Lucia Marcocci

α-Lipoic Acid in Liver Metabolism and Disease

R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved.

Antioxidant Action of Ginkgo Biloba Extract (EGB 761)

Extracts from the leaves of Ginkgo biloba trees have been used therapeutically for centuries in traditional Chinese medicine, and in modern Chinese pharmacopoeias both the leaves and fruits are recommended for treating problems of heart and lungs. EGb 761 is a dry, powdered extract prepared from Ginkgo biloba leaves. It is a standardized mixture of several different chemical constituents; its two major classes of compounds are flavonoid glycosides and terpenoids (Drieu 1986; DeFeudis 1991). The flavonoid fraction is mainly composed of three flavonols: quercetin, kaempferol, and isorhamnetin, which are linked to a sugar(DeFeudis 1991). The terpenoid fraction is composed of ginkgolides and bilobalides(Drieu 1988). It also contains some organic acids, which help make it water-soluble(Drieu 1986).

[46] Antioxidant action of Ginkgo biloba extract EGb 761

Publisher Summary Extracts of Ginkgo biloba leaves (EGb 761) are complex titrated, standardized mixtures of active ingredients introduced into medical practice as polyvalent therapeutic agents. This chapter discusses the radical scavenging properties of EGb 761, in several in vitro systems. The interaction of EGb 761 with superoxide and hydroxyl radicals generated by using the water radiolysis method is presented in the chapter. The method of radiolysis is useful because it specifically generates superoxide and hydroxyl radicals in known quantities, thus reducing the risk of possible interferences. To evaluate the role of terpenes contained in EGb 761, a comparative study is described using CP 202, a similar mixture lacking the terpene fraction. The interaction of EGb 761 with peroxyl radicals is assayed by a chemiluminescent method. The interaction of EGb 761 with nitric oxide is assessed by two different spectrophotometric methods. Finally, the effect of EGb 761 on xanthine oxidase activity is described.

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Cigarette smoke oxidation of human plasma constituents

In vitro exposure of fresh human plasma to cigarette smoke (CS) was used as a model for reactions that could be occurring in CS-exposed respiratory tract lining fluids (RTLFs) and lung parenchyma. The central focus of this model was to characterize the consumption of endogenous plasma antioxidants in relationship to the appearance of oxidized proteins and lipids as a consequence of exposure to CS, or to aldehydes present in CS. The amelioration of CS-induced protein and lipid oxidation in plasma by the addition of selective exogenous antioxidants was also assessed. We found that: (i) exposure of human plasma to gas phase CS causes both lipid peroxidation and protein oxidation, and endogenous ascorbic acid protects against lipid, but not protein, oxidation; (ii) whole CS causes protein oxidation, but does not induce lipid peroxidation; (iii) addition to plasma of aldehydes known to be present in CS causes protein damage, but does not induce either lipid peroxidation or oxidation of ascorbic acid; and (iv) exogenously added dihydrolipoic acid (DHLA) preserves ascorbic acid levels in plasma exposed to the gas phase of CS, and protects, to some extent, against lipid peroxidation; DHLA also protects against protein oxidation, whereas added glutathione (GSH) only protects against protein, but not lipid, oxidation.

Cell Signaling by Protein Carbonylation and Decarbonylation

Reactive oxygen species (ROS) serve as mediators of signal transduction. However, mechanisms of how ROS influence the target molecules to elicit signaling event have not been defined. Our laboratory recently accumulated evidence for the role of protein carbonylation in the mechanism of ROS signaling. This concept originated from experiments in which pulmonary artery smooth muscle cells were treated with endothelin-1 to understand the mechanism of cell growth. Endothelin-1 was found to promote protein carbonylation in an endothelin receptor- and Fenton reaction-dependent manner. Mass spectrometry identified proteins that are carbonylated in response to endothelin-1, including annexin A1. Our experiments generated a hypothesis that endothelin-1-mediated carbonylation and subsequent degradation of annexin A1 promote cell growth. This mechanism was found also to occur in response to other signaling activators such as serotonin and platelet-derived growth factor in smooth muscle cells of pulmonary circulation, systemic circulation, and the airway, as well as in cardiac muscle cells, suggesting the universal role of this pathway. We also discovered a process of decarbonylation that defines transient kinetics of carbonylation signals in certain conditions. We propose that protein carbonylation and decarbonylation serve as a mechanism of signal transduction.

Bcl-2 overexpression in melanoma cells increases tumor progression-associated properties and in vivo tumor growth

In this study, we demonstrated that bcl‐2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis‐related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl‐2 overexpressing derivatives, we found that bcl‐2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP‐2, MMP‐7, MT1‐MMP, and tissue inhibitors of metalloproteases‐1 and ‐2. Moreover, bcl‐2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl‐2 transfectants tumors is significantly associated to an increase in MMP‐2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl‐2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression.

Reactive Oxygen Species and Antioxidants in Pulmonary Hypertension

SIGNIFICANCE Pulmonary hypertension is a devastating disorder without any available treatment strategies that satisfactorily promote the survival of patients. The identification of new therapeutic strategies to treat patients with pulmonary hypertension is warranted. RECENT ADVANCES Human studies have provided evidence that there is increased oxidative stress (lipid peroxidation, protein oxidation, DNA oxidation, and the depletion of small-molecule antioxidants) in patients with pulmonary hypertension. A variety of compounds with antioxidant properties have been shown to have beneficial therapeutic effects in animal models of pulmonary hypertension, possibly supporting the hypothesis that reactive oxygen species (ROS) are involved in the progression of pulmonary hypertension. Thus, understanding the molecular mechanisms of ROS actions could contribute to the development of optimal, antioxidant-based therapy for human pulmonary hypertension. One such mechanism includes action as a second messenger during cell-signaling events, leading to the growth of pulmonary vascular cells and right ventricular cells. CRITICAL ISSUES The molecular mechanisms behind promotion of cell signaling for pulmonary vascular cell growth and right ventricular hypertrophy by ROS are not well understood. Evidence suggests that iron-catalyzed protein carbonylation may be involved. FUTURE DIRECTIONS Understanding precise mechanisms of ROS actions should be useful for designing preclinical animal experiments and human clinical trials of the use of antioxidants and/or other redox compounds in the treatment of pulmonary hypertension.

A New Piece of the Shigella Pathogenicity Puzzle: Spermidine Accumulationby Silencing of the speG Gene

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

Mechanism of protein decarbonylation.

Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling.