Gladys Tapia

Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients.

Oxidative stress is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In the present study, hepatic and plasma oxidative stress-related parameters were measured and correlated with clinical and histological findings in 31 NAFLD patients showing increased body mass index. Liver protein carbonyl content was enhanced by 403% in patients with steatosis (n=15) compared with control values (n=12), whereas glutathione content, superoxide dismutase (SOD) activity and the ferric reducing ability of plasma (FRAP) were decreased by 57%, 48% and 21% (P<0.05) respectively. No changes in microsomal p-nitrophenol hydroxylation and the total content of cytochrome P450 (CYP) or CYP2E1 were observed. Patients with steatohepatitis (n=16) exhibited protein carbonyl content comparable with that of controls, whereas glutathione content, SOD and catalase activities were decreased by 27%, 64% and 48% (P<0.05). In addition, FRAP values in patients with steatohepatitis were reduced by 33% and 15% (P<0.05) when compared with controls and patients with steatosis respectively, whereas p-nitrophenol hydroxylation (52%) and CYP2E1 content (142%) were significantly increased (P<0.05) compared with controls. It is concluded that oxidative stress is developed in the liver of NAFLD patients with steatosis and is exacerbated further in patients with steatohepatitis, which is associated with CYP2E1 induction. Substantial protein oxidation is followed by proteolysis of the modified proteins, which may explain the co-existence of a diminished antioxidant capacity and protein oxidation in the liver of patients with steatohepatitis.

Oxidative stress-mediated hepatotoxicity of iron and copper: role of Kupffer cells.

Iron- or copper-mediated catalysis leads to the generation of reactive oxygen species (ROS) that can attack biomolecules directly, with the consequent enhancement in membrane lipid peroxidation, DNA damage, and protein oxidation. Reactive nitrogen species (RNS) can also be formed, leading to nitration of aromatic structures in addition to the oxidative deterioration of cellular components. Kupffer cells, the resident macrophages of the liver, play significant roles in immunomodulation, phagocytosis, and biochemical attack. Upon stimulation, liver macrophages release biologically active products related to cell injury, namely, ROS, RNS, and both immunomodulatory and fibrogenic cytokines, with production of chemokines and adhesion molecules by other cells of the liver sinusoid. Iron and copper alter the functional status of Kupffer cells by enhancing their respiratory burst activity without modifying particle phagocytosis. This effect is probably due to extra O2 equivalents used in the oxidation of biomolecules and/or in the activating action of iron/copper on nitric oxide synthase, in addition to those employed by NADPH oxidase activity. Changes in gene expression of Kupffer cells may also be accomplished by iron and copper through modulation of the activity of transcription factors such as NF-κB, which signals the production of cytotoxic, proinflammatory, or fibrogenic mediators. Thus, iron/copper-induced hepatotoxicity is a multifactorial phenomenon underlying actions due to the generation of ROS and RNS that may alter essential biomolecules with loss of their biological functions, modulate gene expression of Kupffer cells with production of cytotoxic mediators, or both.

N-3 PUFA Supplementation Triggers PPAR-α Activation and PPAR-α/NF-κB Interaction: Anti-Inflammatory Implications in Liver Ischemia-Reperfusion Injury

Dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to rats preconditions the liver against ischemia-reperfusion (IR) injury, with reduction of the enhanced nuclear factor-κB (NF-κB) functionality occurring in the early phase of IR injury, and recovery of IR-induced pro-inflammatory cytokine response. The aim of the present study was to test the hypothesis that liver preconditioning by n-3 PUFA is exerted through peroxisone proliferator-activated receptor α (PPAR-α) activation and interference with NF-κB activation. For this purpose we evaluated the formation of PPAR-α/NF-κBp65 complexes in relation to changes in PPAR-α activation, IκB-α phosphorylation and serum levels and expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in a model of hepatic IR-injury (1 h of ischemia and 20 h of reperfusion) or sham laparotomy (controls) in male Sprague Dawley rats. Animals were previously supplemented for 7 days with encapsulated fish oil (General Nutrition Corp., Pittsburg, PA) or isovolumetric amounts of saline (controls). Normalization of IR-altered parameters of liver injury (serum transaminases and liver morphology) was achieved by dietary n-3 PUFA supplementation. EPA and DHA suppression of the early IR-induced NF-κB activation was paralleled by generation of PPAR-α/NF-κBp65 complexes, in concomitance with normalization of the IR-induced IκB-α phosphorylation. PPAR-α activation by n-3 PUFA was evidenced by enhancement in the expression of the PPAR-α-regulated Acyl-CoA oxidase (Acox) and Carnitine-Palmitoyl-CoA transferase I (CPT-I) genes. Consistent with these findings, normalization of IR-induced expression and serum levels of NF-κB-controlled cytokines IL-lβ and TNF-α was observed at 20 h of reperfusion. Taken together, these findings point to an antagonistic effect of PPAR-α on NF-κB-controlled transcription of pro-inflammatory mediators. This effect is associated with the formation of PPAR-α/NF-κBp65 complexes and enhanced cytosolic IκB-α stability, as major preconditioning mechanisms induced by n-3 PUFA supplementation against IR liver injury.

Thyroid hormone preconditioning: Protection against ischemia‐reperfusion liver injury in the rat

Recently, we reported that oxidative stress due to 3,3′,5‐triiodothyronine (T3)‐induced calorigenesis up‐regulates the hepatic expression of mediators promoting cell protection. In this study, T3 administration in rats (single dose of 0.1 mg/kg intraperitoneally) induced significant depletion of reduced liver glutathione (GSH), with higher protein oxidation, O2 consumption, and Kupffer cell function (carbon phagocytosis and carbon‐induced O2 uptake). These changes occurred within a period of 36 hours of T3 treatment in animals showing normal liver histology and lack of alteration in serum AST and ALT levels. Partial hepatic ischemia‐reperfusion (IR) (1 h of ischemia via vascular clamping and 20 h reperfusion) led to 11‐fold and 42‐fold increases in serum AST and ALT levels, respectively, and significant changes in liver histology, with a 36% decrease in liver GSH content and a 133% increase in that of protein carbonyls. T3 administration in a time window of 48 hours was substantially protective against hepatic IR injury, with a net 60% and 90% reduction in liver GSH depletion and protein oxidation induced by IR, respectively. Liver IR led to decreased DNA binding of nuclear factor‐κB (NF‐κB) (54%) and signal transducer and activator of transcription 3 (STAT3) (53%) (electromobility shift assay), with 50% diminution in the protein expression of haptoglobin (Western blot), changes that were normalized by T3 preconditioning. Conclusion: T3 administration involving transient oxidative stress in the liver exerts significant protection against IR injury, a novel preconditioning maneuver that is associated with NF‐κB and STAT3 activation and acute‐phase response. (HEPATOLOGY 2007;45:170–177.)

Thyroid hormone-induced oxidative stress triggers nuclear factor-κB activation and cytokine gene expression in rat liver

Nuclear factor-kappaB (NF-kappaB) is a redox-sensitive factor responsible for the transcriptional activation of cytokine-encoding genes. In this study, we show that 3,3,5-triiodothyronine (T(3)) administration to rats activates hepatic NF-kappaB, as assessed by electrophoretic mobility shift assay. This response coincides with the onset of calorigenesis and enhancement in hepatic respiration, and is suppressed by the antioxidants alpha-tocopherol and N-acetylcysteine or by the Kupffer cell inactivator gadolinium chloride. Livers from hyperthyroid rats with enhanced NF-kappaB DNA-binding activity show induced mRNA expression of the NF-kappaB-responsive genes for tumor necrosis factor-alpha (TNF-alpha) and interleukin- (IL-) 10, as evidenced by reverse transcription-polymerase chain reaction assay, which is correlated with increases in the serum levels of the cytokines. T(3) also increased the hepatic levels of mRNA for IL-1alpha and those of IL-1alpha in serum, with a time profile closely related to that of TNF-alpha. It is concluded that T(3)-induced oxidative stress enhances the DNA-binding activity of NF-kappaB and the NF-kappaB-dependent expression of TNF-alpha and IL-10 genes.

