Mitochondria-Derived Reactive Intermediate Species Mediate Asbestos-Induced Genotoxicity and Oxidative Stress–Responsive Signaling Pathways

Background: The incidence of asbestos-induced human cancers is increasing worldwide, and considerable evidence suggests that reactive oxygen species (ROS) are important mediators of these diseases. Our previous studies suggested that mitochondria might be involved in the initiation of oxidative stress in asbestos-exposed mammalian cells. Objective: We investigated whether mitochondria are a potential cytoplasmic target of asbestos using a mitochondrial DNA–depleted (ρ0) human small airway epithelial (SAE) cell model: ρ0 SAE cells lack the capacity to produce mitochondrial ROS. Methods: We examined nuclear DNA damage, micronuclei (MN), intracellular ROS production, and the expression of inflammation-related nuclear genes in both parental and ρ0 SAE cells in response to asbestos treatment. Results: Asbestos induced a dose-dependent increase in nuclear DNA oxidative damage and MN in SAE cells. Furthermore, there was a significant increase in intracellular oxidant production and activation of genes involved in nuclear factor κB and proinflammatory signaling pathways in SAE cells. In contrast, the effects of asbestos were minimal in ρ0 SAE cells. Conclusions: Mitochondria are a major cytoplasmic target of asbestos. Asbestos may initiate mitochondria-associated ROS, which mediate asbestos-induced nuclear mutagenic events and inflammatory signaling pathways in exposed cells. These data provide new insights into the molecular mechanisms of asbestos-induced genotoxicity.


Toxicity of asbestos
The cellular toxicity of asbestos was measured by determining the survival fraction with the CyQuant cell proliferation Assay kit (Invitrogen). Briefly, exponentially growing cells in a 96-well tissue culture plate were treated with graded doses of asbestos fibers (0.5, 1, 2, and 4 µg/cm 2 ) for 72 hours; then culture media were removed and the cellular nuclei acids content of each control and treated group was measured with a fluorescence excitation of 480nm using a Synergy 2 multi-detection Microplate Reader (BioTek Instruments, Inc, Vermont). The surviving fractions (percentage over untreated controls) for treated groups were calculated.

Real-time Quantitative PCR
The mtDNA copy number was determined by real-time SYBR Green PCR using the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems). Genomic DNA was isolated and concentration was measured by spectrophotometry. For each sample, we amplified a 189-bp fragment of the nuclear encoded 18S rRNA gene and a 172-bp fragment of the mtDNA encoded 12S rRNA gene. The primer sequences are as follows: 18S sense, 5'-GGAGTATGGTTGCAAAGCTG-3'; 18S antisense, 5'-CGCTCCACCAACTAAGAACG-3'; 12S sense, 5'-AGAACACTACGAGCCACAGC-3'; and 12S antisense, 5'-ACTTGCGCTTACTTTGTAGCC-3'. All reactions were done in triplicate. The PCR conditions were: 95°C for 15 min followed by 40 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Relative quantification of mtDNA reported as mtDNA/nDNA ratio was done with the comparative threshold cycle (C T ) method as described previously (Partridge et al. 2007;Shao et al. 2006).

Mitochondrial membrane potential
JC-1, a membrane potential-sensitive fluorescent probe (Invitrogen), was used to determine the mitochondrial membrane potential of ρ 0 SAE cells (Liu et al. 2005;Reers et al. 1995). Exponentially growing parental and ρ 0 SAE cells were treated with 10 µmol/L JC-1 in growth medium for 30 min at 37°C. Extra dye was removed by washing with warm PBS and cells were maintained in regular medium on a heated 37°C stage. Cells were immediately visualized and images were captured on a Nikon laser confocal microscope (Nikon eclipse TE2000-U; excitation 488 nm and 543 nm for green and orange fluorescence, respectively).

Oxygen consumption
The oxygen consumption rate in live cells was measured as described previously (King et al. 1992;Partridge et al. 2007). Cells were counted and a minimum of 5 x 10 6 cells were resuspended in 1.5 mL of Opti-MEM (glucose free and sodium pyruvate supplemented medium). Oxygen concentration was monitored over 3 min and recorded for every 10 seconds at 37°C in a Hansatech (MA) Clark's oxygen electrode unit.

