Oxidative Stress in Drug-Induced Liver Injury (DILI): From Mechanisms to Biomarkers for Use in Clinical Practice

Idiosyncratic drug-induced liver injury (DILI) is a type of hepatic injury caused by an uncommon drug adverse reaction that can develop to conditions spanning from asymptomatic liver laboratoryabnormalitiestoacuteliverfailure(ALF)anddeath.Thecellularandmolecularmecha- nisms involved in DILI are poorly understood. Hepatocyte damage can be caused by the metabolic activation of chemically active intermediate metabolites that covalently bind to macromolecules (e.g., proteins, DNA), forming protein adducts—neoantigens—that lead to the generation of oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, which can eventually lead to cell death. In parallel, damage-associated molecular patterns (DAMPs) stimulate the immune response, whereby inflammasomes play a pivotal role, and neoantigen presentation on specific human leukocyte antigen (HLA) molecules trigger the adaptive immune response. A wide array of antioxidant mechanisms exists to counterbalance the effect of oxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which are pivotal in detoxification. These get compromised during DILI, triggering an imbalance between oxidants and antioxidants defense systems, generating oxidative stress. As a result of exacerbated oxidative stress, several danger signals, including mitochondrial damage, cell death, and inflammatory markers, and microRNAs (miRNAs) related to extracellular vesicles (EVs) have already been reported as mechanis- tic biomarkers. Here, the status quo and the future directions in DILI are thoroughly discussed, with a special focus on the role of oxidative stress and the development of new biomarkers.This work was supported by the MINECO Retos SAF2016-78711, EXOHEP-CM S2017/BMD- 3727, NanoLiver-CM Y2018/NMT-4949, ERAB Ref. EA 18/14, AMMF 2018/117, FIS-FEDER PI16_01748, PI19-00883, UMA18-FEDERJA-194, PY18-3364_PY19 and UCM-25-2019. FJC is a Ramón y Cajal Researcher RYC-2014-15242 and a Gilead Liver Research 2018. The research group belongs to the validated Research Groups Ref. 970935 “Liver Pathophysiology” and 920631 “Lymphocyte immunobiology” and IBL-6 (imas12-associated). This article/publication is based upon work from COST Action “CA17112—Prospective European Drug-Induced Liver Injury Network” supported by COST (European Cooperation in Science and Technology); www.cost.eu; accessed 4 March 2021. CIBERehd is funded by ISCiii

Hemp (Cannabis sativa L.) Protein Hydrolysates Promote Anti-Inflammatory Response in Primary Human Monocytes

Hemp seeds have a wide variety of chemical compounds which present biological activity. Specifically, the focus on proteins and bioactive peptides are increasing as alternative sources of nutraceutical uses. In the literature, hemp protein products (HPPs) have reported antioxidant and anti-inflammatory properties. This study aimed to determine the inflammation-related modulatory effects of HPPs on lipopolysaccharide (LPS)-activated primary human monocytes. CD14+ cells were immunomagnetically isolated from buffy coats and the anti-inflammatory activity of hemp protein isolate (HPI) and hydrolysates (HPHs) was evaluated on LPS-stimulated human primary monocytes. The specific markers of inflammation, polarization, and chemoattraction were measured by RT-qPCR and ELISA assays. Our results showed that HPPs decreased the pro-inflammatory mediators (TNF-α, IL-1β, and IL-6) and increased the anti-inflammatory mediators (IL-10 and IL-4). In addition, M1 polarization marker gene expression (CCR7 and iNOS) was downregulated by HPPs and, M2 polarization marker gene expression (CD200R and MRC1) was upregulated. Finally, the mRNA expression of chemotaxis genes (CCR2 and CCL2) was downregulated by HPPs. In conclusion, this study suggests that HPPs may improve chronic inflammatory states and promote regenerative processes by reprogramming monocytes toward M2 polarization phenotypeMinisterio de Ciencia, innovación y Universidades CYTED-2019 119RT056

Critical Review of Gaps in the Diagnosis and Management of Drug-Induced Liver Injury Associated with Severe Cutaneous Adverse Reactions

