Assessing nanotoxicity in cells in vitro

With the advent of nanotechnology, concerns about the potential adverse health effects of nanomaterials have been expressed, especially to workers and users [1, 2]. For these reasons, screening assays are needed to assess a myriad of chemically and physically diverse nanomaterials. Because of the expense of in vivo experiments and public and governmental urging to develop alternatives to animal testing, in vitro models may be more attractive for preliminary testing of nanomaterials to assess their potential toxicologic effects and ability to elicit disease. Human health concerns for nanomaterials are predicated historically by epidemiologic and clinical studies on naturally occurring fibers and particles such as asbestos and silica, respectively. Whereas inhalation of asbestos fibers is associated with the development of nonmalignant (pleural and pulmonary fibrosis or asbestosis) and malignant diseases (lung cancers and mesotheliomas) [3, 4], silica is associated primarily with the development of silicosis, an occupationally-linked pulmonary fibrosis [5]. After decades of research, the complex mechanisms of disease by these minerals are still incompletely understood, but several properties appear important in the long-term health effects of asbestos fibers. These include: 1) respirability or ability to enter the lung; 2) durability, due to intrinsic lack of solubility and/or inability to be cleared by macrophages in the lung, pleura or peritoneum; 3) fibrous geometry; 4) length to width ratio, i.e., longer (> 5 μm) and thinner fibers are more carcinogenic and fibrogenic; and 5) surface properties which play a role in the generation of reactive oxygen or nitrogen species (ROS/RNS) [6]. In addition, both ROS and RNS have been linked to the generation and augmentation of the inflammatory responses to asbestos and silica, and inflammation is thought to be key to the development of fibrosis and many cancers [7]. We recently have shown that stimulation of the inflammasome of human macrophages via NADPH oxidase acts as a sensor for the production of proinflammatory cytokines such as interleukin-1β by asbestos, suggesting that inflammation mediates responses of target cells of lung disease [8]. These studies underscore the importance of effective screening strategies for nanomaterials using multiple cell types, especially since nano-sized particles and fibers may be similar to ultrafine (UF) particles that can penetrate the endothelium of the lung and be transported to distal organs such as the heart and brain [1, 2]. For testing of the pathogenic effects of asbestos and asbestos-like fibers, most in vitro assays have been designed using target cells of the lung and pleura with endpoints such as cytotoxicity, proliferation, and genotoxicity. These phenomena are related to the multiple stages of cancer development which may involve genotoxic (changes to DNA) as well as proliferative events that can lead to the selective expansion of an asbestos-mutated cell population. In this chapter, we review in vitro assays for cytotoxicity, proliferation, genotoxicity, and more robust toxicogenomic approaches that can be used to screen nanomaterials for their potential pathogenic effects. Since the majority of these assays have been standardized in our laboratory using a variety of pathogenic minerals (asbestos, silica) and nonpathogenic particles (fine titanium dioxide [TiO2] or glass beads), we will frequently supplement our discussion of nanomaterials with mention of other mineral/particle types to demonstrate each assay's utility in predicting toxicity. Although cell-free in vitro assays to predict dissolution of nanomaterials in the body are not discussed in detail, they are recommended to predict nanomaterial durability over time, especially since it has been shown that diseases resulting from exposure to other pathogenic materials, such as asbestos, require decades to develop [3, 4].