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Arsenic inhibited cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation
Toxicology 424 (2019) 152225
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Arsenic inhibited cholesterol efflux of THP-1 macrophages via ROSmediated ABCA1 hypermethylation
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Yang Song, Tong Zhou, Yanqiu Zong, Bingyan Gu, Xiaohua Tan, Lei Yang Medical college, Hangzhou Normal University, No.16 Xuelin Road, Xiasha Higher Education Park, Hangzhou, China
A R T I C LE I N FO
A B S T R A C T
Keywords: ABCA1 Arsenic DNMT1 DNA methylation ROS THP-1 macrophages
Growing evidences indicate that epigenetic modification involves in the mechanisms of atherosclerosis, which intersects with oxidative stress pathway. Arsenic is an important environmental contaminant and has been linked to atherosclerosis. However, the exact mechanism is not well understood. In the present study, we analyzed the effect of arsenic on oxidative stress, ABCA1 promoter methylation and cholesterol efflux of THP-1 macrophages. Results showed that arsenic could induce ROS-mediated DNA methyltransferase 1 (DNMT1) transcription and activity up-regulation, causing ABCA1 promoter to be hypermethylated with repressed expression. In addition, arsenic depleted the methyl donor S-adenosylmethionine (SAM) and induced global DNA hypomethylation. Arsenic inhibited cholesterol efflux of THP-1 macrophages, which could be attenuated after pretreatment with NAC or DNMT inhibitor 5-Aza-2′-deoxycytidine, but not with SAM. All of the findings suggest that arsenic inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.
1. Introduction Arsenic, the number one hazardous chemical (ATSDR, 1997), is a metalloid found naturally in soil, water and air. More than 200 million people worldwide were exposed to arsenic via contaminated drinking water (Naujokas et al., 2013). Even in the U.S., approximately 13 million Americans were under similar status of arsenic exposure (National Primary Drinking Water Regulations, 2016). Exceptionally high levels of arsenic could be detected in drinking water in Bangladesh, Taiwan, India, Chile and Argentina (Argos et al., 2010; Nordstrom, 2002). Arsenic exposure was associated with adverse effects on cardiovascular health, including atherosclerosis, hypertension, ischemic heart disease and arteriosclerosis (Moon et al., 2017). Animal models showed that arsenic could accumulate in the vessel wall and induce atherosclerotic lesion formation in the aorta of Apo E knockout mice (Srivastava et al., 2009). However, the exact mechanism is not well elucidated. Mounting studies show that epigenetic modification plays a critical role in the atherosclerosis. DNA methylation is a major reversible epigenetic mechanism by which methyl group (−CH3) is added to the 5carbon position of DNA base cytosine. Aberrant DNA methylation would occur in both global and specific gene promoter, causing genomic instability and the transcription of pro-atherogenic genes (Hai and Zuo, 2016; Muka et al., 2016). ATP-binding cassette transporter A1
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(ABCA1) belongs to the family of ATP-binding cassette transporters, which could transport molecular across the cellular membrane. ABCA1 could be secreted in macrophages and transfers the intracellular cholesterol and phospholipids to the outside of the cell. These chemicals would then combine with apolipoprotein A-I (apo A-I) to form nascent high-density lipoprotein (HDL), which plays a critical role in maintaining normal levels of cholesterol and cardiovascular health (Takata et al., 2005). With numerous CpG islands (CGI) in the promoter, ABCA1 could be hyper-methylated and transcripted depressed, causing plasma HDL-C decrease, foam cell formation and coronary artery disease (Lv et al., 2016; Guay et al., 2012). Ma et al (2016) indicated that ABCA1 methylation could serve as a valuable biomarker for the early detection of atherosclerosis. Although the exact mechanism of aberrant DNA methylation is somewhat controversial, oxidative stress could intersect with DNA methylation, involving in the mechanisms of atherosclerosis (Kim et al., 2013). Arsenic is a metalloid which could be metabolized through repeated reduction and oxidative methylation after consumption reduced glutathione and methyl donor S-adenosyl-methionine (SAM) (Vahter, 1999). In our previous studies, results indicated that arsenic could upregulate oxidation-related enzymes like glutathione-S-transferase and thioredoxin reductase, increase DNA methylation transferase and alter ABCA1 transcription (Tan et al., 2014). We proposed that oxidative stress might play a role in the arsenic-mediated aberrant DNA
Corresponding author. E-mail address: [email protected] (L. Yang).
https://doi.org/10.1016/j.tox.2019.05.012 Received 14 December 2018; Received in revised form 15 April 2019; Accepted 27 May 2019 Available online 28 May 2019 0300-483X/ © 2019 Published by Elsevier B.V.
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We optimized the PCR reaction condition as 95℃ for 2 min followed by 40 cycles of 95℃ for 15 s and 60℃ for 30 s. To test the specificity of the PCR products, an additional program was in progress to obtain the dissociation curves (60-95℃ with a heating rate of 0.1℃ per second). As for the close efficiency of three primers, the relative threshold cycle (Ct) was acquired from that of target gene related to β-actin. And relative quantification of target genes was calculated with the formula 2−△△Ct.
methylation and cholesterol efflux inhibition. Hence, we analyzed the ABCA1 promoter methylation with a focus on redox signaling. 2. Materials and methods 2.1. THP-1 macrophages culture Human THP-1 cells were purchased from Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37℃ in humidified atmosphere containing 5% CO2. THP-1 cells were treated with100 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St Louis, USA) for 48 h and then differentiated into macrophages.
