Clinical pharmacology of cyclooxygenase inhibition and pharmacodynamic interaction with aspirin by floctafenine in Thai healthy subjects.

Floctafenine, a hydroxyquinoline derivative with analgesic properties, is widely used in Thailand and many other countries. The objectives of this study were to evaluate in Thai healthy volunteers: i) the inhibition of whole blood cyclooxygenase(COX)-2 and COX-1 activity by floctafenine and its metabolite floctafenic acid in vitro and ex vivo after dosing with floctafenine; ii) the possible interference of floctafenine administration with aspirin antiplatelet effects. We performed an open-label, cross-over, 3-period study, on 11 healthy Thai volunteers, who received consecutively floctafenine(200mg/TID), low-dose aspirin(81mg/daily) or their combination for 4 days, separated by washout periods. Floctafenine and floctafenic acid resulted potent inhibitors of COX-1 and COX-2 in vitro (floctafenic acid was more potent than floctafenine) showing a slight preference for COX-1. After dosing with floctafenine alone, whole blood COX-1 and COX-2 activities were inhibited ex vivo in a time-dependent fashion which paralleled floctafenic acid plasma concentrations. Aspirin alone inhibited profoundly and persistently platelet COX-1 activity and AA-induced platelet aggregation throughout 24-h dosing interval which was affected by the co-administration of floctafenine. At 24 h after dosing with aspirin and floctafenine, the inhibition of platelet thromboxane(TX)B2 generation and aggregation were significantly(P less than 0.05) lower than that caused by aspirin alone. Therapeutic dosing with floctafenine profoundly inhibited prostanoid biosynthesis through the rapid conversion to floctafenic acid. Floctafenine interfered with the antiplatelet effect of aspirin. Our results suggest that floctafenine should be avoided in patients with cardiovascular disease under treatment with low-dose aspirin.

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family.

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.

Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride.

Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.

Characterization of toad liver glutathione transferase.

The major form of glutathione transferase from the toad liver previously designed as Bufo bufo liver GST-7.6 (A. Aceto, B. Dragani, T. Bucciarelli, P. Sacchetta, F. Martini, S. Angelucci, F. Amicarelli, M. Miranda and C. Di Ilio, Biochem. J. 289 (1993) 417-422) has been characterized. According to its partial amino acid sequence, the toad enzyme may be included in the pi class GST and named bbGST P2-2. However, bbGST P2-2 appears to be immunologically, structurally and kinetically distinct from any other members of pi family, including bbGST P1-1, suggesting that it may constitute a subset of pi class GST. The data support the hypothesis that the transition from aquatic to terrestrial life causes a switch of the GST amphibian pattern promoting the expression of a GST form (bbGST P2-2) able to counteract, with higher efficiency, the toxic effects of reactive metabolites of oxidative metabolism and those of hydrophobic xenobiotics.

Alteration of glutathione transferase subunits composition in the liver of young and aged rats submitted to hypoxic and hyperoxic conditions.

In the present work, we have studied glutathione transferase (GST) activity and GST subunits distribution in the liver of young and aged rats kept under hypoxic or hyperoxic normobaric conditions as model of oxidative stress. A significant decrease of GST activity was detected in young hypoxic rat liver, whereas a significant increase occurred in aged hypoxic liver. No significant alteration of activity was obtained in both young and aged rat livers subjected to hyperoxic treatment. Substrate specificity measurements, SDS/PAGE analysis and reverse-phase HPLC, of GSH-affinity purified fractions were used to study the changes in the GST subunits pattern occurring in the liver of rat as a consequence of hypoxic and hyperoxic treatment. The results demonstrate that young and aged rat liver has a different constitutive GST subunit pattern which are markedly and differentially altered in hypoxia or hyperoxia. The hyperoxic treatment caused an increase of GST subunit 3 in aged, but not in young liver. In aged liver, both the hypoxic and hyperoxic treatment produced a decrease of GST subunit 4. After hypoxic treatment GST subunit 3 significantly increased in both young and aged liver. GST subunit 1a increased in both young and adult liver after hyperoxia. Following hypoxia a decrease of subunit 1a was seen in both young and aged liver. After hypoxic treatment, subunit 6 doubled in young, but not in aged, livers. It was concluded that the alterations in GST subunit expression occurring in the liver as a consequence of hypoxic or hyperoxic treatment respond to the necessity of a better protection of liver against the products of oxidative metabolism.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1.

Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

Irreversible inactivation of calcium-dependent proteinases from rat liver by biological disulfides.

The effect of low-molecular-mass biological disulfides and their related reduced compounds on the activity of two calcium-dependent neutral proteinases (calpains) from rat liver has been investigated. L-Cystine and L-cystamine bring about the inactivation of both enzymes, while the related reduced compounds L-cysteine and L-cysteamine are without effect. Calpain II is more sensitive to the inactivating effect of glutathione disulfide in comparison with calpain I. The inactivation rates of both calpains depend on the concentration of glutathione disulfide. Reduced glutathione, added at physiological concentration (5 mM), neither affects the proteinase activities nor protects the enzymes from the inactivating effect of glutathione disulfide. The enzymes inactivated by biological disulfides cannot be restored by a large excess of a reducing thiolic compound (dithiothreitol). It is suggested that calcium-dependent proteinases might be inactivated also in vivo by enhanced level of glutathione disulfide.

Glyoxalases activity during Bufo bufo embryo development.

In this work we have investigated the expression of glyoxalase I (GLO I) and glyoxalase II (GLO II) activities during Bufo bufo embryo development and in some tissues of both male and female adult animals, in order to study how they correlate with cell proliferation and differentiation. The results show that both the activities are expressed at significant levels from the earliest developmental stages, reaching the highest values at the end of embryonic development (stage 25). The GLO I/GLO II ratio is very high at the beginning of the development and then gradually decreases as the development goes on. These data emphasize the importance of GLO I activity in the phases in which elevated cell division is taking place. In the differentiated tissues, a peculiar sexual dimorphism in both GLO I and GLO II activities, with higher values in female than in male, was found. GLO I embryonic activity levels are comparable to those found in female differentiated tissues, but significantly higher than those detected in male differentiated tissues. On the contrary, the GLO II activities found in the adult tissues were always higher than those found in embryos. These results further support the idea that high GLO I/GLO II ratios are a characteristic of the proliferative status, which assures a good scavenging action against the potentially cytotoxic and cytostatic effect of methylglyoxal.

Time-dependent and tissue-specific variations of glutathione transferase activity during gestation in the mouse.

Glutathione transferases (GSTs; EC. 2.1.5.18) activity was measured in maternal liver and conceptal tissues during gestation. In maternal liver, maximum activity was found at gestational day (GD) 9 after which it slowly decreased up to the end of gestation. The placental GSTs activity at GD18 was three times lower than that found at GD14. Conversely, fetal liver GSTs at GD14 was about 75% that at GD18. It was also observed that GSTs activity at GD9 and GD10 was higher in visceral yolk sac than in embryo proper. Substrate specificity measurements, SDS PAGE analysis and HPLC runs, carried out on GSH-affinity purified fractions, revealed that with the progress of gestation in maternal liver an increase in pi class GSTs subunit occurs, with a concomitant decrease in alpha class GSTs. With respect to the time of gestation, a significant change in alpha, mu and pi class GSTs expression also occurred in fetal liver and in chorioallantoic placenta. It was concluded that during gestation the GSTs system is subjected to a time-dependent and tissue-specific modulation which may play a protective role against developmental toxicants.

Developmental aspects of Bufo bufo embryo glutathione transferases.

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Glutathione peroxidase, glutathione S-transferase and glutathione reductase activities in normal and neoplastic human breast tissue.

Glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-Tr) and glutathione reductase (GSSG-Rx) activities have been determined in normal and neoplastic human breast tissues. Large interindividual variations in the activities of all enzymes tested were found in both tumor and non-tumor specimens. In general a significant increase in the activities of the 3 enzymes was found in tumors, whereas in fibroadenoma they were as high as in healthy tissues. When a comparison was made between normal and neoplastic tissues of the same individual, GSH-Tr and GSSG-Rx activities were found to be higher in 15 and 11 cases, respectively, out of 17. GSG-Px activity was higher in all cases. From measurement of GSG-Px activity with both H202 and cumene hydroperoxide, it was deduced that human breast contains only the selenium-dependent form.

Glyoxalase activities in tumor and non-tumor human urogenital tissues.

Glyoxalase I and glyoxalase II activities have been measured in human tumor and non-tumor samples of 15 kidneys, 15 bladders, 4 testes, 2 adrenals as well as in 4 samples of prostatic adenomas. In all tissues examined glyoxalase I and glyoxalase II activity values varied widely from one patient to another. No significant difference in glyoxalase I activity between the tumor and non-tumor samples was found. When comparison was made between normal and neoplastic tissues of the same patients, glyoxalase I activity was found to be lower in tumor tissues of 10 out of 15 kidneys, and 2 out of 8 bladders and 1 out of 3 testes. A significant (P < 0.004) decrease of glyoxalase II activity was found only in tumor kidney. The possibility of using the present data to predict the relative sensitivity of human tumor tissues to glyoxalase-related chemotherapy is discussed.

Glutathione peroxidase and glutathione reductase activities in cancerous and non-cancerous human kidney tissues.

Selenium-dependent (Se-GSH-Px), selenium-independent (non-Se-GSH-Px) glutathione peroxidase and glutathione reductase (GSSG-Rx) activities have been determined in cancerous and non-cancerous human adult kidney. Large inter-individual variation in the activities of all enzymes tested were found in both tumour and non-tumour specimens. In general a significant decrease in the activities of the three enzymes was found in tumours. When a comparison was made between cancerous and non-cancerous tissues of the same individual, Se-GSH-Px activity was found to be lower in tumour in 17 cases out of 29, and the non-Se-GSH-Px activity in 20. In 20 cases out of 29 GSSG-Rx was found to be lower in tumour. It was concluded that changes in the factors involved in the anti-oxidative protection actually occur in human kidney tumour.

Glutathione transferase isoenzymes in olfactory and respiratory epithelium of cattle.

