Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

Keratitis Fugax Hereditaria Associated With a Novel NLRP3 Mutation in a Non-Finnish Patient.

PURPOSE: The aim of this study was to report a novel heterozygous variant c.1712G>T (p.Gly571Val) in the nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing 3 gene ( NLRP3 ) in a previously unreported non-Finnish individual with keratitis fugax hereditaria (KFH). METHODS: Ophthalmologic examination of the proband was performed with slit-lamp biomicroscopy and anterior segment optical coherence tomography. Saliva was collected as a source of DNA, after which targeted exome sequencing of candidate genes was performed using a commercially available panel. Identified presumed pathogenic variants were confirmed by Sanger sequencing. RESULTS: Slit-lamp examination of the 52-year-old female proband revealed peripheral arcus-like degeneration and bilateral central corneal opacification, observed on anterior segment optical coherence tomography to involve the anterior half of the corneal stroma. Examination of the proband's parents revealed clear corneas in each eye. Genetic testing of the proband identified the presence of a novel heterozygous NLRP3 missense mutation (c.1712G>T, p.Gly571Val), which was confirmed by Sanger sequencing. This mutation was absent in the proband's parents. CONCLUSIONS: Although KFH has been reported only in individuals of Finnish descent and only in association with a missense mutation in exon 1 of NLRP3 , we report an individual of non-Finnish descent with KFH associated with a novel heterozygous variant in exon 2 of NLRP3 . Thus, ophthalmologists should be aware of the ethnic and genetic heterogeneity associated with KFH.

44-year-old Man with Hemoptysis and Hypoxemic Respiratory Failure: A Case Report.

INTRODUCTION: Hemoptysis can be a highly alarming presentation in the emergency department (ED). Even seemingly minor cases may represent potentially lethal underlying pathology. It requires thorough evaluation and careful consideration of a broad differential diagnosis. CASE PRESENTATION: A 44-year-old man presented to the ED with a concern of hemoptysis in the setting of recent fever and myalgias. DISCUSSION: This case takes the reader through how to approach the differential diagnosis and diagnostic work-up of hemoptysis in the ED setting and then reveals the surprising final diagnosis.

Identification of a novel partial deletion of STS associated with pre-Descemet corneal dystrophy and X-linked ichthyosis.

Purpose: Pre-Descemet corneal dystrophy (PDCD) with X-linked ichthyosis (XLI) is associated with mutations in or deletions of the steroid sulfatase gene (STS). As only three cases of genetically confirmed PDCD associated with XLI have been reported, we sought to expand our understanding of the genetic basis of PDCD by screening STS in two previously unreported families. Materials and Methods: The affected individuals underwent cutaneous and slit-lamp examinations. Saliva samples collected from each affected individual served as a source of DNA for the amplification of the 10 coding exons of STS and flanking DNA markers. Results: The slit-lamp examination of three affected men (two of whom were brothers) from two families revealed bilateral punctate posterior corneal stromal opacities anterior to the Descemet membrane. Cutaneous examination demonstrated dry, coarse, scaly ichthyotic changes characteristic of XLI in all individuals. Genetic examination of the STS locus on the X chromosome in Case 1 revealed a deletion that spanned across DNA markers DXS1130-DXS237, which includes all the coding exons (exons 1-10) of STS. Genetic screening of Cases 2 and 3 revealed a partial deletion of the STS locus involving exons 1-7 and flanking DNA marker DXS1130 on the X chromosome. Conclusions: PDCD with XLI may be associated with either partial or complete deletion of STS. Despite the identification of point mutations, partial deletion, and complete deletion of STS in different affected families reported to date, there was no apparent difference in the affected phenotype between the families, suggesting that the identified variants likely all resulted in loss of function of steroid sulfatase.

Raspberry plant stress detection using hyperspectral imaging.