2.6. Western blot assay of ABCA1 and enzyme-linked immunoassay-like analysis of DNMT1 activity THP-1 macrophages were exposed to sodium arsenite as previous description. In other experiment, cells were pre-incubated with 3000 μM NAC or 2 μM 5-Aza-2′-deoxycytidine (5-Aza-Dc, SigmaAldrich, St.Louis, MO, USA) for 1 h followed by treatment with 12 μM sodium arsenite for 48 h. THP-1 macrophages were lysed in RIPA buffer, separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane, which was incubated with ABCA1 (1:1000, Abcam) or β-actin (1:2500) primary antibodies at 4℃ overnight followed by secondary antibodies (Amersham Pharacia, Buckinghamshire, UK) captured with ECL Detection Reagent (Pierce Biotechnology Inc., Rockford, IL, USA). DNMT1 activity was detected with the EpiQuik™ DNMT1 assay kit (Epigentek Group, USA). Briefly, we extracted the nuclear protein and incubated with DNMT1 primary antibody and detected through a colorimetrical ELISA-like reaction with the following formula: DNMT1 change %= (treated sample OD-Blank OD)*100% / (control sample OD- Blank OD)
2.2. Cell viability assay THP-1 macrophages were seeded in a flat-bottom 96-well culture plate and exposed to different concentrations of sodium arsenite (0, 4, 8, 12, 16 or 32 μM) for 48 h. In another experimental group, macrophages were pre-treated with 3000 μM N-acetyl-L-cysteine (NAC, Sigma-Aldrich, St. Louis, MO, USA) for 1 h followed by 12 μM sodium arsenite for 48 h. Then we added CCK-8 solution and measured the absorbance at 450 nm with a microplate reader. 2.3. Reactive oxygen species (ROS) determination and Malondialdehyde (MDA) level THP-1 macrophages were treated with various concentrations of sodium arsenite (0, 4, 8 and 12 μM) for 48 h. In another experimental group, macrophages were pre-incubated with 3000 μM NAC for 1 h followed by 12 μM arsenite for 48 h. We dyed the treated THP-1 macrophages with oxidant-sensing fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) and get the intensity of fluorescence to be detected in a minimum of 10,000 cells in FACS Calibur flow cytometer, which is proportional to the quantity of ROS. Treated cells were pelleted and lysed to assay the MDA level according to the protocol of MDA assay kit (Jiancheng Bioengineering, Nanjing, China).
2.7. S-adenosylmethionine (SAM) content and the global DNA methylation THP-1 macrophages were exposed to sodium arsenite as previous description. In another experimental group, we pre-treated the macrophages with 3000 μM NAC or 8 μM SAM for 1 h followed by treatment with 12 μM sodium arsenite for 48 h. We measured cellular S-adenosylmethionine (SAM) content in a competitive immunoassay with a monoclonal anti-SAM antibody according to Frantzen et al. (1998). Global DNA methylation was detected with the Kit of Methyl Flash Global DNA Methylation (5-mC) Quantification. Duplicate methylated DNA controls and 100 ng macrophages DNA were cultured with the primary 5-methy-cytosine (5-mC) and secondary antibody and detected the absorbance at 450 nm wavelength after coloration. Global methylation was calculated as the formula: 5-mC%=(Sample OD − blank OD)*100%/(Slope×2*100).
2.4. ABCA1 methylation of THP-1 macrophages THP-1 macrophages were exposed to sodium arsenite as previous description. Briefly, genomic DNA from THP-1 macrophages was extracted and bisulfite modified with DNA extraction kit (CoWin Bioscience Co., Beijing, China) and EZ DNA Methylation-Gold Kit™ (Zymo research biotechnology company, USA), respectively. Bisulfitemodified DNA was PCR-amplified and detected with the Kit of pyrosequencing assays (Qiagen). The quality control of pyrosequencing was analyzed with Pyromark Q24 Analysis Software.
2.8. Cholesterol efflux rate and oil red O staining THP-1 macrophages were radio-labeled with 1 μCi/ml 3H-cholesterol and exposed to sodium arsenite as previous description. Radioactivity in culture medium and cells was analyzed in a liquid scintillation counter (LSC) and expressed as counts per minute (CPM). The efflux rate was calculated as dividing the CPM in medium by that in medium and cell. THP-1 foam cells were induced fromTHP-1 macrophages after incubation with 50 mg/l ox-LDL (Sigma-Aldrich) and exposed to sodium arsenite. Fixed THP-1 foam cells were stained with 1% oil red O to distinguish lipid in cytoplasm, which was semi-quantitatively analyzed with Image-Pro Plus 6.0 software (Media Cybernetics Co., USA).
2.5. Quantitative real-time PCR of ABCA1 and DNMT1 We extracted total RNA from THP-1 macrophages. Total RNA (1 μg) was reversely transcribed with Oligo (dT) primers and an RT enzyme mix according to the Kit of high Capacity cDNA Reverse Transcription (TIANGEN). With SYBER Green PCR kit, 2 μl cDNA was amplified with7.2 μl RNase-free water, 10 μl SYBR Green PCR Mix and 0.4 μl 10 μM following primers in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, USA). ABCA1 forward primer: 5′-TTCGCTCTGAGATGAGCACCA-3′ ABCA1 reverse primer: 5′-TTTCAAGCGGGCATAGAACCA-3′ DNMT1 forward primer: 5′-TCCTACGCCATGCCCAGTTTG-3′ DNMT1 reverse primer: 5′-GAAGATGGGCGTCTCATCATCG-3′ β-actin forward primer: 5′-GAGCGGGAAATCGTCCGTGACATT-3′ β-actin reverse primer: 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′
2.9. Statistical analysis Data were reported as mean and standard deviation. Statistical analysis was performed by one-way ANOVA with the statistical package of SPSS (version 12.0). And we considered the difference of P < 0.05 significant. 2
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Fig. 1. Effect of NAC on decreased cellular viability induced by arsenic. (A) Effect of arsenic on the viability of THP-1 macrophages. (B) Effect of NAC on decreased cellular viability induced by arsenic. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
3. Results
3.4. Effect of NAC on increased DNMT1 transcription and activity induced by sodium arsenite
3.1. Effect of NAC on decreased cellular viability induced by sodium arsenite
To further study the epigenetic mechanism underlying ABCA1 hypermethylation, we determined the transcription and activity of DNMT1 after sodium arsenite treatment. As expected, sodium arsenite increased DNMT1 both at the mRNA and protein level by qPCR and ELISA, which could be inhibited by NAC pretreatment (Fig. 4).
As shown in Fig.1A, the survival rate of Thp-1 macrophages was 66.9%, 20.7% and 9.2% after treatment with 12, 16 or 32 μM sodium arsenite, respectively. Based on this result, we used 4, 8 and 12 μM sodium arsenite in subsequent experiments. NAC pre-treatment could inhibit the decreased cellular viability induced by 12 μM sodium arsenite (Fig.1B).
3.5. Effect of NAC or 5-Aza-dC on decreased ABCA1 expression induced by sodium arsenite
3.2. Effect of NAC on ROS generation and MDA level induced by sodium arsenite
To reveal the association of ABCA1 hypermethylation with gene expression, we analyzed ABCA1 expression in the mRNA and protein levels. In line with DNA hypermethylation, sodium arsenite decreased ABCA1 mRNA and protein levels in a dose-response manner with the lowest level to about 20% of the control. Either NAC or 5-Aza-dC pretreatment could inhibit the ABCA1 decrease induced by 12 μM sodium arsenite (Fig. 5).
To determine the effect of arsenic exposure on ROS generation, we assessed arsenic-treated cells in ROS generation and MDA level. As shown in Fig. 2, sodium arsenite induced a dose-dependent increase in ROS generation and MDA level, which could be inhibited by NAC pretreatment.
3.6. Effect of NAC on the S-adenosylmethionine depletion and global DNA hypomethylation induced by sodium arsenite
3.3. Effect of NAC on ABCA1 hypermethylation induced by sodium arsenite A total of 22 methylated sites were epigeno-typed in ABCA1 gene promoter with four CpG islands (Fig. 3A). The CpG sites with low variable methylation (< 10.0% or > 90.0%) were excluded. Hence, we analyzed ABCA1-A locus with 8 CpGs located just upstream the first exon. As shown in Fig. 3B, ABCA1 could be significantly hypermethylated at CpG sites 6 and 8 after exposure to 4 or 8 μM sodium arsenite. Additional CpG sites 1 and 2 were observed to be hypermethylated after exposure to 12 μM sodium arsenite. NAC pretreatment could inhibit hypermethylated CpG sites 1, 6 and 8 induced by 12 μM sodium arsenite.
To elucidate whether the observed aberrant DNA methylation was global or locus-specific, we analyzed the content of methyl donor Sadenosylmethionine (SAM) and global DNA methylation. Fig. 6 showed that arsenic could decrease S-adenosylmethionine, which could be restored by NAC pretreatment. In accordance with the SAM depletion, sodium arsenite decreased the global DNA methylation percentage of Thp-1 macrophage from 0.15-0.2% to 0-0.05%, which could be inhibited after pretreatment with NAC or SAM.
Fig. 2. Effect of NAC on ROS generation and MDA level induced by arsenic. (A) Effect of NAC on ROS generation induced by arsenic. (B) Effect of NAC on MDA level induced by arsenic. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 3. Methylation analysis of ABCA1 by pyrosequencing assays. (A) ABCA1 CpG island proximal promoter region. (B) The methylation status of ABCA1-A locus containing 8 CpG sites. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
glutathione and methyl donor S-adenosyl-methionine (SAM) (Vahter, 1999). Decreased reduced glutathione formed oxidative stress in a cell. Numerous studies testified that arsenic could interfere with antioxidant enzymes and produce a high level of ROS (Ellinsworth, 2015; Guvvala et al., 2018). Our previous research showed that arsenic could up-regulate oxidation-related enzymes like glutathione-S-transferase and thioredoxin reductase (Tan et al., 2014). The present study indicated that arsenic could interfere with normal metabolism of oxygen and increase ROS. MDA as a product of lipid peroxidation is accumulated. DNA methylation is an important epigenetic mechanism under the role of DNA methyltransferases (DNMTs). Although the exact mechanism of aberrant DNA methylation is somewhat controversial, ROS production theory is one of the several speculations raised recently. ROS could up-regulate the transcription of DNMT and form a new DNMT-containing complex (Wu and Ni, 2015), causing the specific gene to be hypermethylated. In humans, DNMT3a and DNMT3b are the methyltransferases for de novo DNA methylation during development (Zhang et al., 2018). DNMT1 played the major role in maintaining the methylation pattern in DNA replication. In the present study, we found
3.7. Effect of NAC, 5-Aza-dC or SAM on cholesterol efflux inhibition and lipid enrichment induced by sodium arsenite In accordance with decreased ABCA1 expression, arsenic could inhibit cholesterol efflux of THP-1 macrophage (Fig. 7). Oil red O staining indicated that arsenic could induce lipid accumulation of THP-1 macrophage-derived foam cells (Fig. 8). Pre-treatment with NAC or 5-AzadC could decrease the cholesterol efflux and lipid enrichment induced by 12 μM sodium arsenite. In contrast, SAM pretreatment did not have any effect. 4. Discussion Mounting researches have indicated the role of DNA methylation in atherosclerosis. In the present study, we found that arsenic could inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation. Arsenic is a metalloid which could be metabolized through repeated reduction and oxidative methylation after consumption reduced
Fig. 4. Effect of NAC on increased DNMT1 transcription and activity induced by sodium arsenite. (A) Effect of NAC on increased DNMT1 transcription induced by sodium arsenite. (B) Effect of NAC on increased DNMT1 activity induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 5. Effect of NAC or 5-Aza-dC on decreased ABCA1 gene and protein expression induced by sodium arsenite. (A) Effect of NAC on decreased ABCA1 transcription induced by sodium arsenite. (B) Effect of 5-Aza-dC on decreased ABCA1 transcription induced by 12 μM sodium arsenite. (C) Effect of NAC on decreased ABCA1 expression induced by sodium arsenite. (D) Effect of 5-Aza-dC on decreased ABCA1 expression induced by 12 μM sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
Fig. 7. Effect of NAC, 5-Aza-dC or SAM on cholesterol efflux inhibition of THP1 macrophages induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
Fig. 6. Effect of NAC on SAM depletion and the global DNA hypomethylation induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 8. Effect of NAC, 5-Aza-dC or SAM on lipid enrichment of THP-1 macrophage-derived foam cells induced by sodium arsenite. (A) Lipid enrichment in THP-1 macrophages. (B) Quantification was performed as described in Methods. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
expression was the possible mechanism underlying cholesterol efflux inhibition in macrophages. ABCA1 hypermethylation may result from aberrant global DNA methylation. To find whether the observed DNA hypermethylation was global, genomic 5-methyl cytosine content was analyzed in the present study. In contrast, arsenic induced a ROS-mediated SAM depletion and global hypomethylation. In the one-carbon metabolism, reactive oxygen species (ROS) cause reduced glutathione decrease, which would shunt of homocysteine into the synthesis pathway of GSH rather than methionine or SAM. In addition, ROS can trigger oxidative DNA lesion 8-hydroxy-2′ -deoxyguanosine (8-OHdG), causing global DNA hypomethylation (Ziech et al., 2011). Another DNA oxidation product, 5hydroxymethylcytosine (5hmC), may achieve DNA hypomethylation through active DNA demethylation processes (Ahsan et al., 2014). As for the role of global methylation in atherosclerosis, existing research results were inconsistent with global hypomethylation by Guarrera et al. (2015); Wei et al. (2014) and Ramos et al. (2016), compared with global hypermethylation by Zaina et al. (2014) and Kim et al. (2010). In the above studies, global DNA methylation was determined in different repetitive elements including LINE-1 and Alu in diverse tissues. Epigenetic modifications will differ in different species, tissues or cells. In the present study, methyl donor SAM did not protect THP-1 macrophages from cholesterol efflux inhibition induced by arsenic, indicating
