Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

Idiosyncratic drug-induced liver injury (IDILI) is a significant source of drug recall and acute liver failure (ALF) in the United States. While current drug development processes emphasize general toxicity and drug metabolizing enzyme- (DME-) mediated toxicity, it has been challenging to develop comprehensive models for assessing complete idiosyncratic potential. In this review, we describe the enzymes and proteins that contain polymorphisms believed to contribute to IDILI, including ones that affect phase I and phase II metabolism, antioxidant enzymes, drug transporters, inflammation, and human leukocyte antigen (HLA). We then describe the various assays that have been developed to detect individual reactions focusing on each of the mechanisms described in the background. Finally, we examine current trends in developing comprehensive models for examining these mechanisms. There is an urgent need to develop a panel of multiparametric assays for diagnosing individual toxicity potential

Biophysical and Biomechanical Properties of Neural Progenitor Cells as Indicators of Developmental Neurotoxicity

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young\u27s modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p \u3c 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p \u3c 0.01) as well as toxicant concentration (p \u3c 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R-2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes

Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

Idiosyncratic drug-induced liver injury (IDILI) is a significant source of drug recall and acute liver failure (ALF) in the United States. While current drug development processes emphasize general toxicity and drug metabolizing enzyme- (DME-) mediated toxicity, it has been challenging to develop comprehensive models for assessing complete idiosyncratic potential. In this review, we describe the enzymes and proteins that contain polymorphisms believed to contribute to IDILI, including ones that affect phase I and phase II metabolism, antioxidant enzymes, drug transporters, inflammation, and human leukocyte antigen (HLA). We then describe the various assays that have been developed to detect individual reactions focusing on each of the mechanisms described in the background. Finally, we examine current trends in developing comprehensive models for examining these mechanisms. There is an urgent need to develop a panel of multiparametric assays for diagnosing individual toxicity potential

Sensitivity of Neural Stem Cell Survival, Differentiation and Neurite Outgrowth Within 3D Hydrogels to Environmental Heavy Metals

© 2015 Elsevier Ireland Ltd. We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM-10. μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥10. nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤100. pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥100. pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead. \u3e. mercury. \u3e. cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways

Enzyme Attached on Polymeric Micelles as a Nanoscale Reactor

Similar to what lipase does, a surface-active enzyme was developed by attaching peroxidase on combshaped polymaleic anhydride-alt-1-tetradecene (PMA-TD) in a microemulsion system composed of n-butyl acetate and buffer solution, and its catalytic characteristics of polyphenol synthesis were investigated in an aqueous solution. The modified peroxidase with PMA-TD tended to form self-assembled aggregates like micelles in the aqueous solution and could be concentrated at solvent/water interfaces without unfolding of the enzyme. The efficiency of conversion of 2,4-dichlorophenol to phenolic oligomers was approximately 2-fold improved with the modified peroxidase compared to native peroxidase. The K m and V max values for the modified peroxidase were 1.5-fold lower and 2-fold higher, respectively. The hydrodynamic diameter of the micelle on the modified peroxidase increased with the reaction time, indicating that phenolic products were accumulated in the hydrophobic interior of micelles. In addition, the molecular weight (MW) of phenolic polymers was much larger in the system with the modified peroxidase. These observations implied that the modified peroxidase with hydrophobic side chains formed micellar structures by solubilization of phenolic products and further polymerization reaction could occur in the hydrophobic interior of the micelles

