TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation.

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Visualizing reactive astrocyte-neuron interaction in Alzheimer's disease using 11C-acetate and 18F-FDG.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Adeno-associated virus (AAV) 9-mediated gene delivery of Nurr1 and Foxa2 ameliorates symptoms and pathologies of Alzheimer disease model mice by suppressing neuro-inflammation and glial pathology.

There is a compelling need to develop disease-modifying therapies for Alzheimer's disease (AD), the most common neuro-degenerative disorder. Together with recent progress in vector development for efficiently targeting the central nervous system, gene therapy has been suggested as a potential therapeutic modality to overcome the limited delivery of conventional types of drugs to and within the damaged brain. In addition, given increasing evidence of the strong link between glia and AD pathophysiology, therapeutic targets have been moving toward those addressing glial cell pathology. Nurr1 and Foxa2 are transcription/epigenetic regulators that have been reported to cooperatively regulate inflammatory and neurotrophic response in glial cells. In this study, we tested the therapeutic potential of Nurr1 and Foxa2 gene delivery to treat AD symptoms and pathologies. A series of functional, histologic, and transcriptome analyses revealed that the combined expression of Nurr1 and Foxa2 substantially ameliorated AD-associated amyloid ß and Tau proteinopathy, cell senescence, synaptic loss, and neuro-inflammation in multiple in vitro and in vivo AD models. Intra-cranial delivery of Nurr1 and Foxa2 genes using adeno-associated virus (AAV) serotype 9 improved the memory and cognitive function of AD model mice. The therapeutic benefits of gene delivery were attained mainly by correcting pathologic glial function. These findings collectively indicate that AAV9-mediated Nurr1 and Foxa2 gene transfer could be an effective disease-modifying therapy for AD.

LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

DDC-Promoter-Driven Chemogenetic Activation of SNpc Dopaminergic Neurons Alleviates Parkinsonian Motor Symptoms.

Parkinson's disease (PD) is a neurodegenerative disorder with typical motor symptoms. Recent studies have suggested that excessive GABA from reactive astrocytes tonically inhibits dopaminergic neurons and reduces the expression of tyrosine hydroxylase (TH), the key dopamine-synthesizing enzyme, in the substantia nigra pars compacta (SNpc). However, the expression of DOPA decarboxylase (DDC), another dopamine-synthesizing enzyme, is relatively spared, raising a possibility that the live but non-functional TH-negative/DDC-positive neurons could be the therapeutic target for rescuing PD motor symptoms. However, due to the absence of a validated DDC-specific promoter, manipulating DDC-positive neuronal activity has not been tested as a therapeutic strategy for PD. Here, we developed an AAV vector expressing mCherry under rat DDC promoter (AAV-rDDC-mCherry) and validated the specificity in the rat SNpc. Modifying this vector, we expressed hM3Dq (Gq-DREADD) under DDC promoter in the SNpc and ex vivo electrophysiologically validated the functionality. In the A53T-mutated alpha-synuclein overexpression model of PD, the chemogenetic activation of DDC-positive neurons in the SNpc significantly alleviated the parkinsonian motor symptoms and rescued the nigrostriatal TH expression. Altogether, our DDC-promoter will allow dopaminergic neuron-specific gene delivery in rodents. Furthermore, we propose that the activation of dormant dopaminergic neurons could be a potential therapeutic strategy for PD.

COVID-19 Prognosis in Association with Antidepressant Use.

INTRODUCTION: Various subtypes of severe acute respiratory syndrome coronavirus 2 and variations among immune systems in different ethnicities need to be considered to understand the outcomes of coronavirus disease 2019 (COVID-19). This study aimed to provide evidence for the association between the use of antidepressants and the severity of COVID-19. METHODS: We used the National Health Information Data-COVID database. Patients with one or more prescriptions of any antidepressant were selected as the exposure group. Detailed analyses were performed to determine the type of medication associated with the prognosis. RESULTS: The use of selective serotonin reuptake inhibitors (SSRIs) was associated with a lower risk of severe outcomes of COVID-19, whereas the use of tricyclic antidepressants (TCAs) increased the risk of poor prognosis of COVID-19. Detailed analyses showed that escitalopram was significantly associated with better clinical outcomes, and nortriptyline was linked to more severe COVID-19 outcomes. CONCLUSION: This study revealed an association between antidepressants and COVID-19 prognosis. SSRIs were significantly associated with a lower risk of severe outcomes, whereas TCAs were related to the poor prognosis of COVID-19.

ANO1/TMEM16A regulates process maturation in radial glial cells in the developing brain.

Neural stem cells (NSCs) are primary progenitor cells in the early developmental stage in the brain that initiate a diverse lineage of differentiated neurons and glia. Radial glial cells (RGCs), a type of neural stem cell in the ventricular zone, are essential for nurturing and delivering new immature neurons to the appropriate cortical target layers. Here we report that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated chloride channel, mediates the Ca2+-dependent process extension of RGCs. ANO1 is highly expressed and functionally active in RGCs of the mouse embryonic ventricular zone. Knockdown of ANO1 suppresses RGC process extension and protrusions, whereas ANO1 overexpression stimulates process extension. Among various trophic factors, brain-derived neurotrophic factor (BDNF) activates ANO1, which is required for BDNF-induced process extension in RGCs. More importantly, Ano1-deficient mice exhibited disrupted cortical layers and reduced cortical thickness. We thus conclude that the regulation of RGC process extension by ANO1 contributes to the normal formation of mouse embryonic brain.