that arsenic could induce oxidative stress, causing DNMT1 to be upregulated and ABCA1 hypermethylated. To find the direct association of aberrant DNA methylation with oxidative stress, we used the antioxidant agent and glutathione inducer N-acetylcysteine (NAC) (Meister and Anderson, 1983), which could antagonize ROS generation, DNMT1 upregulation and ABCA1 hypermethylation induced by 12 μM sodium arsenite. All of the findings indicate that ROS elevation is an early event in arsenic-induced ABCA1 hypermethylation. DNA methylation usually silences transcription. In line with ABCA1 hypermethylation, arsenic could decrease gene and protein expression. Depressed ABCA1 would remove less cholesterol and phospholipids to apolipoprotein A-I (apo A-I), causing less high-density lipoprotein (HDL) to be produced, which is a risk for the atherosclerotic lesion (Takata et al., 2005). ABCA1 deficiency in endothelial cells accelerated atherosclerosis in mice (Westerterp et al., 2016). ABCA1 gene mutation was associated with the occurrence of Tangier disease, coronary heart disease and familial cholesterolemia (Brunham et al., 2015). To find the direct association of DNMT with ABCA1 expression, we used the DNA methyltransferase inhibitor 5-Aza-2-deoxycytidine, which can inhibit the activity of DNA methyltransferase in cells (Goffin and Eisenhauer, 2002). Pretreatment with 5-Aza-2-deoxycytidine would inhibit arsenicinduced ABCA1 depression and cholesterol efflux decrease of THP-1 macrophage. This result may imply that epigenetic silencing of ABCA1 6
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that global DNA methylation might not play a major role. The effect of DNA methylation on gene expression depended on the locus where it occurred (Nelson et al., 2011). Hence, gene-specific methylation may be more informative to study the correlation between DNA methylation, gene expression and health-associated results (Jones, 2012). In conclusion, arsenic could inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.
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Conflict of interest None declared. Acknowledgement This study was funded by National Natural Science Foundation of China (81102161;81573196), the Natural Science Foundation of Zhejiang province (LY14H260004), Public Projects from Science Technology Department of Zhejiang Province (LGD19H260001), Grant from Health Department of Zhejiang Province (201475777), Start-up fund of Hangzhou Normal University (2017QDL004). We are grateful to Yaqian Liu and Yu Zhang for providing us with cell resuscitation. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.tox.2019.05.012. References Ahsan, S., Raabe, E.H., Haffner, M.C., Vaghasia, A., Warren, K.E., Quezado, M., Ballester, L.Y., Nazarian, J., Eberhart, C.G., Rodriguez, F.J., 2014. Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma. Acta Neuropathol. Commun. 2, 59. Argos, M., Kalra, T., Rathouz, P.J., Chen, Y., Pierce, B., Parvez, F., Islam, T., Ahmed, A., Rakibuz-Zaman, M., Hasan, R., Sarwar, G., Slavkovich, V., Geen, A., Graziano, j., Ahsan, H., 2010. Arsenic exposure from drinking water, and all-cause and chronicdisease mortalities in Bangladesh (HEALS): a prospective cohort study. Lancet 376, 252–258. Brunham, L.R., Kang, M.H., Van Karnebeek, C., Sadananda, S.N., Collins, J.A., Zhang, L.H., Sayson, B., Miao, F., Stockler, S., Frohlich, J., Cassiman, D., Rabkin, S., Hayden, M.R., 2015. Clinical, biochemical, and molecular characterization of novel mutations in ABCA1 in families with Tangier disease. JIMD Rep. 18, 51–62. Ellinsworth, D.C., 2015. Arsenic, reactive oxygen, and endothelial dysfunction. J. Pharmacol. Exp. Ther. 353, 458–464. Frantzen, F., Faaren, A.L., Alfheim, I., Nordhei, A.K., 1998. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin. Chem. 44, 311–316. Gad, C., 1997. Agency for toxic substances and disease registry. Asian Am. Pac. Isl. J. Health 5, 121–132. Goffin, J., Eisenhauer, E., 2002. DNA methyltransferase inhibitors-state of the art. Ann. Oncol. 13, 1699–1716. Guarrera, S., Fiorito, G., Onland-Moret, N.C., Russo, A., Agnoli, C., Allione, A., Di Gaetano, C., Mattiello, A., Ricceri, F., Chiodini, P., Polidoro, S., Frasca, G., Verschuren, M.W.M., Boer, J.M.A., Iacoviello, L., van der Schouw, Y.T., Tumino, R., Vineis, P., Krogh, V., Panico, S., Sacerdote, C., Matullo, G., 2015. Gene-specific DNA methylation profiles and LINE-1 hypomethylation are associated with myocardial infarction risk. Clin. Epigenetics 7, 133. Guay, S.P., Brisson, D., Munger, J., Lamarche, B., Gaudet, D., Bouchard, L., 2012. ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia. Epigenetics 7, 464–472. Guvvala, P.R., Ravindra, J.P., Selvaraju, S., Arangasamy, A., Venkata, K.M., 2018. Ellagic
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Arsenic inhibited cholesterol efflux of THP-1 macrophages via ROSmediated ABCA1 hypermethylation
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Yang Song, Tong Zhou, Yanqiu Zong, Bingyan Gu, Xiaohua Tan, Lei Yang Medical college, Hangzhou Normal University, No.16 Xuelin Road, Xiasha Higher Education Park, Hangzhou, China
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A B S T R A C T
Keywords: ABCA1 Arsenic DNMT1 DNA methylation ROS THP-1 macrophages
Growing evidences indicate that epigenetic modification involves in the mechanisms of atherosclerosis, which intersects with oxidative stress pathway. Arsenic is an important environmental contaminant and has been linked to atherosclerosis. However, the exact mechanism is not well understood. In the present study, we analyzed the effect of arsenic on oxidative stress, ABCA1 promoter methylation and cholesterol efflux of THP-1 macrophages. Results showed that arsenic could induce ROS-mediated DNA methyltransferase 1 (DNMT1) transcription and activity up-regulation, causing ABCA1 promoter to be hypermethylated with repressed expression. In addition, arsenic depleted the methyl donor S-adenosylmethionine (SAM) and induced global DNA hypomethylation. Arsenic inhibited cholesterol efflux of THP-1 macrophages, which could be attenuated after pretreatment with NAC or DNMT inhibitor 5-Aza-2′-deoxycytidine, but not with SAM. All of the findings suggest that arsenic inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.
