Signaling pathways involved in isoprostane-mediated fibrogenic effects in rat hepatic stellate cells.

Despite evidence supporting a potential role for F2-isoprostanes (F2-IsoP's) in liver fibrosis, their signaling mechanisms are poorly understood. We have previously provided evidence that F2-IsoP's stimulate hepatic stellate cell (HSC) proliferation and collagen hyperproduction by activation of a modified form of isoprostane receptor homologous to the classic thromboxane receptor (TP). In this paper, we examined which signal transduction pathways are set into motion by F2-IsoP's to exert their fibrogenic effects. HSCs were isolated from rat liver, cultured to their activated myofibroblast-like phenotype, and then treated with the isoprostane 15-F2t-isoprostane (15-F2t-IsoP). Inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined using commercial kits. Mitogen-activated protein kinase (MAPK) and cyclin D1 expression was assessed by Western blotting. Cell proliferation and collagen synthesis were determined by measuring [(3)H]thymidine and [(3)H]proline incorporation, respectively. 15-F2t-IsoP elicited an activation of extracellular-signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), which are known to be also regulated by G-protein-coupled receptors. Preincubation with specific ERK (PD98059), p38 (SB203580), or JNK (SP600125) inhibitors prevented 15-F2t-IsoP-induced cell proliferation and collagen synthesis. 15-F2t-IsoP decreased cAMP levels within 30 min, suggesting binding to the TPß isoform and activation of Giα protein. Also, 15-F2t-IsoP increased IP3 levels within a few minutes, suggesting that the Gq protein pathway is also involved. In conclusion, the fibrogenic effects of F2-IsoP's in HSCs are mediated by downstream activation of MAPKs, through TP binding that couples via both Gqα and Giα proteins. Targeting TP receptor, or its downstream pathways, may contribute to preventing oxidative damage in liver fibrosis.

Ethanol-induced oxidative stress: basic knowledge.

After a general introduction, the main pathways of ethanol metabolism (alcohol dehydrogenase, catalase, coupling of catalase with NADPH oxidase and microsomal ethanol-oxidizing system) are shortly reviewed. The cytochrome P(450) isoform (CYP2E1) specifically involved in ethanol oxidation is discussed. The acetaldehyde metabolism and the shift of the NAD/NADH ratio in the cellular environment (reductive stress) are stressed. The toxic effects of acetaldehyde are mentioned. The ethanol-induced oxidative stress: the increased MDA formation by incubated liver preparations, the absorption of conjugated dienes in mitochondrial and microsomal lipids and the decrease in the most unsaturated fatty acids in liver cell membranes are discussed. The formation of carbon-centered (1-hydroxyethyl) and oxygen-centered (hydroxyl) radicals during the metabolism of ethanol is considered: the generation of hydroxyethyl radicals, which occurs likely during the process of univalent reduction of dioxygen, is highlighted and is carried out by ferric cytochrome P(450) oxy-complex (P(450)-Fe(3+)O(2) (.-)) formed during the reduction of heme-oxygen. The ethanol-induced lipid peroxidation has been evaluated, and it has been shown that plasma F(2)-isoprostanes are increased in ethanol toxicity.

Oxidative stress, isoprostanes and hepatic fibrosis.

An introduction to oxidative stress enlightening the spreading of interest in lipid peroxidation in the 60's and in the identification of cytotoxic aldehydes originating from it is given. The discovery of F2-isoprostanes as specific markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relationship between oxidative stress and collagen hyperproduction has been previously suggested, and since lipid peroxidation products (aldehydes) have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F2-isoprostanes, which can possess receptors for signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period and hepatic collagen content was also increased. Moreover, when hepatic stellate cells (HSC) isolated from normal livers were cultured up to activation and then treated with F2-isoprostanes (8-epi-PGF2alpha) in the concentration range found in the in vivo studies (10(-9) to 10(-8) M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. F2-isoprostanes also increased the production of transforming growth factor-beta1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The hypothesis that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen hyperproduction, seen in this experimental hepatic fibrosis, was reinforced by the demonstration, by using immunoblot analysis, that isoprostane receptors identical or analogous to those for thromboxane A2 (TxA2r) are present in HSC. Immunocytochemical studies showed the major localization of TxA2r in the perinuclear site and its colocalization with alpha-smooth muscle actin.