Role of Autophagy in the Pathogenesis of Diabetes and Therapeutic Potential of Autophagy Modulators in the Treatment of Diabetes and Metabolic Syndrome.

Autophagy is critically involved in the maintenance of intracellular nutrient homeostasis and organelle function. Dysregulated autophagy is likely to play a role in the development of metabolic disorders and diabetes because autophagy is critical in the rejuvenation of dysfunctional or stressed endoplasmic reticulum and mitochondria that play a crucial role in the development of diabetes. Indeed, systemic autophagy insufficiency led to the increased tissue lipid content, aggravated metabolic and finally more severe diabetes when metabolic stress was imposed, suggesting that autophagy insufficiency of dysfunction of lysosome, an effector organelle of autophagy, due to aging, genetic predisposition or environmental factors could be an underlying cause of diabetes. Conversely, autophagy enhancer could improve metabolic profile of obese mice by reducing tissue lipid content and ameliorating metabolic inflammation. Furthermore, clearance of human islet amyloid polypeptide (hIAPP) oligomer and amyloid that accumulate in pancreatic islets of > 90% of diabetes patients was also dependent on autophagy. Consistently, autophagy enhancer could improve glucose profile and ß-cell function of transgenic mice expressing amyloidogenic hIAPP in pancreatic ß-cells, which was accompanied by reduced accumulation of hIAPP oligomer or amyloid, ameliorated ß-cell apoptosis and increased ß-cell mass. These results suggest that autophagy enhancer could be a novel therapeutic modality against diabetes associated with lipid overload and human diabetes characterized by islet amyloid accumulation.

The Application of Mass Spectrometry in Drug Metabolism and Pharmacokinetics.

Drug metabolism and pharmacokinetics (DMPK) are fundamental in drug discovery. New chemical entities (NCEs) are typically evaluated with various in vitro and in vivo assays, which are time-consuming and labor intensive. These experiments are essential in identifying potential new drugs. Recently, mass spectrometry (MS) has played a key role in examining the drug-like properties of NCEs. Quantitative and qualitative mass spectrometry approaches are routinely utilized to obtain high-quality data in an efficient, timely, and cost-effective manner. Especially, liquid chromatography (LC) coupled with MS technology has been refined for metabolite identification (Met ID), which is critical for lead optimization. These qualitative and quantitative MS approaches and their specific utility in DMPK characterization will be described in this chapter.

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice.

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

Minimalistic Principles for Designing Small Molecules with Multiple Reactivities against Pathological Factors in Dementia.

Multiple pathogenic elements, including reactive oxygen species, amyloidogenic proteins, and metal ions, are associated with the development of neurodegenerative disorders. We report minimalistic redox-based principles for preparing compact aromatic compounds by derivatizing the phenylene moiety with various functional groups. These molecular agents display enhanced reactivities against multiple targets such as free radicals, metal-free amyloid-ß (Aß), and metal-bound Aß that are implicated in the most common form of dementia, Alzheimer's disease (AD). Mechanistic studies reveal that the redox properties of these reagents are essential for their function. Specifically, they engage in oxidative reactions with metal-free and metal-bound Aß, leading to chemical modifications of the Aß peptides to form covalent adducts that alter the aggregation of Aß. Moreover, the administration of the most promising candidate significantly attenuates the amyloid pathology in the brains of AD transgenic mice and improves their cognitive defects. Our studies demonstrate an efficient and effective redox-based strategy for incorporating multiple functions into simple molecular reagents.

Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids.

BACKGROUND & AIMS: The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed. METHODS: We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses. RESULTS: Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis. CONCLUSIONS: Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine. LAY SUMMARY: A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.

Developing scalable cultivation systems of hepatic spheroids for drug metabolism via genomic and functional analyses.

Spheroids, a widely used three-dimensional (3D) culture model, are standard in hepatocyte culture as they preserve long-term hepatocyte functionality and enhance survivability. In this study, we investigated the effects of three operation modes in 3D culture - static, orbital shaking, and under vertical bidirectional flow using spheroid forming units (SFUs) on hepatic differentiation and drug metabolism to propose the best for mass production of functionally enhanced spheroids. Spheroids in SFUs exhibited increased hepatic gene expression, albumin secretion, and cytochrome P450 3A4 (CYP3A4) activity during the differentiation period (12 days). SFUs advantages include facilitated mass production and a relatively earlier peak of CYP3A4 activity. However, CYP3A4 activity was not well maintained under dimethyl sulfoxide (DMSO)-free conditions (13-18 days), dramatically reducing drug metabolism capability. Continued shear stimulation without differentiation stimuli in assay conditions markedly attenuated CYP3A4 activity, which was less severe in static conditions. In this condition, SFU spheroids exhibited dedifferentiation characteristics, such as increased proliferation and Notch signaling genes. We found that the dedifferentiation could be overcome by using the serum-free medium formulation. Therefore, we suggest that SFUs represent the best option for the mass production of functionally improved spheroids and so the serum-free conditions should be maintained during drug metabolism analysis.

Rebound burst firing in the reticular thalamus is not essential for pharmacological absence seizures in mice.

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca(2+) channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca(2+) channel, CaV3.3, was deleted (CaV3.3(-/-)). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3(-/-) mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3(-/-) mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

Characterization of fimasartan metabolites in human liver microsomes and human plasma.

1. The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects. 2. We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode. 3. The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards. 4. In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3 min in the presence of NADPH, UDPGA and NADPH + UDPGA, respectively. 5. The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation. 6. In humans orally administered with 120 mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0-8-h pooled plasma sample of each subject. 7. This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.

Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes.

Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.