Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells

Toxicology 300 (2012) 138–148

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Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells Xue-Jiao Yang a , Hui-Yuan Lu a , Zi-Yin Li a , Qian Bian c , Liang-Lin Qiu a , Zhong Li a , Qizhan Liu a , Jianmin Li b , Xinru Wang a , Shou-Lin Wang a,∗ a

Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 140 Hanzhong Rd., Nanjing 210029, PR China Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, 140 Hanzhong Rd., Nanjing 210029, PR China c Jiangsu Provincial Center for Disease Prevention and Control, 172 Jiangsu Rd., Nanjing 210009, PR China b

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Article history: Received 19 April 2012 Received in revised form 24 May 2012 Accepted 16 June 2012 Available online 26 June 2012 Keywords: Cytochrome P450 2A13 Aflatoxin B1 Metabolic activation BEAS-2B cells Cytotoxicity Mitochondrial signaling pathway

a b s t r a c t Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B1 (AFB1 ). However, the role of CYP2A13-mediated AFB1 metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB1 -induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB1 -induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB1 metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB1 induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB1 were significantly remarkable in B2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB1 -induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB1 treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB1 , and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB1 -induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB1 in respiratory diseases mediated by CYP2A13. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Aflatoxin B1 (AFB1 ), a highly substituted coumarin structure that contains a fused dihydrofurofuran moiety, is well known as a potent hepatotoxic and carcinogenic compound. AFB1 is reported to be a ubiquitous contaminate of the human food supply throughout the economically developing world (Groopman et al., 2008). The number of people exposed to high levels of AFB1 worldwide is still unclear, but it is estimated to be at least 500 million (Vineis and Xun, 2009). In China, corn and peanuts might be severely contaminated with aflatoxins, especially AFB1 (Wang and Liu, 2007), and the AFB1

Abbreviations: CYP, cytochrome P450; AFB1 , aflatoxin B1 ; 8-MOP, 8methoxypsoralen; BEAS-2B, immortalized human bronchial epithelial cells; B-2A13, BEAS-2B cells stably expressing CYP2A13; CHO, Chinese Hamster Ovary; PCR, polymerase chain reaction; MTT, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide. ∗ Corresponding author. Tel.: +86 25 8686 2834; fax: +86 25 8652 7613. E-mail address: [email protected] (S.-L. Wang). 0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2012.06.010

content of Chinese peanut butter and sesame paste samples were higher than the levels permitted by Chinese and European Union regulations (Li et al., 2009). AFB1 is implicated in the etiology of human liver cancer and extensive studies have focused on AFB1 -induced hepatocellular carcinoma (Kensler et al., 2011). However, the human respiratory system is also a potential target for AFB1 carcinogenesis. Epidemiological study indicated that the observed mortalities for total-cancer and respiratory cancer were higher than expected in the aflatoxin-exposed group (Hayes et al., 1984), and laboratory evidence showed that AFB1 was bioactivated by cytochromes P450 in isolated lung cells from rabbits and mice, predicted that AFB1 might play an important role in human lung cancer (Massey et al., 2000). In general, aflatoxins are easy to grow under damp conditions, and small particle-like AFB1 can be inhaled into the human respiratory system through breathing. Since the 1980s, airborne aflatoxins in agricultural food production have been studied extensively and agricultural food industry workers are at risk of ingestion, transmucosal absorption, and inhalation of AFB1 released during product preparation or processing (Sorenson et al., 1981; Traverso

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et al., 2010). Typically, lung is a major target organ for inhaled xenobiotics, and the respiratory tract is frequently exposed to high concentrations of these compounds, making it a primary target for toxicity (Zhang et al., 2006). Therefore, AFB1 may be assumed to be associated with respiratory diseases, such as chronic obstructive pulmonary disease and lung cancer. AFB1 is an indirect carcinogen that requires bioactivation by cytochrome P450 (CYP) in the body to be converted into a toxic carcinogen, such as exo-8,9-epoxide, the major and highly active epoxide metabolite of AFB1 (Bedard and Massey, 2006). AFB1 exo8,9-epoxide is capable of reacting with cellular macromolecules, such as DNA, RNA, and proteins. For instance, AFB1 exo-8,9-epoxide can bind to DNA to form AFB1 -DNA adducts and subsequently cause DNA damage cytotoxicity (Gursoy-Yuzugullu et al., 2011), and cellular apoptosis (Ribeiro et al., 2010). CYP enzymes, particularly CYP1A2 and CYP3A4, are critical in the metabolic activation of AFB1 . However, they are mainly expressed in the human liver and rarely expressed in the lungs (Ding and Kaminsky, 2003; Pelkonen and Raunio, 1997). CYP2A13 has been reported to be mainly expressed in the human respiratory system and is the most efficient human CYP enzyme in the metabolic activation of NNK, a major tobacco-specific carcinogen (Su et al., 2000). Our previous study demonstrated that heterologous expression of the CYP2A13 in insect Sf9 cells metabolized AFB1 to potential carcinogenic/toxic epoxides, and CYP2A13 was highly efficient in the metabolic activation of AFB1 in CYP2A13 expressed Chinese Hamster Ovary (CHO) cells (He et al., 2006). In addition, it has been reported that human lung was able to activate AFB1 to form active metabolites which could induce mouse lung tumors (Massey et al., 2000), and the pulmonary P450 enzymes play an essential role in NNK-induced lung carcinogenesis by producing carcinogenic metabolites directly in the target tissue using conditional cytochrome p450 reductase-null mice (Weng et al., 2007). Therefore, CYP2A13 might play an important role in the contribution of airborne AFB1 -induced respiratory diseases because it can metabolize AFB1 in situ and subsequently injure pulmonary cells by inducing necrosis, apoptosis, and malignant transformation. The study of AFB1 -metabolizing enzymes in the lungs is relatively difficult because the lungs have complex tissues that contain many cell types, and the architecture of the lungs is rather complex. Immortalized human bronchial epithelial cells, BEAS-2B, is an SV-40 immortalized cell line that originates from normal human bronchiolar epithelium (NHBE) progenitor cells. It remains nontumorigenic through numerous passages and represent a good model for studying pulmonary injury (Reddel et al., 1988). A previous study have evaluated the metabolic activation of AFB1 using BEAS-2B cells transfected with CYP 1A2 and CYP 3A4 (Van Vleet et al., 2006). However, BEAS-2B cells that stably express CYP2A13 have not been reported. Therefore, in the present study, we used a lentiviral system to establish monoclonal BEAS2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), CYP2A6 (B-2A6), or Vector alone (B-Vector), among which B-1A2, B-2A6, and B-Vector and blank BEAS-2B cells served as controls. Furthermore, we explored the CYP2A13-mediated low concentration of AFB1 -induced cytotoxicity, apoptosis, and the mechanism involved in the formation of AFB1 -DNA adducts and mitochondrial signaling pathways in order to provide a possible clue of the critical role of CYP2A13 in airborne AFB1 -induced respiratory diseases.