Systemic oxidative stress in classic Rett syndrome.

Rett syndrome (RS), a progressive severe neurodevelopmental disorder mainly caused by de novo mutations in the X-chromosomal MeCP2 gene encoding the transcriptional regulator methyl-CpG-binding protein 2, is a leading cause of mental retardation with autistic features in females. However, its pathogenesis remains incompletely understood, and no effective therapy is available to date. We hypothesized that a systemic oxidative stress may play a key role in the pathogenesis of classic RS. Patients with classic RS (n=59) and control subjects (n=43) were evaluated. Oxidative stress markers included intraerythrocyte non-protein-bound iron (NPBI; i.e., free iron), plasma NPBI, F2-isoprostanes (F2-IsoPs, as free, esterified, and total forms), and protein carbonyls. Lung ventilation/perfusion (V/Q) ratio was assessed using a portable gas analyzer, and RS clinical severity was evaluated using standard scales. Significantly increased intraerythrocyte NPBI (2.73-fold), plasma NPBI (x 6.0), free F(2)-IsoP (x1.85), esterified F2-IsoP (x 1.69), total F2-IsoP (x 1.66), and protein carbonyl (x 4.76) concentrations were evident in RS subjects and associated with reduced (-10.53%) arterial oxygen levels compared to controls. Biochemical evidence of oxidative stress was related to clinical phenotype severity and lower peripheral and arterial oxygen levels. Pulmonary V/Q mismatch was found in the majority of the RS population. These data identify hypoxia-induced oxidative stress as a key factor in the pathogenesis of classic RS and suggest new therapeutic approaches based on oxidative stress modulation.

Free iron, total F-isoprostanes and total F-neuroprostanes in a model of neonatal hypoxic-ischemic encephalopathy: neuroprotective effect of melatonin.

Oxidative stress due to free radical formation and initiation of abnormal oxidative reactions is involved in several diseases of newborns, such as hypoxic-ischemic encephalopathy. Melatonin, an endogenously produced indoleamine primarily formed in the pineal gland, is a potent free radical scavenger as well as an indirect antioxidant. The present study was conducted to evaluate the formation of oxidative damage mediators and the possible effect of melatonin treatment in a model of hypoxic-ischemic encephalopathy in 7-day-old rats. Pups were subjected to permanent ligation of the right common carotid artery and exposed for 2.5 hr to a nitrogen-oxygen mixture (92% and 8%, respectively) (hypoxia-ischemia, HI). Melatonin was injected intraperitoneally to a group of rats at the dose of 15 mg/kg 30 min before starting the ischemic procedure (HI-Melatonin). After 24 hr of treatment, in homogenized cerebral cortex, desferoxamine (DFO)-chelatable free iron, total F(2)-isoprostanes and total F(4)-neuroprostanes, originating from the free radical-catalyzed peroxidation of arachidonic and docosahexaenoic acids, respectively, were determined. HI induced a significant increase in DFO-chelatable iron, total F(2)-isoprostanes and F(4)-neuroprostanes in both right and left side of the cerebral cortex. In HI-Melatonin-treated animals the levels of free iron, F(2)-isoprostanes, and F(4)-neuroprostanes were significantly lower than that in HI rats and the values were similar to controls. These data show the important neuroprotective role of melatonin in reducing oxidative damage resulting from HI. Melatonin could represent a potential safe approach to perinatal brain damage in humans.

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease.

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.

Oxidative stress, erythrocyte ageing and plasma non-protein-bound iron in diabetic patients.

