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.

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.

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver.

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis.

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.

Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.

Inactivation, dissociation, and unfolding of the homodimeric glutathione transferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibrium, using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence, far UV circular dichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M the enzyme progressively dissociates into structured monomers. At higher denaturant concentrations the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only by starting from the enzyme denatured at concentrations below 0.5 M. The enzyme denatured at GdmCl concentrations higher than 0.5 M only partially refolds. Globally our results indicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i.e., inactivation of the structured dimer, 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.

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.

Spatial distribution of glutathione, glutathione-related and antioxidant enzymes in cultured mouse embryos.

The present study was undertaken to evaluate the detoxifying capacity of organogenesis-stage murine concepti cultured in vitro. Investigative attention was particularly focused on the embryonic tissue distribution of cytoprotective pathways. Glutathione (GSH) status, GSH-related and antioxidant enzymes were assayed in the embryo proper (EP), visceral yolk sac (VYS) and ectoplacental cone (EC) of 29.44 +/- 1.56 (mean +/- SD) somite pairs concepti. All the tissues displayed significant and comparable concentrations of GSH, further supporting this tripeptide as critical in protection against embryotoxicants. The totality of enzymatic activities was detectable in the selected embryonic compartments. In terms of spatial distribution analysis, maximal activities were found in EC (glutathione peroxidase, glutathione reductase, superoxide dismutase and glyoxalase I and II), and VYS (glutathione transferase and catalase). These results indicate: (1) the organogenesis-stage conceptus, in addition to significant amounts of GSH, expresses constitutive activities of GSH-related and antioxidant enzymes; (2) maximal activity levels are detectable in the embryonic sites which, at the developmental stage selected for assay, serve (VYS) or are evolving to serve (EC) embryo/maternal exchange, and thus represent the primary sites of interaction with foreign compounds.

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.

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.

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.

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.

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.

Structural and functional properties of the 34-kDa fragment produced by the N-terminal chymotryptic cleavage of glutathione transferase P1-1.

Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin generates a 34-kDa GSTP1-1 fragment (a dimer of the 17-kDa subunit composed by residues 48-207) containing the whole C-terminal domain and a part (about 15%) of the N-terminal domain (residues 48-76, i.e., the structural elements beta 3, beta 4, and alpha C). The structural and functional properties of this large fragment have been investigated by analyzing its binding properties to 2-p-toluidinylnaphthalene-6-sulfonate (TNS) extrinsic probe, the TNS displacement technique, and the molecular modeling approach. The results obtained indicated that the 34-kDa GSTP1-1 fragment maintains an hydrophobic pocket with the same structural properties of the corresponding GSTP1-1 hydrophobic binding site. In addition, the 34-kDa GSTP1-1 binds a number of hydrophobic compounds such as 1-chloro-2,4-dinitrobenzene, hemin, and bilirubin with the same affinity of the native enzyme. Being structurally and functionally autonomous, this fragment, mostly constituted by domain II, appears as an independent folding unit in the protein. Nevertheless, in the entire native protein, interdomain interactions occur and are responsible for the major solvent exposure of the H-site in the presence of glutathione.

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 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.

Investigation of intra-domain and inter-domain interactions of glutathione transferase P1-1 by limited chymotryptic cleavage.

Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin performed at 20 degrees and 30 degrees C mainly generates two complementary peptides of 17 kDa and 6 kDa molecular mass with concomitant loss of catalytic capacity. Sequence analysis of these peptides showed that the peptide bond between Tyr47 and Gly48 was cleaved. The analysis of the recently resolved three-dimensional structure of GSTP1-1 [Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federici, G. & Parker, M. W. (1992) J. Mol. Biol. 227, 214-226] suggests that the proteolytically cleaved bond results located in a portion of the polypeptide chain lining the G-site which has been demonstrated to be part of an exposed and flexible region of the N-terminal domain (structural elements alpha B1 and alpha B2) [Aceto, A., Caccuri, A. M., Sacchetta, P., Bucciarelli, T., Dragani, B., Rosato, N., Federici, G. & Di Ilio, C. (1992) Biochem. J. 285, 241-245]. The fragments which are generated by proteolysis at 20 degrees C, remain linked by noncovalent interaction in a complex (nicked GSTP1-1) which is dissociated by incubation at higher temperatures. As shown by circular dichroic analysis, although inactive, nicked GSTP1-1 retains an overall secondary structure closely resembling that of the parent enzyme. However, the fluorescence data of the nicked GSTP1-1 indicate that the Trp38, which is near the chymotrypsin-cleavable bond, becomes exposed in a more polar environment. This indicates that, in the nicked enzyme, the polypeptide portion containing the structural elements alpha B1 and alpha B2 has more freedom of fluctuation. The fact that this polypeptide chain portion contains two essential amino acid residues of the G-site (Trp38 and Lys42) might account for the loss of ability to bind glutathione by the nicked enzyme which is consequently catalytically inactive. Proteolysis performed at 30 degrees C generated a homodimeric 17-kDa fragment. The structural analysis of this fragment suggests that the GSTP1-1 alpha C helix, which is located in the domain I and is thought to be involved in the inter-domain interaction, could exert a critical role in maintaining the native folding of domain II.