Enzyme Attached on Polymeric Micelles as a Nanoscale Reactor

Similar to what lipase does, a surface-active enzyme was developed by attaching peroxidase on combshaped polymaleic anhydride-alt-1-tetradecene (PMA-TD) in a microemulsion system composed of n-butyl acetate and buffer solution, and its catalytic characteristics of polyphenol synthesis were investigated in an aqueous solution. The modified peroxidase with PMA-TD tended to form self-assembled aggregates like micelles in the aqueous solution and could be concentrated at solvent/water interfaces without unfolding of the enzyme. The efficiency of conversion of 2,4-dichlorophenol to phenolic oligomers was approximately 2-fold improved with the modified peroxidase compared to native peroxidase. The K m and V max values for the modified peroxidase were 1.5-fold lower and 2-fold higher, respectively. The hydrodynamic diameter of the micelle on the modified peroxidase increased with the reaction time, indicating that phenolic products were accumulated in the hydrophobic interior of micelles. In addition, the molecular weight (MW) of phenolic polymers was much larger in the system with the modified peroxidase. These observations implied that the modified peroxidase with hydrophobic side chains formed micellar structures by solubilization of phenolic products and further polymerization reaction could occur in the hydrophobic interior of the micelles

3D Cultures of Human Liver Cell Lines Encapsulated in PuraMatrix on a Microarray Chip Platform

A high-throughput cell printing technology has developed to simulate the liver tissue environment using a hydrogel-based chip platform that has potential to shift in vivo drug toxicity models towards in vitro tests. However, the hydrophobic nature of polystyrene chips is not promoting direct adhesion of hydrogels, which created a problem with spot attachment. The main goal of this research is to create a surface chemistry that helps to attach a peptide-based hydrogel, including PuraMatrix, to a polystyrenebased micropillar chip. Seven analogs of maleic anhydride co-polymers were used to coat the micropillar chip to create a functional surface. Then, six ionic solutions were tested for inducing gelation of PuraMatrix. Formation of bubbles and spot detachment on the chip platform was quantified. As a result, an optimum polymer, PMA-OD was selected for surface attachment based on its low bubble formation and high spot attachment. This polymer could easily coat the chip for better gel adhesion. In regards to the gelation of PuraMatrix, poly-L-lysine was the most favorable for spot attachment and cell viability on the chip platform. In future research, encapsulated human liver cells expressing drug metabolizing enzymes will be tested with different drugs to determine mechanisms of drug toxicity.https://engagedscholarship.csuohio.edu/u_poster_2014/1019/thumbnail.jp

High-Content, 3D Cell Culture Assays on a Micropillar/Microwell Chip Platform

High content imaging (HCI) is a multi-parametric assay using multiple fluorescent dyes that are relevant to specific cell functions. The HCI assays provide an insight into the mechanisms of toxic drug responses, thus enhancing predictability of drug toxicity. However, current HCI assays are performed on 2D cell monolayer cultures which are physiologically irrelevant, creating a new opportunity for better predictable 3D HCI assays. The goal of this research is to develop HCI assays on 3D cellular microarrays that can be implemented for various toxicity screening, leading to classification of drug toxicity via investigating profiles of cell injury. As a model system, Hep3B human liver cells were dispensed onto a micropillar chip with a microarray spotter, which were exposed to various concentrations of model drugs. The chip containing the cells was then stained with multiple fluorescent dyes and scanned with a chip scanner to measure different end points. Conclusively, HCI assays performed on the 3D cellular microarrays showed a capability to identify several mechanisms of toxic drug responses. The mechanisms including DNA and mitochondrial impairment, calcium homeostasis, and glutathione conjugation were successfully demonstrated on the micropillar/microwell chip platform. Computational algorithms along with additional assays will be developed for enhanced predictability.https://engagedscholarship.csuohio.edu/u_poster_2014/1017/thumbnail.jp

High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology

Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

Idiosyncratic drug-induced liver injury (IDILI) is a significant source of drug recall and acute liver failure (ALF) in the United States. While current drug development processes emphasize general toxicity and drug metabolizing enzyme- (DME-) mediated toxicity, it has been challenging to develop comprehensive models for assessing complete idiosyncratic potential. In this review, we describe the enzymes and proteins that contain polymorphisms believed to contribute to IDILI, including ones that affect phase I and phase II metabolism, antioxidant enzymes, drug transporters, inflammation, and human leukocyte antigen (HLA). We then describe the various assays that have been developed to detect individual reactions focusing on each of the mechanisms described in the background. Finally, we examine current trends in developing comprehensive models for examining these mechanisms. There is an urgent need to develop a panel of multiparametric assays for diagnosing individual toxicity potential