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2.1. Materials AFB1 , 8-methoxypsoralen (8-MOP), and (−) nicotine were obtained from Sigma–Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO) before use. BEAS-2B cells were purchased from ATCC (Manassas, VA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO-BRL (Grand Island, NY). LipofectamineTM 2000 Transfection Reagent was obtained from Invitrogen (Carlsbad, CA). Antibodies specific for CYP2A6 and CYP1A2 were obtained from Millipore (Billerica, MA), and rabbit anti-human CYP2A13 polyclonal antibody was a generous gift from Prof. Jun-Yan Hong (University of Medicine and Dentistry of New Jersey, Piscataway, NJ). Antibodies specific for caspase-3/9, C-caspase-3/9, C-PARP, Bcl-2, Bax, Bad, p-Bad (ser112 ) and an enhanced chemiluminescence (ECL) immunoblotting assay kit were purchased from Cell Signaling Technology (Danvers, MA). Antibodies specific for aflatoxin B1 -DNA adducts were obtained from Novus Biologicals (Littleton, CO). The PE Annexin V Apoptosis Detection Kit I was obtained from BD Pharmingen (Franklin Lakes, NJ). Antibodies specific for GAPDH and GFP, 3-(4,5)dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT), Hoechst 33258, penicillin, streptomycin, trypsine-EDTA, and bovine serum albumin (BSA) were purchased from Beyotime (Shanghai, China). 2.2. Establishment of BEAS-2B cells that stably express CYP2A13 Plasmids pLJM1 CYP1A2, CYP2A6, and CYP2A13 cDNAs were prepared from a pLJM1 lentivirus vector with the GFP gene that was used in our lab, and were confirmed by polymerase chain reaction (PCR) and DNA sequencing (BGI Sequencing Company). The preparation and infection of the lentiviral products in the cultured BEAS-2B cells were performed as described previously (Lv et al., 2010). Four to seven days post-transfection, the cells were collected and confirmed using an immunoblotting assay with specific antibodies of CYP1A2, CYP2A6, CYP2A13 and GFP. Based on GFP expression levels, the monoclonal transfected cells were screened and identified according to standard protocols with minor modifications (http://www.corning.com). Briefly, the cells were plated into 96-well plates (5 cells/ml) with 200 ␮l growth medium and cultured undisturbed at 37 ◦ C in 5% CO2 for 4–5 days. Single colonies with green fluorescence were then marked under a fluorescence microscope. After 7–10 days, these selected colonies were then subcultured from the wells into larger vessels until the cells grew to ≥90% confluence in a 10 cm plate. Once the colonies screened successfully, the proteins were extracted from each colony for verification and identification using an immunoblotting assay as described previously (Ma et al., 2010). BEAS-2B cells transfected with vector alone were used as controls. 2.3. Cell viability assay BEAS-2B cells that stably express a particular CYP enzyme (1A2, 2A6, or 2A13) were plated into 96-well plates (5000 cells/well) with 200 ␮l growth medium and cultured at 37 ◦ C in 5% CO2 overnight. The cells were then treated with AFB1 at different concentrations of 0–10 ␮M for 24 or 48 h. To investigate the effects of nicotine and 8-MOP on AFB1 -induced cytotoxicity, B-2A13 cells were co-treated with 100 nM AFB1 and different concentrations of nicotine (1, 10, and 100 ␮M) or 8-MOP (0.01, 0.1, and 1 ␮M) for 24, 48, or 72 h. Additionally, B-2A13 cells were pretreated with 100 ␮M nicotine or 1 ␮M 8-MOP for 2 h, rinsed with PBS, and incubated in a medium that contained 100 nM AFB1 for an additional 24, 48 h, or 72 h. The cells treated with 100 ␮M nicotine, 100 nM AFB1 , and 1 ␮M 8-MOP alone served as controls. After the treatments, cell viability was determined using the MTT assay as described previously (Ma et al., 2010). 2.4. Cell apoptosis assay After the cells (B-2A13, B-1A2, B-2A6, and B-Vector) were incubated in six-well plates (3 × 105 cells/well) at 37 ◦ C in 5% CO2 overnight, they were treated with different concentrations of AFB1 that ranged from 0 to 80 nM for 24 h. Additionally, B-2A13 cells were treated with AFB1 at concentrations of 0–80 nM for 12, 24, and 48 h. After the treatments, cellular apoptosis was determined using the Hoechst 33258 assay according to our previous protocol (Ma et al., 2010). Finally, to confirm the above experiments, cellular apoptosis was assessed using a flow cytometry assay as described previously (Ma et al., 2010) after the B-2A13, B-1A2, and B-Vector cells were treated with 5, 20, and 80 nM AFB1 for 24 h. To investigate the effects of nicotine and 8-MOP on AFB1 -induced apoptosis, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 ␮M nicotine or 1 ␮M 8-MOP for 24 h. After the treatment, cellular apoptosis was examined using the Hoechst 33258 assay and flow cytometry assay. 2.5. Aflatoxin B1 -DNA adducts immunohistochemistry assay

2. Material and methods AFB1 is a well known hepatocarcinogen and must be handled with extreme caution. Disposable nitrile gloves were worn while handling AFB1 , and all samples were destroyed in bleach.

