ASSESSMENT OF TOXIC EFFECTS OF DIBUTYL PHTHALATE ON HUMAN LUNG CELL LINE AND POSSIBLE PROTECTIVE EFFECTS OF ASCORBIC ACID AND N-ACETYLCYSTEINE

Abstract

Endocrine-disrupting chemicals (EDCs) are chemicals that affect production, release, biotransformation or excretion of hormones. Dibutylphthalate (DBP) is a phthalate derivative, which used in many fields in industry. Although there is data on testicular and hepatic toxicity of DBP in the literature, there are few studies on lung toxicity. In this study, we aimed to evaluate the possible cytotoxic and oxidative stress-generating effects of DBP in human non-small cell lung cancer cell line (A549 cells). Inhibitor concentration 30 was determined in A549 cells and the effect of intracellular reactive oxygen species (ROS) generating effect was evaluated. Also, possible lipid peroxidation and protein oxidation caused by DBP were investigated, possible alterations in glutathione (GSH), and total antioxidant capacity (TAOC) levels were identified. In the study, the protective effects of ascorbic acid (Asc) and N-acetyl cysteine (NAC) against the possible toxic effects of DBP were determined. Both NAC and Asc along with DBP application reduced ROS and TAOC levels, decreased protein oxidation, and increased total GSH levels due to decreased oxidative stress. According to our data, one of the underlying toxicity mechanisms of DBP is oxidative stress. In addition, Asc and NAC were determined to be protective against oxidative stress caused by DBP.

Keywords

• Ascorbic acid
• Dibutylphthalate (DBP)
• Human lung cancer cells (A549 cells)
• N-acetylcysteine
• Oxidative stress

DİBUTİL FTALATIN İNSAN AKCİĞER HÜCRE HATTINA OLASI TOKSİK ETKİLERİNİN DEĞERLENDİRİLMESİ VE ASKORBİK ASİT VE N-ASETİLSİSTEİNİN OLASI KORUYUCU ETKİLERİ

Abstract

Endokrin bozucu kimyasallar (EBK’ler), hormonların üretimini, salınımını, biyotransformasyonunu ve/veya atılımını etkileyen sentetik veya doğal kimyasal maddelerdir. Dibutilftalat (DBP) endüstride birçok alanda yaygın olarak kullanımı olan bir ftalat türevidir. Literatürde DBP'nin testiküler ve hepatik toksisitesine dair veriler olmakla birlikte, akciğer toksisitesi üzerinde çok az sayıda çalışma bulunmaktadır. Ayrıca, DBP’nin akciğer toksisite mekanizmaları bilinmemektedir. Bu çalışmada, DBP’nin insan küçük hücreli olmayan akciğer kanseri hücre kültürleri (A549 hücreleri) üzerinde olası sitotoksik ve oksidatif stres oluşturucu etkilerinin değerlendirilmesi amaçlanmıştır. Bu kapsamda, A549 hücrelerinde DBP’nin inhibitör konsantrasyon 30 (IC30) dozu belirlenmiş, IC30 dozunun neden olduğu intraselüler reaktif oksijen türleri (ROS)’u arttırıcı etkisi değerlendirilmiştir. Ayrıca, DBP’nin yol açtığı olası lipit peroksidasyon ve protein oksidasyonu incelenmiş, glutatyon (GSH) ve total antioksidan kapasitede (TAOC) düzeylerinde yol açabileceği olası değişiklikler belirlenmiştir. Çalışmada DBP'nin olası toksik etkilerine karşı askorbik asit (Asc) ve N-asetil sisteinin (NAC) olası koruyucu etkileri incelenmiştir. Hem NAC, hem de Asc’nin DBP uygulamasıyla beraber ROS düzeylerini düşürdüğü, protein oksidasyonunu azalttığı, total GSH düzeylerini yükselttiği ve azalan oksidatif strese bağlı olarak her iki antioksidanın da TAOC düzeylerini düşürdüğü belirlenmiştir. Elde edilen bilgiler doğrultusunda, DBP’nin toksik etki mekanizmasının altında yatan nedenlerden birinin oksidatif stres olduğu anlaşılmıştır. Ayrıca, Asc ve NAC’ın DBP’nin neden olduğu oksidatif strese karşı koruyucu oldukları saptanmıştır.

Keywords

• Dibutilftalat (DBP)
• insan akciğer hücreleri (A549 hücreleri)
• askorbik asit
• N-asetilsistein
• oksidatif stres
• sitotoksisite
• reaktif oksijen türleri (ROS)
• Ascorbic acid
• Dibutylphthalate (DBP)
• Human lung cancer cells (A549 cells)
• N-acetylcysteine
• Oxidative stress

References

1. Kabir ER, Rahman MS, Rahman I. A review on endocrin disruptors and their possible impacts on human health. Environmental Toxicology and Pharmacology. 2015;40(1):241-258.
2. Diamanti-Kandarakis E, Bourguignon JP, Guidice LC, et al. Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement. Endocrine Reviews. 2009;30(4):293-342.
3. Braun JM. Early-life exposure to EDCs: role in childhood obesity and neurodevelopment. National Review Endocrinology. 2017;13(3):161-173.
4. Sifakis S, Androutsopoulos VP, Tsatsakis AM, et al. Human exposure to endocrine disrupting chemicals: effects on the male and female reproductive systems. Environmental Toxicology and Pharmacology. 2017;51:56-70.
5. Durmaz E, Erkekoğlu P, Asci A, et al. Urinary phthalate metabolite concentrations in girls with premature thelarche. Environmental Toxicology and Pharmacology. 2018;59:172-181.
6. Ait BY, Shibata E, Saito I, et al. Exposure to house dust phthalates in relation to asthma and allergies in both children and adults. Science of the Total Environment. 2014;485:153-163.
7. Li MC, Chen CH, Guo YL. Phthalate esters and childhood asthma: A systematic review and congener-specific meta-analysis. Environmental Pollutıon. 2017;229:655-660.
8. Upson K, Sathyanarayana S, Roos AJ, et al. Phthalates and risk of endometriosis. Environmental Research. 2013;126:91-97.

