Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

Structural Evolution of Gene Promoters Driven by Primate-Specific KRAB Zinc Finger Proteins.

Krüppel-associated box (KRAB) zinc finger proteins (KZNFs) recognize and repress transposable elements (TEs); TEs are DNA elements that are capable of replicating themselves throughout our genomes with potentially harmful consequences. However, genes from this family of transcription factors have a much wider potential for genomic regulation. KZNFs have become integrated into gene-regulatory networks through the control of TEs that function as enhancers and gene promoters; some KZNFs also bind directly to gene promoters, suggesting an additional, more direct layer of KZNF co-option into gene-regulatory networks. Binding site analysis of ZNF519, ZNF441, and ZNF468 suggests the structural evolution of KZNFs to recognize TEs can result in coincidental binding to gene promoters independent of TE sequences. We show a higher rate of sequence turnover in gene promoter KZNF binding sites than neighboring regions, implying a selective pressure is being applied by the binding of a KZNF. Through CRISPR/Cas9 mediated genetic deletion of ZNF519, ZNF441, and ZNF468, we provide further evidence for genome-wide co-option of the KZNF-mediated gene-regulatory functions; KZNF knockout leads to changes in expression of KZNF-bound genes in neuronal lineages. Finally, we show that the opposite can be established upon KZNF overexpression, further strengthening the support for the role of KZNFs as bona-fide gene regulators. With no eminent role for ZNF519 in controlling its TE target, our study may provide a snapshot into the early stages of the completed co-option of a KZNF, showing the lasting, multilayered impact that retrovirus invasions and host response mechanisms can have upon the evolution of our genomes.

Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

"Can You Deny Her That?" Processes of Governmentality and Socialization of Parents in Elite Women's Gymnastics.

Abusive practices in elite women's artistic gymnastics (WAG) have been the focus of discussions about how to eliminate or reduce them. Both coaches and parents have been named as key actors in bringing about change. Our focus is on parents and their ability to safeguard their daughters in WAG. Parents are not independent actors, however, but are part of a larger web consisting of an entanglement of emotions and technologies and rationalities used by staff, other parents, and athletes, bounded by skill development plans and by coaching expertise and authority. This entanglement may limit the ability of parents to bring about change. We draw on a Deleuzian notion of assemblage, Foucauldian concepts of discourse and governmentality and Ahmed's assertion about the entanglement of discourses and emotions to explore how parents are disciplined into accepting dominant discursive practices of sport clubs for elite athletes. The data were drawn from a project called the Parental Awareness Program (PAP) that was designed to make parents aware of practices in competitive WAG that may not be in their child's best interest. Participants were parents of young gymnasts who had been identified as "talented" and who were members of an elite gymnastics club. The data analysis was based on focus group discussions with a total of 22 parents and semi-structured interviews with 8 parents. The results suggested that although parents problematized many practices during PAP, processes of governmentality involving an assemblage of discourses about coaching expertise, families, talent, enjoyment, long term skill development plans and its associated time demands, together ensured parental consent for dominant practices. The data suggested that a reduction of abusive practices lies in part in critical examinations of skill development plans that are presented as regimes of truth and are kept in place by emotions and the authority accorded to coaching expertise. These processes curtail parents in their ability to safeguard what is in the best pedagogical interest of their daughter.

A hidden layer of structural variation in transposable elements reveals potential genetic modifiers in human disease-risk loci.

Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease-associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease-associated risk loci and in the BCKDK Parkinson's disease-associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.

Prediction of the drug-drug interaction potential of the α1-acid glycoprotein bound, CYP3A4/CYP2C9 metabolized oncology drug, erdafitinib.

Erdafitinib is a potent oral pan-fibroblast growth factor receptor inhibitor being developed as oncology drug for patients with alterations in the fibroblast growth factor receptor pathway. Erdafitinib binds preferentially to α1-acid glycoprotein (AGP) and is primarily metabolized by cytochrome P450 (CYP) 2C9 and 3A4. This article describes a physiologically based pharmacokinetic (PBPK) model for erdafitinib to assess the drug-drug interaction (DDI) potential of CYP3A4 and CYP2C9 inhibitors and CYP3A4/CYP2C9 inducers on erdafitinib pharmacokinetics (PK) in patients with cancer exhibiting higher AGP levels and in populations with different CYP2C9 genotypes. Erdafitinib's DDI potential as a perpetrator for transporter inhibition and for time-dependent inhibition and/or induction of CYP3A was also evaluated. The PBPK model incorporated input parameters from various in vitro and clinical PK studies, and the model was verified using a clinical DDI study with itraconazole and fluconazole. Erdafitinib clearance in the PBPK model consisted of multiple pathways (CYP2C9/3A4, renal, intestinal; additional hepatic clearance), making the compound less susceptible to DDIs. In poor-metabolizing CYP2C9 populations carrying the CYP2C9*3/*3 genotype, simulations shown clinically relevant increase in erdafitinib plasma concentrations. Simulated luminal and enterocyte concentration showed potential risk of P-glycoprotein inhibition with erdafitinib in the first 5 h after dosing, and simulations showed this interaction can be avoided by staggering erdafitinib and digoxin dosing. Other than a simulated ~ 60% exposure reduction with strong CYP3A/2C inducers such as rifampicin, other DDI liabilities were minimal and considered not clinically relevant.

Clinical Investigation on Endogenous Biomarkers to Predict Strong OAT-Mediated Drug-Drug Interactions.

BACKGROUND: Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions early in humans. METHODS: We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition. RESULTS: PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. CONCLUSIONS: The current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction. CLINICAL TRIAL REGISTRATION: EudraCT number: 2016-003923-49.

ZNF91 deletion in human embryonic stem cells leads to ectopic activation of SVA retrotransposons and up-regulation of KRAB zinc finger gene clusters.

Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SINE-VNTR-Alu (SVA) elements are one of three still active TE families; approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting corepressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9-mediated deletion of ZNF91 in human embryonic stem cells, we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild up-regulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Notably, genome-wide derepression of SVAs elicited the communal up-regulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms used by the host genome to sense and counteract TE invasions.

Population pharmacokinetic modeling and simulation to support qualification of pyridoxic acid as endogenous biomarker of OAT1/3 renal transporters.

Renal clearance of many drugs is mediated by renal organic anion transporters OAT1/3 and inhibition of these transporters may lead to drug-drug interactions (DDIs). Pyridoxic acid (PDA) and homovanillic acid (HVA) were indicated as potential biomarkers of OAT1/3. The objective of this study was to develop a population pharmacokinetic model for PDA and HVA to support biomarker qualification. Simultaneous fitting of biomarker plasma and urine data in the presence and absence of potent OAT1/3 inhibitor (probenecid, 500 mg every 6 h) was performed. The impact of study design (multiple vs. single dose of OAT1/3 inhibitor) and ability to detect interactions in the presence of weak/moderate OAT1/3 inhibitors was investigated, together with corresponding power calculations. The population models developed successfully described biomarker baseline and PDA/HVA OAT1/3-mediated interaction data. No prominent effect of circadian rhythm on PDA and HVA individual baseline levels was evident. Renal elimination contributed greater than 80% to total clearance of both endogenous biomarkers investigated. Estimated probenecid unbound in vivo OAT inhibitory constant was up to 6.4-fold lower than in vitro values obtained with PDA as a probe. The PDA model was successfully verified against independent literature reported datasets. No significant difference in power of DDI detection was found between multiple and single dose study design when using the same total daily dose of 2000 mg probenecid. Model-based simulations and power calculations confirmed sensitivity and robustness of plasma PDA data to identify weak, moderate, and strong OAT1/3 inhibitors in an adequately powered clinical study to support optimal design of prospective clinical OAT1/3 interaction studies.

Evolution of Human Brain Size-Associated NOTCH2NL Genes Proceeds toward Reduced Protein Levels.

Ever since the availability of genomes from Neanderthals, Denisovans, and ancient humans, the field of evolutionary genomics has been searching for protein-coding variants that may hold clues to how our species evolved over the last ∼600,000 years. In this study, we identify such variants in the human-specific NOTCH2NL gene family, which were recently identified as possible contributors to the evolutionary expansion of the human brain. We find evidence for the existence of unique protein-coding NOTCH2NL variants in Neanderthals and Denisovans which could affect their ability to activate Notch signaling. Furthermore, in the Neanderthal and Denisovan genomes, we find unusual NOTCH2NL configurations, not found in any of the modern human genomes analyzed. Finally, genetic analysis of archaic and modern humans reveals ongoing adaptive evolution of modern human NOTCH2NL genes, identifying three structural variants acting complementary to drive our genome to produce a lower dosage of NOTCH2NL protein. Because copy-number variations of the 1q21.1 locus, encompassing NOTCH2NL genes, are associated with severe neurological disorders, this seemingly contradicting drive toward low levels of NOTCH2NL protein indicates that the optimal dosage of NOTCH2NL may have not yet been settled in the human population.

Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Widespread correlation of KRAB zinc finger protein binding with brain-developmental gene expression patterns.

