Glutathione transferase omega 1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones

S-(Phenacyl)glutathione reductase (SPG-R) plays a significant role in the biotransformation of reactive alpha-haloketones to nontoxic acetophenones. Comparison of the apparent subunit size, amino acid composition, and catalysis of the reduction of S-(phenacyl)glutathiones indicated that a previously described rat SPG-R (Kitada, M., McLenithan, J. C., and Anders, M. W. (1985) J. Biol. Chem. 260, 11749-11754) is homologous to the omega-class glutathione transferase GSTO1-1. The available data show that the SPG-R reaction is catalyzed by GSTO1-1 and not by other GSTs, including the closely related GSTO2-2 isoenzyme. In the proposed reaction mechanism, the active-site cysteine residue of GSTO1-1 reacts with the S-(phenacyl)glutathione substrate to give an acetophenone and a mixed disulfide with the active-site cysteine; a second thiol substrate (e.g., glutathione or 2-mercaptoethanol) reacts with the active-site disulfide to regenerate the catalytically active enzyme and to form a mixed disulfide. A new spectrophotometric assay was developed that allows the rapid determination of SPG-R activity and specific measurement of GSTO1-1 in the presence of other GSTs. This is the first specific reaction attributed to GSTO1-1, and these results demonstrate the catalytic diversity of GSTO1-1, which, in addition to SPG-R activity, catalyzes the reduction of dehydroascorbate and monomethylarsonate(V) and also possesses thioltransferase and GST activity.     

Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn,Macrobrachium rosenbergii

Phthalate esters (PAEs) have been considered as environmental pollutants and have been subject to control in the United States of America and Japan. The aim of this study was to investigate the effects and toxicity of eight PAEs to hemocytes and the defense functions of giant freshwater prawn (Macrobrachium rosenbergii), including hemocytic adhesion, pseudopodia formation, phenoloxidase (PO) activity, and superoxide anion (O(2)(-)) production, by means of in vitro exposure experiments. After hemocytes were treated separately with eight PAEs at concentrations of 100 microg/ml, the results showed that two PAEs (dipropyl phthalate, DPrP and diethyl phthalate, DEP) increased cells with pseudopodia formation, but decreased adhesive cells; reduction in the percentages of both pseudopodia formation and adhesive cells were detected in the dihexyl phthalate (DHP) and diphenyl phthalate (DPP) experiment groups; and di-(2-ethyl hexyl) phthalate (DEHP) decreased pseudopodia formation, but did not affect the adhesion. In addition, both PO activity and O(2)(-) production were decreased after hemocytes were treated with five PAEs (benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), DEP, DHP and DPrP), respectively. At the same time, microscopy showed that both DPrP and DHP altered morphology of the cell nucleus and led to the presence of vacuoles in cytosol of hemocytes. Using the annexin assay, and after analysis of DNA fragmentation and transmission electron microscopy (TEM), it was found that hemocytes exposed to DHP and DPrP for more than 10 min would primarily die via apoptosis, the fatality correlates with increasing treatment time; and hemocytes treated with either BBP, dicyclohexyl phthalate (DCP), DEP or DPP would primarily die via necrosis. According to these results, we suggest that all eight PAEs examined could damage hemocytes and further influence the defense mechanism of prawns. This study reveals an important precaution for prawn cultivation.     

Tissue distribution and elimination kinetics of polybrominated biphenyls (PBB) from rat tissue

The concentration of polybrominated biphenyl (PBB) in serum in a large number of organs was determined in a population of rats for 36 weeks a single dose of PBB. Groups were killed at 6, 12, 24 and 36 weeks after exposure to PBB (1 mg/100g body wt, i.p.). Growth, weight gain and appearance of the rats and their internal organs were normal. Complex and varied relationships were found in tissue concentrations with time after PBB administration. Serum and fat had apparent first-order elimination kinetics with calculated half-times of 23.1 and 69.3 weeks, respectively. For five other tissues, apparent t 1/2s ranged from 9.0-63 weeks, while for four others, kinetics could not be determined from these 4 time points. It is likely that a substantial residue of PBB will still remain in the body of the rat at the end of its life span because of the persistence of PBB in lipid-rich tissues (adipose, adrenal, and brain).     

Ultrastructural changes during spermatogenesis,biochemical and hormonal evidences of testicular toxicity caused by TBT in freshwater prawn Macrobrachium rosenbergii (De Man, 1879)

The present investigation documents the impact of tributyltin (TBT) on the ultrastructural variation of spermatogenesis in freshwater prawn Macrobrachium rosenbergii. The environmentally realistic concentration of TBT can cause damages to the endocrine and reproductive physiology of crustaceans. In this context, three concentrations viz. 10, 100, and 1000 ng/L were selected and exposed to prawns for 90 days. The TBT exposed prawn exhibited decrease the reproductive activity as evidenced by sperm count and sperm length compared to control. Histopathological results revealed the retarded testicular development, abnormal structure of seminiferous tubule, decrease in the concentration of spermatozoa, diminution of seminiferous tubule membrane, abundance of spermatocytes and vacuolation in testis of treated prawns. Ultrastructural study also confirmed the impairment of spermatogenesis in treated prawns. Furthermore, radioimmunoassay (RIA) clearly documented the reduction of testosterone level in TBT exposed groups. Thus, TBT substantially reduced the level of male sex hormone as well as biochemical constituents which ultimately led to impairment of spermatogenesis in the freshwater male prawn M.rosenbergii. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1171–1181, 2014.     

The toxic effect of phthalate esters on immune responses of giant freshwater prawn (Macrobrachium rosenbergii) via oral treatment

A previous in vitro study has indicated that four phthalate esters (PAEs) could damage hemocytes and decreases the cellular immunity of prawns [Sung, H.H., Kao, W.Y., Su, Y.J., 2003. Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn, Macrobranchium rosenbergii. Aquat. Toxicol. 64, 25-37]. The aim of this study was to investigate the in vivo effect of four PAEs, diethyl phthalate (DEP), dihexyl phthalate (DHP), dipropyl phthalate (DPrP) and diphenyl phthalate (DPP) on the defense system of the giant freshwater prawn, M. rosenbergii. PAE dissolved in corn oil was continuously fed to prawns for 8 days and five immune parameters (total hemocyte count, THC; ratio of granulocytes to hyalinocytes, G/H; intrahemocytic total phenoloxidase activity, PO(T); intracellular superoxide anion (O2-) production; transglutaminase (TGase) activity) were separately detected on days 1, 4 and 8. In addition, mortality was determined on days 4 and 8 after challenging the prawns with Lactobacillus garvieae. In comparison with untreated prawns, the results showed that DHP demonstrated the lowest toxicity in that it only influenced the PO activity and O2- production before 4 days after treatment and caused 6.6% mortality on day 8. DEP decreased G/H, PO(T) and TGase activity on day 1 and reduced THC, G/H and PO(T) and caused 16.6% mortality on day 4; however, on day 8, it increased O2- production and caused no mortality. In the DPrP-treated group, a reduction of all the immune reactions apart from TGase activity and 22.2% mortality were detected on day 4. As for the effect of DPP, results showed that it decreased all the immune parameters apart from THC on days 1 and 4, but caused no mortality on day 4; but on day 8, an increase of O2- production and 17.7% mortality were detected. These results indicated that the immune reactions of prawns were variable due to the different toxic effects of PAEs. In addition, it was found that, on day 8 after treatment, the three PAEs, DHP, DPrP and DPP increased O2- production and did not influence the other four reactions, but mortality was detected in these groups. These results suggest that other physiological responses may also be affected to increase the susceptibility of prawns to pathogens.     

Interactions between Bromobenzene Dose, Glutathione Concentrations, and Organ Toxicities in Single- and Multiple-Treatment Studies

