- Email: [email protected]
Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage
Toxicology and Applied Pharmacology 270 (2013) 114–121
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage Xuejiao Yang a, b, 1, Zhan Zhang a, 1, Xichen Wang a, Yun Wang a, Xiaoming Zhang a, Huiyuan Lu a, Shou-Lin Wang a,⁎ a b
Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166, China Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000, China
a r t i c l e
i n f o
Article history: Received 28 February 2013 Revised 8 April 2013 Accepted 10 April 2013 Available online 18 April 2013 Keywords: Cytochrome P450 2A13 Aflatoxin B1 BEAS-2B Cells DNA adduct DNA damage DNA damage and repair response proteins
a b s t r a c t Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. © 2013 Elsevier Inc. All rights reserved.
Introduction Carcinoma of the lung is currently the leading cause of death due to cancer worldwide (de Groot and Munden, 2012), with the involvement of environmental air pollutants. Epidemiological and laboratory studies have shown that the human respiratory is a target for aflatoxin B1 (AFB1) carcinogenicity (Hayes et al., 1984; Massey et al., 2000). There is a positive association between human lung cancer occurrence and inhalation exposure to AFB1 (Kelly et al., 1997; NTP, 2004). The fungus that produces AFB1 is typically easy to grow under damp conditions, and small-particle AFB1 can be inhaled into the human respiratory system through breathing. Workers with
Abbreviations: CYP450, Cytochrome P450; AFB1, Aflatoxin B1; BEAS-2B cells, immortalized human bronchial epithelial cells; B-2A13 cells, BEAS-2B cells stably expressing CYP2A13; AFB1-FAPY, AFB1-formamidopyrimidine; AFB1-N7-Gua, trans-8,9dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1; 8-MOP, 8-methoxypsoralen; 8-OHdG, 8-hydroxy-deoxyguanosine; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; DSBs, double-strand DNA breaks. ⁎ Corresponding author. Fax: +86 25 8686 8499. E-mail address: [email protected] (S.-L. Wang). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.04.005
occupational exposure to inhaled aflatoxins, particularly in the form of airborne grain dust, are at risk of ingesting, transmucosally absorbing, and inhaling AFB1 released during product preparation or processing (Sorenson et al., 1981; Traverso et al., 2010; Wang et al., 2008). AFB1 is an indirect carcinogen, and its biotransformation plays a crucial role in its disposition, toxicity, and carcinogenicity. As a major naturally occurring liver carcinogen, AFB1 is metabolized mainly by cytochrome P450 (CYP) enzymes, particularly CYP1A2 and CYP3A4, to form AFB1-8,9-epoxides (Kensler et al., 2011). Subsequently, with DNA at the N7 position of guanine, the epoxides form trans-8,9-dihydro8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adducts, and the imidazole ring opens to form stable AFB1-formamidopyrimidine (AFB1-FAPY) adducts (Bedard and Massey, 2006; Brown et al., 2009). The initial AFB1-N7-Gua adducts and AFB1-FAPY adducts individually or collectively represent the likely chemical precursors of the genotoxic effects of AFB1. They serve as an index of biological internal exposure dose or DNA damage (Besaratinia et al., 2009; Egner et al., 2006; Long et al., 2010). Additionally, common oxidative DNA damage that led to 8-hydroxydeoxyguanosine (8-OHdG) lesions was observed in rat hepatic DNA, mouse lung cells, and samples from a biological population following exposure to AFB1 (Guindon et al., 2007; Peng et al., 2007; Shen et al., 1995).
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
In contrast to CYP1A2 and CYP3A4, CYP2A13 is an extrahepatic CYP enzymes that is mainly expressed in the human respiratory system (Ding and Kaminsky, 2003). It was highly efficient in the metabolic activation of AFB1 to form active metabolites (He et al., 2006) shown to play an important role in AFB1-induced cytotoxicity and apoptosis in human bronchial epithelial cells (Yang et al., 2012). Thus, we speculated that CYP2A13 might provide a link between AFB1 and respiratory diseases, especially lung cancer, because it can metabolically activate airborne AFB1 in situ in pulmonary cells and subsequently induce DNA damage and repair, which usually contribute to cellular carcinogenesis. However, to date, few studies have investigated CYP2A13-mediated DNA damage induced by lowconcentration AFB1 in human bronchial epithelial cells. Therefore, the present study used an established line of monoclonal BEAS-2B cells that stably express CYP2A13 (B-2A13) to evaluate the role of CYP2A13 in AFB1-induced DNA damage compared with BEAS-2B cells that stably express CYP1A2 (B-1A2), CYP2A6 (B-2A6), or vector alone (B-vector). Furthermore, AFB1 is well-known to be involved in the formation of covalent adducts with DNA, which can activate DNA damage checkpoint signal pathways in human cells (Gursoy-Yuzugullu et al., 2011). Therefore, several related proteins, including p-ATM, ATM, p-ATR, ATR, p-BRCA1, p-Chk2, p-p53, γH2AX, Ku-80, XLF, Mre11 and Rad50, were selected to investigate DNA damage and repair responses. The results will help provide insights into the relationship between airborne AFB1 pollution and human respiratory diseases, especially lung cancer. Materials and methods Single-cell gel electrophoresis (comet) assay. The comet assay has gained widespread use in various areas including human biomonitoring, genotoxicology, ecological monitoring and as a tool for research into DNA damage or repair in different cell types in response to a range of DNA-damaging agents (Liao et al., 2009). It is a sensitive and rapid method for DNA strand break detection in individual cells. The % of DNA in the tail reflects the break frequency (Shaposhnikov et al., 2009). Cells were plated into six-well plates (3 × 105 cells/well) at 37 °C in 5% CO2 overnight and then treated with different concentrations of AFB1, ranging from 0 to 80 nM, for 24 h. Additionally, B-2A13 cells were treated with AFB1 at concentrations that ranged from 0 to 80 nM for 6, 12, and 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced DNA damage, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, the comet assay was conducted as described previously (Wu et al., 2012). Detection of AFB1-DNA adducts, 8-OHdG, and γH2AX by immunofluorescence. After the cells were incubated in special small laser confocal dishes (3 × 105 cells/well) at 37 °C in 5% CO2 overnight, they were treated with 80 nM AFB1 for 24 h. Additionally, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, γH2AX was detected using an immunofluorescence assay as described previously (Stoimenov et al., 2011). Aflatoxin B1-DNA (AFB1-DNA) adducts and 8-OHdG were detected using a modified immunofluorescence assay as described previously (Gursoy-Yuzugullu et al., 2011). Briefly, after treatment with AFB1, the cells were treated with a buffer that contained 15 mM Na2CO3 and 30 mM NaHCO3 (pH 9.6) at room temperature for 2 h and then were lysed with 100 μg/ml RNAse in Tris buffer (10 mM Trizma Base, 1 mM ethylenediaminetetraacetic acid [EDTA], and 0.4 mol/l NaCl, pH 7.5) at 37 °C for 1 h. After washing with phosphate-buffered saline (PBS), the cells were treated with 10 μg/ml proteinase K at room temperature for 10 min, followed by washing with PBS and 40% ethanol. The cells were then treated with 50 mM NaOH in 40% ethanol for 30 s to denature the DNA and rinsed again with 40% ethanol. After blocking
115