Increased oxidative stress and decreased life span of erythrocytes (RBCs) are repeatedly reported in diabetes. In the aim to elucidate the mechanism of the latter, i.e. the events leading to erythrocyte ageing, this study determined in RBCs from diabetic patients iron release in a free desferrioxamine-chelatable form (DCI), methemoglobin (MetHb) formation, binding of autologous IgG to membrane proteins and in plasma non-protein-bound iron (NPBI), F(2)-Isoprostanes (F(2)-IsoPs) and advanced oxidation protein products (AOPP). DCI and MetHb were higher in diabetic RBCs than in controls and autologous IgG binding occurred in a much higher percentage of diabetic patients than controls. A significant correlation between DCI and IgG binding was found in diabetic RBCs. Plasma NPBI, esterified F(2)-IsoPs and AOPP were higher in diabetic patients and a significant correlation was found between plasma NPBI and intra-erythrocyte DCI. The increased DCI and autologous IgG binding appear to be important factors in the accelerated removal of RBCs from the blood stream in diabetes and the increase in plasma NPBI could play an important role in the increased oxidative stress.

Plasma esterified F2-isoprostanes and oxidative stress in newborns: role of nonprotein-bound iron.

Nonprotein-bound iron (NPBI) and F2-isoprostanes, reliable markers of oxidative stress, are increased in plasma of newborns and inversely correlated to the gestational age. Because NPBI represents a pro-oxidant stimulus in plasma, we test the hypothesis that the entity of lipid peroxidation is related with NPBI concentrations. Plasma levels of free, esterified, and total F2-isoprostanes were investigated in relation to NPBI levels in 59 newborns and 16 healthy adults. The pro-oxidant role of iron was ascertained in vitro, by measuring all the forms of F2-isoprostanes after incubation with ammonium iron sulfate. Significant positive correlations were found between NPBI and total as well as esterified F2-isoprostanes in plasma of the newborns. The addition of ammonium iron sulfate induced a marked increase in all the forms of F2-isoprostanes after 2 hours of incubation. The higher NPBI concentration, the higher F2-isoprostanes levels. An increase NPBI dose dependent in total F2-isoprostanes formation was observed in dialyzed low density lipoprotein from adult plasma. The results clearly show that once NPBI is generated, whatever its source, it is capable of inducing oxidative stress. NPBI-induced oxidative stress may contribute to the morbidity in preterm infants that are particularly susceptible to free radical damage.

F2-isoprostane receptors on hepatic stellate cells.

F2-isoprostanes are considered as the most reliable markers of oxidative stress and can be used to evaluate the oxidative status in a number of human pathologies. Besides being markers of oxidative stress, F2-isoprostanes proved to be mediators of important biological effects and would act through the activation of receptors analogous to those for thromboxane A2. In a previous work, we provided evidence that F(2)-isoprostanes, generated during carbon tetrachloride-induced hepatic fibrosis, mediate hepatic stellate cell (HSC) proliferation and collagen hyperproduction. To investigate whether TxA2 receptor (TxA2r or TPr) is involved in the effects of F2-isoprostanes on HSC, experiments on DNA synthesis were carried out in the presence of 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) or the TxA2r-specific agonist I-BOP ([1S-[1alpha,2alpha(Z),3beta(1E,3S*), 4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid). Both agonists significantly stimulated DNA synthesis, which was almost completely inhibited by the TxA2r-specific antagonist SQ29548 ([1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl] hydrazino] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptanoic acid), suggesting that much of the effect of 8-epi-PGF(2alpha) is mediated by the TxA2r. Further studies showed that increasing concentrations of SQ29548 progressively inhibit DNA synthesis, suggesting a possible competitive antagonism between the two molecules. In addition, we demonstrated that the stimulatory effect of 8-epi-PGF(2alpha) on collagen synthesis could be mediated by TxA2r. The occurrence of TxA2r on HSC was also investigated using western blotting analysis and immunocytochemistry, which reveals that TP is distributed both on plasma membranes and within the cells. Moreover, binding studies indicated the presence of a specific binding site for 3H-SQ29548 on HSC. Competition binding studies indicated that 8-epi-PGF(2alpha) and I-BOP were both able to displace 3H-SQ29548 binding with a very different affinity (K(i)=4.14+/-1.9 x 10(-6) M and K(i)=1.15+/-0.3 x 10(-9) M, respectively), suggesting the involvement of a modified form of isoprostane receptor, homologous to the classic thromboxane A2-binding site in F2-isoprostanes-evoked responses on HSC.