1. Introduction Arsenic, the number one hazardous chemical (ATSDR, 1997), is a metalloid found naturally in soil, water and air. More than 200 million people worldwide were exposed to arsenic via contaminated drinking water (Naujokas et al., 2013). Even in the U.S., approximately 13 million Americans were under similar status of arsenic exposure (National Primary Drinking Water Regulations, 2016). Exceptionally high levels of arsenic could be detected in drinking water in Bangladesh, Taiwan, India, Chile and Argentina (Argos et al., 2010; Nordstrom, 2002). Arsenic exposure was associated with adverse effects on cardiovascular health, including atherosclerosis, hypertension, ischemic heart disease and arteriosclerosis (Moon et al., 2017). Animal models showed that arsenic could accumulate in the vessel wall and induce atherosclerotic lesion formation in the aorta of Apo E knockout mice (Srivastava et al., 2009). However, the exact mechanism is not well elucidated. Mounting studies show that epigenetic modification plays a critical role in the atherosclerosis. DNA methylation is a major reversible epigenetic mechanism by which methyl group (−CH3) is added to the 5carbon position of DNA base cytosine. Aberrant DNA methylation would occur in both global and specific gene promoter, causing genomic instability and the transcription of pro-atherogenic genes (Hai and Zuo, 2016; Muka et al., 2016). ATP-binding cassette transporter A1
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(ABCA1) belongs to the family of ATP-binding cassette transporters, which could transport molecular across the cellular membrane. ABCA1 could be secreted in macrophages and transfers the intracellular cholesterol and phospholipids to the outside of the cell. These chemicals would then combine with apolipoprotein A-I (apo A-I) to form nascent high-density lipoprotein (HDL), which plays a critical role in maintaining normal levels of cholesterol and cardiovascular health (Takata et al., 2005). With numerous CpG islands (CGI) in the promoter, ABCA1 could be hyper-methylated and transcripted depressed, causing plasma HDL-C decrease, foam cell formation and coronary artery disease (Lv et al., 2016; Guay et al., 2012). Ma et al (2016) indicated that ABCA1 methylation could serve as a valuable biomarker for the early detection of atherosclerosis. Although the exact mechanism of aberrant DNA methylation is somewhat controversial, oxidative stress could intersect with DNA methylation, involving in the mechanisms of atherosclerosis (Kim et al., 2013). Arsenic is a metalloid which could be metabolized through repeated reduction and oxidative methylation after consumption reduced glutathione and methyl donor S-adenosyl-methionine (SAM) (Vahter, 1999). In our previous studies, results indicated that arsenic could upregulate oxidation-related enzymes like glutathione-S-transferase and thioredoxin reductase, increase DNA methylation transferase and alter ABCA1 transcription (Tan et al., 2014). We proposed that oxidative stress might play a role in the arsenic-mediated aberrant DNA
Corresponding author. E-mail address: [email protected] (L. Yang).
https://doi.org/10.1016/j.tox.2019.05.012 Received 14 December 2018; Received in revised form 15 April 2019; Accepted 27 May 2019 Available online 28 May 2019 0300-483X/ © 2019 Published by Elsevier B.V.
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We optimized the PCR reaction condition as 95℃ for 2 min followed by 40 cycles of 95℃ for 15 s and 60℃ for 30 s. To test the specificity of the PCR products, an additional program was in progress to obtain the dissociation curves (60-95℃ with a heating rate of 0.1℃ per second). As for the close efficiency of three primers, the relative threshold cycle (Ct) was acquired from that of target gene related to β-actin. And relative quantification of target genes was calculated with the formula 2−△△Ct.
methylation and cholesterol efflux inhibition. Hence, we analyzed the ABCA1 promoter methylation with a focus on redox signaling. 2. Materials and methods 2.1. THP-1 macrophages culture Human THP-1 cells were purchased from Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37℃ in humidified atmosphere containing 5% CO2. THP-1 cells were treated with100 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St Louis, USA) for 48 h and then differentiated into macrophages.
