Toxic Electrophiles Research Articles

Pro-Oxidant Activity of Flavonoids Induces EpRE-Mediated Gene Expression

Flavonoids are important bioactive dietary compounds. They induce electrophile-responsive element (EpRE)-mediated expression of enzymes, such as NAD(P)H-quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs), which are major defense enzymes against electrophilic toxicants and oxidative stress. The induction of EpRE-mediated gene transcription involves the release of the transcription factor Nrf2 from a complex with Keap1, either by a direct interaction of the inducer with Keap1 or by protein kinase C (PKC)-mediated phosphorylation of Nrf2. The inhibition of PKC in Hepa1c1c7 cells, stably transfected with human NQO1-EpRE-controlled luciferase revealed that PKC is not involved in flavonoid-induced EpRE-mediated gene transcription. However, the ability of flavonoids to activate an EpRE-mediated response correlates with their redox properties characterized by quantum mechanical calculations. Flavonoids with a higher intrinsic potential to generate oxidative stress and redox cycling are the most potent inducers of EpRE-mediated gene expression. Modulation of the intracellular glutathione (GSH) level showed that the EpRE-activation by flavonoids increased with decreasing GSH and vice versa, supporting an oxidative mechanism. In conclusion, the pro-oxidant activity of flavonoids can contribute to their health-promoting activity by inducing important detoxifying enzymes, pointing to a beneficial effect of a supposed toxic chemical reaction.

Increased glutathione content in yeastSaccharomyces cerevisiae exposed to NaCl

Glutathione is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. Since an overlap between osmotic and oxidative stress response is already know, the aim of the work was to determine the role of glutathione in yeast stress response to NaCl. YeastSaccharomyces cerevisiae ZIM 2155 was exposed to NaCl in concentration of 1–8% (w/v). Measuring cell cultivability showed a significant decrease appeared in cell exposed to 5–8% NaCl for 1 h. Cultivable cells were about 50% of control. Increased production of reactive oxygen species in cells exposed to 6, 7 and 8% NaCl for 1 h (1.3-fold, 1.9-fold and 2.8-fold increase, respectively) led to elevated glutathione content in reduce d form (119.1%, 122.6%, 141.5%, respectively). Two hours from NaCl addition intracellular oxidant level was slightly elevated compared to 1-h exposure, while glutathione content in reduced form was almost the same. We demonstrated that glutathione plays an important role in yeast stress response to NaCl.

The course of CCl 4 induced hepatotoxicity is altered in mGSTA4-4 null (−/−) mice

Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the α-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the α-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl 4) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl 4 in GSTA4-4 null (−/−) and wild type (+/+) mice. The results show that administration of a single dose of CCl 4 (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both −/− and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in −/− mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of −/− mice. As expected −/− mice did not show any expression of mGSTA4-4; after CCl 4 a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of −/− mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl 4 hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1–6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.

In vivo relevance of two critical levels for NAD(P)H:quinone oxidoreductase (NQO1)-mediated cellular protection against electrophile toxicity found in vitro

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose–response curve revealing a ‘lower protection threshold’ of 0.5 μmol DCPIP/min/mg protein and an ‘upper protection threshold’ at 1 μmol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I 3C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed ‘lower protection threshold’ in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this ‘lower protection threshold’ will require 5–20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.

Facile Access to Isotopically Labelled Valylleucyl Anilides as Biomarkers for the Quantification of Hemoglobin Adducts to Toxic Electrophiles

AbstractAn easy and efficient synthesis of valylleucyl anilide (HValLeuNHPh) and labelled HVal(13C5,15N)LeuNHPh has been developed. Derivatization of these substances with oxirane, acrylonitrile, epichlorohydrin, glyceraldehyde and other aldehydes gives a series of reference substances and internal standards for the quantitative evaluation of human exposure to toxic electrophiles by quantitative determination of their hemoglobin adducts. Coupling of N‐Z‐N‐Me‐Val(13C5,15N)OH with HLeuNHPh followed by hydrogenolysis affords N–Me–Val(13C5,15N)LeuNHPh for quantification of the exposure to methylating agents. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Survey of albumin purification methods for the analysis of albumin-organic toxicant adducts by liquid chromatography-electrospray ionization-tandem mass spectrometry

