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.

The metabolism of Chinese Hamster Ovary (CHO) cells in a production environment has been extensively investigated. However, a key metabolic transition, the switch from lactate production to lactate consumption, remains enigmatic. Though commonly observed in CHO cultures, the mechanism(s) by which this metabolic shift is triggered is unknown. Despite this, efforts to control the switch have emerged due to the association of lactate consumption with improved cell growth and productivity. This review aims to consolidate current theories surrounding the lactate switch. The influence of pH, NAD /NADH, pyruvate availability and mitochondrial function on lactate consumption are explored. A hypothesis based on the cellular redox state is put forward to explain the onset of lactate consumption. Various techniques implemented to control the lactate switch, including manipulation of the culture environment, genetic engineering, and cell line selection are also discussed.

BackgroundDoxorubicin 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 FindingsMice 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/SignificanceThese 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.

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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.