Automated Electrical Stimulation Therapy Accelerates Re-Epithelialization in a Three-Dimensional In Vitro Human Skin Wound Model.

Objective: Electrical Stimulation Therapy (EST) shows promise for the purpose of accelerating wound healing, but the right electrical stimulation parameters and its mode of action remain unclear. We aim to evaluate the effect of a new EST clinical device on epidermal repair using an in vitro human skin wound model. Approach: We scaled up a well-established 3D De-Epidermized Dermis-Human Skin Equivalent (DED-HSE) wound model to fit a clinically used device that delivers preprogrammed microcurrent EST. The impact of EST on re-epithelialization of 4-mm circular epidermal wounds was assessed after 4 and 7 days of treatment, using metabolic activity assay, immunohistochemistry (IHC) staining, and RNA in situ hybridization. Results: EST was successfully applied to the wounded in vitro skin model. Large DED-HSEs retained good cell viability for up to 7 days of EST treatment. Excisional wounds subjected to EST for 4 days consistently exhibited faster closure (mean 65.8%, n = 9) compared to untreated wounds (mean 49.7%, n = 9) (p < 0.05). Wounds exposed to EST exhibited significantly longer epithelial tongues (re-epithelialization mean 50.3%, n = 9) than untreated wounds (mean 26.2%, n = 9) (p < 0.001), suggesting faster keratinocyte migration and proliferation. Increased MMP1 transcription (p < 0.05) in ES-treated periwound suggests a mechanism for enhanced keratinocyte migration. IHC staining showed advanced epidermal proliferation (p63) and differentiation (K10) in EST-exposed wounds (n = 15), as well as stronger attachment of the newly formed epidermis into the dermis compared to untreated controls (n = 15) (p < 0.001). Innovation: We present a novel approach to assess an EST clinical device designed to stimulate wound healing. Using a scaled-up 3D human skin wound model, we could demonstrate the positive effect of EST on epithelial cell responses and shed light on possible mechanism. Conclusion: Our study provides experimental evidence that microcurrent therapy accelerates wound closure and improves the quantity and quality of re-epithelialization.

The mitochondrial superoxide/thioredoxin-2/Ask1 signaling pathway is critically involved in troglitazone-induced cell injury to human hepatocytes.

Although the mechanisms and susceptibility factors of troglitazone-associated idiosyncratic liver injury have not been elucidated, experimental evidence has identified oxidant stress and mitochondrial injury as a potential hazard in vitro. In search of upstream mediators of toxicity, we hypothesized that troglitazone-induced increased mitochondrial generation of superoxide might activate the thioredoxin-2 (Trx2)/apoptosis signal-regulating kinase 1 (Ask1) signaling pathway, leading to cell death, and that, hence, the mitochondrially targeted radical scavenger, mito-carboxy proxyl (CP), would prevent the increase in superoxide net levels and inhibit mitochondrial signaling and cell injury. Immortalized human hepatocytes (HC-04) were exposed to troglitazone (0-100 microM), which caused concentration and time-dependent apoptosis after 12-24 h (ketoconazole-insensitive). We found that troglitazone rapidly dissipated the mitochondrial inner transmembrane potential (DeltaPsi(m)) and independently increased the net levels of mitochondrial superoxide by 5-fold. This was followed by a shift of the redox ratio of mitochondrial Trx2 toward the oxidized state and subsequent activation of Ask1. Cell injury, but not the decrease in DeltaPsi(m), was prevented by cyclosporin A (3 microM), indicating that mitochondrial permeabilization, but not membrane depolarization, was causally involved in cell death. Mito-CP not only decreased troglitazone-induced superoxide levels but also prevented Trx2 oxidation and activation of Ask1 and protected cells from toxic injury. These data indicate that troglitazone, but not its oxidative metabolite(s), produce intramitochondrial oxidant stress that activates the Trx2/Ask1 pathway, leading to mitochondrial permeabilization. Furthermore, the data support our concept that targeted delivery of an antioxidant to mitochondria can inhibit upstream signaling and protect from troglitazone-induced lethal cell injury.

Molecular and functional characterization of drug-metabolizing enzymes and transporter expression in the novel spontaneously immortalized human hepatocyte line HC-04.

In toxicological research, immortalized human hepatocytes provide a useful alternative to primary hepatocytes because interindividual variability in the expression of drug-metabolizing enzymes and drug transporters can largely be eliminated. However, it is essential that the cell line retain the original phenotype. The purpose of this study was to characterize a novel spontaneously immortalized human hepatocyte cell line, HC-04, with respect to the transcript and functional protein expression profile for the major drug-metabolizing enzymes and transmembrane transporters. HC-04 cells retained hepatocyte-specific function including albumin production and ornithine transcarbamoylase and glucose-6-phosphatase activity. Most of the major CYP forms were expressed at basal levels and responsive to inducing agents. In particular, CYP3A4 was expressed abundantly, and HC-04 cells were able to metabolize the CYP3A4 probe, midazolam, at a rate similar to primary human hepatocytes. Furthermore, the major human sulfotransferase and UDP-glucuronosyltransferase forms, as well as members of the ABC and SLC transporter superfamilies, nuclear receptors, and hepatic transcription factors were also expressed. HC-04 cells readily responded to standard hepatotoxicants that are dependent on CYP-mediated bioactivation, while another, tumor-derived cell line remained refractory to the drug challenge. Collectively, HC-04 cells provide a reliable, stable, and reproducible model for biomechanistic studies in drug toxicology.

Mitochondrial abnormalities--a link to idiosyncratic drug hepatotoxicity?

Idiosyncratic drug-induced liver injury (DILI) is a major clinical problem and poses a considerable challenge for drug development as an increasing number of successfully launched drugs or new potential drugs have been implicated in causing DILI in susceptible patient subsets. Although the incidence for a particular drug is very low (yet grossly underestimated), the outcome of DILI can be serious. Unfortunately, prediction has remained poor (both for patients at risk and for new chemical entities). The underlying mechanisms and the determinants of susceptibility have largely remained ill-defined. The aim of this review is to provide both clinical and experimental evidence for a major role of mitochondria both as a target of drugs causing idiosyncratic DILI and as mediators of delayed liver injury. We develop a unifying hypothesis that involves underlying genetic or acquired mitochondrial abnormalities as a major determinant of susceptibility for a number of drugs that target mitochondria and cause DILI. The mitochondrial hypothesis, implying gradually accumulating and initially silent mitochondrial injury in heteroplasmic cells which reaches a critical threshold and abruptly triggers liver injury, is consistent with the findings that typically idiosyncratic DILI is delayed (by weeks or months), that increasing age and female gender are risk factors and that these drugs are targeted to the liver and clearly exhibit a mitochondrial hazard in vitro and in vivo. New animal models (e.g., the Sod2(+/-) mouse) provide supporting evidence for this concept. However, genetic analyses of DILI patient samples are needed to ultimately provide the proof-of-concept.

Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes.

Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca(2+)(c)) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 microM). At 8 h, diclofenac caused increases in [Ca(2+)](c) (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca(2+) chelator) inhibited cell injury, indicating that Ca(2+) plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, DeltaPsi(m) (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca(2+)](c). The CYP2C9 inhibitor sulphaphenazole (10 microM) protected from diclofenac-induced cell injury and prevented increases in [Ca(2+)](c), while it had no effect on the dissipation of the DeltaPsi(m). Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca(2+)](c). In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca(2+)](c), triggering the mPT and precipitating cell death.