Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.

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A GC-TOF-MS method was developed and validated for a metabolic fingerprinting in saliva of smokers and nonsmokers. We validated the method by spiking 37 different metabolites and 6 internal standards to saliva between 0.1 μM and 2 mM. Intraday coefficients of variation (CVs) (accuracies) were on average, 11.9% (85.8%), 8.2% (88.9%), and 10.0% (106.7%) for the spiked levels 25, 50, and 200 μM, respectively (N = 5). Interday CVs (accuracies) were 12.4% (97%), 18.8% (95.5%), and 17.2% (105.9%) for the respective levels of 25, 50, and 200 μM (N = 5). The method was applied to saliva of smokers and nonsmokers, obtained from a 24 h diet-controlled clinical study, in order to identify biomarkers of endogenous origin, which could be linked to smoking related diseases. Automated peak picking, integration, and statistical analysis were conducted by the software tools MZmine, Metaboanalyst, and PSPP. We could identify 13 significantly altered metabolites in smokers (p < 0.05) by matching them against MS libraries and authentic standard compounds. Most of the identified metabolites, including tyramine, adenosine, and glucose-6-phosphate, could be linked to smoking-related perturbations and may be associated with established detrimental effects of smoking.