A metabolomics cell-based approach for anticipating and investigating drug-induced liver injury

In preclinical stages of drug development, anticipating potential adverse drug effects such as toxicity is an important issue for both saving resources and preventing public health risks. Current in vitro cytotoxicity tests are restricted by their predictive potential and their ability to provide mechanistic information. This study aimed to develop a metabolomic mass spectrometry-based approach for the detection and classification of drug-induced hepatotoxicity. To this end, the metabolite profiles of human derived hepatic cells (i.e., HepG2) exposed to different well-known hepatotoxic compounds acting through different mechanisms (i.e., oxidative stress, steatosis, phospholipidosis, and controls) were compared by multivariate data analysis, thus allowing us to decipher both common and mechanism-specific altered biochemical pathways. Briefly, oxidative stress damage markers were found in the three mechanisms, mainly showing altered levels of metabolites associated with glutathione and γ-glutamyl cycle. Phospholipidosis was characterized by a decreased lysophospholipids to phospholipids ratio, suggestive of phospholipid degradation inhibition. Whereas, steatosis led to impaired fatty acids β-oxidation and a subsequent increase in triacylglycerides synthesis. The characteristic metabolomic profiles were used to develop a predictive model aimed not only to discriminate between non-toxic and hepatotoxic drugs, but also to propose potential drug toxicity mechanism(s)

Gut microbiota functions: metabolism of nutrients and other food components

The diverse microbial community that inhabits the human gut has an extensive metabolic repertoire that is distinct from, but complements the activity of mammalian enzymes in the liver and gut mucosa and includes functions essential for host digestion. As such, the gut microbiota is a key factor in shaping the biochemical profile of the diet and, therefore, its impact on host health and disease. The important role that the gut microbiota appears to play in human metabolism and health has stimulated research into the identification of specific microorganisms involved in different processes, and the elucidation of metabolic pathways, particularly those associated with metabolism of dietary components and some host-generated substances. In the first part of the review, we discuss the main gut microorganisms, particularly bacteria, and microbial pathways associated with the metabolism of dietary carbohydrates (to short chain fatty acids and gases), proteins, plant polyphenols, bile acids, and vitamins. The second part of the review focuses on the methodologies, existing and novel, that can be employed to explore gut microbial pathways of metabolism. These include mathematical models, omics techniques, isolated microbes, and enzyme assays

Sample preparation and reporting standards for Metabolomics of Adherent Mammalian Cells

Metabolomics is an analytical technique that investigates the small molecules present within a biological system. Metabolomics of cultured cells allows profiling of the metabolic chemicals involved in a cell type-specific system and the response of that metabolome to external challenges, such as change in environment or exposure to drugs or toxins. The numerous benefits of in vitro metabolomics include a much greater control of external variables and reduced ethical concerns. There is potential for metabolomics of mammalian cells to uncover new information on mechanisms of action for drugs or toxins or to provide a more sensitive, human-specific early risk assessment in drug development or toxicology investigations. One way to achieve stronger biological outcomes from metabolomic data is via the use of these mammalian cultured cell models, particularly in a high-throughput context. With the sensitivity and quantity of data that metabolomics is able to provide, it is important to ensure that the sampling techniques have minimal interference when it comes to interpretation of any observed shifts in the metabolite profile. Here we describe a sampling procedure designed to ensure that the effects seen in metabolomic analyses are explained fully by the experimental factor and not other routine culture-specific activities

Microbial communities in karst groundwater and their potential use for biomonitoring

The structure, diversity and dynamics of microbial communities from a swallow hole draining agricultural land and two connected karst springs (Switzerland) were studied using molecular microbiological methods and related to hydrological and physicochemical parameters. Storm responses and an annual hydrological cycle were monitored to determine the short- and long-term variability, respectively, of bacterial communities. Statistical analysis of bacterial genetic fingerprints (16S rDNA PCR-DGGE) of spring water samples revealed several clusters that corresponded well with different levels of the allochthonous swallow hole contribution. Microbial communities in spring water samples highly affected by the swallow hole showed low similarities among them, reflecting the high temporal variability of the bacterial communities infiltrating at the swallow hole. Conversely, high similarities among samples with low allochthonous contribution provided evidence for a stable autochthonous endokarst microbial community. Three spring samples, representative for low, medium and high swallow hole contribution, were analysed by cloning/sequencing in order to identify the major bacterial groups in the communities. The autochthonous endokarst microbial community was mainly characterized of δ-Proteobacteria, Acidobacteria and Nitrospira species. A high percentage of unknown sequences suggested further that many karst aquifer bacteria are still undiscovered. Finally, the potential use of groundwater biomonitoring using microbial communities is discussed

Calcite Biomineralization by Bacterial Isolates from the Recently Discovered Pristine Karstic Herrenberg Cave

Karstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the genera Arthrobacter, Flavobacterium, Pseudomonas, Rhodococcus, Serratia, and Stenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation, Arthrobacter sulfonivorans and Rhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite. R. globerulus formed idiomorphous crystals with rhombohedral morphology, whereas A. sulfonivorans formed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves

Altered Bile Acid Metabolome in Patients with Nonalcoholic Steatohepatitis

BACKGROUND & AIMS: The prevalence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is increasing at an alarming rate. The role of bile acids in the development and progression of NAFLD to NASH and cirrhosis is poorly understood. This study aimed to quantify the bile acid metabolome in healthy subjects and patients with non-cirrhotic NASH under fasting conditions and after a standardized meal. METHODS: Liquid chromatography tandem mass spectroscopy was used to quantify 30 serum and 16 urinary bile acids from 15 healthy volunteers and 7 patients with biopsy-confirmed NASH. Bile acid concentrations were measured at two fasting and four post-prandial timepoints following a high-fat meal to induce gallbladder contraction and bile acid reabsorption from the intestine. RESULTS: Patients with NASH had significantly higher total serum bile acid concentrations than healthy subjects under fasting conditions (2.2- to 2.4-fold increase in NASH; NASH: 2595–3549 μM and healthy: 1171–1458 μM) and at all post-prandial time points (1.7- to 2.2-fold increase in NASH; NASH: 4444–5898 μM and healthy: 2634–2829 μM). These changes were driven by increased taurine- and glycine-conjugated primary and secondary bile acids. Patients with NASH exhibited greater variability in their fasting and post-prandial bile acid profile. CONCLUSIONS: Results indicate that patients with NASH have higher fasting and post-prandial exposure to bile acids, including the more hydrophobic and cytotoxic secondary species. Increased bile acid exposure may be involved in liver injury and the pathogenesis of NAFLD and NASH