Asbestos increases mitochondrial Ca2+ and H2O2 generation in THP1 cells

Abstract

Asbestosis is an important cause of pulmonary fibrosis. We have shown that hydrogen peroxide (H2O2) released from macrophages contributes to the genesis of fibrosis. Because mitochondrial calcium (Ca2+) influx can increase mitochondrial H2O2 production, we examined if Ca2+ influx and H2O2 generation could be altered in macrophages exposed to asbestos. Using THP1 cells loaded with the Ca2+‐sensitive fluorescent dye Fura‐2, we found that intracellular Ca2+ increased by ~2 fold within 4–8 min after asbestos exposure and remained elevated for 60 min. Cells loaded with MitoTracker Red plus Ca2+‐sensitive Fluo3 (or Ca2+‐indicator Rhod2) showed that mitochondrial Ca2+levels were elevated within min after asbestos exposure. Since mitochondrial Ca2+ 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 Ca2+ entry. Finally, to evaluate the role of mitochondrial Ca2+ entry on H2O2 generation, we treated cells with Ru360, an inhibitor of mitochondrial Ca2+ uniporter (MCU), and found that Ru360 reduced H2O2 generation in cells exposed to asbestos. These findings suggest that asbestos rapidly increases mitochondrial Ca2+ via the MCU, which contributes to mitochondrial H2O2 generation in macrophages. (Supported by 2R01ES015981–06, R01ES014871, VA Merit Review 1BX001135 to A.B.C.)