Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay

Giulia Vietti,Etienne Marbaix,Saloua Ibouraadaten,Christian Bailly,Dominique Lison,Mihaly Palmai-Pallag,Sybille van den Brule,Yousof Yakoub,Ivana Fenoglio

doi:10.1186/1743-8977-10-52

Abstract

BackgroundCarbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts.MethodsThe activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF).ResultsMWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis.ConclusionsIn this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.

Highlights

Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents
MWCNTg 2400 were shorter than NM400 and NM402 and had lower defect and impurity levels
Primary lung fibroblasts were exposed to NM400 and NM402 pre-dispersed in 2% fetal bovine serum (FBS 0.2% final Fetal bovine serum (FBS) concentration during cell exposure), or to human platelet-derived growth factor (PDGF)-BB as a fibrosis-relevant growth factor

Summary

Introduction

Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. Pristine CNT are generally hydrophobic, essentially insoluble in water and, when long enough, expected to be biopersistent. These features contribute to reinforce the concerns about their pathogenic activity [3]. Studies indicated that they were able to induce inflammatory reactions, granulomas, fibrosis, and biochemical changes in rodent lungs when administered intratracheally or intrapharyngeally [4,5,6]. In these studies CNT were shown to be more toxic than quartz, well known for its pulmonary toxicity. Results from inhalation studies revealed the capacity of CNT to induce lung fibrosis [7,8] or not [9,10,11]

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