Graphene oxide induces dose‑dependent lung injury in rats by regulating autophagy

Zhang,Lei; Ouyang,Shuge; Zhang,Hongbo; Qiu,Mingke; Dai,Yuxin; Wang,Shuqing; Wang,Yang; Ou,Jingmin

doi:10.3892/etm.2021.9893

Graphene oxide induces dose‑dependent lung injury in rats by regulating autophagy

Authors:
- Lei Zhang
- Shuge Ouyang
- Hongbo Zhang
- Mingke Qiu
- Yuxin Dai
- Shuqing Wang
- Yang Wang
- Jingmin Ou
View Affiliations

Affiliations: Department of Geotechnical Engineering, College of Civil Engineering, Tongji University, Shanghai 200092, P.R. China, Cambridge International Exam Centre in Shanghai Experimental School, Shanghai 200092, P.R. China, Chongming Branch of Xinhua Hospital Affiliated to The Medical College of Shanghai Jiaotong University, Shanghai 202150, P.R. China, Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, P.R. China
Published online on: March 5, 2021 https://doi.org/10.3892/etm.2021.9893
Article Number: 462

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Graphene is a two‑dimensional structured material with a hexagonal honeycomb lattice composed of carbon atoms. The biological effects of graphene oxide (GO) have been extensively investigated, as it has been widely used in biological research due to its increased hydrophilicity/biocompatibility. However, the exact mechanisms underlying GO‑associated lung toxicity have not yet been fully elucidated. The aim of the present study was to determine the role of GO in lung injury induction, as well as its involvement in oxidative stress, inflammation and autophagy. The results revealed that lower concentrations of GO (5 and 10 mg/kg) did not cause significant lung injury, but the administration of GO at higher concentrations (50 and 100 mg/kg) induced lung edema, and increased lung permeability and histopathological lung changes. High GO concentrations also induced oxidative injury and inflammatory reactions in the lung, demonstrated by increased levels of oxidative products [malondialdehyde(MDA) and 8‑hydroxydeoxyguanosine (8‑OHdG)] and inflammatory factors (TNF‑α, IL‑6, IL‑1β and IL‑8). The autophagy inhibitors 3‑methyladenine (3‑MA) and chloroquine (CLQ) inhibited autophagy in the lung and attenuated GO‑induced lung injury, as demonstrated by a reduced lung wet‑to‑dry weight ratio, lower levels of protein in the bronchoalveolar lavage fluid, and a reduced lung injury score. Furthermore, 3‑MA and CLQ significantly reduced the levels of MDA, 8‑OHdG and inflammatory factors in lung tissue, suggesting that autophagy also mediates the development of oxidative injury and inflammation in the lung. Finally, autophagy was directly inhibited in BEAS‑2B cells by short hairpin RNA‑mediated autophagy protein 5 (ATG5) knockdown, which were then treated with GO. Cell viability, as well as the extent of injury (indicated by lactate dehydrogenase level) and oxidative stress were determined. The results revealed that ATG5 knockdown‑induced autophagic inhibition significantly decreased cellular injury and oxidative stress, suggesting that autophagy induction is a key event that leads to lung injury during exposure to GO. In conclusion, the findings of the present study indicated that GO causes lung injury in a dose‑dependent manner by inducing autophagy.