B-2A13, B-1A2, B-2A6, and B-Vector cells were plated into six-well plates with coverslips (3 × 105 cells/well) at 37 ◦ C in 5% CO2 overnight and then treated with 5, 20, and 80 nM AFB1 for 24 h. After the treatments, using a mouse 6A10 monoclonal antibody against an imidazole ring-opened persistent form of the major N7 -guanine adduct of AFB1 , the AFB1 -DNA adducts were determined using an immunohistochemistry assay with minor modifications as described previously

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Fig. 1. Identification of BEAS-2B cells that stably express a particular CYP enzyme. An immunoblotting assay was performed to assess CYP2A13, CYP1A2, and CYP2A6 protein expression in cell lysates (50 ␮g) using specific antibodies. GFP was used as a transfection reference to evaluate CYP expression levels in each cell line. GAPDH was used as an internal reference. Microsomal proteins (10 ␮g) prepared from Sf9 cells that express CYP1A2, 2A6, and 2A13 were used as a positive control. Blank BEAS-2B cells or cells transfected with vector alone was used as a negative control. (A) Detection of CYP and GFP proteins in transfected BEAS-2B cells before monoclonal selection. (B) Detection of CYP and GFP proteins in monoclonal BEAS-2B cells that stably express a particular CYP enzyme. The numbers represent the different monoclonal cells. The numbers with a box indicate the cells selected for subsequent experiments.

(Gursoy-Yuzugullu et al., 2011). Briefly, after the cells were washed with PBS and fixed in ice-cold methanol, they were treated with a buffer that contained 15 mmol/L Na2 CO3 and 30 mmol/L NaHCO3 (pH 9.6) for 2 h at room temperature, followed by 100 ␮g/ml RNAse treatment at 37 ◦ C for 1 h. After washing with PBS, 10 ␮g/ml proteinase K treatment was performed for 10 min at room temperature, followed by washing with PBS and 40% ethanol. The cells were treated with 50 mM NaOH in 40% ethanol for 30 s to denature the DNA and rinsed again with 40% ethanol. After blocking for 1 h with 5% BSA, the cells were incubated with mouse 6A10 monoclonal antibody overnight at 4 ◦ C. The cells were then incubated in goat-anti-mouse secondary antibodies for 30 min. After washing with PBS, the cells were incubated in SABC reagent for 20 min, stained with DAB, counterstained with hematoxylin, and observed under an Olympus Light Microscope (IX70, Olympus, Japan). AFB1 -DNA adducts expression levels were semi-quantitatively determined by densitometry using Image-Pro Plus 5.0 (IPP 5.0) software (Ojo et al., 2011). 2.6. Detection of proteins related to mitochondrial signaling pathway B-2A13 cells were plated into 10 cm plates (1 × 106 cells/well) in the growth medium and cultured at 37 ◦ C in 5% CO2 overnight. To assess AFB1 -induced changes in apoptosis-related proteins, the cells were treated with AFB1 at different concentrations that ranged from 0 to 80 nM for 24 h. In addition, B-2A13 cells were treated with 80 nM AFB1 for 0, 3, 6, 12, and 24 h to determine whether the proteins changed in a time-dependent manner. To investigate the effects of nicotine and 8-MOP on AFB1 -induced changes in apoptosis-related proteins, the cells were treated with DMSO, 80 nM AFB1 alone, or 80 nM AFB1 combined with 100 ␮M nicotine or 1 ␮M 8-MOP for 24 h. After the treatments, apoptosis-related proteins were determined using immunoblotting assays as described previously (Ma et al., 2010). Briefly, cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Using specific antibodies for caspase-3/9, C-caspase-3/9, C-PARP, Bcl2, Bax, Bad, and p-Bad, the protein immune complexes were detected using an ECL immunoblotting assay kit and exposed to Kodak X-Omat film. GAPDH was used as an internal control. Protein expression levels were semi-quantitatively determined by densitometry and normalized to GAPDH. For the densitometric analyses, the protein bands on the blot were measured by ImageJ software (http://rsb.info.nih.gov/ij) as described previously (Ma et al., 2010). 2.7. Statistical analysis The IC50 values of cell viability were calculated using a modified logit model. The means and standard deviations (SDs) were calculated for all of the investigated parameters, and all of the data are expressed as means ± SD. The data were analyzed

using SPSS 17.0 software for Windows (SPSS, Chicago, IL). Significant differences between the treatment groups and controls were determined using one-way analysis of variance (ANOVA). Significant differences among different treatment groups were determined using the Student–Newman–Keuls test. Values of p < 0.05 were considered statistically significant.

