The effects of genotype × phenotype interactions on silver nanoparticle toxicity in organotypic cultures of murine tracheal epithelial cells

Abstract

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no in vitro studies to date have used organotypic cultures as a high-content in vitro model of the conducting airway to characterize the effects of interactions between host genetic and acquired factors, or gene × phenotype interactions (G×P), on AgNP toxicity. In the present study, we derived organotypic cultures from primary murine tracheal epithelial cells (MTEC) to characterize nominal and dosimetric dose-response relationships for AgNP-induced barrier dysfunction, glutathione (GSH) depletion, reactive oxygen species (ROS) production, lipid peroxidation, and cytotoxicity across two genotypes (A/J and C57BL/6J mice), two phenotypes ("Normal" and "Type 2[T2]-Skewed"), and two exposures (an acute exposure of 24 h and a subacute exposure of 4 hours, every other day, over 5 days [5×4 h]). We characterized the "T2-Skewed" phenotype as an in vitro model of chronic respiratory diseases, which was marked by increased sensitivity to AgNP-induced barrier dysfunction, GSH depletion, ROS production, lipid peroxidation, and cytotoxicity, suggesting that asthmatics are a sensitive population to AgNP exposures in occupational settings. This also suggests that exposure limits, which should be based upon the most sensitive population, should be derived using in vitro and in vivo models of chronic respiratory diseases. This study highlights the importance of considering dosimetry as well as G×P effects when screening and prioritizing potential respiratory toxicants. Such in vitro studies can be used to inform regulatory policy aimed at special protections for all populations.

The effects of gene × environment interactions on silver nanoparticle toxicity in the respiratory system: An adverse outcome pathway

“…Within the AEP framework, we integrated dosimetric data for intracellular uptake and/or localization of Ag mass associated in the lungs of different animal genotypes (Figure 3A). Interestingly, we observed relevant IVIVE from the Nicholas et al to the Scoville et al study, as the levels of intracellular uptake and/or localization of Ag mass associated in our organotypic cultures derived from A/J and C57BL6/J mice translated to a similar pattern of Ag mass associated in the lungs of A/J and C57BL6/J mice (Nicholas et al, 2020a; Scoville et al, 2017).…”

“…Similar levels of Ag mass associated in BEAS‐2B cells have been reported in other in vitro studies (Cronholm et al, 2013; Gliga et al, 2014; Jeannet et al, 2015; Kim et al, 2011, 2014; Nymark et al, 2013; Zhang et al, 2015). In our previous study of G × E effects on AgNP toxicity, we observed the highest levels of Ag mass associated in “AJ:T2‐Skewed” organotypic cultures when normalized by ICP‐MS (Nicholas et al, 2020a).…”

“…Within the AOP framework, we integrated in vitro dose–response data for adverse cellular responses associated with AgNP toxicity, including: oxidative stress (marked by increased ROS production, GSH [glutathione] depletion, lipid peroxidation, and antioxidant enzyme activity) (Figure 2F), mitochondrial dysfunction (marked by increased caspase‐3 activation, apoptosis, and the WNT signaling pathway) (Figure 2G), inflammation (marked by imbalanced T1/T2/[type 17 or pro‐inflammatory] T17 cytokine secretion) (Figure 2H), cytotoxicity (marked by increased LDH secretion) (Figure 2I), and changes in organotypic physiology (marked by decreased barrier function) (Figure 2J) in cell cultures derived from differentially sensitive polymorphisms, genotypes, and phenotypes (Cronholm et al, 2013; Gliga et al, 2014; Jeannet et al, 2015; Kim et al, 2011, 2014; Nicholas et al, 2020a, 2020b; Nymark et al, 2013; Zhang et al, 2015). We constructed plausible KER based on limited or no in vitro dose–response data between “Mitochondrial Function” and “Inflammation” (Mitochondrial Function = p < 0.001; Inflammation = p < 0.001, respectively) as well as “Inflammation” and “Cytotoxicity” (Inflammation = p < 0.001; Cytotoxicity = p < 0.001 – p > 0.05, respectively).…”

“…Previous in vitro studies have used nominal and dosimetric dose–response relationships to define exposure levels corresponding to specific changes in response (Edler, 2014), or a 10% difference in response to represent a shift in one standard deviation from the mean of normally distributed data to reflect all adverse cellular and organ responses (Kavlock, Allen, Faustman, & Kimmel, 1995; Nicholas et al, 2019; Weldon et al, 2016). Using in vitro dose–response data, we derived nominal and dosimetric BMD and their benchmark dose lower 95% confidence limit (BMDL) with linear mixed effects models using the dosimetric dose of Ag mass associated on organotypic cultures as a continuous variable and observed that the most sensitive adverse cellular response was ROS production at acute and subacute exposures (Supplementary Data) (Nicholas et al, 2020a). Using in vivo exposure‐response data, Weldon et al derived nominal and dosimetric BMD (BMDL) with multistage polynomial degree 2 models using the dosimetric dose of Ag mass associated in the lungs of Sprague–Dawley rats from the Sung et al study and used these data to derive human equivalent concentration (HEC) and a health‐based occupational exposure limit (OEL) of 0.19 μg AgNP/m 3 .…”

The Effects of Genotype × Phenotype Interactions on Transcriptional Response to Silver Nanoparticle Toxicity in Organotypic Cultures of Murine Tracheal Epithelial Cells

“…We previously characterized the effects of interactions between host genetic and acquired factors, or gene  environment (G  E) interactions, on AgNP toxicity (Nicholas et al, 2019). Understanding G  E effects is important for identifying sensitive populations, whose underlying genetics or diseases could directly modify their response to AgNP exposures.…”