2.6. Western blot assay of ABCA1 and enzyme-linked immunoassay-like analysis of DNMT1 activity THP-1 macrophages were exposed to sodium arsenite as previous description. In other experiment, cells were pre-incubated with 3000 μM NAC or 2 μM 5-Aza-2′-deoxycytidine (5-Aza-Dc, SigmaAldrich, St.Louis, MO, USA) for 1 h followed by treatment with 12 μM sodium arsenite for 48 h. THP-1 macrophages were lysed in RIPA buffer, separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane, which was incubated with ABCA1 (1:1000, Abcam) or β-actin (1:2500) primary antibodies at 4℃ overnight followed by secondary antibodies (Amersham Pharacia, Buckinghamshire, UK) captured with ECL Detection Reagent (Pierce Biotechnology Inc., Rockford, IL, USA). DNMT1 activity was detected with the EpiQuik™ DNMT1 assay kit (Epigentek Group, USA). Briefly, we extracted the nuclear protein and incubated with DNMT1 primary antibody and detected through a colorimetrical ELISA-like reaction with the following formula: DNMT1 change %= (treated sample OD-Blank OD)*100% / (control sample OD- Blank OD)
2.2. Cell viability assay THP-1 macrophages were seeded in a flat-bottom 96-well culture plate and exposed to different concentrations of sodium arsenite (0, 4, 8, 12, 16 or 32 μM) for 48 h. In another experimental group, macrophages were pre-treated with 3000 μM N-acetyl-L-cysteine (NAC, Sigma-Aldrich, St. Louis, MO, USA) for 1 h followed by 12 μM sodium arsenite for 48 h. Then we added CCK-8 solution and measured the absorbance at 450 nm with a microplate reader. 2.3. Reactive oxygen species (ROS) determination and Malondialdehyde (MDA) level THP-1 macrophages were treated with various concentrations of sodium arsenite (0, 4, 8 and 12 μM) for 48 h. In another experimental group, macrophages were pre-incubated with 3000 μM NAC for 1 h followed by 12 μM arsenite for 48 h. We dyed the treated THP-1 macrophages with oxidant-sensing fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) and get the intensity of fluorescence to be detected in a minimum of 10,000 cells in FACS Calibur flow cytometer, which is proportional to the quantity of ROS. Treated cells were pelleted and lysed to assay the MDA level according to the protocol of MDA assay kit (Jiancheng Bioengineering, Nanjing, China).
2.7. S-adenosylmethionine (SAM) content and the global DNA methylation THP-1 macrophages were exposed to sodium arsenite as previous description. In another experimental group, we pre-treated the macrophages with 3000 μM NAC or 8 μM SAM for 1 h followed by treatment with 12 μM sodium arsenite for 48 h. We measured cellular S-adenosylmethionine (SAM) content in a competitive immunoassay with a monoclonal anti-SAM antibody according to Frantzen et al. (1998). Global DNA methylation was detected with the Kit of Methyl Flash Global DNA Methylation (5-mC) Quantification. Duplicate methylated DNA controls and 100 ng macrophages DNA were cultured with the primary 5-methy-cytosine (5-mC) and secondary antibody and detected the absorbance at 450 nm wavelength after coloration. Global methylation was calculated as the formula: 5-mC%=(Sample OD − blank OD)*100%/(Slope×2*100).
2.4. ABCA1 methylation of THP-1 macrophages THP-1 macrophages were exposed to sodium arsenite as previous description. Briefly, genomic DNA from THP-1 macrophages was extracted and bisulfite modified with DNA extraction kit (CoWin Bioscience Co., Beijing, China) and EZ DNA Methylation-Gold Kit™ (Zymo research biotechnology company, USA), respectively. Bisulfitemodified DNA was PCR-amplified and detected with the Kit of pyrosequencing assays (Qiagen). The quality control of pyrosequencing was analyzed with Pyromark Q24 Analysis Software.
2.8. Cholesterol efflux rate and oil red O staining THP-1 macrophages were radio-labeled with 1 μCi/ml 3H-cholesterol and exposed to sodium arsenite as previous description. Radioactivity in culture medium and cells was analyzed in a liquid scintillation counter (LSC) and expressed as counts per minute (CPM). The efflux rate was calculated as dividing the CPM in medium by that in medium and cell. THP-1 foam cells were induced fromTHP-1 macrophages after incubation with 50 mg/l ox-LDL (Sigma-Aldrich) and exposed to sodium arsenite. Fixed THP-1 foam cells were stained with 1% oil red O to distinguish lipid in cytoplasm, which was semi-quantitatively analyzed with Image-Pro Plus 6.0 software (Media Cybernetics Co., USA).
2.5. Quantitative real-time PCR of ABCA1 and DNMT1 We extracted total RNA from THP-1 macrophages. Total RNA (1 μg) was reversely transcribed with Oligo (dT) primers and an RT enzyme mix according to the Kit of high Capacity cDNA Reverse Transcription (TIANGEN). With SYBER Green PCR kit, 2 μl cDNA was amplified with7.2 μl RNase-free water, 10 μl SYBR Green PCR Mix and 0.4 μl 10 μM following primers in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, USA). ABCA1 forward primer: 5′-TTCGCTCTGAGATGAGCACCA-3′ ABCA1 reverse primer: 5′-TTTCAAGCGGGCATAGAACCA-3′ DNMT1 forward primer: 5′-TCCTACGCCATGCCCAGTTTG-3′ DNMT1 reverse primer: 5′-GAAGATGGGCGTCTCATCATCG-3′ β-actin forward primer: 5′-GAGCGGGAAATCGTCCGTGACATT-3′ β-actin reverse primer: 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′
2.9. Statistical analysis Data were reported as mean and standard deviation. Statistical analysis was performed by one-way ANOVA with the statistical package of SPSS (version 12.0). And we considered the difference of P < 0.05 significant. 2
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Fig. 1. Effect of NAC on decreased cellular viability induced by arsenic. (A) Effect of arsenic on the viability of THP-1 macrophages. (B) Effect of NAC on decreased cellular viability induced by arsenic. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
3. Results
3.4. Effect of NAC on increased DNMT1 transcription and activity induced by sodium arsenite
3.1. Effect of NAC on decreased cellular viability induced by sodium arsenite
To further study the epigenetic mechanism underlying ABCA1 hypermethylation, we determined the transcription and activity of DNMT1 after sodium arsenite treatment. As expected, sodium arsenite increased DNMT1 both at the mRNA and protein level by qPCR and ELISA, which could be inhibited by NAC pretreatment (Fig. 4).
As shown in Fig.1A, the survival rate of Thp-1 macrophages was 66.9%, 20.7% and 9.2% after treatment with 12, 16 or 32 μM sodium arsenite, respectively. Based on this result, we used 4, 8 and 12 μM sodium arsenite in subsequent experiments. NAC pre-treatment could inhibit the decreased cellular viability induced by 12 μM sodium arsenite (Fig.1B).
