Selenoxides inhibit δ-aminolevulinic acid dehydratase

Selenoxides inhibit δ-aminolevulinic acid dehydratase

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  Toxicology Letters 119 (2001) 27–37 Selenoxides inhibit   -aminolevulinic acid dehydratase Marcelo Farina  a,b , Vanderlei Folmer  a , Rodrigo C. Bolzan  a ,Leandro H. Andrade  a , Gilson Zeni  a , Antoˆnio L. Braga  a , Joa˜o B.T. Rocha  a, * a Departamento de Quı´mica ,  Centro de Cieˆncias Naturais e Exatas ,  Uni   ersidade Federal de Santa Maria ,  97105  - 900  , Santa Maria ,  RS  ,  Brazil  b Curso de Farma´cia ,  Centro de Cieˆncias da Sau´de ,  Uni   ersidade Regional Integrada do Alto Uruguai e das Misso˜es ,  Erechim , RS  ,  Brazil  Received 17 July 2000; received in revised form 20 October 2000; accepted 23 October 2000 Abstract The effect of two selenides and their selenoxides on   -aminolevulinic acid dehydratase (  -ALA-D) from liver of adult rats was investigated. In vivo, selenides can be oxidized to selenoxides by flavin-containing monooxygenases(FMO) and selenoxides can regenerate selenides by thiol oxidation. Phenyl methyl selenide (PhSeCH 3 ) and 1-hexynylmethyl selenide (C 4 H 9 C  CSeCH 3 ) were converted to selenoxides by reaction with H 2 O 2 . PhSeCH 3  andC 4 H 9 C  CSeCH 3  had no effect on   -ALA-D up to 400   M. Conversely, their selenoxides inhibited   -ALA-D, andthe IC 50  for enzyme inhibition was about 100 and 70   M, respectively. Partially purified   -ALA-D (P 55 ) from swineliver was also inhibited by these selenoxides. The inhibitory action of selenoxides was antagonized by dithiotreitol(DTT). Moreover,   -ALA-D from a plant source was inhibited by the selenoxides, suggesting a possible involvementof     SH groups in a distinct site of the homologous region implicated in Zn 2 + binding in mammalian   -ALA-D. Afterexposure to PhSeCH 3  (500   mol / kg / day) for 45 or 30 days, the activity of    -ALA-D from liver of mice decreased toabout 50% of the control group. The in vivo inhibitory action of this compound was not antagonized by DTT.PhSeCH 3  and C 4 H 9 C  CSeCH 3  had no effect on the rate of DTT oxidation, but their selenoxides oxidized DTT. Theresults of the present study suggest that hepatic   -ALA-D of rodents is a potential molecular target for selenides asa consequence of their metabolism to selenoxides by FMO. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords :    -Aminolevulinate dehydratase; Selenoxides; Flavin-containing / locate / toxlet 1. Introduction Selenium is an essential trace element that con-stitutes the active center of glutathione peroxidase(Forstrom et al. 1978; Landestein et al., 1979;Wingler and Brigelius-Flohe´, 1999). Moreover,this element is a component of other se-lenoproteins like 5  -deioidinase (Behne and Kyri-akopoulos, 1990) and selenoprotein P (Linder,1990). In view of the fact that selenium was foundto be an essential dietary micronutrient for manymammalian animal species (Oldfield, 1987; Lin- * Corresponding author. Tel.:  + 55-55-2208140; Fax:  + 55-55-2208031. E  - mail address : (J.B.T. Rocha).0378-4274 / 01 / $ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.PII: S0378-4274(00)00296-4  M  .  Farina et al  .  /   Toxicology Letters  119 (2001) 27–37  28 der, 1990), dietary selenium supplementation isaccepted by the scientific community. On theother hand, it is well known that selenium ishighly toxic to several species of mammals likecattle (Hartley and Grant, 1961), sheep (Blodgett and Bevill, 1987), pigs (Penrith, 1995), rats (Anundi et al., 1984) and humans (Cheftel and Truffert, 1972; Frost, 1972; Louria et al., 1972). While the mechanism of selenium toxicity has notbeen clearly elucidated, Painter (1941) emphasizedthat the toxicity of inorganic selenium could berelated to the oxidation of thiols with the con-comitant formation of derivatives of the RSSeSRtype (selenotrisulfides). After the theoreticalPainter proposal, interaction of thiols with inor-ganic selenium was confirmed experimentally byvarious investigators ( Tsen and Tappel, 1958;Ganther, 1968). In fact, inorganic selenium com-pounds (selenite) catalytically increase the oxida-tion of glutathione and this reaction producessuperoxide (O 2  − ) (Seko et al., 1989).Flavin-containing monooxygenases (FMO) areflavoproteins of some mammalian tissues that cat-alyze NADPH- and oxygen-dependent oxidationof a wide variety of xenobiotics (Ziegler, 1988;Cashman, 1995). The catalytic mechanism of theFMO is known in some detail only for the hogliver enzyme (Poulsen and Ziegler, 1979; Beaty and Ballou, 1980), but it is likely that the majorsteps are essentially the same in other mammals.Kinetic studies have indicated that, in the cell, theenzyme is present largely in the hydroperoxyflavinform and, in principle, compounds that can beoxidized by an organic hydroperoxide are poten-tial substrates for this enzyme (Beaty and Ballou,1981). In addition, several studies have demon-strated the monooxygenation of dialkyl- and alky-laryl-selenides to their selenoxides by FMO(Bruice, 1982; Goeger and Ganther, 1994). Se-lenoxides are potent thiol oxidants and can ini-tiate a futile cycle that catalyzes the oxidation of thiols at the expense of NADPH and oxygen(Ziegler et al., 1991).  -Aminolevulinate dehydratase (  -ALA-D) isan essential enzyme in most organisms, catalyzingthe condensation of two molecules of 5-aminolevulinic acid (ALA) to form the monopy-rrole porphobilinogen (Sassa et al., 1989; Jaffe etal., 1995; Sassa, 1998).   -ALA-D is a sulfhydryl-containing enzyme and its activity is highly sensi-tive to the presence of elements such as mercury(Rocha et al., 1993, 1995; Emanuelli et al., 1996, 1998), lead (Rodrigues et al., 1989, 1996; Burns and Godwin, 1991; Flora et al., 1991; Goering,1993), copper (Nelson et al., 1981), and tin (Chibaand Kikuchi, 1984), which possess high affinityfor    SH groups. The enzyme is also inhibited byelements such as aluminum (Schetinger et al.,1999; Vieira et al. 2000), gallium arsenide (Kondoet al., 1996; Flora et al., 1998), and organic sele-nium and tellurium compounds (Barbosa et al.,1998; Maciel et al., 2000) that oxidize    SH groupsor tentatively compete with Zn 2 + .   -ALA-D inhi-bition may impair heme biosynthesis and canresult in the accumulation of ALA, which hassome pro-oxidant activity (Bechara et al., 1993).The selenoxides generated by oxidation of se-lenides via FMO are potent thiol oxidants and,consequently, can oxidize    SH groups of    -ALA-D, causing enzyme inactivation. In the presentstudy, we reported the in vitro effects of onedialkyl- and one alkylaryl-selenide on thesulfhydryl-containing enzyme   -ALA-D from amammalian source. The selenides were reactedwith hydroperoxide in order to form their respec-tive selenoxides and the products of these reac-tions were studied as in vitro inhibitors of   -ALA-D. The effect of exposure to phenylmethyl selenide on   -ALA-D activity was alsostudied in order to identify possible toxic effectsof organoseleno compounds towards thesulfhydryl containing enzyme   -ALA-D. 2. Materials and methods 2  . 1 .  