Humans are increasingly being exposed to nanoparticles from sources such as CoCr nanoparticles in metal on metal bearing orthopaedic implants, and also due to the increasing use of nanotechnology. The potential for nanoparticle exposures during pregnancy to cause developmental toxicity has been shown in vivo, and most commonly affects fetal neural development. This has generally been presumed to be due to the passage of nanoparticles to the fetus. It has however been shown that nanoparticles can cause DNA damage across placental barriers without crossing them. This 'indirect toxicity' is due to nanoparticles being internalised within the barrier and initiating a signalling cascade that is believed to transverse the barrier via connexin 43 gap junctions. In this thesis BeWo trophoblast cells were used to make in vitro models of the placenta to determine whether indirect nanoparticle toxicity could alter neurodevelopment. BeWo barriers were exposed to CoCr nanoparticles and media was harvested from beneath the barrier. This was applied to human neural progenitor cells that were differentiating into neurons and astrocytes. The viability of these cells was.unaffected although a shift to an astrocytic phenotype did occur. The resultant astrocytes had enlarged nuclear and cytoplasmic areas on immunostaining and were believed to be reactive astrocytes. These astrocytes also exhibited high levels of yH2AX foci on immunostaining, representing increased levels of DNA damage. The developing human cortical neurons, in culture with the astrocytes, were also found to have increased levels of yH2AX foci but to a lesser level than found in astrocytes. Near pure neuronal cultures did not however develop increased levels of yH2AX foci, therefore it was believed that the reactive astrocytes were initiating the DNA damage in the developing neurons. Exposures to differentiating embryoid bodies derived from human embryonic stem cells also found that differentiation into neuroectoderm was not affected. These results indicate that nanoparticies may alter neurodevelopment due to triggering signalling within the placenta, and that this is dependent on the presence of astrocytes, with earlier developmental events not being equally affected. Finally further investigations were performed to delineate the initiation and methods of transfer of this DNA damaging signalling in the BeWo barrier. These found that when nanoparticles come into contact with the barriers, impairment of autophagic flux occurs in the upper layers. Successful knockdown of Cx43 in the BeWo barriers was performed using shRNA lentiviral vectors to investigate the transfer of DNA damaging signalling. This however did not prevent morphological changes from developing in astrocytes, suggesting that Cx43 gap junctions may not be the only method of signalling transport involved in indirect nanoparticle toxicity. These results are discussed in relation to our current knowledge of neurodevelopment and the effects that astrocytes have in health and disease. Based on the results in this thesis I believe that limiting exposure to nanoparticles is important in women of childbearing age.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:683558 |
| Date | January 2015 |
| Creators | Hawkins, Simon James |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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