Cytochrome c Oxidase (COX) and succinate dehydrogenase (SDH) activity
COX and SDH activities were measured with biochemical assays as described (King et al. 1967;Partridge et al. 2007;Salviati et al. 2002). COX activity was indicated by the capability of the cell lysates to oxidize reduced cytochrome c at 550 nm (nmol oxidized cytochrome c per min per ml cell lysate) as measured by spectrophotometry. SDH activity was assessed by measuring the oxidization of succinate coupled to the reduction of DCPIP (2,6-dichlorophenolindophenol, an electron acceptor that is blue when oxidized and colorless when reduced) by the SDH reaction at 600 nm with spectrophotometry. The enzyme activity was indicated by nmol oxidized succinate per min per ml cell lysate. Both COX and SDH activities were normalized to mg of protein in the cell lysate.

Intracellular superoxide measurement with dihydroethidium (DHE)
Intracellular superoxide was determined using the fluorescent probe DHE (Invitrogen) as described previously (Zhou et al. 2008). Exponentially growing ρ 0 or parental SAE cells were stained with 2µmol/L DHE in regular medium for 45 min at 37°C. Cells were then trypsinized and suspended in PBS. The DHE fluorescence was measured by flow cytometry in the FL3 channel on a FACSCalibur (Becton Dickinson).  BCL2  CD8A  ICAM1  ITGB1  PLA2G10  SMAD3   BCL2L1  CES1  ICOS  ITGB2  PLA2G1B  SMAD7   BDKRB1  COL4A5  IFNG  KLK1  PLA2G2A  STAT3   BDKRB2  CSF1  IKBKB  KLK14  PLA2G2D  TBX21   C3  CSF2  IL10  KLK15  PLA2G4C  TBXA2R   CACNA1C  CSF3  IL12A  KLK2  PLA2G5  TBXAS1   CACNA1D  CTLA4  IL12B  KLK3  PLA2G7  TFRC   CACNA2D1 CXCL10  IL13  KLKB1  PLCB2  TGFB1   CACNB2  CXCL11  IL15  KNG1  PLCB3  TNF   CACNB4  CXCR3  IL17  LRP2  PLCB4  TNFRSF18   CASP1  CYP1A2  IL18  LTA  PLCD1  TNFRSF1A   CCL19  CYP7A1  IL1A  LTA4H  PLCE1  TNFRSF1B   CCL2  CYSLTR1  IL1B  LTB4R  PLCG1  TNFSF13B   CCL3  ECE1  IL1R1  LTB4R2  PLCG2  Note: IPA-Tox analysis allows assessing the toxicity of asbestos by generating a list of tox functions and/or pathways relevant to asbestos exposure. Ratio = the number of "genes of interest" in the current study that belong to a given pathway, divided by the total number of genes that make up the pathway (obtained from the existing literature); for example, a ratio of 0.025 indicates that 2.5% of the total gene molecules in a given pathway were also found in this study. P-value: the p-value tells the significance of the association between a specific pathway and the genes of interest in this study. Figure 1. Characterization of parental and ρ 0 SAE cells. A, the morphology of parental and ρ 0 SAE cells under optic microscope. Bar, 50µm. Right, enlarged image of boxed area (left). B, growth curves of parental and ρ 0 SAE cells. The population doubling time of each cell line was consistent for cells at different passages. C, mitochondrial morphology. The mitochondria are fragmented in ρ 0 cells but elongated and filamentous in parental cells. The green fluorescence indicates mitochondria stained with an antibody against pyruvate dehydrogenase (PDH), a mitochondrial enzyme. Bar, 10 µm. Images were captured with fluorescence microscopy (Olympus Bh-2 equipped with Olympus MicroSuite FIVE software). D, Toxicity of chrysotile and crocidolite asbestos fibers on ρ 0 and parental SAE cells. The cellular toxicity of asbestos was indicated by survival fraction (percentage of survival over untreated controls) measure with the CyQuant cell proliferation Assay kit. Asbestos and glass fibers exposure duration: 72 hours. Data (mean ± SD) from average of three independent experiments. Supplemental Material, Figure 2. Network of genes mediated by asbestos after 48 hours of treatment in ρ 0 SAE cells. A highly interconnected network of 12 asbestos-responsive genes (P<0.05) was constructed by IPA, based on direct interactions stored in the Ingenuity Knowledge Base, which is a collection of experimentally confirmed relationships between molecules. Color scale bar indicates the fold change in gene expression level, green: down regulation, red: up regulation. Un-colored molecules: expression levels were not examined in the current study. Note: "Ho" is a group/complex suggested by IPA to contain gene(s) examined by our study. "TNF" and "HRH2" were not significantly down-and up-regulated by asbestos according to StatMiner (P value = 0.03 and 0.26, respectively; but suggested by IPA to be affected by asbestos treatment based on the input of 12 interconnected asbestos-responsive genes (this Figure and Supplemental Material, Table 4).