9. Austrilian Department of Public Health. Dibutyl phthalate. 2017 Erişim Linki: https://www.nicnas.gov.au/chemicalinformation/factsheets/chemical-name/dibutyl-phthalate-dbp. [Erişim Tarihi: 04.10.2019].
10. Broe A, Ennis ZN, Pottegard A, et al. Population Exposure to Phthalate-containing Drugs. Basic & Clinical Pharmacology & Toxicology. 2017;121(3):153-158.
11. U.S. Environmental Protection Agency Chemical Assessment Summary National Center for Environmental Assessment, Dibutyl phthalate; CASRN 84-74-2. 1999. Erişim Linki: https://cfpub.epa.Gov/ncea/iris/iris_documents/documents/subst/0038_summary.pdf. [Erişim Tarihi: 12.10.2019].
12. European Chemicals Agency (ECHA), Review Of New Available Information for dibutyl phthalate (DBP) . 2010. Erişim Linki: https://echa.europa.eu/documents/10162/13641/ dbp_echa_review_report_2010_6_en.pdf/64b1253c-9e03-48dd-8546-cb2bee3c3646. [Erişim Tarihi: 02.11.2019]
13. Perez C, Soderholm SC. Some Chemicals Requiring Special Consideration when Deciding Whether to Sample the Particle, Vapor, or Both Phases of an Atmosphere. Applied Occupational and Environmental Hygiene. 2011;6(10):859-864.
14. European Commission, Commission Delegated Directive amending Annex II to Directive 2011/65/EU of the European Parliament and of the Council as regards the list of restricted substances. 2015 Erişim Linki: https://eur-lex.europa.eu/eli/dir del/2015/863/oj#document1. [Erişim tarihi 14.11.2019].
15. Resmi Gazete, Sanayi ve Ticaret Bakanlığı. Bazı Tüketici Ürünlerinin Tehlikeli Kimyasal Madde İçeriğine Yönelik Piyasa Gözetimi ve Denetimine İlişkin Tebliğ . 2011. Erişim Linki: https://www.resmigazete.gov.tr/eskiler/2011/04/20110402-9.htm. [Erişim tarihi: 16.12.2019]
16. Martin A, Sarkar A. Overview on biological implications of metal oxide nanoparticle exposure to human alveolar A549 cell line. Nanotoxicology. 2017;11(6):713-724.
17. Erkekoglu P, Baydar T. Evaluation of the protective effect of ascorbic acid on nitrite- and nitrosamine-induced cytotoxicity and genotoxicity in human hepatoma line. Toxicol Mech Methods. 2010;20(2):45-52.
18. Chao MW, Erkekoglu P, Tseng CY, et al. Wogan GN. Protective effects of ascorbic acid against the genetic and epigenetic alterations induced by 3,5-dimethylaminophenol in AA8 cells. J Appl Toxicol. 2015;35(5):466-77.
19. Lin PY, Chang YJ, Chen YC, et al. Anti-cancer effects of 3,5-dimethylaminophenol in A549 lung cancer cells. PLoS One. 2018;13(10):e0205249.
20. Stockert JC, Horobin RW, Colombo LL, Blazquez-Castro A. Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochemica. 2018;120:159-167.
21. Wojtala A, Bonora M, Malinska D, et al. Methods to Monitor ROS Production by Fluorescence Microscopy and Fluorometry. Methods Enzymology. 2014;542:243-262.
22. Ji H. Lysis of cells for immunoprecipitation. Cold Spring Harb Protoc; 2010;4:1-4.
23. Jardine D, Antolovich M, Prenzler P, et al. Liquid Chromatography-Mass Spectrometry (LC-MS) Investigation of the Thiobarbituric Acid Reactive Substances (TBARS) Reaction. Journal of Agricultural and Food Chemistry. 2012;50:1720-1724.
24. Rahman I, Kode A, Biswas SK. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nature Protocols. 2007;1:3159.
25. Luo S, Wehr NB. Protein carbonylation: avoiding pitfalls in the 2,4-dinitrophenylhydrazine assay. Redox Report. 2009;14(4):159-166.
26. Shahidi F, Zhong Y. Handbook of Antioxidants for Food Preservation. 26th ed. Canada; Woodhead Publishing 2015.
27. Noble JE, Bailey MJ. Quantitaion of protein. Methods Enzymology. 2009;463:73-95.
28. Jepsen KF, Abildtrup A, Larsen ST. Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549. Toxicology In Vitro. 2004;18(3):265-269.
29. Rafael-Vázquez L, García-Trejo S, Aztatzi-Aguilar OG, et al. Exposure to diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) promotes the loss of alveolar epithelial phenotype of A549 cells. Toxicology Letters. 2018;294:135-144.
30. Kwapniewski R, Kozaczka S, Silva MJ, et al. Occupational Exposure to Dibutyl Phthalate Among Manicurists. Journal of occupational and environmental medicine. 2008;50(6):705-711.
31. Wang Y, Zhu H, Kannan K. A Review of Biomonitoring of Phthalate Exposures.Toxics. 2019;7(2):21-49.
32. Batlle DM, Pena OM, Huff RD, et al. Dibutyl phthalate modulates phenotype of granulocytes in human blood in response to inflammatory stimuli. Toxicology Letters. 2018;296:23-30.
33. Odebeatu CC, Taylor T, Fleming LE, et al. Phthalates and asthma in children and adults: US NHANES 2007-2012. Environmental Science Pollution Research International. 2019;26(27):28256-28269.
34. Kim YM, Kim J, Cheong HK, et al. Exposure to phthalates aggravates pulmonary function and airway inflammation in asthmatic children. PLoS One. 2018;18:351-362.
