The effects of Insulin Pre-Administration in Mice Exposed to Ethanol: Alleviating Hepatic Oxidative Injury through Anti-Oxidative,Anti-Apoptotic Activities and Deteriorating Hepatic Steatosis through SRBEP-1c Activation

Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.     

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.     

Diabetes,oxidative stress and therapeutic strategies

Background

Diabetes has emerged as a major threat to health worldwide.

Scope of Review

The exact mechanisms underlying the disease are unknown; however, there is growing evidence that excess generation of reactive oxygen species (ROS), largely due to hyperglycemia, causes oxidative stress in a variety of tissues. Oxidative stress results from either an increase in free radical production, or a decrease in endogenous antioxidant defenses, or both. ROS and reactive nitrogen species (RNS) are products of cellular metabolism and are well recognized for their dual role as both deleterious and beneficial species. In type 2 diabetic patients, oxidative stress is closely associated with chronic inflammation. Multiple signaling pathways contribute to the adverse effects of glucotoxicity on cellular functions. There are many endogenous factors (antioxidants, vitamins, antioxidant enzymes, metal ion chelators) that can serve as endogenous modulators of the production and action of ROS. Clinical trials that investigated the effect of antioxidant vitamins on the progression of diabetic complications gave negative or inconclusive results. This lack of efficacy might also result from the fact that they were administered at a time when irreversible alterations in the redox status are already under way. Another strategy to modulate oxidative stress is to exploit the pleiotropic properties of drugs directed primarily at other targets and thus acting as indirect antioxidants.

Major Conclusions

It appears important to develop new compounds that target key vascular ROS producing enzymes and mimic endogenous antioxidants.

General significance

This strategy might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated with vascular diseases.     

Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.     

Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model

Recently, the treatment of stroke has focused on antioxidant therapies, where oxidative stress is implicated. The preventive and therapeutic potential of plant compounds on ischemic stroke has been intensively studied because many of them contain antioxidant properties. Genistein, one of the active ingredients in soybean, possesses many bioactivities. In this study, we investigated the potential neuroprotective effects of genistein and its possible mechanism of action in a cerebral ischemia mouse model. Mice were pretreated with genistein (2.5, 5, and 10mg/kg) or vehicle orally once daily for 14 consecutive days before transient middle cerebral artery occlusion was performed. Genistein at doses of 2.5-10mg/kg significantly reduced the infarct volume, improved the neurological deficit and prevented cell apoptosis after ischemia. In addition, genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species (ROS) production, enhance the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), and decrease levels of malondialdehyde (MDA) in stroke mice. Moreover, genistein reversed the mitochondria dysfunction after ischemia, as evidenced by decreasing mitochondria ROS levels, preventing cytochrome C release to the cytoplasm and inhibiting caspase-3 activation. Western blotting showed ischemia activated the ROS-dependent nuclear factor-κB (NF-κB) signaling pathway, and genistein suppressed phosphorylation and activation of the NF-κB p65 subunit, as well as the phosphorylation and degradation of the inhibitor protein of κBα (IκBα). Our findings suggested that genistein has a neuroprotective effect in transient focal ischemia, which may involve regulation of mitochondria-dependent apoptosis pathways and suppression of ROS-induced NF-κB activation.     

Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.     

Age-dependent ultrastructural alterations and biochemical response of rat skeletal muscle after hypoxic or hyperoxic treatments

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.     

Antioxidant proteins and reactive oxygen species are decreased in a murine epidermal side population with stem cell-like characteristics

Reactive oxygen species (ROS) and antioxidants are essential to maintain a redox balance within tissues and cells. Intracellular ROS regulate key cellular functions such as proliferation, differentiation and apoptosis through cellular signaling, and response to injury. The redox environment is particularly important for stem/progenitor cells, as their self-renewal and differentiation has been shown to be redox sensitive. However, not much is known about ROS and antioxidant protein function in freshly isolated keratinocytes, notably the different keratinocyte subpopulations. Immunostaining of neonatal cutaneous sections revealed that antioxidant enzymes [catalase, SOD2, gluthatione peroxidase-1 (GPx)] and ROS are localized predominantly to the epidermis. We isolated keratinocyte subpopulations and found lower levels of SOD2, catalase and GPx, as well as decreased SOD and catalase activity in an epidermal side population with stem cell-like characteristics (EpSPs) compared to more differentiated (Non-SP) keratinocytes. EpSPs also exhibited less mitochondrial area, fewer peroxisomes and produced lower levels of ROS than Non-SPs. Finally, EpSPs were more resistant to UV radiation than their progeny. Together, our data indicate ROS and antioxidant levels are decreased in stem-like EpSPs.     

Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease?

Neurodegenerative diseases share various pathological features, such as accumulation of aberrant protein aggregates, microglial activation, and mitochondrial dysfunction. These pathological processes are associated with generation of reactive oxygen species (ROS), which cause oxidative stress and subsequent damage to essential molecules, such as lipids, proteins, and DNA. Hence, enhanced ROS production and oxidative injury play a cardinal role in the onset and progression of neurodegenerative disorders. To maintain a proper redox balance, the central nervous system is endowed with an antioxidant defense mechanism consisting of endogenous antioxidant enzymes. Expression of most antioxidant enzymes is tightly controlled by the antioxidant response element (ARE) and is activated by nuclear factor E2-related factor 2 (Nrf2). In past years reports have highlighted the protective effects of Nrf2 activation in reducing oxidative stress in both in vitro and in vivo models of neurodegenerative disorders. Here we provide an overview of the involvement of ROS-induced oxidative damage in Alzheimer's disease, Parkinson's disease, and Huntington's disease and we discuss the potential therapeutic effects of antioxidant enzymes and compounds that activate the Nrf2-ARE pathway.     

