Effect Of Sodium Citrate And Reduced Glutathione (GSH) On Arginase From Human Fibriod Tissue

1.0 Introduction

1.1 Background Of The Study

Glutathione (GSH) is a water-soluble tripeptide composed of the amino acids glutamine, cysteine, and glycine. The thiol group is a potent reducing agent, rendering GSH the most abundant intracellular small molecule thiol, reaching millimolar concentrations in some tissues. As an important antioxidant, GSH plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione S-transferases (GST) and glutathione peroxidases (GPx). The importance of GSH is evident by the widespread utility in plants, mammals, fungi and some prokaryotic organisms [1]. In addition to detoxification, GSH plays a role in other cellular reactions, including, the glyoxalase system, reduction of ribonucleotides to deoxyribonucleotides, regulation of protein and gene expression via thiol:disulfide exchange reactions [2].

GSH is synthesized de novo from the amino acids glycine, cysteine and glutamic acid. Synthesis of GSH requires the consecutive action of two enzymes, γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase [3], (Fig. 1). γ-GCS is a heterodimer composed of a catalytically active heavy subunit γ-GCS-HS (73 kDa) and a regulatory subunit, γ-GCS- LS (30 kDa) [4,5]. The regulation of γ-GCS is complex. Induction of γ-GCS expression has been demonstrated in response to diverse stimuli in a cell specific manner. The bioavailability of cysteine is rate limiting for the synthesis of GSH. Cysteine and the oxidized form of the amino acid, cystine, are transported into the cell via sodium dependent and independent transporters, respectively [6]. Oxidants (including hyperoxide, H2O2 and electrophilic compounds) promote cystine uptake and a concomitant increase in expression of γ-GCS. The γ-GCS promotor region contains a putative AP-1 binding site, an antioxidant response element (ARE), and an electrophile responsive element [7–9]. The AP-1 site is critical to constitutive expression of the γ-GCS-HS subunit [7]. Post-translational modifications of γ-GCS also influence GSH synthesis [10,11]. Specifically, phosphorylation of γ-GCS leads to the inhibition of GSH synthesis. GSH itself regulates the activity of γ- GCS via a negative feedback mechanism [3]. Hence, GSH depletion increases the rate of GSH synthesis.

The formation of excessive amounts of reactive O2 species (ROS), including peroxide (H2O2) and superoxide anions (O −•) is toxic to the cell. Hence, metabolizing and scavenging systems to remove them are functionally critical and tightly controlled in the cell. GSH peroxidase (GPx) in concert with catalase and superoxide dismutase (SOD) function to protect the cell from damage due to ROS. GPx detoxifies peroxides with GSH acting as an electron donor in the reduction reaction, producing GSSG as an end product. The reduction of GSSG is catalyzed by GSH reductase (GR) in a process that requires NADPH. GR is a member of the flavoprotein disulfide oxidoreductase family and exists as a dimer [2]. Under conditions of oxidative stress, GR is regulated at the level of transcription as well as by posttranslational modifications. Alterations in GR expression and activity have been implicated in cancer and aging [2].

GPx is an 80 kDa protein that is composed of four identical subunits. Five distinct GPx isozymes have been characterized in mammals, (Table 1) [12]. While GPx’s are ubiquitously expressed, individual isoforms are tissue specific. GPx expression is induced by oxidative stress and aberrant expression of GPx’s has been associated with a wide variety of pathologies, including hepatitis [13], HIV [14], and a wide variety of cancers, including skin [15], kidney [16], bowel [17] and breast [18].

1.2 Statement Of Problem

During the life cycle, aerobic organisms are exposed to a number of endogenous and exogenous sources of reactive oxygen species (ROS). To achieve redox homeostasis they developed a powerful antioxidant system (AOS) [1], whose main enzyme components are superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and detoxification enzymes [2,3], as well as processes such as stress response, proliferation, and proteasomal degradation [4,5]. Nrf2 was also identified as an important transcription factor regulating development, progression, and chemoresistance of cancer [6].

Cancer develops over three stages, initiation, promotion, and progression, and oxidative stress is associated with each of them [7]. Endometrial cancer ranks as the fourth most common neoplasm in women from European countries [8]. It has been shown that premalignant changes precede the malignant transformation of the uterus, which is why hyperplasia may be considered as a precursor of endometrial cancer [9–11]. Despite numerous studies, the molecular processes involved in multi-stage development of endometrial cancer are still not completely known.
We have previously shown that gonadotropins influence the antioxidant enzyme (AOE) activity in women with uterine hyperplasia simplex, hyperplasia complex, leiomyoma, and polyps [12–15].

Also, the extent of AOE and lipid hydroperoxides (LOOH) alterations varied with the examined gynecological diagnosis, including adenocarcinoma [16]. The aim of this study was to further clarify the mechanism responsible for the observed AO alterations. Therefore, we examined the changes of protein and mRNA levels of copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and Nrf2 in uterine tissue of patients with benign (polyps and myomas), premalignant (hyperplasia simplex and hyperplasia complex), and malignant (adenocarcinoma) transformations. We also examined correlations of AOE expression with the AOE activity, lipid hydroperoxides (LOOH) level, and level of Nrf2. Results showed significant differences in AO parameters among examined groups of gynecological patients, and indicated their important role in pathophysiological processes in endometrium.

The tripeptide can exist intracellularly in either an oxidized (GSSG) or reduced (GSH) state. Maintaining optimal GSH:GSSG ratios in the cell is critical to survival, hence, tight regulation of the system is imperative. A deficiency of GSH puts the cell at risk for oxidative damage. It is not surprising that an imbalance of GSH is observed in a wide range of pathologies, including, cancer, neurodegenerative disorders, cystic fibrosis (CF), HIV and aging. The role of GSH in disorders will be discussed in this study AS well as the effect on arginase from human fibriod tissue.

1.3 Research Objectives

The objective of this study is to investigate the effect of sodium citrate and reduced glutathione (GSH) on arginase from human fibroid tissue.