Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components

Abstract

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H₂O₂ production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H₂O₂ in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Keywords: Air pollution; Bioenergetics; Extracellular flux; Mitochondria; Quinones.

Figure 1. 1,2-NQ increases basal Oxygen Consumption Rate (OCR, pmol/min)

Bioenergetic effects of 1,2-NQ on BEAS cells were measured using the Cell Mito Stress Test on a XFe96 analyzer. An acute injection of 1,2-NQ was followed by additions of 1 uM oligomycin, 0.25 uM FCCP, and 1 uM antimycin A and rotenone at the indicated times. Data displayed as mean +/− SEM, n ≥ 10. One-sided error bars are used for clarity.

Figure 2. 1,2-NQ-induced OCR increases are independent of mitochondrial electron transport

A. BEAS cells were treated with 1 uM antimycin A and 1 uM rotenone for 13 min prior to addition of DMSO (vehicle), the indicated concentration of 1,2-NQ, or 0.25 uM FCCP. B. The change in OCR after acute injection was calculated from rotenone/antimycin A-pretreated baseline, mean +/− SEM, n = 3, * indicates significant difference from the pretreated baseline (p < 0.05).

Figure 3. Exposure to environmentally relevant redox-cycling quinones increases OCR

BEAS cells were treated with DMSO (vehicle), the indicated concentrations of 1,2-NQ, 1,4-NQ, 9,10-PQ, BQ, or 0.25 uM FCCP following a baseline OCR collection period of 18 min on the extracellular flux analyzer. Shown is the change in OCR at 6 min of exposure relative to basal value. * indicates the compound-induced OCR is significantly different from baseline OCR (p<0.05). For 1,2-NQ (3-50 uM), n ≥ 11. For 1,4-NQ, n ≥ 7. For 9,10-PQ (3-50 uM), n ≥ 6. For 100 uM of all quinones, n=3. For BQ, n =3. For FCCP, n = 4. Data displayed as mean +/− SEM.

Figure 4. Glucose starvation blunts quinone-induced increase in OCR

BEAS cells were starved of glucose (open bars) or provided with 10 mM glucose (closed bars) 2 hours prior to start of assay. BEAS cells were treated with DMSO (vehicle), the indicated concentrations of 1,2-NQ, 1,4-NQ, 9,10-PQ, BQ, or 0.25 uM FCCP following a baseline OCR collection period of 18 min on the extracellular flux analyzer. Shown is the change in OCR relative to basal value, * indicates the compound-induced OCR was significantly (p<0.05) different from baseline OCR. # indicates a significant (p<0.05) difference between glucose starved and supplemented groups. Data displayed as mean +/− SEM. For DMSO, 1,2-NQ and 1,4-NQ, n ≥ 4. For 9,10-PQ and FCCP, n = 3.

Figure 5. Exposure to redox cycling quinones induces H₂O₂ production

BEAS cells were exposed to DMSO (vehicle) or the indicated concentrations of 1,2-NQ, 1,4-NQ, 9,10-PQ, BQ, or H₂O₂ for 18 min. H₂O₂ was measured in apical media as fluorescence at excitation λ= 545 nm, emission λ= 590 nm following a 30 min incubation in the presence of HRP and Amplex Red. n = 3, mean +/− SEM.

Figure 6. Mitochondrial preparations show a diminished 1,2-NQ-induced OCR increase

All values shown as a change from basal OCR observed immediately after addition of DMSO or the indicated concentration of 1,2-NQ. All values were normalized to the mass of mitochondrial protein in each preparation. * indicates the change in OCR was significantly (p<0.05) different from DMSO treatment within each preparation. # indicates the change in OCR was statistically different (p<0.05) between mitochondrial preparations. For intact cells, n ≥ 11; for permeabilized cells, n ≥ 13; for isolated mitochondria, n = 5. Data shown as mean +/− SEM.

Figure 7. 1,2-NQ impairs Complex I-linked respiration in permeabilized cells

A. BEAS cells were permeabilized with XF PMP and treated as indicated with buffer, DMSO,1,2-NQ, 1,4-NQ, 9,10-PQ, BQ for 10 min, followed by an injection of 10 mM pyruvate/2 mM malate or 10 mM glutamate/10 mM malate, in a buffer containing 4 mM ADP, and mitochondrial respiration was measured as OCR. * indicates activity was significantly (p<0.05) different from buffer control. # indicates a significant (p<0.05) difference between glutamate and pyruvate supplementation. For 1,2-NQ, 1,4-NQ, Buffer, and DMSO, n ≥ 4. For 9,10-PQ and BQ, n ≥ 3. B. Complex II-linked mitochondrial respiration measured by OCR after addition of 10 mM succinate/4 mM ADP/2 uM rotenone. BEAS cells were permeabilized with XF PMP and treated as indicated with DMSO,1,2-NQ, 1,4-NQ, 9,10-PQ, BQ for 10 min, and substrate oxidation was measured as OCR. * indicates activity was significantly (p<0.05) different from buffer control. For DMSO and 1,2-NQ, n = 4. For 1,4-NQ and BQ, n = 3. C. BEAS cells were treated with 10 uM or 50 uM 1,2-NQ for 15 min and Complex I activity was assayed as the NADH-dependent reduction of a chromophore over 30 min. * indicates activity was significantly (p<0.05) different from media control, n = 3. Data shown as mean +/− SEM.