gonadotropin; PCNA, proliferating cell nuclear antigen; IHC, immunohistochemistry; BSA, bovine serum albumin; p-PDK1, phosphorylation-phosphoinositide-dependent kinase 1

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