Skip to Content
Merck
  • ZnO nanoparticles-associated mitochondrial stress-induced apoptosis and G2/M arrest in HaCaT cells: a mechanistic approach.

ZnO nanoparticles-associated mitochondrial stress-induced apoptosis and G2/M arrest in HaCaT cells: a mechanistic approach.

Mutagenesis (2019-07-05)
N V Srikanth Vallabani, Souvik Sengupta, Ritesh Kumar Shukla, Ashutosh Kumar
ABSTRACT

Zinc oxide nanoparticles (ZnO NPs) with their wide range of consumer applications in day-to-day life received great attention to evaluate their effects in humans. This study has been attempted to elucidate the DNA damage response mechanism in a dermal model exposed to ZnO NPs through Ataxia Telangiectasia Mutated (ATM)-mediated ChK1-dependent G2/M arrest. Further, viability parameters and mechanism involved in the cell death with special reference to the consequences arising due to DNA damage were explored. Our study showed that ZnO NPs at concentrations 5 and 10 µg/ml induced significant cytotoxic effect in skin cell line. Moreover, the results confirmed generation of reactive oxygen species (ROS) induces the cell death by genotoxic insult, leading to mitochondrial membrane depolarisation and cell cycle arrest. Subsequently, ZnO NPs treatment created DNA damage as confirmed via Comet assay (increase in olive tail moment), micronucleus assay (increase in micronucleus formation), double-strand breaks (increase in ATM and Ataxia Telangiectasia and Rad3 related (ATR) expression), DNA fragmentation and cell cycle (G2/M arrest) studies. Finally, marker proteins analysis concluded the mechanistic approach by demonstrating the key marker expressions HMOX1 and HSP60 (for oxidative stress), cytochrome c, APAF1, BAX, Caspase 9, Caspase 3 and decrease in BCL2 (for activating apoptotic pathway), pATM, ATR and γH2AX (for double-strand breaks), DNA-PK (involved in DNA repair) and decrease in cell cycle regulators. In together, our data revealed the mechanism of ROS generation that triggers apoptosis and DNA damage in HaCaT cell lines exposed to ZnO NPs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Heme Oxygenase-1 Mouse mAb (HO-1-1), liquid, clone HO-1-1, Calbiochem®
Sigma-Aldrich
Anti-Bax Antibody, NT, aa3-16 hBax protein, clone 2D2, clone 2D2, Chemicon®, from mouse
Sigma-Aldrich
Anti-Caspase 3 Antibody, active (cleaved) form, Chemicon®, from rabbit
Sigma-Aldrich
Anti-DNA-PKcs Antibody, clone 3A9.1, clone 3A9.1, from mouse
Sigma-Aldrich
Anti-Cyclin B1 Antibody, clone GNS3 (8A5D12), clone GNS3 (8A5D12), Upstate®, from mouse
Sigma-Aldrich
In Vitro Toxicology Assay Kit, Lactic Dehydrogenase based
Sigma-Aldrich
Anti-Bcl-2 Antibody, clone 1D7.2, clone 1D7.2, from rat
Sigma-Aldrich
Anti-beta-Actin Antibody, clone RM112, clone RM112, from rabbit
Sigma-Aldrich
Anti-Apaf-1 Antibody, clone 18H2, clone 18H2, from rat
Sigma-Aldrich
Anti-ATM phosphoSer1981 Antibody, clone 10H11.E12, clone 10H11.E12, Chemicon®, from mouse