with 5% bovine serum albumin (BSA) for 1 h, the cells were incubated with mouse 6A10 (1:200; Novus Biologicals, NB-600-443H) or goat anti-8-OHdG (1:100; Santa Cruz Biotechnology, SC-130085) monoclonal antibody at 4 °C overnight. The cells were then incubated in Cy3-labeled goat anti-mouse immunoglobulin G (IgG) (1:1000; Beyotime, P0193) or Cy3-labeled donkey anti-goat IgG secondary antibodies (1:1000; Beyotime, P0173) for 30 min. Images were obtained with a Zeiss LSM 710 inverted confocal microscope. Focus maximum projection images were acquired from optical sections 0.50 mm apart with a section thickness of 1.0 mm. The images were processed using Adobe Photoshop. The fluorescence intensity was semi-quantitatively determined using ZEN2008 software (Zeiss, Germany). Cell cycle analysis by flow cytometry. Monoclonal B-2A13, B-1A2, B-2A6, and B-vector cells were prepared and cultured according to our previous study (Yang et al., 2012). For cell cycle analysis, the cells were seeded in a 10 cm dish, incubated overnight, and treated with 80 nM AFB1 for 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced cell cycle changes, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, the cell cycle was analyzed using flow cytometry assay as described previously (Funamizu et al., 2012). Detection of proteins related to DNA damage and repair. B-2A13 cells were plated into 10 cm plates (1 × 106 cells/well) overnight and then treated with AFB1 at different concentrations, ranging from 0 to 80 nM, for 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced changes in proteins related to DNA damage and repair, the cells were treated with 80 nM AFB1 alone or 80 nM AFB1 combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The expression of proteins related to DNA damage and repair was 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 BRCA1, p-BRCA1 (ser1524), ATM, p-ATM (ser1981), ATR, p-ATR (ser428), Chk2, p-Chk2 (thr68), p53, p-p53 (ser15), Mre11, p-Mre11, Ku-80, XLF, Rad50 and γH2AX (ser139), the protein immune complexes were detected using an electrochemiluminescence assay kit and exposed to Kodak X-Omat film. 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). All other antibodies were purchased from Cell Signaling Technology (Danvers, MA). Statistical analysis. The means and standard deviations (SDs) were calculated for all of the investigated parameters, and all of the data are expressed as mean ± 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-NewmanKeuls test. Values of p b 0.05 were considered statistically significant. Results Effects of CYP2A13 on AFB1-induced DNA damage in BEAS-2B cells The single-cell gel electrophoresis (comet) assay was used to evaluate AFB1-induced DNA damage in BEAS-2B cells. As shown in Fig. 1A, DNA damage in B-2A13 cells dose-dependently increased after 24 h of AFB1 treatment. Compared with vehicle control, 10 nM AFB1 significantly induced DNA damage in B-2A13 cells (p b 0.001). DNA damage in B-2A13 cells was much more evident than in B-1A2 cells, whereas no differences between treatment and control were observed in B-2A6 and B-vector cells (Fig. 1A). Additionally, DNA damage in B-2A13 cells
116
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 1. Effect of CYP2A13 on AFB1-induced DNA damage in BEAS-2B cells. DNA damage was detected using the comet assay with a fluorescence microscope. (A) Cells (B-Vector, B-1A2 and B-2A13) were treated with 5–80 nM AFB1 for 24 h. (B) B-2A13 cells were treated with 5–80 nM AFB1 for 6, 12, or 24 h. (C) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The olive tail moment, indicating DNA damage, is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with 80 nM AFB1 alone.
also time-dependently increased with AFB1 treatment. Treatment with 80 nM AFB1 for 6 h or 40 nM AFB1 for 12 h significantly increased DNA damage compared with vehicle control (p b 0.001; Fig. 1B). Similar to our previous study (Yang et al., 2012), nicotine and 8-MOP were used to explore the role of CYP2A13 in AFB1-induced DNA damage. Compared with AFB1 alone, both 100 μM nicotine and 1 μM 8-MOP significantly inhibited AFB1-induced DNA damage (p b 0.001; Fig. 1C). Effects of CYP2A13 in AFB1-induced double-strand DNA breaks in BEAS-2B cells γH2AX, a hallmark of double-strand DNA breaks (DSBs), was detected to identify the type of DNA damage. Compared with vehicle control, 80 nM AFB1 induced greater formation of γH2AX in the nuclei of B-2A13 cells (p b 0.001), whereas no significant changes in γH2AX were found in either B-1A2 or B-2A6 cells (Figs. 2A and B). AFB1 dose-dependently increased γH2AX expression in B-2A13 cells, and nicotine and 8-MOP inhibited the protein expression (Figs. 2C and D). Immunofluorescence assay was used to confirm the effect. Compared with 80 nM AFB1 alone, both 100 μM nicotine and 1 μM 8-MOP obviously inhibited the AFB1-induced formation of γH2AX in the nuclei of B-2A13 cells (p b 0.001; Figs. 2E and F).
Effects of CYP2A13 in AFB1-induced cell cycle changes in BEAS-2B cells As shown in Fig. 3, compared with the vehicle control and B-vector cells, treatment with 80 nM AFB1 for 24 h significantly increased the percentage of cells in the S-phase of the cell cycle in B-2A13 cells (58.4% vs. 34.7%) and B-1A2 cells (46.2% vs. 34.7%; p b 0.001), respectively. The results indicated that AFB1 induced a delay through the S-phase and in the transition from the S- to G2/M-phases in B-2A13 cells. As expected, 100 μM nicotine and 1 μM 8-MOP markedly reversed AFB1-induced cell cycle change (p b 0.001). Effects of CYP2A13 on the formation of AFB1-DNA adducts and 8-OHdG in BEAS-2B cells The formation of the imidazole ring-opened persistent form of major N7-guanine adducts of AFB1 was found in the nuclei of B-2A13 and B-1A2 cells. Treatment with 80 nM AFB1 induced greater formation of AFB1-DNA adducts compared with vehicle control and B-vector cells (p b 0.001). Compared with B-1A2 cells, B-2A13 cells showed much more formation of AFB1-DNA adducts, and the relative fluorescence intensity was approximately nine-times greater than B-1A2 cells (311 vs. 36; p b 0.001; Figs. 4A and B). As predicted, either 100 μM nicotine or
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
117
Fig. 2. Effect of CYP2A13 on AFB1-induced γH2AX expression in BEAS-2B cells. The type of DNA damage was identified by the detection of γH2AX expression using an immunofluorescence assay with a laser scanning confocal microscope. (A–B) Cells were treated with 80 nM AFB1 for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The relative fluorescence intensity was quantified using ZEN 2008 software. (C–D) Cell lysates (100 μg) from B-2A13 cells that were treated with 5–80 nM AFB1 for 24 h (C) or treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h (D) were prepared to determine γH2AX expression. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference. (E–F) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was quantified using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
1 μM 8-MOP significantly reversed the AFB1-induced formation of AFB1-DNA adducts in B-2A13 cells (Figs. 4C and D). Similarly, 8-OHdG was only found in the nuclei of B-2A13 and B-1A2 cells after 80 nM AFB1 treatment (Fig. 5A). Compared with B-1A2 cells, B-2A13 cells showed more 8-OHdG formation, and the relative fluorescence intensity was approximately three-times greater than B-1A2 cells (160 vs. 55; p b 0.001; Fig. 5B). Both 100 μM nicotine and 1 μM 8-MOP markedly reversed the AFB1-induced formation of 8-OHdG (Figs. 5C and D).