35. Kuo PL, Hsu YL, Huang MS, et al. Ginger Suppresses Phthalate Ester-Induced Airway Remodeling. Journal of Agrıcultural and Food Chemistry. 2011;59:3429-3438.
36. Toxic Substances and Disease Registry, Toxicological Profile for Di-n-Butyl Phthalate. 2001. Erişim Linki: https: //www.atsdr. cdc. Gov / toxprofiles/tp135.pdf. [Erişim Tarihi: 16.10.2019].
37. Liu ZH, Li EH, Xu DL, et al. Genetic research and structural dysplasia assessment of anorectal malformations in neonatal male rats induced by di(n-butyl) phthalate. Environmental Toxicology. 2016;31(3):261-268.
38. Zhu YP, Li EH, Sun WL, et al. Maternal exposure to di-n-butyl phthalate (DBP) induces combined anorectal and urogenital malformations in male rat offspring. Reproductive Toxicology.2016;61:169-176.
39. Chen B, Hu X, Zhen X, et al. Effects of dibutyl phthalate and di(2-ethylhexyl) phthalate with their metabolites on CYP2C9*1 and CYP2C19*1 activities in vitro. Journal of Pharmaceutical and Biomedical Analysis. 2018;160:195-201.
40. Walseth F, Toftgard R, Nilsen OG. Phthalate esters I: Effects on cytochrome P-450 mediated metabolism in rat liver and lung, serum enzymatic activities and serum protein levels. Archives of Toxicology. 1982;50(1):1-10.
41. Erkekoğlu P, Rachidi W, Yüzügüllü OG, et al. Induction of ROS, p53, p21 in DEHP- and MEHP-exposed LNCaP cells-protection by selenium compounds. Food Chemical Toxicology. 2011;49(7):1565-1571.
42. Zhang G, Ling X, Liu K, et al. The p-eIF2α/ATF4 pathway links endoplasmic reticulum stress to autophagy following the production of reactive oxygen species in mouse spermatocyte-derived cells exposed to dibutyl phthalate. Free Radical Research. 2016;50(7):698-707.
43. Erkekoğlu P, Rachidi W, De Rosa V, et al. Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)hthalate and mono(2 ethylhexyl)phthalate treatment in LNCaP cells. Free Radical Biology and Medicine. 2010;1549(4):559-566.
44. Rosa V, Erkekoğlu P, Forestier A, et al. Low doses of selenium specifically stimulate the repair of oxidative DNA damage in LNCaP prostate cancer cells. Free Radical Research. 2012;46(2):105-116.
45. Kismali G, Yurdakok DB, Kuzukiran O, et al. Phthalate induced toxicity in prostate cancer cell lines and effects of alpha lipoic acid. Bratislavske Lekarske Listy. 2017;118(8):460-466.
46. Kim JH. Di(2-ethylhexyl) phthalate promotes lung cancer cell line A549 progression via Wnt/β-catenin signaling. J Toxicol Sci. 2019;44(4):237-244.
47. Shi Q, Tang J, Wang L, et al. Combined cytotoxicity of polystyrene nanoplastics and phthalate esters on human lung epithelial A549 cells and its mechanism. Ecotoxicol Environ Saf. 2021;213:112041.
48. Li L, Li HS, Song NN, et al. The immunotoxicity of dibutyl phthalate on the macrophages in mice. Immunopharmacology and Immunotoxicology. 2013;35(2):272-281.
49. Suzukı Y, Matsumoto M. Accumulation of triacylglycerol in tissue culture cells derived from human embryonic lung (L-132 cells) on administration of di-n-butyl phthalate. The Japanese Journal of Experimental Medicine. 1980;50(4):253-261.
50. Kim SH, Kim SS, Kwon O, et al. Effects of dibutyl phthalate and monobutyl phthalate on cytotoxicity and differentiation in cultured rat embryonic limb bud cells; protection by antioxidants. Journal of Toxicology and Environmental Health A. 2002;65:461-472.
51. Zhang G, Yang W, Jiang F, et al. PERK regulates Nrf2/ARE antioxidant pathway against dibutyl phthalate-induced mitochondrial damage and apoptosis dependent of reactive oxygen species in mouse spermatocyte-derived cells. Toxicology Letters. 2019;308:24-33.
52. Albaladejo EP, Fernandes D, Lacorte S, et al. Comparative toxicity, oxidative stress and endocrine disruption potential of plasticizers in JEG-3 human placental cells. Toxicology in Vitro. 2017;38:41-48.
53. Wójtowicz AK, Szychowski KA, Wnuk A, et al. Dibutyl Phthalate (DBP)-Induced Apoptosis and Neurotoxicity are Mediated via the Aryl Hydrocarbon Receptor (AhR) but not by Estrogen Receptor Alpha (ERα), Estrogen Receptor Beta (ERβ), or Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in Mouse Cortical Neurons. Neurotoxicity Research. 2017;31(1):77-89.
54. Cheng L, Li J, Cheng J, et al. Dibutyl phthalate-induced activation of ROS and ERK1/2 causes hepatic and renal damage in Kunming mice. Human and Experimental Toxicology. 2019;38(8):938-950.
55. Yan B, Guo J, Liu X, et al. Oxidative stress mediates dibutyl phthalate induced anxiety-like behavior in Kunming mice. Environmental Toxicology and Pharmacology. 2016;45:45-51.
56. Wang X, Yan X, Yang Y, et al. Dibutyl phthalate-mediated oxidative stress induces splenic injury in mice and the attenuating effects of vitamin E and curcumin. Food and Chemical Toxicology.2020;136:110955.