3. Results 3.1. Identification of BEAS-2B cells that stably express CYP2A13 The recombinant plasmids pLJM1-CYP1A2, pLJM1-CYP2A6, and pLJM1-CYP2A13 were identified by PCR and sequencing. The results showed that pLJM1-CYPs were successfully constructed with no mutations. CYP2A6, 2A13, and 1A2 expression in transfected cells was determined by immunoblotting, and the results showed that a single protein band of each protein was detected in the cells, whereas the cells transfected with vector alone and blank BEAS2B cells showed no bands, suggesting that CYP1A2, CYP2A6, and CYP2A13 were successfully expressed in BEAS-2B cells (Fig. 1A). Based on the similar expression level of GFP, several monoclonal transfected cells were selected and expanded, two of which were identified by immunoblotting. The results showed that both CYP proteins and GFP were successfully expressed in all of the monoclonal cells, in which GFP protein maintained similar expression levels, including the cells transfected with vector alone (Fig. 1B). These data further suggested that BEAS-2B cells stably expressing CYP1A2, 2A6, or CYP2A13 were successfully established. 3.2. Effects of AFB1 on the viability of BEAS-2B cells that express CYP2A13 To demonstrate the role of human CYP2A13 in AFB1 metabolic activation, we used monoclonal transfected cells to determine AFB1 -mediated cytotoxicity. As shown in Fig. 2A, cell viability dosedependently decreased in B-2A13 and B-1A2 cells, whereas B-2A6,

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for 2 h significantly increased cell viability compared with AFB1 treatment alone (p < 0.001). 3.3. Effects of AFB1 on apoptosis in BEAS-2B cells that express CYP2A13

Fig. 2. Effect of AFB1 on the viability of BEAS-2B cells that stably express CYP2A13. The MTT assay was used to determine cell viability. (A) Cells were treated with AFB1 for 24 h. (B) Cells were treated with AFB1 for 48 h. Blank BEAS-2B cells and B-Vector cells were used as negative controls. DMSO (0.02%) was used as a vehicle control. MTT activity is expressed as the mean ± SD of three independent experiments with triplicate samples.

B-Vector, and BEAS-2B blank cells showed no toxicity with AFB1 treatment for 24 h. B-2A13 cells were more sensitive to AFB1 than B-1A2 cells. After 24 h AFB1 treatment, the IC50 value was 590 nM for B-2A13 cells, while 1870 nM for B-1A2 cells. After 48 h AFB1 treatment, the IC50 value for B-2A13 cells was 140 nM, which was one-fifth of the IC50 value for B-1A2 cells (740 nM) and one-twelfth of the IC50 value for B-2A6 cells (3020 nM). No apparent cytotoxic effects were observed in both B-Vector and blank cells after AFB1 treatment (Fig. 2B). These results indicated that AFB1 was metabolically activated by CYP2A13 as well as CYP1A2 and CYP2A6 and induced cytotoxicity. Moreover, CYP2A13 was the most efficient metabolic enzyme of AFB1 , that was consistent with our previous study (He et al., 2006). To further explore the CYP2A13-induced metabolic activity of AFB1 , nicotine and 8-MOP were used. Nicotine can be metabolized by CYP2A13 (von Weymarn et al., 2006) but induce much less cytotoxicity at a concentration of 100 ␮M (Fig. 3A), indicating that nicotine may be useful as a competitive agent to assess AFB1 metabolism. 8-MOP has been reported to be a potent inhibitor/inactivator of the CYP enzyme (von Weymarn et al., 2005), thus it presumably inhibits the activity of CYP2A13 as well. As predicted, both nicotine and 8-MOP significantly and dosedependently inhibited AFB1 -induced cytotoxicity in B-2A13 cells (Fig. 3). For nicotine, cell viability increased with increasing nicotine concentrations compared with AFB1 treatment alone. Treatment with 100 ␮M nicotine for 48 h completely prevented AFB1 -induced cytotoxicity (p < 0.001) (Fig. 3A). Pretreatment with nicotine for 2 h partially increased cell viability, but the effect was not significant. As shown in Fig. 3B, 0.1 ␮M 8-MOP completely inhibited AFB1 induced cytotoxicity (p < 0.001). Pretreatment with 1 ␮M 8-MOP

The Hoechst 33258 assay and flow cytometry assay were used to investigate AFB1 -induced cellular apoptosis in B-2A13 cells. The Hoechst 33258 assay showed that the apoptotic rate of B-2A13 cells dose-dependently increased after 24 h AFB1 treatment. Compared with vehicle treatment, 20 nM AFB1 significantly induced B-2A13 cell apoptosis (p < 0.001). The apoptotic rate of B-2A13 cells was 16.55% after cells were treated with 80 nM AFB1 , whereas no differences in apoptosis were observed in B-1A2, B-2A6, and B-Vector cells after cells were treated with AFB1 as compared to vehicle control (Fig. 4A). The apoptotic rate of B-2A13 cells also timedependently increased with AFB1 treatment. Treatment with 40 nM AFB1 for 24 h or 5 nM AFB1 for 48 h increased the apoptotic rate significantly compared with vehicle (p < 0.01 and 0.001, respectively; Fig. 4B). Interestingly, similar apoptotic rates of B-2A13 cells were induced by treatment with 80 nM AFB1 for 24 h and 48 h, possibly indicating that long-term treatment induced more cellular necrosis than apoptosis (Fig. 4B). The above results were confirmed by flow cytometry. As shown in Fig. 5, AFB1 -induced apoptosis in B-2A13 cells dose-dependently increased, while no changes were observed in B-1A2 and B-Vector cells. The apoptotic rate of B-2A13 cells was significantly higher than vehicle control after 24 h AFB1 treatment at a concentration as low as 5 nM. The apoptotic rate of B-2A13 cells was 30.08% after 80 nM AFB1 treatment for 24 h, whereas the apoptotic rates of B-1A2 and B-Vector cells were approximately 4% (Fig. 5). As discussed above, nicotine and 8-MOP were used to explore AFB1 -induced apoptosis in B-2A13 cells. As shown in Fig. 6, the Hoechst 33258 assay and flow cytometry assay showed that both 100 ␮M nicotine and 1 ␮M 8-MOP markedly inhibited AFB1 induced cellular apoptosis (p < 0.001). When the cells were treated with 80 nM AFB1 alone, the apoptotic rate of B-2A13 cells was 16.55% in the Hoechst 33258 assay. While the apoptotic rates decreased to 3.14% and 2.90%, respectively after the cells were treated with 100 ␮M nicotine or 1 ␮M 8-MOP. These values were similar to the vehicle control (Fig. 6A). The flow cytometry assay revealed similar results. Treatment with AFB1 alone induced an apoptotic rate of 30.08%, whereas treatment with AFB1 plus nicotine and 8-MOP decreased the rate to 3.92% and 3.56%, respectively (Fig. 6B). 3.4. Formation of AFB1 -DNA adducts in BEAS-2B cells that express CYP2A13 An immunohistochemistry assay was used to detect the imidazole ring-opened persistent form of the major N7 -guanine adduct of AFB1 . As shown in Fig. 7, the dose-dependent formation of AFB1 DNA adducts were found in the nuclei of B-2A13 and B-1A2 cells. Treatment with 5 nM AFB1 induced a greater formation of DNA adducts compared with vehicle (p < 0.01, p < 0.001). Compared with B-1A2 cells, B-2A13 cells showed more formations of AFB1 -DNA adducts at each AFB1 concentration (p < 0.01, p < 0.001). As predicted, no significant AFB1 -DNA adducts were found in either B-2A6 or B-Vector cells (Fig. 7). 3.5. Expression of apoptosis-related proteins induced by AFB1 in BEAS-2B cells that express CYP2A13 The mitochondrial signaling pathway plays a critical role in xenobiotic-mediated cytotoxicity, including apoptosis. Several related proteins, such as C-PARP, caspase-3/9, C-caspase-3/9,