View Figures

View References

1	Bianco A: Graphene: Safe or toxic? The two faces of the medal. Angew Chem Int Ed Engl. 52:4986–4997. 2013.PubMed/NCBI View Article : Google Scholar
2	Novoselov KS, Fal'ko VI, Colombo L, Gellert PR, Schwab MG and Kim K: A roadmap for graphene. Nature. 490:192–200. 2012.PubMed/NCBI View Article : Google Scholar
3	Jastrzębska AM, Kurtycz P and Olszyna AR: Recent advances in graphene family materials toxicity investigations. J Nanopart Res. 14(1320)2012.PubMed/NCBI View Article : Google Scholar
4	Loh KP, Bao Q, Eda G and Chhowalla M: Graphene oxide as a chemically tunable platform for optical applications. Nat Chem. 2:1015–1024. 2010.PubMed/NCBI View Article : Google Scholar
5	Rong P, Yang K, Srivastan A, Kiesewetter DO, Yue X, Wang F, Nie L, Bhirde A, Wang Z, Liu Z, et al: Photosensitizer loaded nano-graphene for multimodality imaging guided tumor photodynamic therapy. Theranostics. 4:229–239. 2014.PubMed/NCBI View Article : Google Scholar
6	Lv M, Zhang Y, Liang L, Wei M, Hu W, Li X and Huang Q: Effect of graphene oxide on undifferentiated and retinoic acid-differentiated SH-SY5Y cells line. Nanoscale. 4:3861–3866. 2012.PubMed/NCBI View Article : Google Scholar
7	Yue H, Wei W, Yue Z, Wang B, Luo N, Gao Y, Ma D, Ma G and Su Z: The role of the lateral dimension of graphene oxide in the regulation of cellular responses. Biomaterials. 33:4013–4021. 2012.PubMed/NCBI View Article : Google Scholar
8	Li B, Yang J, Huang Q, Zhang Y, Peng C, Zhang Y, He Y, Shi J, Li W, Hu J and Fan C: Biodistribution and pulmonary toxicity of intratracheally instilled graphene oxide in mice. NPG Asia Materials. 5(e44)2013.
9	Mao L, Hu M, Pan B, Xie Y and Petersen EJ: Biodistribution and toxicity of radio-labeled few layer graphene in mice after intratracheal instillation. Part Fibre Toxicol. 13(7)2016.PubMed/NCBI View Article : Google Scholar
10	Han SG, Kim JK, Shin JH, Hwang JH, Lee JS, Kim TG, Lee JH, Lee GH, Kim KS, Lee HS, et al: Pulmonary responses of sprague-dawley rats in single inhalation exposure to graphene oxide nanomaterials. Biomed Res Int. 2015(376756)2015.PubMed/NCBI View Article : Google Scholar
11	Zhang D, Zhang Z, Liu Y, Chu M, Yang C, Li W, Shao Y, Yue Y and Xu R: The short- and long-term effects of orally administered high-dose reduced graphene oxide nanosheets on mouse behaviors. Biomaterials. 68:100–113. 2015.PubMed/NCBI View Article : Google Scholar
12	Ema M, Gamo M and Honda K: A review of toxicity studies on graphene-based nanomaterials in laboratory animals. Regul Toxicol Pharmacol. 85:7–24. 2017.PubMed/NCBI View Article : Google Scholar
13	Ou L, Song B, Liang H, Liu J, Feng X, Deng B, Sun T and Shao L: Toxicity of graphene-family nanoparticles: A general review of the origins and mechanisms. Part Fibre Toxicol. 13(57)2016.PubMed/NCBI View Article : Google Scholar
14	Liu JH, Yang ST, Wang H, Chang Y, Cao A and Liu Y: Effect of size and dose on the biodistribution of graphene oxide in mice. Nanomedicine (Lond). 7:1801–1812. 2012.PubMed/NCBI View Article : Google Scholar
15	Park EJ, Lee GH, Han BS, Lee BS, Lee S, Cho MH, Kim JH and Kim DW: Toxic response of graphene nanoplatelets in vivo and in vitro. Arch Toxicol. 89:1557–1568. 2015.PubMed/NCBI View Article : Google Scholar
16	Su WC, Ku BK, Kulkarni P and Cheng YS: Deposition of graphene nanomaterial aerosols in human upper airways. J Occup Environ Hyg. 13:48–59. 2016.PubMed/NCBI View Article : Google Scholar
17	Jarosz A, Skoda M, Dudek I and Szukiewicz D: Oxidative stress and mitochondrial activation as the main mechanisms underlying graphene toxicity against human cancer cells. Oxid Med Cell Longev. 2016(5851035)2016.PubMed/NCBI View Article : Google Scholar
18	Kumari R, Mondal T, Bhowmick AK and Das P: Impeded repair of abasic site damaged lesions in DNA adsorbed over functionalized multiwalled carbon nanotube and graphene oxide. Mutat Res Genet Toxicol Environ Mutagen. 804:39–46. 2016.PubMed/NCBI View Article : Google Scholar
19	Zhang W, Sun Y, Lou Z, Song L, Wu Y, Gu N and Zhang Y: In vitro cytotoxicity evaluation of graphene oxide from the peroxidase-like activity perspective. Colloids Surf B Biointerfaces. 151:215–223. 2017.PubMed/NCBI View Article : Google Scholar
20	Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, Hazle JD, Halas NJ and West JL: Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc Natl Acad Sci USA. 100:13549–13554. 2003.PubMed/NCBI View Article : Google Scholar
21	Chen GY, Chen CL, Tuan HY, Yuan PX, Li KC, Yang HJ and Hu YC: Graphene oxide triggers toll-like receptors/autophagy responses in vitro and inhibits tumor growth in vivo. Adv Healthc Mater. 3:1486–1495. 2014.PubMed/NCBI View Article : Google Scholar
22	Chen GY, Meng CL, Lin KC, Tuan HY, Yang HJ, Chen CL, Li KC, Chiang CS and Hu YC: Graphene oxide as a chemosensitizer: Diverted autophagic flux, enhanced nuclear import, elevated necrosis and improved antitumor effects. Biomaterials. 40:12–22. 2015.PubMed/NCBI View Article : Google Scholar
23	Kang Y, Liu J, Wu J, Yin Q, Liang H, Chen A and Shao L: Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12 cell lines apoptosis and cell cycle alterations via the ERK signaling pathways. Int J Nanomedicine. 12:5501–5510. 2017.PubMed/NCBI View Article : Google Scholar
24	Domagala A, Stachura J, Gabrysiak M, Muchowicz A, Zagozdzon R, Golab J and Firczuk M: Inhibition of autophagy sensitizes cancer cells to photofrin-based photodynamic therapy. BMC Cancer. 18(210)2018.PubMed/NCBI View Article : Google Scholar
25	Zhang X, Li M, Wang YB, Cheng Y, Zheng YF, Xi TF and Wei SC: Cell response of nanographene platelets to human osteoblast-like MG63 cells. J Biomed Mater Res A. 102:732–742. 2014.PubMed/NCBI View Article : Google Scholar
26	Chatterjee N, Eom HJ and Choi J: A systems toxicology approach to the surface functionality control of graphene-cell interactions. Biomaterials. 35:1109–1127. 2014.PubMed/NCBI View Article : Google Scholar
27	Liu Y, Luo Y, Wu J, Wang Y, Yang X, Yang R, Wang B, Yang J and Zhang N: Graphene oxide can induce in vitro and in vivo mutagenesis. Sci Rep. 3(3469)2013.PubMed/NCBI View Article : Google Scholar
28	Vallabani NV, Mittal S, Shukla RK, Pandey AK, Dhakate SR, Pasricha R and Dhawan A: Toxicity of graphene in normal human lung cells (BEAS-2B). J Biomed Nanotechnol. 7:106–107. 2011.PubMed/NCBI View Article : Google Scholar
29	De Marzi L, Ottaviano L, Perrozzi F, Nardone M, Santucci S, De Lapuente J, Borras M, Treossi E, Palermo V and Poma A: Flake size-dependent cyto and genotoxic evaluation of graphene oxide on in vitro A549, CaCo2 and vero cell lines. J Biol Regul Homeost Agents. 28:281–289. 2014.PubMed/NCBI
30	Chang Y, Yang ST, Liu JH, Dong E, Wang Y, Cao A, Liu Y and Wang H: In vitro toxicity evaluation of graphene oxide on A549 cells. Toxicol Lett. 200:201–210. 2011.PubMed/NCBI View Article : Google Scholar
31	Wang K, Ruan J, Song H, Zhang J, Wo Y, Guo S and Cui D: Biocompatibility of graphene oxide. Nanoscale Res Lett. 6(8)2011.PubMed/NCBI View Article : Google Scholar
32	Jasim DA, Ménard-Moyon C, Begin D, Bianco A and Kostarelos K: Tissue distribution and urinary excretion of intravenously administered chemically functionalized graphene oxide sheets. Chem Sci. 6:3952–3964. 2015.PubMed/NCBI View Article : Google Scholar
33	Singh Z: Applications and toxicity of graphene family nanomaterials and their composites. Nanotechnol Sci Appl. 9:15–28. 2016.PubMed/NCBI View Article : Google Scholar
34	Li Y, Liu Y, Fu Y, Wei T, Le Guyader L, Gao G, Liu RS, Chang YZ and Chen C: The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways. Biomaterials. 33:402–411. 2012.PubMed/NCBI View Article : Google Scholar
35	Fujimi S, MacConmara MP, Maung AA, Zang Y, Mannick JA, Lederer JA and Lapchak PH: Platelet depletion in mice increases mortality after thermal injury. Blood. 107:4399–4406. 2006.PubMed/NCBI View Article : Google Scholar
36	Zhou H, Zhao K, Li W, Yang N, Liu Y, Chen C and Wei T: The interactions between pristine graphene and macrophages and the production of cytokines/chemokines via TLR- and NF-κB-related signaling pathways. Biomaterials. 33:6933–6942. 2012.PubMed/NCBI View Article : Google Scholar
37	Finkel T: Signal transduction by reactive oxygen species. J Cell Biol. 194:7–15. 2011.PubMed/NCBI View Article : Google Scholar
38	Yan J, Feng Z and Liu J, Shen W, Wang Y, Wertz K, Weber P, Long J and Liu J: Enhanced autophagy plays a cardinal role in mitochondrial dysfunction in type 2 diabetic Goto-Kakizaki (GK) rats: Ameliorating effects of (-)-epigallocatechin-3-gallate. J Nutr Biochem. 23:716–724. 2012.PubMed/NCBI View Article : Google Scholar
39	Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, Mi N, Zhao Y, Liu Z, Wan F, et al: Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 465:942–946. 2010.PubMed/NCBI View Article : Google Scholar
40	Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, Gautier CA, Sou YS, Saiki S, Kawajiri S, Sato F, et al: PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J Cell Biol. 189:211–221. 2010.PubMed/NCBI View Article : Google Scholar