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In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (f u ) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK. We compared three methods to quantify f u : rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log P ow (-0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin. After RED and UF separation, three polar substances (Log P ow < 2) were largely unbound (f u  > 70%), while more lipophilic substances were largely bound (f u  < 33%). Compared to RED or UF, UC resulted in a generally higher f u of lipophilic substances. f u obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in f u higher than the reference data. UF, RED and both UF and UC, resulted in lower f u of Flutamide, Ketoconazole and Colchicine, respectively. For f u quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robert Landsiedel reports financial support was provided by BASF SE. Eric Fabian, Dorothee Funk-Weyer Robert Landsiedel and Dunja Dimitrijevic reports a relationship with BASF SE that includes: employment, equity or stocks, and funding grants., (Copyright © 2023 Elsevier Inc. All rights reserved.)

We demonstrate that a proapoptotic chemical agent, oxidopamine, induces dose dependent changes in chromatin textural patterns which can be quantified using the Gray level co-occurrence matrix (GLCM) method. Peripheral blood (heparin-pretreated) samples were treated with oxidopamine (6-OHDA, 6-hydroxydopamine) to achieve effective concentrations of 100, 200 and 300µM. The samples were smeared on microscope slides and fixated in methanol. The smears were stained using a modification of Feulgen method for DNA visualization. For each stained smear, a sample of 30 lymphocyte chromatin structures were visualized and analyzed. This way, textural parameters for a total of 120 nuclei micrographs were calculated. For each chromatin structure, five different GLCM features were calculated: angular second moment, GLCM entropy, inverse difference moment, GLCM correlation, and GLCM variance. Oxidopamine induced the rise of the values of GLCM entropy and variance, and the reduction of angular second moment, correlation, and inverse difference moment. The trends for GLCM parameter changes were found to be highly significant (p<0.001). These results indicate that GLCM mathematical algorithm might be successfully used in detection and evaluation of discrete early apoptotic structural changes in Feulgen-stained chromatin of peripheral blood lymphocytes that are not detectable using conventional microscopy/cell biology techniques., (Copyright © 2016 Elsevier Ltd. All rights reserved.)