Compounds Dithiothreitol (DTT), 5,5  -dithio-bis(2-ni-trobenzoic acid), magnesium chloride, zinc chlo-ride, 5-aminolevulinic acid, Coomassie brilliantblue G,  p -dimethylaminobenzaldehyde andpurified catalase from bovine liver were obtainedfrom Sigma (St. Louis, MO, USA). Hydrogenperoxide (30%), dimethylsulfoxide (DMSO), ethy-lacetate, potassium permanganate, mono- and  M  .  Farina et al  .  /   Toxicology Letters  119 (2001) 27–37   29 dibasic potassium phosphate, acetic acid, ortho-phosphoric acid, trichloroacetic acid and sodiumchloride were obtained from Merck (Darmstadt,Germany). The selenocompounds phenyl methylselenide and 1-hexynyl methyl selenide were pre-pared according to literature methods (Braga etal., 1996). Their structures are presented inScheme 1. 2  . 2  .  Animals Adult male Wistar rats (aged 2–3 months) andmale mice (aged 2–3 months) from our ownbreeding colony were maintained in a conditionedroom (20–25°C) under natural lighting conditionswith water and food (Guabi-RS, Brasil) ad libi-tum. The hogs used for   -ALA-D purificationwere obtained from the Sector of Swine Cultureof our University. 2  . 3  .  Tissue preparation 2  . 3  . 1 .  Animals Rats and mice were killed by decapitation. Theliver was quickly removed, placed on ice andhomogenized in 7 volumes 150 mM NaCl. Thehomogenate was centrifuged at 4000 ×  g   at 4°Cfor 10 min to yield a low-speed supernatant frac-tion (S 1 ) that was used for enzyme assay. 2  . 3  . 2  .  Plants Cucumber ( Pepino oadai   ) seeds were germi-nated for 5–7 days at 25°C. Leaves were homoge-nized in a medium containing 5 volumes 10 mMTris–HCl buffer (pH 9.0). The homogenate wasthen centrifuged as already described for animaltissue and a low-speed supernatant fraction (S 1 )obtained was used for enzyme assay. 2  . 4  .  Preparation of partially -  purified    - ALA - D Partially-purified   -ALA-D (P 55 ) was obtainedas previously described (Emanuelli et al., 1998). 2  . 5  .  In   i   o exposure to phenyl methyl selenide Mice were injected with phenyl methyl selenide(500   mol / kg / day) once a day, subcutaneously,five times per week for 30 or 45 days. Subcuta-neous route of exposure was selected becausephenyl methyl selenide is highly lipophilic. Thephenyl methyl selenide was diluted in DMSO andinjected at a proportion of 2.5 ml / kg. Controlanimals were injected with DMSO at a proportionof 2.5ml / kg. The animals were killed 24 h after thelast injection and the liver was removed for tissuepreparation. 2  . 6  .  Enzyme assay Mammalian   -ALA-D activity was assayed bythe method of Sassa (1982) by measuring the rateof product (porphobilinogen) formation exceptthat 84 mM potassium phosphate buffer (pH 6.4)and 2.5 mM ALA were used. Moreover, for thepartially purified   -ALA-D, the medium con-tained 100   M  D,L -dithiothreitol. For the plantenzyme, the medium contained 50 mM Tris–HClbuffer (pH 9.0) and 2.5 mM ALA. All experi-ments were carried out after 10 min of pre-incuba-tion. The reaction was started 10 min after theaddition of the enzyme preparation by adding thesubstrate. Incubations were carried out for 1 h at39°C for the mammalian enzyme and for 90 minat 35°C for the plant enzyme. The reactionproduct was determined using modified Ehrlich’sreagent at 555 nm, with a molar absorption coeffi-cient of 6.1 × 10 4 cm − 1 M − 1 for the Ehrlich-por-phobilinogen salt. The reaction rates were linearwith respect to time of incubation and addedprotein for all experimental conditions. 2  . 7  .  