Deriving a point of departure for assessing the skin sensitization risk of wearable device constituents with in vitro methods.

Wearable devices are in contact with the skin for extended periods. As such, the device constituents should be evaluated for their skin sensitization potential, and a Point of Departure (PoD) should be derived to conduct a proper risk assessment. Without historical in vivo data, the PoD must be derived with New Approach Methods (NAMs). To accomplish this, regression models trained on LLNA data that use data inputs from OECD-validated in vitro tests were used to derive a predicted EC3 value, the LLNA value used to classify skin sensitization potency, for three adhesive monomers (Isobornyl acrylate (IBOA), N, N- Dimethylacrylamide (NNDMA), and Acryloylmorpholine (ACMO) and one dye (Solvent Orange 60 (SO60)). These chemicals can be used as constituents of wearable devices and have been associated with causing allergic contact dermatitis (ACD). Using kinetic DPRA and KeratinoSens™ data, the PoDs obtained with the regression model were 180, 215, 1535, and 8325 µg/cm2 for IBOA, SO60, ACMO, and NNDMA, respectively. The PoDs derived with the regression model using NAMs data will enable a proper skin sensitization risk assessment without using animals.

A novel imidazolinone metformin-methylglyoxal metabolite promotes endothelial cell angiogenesis via the eNOS/HIF-1α pathway.

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.

Toxicoproteomic Analysis of Poly(ADP-Ribose)-Associated Proteins Induced by Oxidative Stress in Human Proximal Tubule Cells.

2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ generates reactive oxygen species (ROS), causing DNA-strand breaks, hyperactivation of PARP-1, increases in intracellular calcium ([Ca2+]i), and cell death. PARP-1 catalyzes the attachment of ADP-ribose polymers (PAR) to target proteins. In human kidney proximal tubule cells, ROS-mediated PARP-1 hyperactivation and elevations in [Ca2+]i are reciprocally coupled. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify ROS-induced PAR-associated proteins to further understand their potential role in cell death. PAR-associated proteins were enriched by immunoprecipitation, identified by LC-MS/MS, and relative abundance was obtained by spectral counting. A total of 356 proteins were PAR-modified following TGHQ treatment. A total of 13 proteins exhibited gene ontology annotations related to calcium. Among these proteins, the general transcription factor II-I (TFII-I) is directly involved in the modulation of [Ca2+]i. TFII-I binding to phospholipase C (PLC) leads to calcium influx via the TRPC3 channel. However, inhibition of TRPC3 or PLC had no effect on TGHQ-mediated cell death, suggesting that their loss of function may be necessary but insufficient to cause cell death. Nevertheless, TGHQ promoted a time-dependent translocation of TFII-I from the nucleus to the cytosol concomitant with a decrease in tyrosine phosphorylation in α/ß-TFII-I. Therefore it is likely that ROS have an important impact on the function of TFII-I, such as regulation of transcription, and DNA translesion synthesis. Our data also shed light on PAR-mediated signaling during oxidative stress, and contributes to the development of strategies to prevent PAR-dependent cell death.

From the Cover: ROS-Induced Store-Operated Ca2+ Entry Coupled to PARP-1 Hyperactivation Is Independent of PARG Activity in Necrotic Cell Death.

2,3,5-tris(Glutathion-S-yl)hydroquinone, a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS) causing DNA strand breaks and the subsequent activation of DNA repair enzymes, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative DNA damage, PARP-1 is hyperactivated, resulting in the depletion of NAD+ and ATP with accompanying elevations in intracellular calcium concentrations (iCa2+), and ultimately necrotic cell death. The role of Ca2+ during PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca2+ and PARP-1 during ROS-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Our experiments suggest that store-operated Ca2+ channel (SOC) entry contributes to the coupling of PARP-1 activation to increases in iCa2+ during ROS-induced cell death. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate Ca2+ channels such as TRPM2. However, siRNA knockdown of PARG did not restore cell viability, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. The data indicate that PARP-1 and iCa2+ are coupled through activation of SOC mediated Ca2+ entry in an apparently ADPR-independent fashion; alternative PAR-mediated signaling likely contributes to PARP-dependent necrotic cell death, perhaps via PAR-mediated signaling proteins that regulate iCa2+ homeostasis.

In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope.

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses.