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Effects of mercury and selenite on δ-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats
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Environmental Research 95 (2004) 166–173
Effects of mercury and selenite on d-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats$ Juliano Perottoni,a Luciana P. Lobato,b Aline Silveira,a Joa˜o Batista Teixeira Rocha,a and Tatiana Emanuellia,b, a
Master Science Course in Biochemical Toxicology, Department of Chemistry, Center of Nature and Exacts Sciences, Federal University of Santa Maria, 97105-900 Santa Maria, RS, Brazil b Department of Alimentary Technology and Sciences, Center of Rural Science, Federal University of Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 10 April 2003; received in revised form 14 August 2003; accepted 29 August 2003
Abstract The present study evaluates the effects of Na2SeO3 and HgCl2 on kidney and liver of adult rats. In vivo, HgCl2 (17 mmol/kg, sc) reduced ascorbic acid levels in liver (B15%), whereas in kidney it reduced ALA-D activity (B60%) and ascorbic acid levels (B35%) and increased TBARS content (B50%). Na2SeO3 (17 mmol/kg, sc) exposure increased the content of nonprotein thiol groups in liver (35–60%) and kidney (B50–160%), partially prevented mercury-induced ALA-D inhibition in kidney, and completely prevented a mercury-induced increase of TBARS content and decrease of ascorbic acid levels in kidney. In vitro, HgCl2 and Na2SeO3 inhibited renal and hepatic ALA-D, while HgCl2 increased TBARS in renal and hepatic tissue preparations. Na2SeO3 increased the rate of glutathione oxidation in vitro. Results indicated that Na2SeO3 protected against HgCl2 effects in vivo (prevention of mercury interaction with thiol groups and of mercury-induced oxidative damage). In vitro, Na2SeO3 did not prevent mercury effects, but potentiated ALA-D inhibition by mercury, probably due to its ability to oxidize thiol groups. r 2003 Elsevier Inc. All rights reserved. Keywords: Sodium selenite; Mercuric chloride; 5-Aminolevulinate dehydratase; Thiobarbituric acid-reactive substances; Oxidative stress
1. Introduction Although mercury has long been considered a toxic metal, it possesses a number of important industrial uses, and poisoning from occupational exposure and environmental pollution continues to be a concern (Boischio and Henshel, 1996; Klaassen, 1996). Inorganic
$ This study was supported by CNPq (Grants 420010/01-7 to T.E. and 470539/01-1 to J.B.T.R.), CAPES, and FAPERGS. J.P. was the recipient of the CAPES Fellowship. L.P.L. was the recipient of a FAPERGS Scientific Initiation Fellowship. T.E. and J.B.T.R. are the recipients of the CNPq Research Fellowship (Procs. 550691/02-2 and 523761/95-3). Studies involving animals were conducted according to the guidelines of the Committee on the Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sa˜o Paulo, Brazil. Corresponding author. Departamento de Tecnologia e Cieˆncia dos Alimentos, Centro de Cieˆncias Rurais, Universidade Federal de Santa Maria, Camobi, Santa Maria-RS 97105-900, Brazil. Fax: +55-55-2208353. E-mail address: [email protected] (T. Emanuelli).
0013-9351/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2003.08.007
mercury in the Hg2+ form has a great affinity for SH groups of endogenous biomolecules (Clarkson, 1997). Thus, it is invariably found in cells and tissues attached to thiol-containing proteins and small-molecular-weight thiols such as cysteine and glutathione (GSH). At relatively low toxic or nontoxic doses, mercury increases the renal content of GSH, probably due to the induction of GSH synthesis (Zalups, 2000). However, higher doses of inorganic mercury decrease the renal content of GSH (Zalups, 2000). Thiol-containing enzymes, such as daminolevulinate dehydratase (ALA-D), are inhibited by mercury (Rocha et al., 1995, 2001; Emanuelli et al., 1996). Additionally, mercury can also give rise to free radicals that induce lipid, protein, and DNA oxidation (Lund et al., 1993; Clarkson, 1997). The exact mechanisms underlying free-radical production are still not completely understood, but Woods et al. (1990a, b) have shown that in vitro Hg2+ both hinders the antioxidant potential of glutathione and yields reactive species via thiol complexation. Lipid oxidation yields hydroperoxides and aldehydes, such as malondialdehyde. The
