Abstract
BackgroundExposures to an amphibole fiber in Libby, Montana cause increases in malignant mesothelioma (MM), a tumor of the pleural and peritoneal cavities with a poor prognosis. Affymetrix microarray/GeneSifter analysis was used to determine alterations in gene expression of a human mesothelial cell line (LP9/TERT-1) by a non-toxic concentration (15×106 μm2/cm2) of unprocessed Libby six-mix and negative (glass beads) and positive (crocidolite asbestos) controls. Because manganese superoxide dismutase (MnSOD; SOD2) was the only gene upregulated significantly (p < 0.05) at both 8 and 24 h, we measured SOD protein and activity, oxidative stress and glutathione (GSH) levels to better understand oxidative events after exposure to non-toxic (15×106 μm2/cm2) and toxic concentrations (75×106 μm2/cm2) of Libby six-mix.ResultsExposure to 15×106 μm2/cm2 Libby six-mix elicited significant (p < 0.05) upregulation of one gene (SOD2; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in SOD2. Increased levels of SOD2 mRNA at 24 h were also confirmed in HKNM-2 normal human pleural mesothelial cells by qRT-PCR. SOD2 protein levels were increased at toxic concentrations (75×106 μm2/cm2) of Libby six-mix at 24 h. In addition, levels of copper-zinc superoxide dismutase (Cu/ZnSOD; SOD1) protein were increased at 24 h in all mineral groups. A dose-related increase in SOD2 activity was observed, although total SOD activity remained unchanged. Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence staining and flow cytometry revealed a dose- and time-dependent increase in reactive oxygen species (ROS) production by LP9/TERT-1 cells exposed to Libby six-mix. Both Libby six-mix and crocidolite asbestos at 75×106 μm2/cm2 caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (HO-1) in LP9/TERT-1 and HKNM-2 cells.ConclusionsLibby six-mix causes multiple gene expression changes in LP9/TERT-1 human mesothelial cells, as well as increases in SOD2, increased production of oxidants, and transient decreases in intracellular GSH. These events are not observed at equal surface area concentrations of nontoxic glass beads. Results support a mechanistic basis for the importance of SOD2 in proliferation and apoptosis of mesothelial cells and its potential use as a biomarker of early responses to mesotheliomagenic minerals.
Highlights
Introduction ofMinerals/Agents to Cells Following sterilization under ultraviolet light overnight, minerals were suspended in 1X Hanks’ Balanced Salt Solution (HBSS) at 1 mg/ml, sonicated for 15 min in a water bath sonicator, and triturated 5 times through a 22-gauge needle
The sample of Libby amphibole we used in the studies described here is often referred to as “six-mix” since it includes six different samples collected at the former mine site, and is comprised of a combination of several amphiboles including winchite, richterite and tremolite, as well as other trace elements not classified in this mineral family [19]
TERT-1 cells following Libby six-mix exposure Since SOD2 gene expression was upregulated at both 8 h (4-fold) and 24 h (5-fold), we further examined the expression of SOD protein and enzyme activity using Western blot analysis on whole-cell lysates from LP9/ TERT-1 cells exposed to glass beads, low and high concentrations of Libby six-mix (15 and 75×106 μm2/cm2, respectively), and the phorbol ester, TPA (100 ng/mL)
Summary
Minerals/Agents to Cells Following sterilization under ultraviolet light overnight, minerals were suspended in 1X Hanks’ Balanced Salt Solution (HBSS) at 1 mg/ml, sonicated for 15 min in a water bath sonicator, and triturated 5 times through a 22-gauge needle This suspension was added to cells in medium to achieve the desired surface area-based concentrations. Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence staining and flow cytometry revealed a dose- and timedependent increase in reactive oxygen species (ROS) production by LP9/TERT-1 cells exposed to Libby six-mix. Both Libby six-mix and crocidolite asbestos at 75×106 μm2/cm caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (HO-1) in LP9/TERT-1 and HKNM-2 cells. Lung cancer mortality is elevated in these individuals compared to the remainder of the United States [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
