Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances

p21(Cip1) Protection against Hyperoxia Requires Bcl-X(L) and Is Uncoupled from Its Ability to Suppress Growth

The cyclin-dependent kinase inhibitor p21(Cip1/Waf1/Sdi1) protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p21(1–82NLS)) domain or its carboxy-terminal PCNA-binding (p21(76–164)) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-X(L) declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-X(L) enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-X(L) increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21(+/+) and p21(+/−) mice but not in p21(−/−) mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-X(L) and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-X(L) and is uncoupled from its ability to inhibit proliferation