Liver NF‐κB and AP‐1 DNA Binding in Obese Patients

Oxidative stress and insulin resistance (IR) are major contributors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and in the progression from steatosis to nonalcoholic steatohepatitis (NASH). Our aim was to assess nuclear factor‐κB (NF‐κB) and activating protein‐1 (AP‐1) activation and Toll‐like receptor 4 (TLR4) expression as signaling mechanisms related to liver injury in obese NAFLD patients, and examined potential correlations among them, oxidative stress, and IR. Liver NF‐κB and AP‐1 (electromobility shift assay (EMSA)), TLR4 expression (western blot), ferric reducing ability of plasma (FRAP), and IR evolution (HOMA) were evaluated in 17 obese patients who underwent subtotal gastrectomy with gastro‐jejunal anastomosis in Roux‐en‐Y and 10 nonobese subjects who underwent laparoscopic cholecystectomy (controls). Liver NF‐κB and AP‐1 DNA binding were markedly increased in NASH patients (n = 9; P < 0.05) compared to controls, without significant changes in NAFLD patients with steatosis (n = 8), whereas TLR4 expression was comparable between groups. Hepatic NF‐κB activation was positively correlated with that of AP‐1 (r = 0.79; P < 0.0001); both liver NF‐κB and AP‐1 DNA binding were inversely associated with FRAP (r = −0.43 and r = −0.40, respectively; P < 0.05) and directly correlated with HOMA (r = 0.66 and r = 0.62, respectively, P < 0.001). Data presented show enhanced liver activation of the proinflammatory transcription factors NF‐κB and AP‐1 in obese patients with NASH, parameters that are significantly associated to oxidative stress and IR.

N-3 Long-Chain Polyunsaturated Fatty Acid Supplementation Significantly Reduces Liver Oxidative Stress in High Fat Induced Steatosis

Omega-3 (n-3) long-chain polyunsaturated fatty acids (n-3 LCPUFA) are associated with several physiological functions, suggesting that their administration may prevent non transmissible chronic diseases. Therefore, we investigate whether dietary n-3 LCPUFA supplementation triggers an antioxidant response preventing liver steatosis in mice fed a high fat diet (HFD) in relation to n-3 LCPUFA levels. Male C57BL/6J mice received (a) control diet (10% fat, 20% protein, 70% carbohydrate), (b) control diet plus n-3 LCPUFA (108 mg/kg/day eicosapentaenoic acid plus 92 mg/kg/day docosahexaenoic acid), (c) HFD (60% fat, 20% protein, 20% carbohydrate), or (d) HFD plus n-3 LCPUFA for 12 weeks. Parameters of liver steatosis, glutathione status, protein carbonylation, and fatty acid analysis were determined, concomitantly with insulin resistance and serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels. HFD significantly increased total fat and triacylglyceride contents with macrovesicular steatosis, concomitantly with higher fasting serum glucose and insulin levels, HOMA, and serum TNF-α, IL-1β, and IL-6. Reduced and total liver glutathione contents were diminished by HFD, with higher GSSG/GSH ratio and protein carbonylation, n-3 LCPUFA depletion and elevated n-6/n-3 ratio over control values. These changes were either reduced or normalized to control values in animals subjected to HFD and n-3 LCPUFA, with significant increased hepatic total n-3 LCPUFA content and reduced n-6/n-3 ratio being observed after n-3 LCPUFA supplementation alone. So, repletion of liver n-3 LCPUFA levels by n-3 LCPUFA dietary supplementation in HFD obese mice reduces hepatic lipid content, with concomitant antioxidant and anti-inflammatory responses favouring insulin sensitivity.

