The short-term toxicity and metabolome of Benzene.

A 14-day rat study with plasma metabolomics was conducted to evaluate the toxicity of Benzene. Wistar rats were orally administered Benzene daily at doses of 0, 300 and 1000 mg/kg bw. The study identified liver and kidneys as target organs of Benzene toxicity and found reductions in total white blood cells, absolute lymphocyte and eosinophil cell counts, and increased relative monocyte counts suggesting bone marrow as a target organ. The study also confirmed liver as a target organ using metabolomics, which showed indications of a stress reaction in rats and changes in metabolites suggestive of a metabolic disorder. The metabolomics investigations did not find any other toxicologically relevant modes of action, and the observed metabolite changes were not associated with markers for mitochondrial dysfunction. The study concludes that integration of omics technologies, such as metabolomics, in regulatory toxicity studies is possible, confirms existing knowledge and adds additional information that can be used for mechanistic understanding of observed toxicity.

Use of in vitro metabolomics in NRK cells to help predicting nephrotoxicity and differentiating the MoA of nephrotoxicants.

We describe a strategy using an in vitro metabolomics assay with tubular rat NRK-52E cells to investigate the Modes of Action (MoAs) of nephrotoxic compounds. Chemicals were selected according to their MoAs based on literature information: acetaminophen, 4-aminophenol and S-(trichlorovinyl-)L-cysteine (TCVC), (covalent protein binding); gentamycin, vancomycin, polymycin B and CdCl2 (lysosomal overload) and tenofovir and cidofovir (mitochondrial DNA-interaction). After treatment and harvesting of the cells, intracellular endogenous metabolites were quantified relative to vehicle control. Metabolite patterns were evaluated in a purely data-driven pattern generation process excluding published information. This strategy confirmed the assignment of the chemicals to the respective MoA except for TCVC and CdCl2. Finally, TCVC was defined as unidentified and CdCl2 was reclassified to the MoA "covalent protein binding". Hierarchical cluster analysis of 58 distinct metabolites from the patterns enabled a clear visual separation of chemicals in each MoA. The assay reproducibility was very good and metabolic responses were consistent. These results support the use of metabolome analysis in NRK-52E cells as a suitable tool for understanding and investigating the MoA of nephrotoxicants. This assay could enable the early identification of nephrotoxic compounds and finally reduce animal testing.

Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells.

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.