Interactions between Bromobenzene Dose, Glutathione Concentrations,and Organ Toxicities in Single- and Multiple-Treatment Studies.KLUWE, W. M., MARONPOT, R. R., GREENWELL, A., AND HARRINGTON,F. (1984). Fundam. Appl. Toxicol. 4, 1019–1028. A singleoral dose of 4.0 mmol/kg bromobenzene transiently depleted hepaticand renal reduced nonprotein sulfhydryl group (NPS) concentrations,caused hepatocellular necrosis, and increased serum glutamic-pyruvictransaminase activity in male Fischer 344 rats. The depletionof NPS had partially reversed by 24 hr, and NPS concentrationswere approximately twice normal values by 48 hr post-treatmentWhen the effects of single and repeated (once dairy for 2, 4,or 10 days) treatments with 4.0 mmol/kg were compared, it wasapparent that the severity of hepatotoxicity lessened and thepercentage depletions of hepatic and renal NPS concentrationsdecreased with increasing length of bromobenzene treatment Therewere essentially no signs of toxicity following the tenth treatmentwith 4.0 mmol/kg. Single-treatment studies indicated the followingdose-response; 2.0 mmol/kg bromobenzene depleted liver NPS andwas hepatotoxic 0.5 mmol/ kg caused a lesser depletion of liverNPS and was not (overtly) hepatotoxic, and 0.0625 mmol/ kg wasthe maximum dose that did not deplete liver NPS. The responsesto single and multiple (ten) treatments with these representativedoses were compared. Liver injury was observed after a singlebut not after the tenth daily treatment with 2.0 mmol/kg. Boththe single and the tenth administrations of 2.0 mmol/kg depletedhepatic NPS, but the precentage of depletion was greater afterthe first than after the tenth dose. Liver injury was not detectedwith lower dose regimens. The patterns of NPS depletion in liverand kidney were similar after single or muliple (ten) treatments.The minimum NPS concentrations produced, however, were lowerafter single than after multiple treatments. The molar amountsof liver NPS depleted after the tenth treatment appeared tobe equivalent to or greater than those after the first but priorbromobenzene exposure resulted in a higher concentration oftissue NPS being present at the time of the final treatment.Thus, the minimum tissue concentrations of NPS were greaterafter multiple treatments than after single treatments, despitethe loss of equivalent amounts of NPS. It is concluded fromthese studies that repeated treatment produces resistance tobromobenzene hepatotoxicity. This protective adaptation maybe due to a chemically induced increase in liver glutathioneconcentration     

Glutathione conjugation of the fluorophotometric epoxide substrate, 7-glycidoxycoumarin (GOC), by rat liver glutathione transferase isoenzymes

The fluorophotometric substrate, 7-glycidoxycoumarin (GOC), was examined for the assay of epoxide-glutathione (GSH)-conjugating activities of seven major GSH transferases (GSTs) isolated from rat liver cytosols. GST 7-7 (GST-P), isolated from the liver cytosol of rats bearing hepatic hyperplastic nodules, catalysed the GSH conjugation of GOC at a higher rate than any other examined GST isolated from the normal rat liver cytosol. GSTs 3-3, 3-4 and 4-4 (group 3-4 enzymes) had specific activities towards GOC by one fifth to one third of that of GST 7-7. GSTs 1-1, 1-2 and 2-2 (group 1-2 enzymes) had very low activities towards this epoxide. A kinetic study indicated that GST 7-7 showed the largest kappa cat/Km value for the catalytic reaction of GOC-GSH conjugation among the GSTs. In spite of their much smaller kappa cat values, group 3-4 enzymes showed much larger kappa cat/Km values for GOC than the group 1-2 enzymes, because GOC had a much higher affinity for group 3-4 enzymes than for group 1-2 enzymes. A comparative study was also done with GSH conjugations of styrene 7,8-oxide (STO) and 1-chloro-2,4-dinitrobenzene by the GSTs. Unlike GOC, the conjugation of STO was mediated at rates about twice as high by group 3-4 enzymes than by GST 7-7. STO was also a very poor substrate for group 1-2 enzymes.     

Glutathione transferase zeta: discovery, polymorphic variants, catalysis, inactivation, and properties of Gstz1-/- mice

Glutathione transferase zeta (GSTZ1) is a member of the GST superfamily of proteins that catalyze the reaction of glutathione with endo- and xenobiotics. GSTZ1-1 was discovered by a bioinformatics strategy that searched the human-expressed sequence-tag database with a sequence that matched a putative plant GST. A sequence that was found was expressed and termed GSTZ1-1. In common with other GSTs, GSTZ1-1 showed some peroxidase activity, but lacked activity with most known GST substrates. GSTZ1-1 was also found to be identical with maleylacetoacetate isomerase, which catalyzes the penultimate step in the tyrosine-degradation pathway. Further studies showed that dichloroacetate (DCA) and a range of α-haloalkanoates and α,α-dihaloalkanoates were substrates. A subsequent search of the human-expressed sequence-tag database showed the presence of four polymorphic alleles: 1a, 1b, 1c, and 1d; GSTZ1c was the most common and was designated as the wild-type gene. DCA was shown to be a k(cat) inactivator of human, rat, and mouse GSTZ1-1; human GSTZ1-1 was more resistant to inactivation than mouse or rat GSTZ1-1. Proteomic analysis showed that hGSTZ1-1 was inactivated when Cys-16 was modified by glutathione and the carbon skeleton of DCA. The polymorphic variants of hGSTZ1-1 differ in their susceptibility to inactivation, with 1a-1a being more resistant to inactivation than the other variants. The targeted deletion of GSTZ1 yielded mice that were not phenotypically distinctive. Phenylalanine proved, however, to be toxic to Gstz1(-/-) mice, and these mice showed evidence of organ damage and leucopenia.     

Comparative formation, distribution, and elimination kinetics of diphenylmethoxyacetic acid (a diphenhydramine metabolite) in maternal and fetal sheep.

Deamination to diphenylmethoxyacetic acid (DPMA) is the major route of diphenhydramine (DPHM) clearance in many species. In this study, we assessed the contribution of this pathway to nonplacental DPHM elimination and disposition of DPMA in maternal and fetal sheep. Paired maternal-fetal experiments were conducted in five chronically catheterized pregnant sheep (124-140 days gestation) with an appropriate washout period in between. Both maternal and fetal dosing experiments involved administration of an i.v bolus of deuterium-labeled DPMA ([2H10]-DPMA) combined with a 6-h infusion of DPHM (or a bolus of unlabeled DPMA with an infusion of deuterium-labeled DPHM). Maternal and fetal arterial plasma and urine samples were collected and analyzed for DPMA, [2H10]-DPMA, DPHM, and deuterium-labeled DPHM concentrations using gas chromatography-mass spectrometry. The preformed DPMA (or [2H10]-DPMA) had a substantially lower clearance (maternal: 0.55 +/- 0.18 versus 40.9 +/- 14.0 ml/min/kg; fetal: 0.37 +/- 0.11 versus 285. 6 +/- 122.2 ml/min/kg) and steady-state volume of distribution (Vdss, maternal: 0.10 +/- 0.02 versus 2.1 +/- 1.1 l/kg; fetal: 0.40 +/- 0. 06 versus 13.1 +/- 3.1 l/kg) as compared with the parent drug. The contribution of DPMA formation to maternal and fetal DPHM nonplacental clearance in vivo was 1.78 +/- 2.12% and 0.87 +/- 0.56%, respectively, indicating that DPMA formation is not a major route of DPHM clearance in fetal or maternal sheep. The recoveries of DPMA (or [2H10]-DPMA) in maternal urine were 88.0 +/- 6.5 and 92.1 +/- 7. 4% of the administered dose during maternal and fetal dosing experiments, respectively. Thus, this metabolite does not appear to be secondarily metabolized in fetal or maternal sheep. These findings are in contrast to other species (dog, rhesus monkey, human) where DPMA and its conjugates constitute approximately 40 to 60% of the total DPHM metabolites.     

Glutathione transferase T1 and M1 genotype polymorphism in the normal population of Shanghai

Glutathione transferases are known to be important enzymes in the metabolism of xenobiotics. In humans genetic polymorphisms have been reported for the hGSTM1 and hGSTT1 genes leading to individual differences in susceptibility towards toxic effects, such as cancer. This study describes the distribution of the two polymorphisms of hGSTT1 and hGSTM1 in the normal Chinese population of Shanghai. Out of 219 healthy individuals having been genotyped for GSTT1 and GSTM1, 108 (49%) were identified to be homozygously deficient for the GSTT1 gene and 107 (49%) for the GSTM1 gene. Received: 26 January 1998 / Accepted: 8 April 1998     

Protein farnesyl transferase inhibitors for the treatment of malaria and African trypanosomiasis

Protein farnesyl transferase inhibitors (PFTIs) have been developed as oncology therapeutics but recent studies have supported the use of PFTIs for the treatment of eukaryotic pathogens. Data supporting PFTIs for the treatment of African sleeping sickness caused by Trypanosoma brucei sp, and for the therapy of malaria caused by Plasmodium spp is reviewed. Protein prenylation in T. brucei and P. falciparum has been studied using a variety of techniques, including recombinant and native enzyme assays. Studies have demonstrated farnesylation and geranylgeranylation in these parasites. A variety of PFTIs have shown growth inhibition activity and killing of T. brucei and P. falciparum, yet not all mammalian PFTIs are active on parasitic PFTs. Protein farnesyl transferase as well as protein geranylgeranyl transferase type II enzymatic activities have been demonstrated in T brucei and P. falciparum, but protein geranylgeranyl transferase type I activity may be lacking from these parasites, perhaps explaining the extreme sensitivity of these organisms to PFTIs compared with mammalian cells. Given that PFTIs are relatively non-toxic in short-term administration to humans, PFTIs specific to parasites are not required for therapy. Thus, the challenge in PFTI drug development is not to identify selective antiparasite compounds, but to identify compounds with sufficient potency and pharmacokinetic properties to produce satisfactory drugs for malaria and African sleeping sickness.     

