Evaluation of a Three-Dimensional Primary Human Hepatocyte Spheroid Model: Adoption and Industrialization for the Enhanced Detection of Drug-Induced Liver Injury.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

BACKGROUND: Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Mice were treated with an acute dose of either doxorubicin (DOX) (15 mg/kg) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (25 mg/kg). DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO). Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted. CONCLUSIONS/SIGNIFICANCE: These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these events probably account for the heart damage.

Morphology and electrophysiological properties of hamster spinal dorsal horn neurons that express VGLUT2 and enkephalin.

The excitatory amino acid glutamate mediates transmission at spinal synapses, including those formed by sensory afferent fibers and by intrinsic interneurons. The identity and physiological properties of glutamatergic dorsal horn neurons are poorly characterized despite their importance in spinal sensory circuits. Moreover, many intrinsic spinal glutamatergic synapses colocalize the opioid peptide enkephalin (ENK), but the neurons to which they belong are yet to be identified. Therefore, we used immunohistochemistry and confocal microscopy to investigate expression of the VGLUT2 vesicular glutamate transporter, an isoform reported in nonprimary afferent spinal synapses, and ENK in electrophysiologically identified neurons of hamster spinal dorsal horn. VGLUT2 immunoreactivity was localized in restricted fashion to axon varicosities of neurons recorded from laminae II-V, although the occurrence of immunolabeling in individual varicosities varied widely between cells (39 +/- 36%, n = 31 neurons). ENK colocalized with VGLUT2 in up to 77% of varicosities (17 +/- 21%, n = 21 neurons). The majority of neurons expressing VGLUT2 and/or ENK had axons with dense local terminations or projections consistent with propriospinal functions. VGLUT2 and ENK labeling were not correlated with cellular morphology, intrinsic membrane properties, firing patterns, or synaptic responses to sensory afferent stimulation. However, VGLUT2 expression was significantly higher in neurons with depolarized resting membrane potential. The results are new evidence for a population of dual-function dorsal horn interneurons that might provide another mechanism for limiting excitation within dorsal horn circuits during periods of strong sensory activation.