Glutaminase isoenzymes as key regulators in metabolic and oxidative stress against cancer.

Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation. The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major nonenzymatic cellular antioxidant. Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2. As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment.

Anticancer antioxidant regulatory functions of phytochemicals.

Plant foods are not only a main source of nutrients, but they are also rich in physiologically bioactive bionutrients or phytochemicals. Consumption of fruit and vegetables is associated with a decreased risk of pathological status, including cancer. Reactive oxygen species play a key role in the genesis and development of cancer. Therefore, antioxidant functions of phytonutrients have been thoroughly investigated in the last years in relation to their crucial effect in the pathophysiology associated with neoplasia. This review discusses current knowledge on phytochemicals in relation to their potential as chemopreventive and/or chemotherapeutic molecule against human cancers. Finally, we will outline the use of bioactive phytochemicals on synergistic actions involved in the prevention and treatment of cancer as well as its future prospects.

Natural antioxidants: therapeutic prospects for cancer and neurological diseases.

An all out war is continuously occurring between oxidants and antioxidants inside the cells. This mini-review will provide an updated revision of the function of some natural compounds having main roles in antioxidant function. We will point on some phytochemicals working at two outstanding targets, tumour cells and neurons.

Anti-oxidant enzyme activities and expression and oxidative damage in patients with non-immediate reactions to drugs.

Adverse drug reactions with an immunological basis (ADRIB) may involve activation of other concomitant, non-specific mechanisms, amplifying the specific response and contributing to the severity and duration. One concomitant mechanism could be the generation of reactive oxygen species (ROS) and/or their detoxification by anti-oxidants, including anti-oxidant enzymes. We analysed the activity of the anti-oxidant enzymes Cu/Zn-superoxide dismutase (SOD), catalase (CAT) and cellular glutathione peroxidase (GPX), as well as certain markers of oxidative damage (thiobarbituric acid reactive substances (TBARS) and carbonyl content) in peripheral blood mononuclear cells from patients with non-immediate ADRIB using spectrophotometric methods and the anti-oxidant enzymes expression by quantitative real-time reverse transcription-polymerase chain reaction. SOD activity and expression were increased in all types of non-immediate reactions (urticaria, maculopapular exanthema and toxic epidermal necrolysis). Regarding oxidative damage, TBARS were increased in urticaria and maculopapular exanthema, and carbonyl groups in all types of reactions. Our observations indicate that oxidative damage occurs in non-immediate reactions. Carbonyl stress and the inadequacy of the anti-oxidant defences are probable causes.

Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology.

Reactive Oxygen Species (ROS) are produced during normal cellular function. ROS include hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide. They are very transient species due to their high chemical reactivity that leads to lipid peroxidation and oxidation of DNA and proteins. Under normal conditions, antioxidant systems of the cell minimize the perturbations caused by ROS. When ROS generation is increased to an extent that overcomes the cellular antioxidants, the result is oxidative stress. It is now clear that several biological molecules, which are involved in cell signaling and gene regulation systems are very sensitive to redox statue of the cell. Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. The various antioxidants exert their effect by scavenging superoxide, or by activating of a battery of detoxifying/defensive proteins. The prevention of oxidation is an essential process in all the aerobic organisms, as decreased antioxidant protection may lead to cytotoxicity, mutagenicity and/or carcinogenicity. This article also focuses on the mechanisms by which antioxidants and xenobiotics induce the gene expression of detoxifying enzymes. On the other hand, small molecules that mimic antioxidant enzymes are becoming new tools for the treatment of many diseases.

Allergy to drugs: antioxidant enzymic activities, lipid peroxidation and protein oxidative damage in human blood.

Reactive oxygen species lead to lipid peroxidation and specific oxidation of some specific enzymes, proteins and other macromolecules, thus affecting many intra- and intercellular systems. Recently, antioxidant functions have been linked to anti-inflammatory properties. Cell defences against toxic oxygen include antioxidant enzymes. We studied the enzymic antioxidant capacity in human blood of both erythrocytes and mononuclear cells from patients suffering from an allergic reaction to different drugs. We determined superoxide dismutases (SODs), glutathione peroxidase (GSHPx) and catalase (CAT) activities in each cell type. We also determined the extent of thiobarbituric acid reactive substances (TBARS) and the oxidative damage to proteins, in order to study the correlation between the cellular enzymic activities, the oxidative status and the allergic reaction. In mononuclear cells from allergic patients, SODs and CAT activities were enhanced compared with controls. Conversely, a decrease in GSHPx activity was found. In erythrocytes, higher values for CAT, GSHPx and SODs activities were found in allergic patients. TBARS were also enhanced in both types of cells, and the carbonyl content of serum was equally increased. The respective enzymic imbalances in mononuclear cells and erythrocytes, namely, GSHPx/SOD and CAT/SOD, and their consequences are discussed. To our knowledge, this is the first global study of antioxidant enzyme determinations, including TBARS level and carbonyl content, in patients suffering from allergies to drugs.

Chemical and biological activity of free radical 'scavengers' in allergic diseases.

Reactive oxygen species (ROS) are generated constantly in vivo. They can lead to lipid peroxidation and oxidation of some enzymes, as well as protein oxidation and degradation. Cells possess several biological systems, defined as 'scavengers', to protect themselves from the radical-mediated damage. Immune cells may discharge their arsenal of toxic agents against host tissues, resulting in oxidative damage and inflammation. Therefore, free radical production and disturbance in redox status can modulate the expression of a variety of immune and inflammatory molecules, leading to inflammatory processes, both exacerbating inflammation and effecting tissue damage. Recently, abnormal immunity has been related to oxidative imbalance, and antioxidant functions are linked to anti-inflammatory and/or immunosuppressive properties. Currently, allergy is one of the most important human diseases. We studied the role of the primary antioxidant defence system, constituted by the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, protecting cells from toxic oxygen. We analyzed how they are involved in blood cells detoxification, and how the imbalance of reactive oxygen species is related to inflammation in allergic diseases by affecting immune cells. Finally, we discuss the published data that relates anti-free radical therapy to the management of human allergic diseases.

Role of reactive oxygen species in apoptosis: implications for cancer therapy.

Reactive oxygen species are widely generated in biological systems. Consequently humans have evolved antioxidant defence systems that limit their production. Intracellular production of active oxygen species such as *OH, O2- and H2O2 is associated with the arrest of cell proliferation. Similarly, generation of oxidative stress in response to various external stimuli has been implicated in the activation of transcription factors and to the triggering of apoptosis. Here we review how free radicals induce DNA sequence changes in the form of mutations. deletions, gene amplification and rearrangements. These alterations may result in the initiation of apoptosis signalling leading to cell death, or to the activation of several proto-oncogenes and or the inactivation of some tumour suppressor genes. The regulation of gene expression by means of oxidants, antioxidants and the redox state remains as a promising therapeutic approach. Several anticarcinogenic agents have been shown to inhibit reactive oxygen species production and oxidative DNA damage, inhibiting tumour promotion. In addition, recombinant vectors expressing radical-scavenging enzymes reduce apoptosis. In conclusion, oxidative stress has been implicated in both apoptosis and the pathogenesis of cancer providing contrived support for two notions: free radical reactions may be increased in malignant cells and oxidant scavenging systems may be useful in cancer therapy.

Antioxidant activity levels and oxidative stress as blood markers of allergic response to drugs.

Antioxidant enzymes work together in human blood cells against toxic reactive oxygen species. Although their relationship with several pathophysiologic processes has been stated, not much is known about the connection between antioxidant defence and allergy. This study was designed to determine the enzymatic activities and the oxidative indices in the blood and serum proteins in patients suffering from allergy to drugs. We hypothesize that serum and blood reactions may serve as useful clinical marker for the allergic state. We used enzymatic antioxidant activities, thiobarbituric acid reactive substances, and carbonyl contents of proteins as suitable markers. We determined superoxide dismutases, glutathione peroxidase and catalase activities in each cell type. After antihistaminics plus steroids were given as part of a protocol treatment, enzymatic antioxidant activities, thiobarbituric acid reactive substance levels, and carbonyl contents were used as recovering markers for the disease. We found a relationship between antioxidant enzymatic activities, thiobarbituric acid reactive substance levels, and carbonyl contents for allergic reactions belonging to several type I and type IV allergies, as well as cross-reactive intolerance to nonsteroidal anti-inflammatory drugs and an anaphylactoid reaction to a radiocontrast media. A similar pattern also exists for analogous allergic manifestations and disease-like status.

Antioxidant enzymatic activities in human blood cells after an allergic reaction to pollen or house dust mite.

Several diseases have been related to oxidative stress. Recently, antioxidant functions have also been linked to anti-inflammatory properties. Cell defenses against reactive oxygen species include antioxidant enzymes. We studied the enzymatic antioxidant capacity in human blood of both red blood and mononuclear cells from patients suffering from an allergic reaction to pollen or house dust mite. We determined superoxide dismutases (SODs), glutathione peroxidase (GSHPx) and catalase (CAT) activities in each cell type. We also determined the extent of thiobarbituric acid reactive substances (TBARS), in order to study the correlation between the cellular enzymatic activities, the redox status and the disease. In mononuclear cells from allergic patients, SODs and CAT activities were enhanced compared to controls. Conversely, a decrease in GSHPx activity was found. In erythrocytes, higher values for GSHPx and SODs and similar CAT activities were found in allergic patients and controls. Interestingly, CuZnSOD and MnSOD activities were enhanced in the same proportion for both, erythrocytes and mononuclear cells. TBARS were also enhanced in both types of cells. The respective enzymatic imbalances in mononuclear cells and erythrocytes, namely, GSHPx/SOD and CAT/SOD, and their consequences are discussed. To our knowledge, this is the first global study of antioxidant enzymes, including TBARS level determinations, in allergy.

Antioxidant enzymes and their implications in pathophysiologic processes.

Aerobic organisms possess antioxidant defense systems that deal with reactive oxygen species (ROS) produced as a consequence of aerobic respiration. Reactive oxygen is related to both, the arrest of growth and the start of cell differentiation. Low concentrations of reactive oxygen intermediates may be beneficial or even indispensable in processes such as intracellular messaging and defense against micro-organisms, but higher amounts of active oxygen may be harmful to cells and organisms. A wide array of non-enzymatic and enzymatic antioxidant defenses exists, including superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT). We describe their main characteristics and how these antioxidant enzymes work together against active oxygen. Small deviations from their physiological values may have a dramatic effect on the resistance of cells to oxidative damage to lipids, proteins and DNA. Consequently, toxic oxygen play a role in aging process as well as in a number of human diseases that we list in this review.

Antioxidant enzymes and human diseases.

OBJECTIVES: To describe the importance of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase working together in human cells against toxic reactive oxygen species, their relationship with several pathophysiologic processes and their possible therapeutic implications. CONCLUSIONS: Reactive oxygen species (ROS) are involved in the cell growth, differentiation, progression, and death. Low concentrations of ROS may be beneficial or even indispensable in processes such as intracellular signaling and defense against micro-organisms. Nevertheless, higher amounts of ROS play a role in the aging process as well as in a number of human disease states, including cancer, ischemia, and failures in immunity and endocrine functions. As a safeguard against the accumulation of ROS, several nonenzymatic and enzymatic antioxidant activities exist. Therefore, when oxidative stress arises as a consequence of a pathologic event, a defense system promotes the regulation and expression of these enzymes.

Structure/function relationship studies on the T/S residues 173-177 of rat ODC.

A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.

Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.

The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells.

The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in 'de novo' synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of 'de novo' protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC activity observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.

Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing in vivo.

The antihistaminic (+/-)-chlorpheniramine significantly reduced the progression of Ehrlich carcinoma when it was administered at 0.5 mg/mouse/day from the third day on, after tumour inoculation. The ODC activity of tumour cells was diminished by 70% on day 7 after tumour transplantation, when maximum ODC activity is detected in non-treated tumour growing 'in vivo'. Northern blot analyses indicated that the inhibitory effect of this 1,4-diamine takes place at a post-transcriptional level. Results obtained from serum-free cultured cells indicated that chlorpheniramine inhibits the ODC synthesis rate.

Simultaneous fluorometric determination of intracellular polyamines separated by reversed-phase high-performance liquid chromatography.

A reversed-phase HPLC technique in combination with fluorescent detection is described for simultaneous quantification of the precolumn Dansyl derivatives of intracellular amines. The derivatives were stable for at least one week, kept protected from the light at -20 degrees C. The detection limit was between 1 and 5 pmol for all tested polyamines. Serotonin coeluted with tryptamine. The method has a very good reproducibility for both, retention times and chromatographic peak areas. The average recovery of standard amine solutions added to cellular extracts was estimated to be higher than 90%. The described method enables a rapid, reliable and reproducible quantification of biogenic and related polyamines in biological fluids and tissues.

Diamines interfere with the transport of L-ornithine in Ehrlich-cell plasma-membrane vesicles.

1. L-Ornithine transport by plasma-membrane vesicles isolated from Ehrlich cells is Na(+)-independent and shows a saturable and a diffusional component. 2. Putrescine, histamine, 5-hydroxytryptamine and 2,3-diaminopropane at 55 microM concentration significantly inhibit 0.5 mM-L-ornithine transport at least for the first 10 min of incubation. 3. There is a trans-stimulatory effect of putrescine on L-ornithine transport.

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells.

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 microM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 microM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 microM N-acetyl putrescine, N-acetyl serotonin or N-omega-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistamic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55 microM 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N-N distance (6A) and the charge of one amino group are important chemical characteristics for regulatory effects.