Metabolomics Analysis as a Tool to Measure Cobalt Neurotoxicity: An In Vitro Validation.

In this study, cobalt neurotoxicity was investigated in human astrocytoma and neuroblastoma (SH-SY5Y) cells using proliferation assays coupled with LC-MS-based metabolomics and transcriptomics techniques. Cells were treated with a range of cobalt concentrations between 0 and 200 µM. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed cobalt cytotoxicity and decreased cell metabolism in a dose and time-dependent manner was observed by metabolomics analysis, in both cell lines. Metabolomic analysis also revealed several altered metabolites particularly those related to DNA deamination and methylation pathways. One of the increased metabolites was uracil which can be generated from DNA deamination or fragmentation of RNA. To investigate the origin of uracil, genomic DNA was isolated and analyzed by LC-MS. Interestingly, the source of uracil, which is uridine, increased significantly in the DNA of both cell lines. Additionally, the results of the qRT-PCR showed an increase in the expression of five genes Mlh1, Sirt2, MeCP2, UNG, and TDG in both cell lines. These genes are related to DNA strand breakage, hypoxia, methylation, and base excision repair. Overall, metabolomic analysis helped reveal the changes induced by cobalt in human neuronal-derived cell lines. These findings could unravel the effect of cobalt on the human brain.

Cobalt Neurotoxicity: Transcriptional Effect of Elevated Cobalt Blood Levels in the Rodent Brain.

Metal-on-metal (MoM) hip implants made of cobalt chromium (CoCr) alloy have shown early failure compared with other bearing materials. A consequence of the abnormal wear produced by these prostheses is elevated levels of cobalt in the blood of patients, which can lead to systemic conditions involving cardiac and neurological symptoms. In order to better understand the implications for patients with these implants, we carried out metal content and RNA-Seq analysis of excised tissue from rats treated intraperitonially for 28 days with low concentrations of cobalt. Cobalt blood levels in dosed rats were found to be similar to those seen in some patients with MoM implants (range: 4-38 µg/L Co in blood). Significant accumulation of cobalt was measured in a range of tissues including kidney, liver, and heart, but also in brain tissue. RNA-Seq analysis of neural tissue revealed that exposure to cobalt induces a transcriptional response in the prefrontal cortex (pref. cortex), cerebellum, and hippocampus. Many of the most up- and downregulated genes appear to correspond to choroid plexus transcripts. These results indicate that the choroid plexus could be the brain tissue most affected by cobalt. More specifically, the differentially expressed genes show a disruption of steroidogenesis and lipid metabolism. Several other transcripts also demonstrate that cobalt induces an immune response. In summary, cobalt exposure induces alterations in the brain transcriptome, more specifically, the choroid plexus, which is in direct contact with neurotoxicants at the blood-cerebrospinal fluid barrier.

Cellular trans-differentiation and morphogenesis toward the lymphatic lineage in regenerative medicine.

Lymphedema is a medically irresolvable condition. The lack of therapies addressing lymphatic vessel dysfunction suggests that improved understanding of lymphatic cell differentiation and vessel maturation processes is key to the development of novel, regenerative medicine, and tissue engineering approaches. In this review we provide an overview of lymphatic characterization markers and morphology in development. Further, we describe multiple differentiation processes of the lymphatic system during embryonic, postnatal, and pathogenic development. Using the example of pathogenic Kaposi's sarcoma-associated herpes infection, we illustrate the involvement of the Notch and PI3K pathways for lymphatic transdifferentiation. We also discuss the plasticity of certain cell types and biofactors that enable transdifferentiation toward the lymphatic lineage. Here we argue the importance of pathway-associated induction factors for lymphatic transdifferentiation, including growth factors such as vascular endothelial growth factor receptor-C and interleukins, and the involvement of extracellular matrix characteristics and dynamics for morphological functionality.

Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes.

The toxicity of hexavalent chromium and nickel was investigated using primary cultures of hepatocytes as an in vitro system. Cr VI and Ni are widely used in the steel and orthopaedic implant industry. Although their toxicity has been extensively investigated, the mechanism(s) of action is/are not fully understood. Monolayer cultures of hepatocytes (10(5) cells/cm2) were exposed to various concentrations of Cr VI and Ni for 24 h. Cells were stained with phalloidin-FITC for the detection of the cytoskeletal component, F-actin, and Annexin V-FITC and propidium iodide for the detection of the mode of cell death. Exposure of cells to Cr VI (1, 5, 10 and 50 microM) resulted in the loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Ni, on the other hand, induced detectable damage to the cytoskeleton only at 500 microM. Staining of the cells with Annexin V and propidium iodide showed that Cr VI induces apoptosis at low concentrations (5 microM), and necrosis at higher concentrations (25 and 50 microM). Ni almost exclusively induced necrosis at 500 microM with very few cells undergoing apoptosis. Below this concentration it had no discernable effect on hepatocytes. Damage to the cell cytoskeleton caused by Cr VI may be an early indication of apoptosis in hepatocytes.