4-Nonylphenol (4-NP), a para-substituted phenolic compound mixture comprising a straight or branched carbon chain, is a widespread ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor with potent activities on estrogen nuclear receptors. Constitutive Androstane Receptor (CAR, NR1I3) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in endocrine disruption mechanism. The nonylphenol mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by NP are enigmatic and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR and its variants using molecular dynamics (MD) simulations, cellular experiments with various CAR constructs or CAR ligand binding domain (LBD) mutants, or in differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type (wt)CAR1 as well as CAR3 variant LBDs in MD simulations. Consistently, branched 4-NP activated CAR3 and wtCAR1 LBD more efficiently than linear 4-NP or tert-nonylphenol (22NP). Furthermore, in HepaRG cells, we observed that all NP significantly up-regulated CYP2B6, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and the human CAR-LBD, as well as its dominant variant CAR3 The work could contribute to the safer use of individual NPs in many areas of industry.

Background: Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of ⁶⁸Ga- and ^99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. Results: The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both ⁶⁸Ga- and ^99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. Conclusion: The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin. Graphical