F2-isoprostanes are not just markers of oxidative stress.

F(2)-isoprostanes are not just markers of oxidative stress. The discovery of F(2)-isoprostanes (F(2)-IsoPs) as specific and reliable markers of oxidative stress in vivo is briefly summarized here. F(2)-IsoPs are also agonists of important biological effects, such as the vasoconstriction of renal glomerular arterioles, the retinal vessel, and the brain microcirculature. In addition to the F(2)-IsoPs, E(2)- and D(2)-IsoPs can be formed by rearrangement of H(2)-IsoP endoperoxides and can give rise to cyclopentenone IsoPs, which are very reactive alpha,beta-unsaturated aldehydes. The same type of reactivity is also shown by acyclic gamma-ketoaldehydes formed as products of the IsoP pathway. Because previous studies suggested a relation between oxidative stress and collagen hyperproduction, it was investigated whether collagen synthesis is induced by F(2)-IsoPs, the most proximal products of lipid peroxidation. In contrast to aldehydes, F(2)-IsoPs act through receptors able to elicit definite signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma F(2)-IsoPs were markedly elevated for the entire experimental period; hepatic collagen content was also increased. When hepatic stellate cells from normal liver were cultured up to activation (expression of smooth muscle alpha-actin) and then treated with F(2)-IsoPs in the concentration range found in the in vivo studies (10(-9) to 10(-8) M), a striking increase in DNA synthesis, cell proliferation, and collagen synthesis was observed. Total collagen content was similarly increased. All these stimulatory effects were reversed by the specific antagonist of the thromboxane A(2) receptor, SQ 29 548, whereas the receptor agonist, I-BOP, also had a stimulatory effect. Therefore F(2)-IsoPs generated by lipid peroxidation in hepatocytes may mediate hepatic stellate cell proliferation and collagen hyperproduction seen in hepatic fibrosis.

Isoprostanes and hepatic fibrosis.

After a brief introduction to oxidative stress, the discovery of F(2)-isoprostanes as specific and reliable markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relation between oxidative stress and collagen hyperproduction has been previously suggested and since lipid peroxidation products have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F(2)-isoprostanes the most proximal products of lipid peroxidation. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content was also increased. When hepatic stellate cells from normal liver were cultured up to activation (expression of alpha-smooth muscle-alpha actin) and then treated with F(2)-isoprostanes in the concentration range found in the in vivo studies (10(-9)-10(-8)M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. Moreover, F(2)-isoprostanes increased the production of transforming growth factor-beta1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The data suggest the possibility that F(2)-isoprostanes generated by lipid peroxidation in hepatocytes mediate hepatic stellate cell proliferation and collagen hyperproduction seen in hepatic fibrosis.

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21 percent of initial body weight; serum F2-isoprostane levels decreased by 76 percent; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93 percent, respectively; n-6/n-3 LCPUFA ratio decreased by 51 percent; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164 percent (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.

Polyunsaturated fatty acid pattern in liver and erythrocyte phospholipids from obese patients.

OBJECTIVE: Our aim was to study the fatty acid (FA) composition of liver phospholipids and its relation to that in erythrocyte membranes from patients with obese nonalcoholic fatty liver disease (NAFLD), as an indication of lipid metabolism alterations leading to steatosis. RESEARCH METHODS AND PROCEDURES: Eight control subjects who underwent antireflux surgery and 12 obese patients with NAFLD who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in Roux-en-Y were studied. The oxidative stress status of patients was assessed by serum F2-isoprostanes levels (gas chromatography/negative ion chemical ionization tandem mass spectrometry). Analysis of FA composition of liver and erythrocyte phospholipids was carried out by gas-liquid chromatography. RESULTS: Patients with NAFLD showed serum F2-isoprostanes levels 84% higher than controls. Compared with controls, liver phospholipids from obese patients exhibited significantly 1) lower levels of 20:4n-6, 22:5n-3, 22:6n-3 [docosahexaenoic acid (DHA)], total long-chain polyunsaturated FA (LCPUFA), and total n-3 LCPUFA, 2) higher 22:5n-6 [docosapentaenoic acid (DPAn-6)] levels and n-6/n-3 LCPUFA ratios, and 3) comparable levels of n-6 LCPUFA. Levels of DHA and DPAn-6 in liver were positively correlated with those in erythrocytes (r = 0.77 and r = 0.90, respectively; p < 0.0001), whereas DHA and DPAn-6 showed a negative association in both tissues (r = -0.79, p < 0.0001 and r = -0.58, p < 0.01, respectively), associated with lower DHA/DPAn-6 ratios. DISCUSSION: Obese patients with NAFLD showed marked alterations in the polyunsaturated fatty acid pattern of the liver. These changes are significantly correlated with those found in erythrocytes, thus suggesting that erythrocyte FA composition could be a reliable indicator of derangements in liver lipid metabolism in obese patients.