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Fig. 3. Effect of nicotine and 8-MOP on AFB1 induced-cytotoxicity in BEAS-2B cells that stably express CYP2A13. The MTT assay was used to determine cell viability. (A) B-2A13 cells were treated with 100 nM AFB1 and different concentrations of nicotine for 24, 48, and 72 h or pretreated with nicotine for 2 h and then treated with 100 nM AFB1 for 24, 48, and 72 h. (B) B-2A13 cells were treated with 100 nM AFB1 and different concentrations of 8-MOP for 24, 48, and 72 h or pretreated with 8-MOP for 2 h and then treated with 100 nM AFB1 for 24, 48, and 72 h. Blank BEAS-2B cells and B-Vector cells were used as negative controls. DMSO (0.02%) was used as a vehicle control. MTT activity is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p < 0.001, compared with cells treated with 100 nM AFB1 .

Bcl-2, Bax, p-Bad, and Bad, were selected to investigate the mechanism and find early biomarkers of AFB1 -induced cytotoxicity. As shown in Figs. 8A and S1A and B, C-PARP and C-caspase-3 expression dose-dependently increased, with a marked difference between 20 nM AFB1 and vehicle treatment. Caspase-3 expression significantly decreased. C-caspase-9 expression increased, but no change was observed in caspase-9 expression. Bcl-2 expression dose-dependently decreased, and Bax expression dosedependently increased. Significant differences were observed in Bcl-2 expression after 80 nM AFB1 treatment compared with vehicle and in Bax expression after 20 nM AFB1 treatment compared with vehicle (p < 0.05 and 0.001, respectively). Although no significant change in Bad expression was observed, p-Bad expression dose-dependently decreased (Figs. 8A and S1B). Based on the above changes in protein levels, B-2A13 cells were treated with 80 nM AFB1 for 0, 3, 6, 12, and 24 h. The results showed that C-PARP and C-caspase-3 were expressed in a time-dependent manner. Significant changes were observed as early as 3 h AFB1 treatment (p < 0.01) (Figs. 8B and S1C). Nicotine and 8-MOP were then used to explore the role of apoptosis-related proteins in AFB1 -induced apoptosis in B-2A13 cells. The results showed that 100 ␮M nicotine and 1 ␮M 8-MOP significantly blocked the AFB1 -induced expression of C-PARP and C-caspase-3 (p < 0.001 and 0.05, respectively) and reduced the AFB1 -induced inhibition of Bcl-2 expression, which was consistent with their effects on AFB1 -induced cytotoxicity and apoptosis (Figs. 8C and S1D).

4. Discussion In the present study, we demonstrated that CYP2A13 efficiently activated AFB1 metabolism in BEAS-2B cells that stably express human CYP2A13. AFB1 dose- and time-dependently decreased cell viability and increased cellular apoptosis. The IC50 values in B-2A13 cells were 590 nM with 24 h treatment and 140 nM with 48 h treatment, whereas the IC50 value in B-Vector cells was greater than 10,000 nM, even with 48 h treatment. These data are consistent with our previous studies using CYP2A13-expressed CHO cells (He et al., 2006; Wang et al., 2006). Cellular apoptosis might be more sensitive than cell viability. For instance, treatment with 20 nM AFB1 for 24 h or 5 nM AFB1 for 48 h have already revealed significant increase of cellular apoptosis, but not by the cell viability assay. To further examine the role of CYP2A13 in the metabolic activation of AFB1 , we used nicotine, a competitor of AFB1 in CYP2A13 depletion, and 8-MOP, an inhibitor/inactivator of cytochrome P450 enzymes, to assess cell viability and apoptosis (von Weymarn et al., 2005, 2006). As predicted, both nicotine and 8-MOP inhibited the cytotoxicity of B-2A13 cells induced by AFB1 . As a competitor, high concentrations of nicotine (10–1000 times greater than AFB1 ) competitively depleted CYP2A13 and diminished the CYP2A13induced metabolic activity of AFB1 , resulting in less cytotoxicity. In the present study, 100 ␮M nicotine almost completely eliminated AFB1 -induced cytotoxicity, especially cellular apoptosis. 8-MOP is an inhibitor/inactivator of cytochrome P450 enzymes. 8MOP at concentrations of 0.1 ␮M completely inhibited CYP2A13

