The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.     

Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.     

The role of NADPH oxidase,neuronal nitric oxide synthase and poly(ADP ribose) polymerase in oxidative neuronal death induced in cortical cultures by brain-derived neurotrophic factor and neurotrophin-4/5

Certain neurotrophins promote or induce oxidative neuronal death in cortical cultures. However, the effector mechanisms mediating this phenomenon have not been delineated. In this study, we investigated the possibility that NADPH oxidase and nitric oxide synthase (NOS) function as such effectors. Western blot analysis showed that treatment with brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4/5 increased the levels of NADPH oxidase subunits. Moreover, neurotrophin treatment resulted in membrane translocation of p67phox, a characteristic feature of NADPH oxidase activation. Administration of the specific NADPH oxidase inhibitor, 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), attenuated increases in oxygen free radicals thereby suggesting that NADPH oxidase contributes to the oxidative stress induced by neurotrophins. Furthermore, neuronal death induced by BDNF or NT-4/5 was significantly attenuated by AEBSF. Treatment with BDNF has previously been shown to induce neuronal NOS (nNOS). Our data indicated that inhibitors of nNOS attenuated neuronal death induced by BDNF or NT-4/5, consistent with an active role of nNOS in the mediation of neurotrophin neurotoxicity. As in other models of oxidative cell death, BDNF-induced neuronal death was accompanied by poly(ADP ribose) polymerase (PARP) activation. AEBSF or N-nitro-l-arginine (NNA) reduced BDNF-mediated PARP activation. PARP and poly(ADP ribose) glycohydrolase (PARG) are actively involved in mediating neurotrophin neurotoxicity since inhibitors of PARP and PARG significantly reduced levels of cell death. These results suggest that NADPH oxidase and nNOS contribute to increased oxidative stress, subsequent activation of PARP/PARG, and neuronal death induced by prolonged neurotrophin exposure.     

Liquiritigenin, an aglycone of liquiritin in Glycyrrhizae radix, prevents acute liver injuries in rats induced by acetaminophen with or without buthionine sulfoximine

Glycyrrhizae radix has been used as one of the oldest and most frequently employed botanicals in both western and oriental countries. Previously, we showed that liquiritigenin (LQ), an aglycone of liquiritin in G. radix, exerts cytoprotective effects against heavy metal-induced toxicity in vitro. This study investigated in vivo protective effects of LQ against acute liver injuries induced by acetaminophen (APAP) or APAP plus buthionine sulfoximine (BSO). Liver injuries were assessed by blood biochemistry and histopathology in rats administered with LQ purified from the acid hydrolyates of liquiritin singly (p.o. or i.v., 2-4 days) or in combination with dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB), a synthetic derivative of Schisandrin C in Fructus shizandrae, and exposed to APAP or APAP + BSO. LQ treatments (oral) effectively decreased liver injuries induced by a single dose of APAP, as evidenced by decreases in hepatic necrosis and inflammation as well as plasma alanine aminotransferase and lactate dehydrogenase activities. LQ, when intravenously applied, enhanced hepatoprotective effect with a greater potency. APAP + BSO led to severe liver injuries, resulting in lethality. LQ pretreatments significantly reduced the potentiated liver necrosis, decreasing mortality. In spite of the improvement in blood biochemistry, DDB failed to protect the liver from injuries induced by APAP or APAP + BSO. Combined treatments of rats with LQ and DDB showed some additive protective effect. The present study demonstrates that LQ efficaciously protects the liver from acute injuries induced by APAP or from APAP-induced severe injuries during GSH deficiency, indicating that LQ is one of the principal cytoprotective components comprised in G. radix.     