The large family of KRAB zinc finger (KZNF) genes are transcription factors implicated in recognizing and repressing repetitive sequences such as transposable elements (TEs) in our genome. Through successive waves of retrotransposition-mediated insertions, various classes of TEs have invaded mammalian genomes at multiple timepoints throughout evolution. Even though most of the TE classes in our genome lost the capability to retrotranspose millions of years ago, it remains elusive why the KZNFs that evolved to repress them are still retained in our genome. One hypothesis is that KZNFs become repurposed for other regulatory roles. Here, we find evidence that evolutionary changes in KZNFs provide them not only with the ability to repress TEs, but also to bind to gene promoters independent of TEs. Using KZNF binding site data in conjunction with gene expression values from the Allen Brain Atlas, we show that KZNFs have the ability to regulate gene expression in the human brain in a region-specific manner. Our analysis shows that the expression of KZNFs shows correlation with the expression of their target genes, suggesting that KZNFs have a direct influence on gene expression in the developing human brain. The extent of this regulation and the impact it has on primate brain evolution are still to be determined, but our results imply that KZNFs have become widely integrated into neuronal gene regulatory networks. Our analysis predicts that gene expression networks have been repeatedly innovated throughout primate evolution, continuously gaining new layers of gene regulation mediated by both TEs and KZNFs in our genome. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells.

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.

Apalutamide Absorption, Metabolism, and Excretion in Healthy Men, and Enzyme Reaction in Human Hepatocytes.

In this phase 1 study, the absolute bioavailability and absorption, metabolism, and excretion (AME) of apalutamide, a competitive inhibitor of the androgen receptor, were evaluated in 12 healthy men. Subjects received 240 mg of apalutamide orally plus a 15-minute intravenous infusion of 100 µg of apalutamide containing 9.25 kBq (250 nCi) of 14C-apalutamide (2 hours postdose) for absolute bioavailability assessment or plus one 400-µg capsule containing 37 kBq (1000 nCi) of 14C-apalutamide for AME assessment. Content of 14C and metabolite profiling for whole blood, plasma, urine, feces, and expired air samples were analyzed using accelerator mass spectrometry. Apalutamide absolute oral bioavailability was ≈100%. After oral administration, apalutamide, its N-desmethyl metabolite (M3), and an inactive carboxylic acid metabolite (M4) accounted for most 14C in plasma (45%, 44%, and 3%, respectively). Apalutamide elimination was slow, with a mean plasma half-life of 151-178 hours. The mean cumulative recovery of total 14C over 70 days postdose was 64.6% in urine and 24.3% in feces. The urinary excretion of apalutamide, M3, and M4 was 1.2%, 2.7%, and 31.1% of dose, respectively. Fecal excretion of apalutamide, M3, and M4 was 1.5%, 2.0%, and 2.4% of dose, respectively. Seventeen apalutamide metabolites and six main metabolic clearance pathways were identified. In vitro studies confirmed CYP2C8 and CYP3A4 roles in apalutamide metabolism.

Structurally Conserved Primate LncRNAs Are Transiently Expressed during Human Cortical Differentiation and Influence Cell-Type-Specific Genes.

The cerebral cortex has expanded in size and complexity in primates, yet the molecular innovations that enabled primate-specific brain attributes remain obscure. We generated cerebral cortex organoids from human, chimpanzee, orangutan, and rhesus pluripotent stem cells and sequenced their transcriptomes at weekly time points for comparative analysis. We used transcript structure and expression conservation to discover gene regulatory long non-coding RNAs (lncRNAs). Of 2,975 human, multi-exonic lncRNAs, 2,472 were structurally conserved in at least one other species and 920 were conserved in all. Three hundred eighty-six human lncRNAs were transiently expressed (TrEx) and many were also TrEx in great apes (46%) and rhesus (31%). Many TrEx lncRNAs are expressed in specific cell types by single-cell RNA sequencing. Four TrEx lncRNAs selected based on cell-type specificity, gene structure, and expression pattern conservation were ectopically expressed in HEK293 cells by CRISPRa. All induced trans gene expression changes were consistent with neural gene regulatory activity.

The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles.

Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Long Non-Coding RNAs in Neuronal Aging.

The expansion of long non-coding RNAs (lncRNAs) in organismal genomes has been associated with the emergence of sophisticated regulatory networks that may have contributed to more complex neuronal processes, such as higher-order cognition. In line with the important roles of lncRNAs in the normal functioning of the human brain, dysregulation of lncRNA expression has been implicated in aging and age-related neurodegenerative disorders. In this paper, we discuss the function and expression of known neuronal-associated lncRNAs, their impact on epigenetic changes, the contribution of transposable elements to lncRNA expression, and the implication of lncRNAs in maintaining the 3D nuclear architecture in neurons. Moreover, we discuss how the complex molecular processes that are orchestrated by lncRNAs in the aged brain may contribute to neuronal pathogenesis by promoting protein aggregation and neurodegeneration. Finally, this review explores the possibility that age-related disturbances of lncRNA expression change the genomic and epigenetic regulatory landscape of neurons, which may affect neuronal processes such as neurogenesis and synaptic plasticity.

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study.

Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.