About the Project
Nanotechnology as a modern-day key technology uses nano-sized particles which exhibit novel properties and functions due to their small size and large surface area. Such properties often produce different responses 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; Magdolenova et al., 2014).
These metal nanoparticles sometimes produce ambivalent results in terms of their genotoxic impact on DNA, often depending on size and oxidation state. Nonetheless, at least some of the detrimental effect on the genome is due to oxidative stress. AgNPs used in various industrial and medical applications induce oxidative stress, genotoxicity and apoptosis in cultured cells and animal tissues (Kim and Ryu, 2013). Oxidative stress as the predominant mechanism of toxicity of AgNP was found to be also size-dependent (Carlson et al., 2008). As the uptake of AgNP 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 AgNP induced cytotoxicity, genotoxicity and cell cycle arrest (AshaRani et al., 2009b).
For this project human blood lymphocytes will serve as surrogates for somatic cell and human spermatozoa will be used as one type of germ cell. (Rodent germ cells could be used for detection of phase specificity for other nanoparticles). Selection of the appropriate cytotoxicity assay is vital for the accurate assessment of nanoparticle toxicity. Various assays will be used to study the toxic effects of nanoparticles on cells including lactate dehydrogenase (LDH) leakage, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and identification of cytokine/chemokine production. DNA damage will be evaluated in the Comet assay. The early stage of apoptotic cells will be assessed by the annexin V assay and the last stage of apoptotic cells will be detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) and anti-apoptotic and pro-apoptotic genes such as p53 and bcl2, and DNA repair genes such as ATM and ATR will be analysed by real-time PCR (qPCR) and their protein expression level using the Western blot technique. Superoxide anions will be detected using the nitroblue tetrazolium (NBT) reduction assay.
This project could pave the way to much clearer understanding of the action of metal nanoparticles as well as to finding a suitable approach to ameliorate possible negative effects of these nanoparticles in human cells.
Asharani, P. V., et al., 2009a. Anti-proliferative activity of silver nanoparticles. BMC Cell Biol. 10, 65.
AshaRani, P. V., et al., 2009b. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano. 3, 279-90.
Carlson, C., et al., 2008. Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J Phys Chem B. 112, 13608-19.
Kim, S., Ryu, D. Y., 2013. Silver nanoparticle-induced oxidative stress, genotoxicity and apoptosis in cultured cells and animal tissues. J Appl Toxicol. 33, 78-89.
Magdolenova, Z., et al., 2014. Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles. Nanotoxicology. 8, 233-78.
Najafzadeh, M., et al., 2016. DNA Damage in Healthy Individuals and Respiratory Patients after Treating Whole Blood In vitro with the Bulk and Nano Forms of NSAIDs. Frontiers in molecular biosciences. 3, 50.
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