The distribution of microsomal glutathione transferase among different organelles,different organs,and different organisms

1. In the present study we have used both enzyme assay with 1-chloro-2,4-dinitrobenzene as substrate and immunochemical quantitation to examine the distribution of microsomal glutathione transferase in different organelles, in different organs, and in different organisms.2. This enzyme was found to constitute 3% and 5%, respectively, of the total protein recovered in the microsomal and outer mitochondrial membrane fractions from rat liver, Microsomal glutathione transferase present in other subcellular fractions can be accounted for by contamination by the endoplasmic reticulum.3. In contrast to the situation with rat liver microsomes the glutathione transferase activities of microsomes from extrahepatic tissues of this same animal could not be activated by treatment with N-ethylmaleimide. Nonetheless, significant albeit low levels of a protein with the same molecular weight and immunochemical properties as the rat liver enzyme could be detected in microsomes from several extrahepatic tissues, notably the intestine, the adrenal, and the testis.4. Of those mammals for which fresh liver could be obtained, all demonstrated N-ethylmaleimide-activatable glutathione transferase activity in their liver microsomes. On the other hand, representatives for fish, birds, and amphibia did not demonstrate such activatable transferase activity in their liver microsomes. Toad was the only species that had a notable (twofold) sex difference in their level of hepatic microsomal glutathione transferase activity.     

Pharmacokinetics and distribution of a fluoresceinated glycosaminoglycan, sulodexide, in rats. Part II: Organ distribution in rats

Fluorescein-labelled sulodexide tissue distribution was studied in the rat after intravenous administration at the dosage of 15 mg/kg. A globular fluorescence was observed mainly in the extracellular matrix and a diffuse one was present in liver and kidney parenchyma lasting for a long time, up to 4 h after administration. In particular in the kidney cortex the fluorescent material was present at the higher time intervals. Fluorescence was also observed in the spleen and in the perivascular areas and in the media of the aortic wall.     

Organ distribution and bioaccumulation of microcystins in freshwater fish at different trophic levels from the eutrophic Lake Chaohu, China

This article reports the organ distribution and bioaccumulation of hepatotoxic microcystins (MCs) in freshwater fishes at different trophic levels from the large, shallow, eutrophic Lake Chaohu in September 2003, when there were heavy surface blooms of toxic cyanobacteria. Among all fish, intestines and blood had the highest average content of MC-RR + MC-LR (22.0 and 14.5 microg g(-1) DW, respectively), followed by liver, bile, and kidney (7.77, 6.32, and 5.81 microg g(-1) DW, respectively), whereas muscle had the least (1.81 microg g(-1) DW). MC content in muscle was highest in carnivorous fish (Culter ilishaeformis, 2.22 microg g(-1) DW) and omnivorous fish (Carassius auratus, 1.96 microg g(-1) DW) and was lowest in phytoplanktivorous fish (Hypophthalmichthys molitrix, 1.65 microg g(-1) DW) and herbivorous fish (Parabramis pekinensis 0.660 microg g(-1) DW). However, the amount of MC in the gut of H. molitrix (137 microg g(-1) DW) was more than 20 times that in the other fish (<6.50 microg g(-1) DW). The MCs showed a tendency to accumulate up the food chain, and piscivorous fish at the top of the food chain were at high risk of exposure to MCs in Lake Chaohu. Our study is the first to report MC concentrations in the bile and blood of wild fish. One hundred grams of fish muscle would contain 2.64-49.7 microg of MC-LR equivalent, or about 1.3-25 times the recommended tolerable daily intake of MC-LR by humans, indicating that fish are already severely contaminated by MCs and that the local authorities should warn the public of the risk of poisoning by eating the contaminated fish.     

Regional distribution and kinetics of inhaled pharmaceuticals

Drug biodistribution is often secondary to drug action. However, drugs that have a topical action and are deposited into the airway by inhalation are dependent on effective deposition at the intended site of action. Measurement of the distribution of such drugs in the airway is a useful tool. Distribution data can help to interpret clinical results, to evaluate products relative to each other, to optimize a new drug formulation, and to choose effective drug delivery methods. Imaging of radiotracers is the only means available to measure drug deposition throughout the lungs, nasal passages, and sinuses. There are several approaches to imaging drug deposition. Planar imaging has been the most used method, but SPECT and PET imaging are beginning to be applied effectively. The properties of non-drug tracers, labeling of drugs, evaluation of distribution patterns, and quantification of deposited drugs are important issues that have been addressed. Imaging has been shown to be a powerful technique to evaluate and to speed development of inhaled drugs. This review explores the most recent advances and issues with an emphasis on drug development.