HSA has been shown to react with many organic toxicants to form adducts that are useful biomarkers for exposure. Albumin isolation is an important first step for the analysis of these protein-toxicant adducts. We tested several approaches to isolate albumin from serum treated with an electrophilic organic toxicant known to form adducts with albumin, i.e., sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), in order to evaluate these techniques as purification methods. To select the most efficient isolation strategy, methods were evaluated using gel electrophoresis, total protein quantitation, and peptide-adduct identification by MS. Results suggest that the albumin-rich fractions obtained can be used to identify exposure by quantitating the albumin adducts to electrophilic organic toxicants such as HD. The HiTrap Blue HP albumin isolation system appears to display the most promising results for purifying albumin to detect HD-adducts, exhibiting high purification efficiency, satisfactory albumin recovery, promising specificity, and a higher loading capacity for serum.

Retinoid X Receptor α Regulates the Expression of Glutathione S-transferase Genes and Modulates Acetaminophen-Glutathione Conjugation in Mouse Liver

Nuclear receptors, including constitutive androstane receptor, pregnane X receptor, and retinoid X receptor (RXR), modulate acetaminophen (APAP)-induced hepatotoxicity by regulating the expression of phase I cytochrome P450 (P450) genes. It has not been fully resolved, however, whether they regulate APAP detoxification at the phase II level. The aim of the current study was to evaluate the role of RXRalpha in phase II enzyme-mediated detoxification of APAP. Wild-type and hepatocyte-specific RXRalpha knockout mice were treated with a toxic dose of APAP (500 mg/kg i.p.). Mutant mice were protected from APAP-induced hepatotoxicity, even though basal liver glutathione (GSH) levels were significantly lower in mutant mice compared with those of wild-type mice. High-performance liquid chromatography analysis of APAP metabolites revealed significantly greater levels of APAP-GSH conjugates in livers and bile of mutant mice compared with those of wild-type mice. Furthermore, hepatocyte RXRalpha deficiency altered the gene expression profile of the glutathione S-transferase (Gst) family. Basal expression of 13 of 15 Gst genes studied was altered in hepatocyte-specific RXRalpha-deficient mice. This probably led to enhanced APAP-GSH conjugation and reduced accumulation of N-acetyl-p-benzoquinone imine, a toxic electrophile that is produced by biotransformation of APAP by phase I P450 enzymes. In conclusion, the data presented in this study define an RXRalpha-Gst regulatory network that controls APAP-GSH conjugation. This report reveals a potential novel strategy to enhance the detoxification of APAP or other xenobiotics by manipulating Gst activity through RXRalpha-mediated pathways.

A Study of Glutathione S-transferase pi Expression in Central Nervous System of Subjects with Amyotrophic Lateral Sclerosis Using RNA Extraction from Formalin-Fixed, Paraffin-Embedded Material

The expression of glutathione S-transferase pi (GST pi), an enzyme responsible for inactivation of a large variety of toxic compounds was studied in spinal cord, motor and sensory brain cortex obtained from patients who died in the course of amyotrophic lateral sclerosis (ALS). The studies were performed on formalin-fixed, paraffin-embedded (FFPE) and freshly frozen tissues. The method of RNA isolation from FFPE was modified. A significant decrease of GST pi-mRNA expression was found in cervical spinal cord and motor brain cortex of ALS subjects comparing to analogue control tissues (P<0.01), as well as in motor cortex of ALS subjects comparing to their sensory cortex (P<0.05). In spinal cords the decrease in GST pi-mRNA expression was accompanied by a decrease of GST pi protein level. Results indicated lowered GST pi expression on both mRNA and protein levels in the regions of nervous system affected by ALS. The non-properly inactivated by GST toxic electrophiles and organic peroxides may thus contribute to motor neurons damage.

