Reactive oxygen species,antioxidant mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant treatment

Objective. So far, it is not well established whether oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. Serum levels of proinflammatory cytokines and IL‐2 were also investigated. All these parameters were studied in relation to the clinically most important index of disease progression, namely Performance Status (ECOG PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity, and the reduction of serum levels of IL‐6 and TNF. Design, setting and subjects. We carried out an open non randomized study on 28 advanced stage cancer patients (stage III, 10.7%, and stage IV, 89.3%) with tumours at different (8) sites: all were hospitalized in the Medical Oncology Dept, University of Cagliari Interventions. The patients were divided into 5 groups and a different antioxidant treatment was administered to each group. The selected antioxidants were: alpha lipoic acid 200 mg/day orally, N‐acetylcysteine 1800 mg/day i.v. or carboxycysteine‐lysine salt 2.7 g/day orally, amifostine 375 mg/day i.v., reduced glutathione 600 mg/day i.v., vitamin A 30000 IU/day orally plus vitamin E 70 mg/day orally plus Vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. Results. Our results show that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels and 2 of them (cysteine‐containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the “antioxidant treatment” was found to have an effect both on reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced serum levels of IL‐6 and TNF. Patients in both ECOG PS 0‐1 and ECOG PS 2‐3 responded to antioxidant treatment.     

Obesity,Aerobic Exercise,and Vascular Disease: The Role of Oxidant Stress

Oxidant formation in the vasculature contributes to vascular disease and dysfunction associated with obesity. In contrast, exercise‐dependent production of oxidants may stimulate adaptive responses that protect against the development of such diseases. In this review, we discuss current concepts in the biology of reactive oxygen and nitrogen species and how their function is modulated in the context of vascular disease, obesity, and aerobic exercise.     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.     

Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context

While the chemical nature of reactive oxygen species (ROS) dictates that they are potentially harmful to cells, recent genetic evidence suggests that in planta purely physicochemical damage may be much more limited than previously thought. The most potentially deleterious effect of ROS under most conditions is that at high concentrations they trigger genetically programmed cell suicide events. Moreover, because plants use ROS as second messengers in signal transduction cascades in processes as diverse as mitosis, tropisms and cell death, their accumulation is crucial to plant development as well as defence. Direct ROS signal transduction will ensue only if ROS escape destruction by antioxidants or are otherwise consumed in a ROS cascade. Thus, the major low molecular weight antioxidants determine the specificity of the signal. They are also themselves signal-transducing molecules that can either signal independently or further transmit ROS signals. The moment has come to re-evaluate the concept of oxidative stress. In contrast to this pejorative or negative term, implying a state to be avoided, we propose that the syndrome would be more usefully described as 'oxidative signalling', that is, an important and critical function associated with the mechanisms by which plant cells sense the environment and make appropriate adjustments to gene expression, metabolism and physiology.     

Maternal antioxidant concentrations after uncomplicated pregnancies

Background: To analyse the post-partum concentrations of intra- and extra-cellular blood antioxidants in women with uncomplicated pregnancies.

Methods: Whole blood and plasma thiols, plasma vitamin E and C, serum cholesterol and triglyceride, ferric reducing ability of plasma (FRAP) concentrations were compared between women delivered by caesarean section (n=17) or spontaneous delivery (n=10). A repeated mixed model was used for statistical analysis.

Results: The majority of whole blood thiols increased significantly in both groups the first days post-partum. However, within the caesarean group free cysteine, oxidised cysteine, homocysteine and glutathione and plasma cysteine and homocysteine levels dropped significantly after 24?h, while FRAP levels peaked significantly in this group. Plasma vitamin E levels decreased significantly in both groups within 24 to 48?h after delivery. Independent of the way of delivery whole blood and plasma thiols were significantly increased and vitamin E levels were significantly decreased 3?months post-partum while plasma vitamin C levels and FRAP were unchanged compared to ante-partum levels.

Discussion: Decreased plasma vitamin E levels shortly post-partum are associated with decreased lipid peroxidation. The 24?h post-partum drop of some plasma and whole blood thiols in the caesarean group may be due to prolonged fasting.     