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latter may be measured by reactions with thiobarbituric acid (TBARS) and constitutes a useful index of lipid peroxidation (Ohkawa et al., 1979). Sulfhydryl compounds are considered the most effective agents in the chelation therapy of mercury intoxication (Nielsen and Andersen, 1991). The recommended treatment for intoxication with inorganic or elemental mercury includes dimercaprol or d-penicillamine (Klaassen, 1996). Nevertheless, dimercaprol that possesses a low therapeutic index (Andersen, 1989; Kojima et al., 1989) has been suggested to be neurotoxic (Nogueira et al., 2000) and may occasionally potentiate mercury toxicity (Emanuelli et al., 1996, 1998). Additionally, complexes of Hg2+ with meso-2,3-dimercaptosuccinic acid (Endo and Sakata, 1995; Flora, 1999; Frumkim et al., 2001) and 2,3-dimercaptopropane-1sulfonic acid (Pingree et al., 2001), sulfhydryl compounds that are becoming the drugs of choice for the treatment of heavy metal intoxication, are more potent in vitro inhibitors of hepatic ALA-D than Hg2+ alone (Nogueira et al., unpublished results). Selenium is an essential dietary trace element that plays a crucial role in glutathione peroxidase (Rotruck et al., 1973), thioredoxin reductase (Xia et al., 2003), and other enzymes. It is worth noting that the reduced form of selenium (selenide) presents an affinity constant for mercury even higher than that of sulfhydryl compounds (Clarkson, 1997; Yoneda and Suzuki, 1997). Accordingly, Hg–selenium interactions have been experimentally reported to counteract mercury toxicity (Whanger, 1981; Hoffman and Heinz, 1998; El-Demerdash, 2001). Simultaneous administration of sodium selenite with mercuric chloride usually protects against mercury toxicity in vivo (Naganuma et al., 1984; Rao et al., 1998; El-Demerdash, 2001). Nevertheless, there is some controversy concerning the protection afforded by previous administration of sodium selenite. Some authors have reported an absence of protection (Naganuma et al., 1984) and even an increase of mercury toxicity when selenite has been given 1–2 h before HgCl2 (Magos, 1991). Most studies on Hg–selenium interactions have evaluated selenite protection against mercury-induced glutathione depletion and oxidative damage (Chung et al., 1982; Urano et al., 1997; Yoneda and Suzuki, 1997; Gailer et al., 2000), and, to the best of our knowledge, few studies have evaluated the protective effects of selenite against mercury-induced inhibition of thiol-containing enzymes (Farina et al., 2003a, b). The sulfhydryl-containing enzyme ALA-D, which is susceptible to mercury inhibition (Rocha et al., 1995, 2001; Emanuelli et al., 1996), has recently been demonstrated to be a potential molecular target for selenium intoxication (Maciel et al., 2000; Farina et al., 2001; Jacques-Silva et al., 2001). Therefore, it is of interest to evaluate the combined effect of mercury and selenium on ALA-D activity.
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Considering the unsatisfactory performance of the chelation therapy commonly used in mercury intoxication, it is important to clarify the therapeutic effects of alternative drugs such as sodium selenite. In the present study, we examined whether pretreatment with sodium selenite could provide protection against the effects of HgCl2. ALA-D activity, TBARS production, thiol groups, and ascorbic acid levels were measured in liver and kidney of rats after exposure to sodium selenite and mercuric chloride. The interaction between sodium selenite and mercuric chloride was also investigated in vitro by measuring ALA-D activity and TBARS production in liver and kidney. The effect of sodium selenite on the rate of oxidation of reduced glutathione in vitro has been evaluated as well.
2. Materials and methods 2.1. Animals Adult male Wistar rats from our own breeding colony (250–350 g) were maintained in an air-conditioned room (22–25 C) under natural lighting conditions, with water and food (Guabi, RS, Brasil) ad libitum. Animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sa˜o Paulo, Brazil. 2.2. Chemicals Glacial acetic acid, ortho-phosphoric acid, hydrochloric acid, sodium selenite, sulfuric acid, perchloric acid, ethanol, HgCl2, NaCl, K2HPO4, KH2PO4, 5,50 -dithiobis-(2-nitrobenzoic acid), hydrogen peroxide, and ascorbic acid were obtained from Merck (Rio de Janeiro, RJ, Brazil), and butylated hydroxytoluene, sodium dodecyl sulfate, dimethyl sulfoxide, 2,4-dinitrophenylhydrazine, tris(hydroxymethyl)aminomethane, cysteine, thiobarbituric acid, d-aminolevulinic acid, bovine serum albumin, reduced glutathione, and Coomassie brilliant blue G were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 1,1,3,3-Tetramethoxypropane was obtained from Aldrich (Milwaukee, WI, USA). Tricholoroacetic acid was obtained from Reagen (Rio de Janeiro, RJ, Brazil). p-Dimethylaminobenzaldehyde was obtained from Riedel (Hae¨n, Germany). 2.3. Exposure A nephrotoxic dose of mercuric chloride was chosen based on the studies of Fukino et al. (1984) and Emanuelli et al. (1996), in which acute exposure to a similar dose increased lipid peroxidation and decreased ALA-D activity in renal tissue. Considering that the
