The chemical inducibility of mouse cardiac antioxidants and phase 2 enzymes in vivo

The recognition of the critical involvement of oxidative and electrophilic stress in cardiac disorders has led to extensive investigation of the protective effects of exogenous antioxidants on cardiac injury. On the other hand, another strategy for protecting against oxidative/electrophilic cardiac injury may be through induction of the endogenous antioxidants and phase 2 enzymes in myocardium by chemical inducers. However, our understanding of the chemical inducibility of cardiac antioxidants/phase 2 enzymes in vivo is very limited. In addition, careful studies on the basal levels of a scope of endogenous antioxidants/phase 2 enzymes in myocardium as compared with other tissues, such as liver, are lacking. Accordingly, this study was undertaken to determine the basal levels of endogenous antioxidants/phase 2 enzymes, including superoxide dismutase (SOD), catalase, reduced glutathione (GSH), GSH peroxidase (GPx), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), and investigate the inducibility of the above antioxidants/phase 2 enzymes by the chemoprotectant, 1,2-dithiole-3-thione (D3T), in cardiac as well as hepatic tissues in C57BL/6 mice. Our results demonstrated that in C57BL/6 mice, the levels of catalase, GSH, GPx, GR, and GST were significantly lower in cardiac tissue than in hepatic tissue. The level of total SOD did not differ significantly between mouse heart and liver. Notably, heart contained a much higher NQO1 activity than liver. Immunoblotting and RT-PCR analyses further demonstrated the high expression of NQO1 protein and mRNA in myocardium. Oral administration of D3T at 0.25 and 0.5 mmol/kg body weight for 3 consecutive days resulted in a significant induction of cardiac SOD, catalase, GR, GST, and NQO1. No significant induction of cardiac GSH and GPx was observed with the above D3T treatment. Only GR, GST, and NQO1 in mouse liver were induced by the D3T treatment. Unexpectedly, we observed a significant D3T dose-dependent decrease in hepatic GPx activity. Taken together, this study demonstrates for the first time that: (1) the expression of NQO1 is remarkably high in mouse myocardium though other cardiac antioxidants/phase 2 enzymes are relatively lower as compared with liver; (2) a number of endogenous antioxidants/phase 2 enzymes in mouse cardiac tissue can be significantly induced by D3T following oral administration; and (3) the inducibility of endogenous antioxidants/phase 2 enzymes by D3T differs between mouse cardiac and hepatic tissues. This study provides a basis for future investigation of the cardioprotection of chemically induced endogenous antioxidants and phase 2 enzymes in myocardium in animal models of oxidative/electrophilic cardiac disorders.     

The effect of excimer laser keratectomy on corneal glutathione-related enzymes in rabbits

Glutathione related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Excimer laser is a very useful tool for the treatment of refractive errors and removing superficial corneal opacities. Previous studies have shown that excimer laser may initiate free radical formation in the cornea. In the present study, we evaluated the effect of excimer laser keratectomy on corneal glutathione-related enzyme activities in rabbits. Animals were divided into five groups, and all groups were compared with the controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy (PRK) (group 3), traditional PRK (group 4) and deep traditional PRK (group 5). Corneal glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) activities were measured after 24h. Corneal GPx and GR activities significantly decreased only in group 5 (p < 0.05) but GST activities significantly decreased in all groups when compared with the control group (p < 0.05). In conclusion, excimer laser inhibits the glutathione dependent defense system in the cornea, this effect becomes more prominent after high doses of excimer laser energy and antioxidants may be useful to reduce free radical mediated complications.     

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.     

Transgenic tobacco plants overexpressing cotton glutathione S-transferase (GST) show enhanced resistance to methyl viologen

A GST (EC 2.5.1.18) gene (Gst-cr 1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants overexpressing Gst-cr1 were normal in growth and mature compared with control, but had much higher levels of GST and GPx activities and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (MV). Six antioxidant enzymes, glutathione S-transferase, glutathione peroxidase (EC 1.11.1.9), superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11) were monitored in transgenic lines and non-transgenic control during MV treatments. When they were treated with 0.03 mmol/L of MV, both transgenic lines and control showed a rapid increase in the activities of GST, GPx, SOD, POD, APx, while the activity of CAT seemed to be irregular. The percent of the increase in SOD and POD activities was much higher in control than in transgenic plants. When treated with 0.05 mmol/L of MV, both control and transgenic plants were severely damaged, and the activities of the six enzymes decreased sharply.     

Parameters related to oxygen free radicals in erythrocytes,plasma and epidermis of the hairless rat

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.     

