Virginia Fernández

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.

Effect of acute ethanol intoxication on the content of reduced glutathione of the liver in relation to its lipoperoxidative capacity in the rat

Glutathione is considered to be the most abundant and important intracellular sulfhydryl compound [ 1.21. Both reduced (GSH) and oxidized (GSSG) glutathione are related to several structural and func- tional processes of the cell, and are involved in the protective mechanisms against the deleterious effects of several agents and/or their metabolites [2]. The later function of glutathione has been proposed to be accomplished by either the formation of excretable conjugates [3] or by its participation in the metabolism of peroxides arising from the enhancement of lipo- peroxidative processes [4]. Lipoperoxidation, the oxidative alteration of poly- unsaturated fatty acids that seems to be of importance in the production of liver injury by some hepato- toxins [5,6], has been shown to be increased in the liver following acute [7-l 0] and chronic [8,1 l-141 alcohol ingestion. However, this finding has not been confirmed in the acute model [ 15 171. This report describes the influences of the sex, nutritional status, dosage and the period of intoxication of animals given alcohol acutely on the content of GSH of the liver in relation to its lipoperoxidative capacity. The effect of alcohol on the activity of the enzymes of peroxide metabolism, the other main contributors to the maintenance of the antioxygenic capacity of the hepatocyte [1,4,18], is dealt with in [19]. 2.

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.

CHEMILUMINESCENT AND RESPIRATORY RESPONSES RELATED TO THYROID HORMONE-INDUCED LIVER OXIDATIVE STRESS

Chemiluminescent and respiratory responses were studied in the liver of rats treated with 0.1 mg of triiodothyronine (T3)/kg for 1 to 7 days. Hyperthyroidism resulted in significant increments in the spontaneous chemiluminescence of the in situ liver in animals exhibiting a calorigenic response. Microsomal NADPH-dependent oxygen uptake was enhanced by T3 treatment for 2 days, an effect that was completely abolished by the antioxidant cyanidanol. A similar microsomal antioxidant-sensitive respiratory component was observed in this situation after the addition of t-butyl hydroperoxide (t-BHP). However, basal rates of microsomal oxygen uptake and light emission in liver homogenates and microsomes were decreased by t-BHP, probably related to thyroid hormone-induced diminution in the content of cytochrome P-450 (Fernández et al.) In addition, liver superoxide dismutase and catalase activities as well as the total content of glutathione were depressed by T3. These results indicate that the calorigenic response in the hyperthyroid state is accompanied by the development of an hepatic oxidative stress characterized by enhanced spontaneous chemiluminescence, enhanced NADPH-dependent microsomal respiration and a decreased antioxidant cellular activity.

Hepatic and biliary levels of glutathione and lipid peroxides following iron overload in the rat: Effect of simultaneous ethanol administration

The administration of 125 mg of iron/kg (iron-dextran-Imferon) to fed rats was followed by an increase in the non-hem iron content in plasma and liver over a period of 22 hr, reaching a peak value after 6 hr. Plasma and hepatic iron levels were not modified by ethanol ingestion (5 g/kg). Iron and ethanol treatments enhanced liver lipid peroxidation (malondialdehyde (MDA) formation) by 70 and 35%, respectively, at 6 hr. Since the hepatic MDA formation increased by 92% after the joint iron-ethanol treatment, an additive effect in lipid peroxidation was suggested to occur in this condition. Both iron and ethanol treatments increased biliary levels and release of MDA, in the absence of changes in bile flow. These parameters were further enhanced by the joint iron-ethanol exposure, in that hepatic MDA levels and biliary MDA release were significantly correlated (r = 0.86; p less than 0.05). Plasma MDA levels also increased after iron, ethanol, and iron-ethanol treatments, but they did not reflect the changes in MDA levels in liver. Iron exposure resulted in 26 to 33% decreases in hepatic GSH content at the 6-hr treatment, associated with the peak effect on lipid peroxidation. In this situation, glutathione disulfide (GSSG) levels in liver were not changed, but its biliary release increased by 76%. Hepatic reduced glutathione (GSH) levels were recovered by 18 hr and increased by 23% after 22 hr of iron ingestion. Acute ethanol intake diminished liver GSH content by 30% and enhanced that of GSSG by 73%, thus eliciting a net decrease of 20% in total GSH equivalents (GSH + 2GSSG). Biliary release of total GSH was reduced in this condition. The combined administration of iron and ethanol further influenced the decrease in hepatic GSH and the increase in GSSG levels elicited by the separate treatments, but no alterations in the biliary content and release of total GSH were observed in this situation. These data indicate that iron exposure accentuates the changes in lipid peroxidation and in the glutathione status of the liver cell induced by acute ethanol intoxication.

PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid25-35 fragment, glutamate, and hydrogen peroxide.

This work addresses the differential effects of several oxidative insults on two neuronal cell lines, PC12 and Neuro 2a cells, extensively used as neuronal models in vitro. We measured cellular damage using the cytotoxic assays for MTT reduction and LDH release and found that acetylcholinesterase (AChE)–amyloid–β‐peptide (Aβ) complexes, Aβ25–35 fragment, glutamate and H2O2 were over 200‐fold more toxic to PC12 than to Neuro 2a cells. 17α and 17β estradiol were able to protect both cell types from damage caused by H2O2 or glutamate. By contrast, other insults not related to oxidative stress, such as those caused by the nonionic detergent Triton X‐100 and serum deprivation, induced a similar level of damage in both PC12 and Neuro 2a cells. Considering that the Aβ peptide, H2O2 and glutamate are cellular insults that cause an increase in reactive oxygen species (ROS), the intracellular levels of the antioxidant compound, glutathione were verified. Neuro 2a cells were found to have 4‐ to 5‐fold more glutathione than PC12 cells. Our results suggest that Neuro 2a cells are less susceptible to exposure to AChE–Aβ complexes, Aβ25–35 fragment, glutamate and H2O2 than PC12 cells, due to higher intracellular levels of antioxidant defense factors. J. Neurosci. Res. 56:620–631, 1999. 

Effect of acute ethanol ingestion on lipoperoxidation and on the activity of the enzymes related to peroxide metabolism in rat liver.

Data presented in [l] show that acute ethanol administration to rats stimulates hepatic lipoperoxida. tive processes in conditions of maximal depletion of the content of reduced glutathione (GSH). Apart from the content of tissue GSH, the levels of vitamin E as well as the activity of the enzymes related to peroxide metabolism have been suggested to contribute to the maintenance of the antioxygenic capacity of the liver cell [2-41. This work deals with the influence of acute ethanol ingestion on liver lipoperoxidation in relation to the activity of: (i) Superoxide dismutase and catalase as an antioxidant system preventing lipoperoxide formation by free radicals; (ii) Glutathione (GSH)-peroxidase and glutathione (GSSG)-reductase as a detoxifying system decomposing hydroperoxides and hydrogen peroxide (H,Oz) to inactive metabolites [4].

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.