p-BRCA1, p-Chk2, and p-p53, which was consistent with their effects on AFB1-induced cell cycle changes and DNA damage, while the expression of ATM, ATR, BRCA1, Chk2 and p53 did not change after AFB1 treatment (Fig. S1). Besides, no significant effect of nicotine or 8-MOP was found on the changes in DSB repair-related proteins, such as Ku-80, XLF, Mre11, p-Mre11, and Rad50 (Fig. 6B). The expressions of CYP2A13 and GFP were similar among the B-2A13 cells treated with DMSO, nicotine or 8-MOP. Discussion
Effects of CYP2A13 on AFB1-induced expression of proteins related to DNA damage and repair in BEAS-2B cells As shown in Fig. 6A, the expression of p-ATM, p-ATR, p-BRCA1, p-Chk2, and p-p53 in B-2A13 cells significantly increased as the concentration of AFB1 increased. However, no significant changes were found in the proteins related to DSB repair, such as Ku-80, XLF, Mre11, p-Mre11, and Rad50. As expected, 100 μM nicotine and 1 μM 8-MOP significantly inhibited the AFB1-induced expression of p-ATM, p-ATR,
In the present study, we demonstrated the role of CYP2A13 in the AFB1-induced formation of AFB1-DNA adducts, DNA damage, and subsequent molecular DNA damage and repair responses using monoclonal BEAS-2B cells that stably expressed CYP2A13 (B-2A13). We also confirmed the effects using nicotine (a substrate of CYP2A13) and 8-MOP (an inhibitor/inactivator of cytochrome P450 enzymes) (von Weymarn et al., 2005, 2006). To facilitate the study, we used CYP2A6 and CYP1A2 as references. With 93.5% shared amino acid sequence identity (Smith
118
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 3. Effect of CYP2A13 on AFB1-induced cell cycle changes in BEAS-2B cells. (A) Cells (B-Vector, B-1A2 and B-2A13) were treated with 80 nM AFB1 or B-2A13 cells were treated with 80 nM AFB1 plus 100 μM nicotine or 1 μM 8-MOP for 24 h. The cells were then subjected to Propidium Iodide to detect the cell cycle using a flow cytometry assay. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. (B) The percentage of cells in the S-phase is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
et al., 2007), CYP2A13 is an efficient enzyme in the metabolic activation of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with substrate specificity similar to CYP2A6 (Bao et al., 2005; Su et al., 2000). However, they have large differences in their ability to metabolize xenobiotic/endogenous compounds. CYP2A13 is much more active than CYP2A6 in the metabolic activation of AFB1 (He et al., 2006). CYP1A2 has been shown to be the principle enzyme responsible for AFB1 activation (Gallagher et al., 1996), but it is mainly expressed in the human liver (Pelkonen and Raunio, 1997) rather than in the human respiratory system (Ding and Kaminsky, 2003). As predicted in our previous study (Yang et al., 2012), B-2A13 cells were much more sensitive to AFB1-induced DNA damage, indicating that CYP2A13 was a more efficient metabolic enzyme of AFB1 and might play an important role in airborne AFB1 pollution and human respiratory disease, especially cellular genetic injury-induced lung cancer. AFB1 can generally be metabolized by the CYP2A13 to form AFB1-8,9-epoxide (He et al., 2006), which subsequently binds to DNA to form AFB1-N7-Gua adducts and later AFB1-FAPY adducts, which have been recently used as carcinogenic biomarkers (Carvajal et al., 2012; Long et al., 2010). In the present study, AFB1 induced more AFB1-DNA adducts in B-2A13 cells than in B-1A2 cells, with similar AFB1-DNA adduct formation in B-2A6 cells, B-vector cells, and
vehicle control. Adduct formation was inhibited by nicotine and 8-MOP. 8-OHdG is often used as a marker of oxidative DNA damage and formed by the reaction of hydroxyl radicals (·OH) with guanine residues in DNA (Shen et al., 1995). Previous studies demonstrated elevations of 8-OHdG in AFB1-treated mouse lung cells and human biological samples exposed to AFB1 (Guindon et al., 2007; Peng et al., 2007). Consistent with AFB1-DNA adducts, AFB1 induced much more expression of 8-OHdG, and nicotine and 8-MOP could inhibit 8-OHdG in B-2A13 cells. These results indicate that CYP2A13 might mediate AFB1-DNA adduct and 8-OHdG formation and showed more efficient activation in AFB1 metabolism to AFB1-epoxides. The delayed and defective DNA damage response to AFB1 could be related to the type of DNA and protein adducts that it forms in exposed cells (Shen et al., 1995; Wang and Groopman, 1999). The accumulation of AFB1-DNA adducts can induce DNA damage. Consistent with the AFB1-DNA adduct and 8-OHdG results, AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells, which were much more sensitive than B-1A2 and B-2A6 cells. Nicotine and 8-MOP inhibited AFB1-induced DNA damage in B-2A13 cells. Typically, DSBs and stalled replication forks cause the phosphorylation of neighboring histone 2AX at Ser139 (H2AX for the unphosphorylated form and γH2AX for the phosphorylated form) via ataxia telangiectasia mutated
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
119
Fig. 4. Effect of CYP2A13 on AFB1-induced DNA adduct formation in BEAS-2B cells. (A–B) Cells were treated with 80 nM AFB1 for 24 h and then subjected to the immunofluorescence assay to detect AFB1-DNA adduct formation using a laser scanning confocal microscope. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The relative fluorescence intensity was calculated using ZEN 2008 software. (C–D) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was calculated using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
(ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively (Roos and Kaina, 2012). ATM is the primary kinase that phosphorylates H2AX at DSBs although ATR and DNA-dependent protein kinase (Mah et al., 2010). γH2AX is usually used as a hallmark of DSBs. Our results showed greater expression of γH2AX in B-2A13 cells, which could be inhibited by nicotine and 8-MOP. The present data suggested that CYP2A13 efficiently metabolized AFB1 to generate active epoxides and subsequently form AFB1-DNA adducts, 8-OHdG, and γH2AX, resulting in greater acute damage. Various environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that subsequently activate DNA damage checkpoint signaling pathways in human cells (Kemp et al., 2011). Two of the major regulators of the DNA damage response are the phosphoinositide 3-kinase-related protein kinases ATM and ATR, which respond to different types of DNA damage. ATM is mainly activated by DSBs, whereas ATR is activated at structures that contain single-strand DNA (ssDNA), both of which can be activated by DSBs. Resection of the DSB end leads to a large single-stranded region (Jackson and Bartek, 2009). Activated ATM and ATR phosphorylate their downstream targets, including checkpoint kinase 2 (Chk2), which subsequently activates breast cancer susceptibility gene 1 (BRCA1) and initiates DNA repair. In the present study, ATM, ATR, Chk2, and BRCA1 were upregulated by AFB1 and inhibited by nicotine and 8-MOP. However, DNA repair-related proteins, such as Ku-80, XLF, Mre11, Rad50, and Rad52, were unaffected, suggesting that CYP2A13 mediated more DNA damage than repair with AFB1 treatment. The most deleterious DNA lesions are DSBs. If left unrepaired, they may have severe consequences for cell survival and lead to chromosome aberrations, genomic instability, and cell death. To minimize the detrimental effects of DNA damage on genome stability and cell viability, signal transduction pathways, termed DNA damage checkpoints, delay or prevent cell cycle
progression to allow time for DNA repair (Sancar et al., 2004), which was activated by p53. Both kinases phosphorylate p53 at Ser15 and other sites. The stabilization of p53 is also influenced by Chk2, which is phosphorylated by ATM (Cimprich and Cortez, 2008; Roos and Kaina, 2012). Previous studies showed that AFB1 significantly increased the S-phase cell population in SK-N-MC cells and J774A.1 murine macrophages (Bianco et al., 2012; Ricordy et al., 2002), supporting our results that AFB1 phosphorylated p53 and subsequently induced S-phase arrest in B-2A13 cells. In conclusion, the present study demonstrated that CYP2A13 efficiently contributed to the metabolic activation to AFB1. CYP2A13 played a critical role in the AFB1-induced formation of AFB1-DNA adducts, DNA damage, of DSBs and subsequently activated the ATM/ATR, Chk2, and p53 signaling pathways. Similar to our previous study, because of the high metabolic activity of CYP2A13, low concentrations of AFB1 (i.e., 5 and 10 nM) induced DNA damage in human bronchial epithelial cells. CYP2A13, mainly expressed in the human respiratory system, might be the primary metabolic enzyme involved in AFB1 metabolism in situ in lung epithelial cells and be an important link between environmental airborne AFB1 contaminants and human respiratory disorders, especially carcinoma of the lung. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2013.04.005.
Funding This work was supported by the National Natural Science Foundation of China (30972508, 30771782), the National 973 program (2009CB941701), the Doctoral Fund of Ministry of Education of China (20093234110002), the Six talents peak project of Jiangsu province
120
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 5. Effect of CYP2A13 on AFB1-induced 8-OHdG formation in BEAS-2B cells. (A–B) Cells were treated with 80 nM AFB1 for 24 h and then subjected to the immunofluorescence assay to detect 8-OHdG expression using a laser scanning confocal microscope. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The fluorescence intensity was calculated using ZEN 2008 software. (C–D) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was calculated using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
(DG216D5047), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China. Conflict of interest statement The authors declare that they have no competing financial interests. References
Fig. 6. Effects of CYP2A13 on the expression of proteins related to DNA damage and repair in BEAS-2B cells. (A) B-2A13 cells were treated with 5–80 nM AFB1 for 24 h. (B) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. Cell lysates (100 μg) were prepared to determine protein expression using an immunoblotting assay with antibodies specific for p-ATM, p-ATR, p-Chk2, p-BRCA1, p-p53, Ku-80, XLF, Mre11, p-Mre11, and Rad50. DMSO, nicotine or 8-MOP did not change the expression of CYP2A13 and GFP. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference.
