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Parallel Artificial Membrane Permeability Assay (PAMPA) is an in vitro laboratory method for screening the transmembrane permeability of chemicals. Stemming from medicinal chemistry, PAMPA has the potential for use in the cost-effective high-throughput evaluation of chemicals of environmental concern. However, many chemicals of environmental concern differ substantially from pharmaceuticals in hydrophobicity and volatility. Here, we develop an in silico mass balance model to explore the impacts of chemical properties on chemical mass transfer in PAMPA and PAMPA's applicability to hydrophobic or volatile chemicals of environmental concern. The model's performance is evaluated by agreement between predicted and measured permeabilities of 1383 chemicals. The model predicts that the PAMPA measured permeability can be highly uncertain for hydrophobic chemicals because of considerable retention by the artificial membrane and for volatile chemicals because of substantial volatilization to the headspace. Notably, the permeabilities of hydrophobic chemicals are remarkably sensitive to specific experimental conditions, for example, the frequency of stirring and incubation time, challenging the comparison between measurements under different conditions. For hydrophobic chemicals, the PAMPA measured permeability may largely indicate the permeability of the unstirred water layer over the membrane, instead of the "intrinsic" permeability of the membrane, and therefore, may not be of interest for environmental exposure and risk assessments. The model also predicts that the time for mass transfer of highly hydrophobic chemicals to reach the steady state likely exceeds the incubation time, which violates the steady-state assumption used in calculating permeability from measured concentrations. Overall, our theoretical analysis underscores the importance to consider chemical properties when applying the current design of PAMPA to chemicals of environmental concern. [ABSTRACT FROM AUTHOR]

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling. • Dynamic activation of Nrf2 and NFĸB pathways was investigated using HepG2 reporters. • Soft electrophiles induce the Nrf2 pathway, yet inhibit the NFĸB response. • This opposite regulation is conserved between different hepatocyte test systems. [ABSTRACT FROM AUTHOR]