Effect of Ribose Cysteine Pretreatment on Hepatic and Renal Acetaminophen Metabolite Formation and Glutathione Depletion

Ribose cysteine (2(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid) protects against acetaminophen-induced hepatic and renal toxicity. The mechanism for this protection is not known, but may involve inactivation of the toxic electrophile via enhancement of glutathione (GSH) biosynthesis. Therefore, the goal of this study was to determine if GSH biosynthesis was required for the ribose cysteine protection. Male CD-1 mice were injected with either acetaminophen or acetaminophen and ribose cysteine. The ribose cysteine cotreatment antagonized the acetaminophen-induced depletion of non-protein sulfhydryls in liver as well as GSH in kidney. Moreover, ribose cysteine cotreatment significantly increased the concentration of acetaminophen-cysteine, hepatic acetaminophen-mercapturate in liver and renal acetaminophen-GSH metabolites in kidney 4 hr after acetaminophen. To determine whether protection against acetaminophen-induced liver and kidney damage involved ribose cysteine dependent GSH biosynthesis, buthionine sulfoximine was used to selectively block gamma-glutamylcysteine synthetase (gamma-GCS). Plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen from mice pretreated with buthionine sulfoximine and challenged with acetaminophen indicated that both liver and kidney injury had occurred. While co-treatment with ribose cysteine had previously protected against acetaminophen-induced liver and kidney injury, it did not diminish the acetaminophen-induced damage to either organ in the buthionine sulfoximine-treated mice. In conclusion, ribose cysteine serves as a cysteine prodrug that facilitates GSH biosynthesis and protects against acetaminophen-induced target organ toxicity.

Effect of Ribose Cysteine Pretreatment on Hepatic and Renal Acetaminophen Metabolite Formation and Glutathione Depletion

Abstract: Ribose cysteine (2(R,S)‐D‐ribo‐(1′,2′,3′,4′‐tetrahydroxybutyl)thiazolidine‐4(R)‐carboxylic acid) protects against acetaminophen‐induced hepatic and renal toxicity. The mechanism for this protection is not known, but may involve inactivation of the toxic electrophile via enhancement of glutathione (GSH) biosynthesis. Therefore, the goal of this study was to determine if GSH biosynthesis was required for the ribose cysteine protection. Male CD‐1 mice were injected with either acetaminophen or acetaminophen and ribose cysteine. The ribose cysteine cotreatment antagonized the acetaminophen‐induced depletion of non‐protein sulfhydryls in liver as well as GSH in kidney. Moreover, ribose cysteine cotreatment significantly increased the concentration of acetaminophen‐cysteine, hepatic acetaminophen‐mercapturate in liver and renal acetaminophen‐GSH metabolites in kidney 4 hr after acetaminophen. To determine whether protection against acetaminophen‐induced liver and kidney damage involved ribose cysteine dependent GSH biosynthesis, buthionine sulfoximine was used to selectively block γ‐glutamylcysteine synthetase (γ‐GCS). Plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen from mice pretreated with buthionine sulfoximine and challenged with acetaminophen indicated that both liver and kidney injury had occurred. While co‐treatment with ribose cysteine had previously protected against acetaminophen‐induced liver and kidney injury, it did not diminish the acetaminophen‐induced damage to either organ in the buthionine sulfoximine‐treated mice. In conclusion, ribose cysteine serves as a cysteine prodrug that facilitates GSH biosynthesis and protects against acetaminophen‐induced target organ toxicity.

Regulatory Role of the COX-2 Pathway in the Nrf2-Mediated Anti-Inflammatory Response

Antioxidant responsive element (ARE) mediated gene expression is a pivotal cellular defense mechanism against the toxicity of electrophiles and reactive oxygen species (ROS). Nrf2 belongs to the Cap-N-Collar family of basic region-leucine zipper transcription factors and has emerged as an essential signal transducer that responds to electrophiles and ROS by regulating transcription via the ARE. The ARE-regulated gene battery consists of detoxification enzymes and antioxidant genes and confers protection against oxidative insults such as hyperoxic lung injury and benzo[a]pyrene-induced tumorigenesis in the forestomach. Recent investigations indicated that the ARE gene battery also plays significant roles in the regulation of inflammation. Indeed, many natural or synthetic substances activating the ARE-mediated transcription pathway inhibit pro-inflammatory gene expression. On the other hand, cyclooxygenase-2 (COX-2) exerts anti-inflammatory roles in the protection against inflammation by certain stimuli. Downstream effectors of the COX-2 pathway include cyclopentenone prostaglandins (cyPGs) such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). We recently demonstrated that the Nrf2 pathway is activated by endogenous 15d-PGJ2 during carrageenin-induced inflammation. Thus, cyPGs may act as physiological regulators of Nrf2.

