The effect of metal nanoparticles in human haploid and diploid cells
Summary of Project
The use of metals such as silver (Ag), gold (Au) and copper (Cu) as nanoparticles (NP) in medical and lifestyle applications has exponentially increased in recent years, yet little is still known about their impact on DNA and RNA in somatic as well as in germ cells. As the application of metal nanomaterial is wide spread, exposure to such material is virtually unavoidable. New applications highlighting the potential of nanoparticles are introduced almost every day despite limited knowledge on their genotoxicity and their mode of action.
Nanotechnology as a modern-day key technology utilizes nano-sized particles which exhibit novel properties and functions due to their small size and large surface area, often differing significantly from the corresponding bulk counterpart. The same properties making nanoparticles so distinctive may also be accountable for their potential toxicity. However, despite a growing increase in exposure to nanomaterials currently no clear regulatory guidelines on the testing/evaluation of the toxicological and genotoxicological potential of nanomaterials exists (Arora et al. 2012).
These metal nanoparticles sometimes produce ambivalent results in terms of their genotoxic impact on DNA, often depending on size and oxidation state. Lu et al. for instance did not find any genotoxic effect of Ag NP (Lu et al. 2010). Nonetheless, at least some of the detrimental effect on the genome is due to oxidative stress. Ag NP used in various industrial and medical applications induced oxidative stress, genotoxicity and apoptosis in cultured cells and animal tissues (Kim and Ryu 2013). Oxidative stress as the predominant mechanism of toxicity of Ag NP was found to be also a size-dependent (Carlson et al. 2008). As the uptake of Ag NP occurs mainly via endocytosis and macropinocytosis cellular stress is induced either directly or indirectly through intracellular calcium transients and chromosomal aberrations causing an inhibition of cell proliferation (Asharani et al. 2009a). In cell cultures Ag NP induced cytotoxicity, genotoxicity and cell cycle arrest (AshaRani et al. 2009b).
For this project blood lymphocytes will serve as surrogates for somatic cell and spermatozoa will be used as one type of germ cells. Also, cell cultures are being used to investigate the oxidative effect of metal nanomaterials on DNA and RNA. As oxidative stress can be ameliorated by supplementation of anti-oxidants, compounds like resveratrol, bio-flavonoids and other substances will be used to be co-treated with metal nanoparticles. To identify DNA damage in general the Comet assay as a rapid and inexpensive tool will be used together with the cytokinesis-block micronucleus assay. The latter assay will be extended by using fluorescence in situ hybridization employing pancentromeric probes to detect centromeres in the induced micronuclei. In a recent study, Ag NPs up-regulated the cell cycle checkpoint protein p53, DNA damage repair proteins Rad51 and phosphorylated-H2AX expression in murine embryonic stem cells and embryonic fibroblasts. They also induced cell death measured with the annexin V and MTT assays (Ahamed et al. 2008). Hence, in order to target double-strand breaks (DSB) the gammaH2AX assay will be used for his project; a very sensitive assay that allows the detection of gammaH2AX foci which are equivalent to DSB in the nucleus. For the identification of preferred repair pathways PCR and RNAi can be used. As the genome carries common and rare fragile sites (>110) which are responsible for DNA fragmentation and in some cases for the progression of cancer, such sites (e.g. FRA7) can be used to identify mechanisms of DNA damage.
This project could pave the way to much clearer understand the action of metal nanoparticles as well as to find a suitable approach to ameliorate possible negative effects of these nanoparticles.
At least 2.i Honours degree or equivalent.