Aneuploidy assessed by DNA index influences the effect of iron status on plasma and/or supernatant cytokine levels and progression of cells through the cell cycle in a mouse model

Aneuploidy, a condition associated with altered chromosome number, hence DNA index, is frequently seen in many diseases including cancers and affects immunity. Iron, an essential nutrient for humans, modulates the immune function and the proliferation of normal and cancer cells. To determine whether impaired immunity seen in iron-deficient subjects may be related to aneuploidy, we measured spleen cell DNA index, percent of cells in different phases of the cell cycle, plasma and/or supernatant IL-2, IL-10, IL-12, and interferon-gamma in control, pair-fed, iron-deficient, and iron-replete mice (N = 20–22/group). The test and control diets differed only in iron content (0.09 mmol/kg versus 0.9 mmol/kg) and were fed for 68 days. Mean levels of hemoglobin and liver iron stores of iron-deficient and iron-replete mice were 40–60% lower than those of control and pair-fed mice (P < 0.05). Mean plasma levels of IL-10, interferon-gamma and percent of cells in S + G2/M phases were lower in mice with than in those without aneuploidy (P < 0.05). Lowest plasma IL-12 and interferon-gamma concentrations were observed in iron-deficient mice with aneuploidy. Mean percents of cultures with aneuploidy and DNA indexes were higher in iron-deficient and iron-replete than in control and pair-fed mice likely due to delayed cell division (P < 0.05). Aneuploidy decreased the concentration of IL-2 and interferon-gamma in baseline cultures while it increased that of interferon-gamma in anti-CD3 treated cultures. Aneuploidic indexes negatively correlated with cytokine levels, percents of cells in S + G2/M phases and indicators of iron status (P < 0.05). Although chromosome cytogenetics was not performed, for the first time, we report that increased aneuploidy rate may modulate the immune function during iron-deficiency.     

Studies on the induction of histocompatibility gene mutations in germ cells of mice by chemical mutagens and/or virus-inducing compounds

This work continues earlier studies concerning the use of histocompatibility mutations in mammalian germ cells as a mutagenicity test system (H test). The rate of spontaneous H mutations was re-examined using a new basis for the classification of H mutants. This procedure led to very high frequencies of suspected spontaneous H mutants: among C57Bl/6 mice, 6% and among C3H mice, 9%. F₂ hybrids of a cross between these strains revealed 1% suspected H mutants. Using the same procedure, the sensitivity of the H test was examined with the mutagens ethylnitrosourea, benzo[a]pyrene, 2-acetylaminofluorene (2-AAF), with the solvent dimethyl sulfoxide (DMSO) and with the antibacterial nitrofurantoin. It was possible to demonstrate the mutagenic potential of all mutagens tested as well as their specific action on the different stages of male germ cell development. We succeeded in demonstrating the mutagenicity of 2-AAF for the first time in germ cells of a mammal. In contrast to the negative result with benzopyrene (BP) in the specific locus test, BP induced H mutants even at the very low dose of 2 mg/kg. DMSO was found to induce H mutations in spermatogonia. This extraordinary result is possibly due to the virus-inducing properties of this compound. Nitrofurantoin which is often used in treating bacterial infections of the urinary tract in humans showed a very stage-specific action on maturing spermatids. The value of the H test for mutagenicity testing is discussed with respect to its sensitivity and economy. The very high spontaneous frequency of suspected H mutants and the ease of inducing incraased mutant frequencies by mutagens and by DMSO suggest the possibility that the majority of the histoincompatibilities found in the H test are due to induced antigenic gene products of endogenous viruses. This, however, does not interfere with the applicability of the H test for mutagenicity testing, but rather seems to augment its sensitivity to alkylating mutagens as well as mutagens which probably cause frameshift mutations.Other tests for mutations and/or inherited tumor proneness using mouse germ cells can easily be combined with the H test, because the test animal does not have to be killed, thus reducing the cost of the test.     