Vitamin E But Not 17β-Estradiol Protects against Vascular Toxicity Induced by β-Amyloid Wild Type and the Dutch Amyloid Variant

Amyloid β-peptide (Aβ) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimers disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in Aβ yielding AβGlu22→Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated Aβ on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by Aβ1–40 Glu22→Gln and Aβ1–40 wild-type fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that AβGlu22→Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of Aβ fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. Aβ1–40 wild-type–AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of Aβ fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17β-estradiol on vascular damage induced by Aβwild-type and AβGlu22→Gln. Our data indicate that vitamin E attenuated significantly the Aβ-mediated cytotoxicity on vascular cells, although 17β-estradiol and vitamin C failed to inhibit the cytotoxicity induced by Aβ fibrils.

Protection against in vivo liver ischemia-reperfusion injury by n-3 long-chain polyunsaturated fatty acids in the rat

Abstract N-3 polyunsaturated fatty acids (n-3 PUFA) affect inflammatory processes. This study evaluated the effects of dietary supplementation with fish oil on hepatic ischemia-reperfusion (IR) injury in the rat. Parameters of liver injury (serum transaminases and histology) and oxidative stress (serum 8-isoprostanes and hepatic GSH and GSSG), were correlated with NF-κB DNA binding and FA composition and inflammatory cytokine release. N-3 PUFA supplementation significantly increased liver n-3 PUFA content and decreased n-6/n-3 PUFA ratios. IR significantly modified liver histology and enhanced serum transaminases, 8-isoprotanes and inflammatory cytokines, with net reduction in liver GSH levels and net increment in those of GSSG. Early increase (3 h) and late reduction (20 h) in NF-κB activity was induced. All IR-induced changes were normalized by n-3 PUFA supplementation. In conclusion, prevention of liver IR-injury was achieved by n-3 PUFA supplementation, with suppression of oxidative stress and recovery of pro-inflammatory cytokine homeostasis and NF-κB functionality lost during IR.

Influence of C-phycocyanin on hepatocellular parameters related to liver oxidative stress and Kupffer cell functioning

Abstract. Objectives: Kupffer cells, liver macrophages involved in immunomodulation, phagocytosis, and biochemical attack, can induce cytotoxicity and inflammation when their activity is exacerbated. The aim of this study was to evaluate the effects of C-phycocyanin on Kupffer cell functioning considering its antioxidant and anti-inflammatory properties.¶Materials and methods: Actions of C-phycocyanin on colloidal carbon phagocytosis, carbon-induced respiratory burst activity, and sinusoidal lactate dehydrogenase (LDH) release were studied in isolated perfused mouse liver. The influence of C-phycocyanin on tumor necrosis factor-α (TNF-α) and nitrite levels in serum and liver nitric oxide synthase (NOS) activity was assessed in rats subjected to thyroid hormone (T3) administration, a condition known to underlie hepat-ic oxidative stress comprising an increased Kupffer cell activity.¶Results: C-phycocyanin elicited a concentration-dependent inhibition of carbon phagocytosis and carbon-induced O2 uptake (IC50 = 0.2 mg/ml) by perfused livers, with a 52 % diminution in the carbon-induced sinusoidal release of LDH being found at a concentration of 0.25 mg/ml. Thyroid calorigenesis induced an 82-fold increase in serum TNF-α levels, an effect that was suppressed by pretreatment with C-phycocyanin, the antioxidant α-tocopherol, and by the Kupffer cell inactivator gadolinium chloride. C-phycocyanin also suppressed the T3-induced increases in serum nitrite levels (234 %) and in the activity of hepatic NOS (75 %).¶Conclusions: C-phycocyanin significantly decreases Kupffer cell phagocytosis and the associated respiratory burst activity, effects that may contribute to the abolition of oxidative stress-induced TNF-α response and NO production by hyperthyroid state.