High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells.

Drug-induced liver injury (DILI) results in the termination of drug development or withdrawal of a drug from the market. The establishment of a predictive, high-throughput preclinical test system to evaluate potential clinical DILI is therefore required. Here, we established a high content analysis (HCA) assay in human hepatocyte cell lines such as the HepaRG with normal expression levels of CYP enzymes and HepG2 with extremely low expression levels of CYP enzymes. Clinical DILI or non-DILI compounds were evaluated for reactive oxygen species (ROS) production, glutathione (GSH) consumption, and mitochondrial membrane potential (MMP) attenuation. A proportion of DILI compounds induced ROS generation, GSH depletion, and MMP dysfunction, which was consistent with reported mechanisms of DILI of these compounds. In particular, DILI compounds that deplete GSH via reactive metabolites exhibited a more marked decrease in intracellular GSH or increase in ROS production in HepaRG cells than in HepG2 cells. Comparison of the two cell lines with different levels of CYP expression might help clarify the contribution of metabolism to hepatocyte toxicity. These results suggest that the HCA assay in HepaRG and HepG2 cells might help improve the accuracy of evaluating clinical DILI potential during drug screening.

Hydroxyproline, a serum biomarker candidate for gastric ulcer in rats: a comparison study of metabolic analysis of gastric ulcer models induced by ethanol, stress, and aspirin.

Gastrointestinal symptoms are a common manifestation of adverse drug effects. Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed drugs that induce the serious side effect of gastric mucosal ulceration. Biomarkers for these side effects have not been identified and ulcers are now only detectable by endoscopy. We previously identified five metabolites as biomarker candidates for NSAID-induced gastric ulcer using capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic analysis of serum and stomach from rats. Here, to clarify mechanism of changes and limitations of indications of biomarker candidates, we performed CE-MS-based metabolomic profiling in stomach and serum from rats with gastric ulcers induced by ethanol, stress, and aspirin. The results suggest that a decrease in hydroxyproline reflects the induction of gastric injury and may be useful in identifying gastric ulcer induced by multiple causes. While extrapolation to humans requires further study, hydroxyproline can be a new serum biomarker of gastric injury regardless of cause.

Metabolic profiling to identify potential serum biomarkers for gastric ulceration induced by nonsteroid anti-inflammatory drugs.

Nonsteroid anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs currently available. The most frequently reported serious side effects associated with NSAIDs are gastric mucosal ulceration and gastric hemorrhage. Presently, these side effects are only detectable by endoscopy, however, and no biomarkers have yet been identified. The ability to identify serum biomarkers would likely improve the safety of NSAID use. In this study we performed capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic profiling in stomach extract and serum from rats administered NSAIDs. Results showed drug-induced decreases in levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, o-acetyl carnitine, proline, and hydroxyproline. We consider that these changes are due to NSAID-induced depression of mitochondrial function and activation of collagenase by lesions in the stomach. In addition, four of these changes in metabolite levels in the stomach were significantly correlated with changes in the serum. While further study is needed to clarify the mechanism of change in the level of these biomarkers, limitation of indications, and extrapolation to humans, these new serum biomarker candidates of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.

Combination treatment of tribochemical treatment and phosphoric acid ester monomer of zirconia ceramics enhances the bonding durability of resin-based luting cements.

The aim of the present study was to evaluate the bonding durability of resin-based luting cement to partially stabilized tetragonal zirconia (Y-TZP) achieved by combination treatment of tribochemical (TBC) treatment and two different phosphate acid ester monomers. Two phosphate acid ester monomers (EP: Epricord opaque primer, AZ: AZ primer) were applied to each surface modification followed by application of resin-based luting cement (Rely-X ARC). Bonding specimens were placed in deionized water at 37 degrees C and stored for 24 h. The other groups were subjected to 30,000 cycles of a thermal stress for the durability test. Shear bond tests were done using a universal testing machine at 1 mm/min. Shear bond strengths of combination treatments using EP and AZ on TBC treatment after thermal stress showed no significant difference (p>0.05) compared with those of storage after 24 h. Combination treatment using phosphoric acid ester monomer could achieve a durable bond.

[Role of biomarkers in toxicity treatment "metabonomics" as a new biomarker discovery method].

For the treatment of clinical toxicity, investigations to determine the offending substance and rapid treatment are required. Particularly in the case of drug development, the side-effect biomarkers anticipated in a clinical study are based on various toxicological information gleaned from non-clinical studies. In fact, drug development may be discontinued if no biomarkers can be detected using conventional clinical laboratory methodology; therefore, new approaches for finding biomarkers are needed. The use of molecular toxicological methods using omics technology is expected to be an effective future approach. Metabonomics is the omics approach that inspects the movement of endogenous metabolites comprehensively and searches for a toxicological mechanism or biomarker. It is expected to become a useful biomarker discovery tool; in fact, reports about new biomarker discoveries made using metabonomics have already been published; however, the rate of metabolite identification and metabolism map development are not yet sufficient. Therefore, the development of a database containing this type of information as well as clinical information is necessary to be able to apply this technology to toxicological biomarker discovery. Further, studies for translation from the non-clinical to the clinical setting are very important for discovering useful metabonomic side-effect biomarkers. Therefore, building new collaborative relationships between pharmaceutical companies, doctors, medical technologists, and diagnostic agent companies is considered to be important.