title: Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and -OH and -COOH functionalized multi-wall carbon nanotubes.
abstract: Toxic effects were reported for pristine-multi-wall carbon nanotubes (p-MWCNTs) while the role of the functionalization on MWCNT-induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS-2B) cells exposed to p-MWCNTs, MWCNTs-OH and MWCNTs-COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT-induced toxicity. In A549 cells, p-MWCNTs and MWCNTs-OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs-COOH were confined inside filled cytoplasmic vesicles. WST-1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS-2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs-COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs-interference. Pristine and MWCNTs-COOH induced membrane damage, particularly in BEAS-2B cells. MWCNTs-COOH induced interleukin-6 (IL-6) and IL-8 release in A549 cells whereas p-MWCNTs induced IL-8 release in BEAS-2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p-MWCNTs disrupting the membrane and vesicle-confined MWCNTs-COOH inducing inflammation. WST-1 was more reliable than MTT to test MWCNT-toxicity. BEAS-2B cells were more susceptible then A549 cells, particularly to MWCNT-COOH cytotoxicity. Our results confirm the toxicity of p-MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action.
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Cinzia Lucia
authors.initials: CL
authors.lastname: Ursini
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Maiello
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Aureliano
authors.initials: A
authors.lastname: Ciervo
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Fresegna
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Buresti
authors.affiliation: ISS- Istituto Superiore di Sanità, Department of Technology and Health, Viale Regina Elena 299, 00161, Rome, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Superti
authors.affiliation: ISS- Istituto Superiore di Sanità, Department of Technology and Health, Viale Regina Elena 299, 00161, Rome, Italy.
authors.forename: Magda
authors.initials: M
authors.lastname: Marchetti
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Iavicoli
authors.affiliation: INAIL-Italian Workers' Compensation Authority - Research Area, Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, via Fontana Candida 1, 00040, Monteporzio Catone, Rome, Italy.
authors.forename: Delia
authors.initials: D
authors.lastname: Cavallo
mesh_terms: D001681:Biological Assay; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006900:Hydroxylation; D018836:Inflammation Mediators / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D037742:Nanotubes, Carbon / Q000737:chemistry / Q000633:toxicity*; D011014:Pneumonia / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D018570:Risk Assessment; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C508606:CXCL8 protein, human; D002264:Carboxylic Acids; C508600:IL6 protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D037742:Nanotubes, Carbon; D014409:Tumor Necrosis Factor-alpha; D007770:L-Lactate Dehydrogenase
keywords: cytotoxicity; functionalized MWCNTs; inflammatory effects; lung cells; uptake