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Fig. 4. Effect of AFB1 on cellular apoptosis in BEAS-2B cells that express CYP2A13. The Hoechst 33258 staining assay was used to determine cellular apoptosis. (A) B-2A13, B-1A2, and B-2A6 cells were treated with 5–80 nM AFB1 for 24 h and then subjected to the Hoechst 33258 staining assay to detect apoptotic cells using a fluorescence microscope. (B) B-2A13 cells were treated with 5–80 nM AFB1 for 12, 24, and 48 h and then subjected to the Hoechst 33258 staining assay to detect apoptotic cells using a fluorescence microscope. B-Vector cells were used as a negative control, and DMSO (0.02%) was used as a vehicle control. The percentage of apoptotic cells is expressed as the mean ± SD of three independent experiments with triplicate samples. **p < 0.01, ***p < 0.001, compared with cells treated with DMSO.

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Fig. 5. Effect of AFB1 on cellular apoptosis in BEAS-2B cells that express CYP2A13. The flow cytometry assay was used to determine cellular apoptosis. B-2A13 and B-1A2 cells were treated with 5–80 nM AFB1 for 24 h and then subjected to Annexin V-7-AAD to detect apoptotic cells using a flow cytometry assay. B-Vector cells were used as a negative control, and DMSO (0.02%) was used as a vehicle control. The percentage of apoptotic cells is expressed as the mean ± SD of three independent experiments with triplicate samples. **p < 0.01, ***p < 0.001, compared with cells treated with DMSO.

activity and returned cytotoxicity to the level of controls. Pretreatment with 1 ␮M 8-MOP for 2 h blocked AFB1 -induced cytotoxicity. These results confirm that CYP2A13 plays an important role in the metabolic activation of AFB1 . Although CYP3A4 and CYP1A2 are the major AFB1 -metabolizing enzymes, CYP1A2 in the human lung appears to play a more important role in the bioactivation of low concentrations of AFB1 (Van Vleet et al., 2002, 2006). CYP2A6 shares 93.5% amino acid sequence identity with CYP2A13, and both proteins are composed of 494 amino acid residues (Smith et al., 2007). However, they have large differences in their ability to metabolize xenobiotic/endogenous compounds. CYP2A13 has much higher activity in NNK metabolism, but its activity in metabolizing coumarin is only one-tenth of CYP2A6 (Su et al., 2000). Our previous study found that CYP2A13 had much high metabolic activation than CYP2A6 in CHO cells (He et al., 2006). CYP1A2 and CYP3A4 are mainly expressed in the human liver (Pelkonen and Raunio, 1997) rather than in the human respiratory system (Ding and Kaminsky, 2003) where CYP2A13 is mainly expressed (Su et al., 2000; Zhu et al., 2006). In the present study, CYP1A2 and CYP2A6 were used as reference enzymes to

assess the critical role of CYP2A13 in the AFB1 -induced injury of pulmonary cells. Compared with CYP1A2 and 2A6, CYP2A13 showed much more metabolic activation. In monoclonal BEAS-2B cells that stably express specific CYP at similar expression levels as GFP protein, the IC50 value in B-2A13 cells (140 nM) was lower as compared to B-1A2 cells (740 nM) and B-2A6 cells (3020 nM) after 48 h AFB1 treatment. AFB1 induced more apoptosis in B-2A13 cells as compared to B-1A2 or B-2A6 cells. Furthermore, AFB1 induced more AFB1 -DNA adducts in B-2A13 cells than in B-1A2 cells and B-2A6 cells. Nicotine and 8-MOP could expectedly reduce the formation of AFB1 -DNA adducts in B-2A13 cells (data not shown). Our results showed that CYP2A13 is much more active than CYP1A2 and CYP2A6 in the metabolic activation of AFB1 in human lung epithelial cells, suggesting CYP2A13 might be the primary metabolic enzyme involved in AFB1 metabolism in situ and possibly play an important role in environmental contaminants induced respiratory disorders. AFB1 is bioactivated by P450 enzymes to generate its metabolites, including the active type of AFB1 exo- and endo-8,9-epoxides. AFB1 exo-8,9-epoxide can react with DNA, forming trans8,9-dihydro-8-(N7 -guanyl)-9-hydroxyaflatoxin B1 (AFB1 -N7 -Gua)

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Fig. 6. Effects of nicotine and 8-MOP on AFB1 -induced apoptosis in BEAS-2B cells that express CYP2A13. The Hoechst 33258 staining assay and flow cytometry assay were used to determine apoptosis in B-2A13 cell treated with 80 nM AFB1 alone or combined with 100 ␮M nicotine or 1 ␮M 8-MOP for 24 h. (A) Cellular apoptosis was analyzed using the Hoechst 33258 staining assay. (B) Cellular apoptosis was analyzed using a flow cytometry assay with Annexin V-7-AAD. DMSO (0.02%) was used as a vehicle control. The percentage of apoptotic cells is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p < 0.001, compared with cells treated with AFB1 alone.