Glutathione transferase (GST) was investigated in the olfactory and respiratory epithelium of cattle. A significantly more abundant GST in terms of either protein amount or activity was found in the olfactory rather than in the respiratory epithelium. No apparent qualitative differences in the isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC profiles were noted in the reduced glutathione (GSH) affinity purified GST pool of olfactory and respiratory epithelium. Both tissues have at least six GST isoenzymes with isoelectric point values of 4.9 (peak I), 5.3 (peak II), 5.95 (peak III), 6.5 (peak IV), 7.1 (peak V) and 9.3 (peak VI). From both tissues at least seven different GST subunits can be resolved by HPLC analysis. The GST isoenzymes having pI at 5.3 and 9.3 were predominantly expressed in the olfactory than in the respiratory epithelium. These latter forms conjugate GSH efficiently with alkenals and hydroperoxides, respectively. Kinetic, immunological and structural properties, including HPLC analysis and N-terminal region amino acid sequence seem to indicate that the bovine nasal mucosa tissue in addition to a GST subunit which is orthologue to rat subunit 8 (alpha class) express tissues specific subunits.

Interaction of glutathione transferase P1-1 with captan and captafol.

Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.

Glutathione-S-transferase activity from rat placenta.

Glutathione S-transferase activity significantly decreased in the rat placenta from the 16th to the 20th day of gestation. Isoelectric focusing of rat placenta supernatant yielded essentially a single peak of glutathione S-transferase activity with I-chloro-2,4-dinitrobenzene as substrate, centred on pH 7.45. Substrate specificity measurements, as well as inhibitory studies, revealed pronounced differences between rat and human placental enzymes. Whether the biochemical differences between rat and human GSH-Trs are reflected in physiological differences remains to be ascertained. The sodium dodecyl sulphate (SDS) gel electrophoresis data on subunit composition showed that the rat enzyme is composed of two identical subunits whose molecular mass closely approaches that of human transferase.

Developmental aspects of detoxifying enzymes in fish (Salmo iridaeus).

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism. A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development. In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney. No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.

Binding of pesticides to alpha, mu and pi class glutathione transferase.

The binding of fluorodifen, fenarimol, acifluorfen, 2,4-DES, methyl parathion, paraquat and pyrazophos by alpha, mu and pi class glutathione transferases (GST) was determined by the 2-p-toluidinylnaphthalene-6-sulphonate (TNS) binding fluorescence inhibition technique. Although all the 3 GST classes appear to be capable of binding the pesticides investigated, mu class exhibited somewhat higher affinity than the alpha and pi classes.

Purification and characterization of three Pi class glutathione transferase from monkey (Macaca fascicularis) placenta.

Three forms of glutathione transferase (GST) with an apparent isoelectric point of pH 4.65 (GST I), 4.75 (GST II) and 4.9 (GST III) were resolved from the monkey (Macaca fascicularis) placenta after GSH-affinity chromatography followed by chromatofocusing. Substrate specificity, immunological reactivity, as well as N-terminal aminoacid sequences indicate that the three enzymes belongs to the pi class of GST. Reverse phase HPLC analysis indicates that the three GST arise from the combination of two different subunits eluting respectively at 29.60 +/- 0.10 min and 32.43 +/- 0.13 min. GST I is an homodimer of the 29.60 +/- 0.10 min subunit, GST III is an homodimer of the 32.43 +/- 0.13 min subunit, whereas the GST II is an heterodimer of the 29.60 +/- 0.10 min and 32.43 +/- 0.13 min subunits. Our results strongly suggest that unlike human, multiple forms of pi class GST exist in monkey placenta.

Proteolysis in human erythrocytes is triggered only by selected oxidative stressing agents.

Human erythrocytes have been treated with different agents producing oxidative stress. Diamide, tetrathionate, chromate, cystamine and iodate preferentially influenced the cellular redox state by oxidation of free and protein thiol groups leaving the redox state of hemoglobin virtually unaffected. None of these compounds was able to stimulate the proteolysis. By contrast, phenylhydrazine, nitrite, hydrogen peroxide, ter-butylhydroperoxide, cumene hydroperoxide and copper-ascorbate caused a noticeable oxidation of hemoglobin to methemoglobin. These latter agents, except nitrite and copper-ascorbate, triggered proteolysis. Identical results have been obtained in a ghost-free hemolysate. The fraction containing the proteolytic activity was isolated from hemolysate and tested on native or oxidant-treated hemoglobin. The proteolysis was stimulated by all agents able to produce methemoglobin. It is concluded that proteolysis correlated to an unbalance of cellular redox state. The results obtained with isolated and recombined fractions suggests that increased proteolysis does not depend on the removal of the effect of protease(s) inhibitor(s). Since all agents stimulating proteolysis are able to generate free radicals, it seems that protein breakdown is triggered by the direct effect of these intermediates on proteins (mostly hemoglobin) without the involvement of radical species produced in the membranes by action of organic hydroperoxides. In addition, since nitrite and copper-ascorbate, which also oxidize hemoglobin by radical generation, are unable to stimulate proteolysis, it should be concluded that protein degradation induced by oxidative stress is a complex event induced only by specific agents.