Genetic combining ability of glucoraphanin level and other horticultural traits of broccoli

Broccoli (Brassica oleracea L., Italica Group) is a source of glucosinolates and their respective isothiocyanate metabolites that are believed to have chemoprotective properties in humans. Glucoraphanin (4-methylsulfinyl-butyl glucosinolate) is a predominant glucosinolate of broccoli. Its cognate isothiocyanate, sulforaphane, has proven a potent inducer of phase II detoxification enzymes that protect cells against carcinogens and toxic electrophiles. Little is known about the genetic combining ability for glucosinolate levels or the types of genetic variation (i.e., additive vs. dominance) that influence those levels in broccoli. In this study, a diallel mating design was employed in two field experiments to estimate combining abilities for glucoraphanin content. The diallel population was developed by crossing nine doubled-haploid (inbred) parents in all possible combinations (36), excluding the reciprocals. Horticultural traits of all entries were assessed on a plot basis. In fall 2001, glucoraphanin concentration of broccoli heads ranged from 0.83 to 6.00 μmol/gdw, and in spring 2002, ranged from 0.26 to 7.82 μmol/gdw. In both years, significant general combining ability was observed for glucoraphanin concentration and total head content, days from transplant to harvest, head weight, and stem diameter. Conversely, no significant specific combining ability was observed for any trait in either year. Results indicate that a given inbred will combine with others to make hybrids with relatively predictable levels of head glucoraphanin as well as, other important horticultural traits. This should allow identification of inbreds that typically contribute high glucoraphanin levels when hybridized with others.

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain.

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain.

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.

Role of Nicotinamide Quinone Oxidoreductase 1 (NQO1) in Protection against Toxicity of Electrophiles and Reactive Oxygen Intermediates

This chapter elaborates the role of nicotinamide quinone oxidoreductase 1 (NQO1) in protection against toxicity of electrophiles and reactive oxygen intermediates. NQO1 is a broadly distributed FAD-dependent flavoprotein that catalyzes the reduction of a wide variety of quinones, quinone imines, nitroaromatics, and azo dyes. Its notable characteristics include: equal efficiency of NADH and NADPH as electron donors; an obligatory two-electron reduction mechanism without semiquinone intermediates; potent inhibition by dicumarol and similar anticoagulants which has been a valuable tool for assessing the functional role of this enzyme; and induction by a plethora of chemically diverse inducers that activate the regulatory antioxidant response element (ARE) of the NQO1 gene. The physiological functions of NQO1 are described in this chapter. Evidences are presented for the fact that NQO1 protects against electrophile toxicity, oxidative stress, and neoplasia. Regulation of NQO1 by antioxidant response elements (ARE) and consequences of disruption of the NQO1 Gene are discussed in the chapter. The chapter explores the epidemiological links of NQO1 polymorphism to the risk of developing cancer.

Induction of NAD(P)H:quinone Reductase by Probucol: A Possible Mechanism for Protection against Chemical Carcinogenesis and Toxicity