Synthesis and structure of iron (III) and iron (II) complexes in S4P2 environment created by diethyldithiocarbamate and 1,2-bis(diphenylphosphino)ethane chelation: Investigation of the electronic structure of the complexes by Mössbauer and magnetic studies

Iron (II) and iron (III) complexes, [Fe^II(DEDTC)₂(dppe)] · CH₂Cl₂ (1), [Fe^II(ETXANT)₂(dppe)] (2) (DEDTC = diethyldithiocarbamate, ETXANT = ethyl xanthate, dppe = 1,2-bis (diphenylphosphino) ethane), and [Fe^III(DEDTC)₂(dppe)] [Fe^IIICl₄] (3) have been synthesized and characterized. Since 3 contains two magnetic centers, an anion metathesis reaction has been conducted to replace the tetrahedral FeCl₄⁻ by a non-magnetic BPh₄⁻ ion producing [Fe^III(DEDTC)₂(dppe)]BPh₄ (4) for the sake of unequivocal understanding of the magnetic behavior of the cation of 3. With the similar end in view, the well-known FeCl₄⁻ ion, the counter anion of 3, is trapped as PPh₄[Fe^IIICl₄] (5) and its magnetic property from 298 to 2 K has been studied. Besides the spectroscopic (IR, UV-Vis, NMR, EPR, Mass and XPS) characterization of the appropriate compounds, especially 2, others viz. 1, 3 and 4 have been structurally characterized by X-ray crystallography. While Fe^II complexes, 1 and 2, are diamagnetic, the Fe^III systems, namely the cations of 3, and 4 behave as low-spin (S = 1/2) paramagnetic species from 298 to 50 K. Below 50 K 3 shows gradual increase of χ_MT up to 2 K suggesting ferromagnetic behavior while 4 exhibits gradual decrease of magnetic moment from 60 to 2 K, indicating the occurrence of weak antiferromagnetic interaction. These conclusions are supported by the Mössbauer studies of 3 and 4. The Mössbauer pattern of 1 exhibits a doublet site for diamagnetic (2-400 K) Fe^II. The compounds 1, 2 and 4 encompass interesting cyclic voltammetric responses involving Fe^II, Fe^III and Fe^IV.     

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND: Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS: We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION: We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.     

Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3

Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10-60 microg/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 microg/ml at 24 h and 31.5 microg/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 microg/ml GLA for 48 h and with 40 microg/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pan-caspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation.     

Hydrogen sulfide protects SH-SY5Y neuronal cells against d-galactose induced cell injury by suppression of advanced glycation end products formation and oxidative stress

d-Galactose is widely used as an agent to cause aging effects in experimental animals. The present study aims to investigate the effects of hydrogen sulfide (H₂S) in human neuroblastoma SH-SY5Y cells exposed to d-galactose. Cells were pretreated with NaHS, an H₂S donor, and then exposed to d-galactose (25–400 mM for 48 h). We found that NaHS pretreatment significantly reversed the d-galactose-induced cell death and cellular senescence. MTT assay shows that NaHS significantly increased cell viability from 62.31 ± 1.29% to 72.34 ± 0.46% compared with d-galactose (200 mM) treatment group. The underlying mechanism appeared to involve a reduction by NaHS in the formation of advanced glycation end products (AGEs), which are known to contribute to the progression of age-related diseases. In addition, NaHS decreased the elevation of reactive oxygen species from 151.17 ± 2.07% to 124.8 ± 2.89% and malondialdehyde from 1.72 ± 0.07 to 1.10 ± 0.08 (nmol/mg protein) in SH-SY5Y cells after d-galactose exposure. NaHS also stimulated activities of superoxide dismutase from 0.42 ± 0.05 to 0.73 ± 0.04 (U/mg protein) and glutathione peroxidase from 3.98 ± 0.73 to 14.73 ± 0.77 (nmol/min/mg protein) and upregulated the gene expression levels of copper transport protein ATOX1, glutathione synthetase (GSS) and thioredoxin reductase 1 (TXNRD1) while down-regulated aldehyde oxidase 1 (AOX1). In summary, our data indicate that H₂S may have potentially anti-aging effects through the inhibition of AGEs formation and reduction of oxidative stress.