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Asbestos increases mitochondrial Ca2+ and H2O2 generation in THP1 cells
Author(s) -
Ryan Alan James,
Carter Brent A
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1166.15
Subject(s) - uniporter , mitochondrion , chemistry , mitochondrial ros , asbestos , inner mitochondrial membrane , reactive oxygen species , calcium , hydrogen peroxide , microbiology and biotechnology , biophysics , biology , cytosol , biochemistry , materials science , organic chemistry , metallurgy , enzyme
Asbestosis is an important cause of pulmonary fibrosis. We have shown that hydrogen peroxide (H 2 O 2 ) released from macrophages contributes to the genesis of fibrosis. Because mitochondrial calcium (Ca 2+ ) influx can increase mitochondrial H 2 O 2 production, we examined if Ca 2+ influx and H 2 O 2 generation could be altered in macrophages exposed to asbestos. Using THP1 cells loaded with the Ca 2+ ‐sensitive fluorescent dye Fura‐2, we found that intracellular Ca 2+ increased by ~2 fold within 4–8 min after asbestos exposure and remained elevated for 60 min. Cells loaded with MitoTracker Red plus Ca 2+ ‐sensitive Fluo3 (or Ca 2+ ‐indicator Rhod2) showed that mitochondrial Ca 2+ levels were elevated within min after asbestos exposure. Since mitochondrial Ca 2+ uptake is driven by a negative mitochondrial membrane potential (ΔΨ), we evaluated ΔΨ in cells loaded with JC‐1. We found that ΔΨ was reduced by ~25–50% in cells exposed to asbestos, a finding consistent with expected net charge changes associated with mitochondrial Ca 2+ entry. Finally, to evaluate the role of mitochondrial Ca 2+ entry on H 2 O 2 generation, we treated cells with Ru360, an inhibitor of mitochondrial Ca 2+ uniporter (MCU), and found that Ru360 reduced H 2 O 2 generation in cells exposed to asbestos. These findings suggest that asbestos rapidly increases mitochondrial Ca 2+ via the MCU, which contributes to mitochondrial H 2 O 2 generation in macrophages. (Supported by 2R01ES015981–06, R01ES014871, VA Merit Review 1BX001135 to A.B.C.)