Volume 140, Issue 7, Pages 2116-2125 (June 2011) Reactive Oxygen Species Induced by Bile Acid Induce Apoptosis and Protect Against Necrosis in Pancreatic Acinar Cells  David M. Booth, John A. Murphy, Rajarshi Mukherjee, Muhammad Awais, John P. Neoptolemos, Oleg V. Gerasimenko, Alexei V. Tepikin, Ole H. Petersen, Robert Sutton, David N. Criddle  Gastroenterology  Volume 140, Issue 7, Pages 2116-2125 (June 2011) DOI: 10.1053/j.gastro.2011.02.054 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Increases in [ROS]I induced by TLC-S in isolated pancreatic acinar cells. (A) Transmitted light and DCFDA whole cell fluorescence (green) images of murine cells showing increases in [ROS]I induced by 500 μmol/L TLC-S; increases in [ROS]I induced by 30 μmol/L MEN are shown as positive control. (B) Mean data from murine cells showing no increase in [ROS]I detected using DCFDA from 200 μmol/L TLC-S (n = 11), whereas 500 μmol/L TLC-S produced a sustained increase in [ROS]I (n = 14, see inset). (C) Mean data from human cells showing an increase in [ROS]I from 500 μmol/L TLC-S (n = 12), abolished by 10 mmol/L NAC (n = 10; no NAC during MEN to check ROS generation). Inset shows typical increase of NADPH level following initial increase in [Ca2+]C (see also Figure 4). Data normalized from basal fluorescence levels (F/F0). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Distribution of oxidant scavenging enzyme NQO1 and effects of NQO1 inhibition, Ca2+ chelation, or NADPH oxidase inhibition on increase in [ROS]I induced by TLC-S. (A) Similar cytosolic distribution of NQO1 immunofluorescence (green) in human and murine cells (F-actin [red] and Hoechst [blue] counterstains). (B) Inhibition of NQO1 with DMN (30 μmol/L) unmasked ROS generation by 200 μmol/L TLC-S (detected using DCFDA fluorescence) (green) (n = 11). Mean data (inset) show ROS production induced by 200 μmol/L (n = 11) and 500 μmol/L (n = 12) TLC-S was greater in the presence of DMN (30 μmol/L). (C) ROS generation by 200 μmol/L TLC-S with DMN (30 μmol/L) was completely blocked by L-BAPTA pretreatment, whereas ROS generation by MEN (30 μmol/L) was unaffected (control, n = 11; L-BAPTA, n = 14). (D) Increases in [ROS]I from TLC-S or MEN were not inhibited by the NADPH oxidase inhibitor diphenyliodonium chloride (DPI; 10 μmol/L; control, n = 15; DPI, n = 12). Mean data normalized from basal fluorescence levels (F/F0). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Effects of TLC-S on [Ca2+]C (blue), [Ca2+]M (blue), NADPH levels (red), and [ROS]M (magenta); data (A–C) are normalized from basal fluorescence levels (F/F0). (A) TLC-S 500 μmol/L induced sustained nonoscillatory signals of [Ca2+]C (n = 20 of 20, left panel) with a small initial increase and then a sustained decrease in NADPH levels. Corresponding increases in [Ca2+]M (Rhod-2, blue) and decreases in NADPH (red) were also sustained (n = 20 of 20, right panel, see inset for representative images), indicating mitochondrial Ca2+ overload. (B) Characteristic changes of ROS and NADPH after 500 μmol/L TLC-S, contrasted with inhibition of electron transport and ROS production by transport of 10 μmol/L antimycin A and 5 μmol/L rotenone (A+R), identifying mitochondria as a likely site of ROS production. (C) Representative images of cells after 200 μmol/L TLC-S for 30 minutes showing mitochondria (MitoTracker, yellow) and increases in [ROS]M (MitoSOX, magenta; colocalized fluorescence, pink) (n = 52). Inhibition of electron transport abolished increases in [ROS]M (n = 47). (D) Mitochondrial ROS production after 200 μmol/L TLC-S: left panel shows a line (with 2 angles) of 18 μM drawn through perigranular mitochondria (MitoTracker) before 30 minutes of 200 μmol/L TLC-S, after which images were recorded (MitoTracker and MitoSOX; inset shows differential interference contrast image of cells); right panel shows fluorescence changes along the same line. Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Effects of agents changing [ROS]I and [ROS]M on cell death induced by TLC-S and MEN (data expressed as means ± SE; n = cells). (A) Representative images of caspase activation (fluorescent general caspase substrate) (green), plasma membrane rupture (PI) (red), and all cells (nuclear stain Hoechst) (blue). After 30 minutes of TLC-S, dose-dependent increases in caspase activation and PI uptake occurred, while S-BAPTA only reduced PI uptake; plot shows TLC-S–induced increases in caspase activation and PI uptake. S-BAPTA significantly reduced PI uptake, not caspase activation, whereas L-BAPTA plus 20 mmol/L caffeine prevented increases in PI uptake and caspase activation (384–2010 cells per column). (B) NAC (10 mmol/L) markedly reduced caspase activation, whereas PI and total death pathway activation (from 500 μmol/L TLC-S) were increased (145–1446 cells). (C) DMN (30 μmol/L) increased caspase activation induced by both concentrations of TLC-S (significantly at 500 μmol/L) while reducing PI uptake (186–1446 cells). (D) NAC (10 mmol/L) prevented caspase activation from MEN (30 μmol/L; 307–632 cells). (E) Increases in [ROS]M after 30 minutes of 200 μmol/L TLC-S, and effect of mitochondrial electron transport inhibition (A+R), showing no increase in [ROS]M (31–52 cells; *P < .01 vs TLCS alone). (F) Inhibition of increases in [ROS]M with either 1 μmol/L mitoquinone (MQ) or antimycin A plus rotenone prevented TLC-S–induced caspase activation with no effect on PI uptake; decylquinone (DQ), a redox neutral quinone targeting mitochondria, was without effect (479–2489 cells). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Neither inhibition (3-methyladenine [3MA]) nor stimulation (rapamycin [Rap]) of autophagy reduced cell death pathway activation from TLC-S in acinar cells. (A) Inhibition of autophagy did not alter caspase activation or PI uptake induced by TLC-S (582–1524 cells). (B) Autophagosome marker LC3b (green immunofluorescence) was mainly basolateral following 16-hour culture; 200 μmol/L TLC-S, 100 nmol/L RAP, or both led to LC3b detection in central and apical regions; 5 mmol/L 3MA led to a loss of all but basolateral immunofluorescence (30 cells/condition). (C) Representative images of cells treated with 200 μmol/L TLC-S showing either chromatin condensation without plasma membrane rupture (AO fluorescence in nuclei and cytoplasm but no nuclear ethidium bromide [EB], 4 left images) or less chromatin condensation with obvious plasma membrane rupture (weak nuclear and no cytoplasmic AO with marked nuclear EB fluorescence, 4 right images); nuclear Hoechst staining was used for cell counts. (D) Proportion of cells showing either pattern of cell death pathway activation described in B induced by 200 μmol/L TLC-S. DMN (30 μmol/L) increased chromatin condensation (AO) whereas NAC (10 mmol/L) reduced chromatin condensation and increased plasma membrane rupture (EB), consistent with Figure 4. Neither RAP nor 3MA significantly altered total cell death pathway activation (1316–2128 cells). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Effects of 200 μmol/L TLC-S on [Ca2+]C (blue), IClCa (black), and NADPH levels (red) in whole-cell patch-clamped (at −30 mV) cells, with either (A) 0 mmol/L ATP or (B) 4 mmol/L ATP in the pipette solution. Without supplementary ATP, TLC-S induced oscillatory increases in [Ca2+]C and then a sustained elevation, mirrored by large inward Cl− currents, decreased NADPH levels, and in (C) PI uptake. With supplementary ATP, although NADPH levels declined, no sustained increases in [Ca2+]C or in (C) PI uptake were observed (n = 6 of 6). Data normalized from basal fluorescence levels (F/F0) and absolute current changes (pA). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Roles of [ROS]I, [Ca2+]C, and [Ca2+]M in TLC-S–induced pancreatitis. Driven by increases in [Ca2+]C and [Ca2+]M, increases in [ROS]I trigger apoptosis, removing deleterious enzyme-rich products. Large increases in [Ca2+]C and [Ca2+]M, however, cause cellular injury, cytokine release and necrosis through mitochondrial inhibition, and impaired ATP production. Inflammation is driven by increases in [ROS]I, but paradoxically this protective response exacerbates acute pancreatitis (AP), driving the systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Gastroenterology 2011 140, 2116-2125DOI: (10.1053/j.gastro.2011.02.054) Copyright © 2011 AGA Institute Terms and Conditions