Monitoring plant responses to stress is an ongoing challenge for crop breeders, growers, and agronomists. The measurement of below-ground stress is particularly challenging as plants do not always show visible signs of stress in the above-ground organs, particularly at early stages. Hyperspectral imaging is a technique that could be used to overcome this challenge if associations between plant spectral data and specific stresses can be determined. In this study, three genotypes of red raspberry plants grown under controlled conditions in a glasshouse were subjected to below-ground biotic stresses (root pathogen Phytophthora rubi and root herbivore Otiorhynchus sulcatus) or abiotic stress (soil water availability) and regularly imaged using hyperspectral cameras over this period. Significant differences were observed in plant biophysical traits (canopy height and leaf dry mass) and canopy reflectance spectrum between the three genotypes and the imposed stress treatments. The ratio of reflectance at 469 and 523 nm showed a significant genotype-by-treatment interaction driven by differential genotypic responses to the P. rubi treatment. This indicates that spectral imaging can be used to identify variable plant stress responses in raspberry plants.

Novel DCN Mutation in Armenian Family With Congenital Stromal Corneal Dystrophy.

PURPOSE: Congenital stromal corneal dystrophy (CSCD) is a rare congenital, dominantly inherited disorder characterized by diffuse stromal opacification associated with mutations in the decorin gene ( DCN ). As only 5 families with genetically confirmed CSCD have been reported, the identification of a novel pedigree provides the opportunity to better characterize the phenotype and genetic basis. METHODS: An Armenian family with individuals in 4 consecutive generations demonstrated clinical features consistent with CSCD. Consented individuals underwent slit lamp examination, optical coherence tomography, and confocal microscopy. Genomic DNA was collected from saliva and all coding and adjacent intronic regions of DCN were sequenced. In silico analysis was performed for identified mutation(s). Excised corneal tissue underwent light, electron microscopic, and immunohistochemical evaluation. RESULTS: Affected individuals demonstrated bilateral, diffuse, panstromal corneal opacification. Three of the 6 individuals diagnosed with CSCD underwent genetic analysis; all demonstrated a novel heterozygous frameshift deletion in exon 8 of DCN (p.His317Thrfs*11), predicted to cause a 33 amino acid truncation and to be damaging and disease causing by SIFT and MutationTaster. Light and electron microscopic examination of an excised cornea demonstrated increased corneal thickness, stromal scarring, keratocyte loss, and an irregularity of lamellar collagen spacing and fibril formation. Immunofluorescent examination demonstrated increased DCN immunostaining, predominantly in the widened interlamellar spaces. CONCLUSIONS: We report only the sixth pedigree with genetically confirmed CSCD, associated with a novel DCN frameshift mutation. The clinical evaluation, multimodal imaging, and histopathologic assessment in this family with CSCD broaden our understanding of this rare corneal disease.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds.

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress.

BACKGROUND: Tissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear. RESULTS: Here, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations. CONCLUSIONS: The liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.

In silico approaches in organ toxicity hazard assessment: current status and future needs in predicting liver toxicity.

Hepatotoxicity is one of the most frequently observed adverse effects resulting from exposure to a xenobiotic. For example, in pharmaceutical research and development it is one of the major reasons for drug withdrawals, clinical failures, and discontinuation of drug candidates. The development of faster and cheaper methods to assess hepatotoxicity that are both more sustainable and more informative is critically needed. The biological mechanisms and processes underpinning hepatotoxicity are summarized and experimental approaches to support the prediction of hepatotoxicity are described, including toxicokinetic considerations. The paper describes the increasingly important role of in silico approaches and highlights challenges to the adoption of these methods including the lack of a commonly agreed upon protocol for performing such an assessment and the need for in silico solutions that take dose into consideration. A proposed framework for the integration of in silico and experimental information is provided along with a case study describing how computational methods have been used to successfully respond to a regulatory question concerning non-genotoxic impurities in chemically synthesized pharmaceuticals.

"Before the training I just assumed they've done something bad": Reporting on professional training for pharmacy assistants and pharmacy dispensary technicians on medically assisted treatment of opioid dependence.