Maternal antioxidant concentrations after uncomplicated pregnancies

Background: To analyse the post-partum concentrations of intra- and extra-cellular blood antioxidants in women with uncomplicated pregnancies.

Methods: Whole blood and plasma thiols, plasma vitamin E and C, serum cholesterol and triglyceride, ferric reducing ability of plasma (FRAP) concentrations were compared between women delivered by caesarean section (n=17) or spontaneous delivery (n=10). A repeated mixed model was used for statistical analysis.

Results: The majority of whole blood thiols increased significantly in both groups the first days post-partum. However, within the caesarean group free cysteine, oxidised cysteine, homocysteine and glutathione and plasma cysteine and homocysteine levels dropped significantly after 24 h, while FRAP levels peaked significantly in this group. Plasma vitamin E levels decreased significantly in both groups within 24 to 48 h after delivery. Independent of the way of delivery whole blood and plasma thiols were significantly increased and vitamin E levels were significantly decreased 3 months post-partum while plasma vitamin C levels and FRAP were unchanged compared to ante-partum levels.

Discussion: Decreased plasma vitamin E levels shortly post-partum are associated with decreased lipid peroxidation. The 24 h post-partum drop of some plasma and whole blood thiols in the caesarean group may be due to prolonged fasting.     

Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines

Recent evidence suggests that reactive oxygen species (ROS) play an important role in the pathogenesis of various illnesses, and the ROS and antioxidant enzymes are highly associated with cell differentiation and diseases. In this study, we tested the hypothesis that specific antioxidant enzymes are differentially expressed in hepatocellular carcinoma (HCC) cell lines with various degrees of differentiation. We compared the expression of several antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GRx), and glutathione peroxidase (GPx) in five HCC cell lines with well (Hep G2 and Hep 3B) or poor (HA22T/VGH, HA55T/VGH, and SK-Hep-1) differentiation. Our results showed that both well-differentiated HCC cell lines expressed extremely higher CAT and GRx enzyme activities than all three poorly differentiated ones. Moreover, the protein and mRNA levels of CAT were much higher in two well-differentiated HCC cell lines than in all three poorly differentiated ones. Both well-differentiated HCC cell lines also showed a higher protein or mRNA expression of Cu/ZnSOD and MnSOD than three poorly differentiated ones. Our results demonstrate that specific antioxidant enzymes (especially, CAT and GRx) are differentially expressed in HCC cell lines with well or poor differentiation. These findings suggest that CAT and GRx are two potential differentiation markers for HCC.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise

Hydrogen peroxide (H₂O₂) could induce oxidative damage at long distance from its generation site and it is also an important signalling molecule that induces some genes related to oxidative stress. Our objective was to study the plasma and blood cells capability to detoxify H₂O₂ after intense exercise and its correlation with oxidative damage. Blood samples were taken from nine professional cycling, participating in a mountain stage, under basal conditions and 3 h after the competition. Catalase and glutathione peroxidase activities decreased (40 and 50% respectively) in neutrophils after the cycling stage, while glutathione peroxidase increased (87%) in lymphocytes. Catalase protein levels and catalase specific activity maintained basal values after the stage in plasma. Catalase protein levels decreased (48%) in neutrophils and its specific activity increased up to plasma values after exercise. Myeloperoxidase (MPO) increased (39%) in neutrophils after the cycling stage. Exercise-induced hemolysis and lymphopenia inversely correlated with cellular markers of oxidative stress. Plasma malondialdehyde (MDA) directly correlated with neutrophil MPO activity and erythrocytes MDA. Intense exercise induces oxidative damage in blood cells as erythrocytes and lymphocytes, but not in neutrophils.     

Interaction of metals with peroxidase--mediated luminol-enhanced, chemiluminescence (PLmCL).

