Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

The number of legacy chemicals without toxicity reference values combined with the rate of new chemical development is overwhelming the capacity of the traditional risk assessment paradigm. More efficient approaches are needed to quantitatively estimate chemical risks. In this study, rats were dosed orally with multiple doses of six chemicals for 5 days and 2, 4, and 13 weeks. Target organs were analyzed for traditional histological and organ weight changes and transcriptional changes using microarrays. Histological and organ weight changes in this study and the tumor incidences in the original cancer bioassays were analyzed using benchmark dose (BMD) methods to identify noncancer and cancer points of departure. The dose-response changes in gene expression were also analyzed using BMD methods and the responses grouped based on signaling pathways. A comparison of transcriptional BMD values for the most sensitive pathway with BMD values for the noncancer and cancer apical endpoints showed a high degree of correlation at all time points. When the analysis included data from an earlier study with eight additional chemicals, transcriptional BMD values for the most sensitive pathway were significantly correlated with noncancer (r = 0.827, p = 0.0031) and cancer-related (r = 0.940, p = 0.0002) BMD values at 13 weeks. The average ratio of apical-to-transcriptional BMD values was less than two, suggesting that for the current chemicals, transcriptional perturbation did not occur at significantly lower doses than apical responses. Based on our results, we propose a practical framework for application of transcriptomic data to chemical risk assessment.

Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, gammadelta(+) T cells, and CD8(+) T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1-4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.