BACKGROUND: A study that examined the lived experiences of Medically Assisted Treatment of Opioid Dependence (MATOD) consumers suggested that they had experienced discrimination and stigma in pharmacies in regional Victoria, Australia. To address this, the need for professional training opportunities for Pharmacy Assistants (PAs) and Pharmacy Dispensary Technicians (PTDs) had been emphasised. A research project was undertaken to develop training modules using Social Determinants of Health (SDH) for PAs and PDTs involved in providing MATOD pharmacy services in regional Victoria, and to evaluate their effectiveness. OBJECTIVES: The study aimed to examine and evaluate changes in attitudes and practices amongst PAs and PDTs involved in MATOD services in regional Victoria, Australia and who had undertaken the training modules. METHODS: The paper reports primarily on the in-depth qualitative interviews that were completed after the training with PAs and PTDs. Thematic analysis was employed to code the data. RESULTS: Thematic analysis generated five themes: understanding of the professional role of PAs and PTDs, initial judgements concerning MATOD consumers, reflection on the SDH, training content analysis, and the post-training impact upon Professional Practice. These themes reflected participants' insights with regards both to MATOD consumers and the impact the training itself had had on their professional practice. CONCLUSION: "Consciousness-raising" from participation in the training can positively influence the development of participants' professional attitudes and practices with regards to MATOD service delivery. This development supports that training informed by SDH and ideas of critical reflection can help facilitate the creation of knowledge around the social construction of health and increased understanding of the impacts of language-use, attitudes and behaviour.

Transfer of hepatocellular microRNA regulates cytochrome P450 2E1 in renal tubular cells.

BACKGROUND: Extracellular microRNAs enter kidney cells and modify gene expression. We used a Dicer-hepatocyte-specific microRNA conditional-knock-out (Dicer-CKO) mouse to investigate microRNA transfer from liver to kidney. METHODS: Dicerflox/flox mice were treated with a Cre recombinase-expressing adenovirus (AAV8) to selectively inhibit hepatocyte microRNA production (Dicer-CKO). Organ microRNA expression was measured in health and following paracetamol toxicity. The functional consequence of hepatic microRNA transfer was determined by measuring the expression and activity of cytochrome P450 2E1 (target of the hepatocellular miR-122), and by measuring the effect of serum extracellular vesicles (ECVs) on proximal tubular cell injury. In humans with liver injury we measured microRNA expression in urinary ECVs. A murine model of myocardial infarction was used as a non-hepatic model of microRNA release. FINDINGS: Dicer-CKO mice demonstrated a decrease in kidney miR-122 in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNA increased kidney cytochrome P450 2E1 expression and activity. Serum ECVs from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin toxicity. miR-122 increased in urinary ECVs during human hepatotoxicity. Transfer of microRNA was not restricted to liver injury -miR-499 was released following cardiac injury and correlated with an increase in the kidney. INTERPRETATION: Physiological transfer of functional microRNA to the kidney is increased by liver injury and this signalling represents a new paradigm for understanding the relationship between liver injury and renal function. FUNDING: Kidney Research UK, Medical Research Scotland, Medical Research Council.

HepG2 (C3A) spheroids show higher sensitivity compared to HepaRG spheroids for drug-induced liver injury (DILI).

High-throughput, automation-friendly and therapeutically-predictive assays are needed in early drug discovery in order to prioritise compounds and reduce the risk of new drugs causing Drug-Induced Liver Injury (DILI). We evaluated the suitability of high-throughput 3D liver spheroid models of HepG2 (C3A clone) and HepaRG cell lines to predict DILI in early drug development. Spheroids were formed in 384-well ultra-low-attachment plates and dosed via direct acoustic droplet ejection at nine half-log spaced concentrations per compound. Spheroid viability was quantified with an ATP endpoint after a 4-day incubation with 150 drugs with known DILI liability. We derived a margin of safety for each cell line defined as the ratio between the IC50 values generated for each compound to their maximum plasma concentration Cmax which resulted in optimal classification accuracy. The margin of safety can be used to estimate a maximum safe Cmax for compounds in early drug discovery for which Cmax is not yet known. Both cell lines had similar level of accuracy in predicting DILI, with HepG2 spheroids being more sensitive. HepG2 spheroids had a sensitivity of 58% and a specificity of 83%, while HepaRG spheroids had a sensitivity of 47% and specificity of 86%. Ninety-nine of the 150 compounds were used to compare the relative sensitivities of HepG2 and HepaRG spheroids. HepaRG spheroids were more sensitive to 7 compounds and HepG2 spheroids were more sensitive to 34 compounds. In conclusion, across a diverse group of drugs HepG2 spheroids were more predictive of DILI compared to HepaRG spheroids.

Morphological and Mechanistic Aspects of Thiourea-Induced Acute Lung Injury and Tolerance in the Rat.