Dietary antioxidants protect laboratory animals against induction of tumours by a variety of chemical carcinogens. Among possible mechanism, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes, including quinone reductase and glutathione S-transferase (GSH transferase). Probucol is used cholesterol-lowering drug used in the clinic, with pronounced antioxidant effect that protect against chemical carcinogenesis and toxicity. In the present study we therefore examined the ability of probucol to induce activities of quinone reductase in the cytosolic fractions of various tissues of mice. Quinone reductase activity was increased significantly in 6 of 8 tissues examined from probucol-fed mice. The greatest proportionate increase, to 1.8 times control levels, was observed in liver. Probucol also increased quinone reductase activities of forestomach, heart, kidney, lungs and spleen. Quinone reductase is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against toxicity of quinones. It is induced in many tissues coordinately with other enzymes that protect against electrophilic toxicity. The protective effects of probucol appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the non-oxidative metabolism of a wide variety of xenobiotics. The induction of such enzyme, quinone reductase by probucol suggests the potential value of this compound as a protective agent against chemical carcinogenesis and other forms of electrophilic toxicity. The significance of these results can be implicated in relation to cancer chemopreventive effects of probucol in various target organs.

Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: possible role in protection against chemical carcinogenesis and toxicity.

Dietary antioxidants protect laboratory animals against the induction of tumours by a variety of chemical carcinogens. Among possible mechanism of protection against chemical carcinogenesis could be mediated via-antioxidant-dependent induction of detoxifying enzymes. Curcumin, a yellow pigment from Curcuma longa, is a major component of turmeric and is commonly used as a spice and food colouring material and exhibits antiinflammatory antitumour, and antioxidant properties. In this study we therefore investigated the effect of dietary supplementation of curcumin on the activities of antioxidant and phase II-metabolizing enzymes involved in detoxification, and production of reactive oxygen species were quantified in ddY male mice. Dietary supplementation of curcumin (2%, w/v) to male ddY mice for 30 days significantly increased the activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and catalase to 189%, 179%, 189%, and 181% in liver and 143%, 134%, 167% and 115% in kidney respectively as compared with corresponding normal diet fed control (P<0.05-0.001). Parallel to these changes, curcumin feeding to mice also resulted in a considerable enhancement in the activity of phase II-metabolizing enzymes viz. glutathione S-transferase and quinone reductase to 1.7 and 1.8 times in liver and 1.1 and 1.3 times in kidney respectively as compared with corresponding normal diet fed control (P<0.05-0.01). In general, the increase in activities of antioxidant and phase II-metabolizing enzymes was more pronounced in liver as compared to kidney. The induction of such detoxifying enzymes by curcumin suggest the potential value of this compound as protective agent against chemical carcinogenesis and other forms of electrophilic toxicity. The significance of these results can be implicated in relation to cancer chemopreventive effects of curcumin against the induction of tumours in various target organs.

Tumor necrosis factor-alpha inhibits glutathione S-transferase-alpha expression in cultured porcine Sertoli cells.

Glutathione S-transferases (GSTs) are a family of soluble enzymes of detoxification that use reduced glutathione in conjugation and reduction reactions. Toxic electrophiles are substrates for the GSTs. GSTalpha is expressed at high levels in different tIssues such as the testis. Among the different GSTs present in the testis, GSTalpha is specifically expressed in Leydig and Sertoli cells known to be under the control of hormonal and local regulatory factors. The present study investigated the regulatory action of tumor necrosis factor-alpha (TNFalpha) on basal and hormone (FSH and testosterone)-stimulated GSTalpha expression in cultured Sertoli cells. Treatment with TNFalpha (0-20 ng/ml, 48 h) induced a decrease in basal GSTalpha mRNA levels in a dose-dependent manner (fivefold decrease; P<0.001). The maximal and half maximal effects were observed at 20 ng/ml and 7 ng/ml respectively. The inhibitory effect of TNFalpha was also time-dependent with a maximal inhibitory effect (threefold decrease; P<0.001) observed at 48 h. The inhibitory effect of the cytokine was also observed on basal GSTalpha protein (28 kDa) levels. TNFalpha also inhibited the hormone-stimulated GSTalpha expression in Sertoli cells. The treatment of cultured Sertoli cells with both FSH and TNFalpha (100 ng/ml and 10 ng/ml respectively, 48 h) resulted in a complete suppression of the stimulatory action of FSH on GSTalpha mRNA levels. Similarly, in Sertoli cells treated with testosterone or its non-aromatizable metabolite dihydrotestosterone (100 ng/ml, 24 h), TNFalpha reduced the hormone-stimulated GSTalpha mRNA and protein levels. TNFalpha inhibited basal GSTalpha expression without affecting mRNA stability. Indeed, the decay curves (mRNA half-life time=18 h) for the GSTalpha basal mRNA levels in Sertoli cells was similar in the absence or presence of TNFalpha (10 ng/ml, 48 h). Testosterone increased GSTalpha mRNA without affecting the enzyme mRNA stability. TNFalpha antagonized the androgen-stimulated GSTalpha mRNA levels without affecting the enzyme mRNA stability, suggesting that the interaction between the androgen and the cytokine is mostly exerted at a transcriptional level. FSH increased GSTalpha mRNA levels through an increase in mRNA stability (increased mRNA half-life times to 119 h). TNFalpha antagonized the stimulatory effect of FSH on GSTalpha mRNA levels by antagonizing the stabilizing effect exerted by the hormone on GSTalpha mRNA. Together, these results suggest that the increase in the cytokine levels within the testis would alter the detoxification processes against genotoxic products during spermatogenesis.

