Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells

Abstract

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk assessment based on in vitro and in silico testing requires utilizing toxicity pathway information to distinguish adverse outcomes from recoverable adaptive events. Little work has focused on oxidative stresses in human airway for the purposes of predicting adverse responses. We hypothesize that early gene expression-mediated molecular changes could be used to delineate adaptive and adverse responses to environmentally-based perturbations. Here, we examined cellular responses of the tracheobronchial airway to zinc (Zn) exposure, a model oxidant. Airway derived BEAS-2B cells exposed to 2–10 μM Zn2+ elicited concentration- and time-dependent cytotoxicity. Normal, adaptive, and cytotoxic Zn2+ exposure conditions were determined with traditional apical endpoints, and differences in global gene expression around the tipping point of the responses were used to delineate underlying molecular mechanisms. Bioinformatic analyses of differentially expressed genes indicate early enrichment of stress signaling pathways, including those mediated by the transcription factors p53 and NRF2. After 4 h, 154 genes were differentially expressed (p < 0.01) between the adaptive and cytotoxic Zn2+ concentrations. Nearly 40% of the biomarker genes were related to the p53 signaling pathway with 30 genes identified as likely direct targets using a database of p53 ChIP-seq studies. Despite similar p53 activation profiles, these data revealed widespread dampening of p53 and NRF2-related genes as early as 4 h after exposure at higher, unrecoverable Zn2+ exposures. Thus, in our model early increased activation of stress response pathways indicated a recoverable adaptive event. Overall, this study highlights the importance of characterizing molecular mechanisms around the tipping point of adverse responses to better inform HTS paradigms.

Transcriptome Analysis Reveals Distinct Responses to Physiologic versus Toxic Manganese Exposure in Human Neuroblastoma Cells

“…In recent years, studies have shown that microarray analysis and deep-sequencing technologies such as RNA-Seq analysis can be exploited to determine adaptive pathways, which regulate responses to stressors (Pillai et al, 2014; Currier et al, 2016; Zheng et al, 2018). Previous microarray studies linking toxicity and adaptive transcriptomic and proteomic responses in green algae, Chlamydomonas reinhardtii , exposed to silver showed that the cells initiate a defense response to combat oxidative stress and eliminate silver via efflux transporters (Pillai et al, 2014).…”

“…Previous microarray studies linking toxicity and adaptive transcriptomic and proteomic responses in green algae, Chlamydomonas reinhardtii , exposed to silver showed that the cells initiate a defense response to combat oxidative stress and eliminate silver via efflux transporters (Pillai et al, 2014). Another microarray study used a similar strategy to identify enrichment in p53 signaling and NRF2-related genes in adaptive and adverse responses to zinc in human bronchial epithelial cells (Currier et al, 2016). For Mn exposures, there is no information on Mn dose-response effects on the gene expression profile with a graded model from non-toxic to toxic exposures.…”

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

“…Total p53 did not demonstrate a clear treatment response in this model system. We have previously observed activation of p53 phosphorylation in the absence of increased levels of total p53 under similar conditions in zinc-treated BEAS-2B cells 14 .…”