Thiourea-based molecules cause pulmonary edema when administered to rats at relatively low doses. However, rats survive normally lethal doses after prior exposure to a lower, nonlethal dose; this phenomenon is known as tolerance. The present study investigated the morphological and functional aspects of acute lung injury (ALI) induced by methylphenylthiourea (MPTU) in the Wistar rat and the pulmonary response involved in prevention of the injury. We identified pulmonary endothelial cells as the main target of acute MPTU injury; they exhibited ultrastructural alterations that can result in increased vascular permeability. In tolerant rats, the lungs showed only transient endothelial changes, at 24-hour post dosing, and mild type II pneumocyte hyperplasia on day 7 post dosing. They exhibited glutathione levels similar to the controls and increased expression of flavin-containing monooxygenase 1 (FMO1), the enzyme responsible for bioactivation of small thioureas in the laboratory rat. Incubation of rat pulmonary microsomal preparations with MPTU inhibited FMO activity, indicating that tolerance is related to irreversible inhibition of FMOs. The rat model of thiourea-induced pulmonary toxicity and tolerance represents an interesting approach to investigate certain aspects of the pathogenesis of ALI and therapeutic approaches to lung diseases, such as acute respiratory distress syndrome.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Differences in Cardiovascular Care Between Adults With and Without Opioid Prescriptions in the United States.

Background Patients prescribed opioids often have chronic conditions that increase their risk of adverse cardiovascular outcomes, but little is known about the primary preventive cardiovascular care these patients receive. Methods and Results We analyzed data from the 2014 to 2016 National Ambulatory Medical Care Survey to evaluate physicians' provision of primary preventive cardiovascular care to adults with and without opioid prescriptions. We included all visits made by adults 40 to 79 years old with at least 1 cardiovascular risk factor but no existing atherosclerotic cardiovascular disease. There were ≈32 million visits by adults who were prescribed opioids and ≈167 million visits by adults not prescribed opioids on an annual basis. The prevalence of primary preventive care was modest in patients with versus those without opioid prescriptions, respectively: (1) statins for patients with dyslipidemia (52.1% versus 46.3%); (2) statins for patients with diabetes mellitus (49.1% versus 37.9%); (3) antihypertensive agents for patients with hypertension (76.5% versus 65.8%); (4) diet/exercise counseling (40.5% versus 45.3%); and (5) smoking cessation therapy (25.3% versus 19.3%). In multivariate analyses, opioid use was associated with higher rates of statin therapy in patients with diabetes mellitus (adjusted relative risk [aRR], 1.25; 95% CI, 1.06-1.47; P=0.007) and antihypertensive medication in patients with hypertension (aRR 1.14; 95% CI, 1.06-1.22; P<0.001). Conclusions Overall adherence to guideline-recommended primary preventive cardiovascular care during ambulatory visits was suboptimal. Findings show that patients prescribed opioids versus those without opioid prescriptions were more likely to receive statin therapy and antihypertensive agents in the setting of diabetes mellitus and hypertension, respectively. Ongoing efforts to bridge these gaps in primary prevention of cardiovascular disease remain a high priority.

Determining organ weight toxicity with Bayesian causal models: Improving on the analysis of relative organ weights.

Regulatory authorities require animal toxicity tests for new chemical entities. Organ weight changes are accepted as a sensitive indicator of chemically induced organ damage, but can be difficult to interpret because changes in organ weight might reflect chemically-induced changes in overall body weight. A common solution is to calculate the relative organ weight (organ to body weight ratio), but this inadequately controls for the dependence on body weight - a point made by statisticians for decades, but which has not been widely adopted. The recommended solution is an analysis of covariance (ANCOVA), but it is rarely used, possibly because both the method of statistical correction and the interpretation of the output may be unclear to those with minimal statistical training. Using relative organ weights can easily lead to incorrect conclusions, resulting in poor decisions, wasted resources, and an ethically questionable use of animals. We propose to cast the problem into a causal modelling framework as it directly assesses questions of scientific interest, the results are easy to interpret, and the analysis is simple to perform with freely available software. Furthermore, by taking a Bayesian approach we can model unequal variances, control for multiple testing, and directly provide evidence of safety.

Multiscale modelling of drug transport and metabolism in liver spheroids.

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo. While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity.

In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-ß. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC50 at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms.