In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes.

BACKGROUND: Cardiotoxicity remains one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to cardiac tissue. RESEARCH DESIGN AND METHODS: Non-clinical models with better predictive value need to be established to improve cardiac safety pharmacology. To this end, high-throughput RNA sequencing (ScreenSeq) was combined with high-content imaging (HCI) and Ca2+ transience (CaT) to analyze compound-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). RESULTS: Analysis of hiPSC-CMs treated with 33 cardiotoxicants and 9 non-cardiotoxicants of mixed therapeutic indications facilitated compound clustering by mechanism of action, scoring of pathway activities related to cardiomyocyte contractility, mitochondrial integrity, metabolic state, diverse stress responses and the prediction of cardiotoxicity risk. The combination of ScreenSeq, HCI and CaT provided a high cardiotoxicity prediction performance with 89% specificity, 91% sensitivity and 90% accuracy. CONCLUSIONS: Overall, this study introduces mechanism-driven risk assessment approach combining structural, functional and molecular high-throughput methods for pre-clinical risk assessment of novel compounds.

The evolution of strategies to minimise the risk of human drug-induced liver injury (DILI) in drug discovery and development.

Early identification of toxicity associated with new chemical entities (NCEs) is critical in preventing late-stage drug development attrition. Liver injury remains a leading cause of drug failures in clinical trials and post-approval withdrawals reflecting the poor translation between traditional preclinical animal models and human clinical outcomes. For this reason, preclinical strategies have evolved over recent years to incorporate more sophisticated human in vitro cell-based models with multi-parametric endpoints. This review aims to highlight the evolution of the strategies adopted to improve human hepatotoxicity prediction in drug discovery and compares/contrasts these with recent activities in our lab. The key role of human exposure and hepatic drug uptake transporters (e.g. OATPs, OAT2) is also elaborated.

An in vitro method to assess toxicity of waterborne metals to fish.

The transcription of metal-responsive genes in the rainbow trout (Oncorhynchus mykiss) gill tissue can be used to detect effects of bioreactive metals in natural waters. Here we take advantage of an in vitro gill epithelium, which can be directly exposed to test water samples. The in vitro gill epithelial model mimics the molecular response of in vivo gill epithelial cells to waterborne contaminants. The same culture system can detect trace metals and organic waterborne contaminants. Furthermore, combining this epithelial model with transcriptomic profiling yields an extremely discriminatory biomonitoring tool able to detect and differentiate waterborne metal contaminants. The bioreactive fraction of metal in the water sample is detected using the cells naturally occurring metal sensor, metal-responsive transcription factor 1 (MTF1), which acts upon Metal Response Elements (MRE's) in the enhancer region of metal regulated genes. Induction of the MTF1 responsive genes, metallothionein-A (MTA), metallothionein-B (MTB), and zinc transporter 1 (ZnT-1) in the cell culture was strongly dependent of the concentrations of bioreactive zinc and silver in the test water. Importantly, gene expression in cell culture reflected animal toxicity, measured as inhibition of Ca(2+) and Na(+) influx, in live rainbow trout exposed to the same waters. A cDNA microarray was deployed to determine the differential profiles of transcripts characteristic of exposure to silver, copper or cadmium within this in vitro system. These experiments illustrated the potential power of combining the in vitro gill model epithelium with genetic profiling for accurate characterisation and identification of bioreactive toxicants in waterborne samples.

Cortisol stimulates the zinc signaling pathway and expression of metallothioneins and ZnT1 in rainbow trout gill epithelial cells.

Intracellular zinc signaling is important in the control of a number of cellular processes. Hormonal factors that regulate cellular zinc influx and initiate zinc signals are poorly understood. The present study investigates the possibility for cross talk between the glucocorticoid and zinc signaling pathways in cultured rainbow trout gill epithelial cells. The rainbow trout metallothionein A (MTA) gene possesses a putative glucocorticoid response element and multiple metal response elements 1042 base pairs upstream of the start codon, whereas metallothionein B (MTB) and zinc transporter-1 (ZnT1) have multiple metal response elements but no glucocorticoid response elements in this region. Cortisol increased MTA, MTB, and ZnT1 gene expression, and this stimulation was enhanced if cells were treated with cortisol together with zinc. Cells treated with zinc showed increased zinc accumulation, transepithelial zinc influx (apical to basolateral), and intracellular labile zinc concentrations. These responses were also significantly enhanced in cells pretreated with cortisol and zinc. The cortisol-mediated effects were blocked by the glucocorticoid receptor (GR) antagonist RU-486, indicating mediation via a GR. In reporter gene assays, zinc stimulated MTA promoter activity, whereas cortisol did not. Furthermore, cortisol significantly reduced zinc-stimulated MTA promoter activity in cells expressing exogenous rainbow trout GR. These results demonstrate that cortisol enhances cellular zinc uptake, which in turn stimulates expression of MTA, MTB, and ZnT1 genes.

Influence of culture conditions on metal-induced responses in a cultured rainbow trout gill epithelium.

A primary culture technique for rainbow trout (Oncorhynchus mykiss) gill cells was optimized to better represent the intact gill in vivo in response to waterborne toxic metals. Modifications in cell seeding density and culture conditions resulted in a gill epithelial cell culture model, which displayed classic in vivo responses to toxic metals. Metallothionein-A (MTA), metallothionein-B (MTB), zinc transporter-1 (ZnT-1), glutathione-S-transferase (GST), and glucose-6-phosphate-dehydrogenase (G6PD) all showed dose-dependent increases in expression at the mRNA level in response to waterborne zinc. Of these genes, the change in zinc-induced expression relative to the control was greatest for MTA, MTB, and ZnT-1. MT expression was also induced by silver, lead, copper, and cadmium. Cells cultured with freshwater on the apical side maintained the net transepithelial influx of Ca2+ displayed by freshwater trout gills in vivo, and there was an active inward movement of Ca2+. Waterborne zinc applied to the apical compartment reduced the net uptake of Ca2+ by stimulating the efflux component. The use of endogenous metal-responsive gene expression and inhibition of ion transport in the developed cell culture system will facilitate studies of metal-gill interactions and may prove to have future practical applications within biomonitoring of natural waters.

Dietary phenolic antioxidants, caffeic acid and Trolox, protect rainbow trout gill cells from nitric oxide-induced apoptosis.

Caffeic acid (CA) and Trolox are phenolic acids that have beneficial antioxidant effect, but the underlying mechanisms involved are not fully understood. The extent to which CA and Trolox protect against sodium nitroprusside (SNP)-induced oxidative cell injury was investigated in cultured rainbow trout gill cells. The cells exposed to SNP for 24 h displayed a dose-dependent leakage of lactate dehydrogenase (LDH) and decreased cell viability as indicated by the MTT assay (mitochondrial dehydrogenase activity). Both effects were prevented by treatment with 50 microM CA or Trolox. CA or Trolox, protected against SNP-induced caspase-3 activation and DNA fragmentation, indicating a reduction of apoptosis. Thus, the results indicate that SNP induced cell death is caspase-3 related apoptosis and the treatment with CA inhibited the apoptotic pathway. In addition, we studied the effect of CA and Trolox on expression of zinc-responsive antioxidant genes such as metallothioneins (MT), glutathione-S-transferase (GST Class pi) and glucose-6-phosphate dehydrogenase (G6PD) in cultured gill cells. CA, 100 microM, increased accumulation of mRNA for MTA, MTB, GST and G6PD in cells. Thus, in addition to its ability to sequester free radicals, CA may protect against oxidative stress through expression of zinc-induced antioxidant proteins. Because of these properties we suggest that CA could be a beneficial additive to fish feeds in aquaculture.

ZINC-mediated gene expression offers protection against H2O2-induced cytotoxicity.

The ability of zinc to mobilize defense against reactive oxygen species (ROS) and H2O2-induced apoptosis was studied using a primary culture of rainbow trout gill cells. Gill cells were pretreated for 24 h with 100 microM ZnSO4 followed by 24-h exposure to 100 or 200 microM H2O2, or were subjected to 100 microM ZnSO4 together with 100 or 200 microM H2O2. Metallothionein-A (MTA) and metallothionein-B (MTB) mRNA levels were increased after treatment with zinc or H2O2, separately or in combination. Similarly, mRNA for glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD) were increased in response to either zinc or H2O2, or after sequential treatments with zinc followed by H2O2. The stimulatory effects of zinc or H2O2 on MTA, MTB, GST, and G6PD mRNA levels could be blocked by addition of the membrane permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that H2O2-induced upregulation of these genes is zinc-dependent. Pretreatment with zinc protected the cells from subsequent cell damage and apoptosis, as assessed by lactate dehydrogenase leakage, mitochondrial dehydrogenase activity (MTT assay), caspase-3 activity, and DNA fragmentation. In contrast, when gill cells were coincubated with zinc and H2O2 at the same time, H2O2 toxicity was higher than after treatment with H2O2 alone. It is concluded that zinc had a direct pro-oxidant effect when administered together with H2O2, but that pretreatment of zinc inhibited cytotoxicity and apoptosis through an indirect antioxidant action. We propose that the antioxidant action is manifested through zinc-dependent expression of several genes encoding antioxidant proteins (e.g., MTA, MTB, G6PD, and GST). Furthermore, the apparent zinc-dependency of H2O2-induced expression of antioxidant genes suggests that zinc might act as a physiological signal to mediate the response to oxidative stress.

Nutritive metal uptake in teleost fish.

Transition metals are essential for health, forming integral components of proteins involved in all aspects of biological function. However, in excess these metals are potentially toxic, and to maintain metal homeostasis organisms must tightly coordinate metal acquisition and excretion. The diet is the main source for essential metals, but in aquatic organisms an alternative uptake route is available from the water. This review will assess physiological, pharmacological and recent molecular evidence to outline possible uptake pathways in the gills and intestine of teleost fish involved in the acquisition of three of the most abundant transition metals necessary for life; iron, copper, and zinc.