Age and gender dependent levels of glutathione and glutathione S-transferases in human lymphocytes

Glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals including chemical carcinogens. Human cytosolic GSTs are divided into four major classes; alpha, mu, pi and theta. This study was performed to evaluate the influence of age and gender on the GST isoenzyme expression and glutathione (GSH) content in lymphocytes. Blood was collected from 124 healthy controls, which were divided into age groups of 20-40 years (21 females, 20 males), 40-60 years (20 females, 21 males) and 60-80 years (20 females, 22 males). Lymphocytes were isolated by density centrifugation on Histopaque-1077. After homogenization, cytosolic fractions were isolated. Herein, GST isoenzyme levels were determined by densitometrical analysis of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high performance liquid chromatography after conjugation with monobromobimane. Spearman rank correlation and Wilcoxon rank sum tests were used for statistical evaluation. Lymphocytic GSTmu and pi levels were not correlated with age or gender. GSTalpha was not detectable in lymphocytes. GSH contents were not different in males and females, but decreased with age in both males and females. In age group 60-80, GSH content was significantly lower as compared with age groups 20-40 and 40-60 in both sexes. Since high GSH is an essential factor in the detoxification of many compounds, these data indicate that the detoxification potential of the GSH/GST system in lymphocytes may decrease with age in man.     

Effects of the antiproliferative cyclopentenone prostaglandin A1 on glutathione metabolism in human cancer cells in culture

Homeostatic mechanisms for the maintenance of glutathione (GSH) are fundamental in the provision of a cellular defense against electrophilic/oxidant challenges. Cyclopentenone prostaglandins (CP-PGs) are powerful antiproliferative endogenous substances that may act as electrophilic regulating compounds, by virtue of the presence of an alpha,beta-unsaturated carbonyl group in the cyclopentane ring. Nevertheless, differential resistance to CP-PG cytotoxic/cytostatic effect has been reported in different cell types. It is reported that the activity/expression of gamma-glutamylcysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis) can be inducibly activated by electrophiles, including CP-PGs. The response of the human cancer strains HEp-2 (larynx carcinoma) and HL-60 (promyelocytic leukemia) cells to treatment with the CP-PG PGA1 in culture was investigated by evaluating the time-course of GSH synthesis and activity of enzymes of GSH metabolism, other than gamma-GCS, after PGA1 addition. HEp-2 cells, being more resistant to PGA1 cytotoxic and cytostatic effects, have basal GSH levels that were 2.4-fold higher than that of HL-60 cells. The activities of GSH S-transferase (GST), glutathione reductase (GSRd) and glutathione peroxidase (GSPx) are constitutively higher in HL-60 cells than in HEp-2 cells (respectively, 17.0-, 28.5- and 12.3-fold). When challenged with PGA1, both cell types exhibited a dose-dependent rise in GSH content that was maximal 18 h after PGA1 addition and was preceded by a rise in GST and GSRd activities in both cell types (at 12 h). GSPx activity increased only in HEp-2 (PGA1 evoked a 93.4%-inhibition in HL-60 cells). Moreover only HEp-2 cells exhibited early capacity to enhance GSH content (1-2 h just after PGA1 addition). These results and earlier data showing that leukemia cells are sensitive to CP-PG treatment suggest that deficiencies in GSH metabolism may be strategically in therapeutic approaches to the treatment of human leukemias.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.     

Mechanism of inhibition of benzo[a]pyrene-induced forestomach cancer in mice by dietary curcumin

Curcumin (diferuloylmethane), the major yellow pigment in turmeric, has been shown to inhibit benzo[a]pyrene (BaP)-induced forestomach cancer in mice through mechanism(s) not fully understood. It is well known that while cytochrome P4501A1 (CYP1A1) and epoxide hydrolase (EH) are important in the conversion of BaP to its activated form, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BaPDE], the detoxification of (+)-anti-BaPDE is accomplished by glutathione (GSH) S-transferases (GST). Therefore, it seems reasonable to postulate that curcumin may exert anti-carcinogenic activity either by inhibiting activation of BaP or (and) by enhancing the detoxification of (+)-anti-BaPDE. Administration p.o. of 2% curcumin in the diet to female A/J mice for 14 days, which has been shown to cause a significant inhibition in BaP-induced forestomach tumorigenesis, resulted in a modest but statistically significant reduction in hepatic ethoxyresorufin O-deethylase (EROD) activity, a reaction preferentially catalyzed by CYP1A1. While EROD activity could not be detected in the forestomach of either control or treated mice, curcumin feeding caused a statistically significant increase (approximately 2.3-fold) in hepatic EH and GST activities. Hepatic and forestomach GSH levels, and forestomach EH and GST activities were not affected by curcumin treatment. Even though the levels of various hepatic GST isoenzymes were significantly increased upon curcumin feeding, maximum induction was noticed for the pi class isoenzyme (mGSTP1-1), which among murine hepatic GSTs is highly efficient in the detoxification of (+)-anti-BaPDE. In conclusion, the results of the present study suggest that curcumin may inhibit BaP-induced forestomach cancer in mice by affecting both activation as well as inactivation pathways of BaP metabolism in the liver.     

Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress

Expression of the MRP1 gene encoding the GS-X pump and of the gamma-GCSh gene encoding the heavy (catalytic) subunit of the gamma-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like gamma-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1, 4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably gamma-GCSh-transfected cell lines down-regulated endogenous MRP1 and gamma-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and gamma-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and gamma-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and gamma-GCSh expression.     

Localization of glutathione S-transferases alpha and pi in human embryonic tissues at 8 weeks gestational age

Glutathione S-transferases (GST) are a family of enzymes involved in the detoxification of xenobiotics. In humans, GST are divided into four different classes, alpha, mu, pi and theta, with partly overlapping substrate specificity and a tissue-specific expression pattern. We studied the cellular distribution of GST alpha and pi in a variety of human embryonic organs obtained from an extra-uterine monozygotic twin pregnancy at 8 weeks' gestational age. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Three 4 microm thick sections were used, one for routine haematein and eosin staining, the others for immunohistochemical determination using monoclonal and polyclonal antibodies against GST alpha and pi, respectively. Both GST alpha and pi were present in hepatocytes, gastrointestinal epithelium, adrenal gland medulla, and tela chorioidea in the telencephalon. GST pi, but not alpha, was found in the epithelium of pancreatic and pulmonary glands, trachea, nephrons and urinary collecting ducts, as well as in the pia mater of the telencephalon and in developing nerve tissue in the gastrointestinal muscularis mucosae. In summary, we have demonstrated that immunoreactive protein for both GST alpha and pi is expressed in the human embryo at 8 weeks' gestational age. The early expression of GST alpha and pi in the epithelia of the urinary and digestive tracts and the respiratory system supports the importance of GST in the detoxification of potentially toxic or carcinogenic compounds. Our results suggest that the embryo itself is capable of detoxifying noxious compounds that are generated intracellularly or that cross the trophoblastic tissue.     

cis-diamminedichloroplatinum(ii) resistance in vitro and in vivo in human embryonal carcinoma cells

In the embryonal carcinoma cell line Tera and its 3.7-fold cis-diamminedichloroplatinum(II) (CDDP)-resistant subline, Tera-CP, parameters were studied that might have changed in relation to induction of CDDP resistance. Phenotypes of both lines were embryonal carcinoma. Karyotypes were related with a decreased mean number of chromosomes and fewer copies of the short arm of chromosome 12 in Tera-CP. Tera-CP showed cross-resistance for melphalan and 4-hydroperoxycyclophosphamide and had an 1.4-fold increased glutathione (GSH) level, a 1.5-fold increased glutathione S-transferase (GST) activity, and a 1.4-fold increased GST pi expression compared to Tera. Tera-CP was cross-resistant to 5-fluorouracil, but thymidylate synthase activity was not increased. Topoisomerase I and II activities and c-myc RNA and protein expression were the same in both lines. Platinum accumulation was equal in both lines, and platinum-DNA binding was lower in Tera-CP than in Tera. Both cell lines were xenografted into nude mice and tumors showed marked differentiation. Tera-CP tumors were 2.8-fold resistant to CDDP compared to Tera tumors. In new cell lines derived from xenografts of Tera and Tera-CP CDDP sensitivity, GST activity and GSH level corresponded with their sensitivity and resistant origin. Tera-CP is a model of in vitro and in vivo CDDP resistance with the GSH/GST detoxifying system as an important mechanism. CDDP resistance could be induced without a concomitant increase in differentiation.     

A homology model for rat mu class glutathione S-transferase 4-4

Glutathione S-transferases (GSTs) are an important class of phase II (de)toxifying enzymes, catalyzing the conjugation of glutathione (GSH) to electrophilic species. Recently, a number of cytosolic GSTs was crystallized. In the present study, molecular modeling techniques have been used to derive a three-dimensional homology model for rat GST 4-4 based upon the crystal structure of rat GST 3-3, both members of the mu class. GST 3-3 and GST 4-4 isoenzymes share a sequence homology of 88%. GST 4-4 distinguishes itself from GST 3-3 in being much more efficient and stereoselective in the nucleophilic addition of GSH to epoxides and alpha,beta-unsaturated ketones. GST 3-3, however, is much more efficient in catalyzing nucleophilic aromatic substitution reactions. In this study, several known substrates of GST 4-4 were selected and their GSH conjugates docked into the active site of GST 4-4. GSH conjugates of phenanthrene 9(S),10(R)-oxide and 4,5-diazaphenanthrene 9(S),10(R)-oxide were docked into the active site of both GST 3-3 and GST 4-4. From these homology modeling and docking data, the difference in stereoselectivity between GST 3-3 and GST 4-4 for the R- and S-configured carbons of the oxirane moiety could be rationalized. The data acquired from a recently derived small molecule model for GST 4-4 substrates were compared with the results of the present protein homology model of GST 4-4. The energy optimized positions of the conjugates in the protein model agreed very well with the original relative positions of the substrates within the substrate model, confirming the usefulness of small molecule models in the absence of structural protein data. The protein homology model, together with the substrate model, will be useful to further rationalize the substrate selectivity of GST 4-4, and to identify new potential GST 4-4 substrates.     