Bao, Z., He, X.Y., Ding, X., Prabhu, S., Hong, J.Y., 2005. Metabolism of nicotine and cotinine by human cytochrome P450 2A13. Drug Metab. Dispos. 33, 258–261. Bedard, L.L., Massey, T.E., 2006. Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241, 174–183. Besaratinia, 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). Bianco, G., Russo, R., Marzocco, S., Velotto, S., Autore, G., Severino, L., 2012. Modulation of macrophage activity by aflatoxins B1 and B2 and their metabolites aflatoxins M1 and M2. Toxicon 59, 644–650. Brown, K.L., Voehler, M.W., Magee, S.M., Harris, C.M., Harris, T.M., Stone, M.P., 2009. Structural perturbations induced by the alpha-anomer of the aflatoxin B(1) formamidopyrimidine adduct in duplex and single-strand DNA. J. Am. Chem. Soc. 131, 16096–16107. Carvajal, M., Berumen, J., Guardado-Estrada, M., 2012. The presence of aflatoxin B(1)FAPY adduct and human papilloma virus in cervical smears from cancer patients in Mexico. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29, 258–268. Cimprich, K.A., Cortez, D., 2008. ATR: an essential regulator of genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627. de Groot, P., Munden, R.F., 2012. Lung cancer epidemiology, risk factors, and prevention. Radiol. Clin. North Am. 50, 863–876. 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. 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.
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121 Funamizu, N., Lacy, C.R., Fujita, K., Furukawa, K., Misawa, T., Yanaga, K., Manome, Y., 2012. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. PLoS One 7, e37424. Gallagher, E.P., Kunze, K.L., Stapleton, P.L., Eaton, D.L., 1996. The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4. Toxicol. Appl. Pharmacol. 141, 595–606. Guindon, K.A., Bedard, L.L., Massey, T.E., 2007. Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1). Toxicol. Sci. 98, 57–62. 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. Jackson, S.P., Bartek, J., 2009. The DNA-damage response in human biology and disease. Nature 461, 1071–1078. Kelly, J.D., Eaton, D.L, Guengerich, F.P., Coulombe Jr., R.A., 1997. Aflatoxin B1 activation in human lung. Toxicol. Appl. Pharmacol. 144, 88–95. Kemp, M.G., Lindsey-Boltz, L.A., Sancar, A., 2011. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA. J. Biol. Chem. 286, 19237–19246. 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), S28–S48. Liao, W., McNutt, M.A., Zhu, W.G., 2009. The comet assay: a sensitive method for detecting DNA damage in individual cells. Methods 48, 46–53. 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 B1-related hepatocellular carcinoma in the Guangxi population. Hepatology 52, 1301–1309. Ma, J., Lu, H.Y., Xia, Y.K., Dong, H.B., Gu, A.H., Li, Z.Y., Li, Z., Chen, A.M., Wang, X.R., Wang, S.L., 2010. BCL2 Ala43Thr Is a functional variant associated with protection against azoospermia in a Han-Chinese population. Biol. Reprod. 83, 656–662. Mah, L.J., El-Osta, A., Karagiannis, T.C., 2010. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24, 679–686. Massey, T.E., Smith, G.B., Tam, A.S., 2000. Mechanisms of aflatoxin B1 lung tumorigenesis. Exp. Lung Res. 26, 673–683. National Toxicology Program., 2004. NTP 11th Report on Carcinogens. Rep. Carcinog. 11, 1–A32. Pelkonen, O., Raunio, H., 1997. Metabolic activation of toxins: tissue-specific expression and metabolism in target organs. Environ. Health Perspect. 105 (Suppl. 4), 767–774. Peng, T., Li, L.Q., Peng, M.H., Liu, Z.M., Liu, T.W., Guo, Y., Xiao, K.Y., Qin, Z., Ye, X.P., Mo, X.S., Yan, L.N., Lee, B.L., Shen, H.M., Tamae, K., Wang, L.W., Wang, Q., Khan, K.M., Wang, K.B., Liang, R.X., Wei, Z.L., Kasai, H., Ong, C.N., Santella, R.M., 2007. Evaluation of oxidative stress in a group of adolescents exposed to a high level of aflatoxin B1—a multi-center and multi-biomarker study. Carcinogenesis 28, 2347–2354.
121
Ricordy, R., Gensabella, G., Cacci, E., Augusti-Tocco, G., 2002. Impairment of cell cycle progression by aflatoxin B1 in human cell lines. Mutagenesis 17, 241–249. Roos, W.P., Kaina, B., 2012. DNA damage-induced apoptosis: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. http:// dx.doi.org/10.1016/j.canlet.2012.01.007. Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K., Linn, S., 2004. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85. Shaposhnikov, S., Frengen, E., Collins, A.R., 2009. Increasing the resolution of the comet assay using fluorescent in situ hybridization—a review. Mutagenesis 24, 383–389. Shen, H.M., Ong, C.N., Lee, B.L., Shi, C.Y., 1995. Aflatoxin B1-induced 8hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16, 419–422. Smith, B.D., Sanders, J.L., Porubsky, P.R., Lushington, G.H., Stout, C.D., Scott, E.E., 2007. Structure of the human lung cytochrome P450 2A13. J. Biol. Chem. 282, 17306–17313. Sorenson, W.G., Simpson, J.P., Peach III, M.J., Thedell, T.D., Olenchock, S.A., 1981. Aflatoxin in respirable corn dust particles. J. Toxicol. Environ. Health 7, 669–672. Stoimenov, I., Schultz, N., Gottipati, P., Helleday, T., 2011. Transcription inhibition by DRB potentiates recombinational repair of UV lesions in mammalian cells. PLoS One 6, e19492. Su, T., Bao, Z., Zhang, Q.Y., Smith, T.J., Hong, J.Y., Ding, X., 2000. Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 60, 5074–5079. Traverso, A., Bassoli, V., Cioe, A., Anselmo, S., Ferro, M., 2010. Assessment of aflatoxin exposure of laboratory worker during food contamination analyses. Assessment of the procedures adopted by an A.R.P.A.L. laboratory (Liguria Region Environmental Protection Agency). Med. Lav. 101, 375–380. von Weymarn, L.B., Zhang, Q.Y., Ding, X., Hollenberg, P.F., 2005. Effects of 8-methoxypsoralen on cytochrome P450 2A13. Carcinogenesis 26, 621–629. von Weymarn, L.B., Brown, K.M., Murphy, S.E., 2006. Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism. J. Pharmacol. Exp. Ther. 316, 295–303. Wang, J.S., Groopman, J.D., 1999. DNA damage by mycotoxins. Mutat. Res. 424, 167–181. Wang, Y., Chai, T., Lu, G., Quan, C., Duan, H., Yao, M., Zucker, B.A., Schlenker, G., 2008. Simultaneous detection of airborne aflatoxin, ochratoxin and zearalenone in a poultry house by immunoaffinity clean-up and high-performance liquid chromatography. Environ. Res. 107, 139–144. Wu, Y., Qi, X., Gong, L., Xing, G., Chen, M., Miao, L., Yao, J., Suzuki, T., Furihata, C., Luan, Y., Ren, J., 2012. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation. PLoS One 7, e35010. Yang, X.J., Lu, H.Y., Li, Z.Y., Bian, Q., Qiu, L.L., Li, Z., Liu, Q., Li, J., Wang, X., Wang, S.L., 2012. Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells. Toxicology 300, 138–148.