Reaction of selenides with hydroperoxide The selenides (28.5   M–2.85 mM) dissolved in10   l ethanol were mixed with 25   l H 2 O 2  to givea final concentration of 30 mM. The reaction was Scheme 1. Structures of selenides.  M  .  Farina et al  .  /   Toxicology Letters  119 (2001) 27–37  30 carried out for 1 h at 39°C and stopped by addingcatalase (200 U). The same tubes were used forthe enzyme assay and the final concentrations of the chalcogens ranged from 4 to 400   M. Theabsence of H 2 O 2  was assessed in parallel tubes bytritiating with potassium permanganate (KMnO 4 ). 2  . 8  .  Determination of the rate of DTT oxidation Reaction of selenides with H 2 O 2  were carriedout as described in Section 2.7, except that H 2 O 2 (125 mM) and selenides (0.7–14 mM) were used,and the final concentrations of chalcogens rangedfrom 0.1 to 2 mM. After catalase addition, 84mM potassium phosphate buffer (pH 6.4) wasadded. DTT (2 mM) oxidation was evaluated bymeasuring the disappearance of     SH groups bythe method of Ellman (1959). Incubation at 39°Cwas initiated by adding DTT in a total volume of 250   l. Aliquots of 50   l were sampled at 0, 30and 60 min to determine the amount of     SHgroups at 412 nm. 2  . 9  .  Protein quantification Protein was measured by the method of Brad-ford (1976) using bovine serum albumin asstandard. 2  . 10  .  IC  50   determination The IC 50  for in vitro inhibition of    -ALA-Dwas calculated by the method of Dixon and Webb(1964). 2  . 11 .  1 H  - NMR studies The selenides were incubated with H 2 O 2  at aratio 1:10 (mol / mol) at 39°C for 24 h. The organicphase was extracted with ethylacetate and ana-lyzed with a Brucker DPX-200 (200 MH Z ) spec-trometer for solution in CDCl 3  withtetramethylsilane as internal standard. 2  . 12  .  Statistical analysis In vivo experiments and the enzymatic assayswith varying DTT concentrations were analyzed Table 1IC 50  (  M) values for   -ALA-D inhibition by PhSeCH 3  andC 4 H 9 C  CSeCH 3  and their reaction products with H 2 O 2a Rat (S 1 )Compound Plant (S 1 )Hog (P 55 )  400  400   400PhSeCH 3 97104PhSeCH 3 + H 2 O 2  61  400  400  400C 4 H 9 C  CSeCH 3 21072C 4 H 9 C  CSeCH 3  123 + H 2 O 2a Reaction of selenides with H 2 O 2  was carried out for 1 hand the remaining H 2 O 2  was eliminated by adding catalase(200 U). Enzyme pre-incubation with selenides or productsfrom the reaction of selenides with H 2 O 2  was started byadding the supernatant from rat (S 1 ) or partially purified hogliver   -ALA-D (P 55 ) to a medium containing 84 mM potas-sium phosphate buffer (pH 6.4). For plant   -ALA-D, superna-tants from cucumber leaves (S 1 ) was added to a mediumcontaining 50 mM Tris–HCl buffer (pH 9.0). The reaction wasstarted 10 min later by adding ALA (2.5 mM). Data areexpressed as the mean of five to seven independent experi-ments. S.E.M. was less than 15% of respective means. by one-way analysis of variance (ANOVA) fol-lowed by Duncan’s Multiple Range Test whenappropriate. The data of experiments with a fixedDTT concentration were analyzed by three-wayanalysis of variance. All other results were ana-lyzed by two-way ANOVA, followed by Duncan’sMultiple Range Test when appropriate. Differ-ences between groups were considered to be sig-nificant when  P  0.05. 3. Results 3  . 1 .  Selenocompounds × H  2  O 2  ×  - ALA - Dacti   ity The compounds phenyl methyl selenide(PhSeCH 3 ) and 1-hexynyl methyl selenide(C 4 H 9 C  CSeCH 3 ) did not inhibit hepatic   -ALA-D. However, the products of reaction of phenyl methyl selenide or 1-hexynyl selenide withH 2 O 2  inhibited   -ALA-D from rat liver with IC 50 values in the micromolar range (Table 1). Statisti-cal analysis indicated a significant ( P  0.01) in-teraction between selenide and H 2 O 2 , indicatingthat the reactions of the selenides with H 2 O 2  M  .  