Cobalt cardiotoxicity - effects on the contractile and non-contractile cells of the heart

Exposure to cobalt is known to cause cardiotoxicity and a common source of cobalt exposure is from metal-on-metal bearings used in prosthetic joint replacements. Acute and chronic effects of cobalt at a cellular level in the heart are not well understood. This study investigated the effects of cobalt (CoCl2) treatment on contractile and non-contractile cells of the heart. We have used isolated adult rat ventricular papillary muscles to investigate the effects of CoCl2 on basal and isoprenaline-stimulated contractile responses. In addition, we used freshly isolated primary adult rat ventricular fibroblasts maintained in short-term culture to assess the effects of CoCl2 on cell viability and proliferation. Stimulation of isolated ventricular papillary muscles with the positive inotrope isoprenaline (1uM) resulted in a consistent increase (~35% increase) over the basal contractile response as expected. Following treatment with CoCl2 (1uM) for 4h, there was a dramatic reduction in both the basal and isoprenaline-stimulated contractile responses (both~40% reduction). This effect was not due to a time-dependent decrease in contractility since in separate parallel control preparations consistent increases in contraction were observed following 1uM isoprenaline challenges at time zero and after 4h without CoCl2. Examination of the effects of CoCl2 on cardiac fibroblast proliferation and viability was performed using a range of assays. To assess effects on proliferation, MTT, neutral red and crystal violet assays were all used to compare effects of increasing concentrations of CoCl2 on the Swiss 3T3 fibroblast cell line and primary cardiac fibroblasts. Over 72h, increasing CoCl2 concentrations (up to 500uM) resulted in decreased proliferation. The MTT and Crystal violet assays showed the most reproducible results with IC50 values for CoCl2 in the range of ~300uM. Interestingly, further experiments using BrdU incorporation to assess proliferation suggested that cardiac fibroblasts were more sensitive to CoCl2 treatment than Swiss 3T3s. In the former, after either 48h or 72h there was ~80% reduction in proliferation with 25uM CoCl2 and almost no proliferation following 100–150uM CoCl2. Cell viability in increasing concentrations of CoCl2 (up to 500uM) was assessed using CFDA and propidium iodide staining. The ratio of live:dead cells decreased dramatically with increasing CoCl2. Phalloidin-FITC was also used to examine cell viability and structure following treatment. With increasing CoCl2 there was evidence for increased disruption of actin filaments. In conclusion, short-term low dose CoCl2 treatment of ventricular preparations results in compromised contractile function. Treatment of non-contractile cardiac fibroblasts with higher concentrations results in decreased ability of cells to proliferate as well as long-term cell damage and death. It is likely that the cardiotoxic effects of CoCl2 are manifest in both contractile and non-contractile cells of the heart. The underlying cellular mechanisms involved have yet to be established

BDNF Val66Met polymorphism attenuates explosive jump fatigue differentially after trans-spinal anodal direct current stimulation

Non-invasive anodal trans-spinal direct current stimulation (tsDCS) can modulate central nervous system activity (1-5) with effects lasting for at least one hour post stimulation. In healthy subjects we observed tsDCS to alter the performance of repeated maximal effort explosive countermovement vertical jumps through effects on motor fatigue mechanisms and coordination (6). However, there is significant variability between subjects. Brain-derived neurotrophic factor (BDNF) is a key mediator of activity-based neuroplasticity. Carriers of the BDNF Val66Met single nucleotide polymorphism (Met SNP; rs6265) secrete less BDNF (7) and have altered neuroplastic responses to tsDCS (8) compared to normal Val66Val carriers. Accordingly, we are investigating if any association can be identified between BDNF genotype and changes in repeated jump performance following sham and active anodal tsDCS

Metabolomics-guided isolation of anti-trypanosomal compounds from endophytic fungi of the mangrove plant Avicennia lanata

Endophytic fungi have been explored not just for their ecological functions but also for their secondary metabolites as a new source of these pharmacologically active natural products. Accordingly, many structurally unique and biologically active compounds have been obtained from the cultures of endophytic fungi. Fusarium sp. and Lasiodiplodia theobromae were isolated from the root and stem of the mangrove plant Avicennia lanata, respectively, collected from Terengganu, Malaysia. High-resolution mass spectrometry and NMR spectroscopy were used as metabolomics profiling tools to identify and optimize the production of bioactive secondary metabolites in both strains at different growth stages and culture media. The spectral data was processed by utilizing the MZmine 2.2, a quantitative expression analysis software and an in house MS-Excel macro coupled with the Dictionary of Natural Products databases for dereplication studies. The investigation for the potential bioactive metabolites from a 15-day rice culture of Fusarium sp. yielded four 1,4-naphthoquinone with naphthazarin structures (1-4). On the other hand, the endophytic fungus L. theobromae grown on the 15-day solid rice culture produced dihydroisocoumarins (5 to 8). All the isolated compounds (1 to 8) showed significant activity against Trypanosoma brucei brucei with MIC values of 0.32-12.5 μM. Preliminary cytotoxicity screening against normal prostate cells (PNT2A) was also performed. All compounds exhibited low cytotoxicity, with compounds 3 and 4 showing the lowest cytotoxicity of only 22.3% and 38.6% of the control values at 100 μg/mL, respectively. Structure elucidation of the isolated secondary metabolites was achieved using 1D and 2D-NMR and HRESI-MS as well as comparison with literature data

In vitro analyses of the toxicity, immunological, and gene expression effects of cobalt-chromium alloy wear debris and Co ions derived from metal-on-metal hip implants

Joint replacement has proven to be an extremely successful and cost-effective means of relieving arthritic pain and improving quality of life for recipients. Wear debris-induced osteolysis is, however, a major limitation and causes orthopaedic implant aseptic loosening, and various cell types including macrophages, monocytes, osteoblasts, and osteoclasts, are involved. During the last few years, there has been increasing concern about metal-on-metal (MoM) hip replacements regarding adverse reactions to metal debris associated with the MoM articulation. Even though MoM-bearing technology was initially aimed to extend the durability of hip replacements and to reduce the requirement for revision, they have been reported to release at least three times more cobalt and chromium ions than metal-on-polyethylene (MoP) hip replacements. As a result, the toxicity of metal particles and ions produced by bearing surfaces, both locally in the periprosthetic space and systemically, became a concern. Several investigations have been carried out to understand the mechanisms responsible for the adverse response to metal wear debris. This review aims at summarising in vitro analyses of the toxicity, immunological, and gene expression effects of cobalt ions and wear debris derived from MoM hip implants

The effects of hydration media on the characteristics of non-ionic surfactant vesicles (NISV) prepared by microfluidics

Non-ionic surfactant vesicles (NISV) are colloidal particles that provide a useful delivery system for drugs and vaccines. One of the methods that is used for NISV preparation is microfluidics in which the lipid components dissolved in organic phase are mixed with an aqueous medium to prepare the particles through self-assembly of the lipids. In this work, we examined the effect of using different types of aqueous media on the characteristics of the NISV prepared by microfluidics. Five aqueous media were tested: phosphate buffered saline, HEPES buffer, Tris buffer, normal saline and distilled water. The resulting particles were tested for their physical characteristics and cytotoxicity. The aqueous media were found to have significant effects on the physical characteristics of the particles, as well as their overall stability under different conditions and their cytotoxicity to different human cell lines. Careful consideration should be taken when choosing the aqueous media for preparing NISV through microfluidics. This is an important factor that will also have implications with respect to the entrapped material, but which in addition may help to design vesicles for different uses based on changing the preparation medium

In vitro effects of cobalt ions on CNS derived cell lines

Several neurological symptoms associated with the toxic action of cobalt ions have been reported among patients with metal-on-metal (MoM) hip prostheses made of cobalt chromium (CoCr) alloy. The sporadic nature of these manifestations, combined with the fact that the medical evidence is relatively new, have contributed to poor understanding of the impact of cobalt poisoning on the brain. In the present study, we characterise the cytotoxicity of cobalt ions in human U 373 astrocytoma and SH-SY5Y neuroblastoma cell lines. Metal ion uptake with different cobalt chloride concentrations (0 to 500μM) for three time-points (24, 48 and 72h) was measured using inductively coupled plasma mass spectrometry (ICP-MS), while cell viability was tested with MTT and Neutral Red (NR) assays, and by microscopy. The results show that cobalt uptake is dose and time-dependent (up to 415.23, and 69.47μg/L for astrocytes and neurons, respectively), which corresponds with the significant decrease in cell viability (p<.05) at high cobalt concentrations both for the MTT and NR assays. IC50 values were 438.27±37.73 and 267.36±14.57μM at 48h for astrocytes and neurons, respectively (similar values for 72h), with the MTT assay more sensitive at detecting toxicity, suggesting involvement of redox mechanisms. Morphological changes, such as extensive vacuolization of the cytosol, usually associated with autophagy, were observed in the course of cell death. These results indicate that exposure to cobalt at high concentrations could have deleterious effects on brain cells. Future focus will be on the mechanism(s) responsible for cobalt uptake, which may provide a therapeutic intervention for MoM patients

The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E2 (PGE2) is responsible for inflammatory symptoms. However, PGE2 also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE2 receptors, EP1 – EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R - EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells. Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE2, alongside both selective agonists of EP1 – EP4 receptors, were assessed on LPS-induced TNF-α release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of TNF-α protein was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs. PGE2 and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulate expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE2 on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE2. This indicates that PGE2 suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory response

Exosomes : fighting cancer with cancer

Exosomes are nanovesicles secreted by many cells, including cancer cells. Extensive research has been carried out to validate potential applications of exosomes and to evaluate their efficiency in a wide range of diseases, including cancer. The current knowledge on the origin, biogenesis and composition of exosomes is described. This review then focuses on the use of exosomes in cancer diagnostics and therapeutics

Investigating cobalt toxicity in the context of joint replacement patients - cobalt uptake in primary cardiac fibroblasts and in 3T3 cells

Cobalt leaches out from cobalt/chromium metal-on-metal hip implants into patient blood, and its effects are thought to be toxic. There has been a 5% estimated incidence of adverse effects, including toxicity to the heart, in joint implant patients over the last 40 years. This was investigated by examination of the effects of CoCl2 on cell proliferation and viability performed using a range of assays. To assess effects on proliferation, MTT, neutral red and crystal violet assays were all used to compare effects of increasing concentrations of CoCl2 on the Swiss 3T3 fibroblast cell line (3T3s) and primary cardiac fibroblasts (CFs). CoCl2 induced toxicity in both 3T3s and CFs in a time- and dose-dependent manner with IC50 values for CoCl2 in the range of ~300 µM in both cells. Over 72h, increasing CoCl2 concentrations (up to 500 µM) resulted in decreased proliferation. Interestingly, in terms of proliferation, the 3T3s were more tolerant of CoCl2 than CFs. Uptake of CoCl2 into the 3T3s and CFs was measured by detecting intracellular metal content using ICP-MS. Cells were cultured and exposed to various concentrations of CoCl2 (0-72 ppm) and different exposure times (24, 48 and 72 h). Analysis of cobalt content of cells revealed that with increasing medium concentration of CoCl2 intracellular Co concentration on both 3T3s and CFs increased, to a range between 0-50 ppb and 0-120 ppb, respectively. Uptake into CFs was greater than into the 3T3s, and this at least partly explains the difference in toxicity between the two cell types