(Wang and Groopman, 1999). The presence of a positive change on the imidazole ring of AFB1 -N7 -Gua makes this adduct fairly labile, and it undergoes three reactions in vitro, one of which is a stable AFB1 -formamidopyrimmidine adduct (AFB1 -FAPY) (Bedard and Massey, 2006). AFB1 -N7 -Gua and AFB1 -FAPY were served as biologically internal exposure dose or DNA damage effect index because the formation of this adduct lies on the causal pathway to aflatoxin induced HCC (Besaratinia et al., 2009; Egner et al., 2006; Long et al., 2010). AFB1 -N7 -Gua and AFB1 -FAPY were repaired primarily by NER in bacteria, yeast and mammals (Alekseyev et al., 2004; Guo et al., 2005; Takahashi et al., 2002). DNA adducts provoke severe steric alterations in DNA that impair DNA-dependent metabolic process, including DNA replication and transcription (Roos and Kaina, 2006). DNA replication through such DNA lesions can lead to secondary DNA damage, most notably double-strand breaks (DSBs). A previous study showed that HepG2 cells treated with AFB1 induced DNA adducts, 8-hydroxyguanine lesions, and DNA strand breaks, and AFB1 -induced DNA damage might trigger a checkpoint response that is compatible with a DSB-type response that involves Ataxia-telangiectasia mutated (ATM) (Gursoy-Yuzugullu et al., 2011) and might in turn stimulates the transcription of the p73 gene and increases p73 proteins. p73 is pro-apoptotic, even in the absence of p53. It has also been shown to activate the p53 (p73)

target gene that encodes NOXA, causing mitochondrial dysfunction, cytotoxicity, and apoptosis (Roos and Kaina, 2006). Study had showed that BEAS-2B cells transfected with CYP1A2 (B-1A2 cells) can metabolize AFB1 and form AFB1 -DNA adducts (Van Vleet et al., 2002), but no DNA laddering, an indicator of apoptotic cell death in AFB1 (0 to 15 ␮M) (Van Vleet et al., 2006). All the studies suggested that DNA adducts might form before apoptosis, and could be a sensitive biomarker in AFB1 -induced cytotoxicity. In present study, DNA adducts rather than apoptosis were detected in 80 nM AFB1 treated B-1A2 cells might be associated with the low concentration of AFB1 treatments while they were detected in B-2A13 cells, which suggested that CYP2A13 could efficiently metabolize AFB1 to generate active epoxides and subsequently form AFB1 -DNA adducts, resulting in greater cytotoxicity. The commitment of a cell to enter a programmed cell death pathway represents a delicate balance between pro- and antiapoptotic signals. Mitochondria are involved in the active control of cell death processes. The Bcl-2 family of proteins is a key player in the mitochondria-mediated apoptotic pathway (Martinou and Youle, 2011). Anti-apoptotic Bcl-2-like proteins (e.g., Bcl-2, Bcl-x L, Bcl-w, Mcl-1, and A1/Bfl-1) prevent cytochrome c release by forming heterodimer complexes with pro-apoptotic Bcl-2 proteins (e.g., Bax and Bak), whereas pro-apoptotic Bax-like proteins (e.g., Bax,

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Fig. 7. Effect of AFB1 on the formation of AFB1 -DNA adducts in BEAS-2B cells that express CYP2A13. (A) Cells were treated with 5–80 nM AFB1 for 24 h and then subjected to an immunohistochemistry assay to detect the imidazole ring-opened persistent form of the major N7 -guanine adduct of AFB1 (AFB1 -DNA adducts) after the cells were stained with DAB and counterstained with hematoxylin. The cells were observed under an Olympus Light Microscope. (B) AFB1 -DNA adducts expression levels were semiquantitatively determined by intensity of immunostaining using Image-Pro Plus 5.0 (IPP 5.0) software. Vector cells were used as a negative control. DMSO (0.02%) was used as a vehicle control. The experiments were repeated three times independently. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. **p < 0.01, ***p < 0.001, compared with cells treated with DMSO only. ## p < 0.01, ### p < 0.001, compared with B-1A2 cells.

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Bak, and Bok/Mtd) facilitate the release of apoptogenic molecules from mitochondria to the cytosol (Kroemer et al., 2007). Caspases are central mediators of apoptosis (Denault and Salvesen, 2008), and caspase-3 plays a critical role in the execution of the apoptotic process (Chen et al., 2001). In the mitochondrial pathway of apoptosis, this caspase is complex called apoptosome, including Apaf-1, caspase-9, dATP, and cytochrome c (Zou et al., 1999). When mitochondrial membrane permeability is disrupted, cytochrome c is released from the mitochondria to cytosol during apoptosis. The leakage of cytosol cytochrome c can lead to the activation of caspase-3 and caspase-9. C-PARP is a caspase-3 substrate, the cleavage of which is indicative of the activation of the apoptotic pathway. Previous reports demonstrated that AFB1 increased Bax, C-caspase-3 (Duan et al., 2005; Van Vleet et al., 2006) and led to cellular apoptosis via a process that involves mitochondrial damage (Reddy et al., 2006). In the present study, consistent with AFB1 induced cytotoxicity, cellular apoptosis, and DNA adducts, C-PARP, C-caspase-3 and Bax expression increased and caspase-3, Bcl-2, and p-Bad expression decreased in B-2A13 cells. C-PARP and Ccaspase-3 significantly increased with 3 h AFB1 treatment before cytotoxicity and apoptosis occurred. As expected, nicotine and 8MOP reversed the AFB1 -induced changes in C-PARP, C-caspase-3, and Bcl-2 expression in B-2A13 cells. The present results indicate that AFB1 increased the Bax/Bcl-2 ratio and activated PARP, caspase-3, and caspase-9 expression in B-2A13 cells, demonstrating that cytotoxicity mediated by CYP2A13 metabolic activity after AFB1 treatment is associated with the mitochondrial signaling pathway. BEAS-2B cells remain nontumorigenic through numerous passages and represent a good model for studying pulmonary carcinogenesis because they originate from cells targeted by chemical carcinogens in the lung (Reddel et al., 1988). Several drug-metabolizing CYP enzymes were not detected in BEAS-2B cells (Nichols et al., 2003; Van Vleet et al., 2006) because the lack of CYP expression in this cell line might result from immortalization by the SV40 large T antigen. BEAS-2B cells transfected with cDNA for CYP1A2, CYP2A6, and CYP3A4 have been previously used as a cellular model (Nichols et al., 2003; Van Vleet et al., 2006). In the present study, we first established BEAS-2B cells that stably express human CYP2A13 using lentiviral protein expression technology because lentivirus-encoded genes can be efficiently integrated into the host genome (Naldini, 1998). Additionally, the expression of fused GFP protein in each transfected cell was used to screen and construct monoclonal cells with similar expression profiles to compare cytotoxicity among the cells that stably express human CYP2A13, CYP1A2, and CYP2A6 and the vector control. B-2A13, B-1A2, and B-2A6 cells should provide suitable models to explore AFB1 -induced lung carcinogens or other procarcinogens that require CYP2A13-mediated metabolic activation. Furthermore, using this model, we clearly demonstrated that CYP2A13 efficiently activated AFB1 metabolism, subsequently formed AFB1 -DNA adducts, and caused cytotoxicity and apoptosis via the mitochondrial signaling pathway. The present results may be helpful for exploring the etiology of airborne chemical contamination-induced human respiratory diseases, especially at a very low exposure concentration.

