Energy metabolism modulation by biguanides in comparison with rotenone in rat liver and heart.

The biguanide metformin, a widely used antidiabetic drug, has received great interest in oncology research in recent years after an epidemiological study showed a link between metformin treatment and a reduced cancer risk in diabetic patients. Since mitochondrial metabolism has become a target for possible cancer therapeutic approaches, especially for tumors relying on oxidative metabolism, mitochondrial complex I inhibition is under discussion to be responsible for the anti-cancer effect of metformin. Rotenone, a well-known strong mitochondrial complex I inhibitor, yet associated with toxic effects, has also shown anti-cancer activity. Thus, we compared metformin and phenformin, another biguanide previously on the market as antidiabetic, with rotenone, to elucidate potential mechanisms rendering biguanides apparently less toxic than rotenone. Therefore, we conducted in vivo rat studies with metformin and phenformin, based on an experimental design previously described for mechanistic investigations of the effects of rotenone, including blood and tissue analysis, histopathology and gene expression profiling. These investigations show that the mechanistic profile of phenformin appears similar to that of rotenone, yet at a quantitatively reduced level, whereas metformin displays only transient similarities after one day of treatment. A potential reason may be that metformin, but not rotenone or phenformin, self-limits its entry into mitochondria due to its molecular properties. Thus, our detailed molecular characterization of these compounds suggests that inhibition of mitochondrial functions can serve as target for an anti-cancer mode of action, but should be self-limited or balanced to some extent to avoid exhaustion of all energy stores.

Mechanistic Investigations of the Mitochondrial Complex I Inhibitor Rotenone in the Context of Pharmacological and Safety Evaluation.

Inhibitors of the mitochondrial respiratory chain complex I are suggested to exert anti-tumor activity on those tumors relying on oxidative metabolism and are therefore of interest to oncology research. Nevertheless, the safety profile of these inhibitors should be thoroughly assessed. Rotenone, a proven complex I inhibitor, has shown anti-carcinogenic activity in several studies. In this context rotenone was used in this study as a tool compound with the aim to identify suitable biomarker candidates and provide enhanced mechanistic insights into the molecular and cellular effects of complex I inhibitors. Rats were treated with 400 ppm rotenone daily for 1, 3 or 14 consecutive days followed by necropsy. Classical clinical endpoints, including hematology, clinical chemistry and histopathology with supporting investigations (FACS-analysis, enzymatic activity assays) were examined as well as gene expression analysis. Through these investigations, we identified liver, bone marrow and bone as target organs amongst approx. 40 organs evaluated at least histopathologically. Our results suggest blood analysis, bone marrow parameters, assessment of lactate in serum and glycogen in liver, and especially gene expression analysis in liver as useful parameters for an experimental model to help to characterize the profile of complex I inhibitors with respect to a tolerable risk-benefit balance.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to ß1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Automatic counting and positioning of 5-bromo-2-deoxyuridine (BrdU) positive cells in cortical layers of rat brain slices.

5-Bromo-2-deoxyuridine (BrdU) staining is often used to evaluate cortical layer formation during mammalian brain development. This method allows the quantification of newly generated cells and therefore the study of the effects of xenobiotics or genetic factors on proliferation, cell death and migration behavior in a quantitative manner. However, these endpoints are generally assessed by time-consuming manual evaluation. In the present work, we introduce a novel procedure to identify and quantify BrdU(+) cells within cortical layers, using the commercially available vHCS-Scan V.6.3.1 software to identify BrdU(+) cell coordinates and the novel program 'BrdeLuxe' to define cortical layers and quantitatively assign BrdU(+) cells to them. This procedure is compared to BrdU(+) cell counting with the freeware 'ImageJ' in respect to the manual evaluation, all by two different researchers. BrdeLuxe shows high accuracy and precision for the determination of total number of BrdU(+) cells compared to the manual counting, while ImageJ does not reach such results. Accuracy and precision are also higher for employing the BrdeLuxe program to evaluate the percentage of BrdU(+) cells per brain layer compared to ImageJ. In terms of running time, BrdeLuxe is the fastest method of the three making it more suitable for multiple brain slices analyses.