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage Xuejiao Yang a, b, 1, Zhan Zhang a, 1, Xichen Wang a, Yun Wang a, Xiaoming Zhang a, Huiyuan Lu a, Shou-Lin Wang a,⁎ a b
Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166, China Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000, China
a r t i c l e
i n f o
Article history: Received 28 February 2013 Revised 8 April 2013 Accepted 10 April 2013 Available online 18 April 2013 Keywords: Cytochrome P450 2A13 Aflatoxin B1 BEAS-2B Cells DNA adduct DNA damage DNA damage and repair response proteins
a b s t r a c t Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. © 2013 Elsevier Inc. All rights reserved.
Introduction Carcinoma of the lung is currently the leading cause of death due to cancer worldwide (de Groot and Munden, 2012), with the involvement of environmental air pollutants. Epidemiological and laboratory studies have shown that the human respiratory is a target for aflatoxin B1 (AFB1) carcinogenicity (Hayes et al., 1984; Massey et al., 2000). There is a positive association between human lung cancer occurrence and inhalation exposure to AFB1 (Kelly et al., 1997; NTP, 2004). The fungus that produces AFB1 is typically easy to grow under damp conditions, and small-particle AFB1 can be inhaled into the human respiratory system through breathing. Workers with
Abbreviations: CYP450, Cytochrome P450; AFB1, Aflatoxin B1; BEAS-2B cells, immortalized human bronchial epithelial cells; B-2A13 cells, BEAS-2B cells stably expressing CYP2A13; AFB1-FAPY, AFB1-formamidopyrimidine; AFB1-N7-Gua, trans-8,9dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1; 8-MOP, 8-methoxypsoralen; 8-OHdG, 8-hydroxy-deoxyguanosine; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; DSBs, double-strand DNA breaks. ⁎ Corresponding author. Fax: +86 25 8686 8499. E-mail address: [email protected] (S.-L. Wang). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.04.005
occupational exposure to inhaled aflatoxins, particularly in the form of airborne grain dust, are at risk of ingesting, transmucosally absorbing, and inhaling AFB1 released during product preparation or processing (Sorenson et al., 1981; Traverso et al., 2010; Wang et al., 2008). AFB1 is an indirect carcinogen, and its biotransformation plays a crucial role in its disposition, toxicity, and carcinogenicity. As a major naturally occurring liver carcinogen, AFB1 is metabolized mainly by cytochrome P450 (CYP) enzymes, particularly CYP1A2 and CYP3A4, to form AFB1-8,9-epoxides (Kensler et al., 2011). Subsequently, with DNA at the N7 position of guanine, the epoxides form trans-8,9-dihydro8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adducts, and the imidazole ring opens to form stable AFB1-formamidopyrimidine (AFB1-FAPY) adducts (Bedard and Massey, 2006; Brown et al., 2009). The initial AFB1-N7-Gua adducts and AFB1-FAPY adducts individually or collectively represent the likely chemical precursors of the genotoxic effects of AFB1. They serve as an index of biological internal exposure dose or DNA damage (Besaratinia et al., 2009; Egner et al., 2006; Long et al., 2010). Additionally, common oxidative DNA damage that led to 8-hydroxydeoxyguanosine (8-OHdG) lesions was observed in rat hepatic DNA, mouse lung cells, and samples from a biological population following exposure to AFB1 (Guindon et al., 2007; Peng et al., 2007; Shen et al., 1995).
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
In contrast to CYP1A2 and CYP3A4, CYP2A13 is an extrahepatic CYP enzymes that is mainly expressed in the human respiratory system (Ding and Kaminsky, 2003). It was highly efficient in the metabolic activation of AFB1 to form active metabolites (He et al., 2006) shown to play an important role in AFB1-induced cytotoxicity and apoptosis in human bronchial epithelial cells (Yang et al., 2012). Thus, we speculated that CYP2A13 might provide a link between AFB1 and respiratory diseases, especially lung cancer, because it can metabolically activate airborne AFB1 in situ in pulmonary cells and subsequently induce DNA damage and repair, which usually contribute to cellular carcinogenesis. However, to date, few studies have investigated CYP2A13-mediated DNA damage induced by lowconcentration AFB1 in human bronchial epithelial cells. Therefore, the present study used an established line of monoclonal BEAS-2B cells that stably express CYP2A13 (B-2A13) to evaluate the role of CYP2A13 in AFB1-induced DNA damage compared with BEAS-2B cells that stably express CYP1A2 (B-1A2), CYP2A6 (B-2A6), or vector alone (B-vector). Furthermore, AFB1 is well-known to be involved in the formation of covalent adducts with DNA, which can activate DNA damage checkpoint signal pathways in human cells (Gursoy-Yuzugullu et al., 2011). Therefore, several related proteins, including p-ATM, ATM, p-ATR, ATR, p-BRCA1, p-Chk2, p-p53, γH2AX, Ku-80, XLF, Mre11 and Rad50, were selected to investigate DNA damage and repair responses. The results will help provide insights into the relationship between airborne AFB1 pollution and human respiratory diseases, especially lung cancer. Materials and methods Single-cell gel electrophoresis (comet) assay. The comet assay has gained widespread use in various areas including human biomonitoring, genotoxicology, ecological monitoring and as a tool for research into DNA damage or repair in different cell types in response to a range of DNA-damaging agents (Liao et al., 2009). It is a sensitive and rapid method for DNA strand break detection in individual cells. The % of DNA in the tail reflects the break frequency (Shaposhnikov et al., 2009). Cells were plated into six-well plates (3 × 105 cells/well) at 37 °C in 5% CO2 overnight and then treated with different concentrations of AFB1, ranging from 0 to 80 nM, for 24 h. Additionally, B-2A13 cells were treated with AFB1 at concentrations that ranged from 0 to 80 nM for 6, 12, and 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced DNA damage, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, the comet assay was conducted as described previously (Wu et al., 2012). Detection of AFB1-DNA adducts, 8-OHdG, and γH2AX by immunofluorescence. After the cells were incubated in special small laser confocal dishes (3 × 105 cells/well) at 37 °C in 5% CO2 overnight, they were treated with 80 nM AFB1 for 24 h. Additionally, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, γH2AX was detected using an immunofluorescence assay as described previously (Stoimenov et al., 2011). Aflatoxin B1-DNA (AFB1-DNA) adducts and 8-OHdG were detected using a modified immunofluorescence assay as described previously (Gursoy-Yuzugullu et al., 2011). Briefly, after treatment with AFB1, the cells were treated with a buffer that contained 15 mM Na2CO3 and 30 mM NaHCO3 (pH 9.6) at room temperature for 2 h and then were lysed with 100 μg/ml RNAse in Tris buffer (10 mM Trizma Base, 1 mM ethylenediaminetetraacetic acid [EDTA], and 0.4 mol/l NaCl, pH 7.5) at 37 °C for 1 h. After washing with phosphate-buffered saline (PBS), the cells were treated with 10 μg/ml proteinase K at room temperature for 10 min, followed by washing with PBS and 40% ethanol. The cells were then treated with 50 mM NaOH in 40% ethanol for 30 s to denature the DNA and rinsed again with 40% ethanol. After blocking
115
with 5% bovine serum albumin (BSA) for 1 h, the cells were incubated with mouse 6A10 (1:200; Novus Biologicals, NB-600-443H) or goat anti-8-OHdG (1:100; Santa Cruz Biotechnology, SC-130085) monoclonal antibody at 4 °C overnight. The cells were then incubated in Cy3-labeled goat anti-mouse immunoglobulin G (IgG) (1:1000; Beyotime, P0193) or Cy3-labeled donkey anti-goat IgG secondary antibodies (1:1000; Beyotime, P0173) for 30 min. Images were obtained with a Zeiss LSM 710 inverted confocal microscope. Focus maximum projection images were acquired from optical sections 0.50 mm apart with a section thickness of 1.0 mm. The images were processed using Adobe Photoshop. The fluorescence intensity was semi-quantitatively determined using ZEN2008 software (Zeiss, Germany). Cell cycle analysis by flow cytometry. Monoclonal B-2A13, B-1A2, B-2A6, and B-vector cells were prepared and cultured according to our previous study (Yang et al., 2012). For cell cycle analysis, the cells were seeded in a 10 cm dish, incubated overnight, and treated with 80 nM AFB1 for 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced cell cycle changes, B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. After treatment, the cell cycle was analyzed using flow cytometry assay as described previously (Funamizu et al., 2012). Detection of proteins related to DNA damage and repair. B-2A13 cells were plated into 10 cm plates (1 × 106 cells/well) overnight and then treated with AFB1 at different concentrations, ranging from 0 to 80 nM, for 24 h. To investigate the effects of nicotine and 8-MOP on AFB1-induced changes in proteins related to DNA damage and repair, the cells were treated with 80 nM AFB1 alone or 80 nM AFB1 combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The expression of proteins related to DNA damage and repair was 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 BRCA1, p-BRCA1 (ser1524), ATM, p-ATM (ser1981), ATR, p-ATR (ser428), Chk2, p-Chk2 (thr68), p53, p-p53 (ser15), Mre11, p-Mre11, Ku-80, XLF, Rad50 and γH2AX (ser139), the protein immune complexes were detected using an electrochemiluminescence assay kit and exposed to Kodak X-Omat film. 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). All other antibodies were purchased from Cell Signaling Technology (Danvers, MA). Statistical analysis. The means and standard deviations (SDs) were calculated for all of the investigated parameters, and all of the data are expressed as mean ± 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-NewmanKeuls test. Values of p b 0.05 were considered statistically significant. Results Effects of CYP2A13 on AFB1-induced DNA damage in BEAS-2B cells The single-cell gel electrophoresis (comet) assay was used to evaluate AFB1-induced DNA damage in BEAS-2B cells. As shown in Fig. 1A, DNA damage in B-2A13 cells dose-dependently increased after 24 h of AFB1 treatment. Compared with vehicle control, 10 nM AFB1 significantly induced DNA damage in B-2A13 cells (p b 0.001). DNA damage in B-2A13 cells was much more evident than in B-1A2 cells, whereas no differences between treatment and control were observed in B-2A6 and B-vector cells (Fig. 1A). Additionally, DNA damage in B-2A13 cells
116
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 1. Effect of CYP2A13 on AFB1-induced DNA damage in BEAS-2B cells. DNA damage was detected using the comet assay with a fluorescence microscope. (A) Cells (B-Vector, B-1A2 and B-2A13) were treated with 5–80 nM AFB1 for 24 h. (B) B-2A13 cells were treated with 5–80 nM AFB1 for 6, 12, or 24 h. (C) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The olive tail moment, indicating DNA damage, is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with 80 nM AFB1 alone.
also time-dependently increased with AFB1 treatment. Treatment with 80 nM AFB1 for 6 h or 40 nM AFB1 for 12 h significantly increased DNA damage compared with vehicle control (p b 0.001; Fig. 1B). Similar to our previous study (Yang et al., 2012), nicotine and 8-MOP were used to explore the role of CYP2A13 in AFB1-induced DNA damage. Compared with AFB1 alone, both 100 μM nicotine and 1 μM 8-MOP significantly inhibited AFB1-induced DNA damage (p b 0.001; Fig. 1C). Effects of CYP2A13 in AFB1-induced double-strand DNA breaks in BEAS-2B cells γH2AX, a hallmark of double-strand DNA breaks (DSBs), was detected to identify the type of DNA damage. Compared with vehicle control, 80 nM AFB1 induced greater formation of γH2AX in the nuclei of B-2A13 cells (p b 0.001), whereas no significant changes in γH2AX were found in either B-1A2 or B-2A6 cells (Figs. 2A and B). AFB1 dose-dependently increased γH2AX expression in B-2A13 cells, and nicotine and 8-MOP inhibited the protein expression (Figs. 2C and D). Immunofluorescence assay was used to confirm the effect. Compared with 80 nM AFB1 alone, both 100 μM nicotine and 1 μM 8-MOP obviously inhibited the AFB1-induced formation of γH2AX in the nuclei of B-2A13 cells (p b 0.001; Figs. 2E and F).
Effects of CYP2A13 in AFB1-induced cell cycle changes in BEAS-2B cells As shown in Fig. 3, compared with the vehicle control and B-vector cells, treatment with 80 nM AFB1 for 24 h significantly increased the percentage of cells in the S-phase of the cell cycle in B-2A13 cells (58.4% vs. 34.7%) and B-1A2 cells (46.2% vs. 34.7%; p b 0.001), respectively. The results indicated that AFB1 induced a delay through the S-phase and in the transition from the S- to G2/M-phases in B-2A13 cells. As expected, 100 μM nicotine and 1 μM 8-MOP markedly reversed AFB1-induced cell cycle change (p b 0.001). Effects of CYP2A13 on the formation of AFB1-DNA adducts and 8-OHdG in BEAS-2B cells The formation of the imidazole ring-opened persistent form of major N7-guanine adducts of AFB1 was found in the nuclei of B-2A13 and B-1A2 cells. Treatment with 80 nM AFB1 induced greater formation of AFB1-DNA adducts compared with vehicle control and B-vector cells (p b 0.001). Compared with B-1A2 cells, B-2A13 cells showed much more formation of AFB1-DNA adducts, and the relative fluorescence intensity was approximately nine-times greater than B-1A2 cells (311 vs. 36; p b 0.001; Figs. 4A and B). As predicted, either 100 μM nicotine or
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
117
Fig. 2. Effect of CYP2A13 on AFB1-induced γH2AX expression in BEAS-2B cells. The type of DNA damage was identified by the detection of γH2AX expression using an immunofluorescence assay with a laser scanning confocal microscope. (A–B) Cells were treated with 80 nM AFB1 for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The relative fluorescence intensity was quantified using ZEN 2008 software. (C–D) Cell lysates (100 μg) from B-2A13 cells that were treated with 5–80 nM AFB1 for 24 h (C) or treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h (D) were prepared to determine γH2AX expression. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference. (E–F) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was quantified using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
1 μM 8-MOP significantly reversed the AFB1-induced formation of AFB1-DNA adducts in B-2A13 cells (Figs. 4C and D). Similarly, 8-OHdG was only found in the nuclei of B-2A13 and B-1A2 cells after 80 nM AFB1 treatment (Fig. 5A). Compared with B-1A2 cells, B-2A13 cells showed more 8-OHdG formation, and the relative fluorescence intensity was approximately three-times greater than B-1A2 cells (160 vs. 55; p b 0.001; Fig. 5B). Both 100 μM nicotine and 1 μM 8-MOP markedly reversed the AFB1-induced formation of 8-OHdG (Figs. 5C and D).