Toxic dose of a simple phenolic antioxidant, protocatechuic acid, attenuates the glutathione level in ICR mouse liver and kidney.

It has previously been reported that a toxic dose of protocatechuic acid (PA), a naturally occurring simple phenolic antioxidant in dietary plant foodstuff, has a potential to enhance tumorigenesis and induce contact hypersensitivity in mouse skin. In this study, the modifying effect of a toxic dose of PA on the glutathione (GSH) level in mouse liver and kidney was examined. Intraperitoneal administration of PA (500 mg/kg) caused significant hepatic and nephrotic GSH depletion. Interestingly, slight but significant hepatotoxicity and nephrotoxicity, characterized by the enhancement of plasmic alanine aminotrasferase (ALT) activity and urea level, respectively, were also observed. The subchronic administration of PA (0.1% in drinking water) for 60 days showed not only a significant decrease in the GSH level in kidney but also a significant enhancement of ALT activity in plasma. The protective role of GSH for acute hepatotoxicity using GSH-depleted mice administered a GSH synthesis inhibitor buthionine sulfoximine was also demonstrated. Thus, it is suggested that overdoses of PA can disturb the detoxification of other electrophilic toxicants including ultimate carcinogens.

Gonadotropin regulation of glutathione synthesis in the rat ovary

Glutathione (GSH), an antioxidant and conjugator of electrophilic toxicants, prevents toxicant-mediated destruction of ovarian follicles and oocytes. Ovarian GSH has previously been shown to change with estrous cycle stage in rats, suggesting that the gonadotropin hormones may regulate ovarian GSH synthesis. The present studies tested the hypotheses that [1] estrous cycle-related changes in ovarian GSH result from cyclic changes in protein and mRNA expression of the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase (GCL, also called γ-glutamylcysteine synthetase), and [2] that these changes result from gonadotropin-mediated regulation of GCL subunit expression. In the first experiment, ovaries were harvested from cycling adult female rats on each stage of the estrous cycle. In the second experiment immature female rats were injected with pregnant mare’s serum gonadotropin (PMSG) to stimulate follicular development or with vehicle and killed 8, 24, or 48 h later. In both experiments the ovaries were harvested for [1] total GSH assay, [2] Western analysis for GCL catalytic (GCLc) and regulatory (GCLm) subunit protein levels, or [3] Northern analysis for Gclc and Gclm mRNA levels. Ovarian GSH concentrations and Gclc and Gclm mRNA levels, but not GCL subunit protein levels, varied significantly with estrous cycle stage. PMSG administration significantly increased ovarian GSH concentrations 24 and 48 h later. GCLm protein levels increased significantly at 24h and 48h following PMSG. GCLc protein levels did not increase significantly following PMSG. Gcl subunit mRNA levels were not significantly increased at any time point by the planned ANOVA; however, an increase in Gclc at 48 h was identified by t-testing. These results support the hypothesis that gonadotropins regulate ovarian GSH synthesis by modulating GCL subunit expression.