Cobalt toxicity, arising from the release of metal nanoparticles and ions during wear of Co/Cr metal-on-metal (MoM) hip implants has become a recognised internal source of Co poisoning. Co ions enter the bloodstream during wear and corrosion processes of MoM implants, and then travel throughout the body causing systemic toxicity. Cardiac toxic manifestations have been shown to be among the most prevalent systemic symptoms following exposure. In this thesis we have investigated the effects of short and/or long term CoCl2 exposure on the Swiss 3T3 fibroblast cell line (3T3s) and primary adult cardiac fibroblasts (CFs) in vitro. We have measured effects on cell viability, morphology, proliferation, assessed intracellular Co uptake, and detected and quantified protein expression by western blotting. Further, we also studied the effects of chronic in vivo CoCl2 treatment (4 weeks of daily 1mg/kg intraperitoneal injections) of rats carrying out echocardiography, and determining the Co distribution into various organs. The expression of potential transporter proteins, TRPC6, TRPM7, DMT1, and of CAMKIIδ, a serine/threonine kinase with pivotal roles in cardiovascular function through regulation of Ca2+ handling and excitation-contraction coupling, were also investigated. To complete the in vitro and in vivo studies, the molecular mechanism(s) underlying the overall changes were screened by RNA-Seq analysis, and gene expression was subsequently validated by quantitative real-time-PCR (RT-qPCR) and western blotting. In terms of proliferation of 3T3 cells and CFs treated with CoCl2, the CFs were much more sensitive than 3T3 cells with IC50 values for CoCl2 in the range of ~20 μM inCFs and ~250 μM in 3T3 cells. Using phalloidin to stain the actin inside cells showed that in both types of cells actin fibres were disrupted, and the membrane formed blebs at high Co concentrations. Co uptake, evaluated at the cellular level by using inductively coupled plasma mass spectrometry (ICP-MS), revealed 3 to 4-times greater Co uptake into CFs than 3T3 cells at 48 h. In vivo studies using echocardiography showed evidence of altered cardiac function in Co treated rats after 28 days exposure. Reduction of the percentage of fractional shortening (%FS), from 60.29±0.53%, to 54.01±0.90% n=6, p < 0.05, (Co-treated compared to normal rats) occurred. This could imply early indications of cardiac dysfunction. Co accumulated in the organs of the rats and significant increases in Co ion levels (compared with control untreated animals) were detected in liver, kidney and heart (1,839.86±177.30, 1,536.01±83.95, and 307.82±35.74 μg/L respectively) by ICP-MS after 7 days exposure. Liver and kidney are the primary organs of excretion, and higher concentrations than other organs might be expected. However, these data provide strong evidence that Co accumulates in the hearts of the treated rats and this may result in cardiac dysfunction. Western blot analysis of CFs, and heart tissue lysates from animals treated with CoCl2 for 28 days, showed a significantly increased level of protein expression for CaMKIIδ, TRPC6 and TRPM7. In contrast, in 3T3 cells, the expression of TRPs was decreased at high concentrations of Co. TRP transport channel proteins are likely to act as ubiquitous metal ion flux pathways, and may play a role in uptake of Co2+ into the heart enabling it to contribute to compromised heart function. Gene expression analysis of Camkiid, Dmt1, Trpc6, Trpm7, and Trpv1 using RNA-Seq and RT-qPCR, found all had increased expression levels in heart RNA after 28 days exposure. RT-qPCR analysis showed there were increased levels in 4 out of the 5 selected genes (Camkiid, Dmt1, Trpc6, and Trpm7) in CFs after 72 h Co treatment. In contrast, Trpc6 mRNA expression was absent in 3T3 cells using RNA-Seq and RT-qPCR. These results suggest the differential uptake of Co2+ between CFs and 3T3 cells might be mediated through Trpc6. Sequestration of Co into cardiac cells in vitro and in vivo, suggests that in vivo the metal may accumulate in the hearts of MoM patients with high circulating Co blood levels. This may lead to systemic adverse effects including cardiotoxicity. Inhibition or downregulation of Co uptake mechanisms into cardiac cells may offer a therapeutic intervention to minimise adverse effects of Co in patients with MoM implants. We recommend careful monitoring of cardiac function in MoM patients, in addition to the blood metal ion level monitoring and scans already instigated by MHRA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:750631 |
| Date | January 2018 |
| Creators | Laovitthayanggoon, Sarunya |
| Publisher | University of Strathclyde |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=29539 |
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