CLINICAL TRIAL PARTICIPATION AFTER ACUTE CORONARY SYNDROME AND ASSOCIATED OUTCOMES: INSIGHT FROM THE ACTION REGISTRY-GWTG

Background/Aims: In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50 mu mol/L) or H(2)O(2) (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining. Results: (1) Menadione (50 mu mol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 mu mol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. (2) H(2)O(2) increases necrotic cell death at high concentration or when H(2)O(2) detoxification is impaired. H202 does not activate MAPKs signalling. (3) PEG-SOD prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death. Conclusions: In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Hormesis in Cholestatic Liver Disease; Preconditioning with Low Bile Acid Concentrations Protects against Bile Acid-Induced Toxicity

Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis. To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions. HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1-50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed. Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning. Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transpor

GCDCA, TCA and TUDCA preconditioning are most protective against GCDCA-induced apoptosis, compared to preconditioning with CDCA, CA or UDCA.

<p>HepG2.rNtcp cells were preconditioned with CDCA, GCDCA, CA, TCA, UDCA or TUDCA (25 μM / 24 h), without challenge with GCDCA (Control, black bars) or challenged with 200 μM GCDCA for 4 h (grey bars). Caspase-3/7 activity is shown as fold change compared to control (no preconditioning and no GCDCA challenge), control values were set as one. Experiment where performed n = 3–10 depending on the condition. Error bars represent the SEM. *** = P<0.001, No challenge versus GCDCA challenge counterpart. # = P<0.05, ## = P<0.01, ### = P<0.001, compared to no preconditioning (24 hr) / GCDCA challenge.</p

The protective effect of GCDCA preconditioning is time- and concentration-dependent.

<p>HepG2.rNtcp cells were preconditioned with GCDCA for different periods and in different concentrations. <b>(A)</b> Cells were preconditioned with 25 μM GCDCA for 2, 6, 12, 24, and 48 h, without challenge (control, black bars) or challenged 200 μM GCDCA for 4 h (grey bars). <b>(B)</b> Cells were preconditioned with 0.25, 1, 5, or 25 μM GCDCA for 24 h without challenge (control, black bars) or challenged 200 μM GCDCA for 4 h (grey bars). Caspase-3/7 activity is shown as fold change compared to control (no preconditioning and no GCDCA challenge), control values were set as one. Experiments were performed in n = 3 for each condition. Error bars represent the SEM. * = P<0.05, *** = P<0.001 No challenge versus GCDCA challenge counterpart. ### = P<0.001 compared to no preconditioning (24 h) / no GCDCA challenge.</p

GCDCA preconditioning does not increase the efflux of GCDCA after the challenge.

<p>HepG2.rNtcp cells were preconditioned with GCDCA (25 μM / 24 h) without challenge with GCDCA (Control, black bars) or challenged with the pro-apoptotic concentration (200 μM) GCDCA for 4 h (grey bars). UHPLC-MS/MS was used to determine bile acid concentrations, intracellular GCDCA concentrations are shown as relative peak area. Error bars represent the SEM, experiment was performed in n = 3. ** = P<0.01, No challenge vs GCDCA challenge counterpart.</p

Bile acid preconditions does not induce a shift from apoptosis to necrosis when HepG2.rNtcp cells are exposed to 200 μM GCDCA.

<p>HepG2.rNtcp cells were preconditioned with CDCA, GCDCA, CA, TCA, UDCA or TUDCA (25 μM / 24 h), without challenge with GCDCA (Control, black bars) or challenged with the pro-apoptotic concentration (200 μM) GCDCA for 4 h (grey bars). LDH activity was measured in medium alone and in medium from digitonin-treated cells (100% leakage). LDH leakage is shown as percentage of total LDH. Experiments were performed in n = 3 for each condition. Error bars represent the SEM.</p

Refractive index tomography for drug-induced liver injury analysis

Refractive index tomography compatible with conventional microscope is used for analyzing primary rat hepatocytes injury induced by pharmacological treatments. We found that mitochondria malfunctioning is correlated with refractive index variations