3.5. Effect of NAC or 5-Aza-dC on decreased ABCA1 expression induced by sodium arsenite
3.2. Effect of NAC on ROS generation and MDA level induced by sodium arsenite
To reveal the association of ABCA1 hypermethylation with gene expression, we analyzed ABCA1 expression in the mRNA and protein levels. In line with DNA hypermethylation, sodium arsenite decreased ABCA1 mRNA and protein levels in a dose-response manner with the lowest level to about 20% of the control. Either NAC or 5-Aza-dC pretreatment could inhibit the ABCA1 decrease induced by 12 μM sodium arsenite (Fig. 5).
To determine the effect of arsenic exposure on ROS generation, we assessed arsenic-treated cells in ROS generation and MDA level. As shown in Fig. 2, sodium arsenite induced a dose-dependent increase in ROS generation and MDA level, which could be inhibited by NAC pretreatment.
3.6. Effect of NAC on the S-adenosylmethionine depletion and global DNA hypomethylation induced by sodium arsenite
3.3. Effect of NAC on ABCA1 hypermethylation induced by sodium arsenite A total of 22 methylated sites were epigeno-typed in ABCA1 gene promoter with four CpG islands (Fig. 3A). The CpG sites with low variable methylation (< 10.0% or > 90.0%) were excluded. Hence, we analyzed ABCA1-A locus with 8 CpGs located just upstream the first exon. As shown in Fig. 3B, ABCA1 could be significantly hypermethylated at CpG sites 6 and 8 after exposure to 4 or 8 μM sodium arsenite. Additional CpG sites 1 and 2 were observed to be hypermethylated after exposure to 12 μM sodium arsenite. NAC pretreatment could inhibit hypermethylated CpG sites 1, 6 and 8 induced by 12 μM sodium arsenite.
To elucidate whether the observed aberrant DNA methylation was global or locus-specific, we analyzed the content of methyl donor Sadenosylmethionine (SAM) and global DNA methylation. Fig. 6 showed that arsenic could decrease S-adenosylmethionine, which could be restored by NAC pretreatment. In accordance with the SAM depletion, sodium arsenite decreased the global DNA methylation percentage of Thp-1 macrophage from 0.15-0.2% to 0-0.05%, which could be inhibited after pretreatment with NAC or SAM.
Fig. 2. Effect of NAC on ROS generation and MDA level induced by arsenic. (A) Effect of NAC on ROS generation induced by arsenic. (B) Effect of NAC on MDA level induced by arsenic. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 3. Methylation analysis of ABCA1 by pyrosequencing assays. (A) ABCA1 CpG island proximal promoter region. (B) The methylation status of ABCA1-A locus containing 8 CpG sites. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
glutathione and methyl donor S-adenosyl-methionine (SAM) (Vahter, 1999). Decreased reduced glutathione formed oxidative stress in a cell. Numerous studies testified that arsenic could interfere with antioxidant enzymes and produce a high level of ROS (Ellinsworth, 2015; Guvvala et al., 2018). Our previous research showed that arsenic could up-regulate oxidation-related enzymes like glutathione-S-transferase and thioredoxin reductase (Tan et al., 2014). The present study indicated that arsenic could interfere with normal metabolism of oxygen and increase ROS. MDA as a product of lipid peroxidation is accumulated. DNA methylation is an important epigenetic mechanism under the role of DNA methyltransferases (DNMTs). Although the exact mechanism of aberrant DNA methylation is somewhat controversial, ROS production theory is one of the several speculations raised recently. ROS could up-regulate the transcription of DNMT and form a new DNMT-containing complex (Wu and Ni, 2015), causing the specific gene to be hypermethylated. In humans, DNMT3a and DNMT3b are the methyltransferases for de novo DNA methylation during development (Zhang et al., 2018). DNMT1 played the major role in maintaining the methylation pattern in DNA replication. In the present study, we found
3.7. Effect of NAC, 5-Aza-dC or SAM on cholesterol efflux inhibition and lipid enrichment induced by sodium arsenite In accordance with decreased ABCA1 expression, arsenic could inhibit cholesterol efflux of THP-1 macrophage (Fig. 7). Oil red O staining indicated that arsenic could induce lipid accumulation of THP-1 macrophage-derived foam cells (Fig. 8). Pre-treatment with NAC or 5-AzadC could decrease the cholesterol efflux and lipid enrichment induced by 12 μM sodium arsenite. In contrast, SAM pretreatment did not have any effect. 4. Discussion Mounting researches have indicated the role of DNA methylation in atherosclerosis. In the present study, we found that arsenic could inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation. Arsenic is a metalloid which could be metabolized through repeated reduction and oxidative methylation after consumption reduced
Fig. 4. Effect of NAC on increased DNMT1 transcription and activity induced by sodium arsenite. (A) Effect of NAC on increased DNMT1 transcription induced by sodium arsenite. (B) Effect of NAC on increased DNMT1 activity induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 5. Effect of NAC or 5-Aza-dC on decreased ABCA1 gene and protein expression induced by sodium arsenite. (A) Effect of NAC on decreased ABCA1 transcription induced by sodium arsenite. (B) Effect of 5-Aza-dC on decreased ABCA1 transcription induced by 12 μM sodium arsenite. (C) Effect of NAC on decreased ABCA1 expression induced by sodium arsenite. (D) Effect of 5-Aza-dC on decreased ABCA1 expression induced by 12 μM sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
Fig. 7. Effect of NAC, 5-Aza-dC or SAM on cholesterol efflux inhibition of THP1 macrophages induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
Fig. 6. Effect of NAC on SAM depletion and the global DNA hypomethylation induced by sodium arsenite. Data were representative of three independent experiments and presented as mean ± standard deviation. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
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Fig. 8. Effect of NAC, 5-Aza-dC or SAM on lipid enrichment of THP-1 macrophage-derived foam cells induced by sodium arsenite. (A) Lipid enrichment in THP-1 macrophages. (B) Quantification was performed as described in Methods. (*) and (#) indicated significant difference (P < 0.05) compared with control and 12μM arsenic group, respectively.