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse

Abstract

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H₂O₂ accumulation), are presented. Although in most instances accumulation of H₂O₂ affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.     

Antioxidant enzyme responses of plants to heavy metal stress

Heavy metal pollutions caused by natural processes or anthropological activities such as metal industries, mining, mineral fertilizers, pesticides and others pose serious environmental problems in present days. Evidently there is an urgent need of efficient remediation techniques that can tackle problems of such extent, especially in polluted soil and water resources. Phytoremediation is one such approach that devices effective and affordable ways of engaging suitable plants to cleanse the nature. Excessive accumulation of metal in plant tissues are known to cause oxidative stress. These, in turn differentially affect other plant processes that lead to loss of cellular homeostasis resulting in adverse affects on their growth and development apart from others. Plants have limited mechanisms of stress avoidance and require flexible means of adaptation to changing. A common feature to combat stress factors is synchronized function of antioxidant enzymes that helps alleviating cellular damage by limiting reactive oxygen species (ROS). Although, ROS are inevitable byproducts from essential aerobic metabolisms, these are needed under sub-lethal levels for normal plant growth. Understanding the interplay between oxidative stress in plants and role of antioxidant enzymes can result in developing plants that can overcome oxidative stress with the expression of antioxidant enzymes. These mechanisms have been proving to have immense potential for remediating these metals through the process of phytoremediation. The aim of this review is to assemble our current understandings of role of antioxidant enzymes of plants subjected to heavy metal stress.     

Semecarpus anacardium nut extract promotes the antioxidant defence system and inhibits anaerobic metabolism during development of lymphoma

Antioxidants are substances that fight against ROS (reactive oxygen species) and protect the cells from their damaging effects. Production of ROS during cellular metabolism is balanced by their removal by antioxidants. Any condition leading to increased levels of ROS results in oxidative stress, which promotes a large number of human diseases, including cancer. Therefore antioxidants may be regarded as potential anticarcinogens, as they may slow down or prevent development of cancer by reducing oxidative stress. Fruits and vegetables are rich source of antioxidants. Moreover, a number of phytochemicals present in medicinal plants are known to possess antioxidant activity. Therefore the aim of the present study was to investigate antioxidant activity of the aqueous extract of nuts of the medicinal plant Semecarpus anacardium in AKR mouse liver during the development of lymphoma. Antioxidant action was monitored by the activities of antioxidant enzymes catalase, superoxide dismutase and glutathione transferase. The effect of S. anacardium was also studied by observing the activity of LDH (lactate dehydrogenase), an enzyme of anaerobic metabolism. LDH activity serves as a tumour marker. The activities of antioxidant enzymes decreased gradually as lymphoma developed in mouse. However, LDH activity increased progressively. Administration of the aqueous extract of S. anacardium to lymphoma-transplanted mouse led to an increase in the activities of antioxidant enzymes, whereas LDH activity decreased significantly, indicating a decrease in carcinogenesis. The aqueous extract was found to be more effective than doxorubicin, a classical anticarcinogenic drug, with respect to its action on antioxidant enzymes and LDH in the liver of mice with developing lymphomas.     

Arsenic induced oxidative stress in plants

Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in the plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues adversely affects the plant metabolism and leads to various physiological and structural disorders. Photosynthetic apparatus, cell division machinery, energy production, and redox status are the major section of plant system that are badly affected by As (V). Similarly As (III) can react with thiol (-SH) groups of enzymes and inhibits various metabolic processes. Arsenic is also known to induce oxidative stress directly by generating reactive oxygen species (ROS) during conversion of its valence forms or indirectly by inactivating antioxidant molecules through binding with their -SH groups. As-mediated oxidative stress causes cellular, molecular and physiological disturbances in various plant species. Activation of enzymatic antioxidants namely, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), Glutathione s-transferase, glutathione peroxidase (GPX) as well as non antioxidant compounds such as, ascorbate, glutathione, carotenoids are reported to neutralize arsenic mediated oxidative stress. Understanding of biochemistry of arsenic toxicity would be beneficial for the development of arsenic tolerant crops and other economically important plants.     

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.     

Ellagic acid inhibits PKC signaling by improving antioxidant defense system in murine T cell lymphoma

Antioxidants protect the cells from the damaging effects of reactive oxygen species (ROS). Production of ROS during cellular metabolism is balanced by their removal by antioxidants. Any condition leading to increased levels of ROS results in oxidative stress, which participates in multistage carcinogenesis by causing oxidative DNA damage, mutations in the proto-oncogenes and tumor suppressor genes. Antioxidant defense system is required to overcome the process of carcinogenesis generated by ROS. Antioxidant enzymes are major contributors to endogenous antioxidant defense system. Protein kinase C (PKC) is generally involved in cell proliferation and its over expression leads to abnormal tumor growth. Out of three classes of PKC, classical PKC is mainly involved in cell proliferation and tumor growth. Classical PKC initiates signaling pathway and leads to activation of a number of downstream protein via activation of NF-κB. Therefore any agent which can promotes the endogenous antioxidant defense system should be able to down regulate PKC and NF-κB activation and thus may be useful in reducing cancer progression. To investigate this hypothesis we have tested the effect of antioxidant ellagic acid on antioxidant enzymes and PKC signaling in Dalton’s lymphoma bearing (DL) mice. DL mice were treated with three different doses of ellagic acid. The treatment significantly increases the activity and expression of antioxidant enzymes and down regulates the expression of classical isozymes of PKC as well as the activation of NF-κB, indicating that ellagic acid improves antioxidant defense system and PKC signaling via NF-κB which may contribute to its cancer preventive role.     

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

Abstract

γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.