Oxidative stress and autologous immunoglobulin G binding to band 3 dimers in newborn erythrocytes.

Since birth-induced oxidative stress (OS) results in the removal of erythrocytes from the blood stream, we studied the binding of autologous IgG to erythrocyte band 3 dimers (the 170-kDa band, which marks the erythrocytes for removal) in preterm and term newborns and in adults. The 170-kDa band was present in as much as 74% of preterm, in 21% of term newborns, and in 10% of adults. During erythrocyte ageing "in vitro" (0, 24, and 48 h aerobic incubation), the appearance of the band occurred much faster with erythrocytes from newborns (particularly preterm) than with those from adults. When the blots for the 170-kDa band were quantified by scanning densitometry, it was seen that the 0 time values were significantly higher in preterm compared to term and adult values. After aerobic incubation a progressive increase in the optical density was observed in each group and the densities were higher in preterm than in the other groups. The course of iron release during the various incubations was analogous to that of the 170-kDa band blots, and significant correlations were found at 0 and 48 h. Methemoglobin formation roughly paralleled iron release. Esterified F(2)-isoprostanes (markers of OS) and O(2)(-) production in the nonincubated (0 time) erythrocytes were much higher in newborn (preterm and term) than in adult erythrocytes. Plasma free F(2)-isoprostanes were significantly higher in preterms than in terms and in terms than in adults. Plasma non-protein-bound iron (NPBI) was higher in preterm than in term newborns and not detectable in adults. In conclusion dimers of band 3 with autologous IgG are found under conditions in which OS can be detected in erythrocytes or in plasma: namely in newborns or in aged erythrocytes.

F2-isoprostanes stimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis?

Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F2-isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl4-induced hepatic fibrosis, plasma F2-isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F2-isoprostanes in the concentration range found in the in vivo studies (10(-9)-10(-8) M), a striking increase in DNA synthesis (reversed by the thromboxane A2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F2-isoprostanes markedly increased the production of transforming growth factor-beta1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.

Plasma F2-isoprostanes are elevated in newborns and inversely correlated to gestational age.

F(2)-isoprostanes, prostaglandin F(2)-like compounds formed by free radical-catalyzed lipid peroxidation, are considered the most reliable markers of oxidative stress. It has been repeatedly suggested that newborns are exposed to conditions of oxidative stress resulting from the change from a low oxygen pressure in utero to a high oxygen pressure at birth. We measured the levels of F(2)-isoprostanes in plasma of newborns by gas chromatography/mass spectrometry and we found that F(2)-isoprostanes are significantly higher in term newborns compared to healthy adults. The greatest values were found in preterm newborns in whom F(2)-isoprostanes are even higher than in term babies. Moreover a significant inverse correlation was found between the plasma levels of isoprostanes and the gestational age. A quite normal level of isoprostanes was found in the mothers both at delivery and during pregnancy. Placental total F(2)-isoprostanes (sum of free plus esterified) were significantly higher in preterm compared to term deliveries and such a difference might account for the difference in plasma isoprostanes. Plasma non-protein-bound iron is higher in preterm than in term newborns, even if no correlation was found with plasma F(2)-isoprostanes. Erythrocyte desferrioxamine-chelatable iron content (0 time) and release (24 h of aerobic incubation) are higher in newborns than in adults and in preterm than in term newborns, but again no correlation was found with plasma F(2)-isoprostanes. The marked increase in plasma isoprostanes suggests that oxidative stress is a feature of the physiopathological changes seen in the perinatal period.

Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation.

Iron is released in a desferrioxamine (DFO)-chelatable form when erythrocytes are challenged by an oxidative stress. The release is increased when an accelerated removal of erythrocytes occurs such as in perinatal period, in which iron release is greater in hypoxic than in non-hypoxic newborns. This suggests that an hypoxic environment at birth promotes iron release. To test this possibility, iron release in a model of hypoxia, hypoxia-reoxygenation and normoxia was studied in newborn and adult erythrocytes. In newborn erythrocytes, hypoxia induced a much greater iron release compared to an equal period of normoxia. In adult erythrocytes, hypoxia also induced a greater iron release as compared to normoxia, but it was much lower than that seen with newborn erythrocytes. Methemoglobin (MetHb) formation roughly paralleled iron release. The phenylhydrazine-promoted superoxide anion (O(2)?(-)) production was greater with normoxic but lower with hypoxic erythrocytes from newborns as compared to that from adults. This discrepancy between iron release and O(2)?(-) production may be explained by the shift towards MetHb in hemoglobin autoxidation. Iron diffusion out of the erythrocytes was much higher with hypoxic erythrocytes from newborns as compared to that from adults. Also the binding of autologous IgG to band 3 dimers (AIgGB) is much greater with hypoxic erythrocytes from newborns as compared to that from adults, suggesting that the level of iron release is related to the extent of band 3 clustering and that hypoxia accelerates removal of erythrocytes from bloodstream in in vivo condition.

Role of caspases-3 and -7 in Apaf-1 proteolytic cleavage and degradation events during cisplatin-induced apoptosis in melanoma cells.

Apoptosis protease-activating factor-1 (Apaf-1), the central element in the mitochondrial pathway of apoptosis, is frequently absent or poorly expressed in metastatic melanomas, a tumor type showing a low degree of spontaneous apoptosis and a poor response to conventional therapies. In the present study, we used the Apaf-1-positive Me665/2/21 melanoma cell line to investigate the fate of Apaf-1 during cisplatin-induced apoptosis. As novel findings described for the first time in melanoma cells, we observed that Apaf-1 was markedly decreased during apoptosis, already at early stages of cell damage; concurrently, an immunoreactive N-terminal fragment of congruent with 26 kDa was evident. In spite of the remarkable decrease of Apaf-1 in apoptotic cells, caspase-9 was found to be processed and enzymatically active. Both Apaf-1 depletion and its proteolytic cleavage were markedly prevented in presence of the caspase-3/-7 inhibitor ac-DEVD-CHO. In presence of ac-DEVD-CHO, caspase-9 activity was also inhibited, along with a partially different pattern of caspase-9 processing forms. Unexpectedly, the inhibition afforded by ac-DEVD-CHO on several components, that is, caspase-3/-7 and caspase-9 activities, and Apaf-1 proteolytic degradation, did not abrogate the apoptotic morphology and cell detachment, nor the proteolytic degradation of crucial targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. Together, our results suggest that caspase-3 and -7, proved to be dispensable for the above apoptosis-associated events, play a role on Apaf-1 handling and possibly on apoptosome function.

Ion trap tandem mass spectrometric determination of F2-isoprostanes.

A new tandem mass spectrometry (MS/MS) method based on the use of an ion trap mass spectrometer for the identification and quantitation of F(2)-isoprostanes has been developed. It consists of two solid-phase extractions and two derivation steps followed by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis. This method is highly selective and sensitive and it has been successfully applied to biological samples.

Redox modulation of NF-kappaB nuclear translocation and DNA binding in metastatic melanoma. The role of endogenous and gamma-glutamyl transferase-dependent oxidative stress.

AIMS AND BACKGROUND: The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity--expressed by a number of malignancies, including melanoma--can act as a basal source of superoxide, hydrogen peroxide and other prooxidants. METHODS: In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme--through the promotion of redox processes--may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kappaB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies). RESULTS: Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status. CONCLUSIONS: Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.