The peroxidase-mediated luminol-enhanced chemiluminescence (PLmCL) method has been used to study the in vitro effect of contaminants such as heavy metals on the reactive oxygen species production by immunocytes. We were interested to know whether metals could directly affect peroxidase-mediated luminescence, taking horseradish peroxidase (HRP) as a model enzyme, since this could contribute to the inhibition of immunocyte LmCL. Copper inhibited PLmCL in a dose-dependent manner, while cadmium, iron, silver and lead only partly decreased the signal in the concentration range tested. In contrast, zinc enhanced the signal at high concentrations. Eventually, chromium, mercury and aluminium did not affect PLmCL. It is suggested that these effects reflect the ability of the metals to interact with the active site of the peroxidase. These results demonstrate that such interactions have to be considered when interpreting the effects of metals on immunocytes using the LmCL method.     

Effects of post-injury hypothermia and nerve growth factor infusion on antioxidant enzyme activity in the rat: implications for clinical therapies

The pathological sequelae of traumatic brain injury (TBI) include increased oxidative stress due to the production of reactive oxygen species (ROS). Regulation of ROS levels following TBI is determined primarily by antioxidant enzyme activity that in turn can be influenced by nerve growth factor (NGF). Hypothermia is one of the current therapies designed to combat the deleterious effects of TBI. However, it has been shown to suppress post-trauma increases in NGF levels in rat brain. The present study sought to determine whether post-injury hypothermia also impairs the antioxidant response to injury, and if such an effect could be reversed by infusion of exogenous NGF. We employed a lateral controlled cortical impact injury model in rat, followed by moderate hypothermia treatment with supplemental intracerebroventricular infusion of NGF or vehicle. The time course of changes in post-injury/intervention levels of NGF and activity of three major enzymes responsible for ROS scavenging, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), was determined in the hippocampus. Relative to levels in injured, normothermic animals, hypothermia treatment not only suppressed NGF levels, but also attenuated CAT and GPx activity, and increased SOD activity. Infusion of NGF in injured, hypothermia-treated animals was ineffective in restoring hippocampal antioxidant enzymes activity to levels produced after injury under normothermic conditions, although it was able to increase septal cholinergic (choline acetyltransferase) enzyme activity. These results have implications for clinical treatment of TBI, demonstrating that moderate hypothermia suppresses NGF and the antioxidant response after TBI; the latter cannot be countered by exogenous NGF administration.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Evidence that peroxiredoxins are novel members of the thioredoxin fold superfamily.

Peroxiredoxins catalyze reduction of hydrogen peroxide or alkyl peroxide, to water or the corresponding alcohol. Detailed analysis of their sequences indicates that these enzymes possess a thioredoxin (Trx)-like fold and consequently are homologues of both thioredoxin and glutathione peroxidase (GPx). Sequence- and structure-based multiple sequence alignments indicate that the peroxiredoxin active site cysteine and GPx active site selenocysteine are structurally equivalent. Homologous peroxiredoxin and GPx enzymes are predicted to catalyze equivalent reactions via similar reaction intermediates.     

The changes in the antioxidant status of heart during experimental hypomagnesemia in balb/c mice

The present experiment was performed to assess if hypomagnesemia can influence antioxidant status in mice heart. The results could explain possibly a free radical theory of heart damage in magnesium deficiency. We used a rodent model of hypomagnesemia. The magnesium sufficient group received a standard diet whereas a magnesium deficient group received the diet containing a trace amount of magnesium. The activities of the most important antioxidant enzymes – catalase, glutathione peroxidase and superoxide dismutase were assessed in mice heart and liver in a time dependent manner, on the 10th and the 20th day of experiment. The level of magnesium in plasma of animals receiving the magnesium deficient diet dropped twice after the 8th day and four times after the 13th day and then reached a plateau value. The activity of catalase in heart in the magnesium deficient group increased gradually and was significantly (P<0.05) elevated by 27% on the 20th day of experiment whereas the superoxide dismutase activity was significantly decreased by 17% on the 20th day. Glutathione peroxidase activity was insignificantly elevated. The alterations of antioxidant enzyme activities in the heart indicate cardiomyocytes's exposure to oxidative stress, which can be responsible for the cardiac lesions observed during hypomagnesemia.     

Differential effects of vitamin E and three hydrophilic antioxidants on the actinomycin D-induced and colcemid-accelerated apoptosis in human leukemia CMK-7 cell line

The actinomycin D (AD)-induced apoptosis in human leukemia CMK-7 cell line is greatly accelerated by microtubule disruption with colcemid (CL). We studied the effect of antioxidants on this apoptosis in order to learn how the universal signal mediators, reactive oxygen species (ROS), are involved. Caspase-3 activation and DNA fragmentation were both suppressed by vitamin E (VE), t-butylhydroxyanisole, and luteolin. The ROS formation in the AD treatment was evidenced by flow cytometry, and further supported by suppression of caspase-3 activation by superoxide radical-forming enzyme inhibitors (TTFA, rotenone, and DPI). The inhibition of apoptosis by VE was completed during the initial 1-h treatment with AD, but it did not appear when VE was added with CL to washed cells after AD treatment. Luteolin, an iron chelator PDTC, and a water-soluble VE analogue, trolox, inhibited the apoptosis when added with CL after the AD treatment. Western blot analysis showed that the proteolytic cleavage of procaspase-9 and procaspase-3 were both inhibited when VE was added with AD or when luteolin was added with CL, and that the cytochrome c liberation was suppressed by both antioxidants. This result implies that the ROS are initially formed in lipophilic environments (e.g. mitochondrial membrane) and then they diffuse into an aqueous environment (i.e. cytoplasm) where they promote the apoptotic process in combination with the cytoskeletal disruption. Thus, the different antioxidants are effective to scavenge ROS for preventing the apoptosis in its different phases.     

A comparative study on effect of dietary selenium and vitamin E on some antioxidant enzyme activities of liver and brain tissues

Since selenium and vitamin E have been increasingly recognized as an essential element in biology and medicine, current research activities in the field of human medicine and nutrition are devoted to the possibilities of using these antioxidants for the prevention or treatment of many diseases. The present study was aimed at investigating and comparing the effects of dietary antioxidants on glutathione reductase and glutathione peroxidase activities as well as free and protein-bound sulfhydryl contents of rat liver and brain tissues. For 12–14 wk, both sex of weanling rats were fed a standardized selenium-deficient and vitamin E-deficient diet, a selenium-excess diet, or a control diet. It is observed that glutathione reductase and glutathione peroxidase activities of both tissues of the rats fed with a selenium-deficient or excess diet were significantly lower than the values of the control group. It is also shown that free and bound sulfhydryl concentrations of these tissues of both experimental groups were significantly lower than the control group. The percentage of glutathione reductase and glutathione peroxidase activities of the deficient group with respect to the control were 50% and 47% in liver and 66% and 61% in the brain, respectively; while these values in excess group were 51% and 69% in liver and 55% and 80% in brain, respectively. Free sulfhydryl contents of the tissues in both experimental groups showed a parallel decrease. Furthermore, the decrease in protein-bound sulfhydryl values of brain tissues were more pronounced than the values found for liver. It seems that not only liver but also the brain is an important target organ to the alteration in antioxidant system through either a deficiency of both selenium and vitamin E or an excess of selenium alone in the diet.     

Glutathione peroxidase‐1 overexpression reduces oxidative stress,and improves pathology and proteome remodeling in the kidneys of old mice

This study investigated the direct roles of hydrogen peroxide (H₂O₂) in kidney aging using transgenic mice overexpressing glutathione peroxidase‐1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H₂O₂ by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti‐aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label‐free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.     

Interaction of exercise and adenosine receptor agonist and antagonist on rat heart antioxidant defense system

This study investigated the interactive effects of acute exercise and adenosine receptor agonist and antagonist on antioxidant enzyme activities, glutathione and lipid peroxidation in the heart of the rat. Male Fisher-344 rats were divided into six groups and treated as follows: (1) saline control; (2) acute exercise (100% VO_2max); (3) R-Phenyl isopropyl adenosine (R-PIA) (3.46 mol/kg, i.p.); (4) theophylline (1.70 mol/kg, i.p.) plus acute exercise; (5) theophylline plus R-PIA; and (6) theophylline. Animals were sacrificed 1 h after treatments; hearts were isolated and analyzed. The results show that acute exercise as well as adenosine receptor agonist R-PIA significantly enhanced cardiac superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) activity by 36–135% and 16–51%, respectively. Adenosine receptor agonist R-PIA significantly decreased cardiac GSSG concentration and enhanced GSH/GSSG ratio by 22 and 30%, respectively. Whereas theophylline treatment blocked the activation of antioxidant enzyme activities enhanced by acute exercise and R-PIA. Theophylline treatment significantly increased lipid peroxidation by 43% in the heart of exercised rats. The study concluded that the adenosine receptors are involved in the upregulation of cardiac antioxidant defense system and attenuation of lipid peroxidation due to acute exercise in rats. (Mol Cell Biochem 270: 209–214, 2005)     

