The integrated stress response-related expression of CHOP due to mitochondrial toxicity is a warning sign for DILI liability.

BACKGROUND AND AIMS: Drug-induced liver injury (DILI) is one of the most frequent reasons for failure of drugs in clinical trials or market withdrawal. Early assessment of DILI risk remains a major challenge during drug development. Here, we present a mechanism-based weight-of-evidence approach able to identify certain candidate compounds with DILI liabilities due to mitochondrial toxicity. METHODS: A total of 1587 FDA-approved drugs and 378 kinase inhibitors were screened for cellular stress response activation associated with DILI using an imaging-based HepG2 BAC-GFP reporter platform including the integrated stress response (CHOP), DNA damage response (P21) and oxidative stress response (SRXN1). RESULTS: In total 389, 219 and 104 drugs were able to induce CHOP-GFP, P21-GFP and SRXN1-GFP expression at 50 µM respectively. Concentration response analysis identified 154 FDA-approved drugs as critical CHOP-GFP inducers. Based on predicted and observed (pre-)clinical DILI liabilities of these drugs, nine antimycotic drugs (e.g. butoconazole, miconazole, tioconazole) and 13 central nervous system (CNS) agents (e.g. duloxetine, fluoxetine) were selected for transcriptomic evaluation using whole-genome RNA-sequencing of primary human hepatocytes. Gene network analysis uncovered mitochondrial processes, NRF2 signalling and xenobiotic metabolism as most affected by the antimycotic drugs and CNS agents. Both the selected antimycotics and CNS agents caused impairment of mitochondrial oxygen consumption in both HepG2 and primary human hepatocytes. CONCLUSIONS: Together, the results suggest that early pre-clinical screening for CHOP expression could indicate liability of mitochondrial toxicity in the context of DILI, and, therefore, could serve as an important warning signal to consider during decision-making in drug development.

Protectiveness of NAM-based hazard assessment - which testing scope is required?

Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.

Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.

Correction for both common and rare cell types in blood is important to identify genes that correlate with age.

BACKGROUND: Aging is a multifactorial process that affects multiple tissues and is characterized by changes in homeostasis over time, leading to increased morbidity. Whole blood gene expression signatures have been associated with aging and have been used to gain information on its biological mechanisms, which are still not fully understood. However, blood is composed of many cell types whose proportions in blood vary with age. As a result, previously observed associations between gene expression levels and aging might be driven by cell type composition rather than intracellular aging mechanisms. To overcome this, previous aging studies already accounted for major cell types, but the possibility that the reported associations are false positives driven by less prevalent cell subtypes remains. RESULTS: Here, we compared the regression model from our previous work to an extended model that corrects for 33 additional white blood cell subtypes. Both models were applied to whole blood gene expression data from 3165 individuals belonging to the general population (age range of 18-81 years). We evaluated that the new model is a better fit for the data and it identified fewer genes associated with aging (625, compared to the 2808 of the initial model; P ≤ 2.5⨯10-6). Moreover, 511 genes (~ 18% of the 2808 genes identified by the initial model) were found using both models, indicating that the other previously reported genes could be proxies for less abundant cell types. In particular, functional enrichment of the genes identified by the new model highlighted pathways and GO terms specifically associated with platelet activity. CONCLUSIONS: We conclude that gene expression analyses in blood strongly benefit from correction for both common and rare blood cell types, and recommend using blood-cell count estimates as standard covariates when studying whole blood gene expression.

Regulation of the mitochondrial permeability transition pore and its effects on aging.

Aging is an evolutionarily conserved process and is tightly connected to mitochondria. To uncover the aging molecular mechanisms related to mitochondria, different organisms have been extensively used as model systems. Among these, the budding yeast Saccharomyces cerevisiae has been reported multiple times as a model of choice when studying cellular aging. In particular, yeast provides a quick and trustworthy system to identify shared aging genes and pathway patterns. In this viewpoint on aging and mitochondria, I will focus on the mitochondrial permeability transition pore (mPTP), which has been reported and proposed as a main player in cellular aging. I will make several parallelisms with yeast to highlight how this unicellular organism can be used as a guidance system to understand conserved cellular and molecular events in multicellular organisms such as humans. Overall, a thread connecting the preservation of mitochondrial functionality with the activity of the mPTP emerges in the regulation of cell survival and cell death, which in turn could potentially affect aging and aging-related diseases.

Nicotinamide supplementation phenocopies SIR2 inactivation by modulating carbon metabolism and respiration during yeast chronological aging.

Nicotinamide (NAM), a form of vitamin B3, is a byproduct and noncompetitive inhibitor of the deacetylation reaction catalyzed by Sirtuins. These represent a family of evolutionarily conserved NAD+-dependent deacetylases that are well-known critical regulators of metabolism and aging and whose founding member is Sir2 of Saccharomyces cerevisiae. Here, we investigated the effects of NAM supplementation in the context of yeast chronological aging, the established model for studying aging of postmitotic quiescent mammalian cells. Our data show that NAM supplementation at the diauxic shift results in a phenocopy of chronologically aging sir2Δ cells. In fact, NAM-supplemented cells display the same chronological lifespan extension both in expired medium and extreme Calorie Restriction. Furthermore, NAM allows the cells to push their metabolism toward the same outcomes of sir2Δ cells by elevating the level of the acetylated Pck1. Both these cells have the same metabolic changes that concern not only anabolic pathways such as an increased gluconeogenesis but also respiratory activity in terms both of respiratory rate and state of respiration. In particular, they have a higher respiratory reserve capacity and a lower non-phosphorylating respiration that in concert with a low burden of superoxide anions can affect positively chronological aging.