Farina et al  .  /   Toxicology Letters  119 (2001) 27–37   31 generated more potent products that inhibited  -ALA-D from rat liver.  -ALA-D was partially purified to assess if endogenous liver substances existing in S 1  wouldcontribute to the inactivation of    -ALA-D bythese selenocompounds. The inhibitory effects of phenyl methyl selenide and 1-hexynyl methyl sele-nide on   -ALA-D activity from hog (P 55 ) weresimilar to those obtained using S 1  (Table 1). Asobserved with S 1 , the selenides did not inhibit thepartially purified enzyme and the products of thereactions of these compounds with H 2 O 2  inhibitedALA-D with IC 50  values in the micromolar range(Table 1).  -ALA-D from mammals possesses at least twosites that contain cysteinyl residues. One of thesesites binds zinc with relatively low affinity (B site),while the other binds zinc with relatively highaffinity (A site) (Dent et al. 1990). In plants, thesite homologous to mammalian B site is charac-terized by the substitution of cysteinyl residues byacidic amino acids, which apparently renders theenzyme less susceptible to oxidation (Jaffe et al.,1995). Similarly to that obtained with mammalianenzyme, the products of the reaction of the se-lenides with H 2 O 2  inhibited   -ALA-D from cu-cumber leaves. The IC 50  values for   -ALA-Dinhibition by the products of reaction betweenH 2 O 2  and phenyl methyl selenide and 1-hexynylmethyl selenide were in the micromolar range(Table 1).The interaction between selenocompounds and   SH groups has been frequently reported (Tsenand Tappel, 1958; Ganther, 1968; Barbosa et al.,1998; Maciel et al., 2000). Moreover, selenoxidesare rapidly reduced by glutathione (GSH), yield-ing oxidized glutathione and the correspondingselenide (Chen and Ziegler, 1994; Goeger andGanther, 1994). Involvement of cysteinyl groupsin   -ALA-D inhibition by the products of thereaction between the selenides and H 2 O 2  wereexamined by testing the effect of DTT on   -ALA-D. Addition of DTT (2 mM) increased   -ALA-Dactivity by 28–35% and protected   -ALA-D fromthe inhibition caused by the products of reactionbetween the selenides and H 2 O 2  (Fig. 1A,B). Theincrease in   -ALA-D activity caused by DTT isdue to reactivation of enzyme molecules that oxi-dized during tissue preparation. Similar protec-tion by DTT against the inhibitory effects of products of reaction of selenides with H 2 O 2  wasobserved when plant   -ALA-D was used (datanot shown). 3  . 2  .  1 H  - NMR studies for chemical characterization of the selenoxides 1 H-NMR studies were carried out in order tocharacterize the chemical structure of products of  Fig. 1. Effect of PhSeCH 3  (A) and C 4 H 9 C  CSeCH 3  (B), andof their products of reaction with H 2 O 2  on   -ALA-D fromliver. Reaction of selenides with H 2 O 2  was carried out for 1 hand the remaining H 2 O 2  was eliminated by adding catalase(200 U). Enzyme pre-incubation with selenides or productsfrom the reaction of selenides with H 2 O 2  was started byadding the liver supernatant (S1) to a medium containing 84mM potassium phosphate buffer (pH 6.4) in the absence or inthe presence of 2 mM DTT. The   -ALA-D reaction wasstarted 10 min later by adding ALA (to give a final concentra-tion of 2.5 mM and a volume of 0.25 ml). Data are expressedas mean  S.E.M. for three independent experiments.
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