Major differences among chemopreventive organoselenocompounds in the sustained elevation of cytoprotective genes

Cytoprotective enzyme elevation through the nuclear erythroid 2 p45‐related factor 2 (Nrf2)–Kelch‐like ECH‐associated protein 1/antioxidant response element pathway has been promulgated for cancer prevention. This study compares the redox insult and sustained cytoprotective enzyme elevation by organoselenocompounds and sulforaphane (SF) in lung cells. SF elicited a rise in reactive oxygen species (ROS) and drop in glutathione (GSH) at 2 h; nuclear accumulation of Nrf2 at 4 h; and a GSH rebound and elevation in NAD(P)H quinone oxidoreductase (NQO1), thioredoxin reductase (TR1), and glutamate–cysteine ligase (GCL) at 24 h. Selenocystine (SECY) elicited a similar 24 h response, despite lesser earlier time‐point changes. 2‐Cyclohexylselenazolidine‐4‐carboxylic acid effects were similar to SECY's but with a larger Nrf2 change and the largest 24 h increase in GSH, GCL, TR1, and NQO1 of any compound investigated. Selenomethionine elicited a similar acute rise in ROS, but lesser depletion of GSH, no 4 h increase in nuclear Nrf2, only minor 24 h elevations in TR1 and NQO1, and a GCL elevation insufficient to elevate GSH. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:344–353, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21417     

Induction of NAD(P)H quinone oxidoreductase and glutathione S-transferase activities in livers of female August-Copenhagen Irish rats treated chronically with estradiol: comparison with the Sprague-Dawley rat

Estradiol (E2) has been linked to both, protection against damage associated with chronic diseases or exposure to chemicals, and to the incidence of cancer. In its protective role, E2 appears to attenuate oxidative stress while as a carcinogen, E2 damages macromolecules via formation of reactive catechol metabolites. Alterations in the expression of antioxidant and xenobiotic metabolizing enzymes upon administration of pharmacological doses of E2 have been previously identified, but the effect of chronic exposure to low concentrations of E2 on activities of those enzymes in liver is unclear. The August-Copenhagen Irish (ACI) rat is more sensitive to estrogen-induced carcinogenesis than the Sprague-Dawley rat. Accordingly, the effect of treatment of female ACI and Sprague-Dawley rats for 6 weeks with E2 on activities of NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase, glutathione S-transferase (GST), phenol sulfotransferase (SULT1A1), cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) was studied. Basal expression of these enzymes was similar in livers from both strains prior to exposure to E2. However, only NQO1 and GST activity was increased (3- and 2.5-fold, respectively) in liver cytosol of ACI rats treated with E2. In contrast, only NQO1 activity was increased modestly in livers of Sprague-Dawley rats. Other enzymes were not significantly affected in the livers of ACI or Sprague-Dawley rats following chronic treatment with E2. The selective induction of NQO1 and GST activity suggests that under physiological conditions, E2 may protect against oxidative stress via elevation of these antioxidant enzymes. The marked induction of NQO1 and GST in the ACI rat indicates a potential for this strain to be used as a model to study the E2-mediated modulation of these enzymes in tissues that are either sensitive to E2 carcinogenesis or to its protective effects.     

Protective effect of <Emphasis Type="Italic">Spirulina</Emphasis> against 4-nitroquinoline-1-oxide induced toxicity

In the present study, we focused on the protective effect of Spirulina against 4-nitroquinoline-1-oxide (4NQO) induced hepato and nephrotoxicity in the experimental rats. The 4NQO administration resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. It also increased the oxidative stress resulting in increased levels of the lipid peroxidation with a concomitant decline in the levels of non enzymic [reduced glutathione (GSH)] and enzymic antioxidants [(Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney. Oral pretreatment with aqueous extract of Spirulina prevented 4NQO induced changes in the levels of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney. The antioxidant responsiveness mediated by Spirulina may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning and provide a scientific rationale for the use of Spirulina.     

Mitochondrial antioxidant defence in radio-resistantLepidopteran insect cells

Cells isolated from Lepidopteran insects (butterfly and moths) display very high radioresistance as compared to mammals and other insect species. Since free radical induced mitochondrial damage under stress conditions is very crucial for cellular fate determination, antioxidant system is the major protective modality required to minimize stress-induced damage and to modulate cellular sensitivity. In this study, we predict the mitochondrial localization potential and co-existence of important antioxidant enzymes in insect cells and compare with other radiosensitive (mammals, Dipteran insects) and radioresistant (nematodes) species. Our study clearly demonstrates the inter-species variation in then localization potential of various antioxidant enzymes. A higher mitochondrial localization potential as a function of mitoprot score was evident for all important antioxidant enzymes in the lepidopteran insect Bombyx mori (Mn-SOD, 0.694; GPx, 0.862; TRPx, 0.997; TR, 0.9), besides an unusual mitochondrial localization prediction for catalase (0.453). We further found coexistence of glutathione and thioredoxin system in the mitochondria of lepidopteran insects as also reported in various plant species. On the basis of above observations, we hypothesize that a strong mitochondrial antioxidant enzyme system including the unusual coexistence of catalase, glutathione and thioredoxin system may help minimize the free radical mediated damage to mitochondria and can contribute to the intrinsic radioresistance of lepidopteran insects.     