Drug-induced liver injury (DILI) encompasses the unexpected damage that drugs can cause to the liver. DILI may develop in the context of an immunoallergic syndrome with cutaneous manifes- tations, which are sometimes severe (SCARs). Nevirapine, allopurinol, anti-epileptics, sulfonamides, and antibiotics are the most frequent culprit drugs for DILI associated with SCARs. Interestingly, alleles HLA-B*58:01 and HLA-A*31:01 are associated with both adverse reactions. However, there is no consensus about the criteria used for the characterization of liver injury in this context, and the different thresholds for DILI definition make it difficult to gain insight into this complex disorder. Moreover, current limitations when evaluating causality in patients with DILI associated with SCARs are related to the plethora of causality assessment methods and the lack of consensual complementary tools. Finally, the management of this condition encompasses the treatment of liver and skin injury. Although the use of immunomodulant agents is accepted for SCARs, their role in treating liver injury remains controversial. Further randomized clinical trials are needed to test their efficacy and safety to address this complex entity. Therefore, this review aims to identify the current gaps in the definition, diagnosis, prognosis, and management of DILI associated with SCARs, proposing different strategies to fill in these gaps.Instituto de Salud Carlos III: PI18/01804 Instituto de Salud Carlos III: PI19-00883; Instituto de Salud Carlos III: PT 20/00127; Instituto de Salud Carlos III: UMA18-FEDERJA-194; Instituto de Salud Carlos III: PY18-3364; Consejería de Salud de Andalucía: PI-0310-2018, PEMP-0127-2020; Instituto de Salud Carlos III: Rio Hortega CM17/00243; Instituto de Salud Carlos III: Sara Borrell CD20/00083; Instituto de Salud Carlos III: Sara Borrell CD21/00198

Antihypertensive and Antioxidant Activity of Chia Protein Techno-Functional Extensive Hydrolysates

Twelve high-quality chia protein hydrolysates (CPHs) were produced from chia protein isolate (CPI) in a pilot plant of vegetable proteins. To obtain functional hydrolysate, four CPHs were hydrolyzed by the action of Alcalase, an endoprotease, and the other eight CPHs were hydrolyzed by the action of Flavourzyme, an exoprotease. Alcalase-obtained CPHs showed significant antihypertensive properties particularly, the CPH obtained after 15 min of hydrolysis with Alcalase (CPH15A), which showed a 36.2% hydrolysis degree. In addition, CPH15A increased the antioxidant capacity compared to CPI. The CPH15A physicochemical composition was characterized and compared to chia defatted flour (CDF) and CPI, and its techno-functional properties were determined by in vitro experiments through the analysis of its oil absorption capacity, as well as the capacity and stability of foaming and emulsifying, resulting in an emulsifier and stabilizer better than the intact protein. Therefore, the present study revealed that CPH15A has potent antihypertensive and antioxidant properties and can constitute an effective alternative to other plant protein ingredients sources that are being used in the food industry.Spanish Ministry of Science, Innovation, and Universities grant CYTED-2019/119RT056

Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease

Background Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal β-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. Results Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. Conclusion These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.info:eu-repo/semantics/publishedVersio

Control compounds for preclinical drug-induced liver injury assessment: Consensus-driven systematic review by the ProEuroDILI network

Background & Aims Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). Methods Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified ‘Toxicological Data Reliability Assessment Tool’. Results A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. Conclusions Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed.Funding for open access charge: Universidad de Málaga / CBUA

Epigenetic inactivation of the splicing RNA-binding protein CELF2 in human breast cancer

Altres ajuts: This work was co-finaced by the European Development Regional Fund, "A way to achieve Europe" ERDF; the Cellex Foundation; and "la Caixa" Banking Foundation (LCF/PR/PR15/ 11100003).Human tumors show altered patterns of protein isoforms that can be related to the dysregulation of messenger RNA alternative splicing also observed in transformed cells. Although somatic mutations in core spliceosome components and their associated factors have been described in some cases, almost nothing is known about the contribution of distorted epigenetic patterns to aberrant splicing. Herein, we show that the splicing RNA-binding protein CELF2 is targeted by promoter hypermethylation-associated transcriptional silencing in human cancer. Focusing on the context of breast cancer, we also demonstrate that CELF2 restoration has growth-inhibitory effects and that its epigenetic loss induces an aberrant downstream pattern of alternative splicing, affecting key genes in breast cancer biology such as the autophagy factor ULK1 and the apoptotic protein CARD10. Furthermore, the presence of CELF2 hypermethylation in the clinical setting is associated with shorter overall survival of the breast cancer patients carrying this epigenetic lesion

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License.Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.This work was supported by FIS PI10/00543 grant, FIS EC08/00076 grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS 111242 grant, Servicio Andaluz de Salud Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, and by AEPMI (Asociación de Enfermos de Patología Mitocondrial).Peer reviewe