expression was the possible mechanism underlying cholesterol efflux inhibition in macrophages. ABCA1 hypermethylation may result from aberrant global DNA methylation. To find whether the observed DNA hypermethylation was global, genomic 5-methyl cytosine content was analyzed in the present study. In contrast, arsenic induced a ROS-mediated SAM depletion and global hypomethylation. In the one-carbon metabolism, reactive oxygen species (ROS) cause reduced glutathione decrease, which would shunt of homocysteine into the synthesis pathway of GSH rather than methionine or SAM. In addition, ROS can trigger oxidative DNA lesion 8-hydroxy-2′ -deoxyguanosine (8-OHdG), causing global DNA hypomethylation (Ziech et al., 2011). Another DNA oxidation product, 5hydroxymethylcytosine (5hmC), may achieve DNA hypomethylation through active DNA demethylation processes (Ahsan et al., 2014). As for the role of global methylation in atherosclerosis, existing research results were inconsistent with global hypomethylation by Guarrera et al. (2015); Wei et al. (2014) and Ramos et al. (2016), compared with global hypermethylation by Zaina et al. (2014) and Kim et al. (2010). In the above studies, global DNA methylation was determined in different repetitive elements including LINE-1 and Alu in diverse tissues. Epigenetic modifications will differ in different species, tissues or cells. In the present study, methyl donor SAM did not protect THP-1 macrophages from cholesterol efflux inhibition induced by arsenic, indicating
that arsenic could induce oxidative stress, causing DNMT1 to be upregulated and ABCA1 hypermethylated. To find the direct association of aberrant DNA methylation with oxidative stress, we used the antioxidant agent and glutathione inducer N-acetylcysteine (NAC) (Meister and Anderson, 1983), which could antagonize ROS generation, DNMT1 upregulation and ABCA1 hypermethylation induced by 12 μM sodium arsenite. All of the findings indicate that ROS elevation is an early event in arsenic-induced ABCA1 hypermethylation. DNA methylation usually silences transcription. In line with ABCA1 hypermethylation, arsenic could decrease gene and protein expression. Depressed ABCA1 would remove less cholesterol and phospholipids to apolipoprotein A-I (apo A-I), causing less high-density lipoprotein (HDL) to be produced, which is a risk for the atherosclerotic lesion (Takata et al., 2005). ABCA1 deficiency in endothelial cells accelerated atherosclerosis in mice (Westerterp et al., 2016). ABCA1 gene mutation was associated with the occurrence of Tangier disease, coronary heart disease and familial cholesterolemia (Brunham et al., 2015). To find the direct association of DNMT with ABCA1 expression, we used the DNA methyltransferase inhibitor 5-Aza-2-deoxycytidine, which can inhibit the activity of DNA methyltransferase in cells (Goffin and Eisenhauer, 2002). Pretreatment with 5-Aza-2-deoxycytidine would inhibit arsenicinduced ABCA1 depression and cholesterol efflux decrease of THP-1 macrophage. This result may imply that epigenetic silencing of ABCA1 6
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that global DNA methylation might not play a major role. The effect of DNA methylation on gene expression depended on the locus where it occurred (Nelson et al., 2011). Hence, gene-specific methylation may be more informative to study the correlation between DNA methylation, gene expression and health-associated results (Jones, 2012). In conclusion, arsenic could inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.
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Conflict of interest None declared. Acknowledgement This study was funded by National Natural Science Foundation of China (81102161;81573196), the Natural Science Foundation of Zhejiang province (LY14H260004), Public Projects from Science Technology Department of Zhejiang Province (LGD19H260001), Grant from Health Department of Zhejiang Province (201475777), Start-up fund of Hangzhou Normal University (2017QDL004). We are grateful to Yaqian Liu and Yu Zhang for providing us with cell resuscitation. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.tox.2019.05.012. References Ahsan, S., Raabe, E.H., Haffner, M.C., Vaghasia, A., Warren, K.E., Quezado, M., Ballester, L.Y., Nazarian, J., Eberhart, C.G., Rodriguez, F.J., 2014. Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma. Acta Neuropathol. Commun. 2, 59. Argos, M., Kalra, T., Rathouz, P.J., Chen, Y., Pierce, B., Parvez, F., Islam, T., Ahmed, A., Rakibuz-Zaman, M., Hasan, R., Sarwar, G., Slavkovich, V., Geen, A., Graziano, j., Ahsan, H., 2010. Arsenic exposure from drinking water, and all-cause and chronicdisease mortalities in Bangladesh (HEALS): a prospective cohort study. Lancet 376, 252–258. Brunham, L.R., Kang, M.H., Van Karnebeek, C., Sadananda, S.N., Collins, J.A., Zhang, L.H., Sayson, B., Miao, F., Stockler, S., Frohlich, J., Cassiman, D., Rabkin, S., Hayden, M.R., 2015. Clinical, biochemical, and molecular characterization of novel mutations in ABCA1 in families with Tangier disease. JIMD Rep. 18, 51–62. Ellinsworth, D.C., 2015. Arsenic, reactive oxygen, and endothelial dysfunction. J. Pharmacol. Exp. Ther. 353, 458–464. Frantzen, F., Faaren, A.L., Alfheim, I., Nordhei, A.K., 1998. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin. Chem. 44, 311–316. Gad, C., 1997. Agency for toxic substances and disease registry. Asian Am. Pac. Isl. J. Health 5, 121–132. Goffin, J., Eisenhauer, E., 2002. DNA methyltransferase inhibitors-state of the art. Ann. Oncol. 13, 1699–1716. Guarrera, S., Fiorito, G., Onland-Moret, N.C., Russo, A., Agnoli, C., Allione, A., Di Gaetano, C., Mattiello, A., Ricceri, F., Chiodini, P., Polidoro, S., Frasca, G., Verschuren, M.W.M., Boer, J.M.A., Iacoviello, L., van der Schouw, Y.T., Tumino, R., Vineis, P., Krogh, V., Panico, S., Sacerdote, C., Matullo, G., 2015. Gene-specific DNA methylation profiles and LINE-1 hypomethylation are associated with myocardial infarction risk. Clin. Epigenetics 7, 133. Guay, S.P., Brisson, D., Munger, J., Lamarche, B., Gaudet, D., Bouchard, L., 2012. ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia. Epigenetics 7, 464–472. Guvvala, P.R., Ravindra, J.P., Selvaraju, S., Arangasamy, A., Venkata, K.M., 2018. Ellagic
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