A subfamily 2 homo-dimeric glutathione S-transferase mYrs-mYrs of class theta in mouse liver cytosol

A homo-dimeric subfamily 2 glutathione (GSH) S-transferase (GST) mYrs-mYrs of the class theta was isolated from mouse liver cytosol and purified to homogeneity. The first 28 N-terminal amino acid sequence of the GST was completely identical to that of rat subfamily 2 GST Yrs-Yrs of the class theta. GST mYrs-mYrs cross-reacted with anti-rat GST Yrs-anti-sera but not with anti-sera raised against rat GSTs Ya-Ya (alpha), Yb1-Yb1 (mu), and Yp-Yp (pi) and represented more than 95% of the mouse liver cytosolic GST activity to scavenge the reactive sulfate ester 5-sulfoxymethylchrysene of the potent carcinogen 5-hydroxymethylchrysene. The mouse class theta GST had little activity toward 1-chloro-2,4-dinitrobenzene and was unretainable on GSH and an S-hexyl-GSH affinity columns. GST mYrs-mYrs had a much higher GSH peroxidase activity toward fatty acid hydroperoxides than did the other classes of mouse GSTs.     

Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.     

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.     

Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.     

Localization of neutral endopeptidase in the ovine uterus and conceptus during the oestrous cycle and early pregnancy

Allylamine (AA) is an electrophilic amine with a long history of experimental usage because of its extremely potent and relatively specific cardiovascular toxicity; it has been utilized in a variety of experimental models attempting to mimic human atherosclerotic lesions, myocardial infarction, and vascular injury. Even though the exact mechanisms by which AA causes vascular lesions remain unresolved, recent studies on the acute effects of AA exposure in rats strongly suggest that deamination to the aldehyde acrolein, oxidative stress, and the resultant increase in lipid peroxidation, generation of .OH radicals, and acute depletion of glutathione (GSH) may be some of the causative factors in AA-induced vascular lesions. Since glutathione S-transferase 8-8 (GST8-8) of rat belongs to a distinct subgroup of GST isozymes involved in the detoxification of products of lipid peroxidation, we designed studies to examine the effects of AA exposure on this GST isoform in rat aorta using Western blotting and immunohistochemical techniques. The results of these studies demonstrate that GST8-8 is expressed in rat aorta and is dramatically induced upon AA exposure. By immunohistochemistry, GST8-8 was localized in the smooth muscle cells of the vascular media which is believed to be the site of metabolism of AA. A significant increase in gamma-glutamylcysteine synthetase activity and GST activity toward 4-hydroxynonenal and acrolein, which are preferred substrates of GST8-8, was seen as early as 3 days following AA treatment. Alterations in GSH and other GSH-related enzymes at 3 and 10 days support the concept that--upon AA exposure--aortic defense mechanisms respond early and induction of GSH biosynthesis and rat GST8-8 occur to alleviate the toxic effects of acrolein, a major, genotoxic product of AA metabolism. The presence of GST8-8 in the vasculature, which is constantly exposed to products of lipid peroxidation, and its induction by AA, suggest that GST8-8 plays a key role in protecting blood vessels against oxidative stress and hence, may be involved in the atherogenic process.     

Rape flashbacks: constructing a new narrative

1. Radiotherapy has attracted increasing interest in recent years. It is known that ionizing radiation induces oxygen radical injury, whereas oxidative stress by the radiation can cause cellular responses to defense cellular injury. In this study, the metabolism of antioxidants in response to ionizing radiation to the brain was studied in the brain using experimental rabbits. 2. Ionizing radiation to the hemicerebrum caused an increase in the levels of glutathione (GSH) and the activity of a GSH synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Ionizing radiation also induced DNA-damage estimated by the formation of 8-hydroxydeoxyguanosine. These changes were dependent on the radiation dose. 3. Previous intrathecal-administration of buthionine sulfoximine (100 microM), a specific inhibitor of gamma-GCS, increased DNA damage by radiation in the radiated hemicerebrum. That of S-methyl GSH, on the other hand, resulted in a significant reduction of DNA damage by radiation. 4. These results suggest that synthesis of GSH and Cu,Zn-SOD is responsive to ionizing radiation and this induction of antioxidants may play a role in reducing tissue damage in radiotherapy.