The KEAP1-NRF2-ARE signaling pathway plays a central role in mediating the adaptive cellular stress response to oxidative and electrophilic chemicals. This canonical pathway has been extensively studied and reviewed in the past two decades, but rarely was it looked at from a quantitative signaling perspective. Signal amplification, i.e., ultrasensitivity, is crucially important for robust induction of antioxidant genes to appropriate levels that can adequately counteract the stresses. In this review article, we examined a number of well-known molecular events in the KEAP1-NRF2-ARE pathway from a quantitative perspective with a focus on how signal amplification can be achieved. We illustrated, by using a series of mathematical models, that redox-regulated protein sequestration, stabilization, translation, nuclear trafficking, DNA promoter binding, and transcriptional induction – which are embedded in the molecular network comprising KEAP1, NRF2, sMaf, p62, and BACH1 – may generate highly ultrasensitive NRF2 activation and antioxidant gene induction. The emergence and degree of ultrasensitivity depend on the strengths of protein-protein and protein-DNA interaction and protein abundances. A unique, quantitative understanding of signal amplification in the KEAP1-NRF2-ARE pathway will help to identify sensitive targets for the prevention and therapeutics of oxidative stress-related diseases and develop quantitative adverse outcome pathway models to facilitate the health risk assessment of oxidative chemicals.

Chemical toxicity testing is moving steadily toward a human cell and organoid-based in vitro approach for reasons including scientific relevancy, efficiency, cost, and ethical rightfulness. Inferring human health risk from chemical exposure based on in vitro testing data is a challenging task, facing various data gaps along the way. This review identifies these gaps and makes a case for the in silico approach of computational dose-response and extrapolation modeling to address many of the challenges. Mathematical models that can mechanistically describe chemical toxicokinetics (TK) and toxicodynamics (TD), for both in vitro and in vivo conditions, are the founding pieces in this regard. Identifying toxicity pathways and in vitro point of departure (PoD) associated with adverse health outcomes requires an understanding of the molecular key events in the interacting transcriptome, proteome, and metabolome. Such an understanding will in turn help determine the sets of sensitive biomarkers to be measured in vitro and the scope of toxicity pathways to be modeled in silico. In vitro data reporting both pathway perturbation and chemical biokinetics in the culture medium serve to calibrate the toxicity pathway and virtual tissue models, which can then help predict PoDs in response to chemical dosimetry experienced by cells in vivo. Two types of in vitro to in vivo extrapolation (IVIVE) are needed. (1) For toxic effects involving systemic regulations, such as endocrine disruption, organism-level adverse outcome pathway (AOP) models are needed to extrapolate in vitro toxicity pathway perturbation to in vivo PoD. (2) Physiologically-based toxicokinetic (PBTK) modeling is needed to extrapolate in vitro PoD dose metrics into external doses for expected exposure scenarios. Linked PBTK and TD models can explore the parameter space to recapitulate human population variability in response to chemical insults. While challenges remain for applying these modeling tools to support in vitro toxicity testing, they open the door toward population-stratified and personalized risk assessment.

Thyroid hormones (THs), including T4 and T3, are produced and released by the thyroid gland under the stimulation of thyroid-stimulating hormone (TSH). The homeostasis of THs is regulated via the coordination of the hypothalamic-pituitary-thyroid axis, plasma binding proteins, and local metabolism in tissues. TH synthesis and secretion in the thyrocytes-containing thyroid follicles are exquisitely regulated by an elaborate molecular network comprising enzymes, transporters, signal transduction machineries, and transcription factors. In this article, we synthesized the relevant literature, organized and dissected the complex intrathyroidal regulatory network into structures amenable to functional interpretation and systems-level modeling. Multiple intertwined feedforward and feedback motifs were identified and described, centering around the transcriptional and posttranslational regulations involved in TH synthesis and secretion, including those underpinning the Wolff-Chaikoff and Plummer effects and thyroglobulin-mediated feedback regulation. A more thorough characterization of the intrathyroidal network from a systems biology perspective, including its topology, constituent network motifs, and nonlinear quantitative properties, can help us to better understand and predict the thyroidal dynamics in response to physiological signals, therapeutic interventions, and environmental disruptions.

Flutamide is a widely used nonsteroidal antiandrogen for prostate cancer therapy, but its clinical application is restricted by the concurrent liver injury. Increasing evidence suggests that flutamide‐induced liver injury is associated with oxidative stress, though the precise mechanism is poorly understood. Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a master transcription factor regulating endogenous antioxidants including heme oxygenase‐1 (HO‐1). This study was designed to delineate the role of Nrf2/HO‐1 in flutamide‐induced hepatic cell injury. Our results showed that flutamide concentration dependently induced cytotoxicity, hydrogen peroxide accumulation, and mitochondrial dysfunction as indicated by mitochondrial membrane potential loss and ATP depletion. The protein expression of Nrf2 and HO‐1 was induced by flutamide at 12.5 μM but was downregulated by higher concentrations of flutamide. Silencing either Nrf2 or HO‐1 was found to aggravate flutamide‐induced hydrogen peroxide accumulation and mitochondrial dysfunction as well as inhibition of the Nrf2 pathway. Moreover, preinduction of HO‐1 by Copp significantly attenuated flutamide‐induced oxidative stress and mitochondrial dysfunction, while inhibition of HO‐1 by Snpp aggravated these deleterious effects. These findings suggest that flutamide‐induced hepatic cell death and mitochondrial dysfunction is assoicated with inhibition of Nrf2‐mediated HO‐1. Pharmacologic intervention of Nrf2/HO‐1 may provide a promising therapeutic approach in flutamide‐induced liver injury.