Background Systemic lupus erythematosus (SLE) patients exhibit T-cell dysfunction which can be regulated through the mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione. Therefore, the safety, tolerance, and efficacy of glutathione-precursor N-acetylcysteine (NAC) were examined in this randomized double-blind placebo-controlled study. Methods 36 SLE patients received daily placebo or 1.2 g, 2.4 g or 4.8 g of NAC. Disease activity was monthly evaluated by BILAG, SLEDAI and fatigue assessment scale (FAS) before, during, and after 3-month treatment. Δψm and mTOR were assessed by flow cytometry. 42 healthy subjects matched for patients’ age, gender, and ethnicity were studied as controls. Results NAC was tolerated by all patients up to 2.4 g/day while 33% of those receiving 4.8 g/day had reversible nausea. Placebo or 1.2 g/day NAC did not influence disease activity. Considered together, 2.4 g and 4.8 g NAC reduced: 1) SLEDAI after 1 month (p=0.0007), 2 months (p=0.0009), 3 months (p=0.0030) and 4 months (p=0.0046); 2) BILAG after 1 month (p=0.029) and 3 months (p=0.0009); and 3) FAS after 2 months (p=0.002) and 3 months (p=0.004). NAC increased Δψm (p=0.0001) in all T cells, it profoundly reduced mTOR activity (p=0.0001), enhanced apoptosis (p=0.0004) and reversed expansion of CD4−/CD8− T cells (1.35 ± 0.12-fold; p=0.008), stimulated Foxp3 expression in CD4+/CD25+ T cells (p=0.045), and reduced anti-DNA production (p=0.049). Conclusions This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1 -/-and Taldo1 +/-mice spontaneously developed HCC, and Taldo1 -/-mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1 -/-livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced β-catenin phosphorylation and enhanced c-Jun expression in Taldo1 -/-livers reflected adaptation to oxidative stress. Taldo1 -/-hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1 -/-mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

No abstract

Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within the immune system. Activation, proliferation, or death of cells of the immune system is dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (∆ψ (m)) reflects the energy stored in the electrochemical gradient across the inner mitochondrial membrane which, in turn, is used by F(0)F(1)-ATPase to convert ADP to ATP during oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion represent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients with SLE exhibit elevated ∆ψ (m), i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alkalinization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox--sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key sensor of MHP and mediator of enhanced Ca(2+) flux in lupus T cells.

Properly assessing mitochondrial health is crucial to understand their role in disease. MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨm). Here, we utilized flow cytometry to evaluate the performance of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC) in human peripheral blood lymphocytes (PBL). In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined under oxidative and nitrosative stress induced by rotenone and NOC-18 while N-acetylcysteine (NAC) was employed as an antioxidant. Production of reactive oxygen species (ROS) and ΔΨm were monitored in parallel. With respect to representation of mitochondrial mass, neither MTG nor NAO was affected by ΔΨm. However, MTG showed significant correlation with cytosolic and mitochondrial ROS production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for assessment of mitochondrial mass by flow cytometry during oxidative stress.