Fig. 8. Effect of AFB1 on the expression of apoptosis-related proteins in BEAS-2B cells that express CYP2A13. Cell lysates (50 ␮g) were prepared to determine the expression of apoptosis-related proteins in B-2A13 cells using an immunoblotting assay. (A) B-2A13 cells were treated with 5–80 nM AFB1 for 24 h and then subjected to immunoblotting with antibodies specific for CPARP, caspase-3/9, C-caspase-3/9, Bcl-2, Bax, p-Bad, and Bad. (B) B-2A13 cells were

treated with 80 nM AFB1 for 0, 3, 6, 12, and 24 h and then subjected to immunoblotting with antibodies specific for C-PARP, C-caspase-3/9, and caspase-9. (C) B-2A13 cells treated with 80 nM AFB1 alone or combined with 100 ␮M nicotine or 1 ␮M 8-MOP for 24 h and then subjected to immunoblotting with antibodies specific for C-PARP, C-caspase-3, Bcl-2, and Bax. DMSO (0.02%) was used as a vehicle control.

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Funding This work was supported by National Natural Science Foundation of China (30771782, 30972508), National 973 program (2009CB941701), Enviromnental protection research special funds for public welfare projects (200909054), Six talents peak project of Jiangsu province (DG216D5047) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China. Conflicts of interest None. Acknowledgements The authors wish to thank Prof. Jun-Yan Hong (University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA) for the generous gift of CYP2A13 cDNA plasmids and the specific antibody. We also thank Dr. Weimin Gao (Texas Tech University, Lubbock, TX, USA) for the major revision of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2012.06.010. References Alekseyev, Y.O., Hamm, M.L., Essigmann, J.M., 2004. Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo. Carcinogenesis 25, 1045–1051. Bedard, L.L., Massey, T.E., 2006. Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241, 174–183. BesaratiniaF A., Kim, S.I., Hainaut, P., Pfeifer, G.P., 2009. In vitro recapitulating of TP53 mutagenesis in hepatocellular carcinoma associated with dietary aflatoxin B1 exposure. Gastroenterology 137, 1127–1137, 1137 e1121–1125. Chen, T.A., Yang, F., Cole, G.M., Chan, S.O., 2001. Inhibition of caspase-3-like activity reduces glutamate induced cell death in adult rat retina. Brain Res. 904, 177–188. Denault, J.B., Salvesen, G.S., 2008. Apoptotic caspase activation and activity. Methods Mol. Biol. 414, 191–220. Ding, X., Kaminsky, L.S., 2003. Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu. Rev. Pharmacol. Toxicol. 43, 149–173. Duan, X.X., Ou, J.S., Li, Y., Su, J.J., Ou, C., Yang, C., Yue, H.F., Ban, K.C., 2005. Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis. World J. Gastroenterol. 11, 4740–4744. Egner, P.A., Groopman, J.D., Wang, J.S., Kensler, T.W., Friesen, M.D., 2006. Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem. Res. Toxicol. 19, 1191–1195. Groopman, J.D., Kensler, T.W., Wild, C.P., 2008. Protective interventions to prevent aflatoxin-induced carcinogenesis in developing countries. Annu. Rev. Public Health 29, 187–203. Guo, Y., Breeden, L.L., Zarbl, H., Preston, B.D., Eaton, D.L., 2005. Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol. Cell. Biol. 25, 5823–5833. Gursoy-Yuzugullu, O., Yuzugullu, H., Yilmaz, M., Ozturk, M., 2011. Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation. Liver Int. 31, 561–571. Hayes, R.B., van Nieuwenhuize, J.P., Raatgever, J.W., ten Kate, F.J., 1984. Aflatoxin exposures in the industrial setting: an epidemiological study of mortality. Food Chem. Toxicol. 22, 39–43. He, X.Y., Tang, L., Wang, S.L., Cai, Q.S., Wang, J.S., Hong, J.Y., 2006. Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract. Int. J. Cancer 118, 2665–2671. Kensler, T.W., Roebuck, B.D., Wogan, G.N., Groopman, J.D., 2011. Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol. Sci. 120 (Suppl. 1), S28L 48. Kroemer, G., Galluzzi, L., Brenner, C., 2007. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163. Li, F.Q., Li, Y.W., Wang, Y.R., Luo, X.Y., 2009. Natural occurrence of aflatoxins in Chinese peanut butter and sesame paste. J. Agric. Food Chem. 57, 3519–3524. Long, X.D., Ma, Y., Zhou, Y.F., Ma, A.M., Fu, G.H., 2010. Polymorphism in xeroderma pigmentosum complementation group C codon 939 and aflatoxin

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