Drug induced mitochondrial dysfunction has been implicated in organ toxicity and the withdrawal of drugs or black box warnings limiting their use. The development of highly specific and sensitive in vitro assays in early drug development would assist in detecting compounds which affect mitochondrial function. Here we report the combination of two in vitro assays for the detection of drug induced mitochondrial toxicity. The first assay measures cytotoxicity after 24h incubation of test compound in either glucose or galactose conditioned media (Glu/Gal assay). Compounds with a greater than 3-fold toxicity in galactose media compared to glucose media imply mitochondrial toxicity. The second assay measures mitochondrial respiration, glycolysis and a reserve capacity with mechanistic responses observed within one hour following exposure to test compound. In order to assess these assays a total of 72 known drugs and chemicals were used. Dose-response data was normalised to 100× Cmax giving a specificity, sensitivity and accuracy of 100%, 81% and 92% respectively for this combined approach.

Aflatoxin B1 (AFB1), a common dietary contaminant, is a major risk factor of hepatocellular carcinoma (HCC). Early onset of HCC in some countries in Africa and South-East Asia indicates the importance of early life exposure. Placenta is the primary route for various compounds, both nutrients and toxins, from the mother to the fetal circulation. Furthermore, placenta contains enzymes for xenobiotic metabolism. AFB1, AFB1-metabolites, and AFB1-albumin adducts have been detected in cord blood of babies after maternal exposure during pregnancy. However, the role that the placenta plays in the transfer and metabolism of AFB1 is not clear. In this study, placental transfer and metabolism of AFB1 were investigated in human placental perfusions and in in vitro studies. Eight human placentas were perfused with 0.5 or 5microM AFB1 for 2-4 h. In vitro incubations with placental microsomal and cytosolic proteins from eight additional placentas were also conducted. Our results from placental perfusions provide the first direct evidence of the actual transfer of AFB1 and its metabolism to aflatoxicol (AFL) by human placenta. In vitro incubations with placental cytosolic fraction confirmed the capacity of human placenta to form AFL. AFL was the only metabolite detected in both perfusions and in vitro incubations. Since AFL is less mutagenic, but putatively as carcinogenic as AFB1, the formation of AFL may not protect the fetus from the toxicity of AFB1.

Exposure to cigarette smoke is a leading cause of lung diseases including chronic obstructive pulmonary disease and cancer. Cigarette smoke is a complex aerosol containing over 6000 chemicals and thus it is difficult to determine individual contributions to overall toxicity as well as the molecular mechanisms by which smoke constituents exert their effects. We selected three well-known harmful and potentially harmful constituents (HPHCs) in tobacco smoke, acrolein, formaldehyde and catechol, and established a high-content screening method using normal human bronchial epithelial cells, which are the first bronchial cells in contact with cigarette smoke. The impact of each HPHC was investigated using 13 indicators of cellular toxicity complemented with a microarray-based whole-transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. HPHCs were evaluated over a wide range of concentrations and at different exposure time points (4, 8, and 24 h). By high-content screening, the toxic effects of the three HPHCs could be observed only at the highest doses. Whole-genome transcriptomics unraveled toxicity mechanisms at lower doses and earlier time points. The most prevalent toxicity mechanisms observed were DNA damage/growth arrest, oxidative stress, mitochondrial stress, and apoptosis/necrosis. A combination of multiple toxicological end points with a systems-based impact assessment allows for a more robust scientific basis for the toxicological assessment of HPHCs, allowing insight into time- and dose-dependent molecular perturbations of specific biological pathways. This approach allowed us to establish an in vitro systems toxicology platform that can be applied to a broader selection of HPHCs and their mixtures and can serve more generally as the basis for testing the impact of other environmental toxicants on normal bronchial epithelial cells.

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