p-BRCA1, p-Chk2, and p-p53, which was consistent with their effects on AFB1-induced cell cycle changes and DNA damage, while the expression of ATM, ATR, BRCA1, Chk2 and p53 did not change after AFB1 treatment (Fig. S1). Besides, no significant effect of nicotine or 8-MOP was found on the changes in DSB repair-related proteins, such as Ku-80, XLF, Mre11, p-Mre11, and Rad50 (Fig. 6B). The expressions of CYP2A13 and GFP were similar among the B-2A13 cells treated with DMSO, nicotine or 8-MOP. Discussion
Effects of CYP2A13 on AFB1-induced expression of proteins related to DNA damage and repair in BEAS-2B cells As shown in Fig. 6A, the expression of p-ATM, p-ATR, p-BRCA1, p-Chk2, and p-p53 in B-2A13 cells significantly increased as the concentration of AFB1 increased. However, no significant changes were found in the proteins related to DSB repair, such as Ku-80, XLF, Mre11, p-Mre11, and Rad50. As expected, 100 μM nicotine and 1 μM 8-MOP significantly inhibited the AFB1-induced expression of p-ATM, p-ATR,
In the present study, we demonstrated the role of CYP2A13 in the AFB1-induced formation of AFB1-DNA adducts, DNA damage, and subsequent molecular DNA damage and repair responses using monoclonal BEAS-2B cells that stably expressed CYP2A13 (B-2A13). We also confirmed the effects using nicotine (a substrate of CYP2A13) and 8-MOP (an inhibitor/inactivator of cytochrome P450 enzymes) (von Weymarn et al., 2005, 2006). To facilitate the study, we used CYP2A6 and CYP1A2 as references. With 93.5% shared amino acid sequence identity (Smith
118
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 3. Effect of CYP2A13 on AFB1-induced cell cycle changes in BEAS-2B cells. (A) Cells (B-Vector, B-1A2 and B-2A13) were treated with 80 nM AFB1 or B-2A13 cells were treated with 80 nM AFB1 plus 100 μM nicotine or 1 μM 8-MOP for 24 h. The cells were then subjected to Propidium Iodide to detect the cell cycle using a flow cytometry assay. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. (B) The percentage of cells in the S-phase is expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
et al., 2007), CYP2A13 is an efficient enzyme in the metabolic activation of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with substrate specificity similar to CYP2A6 (Bao et al., 2005; Su et al., 2000). However, they have large differences in their ability to metabolize xenobiotic/endogenous compounds. CYP2A13 is much more active than CYP2A6 in the metabolic activation of AFB1 (He et al., 2006). CYP1A2 has been shown to be the principle enzyme responsible for AFB1 activation (Gallagher et al., 1996), but it is mainly expressed in the human liver (Pelkonen and Raunio, 1997) rather than in the human respiratory system (Ding and Kaminsky, 2003). As predicted in our previous study (Yang et al., 2012), B-2A13 cells were much more sensitive to AFB1-induced DNA damage, indicating that CYP2A13 was a more efficient metabolic enzyme of AFB1 and might play an important role in airborne AFB1 pollution and human respiratory disease, especially cellular genetic injury-induced lung cancer. AFB1 can generally be metabolized by the CYP2A13 to form AFB1-8,9-epoxide (He et al., 2006), which subsequently binds to DNA to form AFB1-N7-Gua adducts and later AFB1-FAPY adducts, which have been recently used as carcinogenic biomarkers (Carvajal et al., 2012; Long et al., 2010). In the present study, AFB1 induced more AFB1-DNA adducts in B-2A13 cells than in B-1A2 cells, with similar AFB1-DNA adduct formation in B-2A6 cells, B-vector cells, and
vehicle control. Adduct formation was inhibited by nicotine and 8-MOP. 8-OHdG is often used as a marker of oxidative DNA damage and formed by the reaction of hydroxyl radicals (·OH) with guanine residues in DNA (Shen et al., 1995). Previous studies demonstrated elevations of 8-OHdG in AFB1-treated mouse lung cells and human biological samples exposed to AFB1 (Guindon et al., 2007; Peng et al., 2007). Consistent with AFB1-DNA adducts, AFB1 induced much more expression of 8-OHdG, and nicotine and 8-MOP could inhibit 8-OHdG in B-2A13 cells. These results indicate that CYP2A13 might mediate AFB1-DNA adduct and 8-OHdG formation and showed more efficient activation in AFB1 metabolism to AFB1-epoxides. The delayed and defective DNA damage response to AFB1 could be related to the type of DNA and protein adducts that it forms in exposed cells (Shen et al., 1995; Wang and Groopman, 1999). The accumulation of AFB1-DNA adducts can induce DNA damage. Consistent with the AFB1-DNA adduct and 8-OHdG results, AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells, which were much more sensitive than B-1A2 and B-2A6 cells. Nicotine and 8-MOP inhibited AFB1-induced DNA damage in B-2A13 cells. Typically, DSBs and stalled replication forks cause the phosphorylation of neighboring histone 2AX at Ser139 (H2AX for the unphosphorylated form and γH2AX for the phosphorylated form) via ataxia telangiectasia mutated
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
119
Fig. 4. Effect of CYP2A13 on AFB1-induced DNA adduct formation in BEAS-2B cells. (A–B) Cells were treated with 80 nM AFB1 for 24 h and then subjected to the immunofluorescence assay to detect AFB1-DNA adduct formation using a laser scanning confocal microscope. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The relative fluorescence intensity was calculated using ZEN 2008 software. (C–D) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was calculated using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
(ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively (Roos and Kaina, 2012). ATM is the primary kinase that phosphorylates H2AX at DSBs although ATR and DNA-dependent protein kinase (Mah et al., 2010). γH2AX is usually used as a hallmark of DSBs. Our results showed greater expression of γH2AX in B-2A13 cells, which could be inhibited by nicotine and 8-MOP. The present data suggested that CYP2A13 efficiently metabolized AFB1 to generate active epoxides and subsequently form AFB1-DNA adducts, 8-OHdG, and γH2AX, resulting in greater acute damage. Various environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that subsequently activate DNA damage checkpoint signaling pathways in human cells (Kemp et al., 2011). Two of the major regulators of the DNA damage response are the phosphoinositide 3-kinase-related protein kinases ATM and ATR, which respond to different types of DNA damage. ATM is mainly activated by DSBs, whereas ATR is activated at structures that contain single-strand DNA (ssDNA), both of which can be activated by DSBs. Resection of the DSB end leads to a large single-stranded region (Jackson and Bartek, 2009). Activated ATM and ATR phosphorylate their downstream targets, including checkpoint kinase 2 (Chk2), which subsequently activates breast cancer susceptibility gene 1 (BRCA1) and initiates DNA repair. In the present study, ATM, ATR, Chk2, and BRCA1 were upregulated by AFB1 and inhibited by nicotine and 8-MOP. However, DNA repair-related proteins, such as Ku-80, XLF, Mre11, Rad50, and Rad52, were unaffected, suggesting that CYP2A13 mediated more DNA damage than repair with AFB1 treatment. The most deleterious DNA lesions are DSBs. If left unrepaired, they may have severe consequences for cell survival and lead to chromosome aberrations, genomic instability, and cell death. To minimize the detrimental effects of DNA damage on genome stability and cell viability, signal transduction pathways, termed DNA damage checkpoints, delay or prevent cell cycle
progression to allow time for DNA repair (Sancar et al., 2004), which was activated by p53. Both kinases phosphorylate p53 at Ser15 and other sites. The stabilization of p53 is also influenced by Chk2, which is phosphorylated by ATM (Cimprich and Cortez, 2008; Roos and Kaina, 2012). Previous studies showed that AFB1 significantly increased the S-phase cell population in SK-N-MC cells and J774A.1 murine macrophages (Bianco et al., 2012; Ricordy et al., 2002), supporting our results that AFB1 phosphorylated p53 and subsequently induced S-phase arrest in B-2A13 cells. In conclusion, the present study demonstrated that CYP2A13 efficiently contributed to the metabolic activation to AFB1. CYP2A13 played a critical role in the AFB1-induced formation of AFB1-DNA adducts, DNA damage, of DSBs and subsequently activated the ATM/ATR, Chk2, and p53 signaling pathways. Similar to our previous study, because of the high metabolic activity of CYP2A13, low concentrations of AFB1 (i.e., 5 and 10 nM) induced DNA damage in human bronchial epithelial cells. CYP2A13, mainly expressed in the human respiratory system, might be the primary metabolic enzyme involved in AFB1 metabolism in situ in lung epithelial cells and be an important link between environmental airborne AFB1 contaminants and human respiratory disorders, especially carcinoma of the lung. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2013.04.005.
Funding This work was supported by the National Natural Science Foundation of China (30972508, 30771782), the National 973 program (2009CB941701), the Doctoral Fund of Ministry of Education of China (20093234110002), the Six talents peak project of Jiangsu province
120
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121
Fig. 5. Effect of CYP2A13 on AFB1-induced 8-OHdG formation in BEAS-2B cells. (A–B) Cells were treated with 80 nM AFB1 for 24 h and then subjected to the immunofluorescence assay to detect 8-OHdG expression using a laser scanning confocal microscope. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. The fluorescence intensity was calculated using ZEN 2008 software. (C–D) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. The relative fluorescence intensity was calculated using ZEN 2008 software. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. ***p b 0.001, compared with cells treated with DMSO; ###p b 0.001, compared with cells treated with AFB1 alone.