The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger

Free radicals are by-products of metabolism and exist in a homeostasis between generation and scavenging in vivo. Excessive free radicals cause various diseases, including nervous system diseases. Neuroglobin (Ngb), a nervous system-specific oxygen-binding protein, has been suggested to be a potential free radical scavenger in the nervous system in vivo; however, its underlying mechanism remains unclear. In this study, we investigated the antioxidant potential and free radical scavenging properties of recombinant human Ngb (rhNgb) in vitro. Interestingly, we found that the rhNgb protein itself has a direct and distinct antioxidant capacity and can efficiently scavenge a variety of free radicals, including the [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] (ABTS) cation, superoxide anion, hydrogen peroxide, and hydroxyl radical. The capacity of rhNgb to scavenge the superoxide anion and hydrogen peroxide was even comparable to that of vitamin C. In addition, rhNgb had Fe(2+) chelating activity but hemoglobin did not. In conclusion, our results indicated that the rhNgb protein itself has antioxidant and free radical scavenging activities, providing fundamental evidence for the neuroprotective function of Ngb. These data provide key information for the origin of the neuroprotective and physiological role of Ngb and will promote the treatment of reactive oxygen species (ROS)-related diseases using this novel oxygen-binding globin.     

Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action

The present investigation focused, firstly, on the effects of oral administration of thymoquinone (TQ) on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase activity in hepatic, cardiac and kidney tissues of normal mice. Superoxide dismutase (SOD; E.C:1.15.1.1), catalase (CAT; E.C:1.11.1.6), glutathione peroxidase (GSH-Px; E.C:1.11.1.9), glutathione-S-transferase (GST; E.C:2.5.1.18), and DT-diaphorase (E.C:1.6.99.2) enzyme activities in each tissue type were determined. Treatment of mice with the different doses of TQ (25, 50 and 100 mg kg(-1) day(-1) orally) for 5 successive days, produced significant reductions in hepatic SOD, CAT and GSH-Px activities. In addition cardiac SOD activity was markedly inhibited with the higher doses of TQ, (namely 50 and 100 mg kg(-1)). Moreover, TQ (100 mg kg(-1)) significantly reduced hepatic and cardiac lipid peroxidation as compared with the respective control group. Conversely, TQ (50,100 mg kg(-1)) and TQ (100 mg kg(-1)) enhanced cardiac and renal DT-diaphorase activity respectively. However, the selected doses of TQ neither produced any change in GST activity nor influenced reduced glutathione content in all tissues studied. TQ was tested, secondly, as a substrate for hepatic, cardiac and renal DT-diaphorase of normal mice in the presence of NADPH. Kinetic parameters for the reduction of TQ to dihydrothymoquinone (DHTQ) indicated that DT-diaphorase of different tissues can efficiently reduce TQ to DHTQ. K(m) and V(max) values revealed that hepatic DT-diaphorase exhibited the higher values, while the lower values were associated with renal DT-diaphorase. TQ and DHTQ were tested, thirdly, as specific scavengers for superoxide anion (generated biochemically) or as general scavengers for free radicals (generated photochemically). The results revealed that TQ and DHTQ acted not only as superoxide anion scavengers but also as general free radical scavengers. The IC(50) for TQ and DHTQ in biochemical and photochemical assays were in the nanomolar and micromolar range respectively. Our data may explain at least partly the reported beneficial in vivo protective effects of TQ through the combined antioxidant properties of TQ and its metabolite DHTQ.