Activities of conjugating and antioxidant enzymes following endotoxin exposure

Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S‐transferase (GST), and UDP‐glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague–Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S‐transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin‐treated animals. Changes in the conjugative enzymes and the free‐radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 63–69, 1999     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes

Activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glucose-6-phophate dehydrogenase (G6PDH) were measured in four tissues of goldfish, Carassius auratus L., over 1-12 h of high temperature (35 degrees C) exposure followed by 4 or 24 h of lower temperature (21 degrees C) recovery. SOD activity was strongly affected by heat shock, increasing 4-fold in brain, liver, and kidney, but was mainly reversed at recovery. In some tissues, activities of SOD, catalase, GPx, and G6PDH decreased significantly after 1 h heat shock exposure suggesting that thermal inactivation possibly occurred, but were renewed at further exposure. In many cases, 4 h of return to the initial temperature decreased enzyme activities. High correlation coefficients between SOD activities and levels of lipid peroxidation products suggest that these products might be involved in up-regulation of antioxidant defense. Several enzymes (SOD, GST, GR) responded to stress in coordinated manner.     

Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.     

The effect of ovarian hormones on antioxidant enzyme activities in the brain of male rats

The brain is widely responsive to gonadal hormones. The functional significance of ovarian hormones in the brain is evident from biochemical studies indicating that estradiol or progesterone treatment of testectomized rats produces changes of antioxidant enzyme activities. The effect of estradiol benzoate (EB) and progesterone (P) in the control of antioxidant (AO) enzyme activities was studied in the brain of adult male Wistar rats. The activities of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and glutathione reductase (GR) were measured in appropriate subcellular fractions, prepared from brains of animals belonging to various experimental groups. These groups were designed with the intention to follow changes in enzyme activities 2 h or 24 h after systemic administration of 5 microg EB or 2 mg P to testectomized (TX) animals. The obtained results show that both EB and P increase CAT activity, whereas EB decreases GSH-Px, GST and GR activities. These findings clearly show the modulatory role of EB and P in the control of enzymes responsible for the protection of rat nerve cells against oxidative damage caused by free oxygen radicals.     

Induction of drug metabolizing enzymes by 1,7‐phenanthroline and oltipraz in mice is unrelated to Ah‐responsiveness

The protective effect of several classes of compounds against the toxic and neoplastic effects of xenobiotics has been attributed to the induction of noncytochrome P450 (P450) drug metabolizing enzymes. Glutathione S‐transferases (GST), NAD(P)H:quinone oxidoreductase (QOR), and UDP‐glucuronosyltransferases (UGT) play a prominent role in detoxification and can be induced by oltipraz and other N‐heterocyclic compounds in rats. In contrast to the induction of these enzymes by aryl hydrocarbon (Ah)‐receptor agonists, induction by oltipraz and 1,7‐phenanthroline is not accompanied by CYP1A induction. This study investigated the induction of drug metabolizing enzymes following administration of oltipraz and 1,7‐phenanthroline in four mouse strains (C57B6A‐J, Frings × C57B6J, Frings, CF‐1) exhibiting varying degrees of responsiveness to an Ah‐receptor agonist. The relative Ah responsiveness was determined in all strains by the induction of hepatic Cyp1a after three daily doses of 3‐methylcholanthrene (20 mg/kg). After treatment with 1,7‐phenanthroline and oltipraz (150 mg/kg i.g.) daily for 3 days, all strains showed similar induction of GST and QOR activities for each inducer. Both compounds were equally effective in elevating GST activity, but 1,7‐phenanthroline was more effective than oltipraz in elevating QOR activity. In addition to GST and QOR changes, 1,7‐phenanthroline significantly elevated UGT (1‐naphthol) activity in the Frings strain. Neither compound produced significant changes in Cyp1a parameters. The independence of 1,7‐phenanthroline and oltipraz induction of GST and QOR from Cyp1a‐responsiveness is in line with the concept that N‐heterocycle‐containing inducers act by mechanisms other than an Ah‐receptor‐dependent pathway in which the P450 response has been masked or prevented. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 77‐82, 1999     

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.