(DG216D5047), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China. Conflict of interest statement The authors declare that they have no competing financial interests. References
Fig. 6. Effects of CYP2A13 on the expression of proteins related to DNA damage and repair in BEAS-2B cells. (A) B-2A13 cells were treated with 5–80 nM AFB1 for 24 h. (B) B-2A13 cells were treated with 80 nM AFB1 alone or combined with 100 μM nicotine or 1 μM 8-MOP for 24 h. Dimethylsulfoxide (DMSO; 0.02%) was used as a vehicle control. Cell lysates (100 μg) were prepared to determine protein expression using an immunoblotting assay with antibodies specific for p-ATM, p-ATR, p-Chk2, p-BRCA1, p-p53, Ku-80, XLF, Mre11, p-Mre11, and Rad50. DMSO, nicotine or 8-MOP did not change the expression of CYP2A13 and GFP. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference.
Bao, Z., He, X.Y., Ding, X., Prabhu, S., Hong, J.Y., 2005. Metabolism of nicotine and cotinine by human cytochrome P450 2A13. Drug Metab. Dispos. 33, 258–261. Bedard, L.L., Massey, T.E., 2006. Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241, 174–183. Besaratinia, 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). Bianco, G., Russo, R., Marzocco, S., Velotto, S., Autore, G., Severino, L., 2012. Modulation of macrophage activity by aflatoxins B1 and B2 and their metabolites aflatoxins M1 and M2. Toxicon 59, 644–650. Brown, K.L., Voehler, M.W., Magee, S.M., Harris, C.M., Harris, T.M., Stone, M.P., 2009. Structural perturbations induced by the alpha-anomer of the aflatoxin B(1) formamidopyrimidine adduct in duplex and single-strand DNA. J. Am. Chem. Soc. 131, 16096–16107. Carvajal, M., Berumen, J., Guardado-Estrada, M., 2012. The presence of aflatoxin B(1)FAPY adduct and human papilloma virus in cervical smears from cancer patients in Mexico. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29, 258–268. Cimprich, K.A., Cortez, D., 2008. ATR: an essential regulator of genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627. de Groot, P., Munden, R.F., 2012. Lung cancer epidemiology, risk factors, and prevention. Radiol. Clin. North Am. 50, 863–876. 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. 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.
X. Yang et al. / Toxicology and Applied Pharmacology 270 (2013) 114–121 Funamizu, N., Lacy, C.R., Fujita, K., Furukawa, K., Misawa, T., Yanaga, K., Manome, Y., 2012. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. PLoS One 7, e37424. Gallagher, E.P., Kunze, K.L., Stapleton, P.L., Eaton, D.L., 1996. The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4. Toxicol. Appl. Pharmacol. 141, 595–606. Guindon, K.A., Bedard, L.L., Massey, T.E., 2007. Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1). Toxicol. Sci. 98, 57–62. 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. Jackson, S.P., Bartek, J., 2009. The DNA-damage response in human biology and disease. Nature 461, 1071–1078. Kelly, J.D., Eaton, D.L, Guengerich, F.P., Coulombe Jr., R.A., 1997. Aflatoxin B1 activation in human lung. Toxicol. Appl. Pharmacol. 144, 88–95. Kemp, M.G., Lindsey-Boltz, L.A., Sancar, A., 2011. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA. J. Biol. Chem. 286, 19237–19246. 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), S28–S48. Liao, W., McNutt, M.A., Zhu, W.G., 2009. The comet assay: a sensitive method for detecting DNA damage in individual cells. Methods 48, 46–53. 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 B1-related hepatocellular carcinoma in the Guangxi population. Hepatology 52, 1301–1309. Ma, J., Lu, H.Y., Xia, Y.K., Dong, H.B., Gu, A.H., Li, Z.Y., Li, Z., Chen, A.M., Wang, X.R., Wang, S.L., 2010. BCL2 Ala43Thr Is a functional variant associated with protection against azoospermia in a Han-Chinese population. Biol. Reprod. 83, 656–662. Mah, L.J., El-Osta, A., Karagiannis, T.C., 2010. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24, 679–686. Massey, T.E., Smith, G.B., Tam, A.S., 2000. Mechanisms of aflatoxin B1 lung tumorigenesis. Exp. Lung Res. 26, 673–683. National Toxicology Program., 2004. NTP 11th Report on Carcinogens. Rep. Carcinog. 11, 1–A32. Pelkonen, O., Raunio, H., 1997. Metabolic activation of toxins: tissue-specific expression and metabolism in target organs. Environ. Health Perspect. 105 (Suppl. 4), 767–774. Peng, T., Li, L.Q., Peng, M.H., Liu, Z.M., Liu, T.W., Guo, Y., Xiao, K.Y., Qin, Z., Ye, X.P., Mo, X.S., Yan, L.N., Lee, B.L., Shen, H.M., Tamae, K., Wang, L.W., Wang, Q., Khan, K.M., Wang, K.B., Liang, R.X., Wei, Z.L., Kasai, H., Ong, C.N., Santella, R.M., 2007. Evaluation of oxidative stress in a group of adolescents exposed to a high level of aflatoxin B1—a multi-center and multi-biomarker study. Carcinogenesis 28, 2347–2354.
121
Ricordy, R., Gensabella, G., Cacci, E., Augusti-Tocco, G., 2002. Impairment of cell cycle progression by aflatoxin B1 in human cell lines. Mutagenesis 17, 241–249. Roos, W.P., Kaina, B., 2012. DNA damage-induced apoptosis: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. http:// dx.doi.org/10.1016/j.canlet.2012.01.007. Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K., Linn, S., 2004. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85. Shaposhnikov, S., Frengen, E., Collins, A.R., 2009. Increasing the resolution of the comet assay using fluorescent in situ hybridization—a review. Mutagenesis 24, 383–389. Shen, H.M., Ong, C.N., Lee, B.L., Shi, C.Y., 1995. Aflatoxin B1-induced 8hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16, 419–422. Smith, B.D., Sanders, J.L., Porubsky, P.R., Lushington, G.H., Stout, C.D., Scott, E.E., 2007. Structure of the human lung cytochrome P450 2A13. J. Biol. Chem. 282, 17306–17313. Sorenson, W.G., Simpson, J.P., Peach III, M.J., Thedell, T.D., Olenchock, S.A., 1981. Aflatoxin in respirable corn dust particles. J. Toxicol. Environ. Health 7, 669–672. Stoimenov, I., Schultz, N., Gottipati, P., Helleday, T., 2011. Transcription inhibition by DRB potentiates recombinational repair of UV lesions in mammalian cells. PLoS One 6, e19492. Su, T., Bao, Z., Zhang, Q.Y., Smith, T.J., Hong, J.Y., Ding, X., 2000. Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 60, 5074–5079. Traverso, A., Bassoli, V., Cioe, A., Anselmo, S., Ferro, M., 2010. Assessment of aflatoxin exposure of laboratory worker during food contamination analyses. Assessment of the procedures adopted by an A.R.P.A.L. laboratory (Liguria Region Environmental Protection Agency). Med. Lav. 101, 375–380. von Weymarn, L.B., Zhang, Q.Y., Ding, X., Hollenberg, P.F., 2005. Effects of 8-methoxypsoralen on cytochrome P450 2A13. Carcinogenesis 26, 621–629. von Weymarn, L.B., Brown, K.M., Murphy, S.E., 2006. Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism. J. Pharmacol. Exp. Ther. 316, 295–303. Wang, J.S., Groopman, J.D., 1999. DNA damage by mycotoxins. Mutat. Res. 424, 167–181. Wang, Y., Chai, T., Lu, G., Quan, C., Duan, H., Yao, M., Zucker, B.A., Schlenker, G., 2008. Simultaneous detection of airborne aflatoxin, ochratoxin and zearalenone in a poultry house by immunoaffinity clean-up and high-performance liquid chromatography. Environ. Res. 107, 139–144. Wu, Y., Qi, X., Gong, L., Xing, G., Chen, M., Miao, L., Yao, J., Suzuki, T., Furihata, C., Luan, Y., Ren, J., 2012. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation. PLoS One 7, e35010. Yang, X.J., Lu, H.Y., Li, Z.Y., Bian, Q., Qiu, L.L., Li, Z., Liu, Q., Li, J., Wang, X., Wang, S.L., 2012. Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells. Toxicology 300, 138–148.