Bridging inhaled aerosol dosimetry to physiologically based pharmacokinetic modeling for toxicological assessment: nicotine delivery systems and beyond.

One of the challenges for toxicological assessment of inhaled aerosols is to accurately predict their deposited and absorbed dose. Transport, evolution, and deposition of liquid aerosols are driven by complex processes dominated by convection-diffusion that depend on various factors related to physics and chemistry. These factors include the physicochemical properties of the pure substance of interest and associated mixtures, the physical and chemical properties of the aerosols generated, the interplay between different factors during transportation and deposition, and the subject-specific inhalation topography. Several inhalation-based physiologically based pharmacokinetic (PBPK) models have been developed, but the applicability of these models for aerosols has yet to be verified. Nicotine is among several substances that are often delivered via the pulmonary route, with varied kinetics depending upon the route of exposure. This was used as an opportunity to review and discuss the current knowledge and state-of-the-art tools combining aerosol dosimetry predictions with PBPK modeling efforts. A validated tool could then be used to perform for toxicological assessment of other inhaled therapeutic substances. The Science Panel from the Alliance of Risk Assessment have convened at the "Beyond Science and Decisions: From Problem Formulation to Dose-Response Assessment" workshop to evaluate modeling approaches and address derivation of exposure-internal dose estimations for inhaled aerosols containing nicotine or other substances. The discussion involved PBPK model evaluation criteria, challenges, and choices that arise in such a model design, development, and application as a computational tool for use in human toxicological assessments.

Systems toxicology meta-analysis of in vitro assessment studies: biological impact of a candidate modified-risk tobacco product aerosol compared with cigarette smoke on human organotypic cultures of the aerodigestive tract.

Systems biology combines comprehensive molecular analyses with quantitative modeling to understand the characteristics of a biological system as a whole. Leveraging a similar approach, systems toxicology aims to decipher complex biological responses following exposures. This work reports a systems toxicology meta-analysis in the context of in vitro assessment of a candidate modified-risk tobacco product (MRTP) using three human organotypic cultures of the aerodigestive tract (buccal, bronchial, and nasal epithelia). Complementing a series of functional measures, a causal network enrichment analysis of transcriptomic data was used to compare quantitatively the biological impact of aerosol from the Tobacco Heating System (THS) 2.2, a candidate MRTP, with 3R4F cigarette smoke (CS) at similar nicotine concentrations. Lower toxicity was observed in all cultures following exposure to THS2.2 aerosol compared with 3R4F CS. Because of their morphological differences, a smaller exposure impact was observed in the buccal (stratified epithelium) compared with the bronchial and nasal (pseudostratified epithelium). However, the causal network enrichment approach supported a similar mechanistic impact of CS across the three cultures, including the impact on xenobiotic, oxidative stress, and inflammatory responses. At comparable nicotine concentrations, THS2.2 aerosol elicited reduced and more transient effects on these processes. To demonstrate the benefits of additional data modalities, we employed a newly established targeted mass-spectrometry marker panel to further confirm the reduced cellular stress responses elicited by THS2.2 aerosol compared with 3R4F CS in the nasal culture. Overall, this work demonstrates the applicability and robustness of the systems toxicology approach for in vitro inhalation toxicity assessment.

Identification of gene expression signature for cigarette smoke exposure response--from man to mouse.

Gene expression profiling data can be used in toxicology to assess both the level and impact of toxicant exposure, aligned with a vision of 21st century toxicology. Here, we present a whole blood-derived gene signature that can distinguish current smokers from either nonsmokers or former smokers with high specificity and sensitivity. Such a signature that can be measured in a surrogate tissue (whole blood) may help in monitoring smoking exposure as well as discontinuation of exposure when the primarily impacted tissue (e.g., lung) is not readily accessible. The signature consisted of LRRN3, SASH1, PALLD, RGL1, TNFRSF17, CDKN1C, IGJ, RRM2, ID3, SERPING1, and FUCA1. Several members of this signature have been previously described in the context of smoking. The signature translated well across species and could distinguish mice that were exposed to cigarette smoke from ones exposed to air only or had been withdrawn from cigarette smoke exposure. Finally, the small signature of only 11 genes could be converted into a polymerase chain reaction-based assay that could serve as a marker to monitor compliance with a smoking abstinence protocol.

Biological impact of cigarette smoke compared to an aerosol produced from a prototypic modified risk tobacco product on normal human bronchial epithelial cells.

Cigarette smoking causes serious and fatal diseases. The best way for smokers to avoid health risks is to quit smoking. Using modified risk tobacco products (MRTPs) may be an alternative to reduce the harm caused for those who are unwilling to quit smoking, but little is known about the toxic effects of MRTPs, nor were the molecular mechanisms of toxicity investigated in detail. The toxicity of an MRTP and the potential molecular mechanisms involved were investigated in high-content screening tests and whole genome transcriptomics analyses using human bronchial epithelial cells. The prototypic (p)MRTP that was tested had less impact than reference cigarette 3R4F on the cellular oxidative stress response and cell death pathways. Higher pMRTP aerosol extract concentrations had impact on pathways associated with the detoxification of xenobiotics and the reduction of oxidative damage. A pMRTP aerosol concentration up to 18 times higher than the 3R4F caused similar perturbation effects in biological networks and led to the perturbation of networks related to cell stress, and proliferation biology. These results may further facilitate the development of a systems toxicology-based impact assessment for use in future risk assessments in line with the 21st century toxicology paradigm, as shown here for an MRTP.

Genomic impact of cigarette smoke, with application to three smoking-related diseases.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

High-resolution crystal structure and in vivo function of a kinesin-2 homologue in Giardia intestinalis.

A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis-deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of G. intestinalis wild-type kinesin-2 (GiKIN2a) motor domain, with its docked neck linker and the hydrolysis-deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg(2+) at 1.6 and 1.8 A resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. G. intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes, with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in G. intestinalis.