Related Experiment Videos
Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis
Arti Shukla1, Michael Jung, Maria Stern
1Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Abstract:
To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.
Insights
Asbestos exposure causes mitochondrial DNA damage and cell death through oxidative stress. Protecting mitochondria with DNA repair enzymes can increase cell survival, indicating mitochondria are key targets.
Area of Science:
- Cell Biology
- Toxicology
- Molecular Biology
Background:
- Asbestos is a known human carcinogen.
- The precise mechanisms of asbestos-induced cellular injury are not fully understood.
- Mitochondria play critical roles in cellular energy production and apoptosis.
Purpose of the Study:
- To investigate the role of mitochondria in asbestos-mediated cell injury.
- To determine if asbestos-induced DNA damage is oxidant-dependent.
- To explore the contribution of mitochondrial pathways to asbestos-induced apoptosis.
Main Methods:
- Quantitative PCR to assess mitochondrial and nuclear DNA damage.
- Confocal scanning laser microscopy to detect oxidant stress.
- Measurement of mRNA levels for mitochondrial genes.
- Assay of formazan production to evaluate mitochondrial function.
- Analysis of apoptotic gene expression and cell counts.
- Inhibition studies using caspase-9 inhibitor and catalase.
- Mitochondrial targeting of DNA repair enzyme (8-oxoguanine DNA glycosylase) in HeLa cells.
Main Results:
- Mitochondrial DNA damage occurred at lower asbestos concentrations than nuclear DNA damage.
- Asbestos exposure induced significant oxidant stress in mesothelial cells.
- Decreased mRNA levels of key mitochondrial respiratory chain components were observed.
- Mitochondrial dysfunction, increased apoptosis, and altered apoptotic gene expression followed asbestos exposure.
- Asbestos-induced apoptosis was reduced by caspase-9 inhibition, catalase, and mitochondrial DNA repair enzyme expression.
Conclusions:
- Mitochondria are initial targets of asbestos-induced DNA damage.
- Asbestos-mediated cell injury and apoptosis are driven by an oxidant-dependent mitochondrial pathway.
- Targeting mitochondrial DNA repair offers a potential strategy to mitigate asbestos toxicity.