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molar ratio of Hg2+ to Se is 1:1 (Sasakura and Suzuki, 1998), the dose of sodium selenite was equimolar to that of mercuric chloride. Sodium selenite and HgCl2 solutions were prepared in 120 mM NaCl and 10 mM phosphate buffer, pH 7.4. For the in vivo studies, rats (25) received a subcutaneous injection (1 mL/kg body wt) of vehicle (120 mM NaCl, 10 mM phosphate buffer, pH 7.4) or sodium selenite (17 mmol/kg). After 6 hours, animals were subcutaneously injected (1 mL/kg body wt) with vehicle or HgCl2 (17 mmol/kg). Rats were anaesthetized with ether and killed by decapitation 12 h after the last injection to determine ALA-D activity, TBARS, thiol groups, and ascorbic acid content. 2.4. ALA-D activity Kidneys and livers of treated animals were quickly removed, placed on ice, and homogenized in 150 mM NaCl of 7 and 10 vol, respectively. The homogenate was centrifuged at 4000g at 4 C for 10 min to yield a lowspeed supernatant fraction (S1) that was used for enzyme assay. The enzyme assay was carried out as described by Sassa (1982) by measuring the rate of product (porphobilinogen) formation, except that 200 mM potassium phosphate buffer, pH 6.4, and 2.5 mM d-aminolevulinic acid were used. The reaction was started 10 min after the addition of the enzyme preparation (0.8–1.1 mg of protein) by the addition of the substrate and carried out for 90 min at 39 C. The reaction product was determined using a modified Ehrlich’s reagent at 555 nm with a molar absorption coefficient of 6.1 104 for the Ehrlich-porphobilinogen salt. In vitro ALA-D assays were carried out as described above, except that untreated animals were used and HgCl2 (0–100 mM) and/or sodium selenite (0–40 mM) was included in the incubation assay. 2.5. TBARS determination Kidneys and livers of treated animals were homogenized in 7 vol of a medium containing 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4, and 7.2 mM butylated hydroxytoluene to prevent further oxidation. The homogenate was centrifuged at 4000g at 4 C for 10 min and immediately used to determine thiobarbituric acid-reactive substances. TBARS were assayed as described by Ohkawa et al. (1979). Briefly, samples were incubated at 100 C for 120 min in a medium containing 0.45% sodium dodecyl sulfate, 100 mM hydrochloric acid, 1.4 M acetic acid, pH 3.4, and 0.3% thiobarbituric acid. After centrifugation, the reaction product was determined at 532 nm using 1,1,3,3-tetramethoxypropane as a standard. In vitro TBARS determination was carried out as described above, except that untreated animals were used, butylated hydroxytoluene was omitted, and tissue
supernatants were preincubated at 37 C for 60 min in a medium containing HgCl2 (0–400 mM) and/or sodium selenite (0–40 mM) before the TBARS assay was carried out. 2.6. Thiol group determination Thiol groups were determined as described by Ellman (1959) at 412 nm, after reaction with 5,50 -dithio-bis-(2nitrobenzoic) acid. Tissues of treated animals were homogenized in 7 vol of 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4, and centrifuged at 4000g at 4 C for 10 min, and supernatants were used to determine the content of total thiol groups. Nonprotein thiol groups were determined in the fraction obtained after dilution of the supernatants with 1 vol of 4% trichloroacetic acid followed by centrifugation and neutralization with 1 M tris(hydroxymethyl)aminomethane. A standard curve using cysteine was used to calculate the content of total and nonprotein thiol groups in the tissue samples. 2.7. Ascorbic acid determination Ascorbic acid determination was performed as described by Benderitter et al. (1998) and Jacques-Silva et al. (2001). Briefly, tissues of treated animals were homogenized in 7 vol of 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4 and centrifuged at 4000g at 4 C for 10 min, and protein was removed by dilution with 1 vol of 4% trichloroacetic acid followed by centrifugation. Part of the sample was incubated at 37 C in a medium containing 4.5 mg/mL dinitrophenylhydrazine, 0.6 mg/mL thiourea, 0.075 mg/mL CuSO4, and 0.675 mol/L H2SO4 (final volume of 1 mL). After 3 h, 1 mL 65% H2SO4 was added and the samples were read at 520 nm. A standard curve was constructed using ascorbic acid. 2.8. Determination of the rate of GSH oxidation The rate of GSH oxidation was followed by the disappearance of –SH groups as described by Ellman (1959). Reduced –SH groups of GSH were quantified at 0, 10, 20, 30, and 60 min after the addition of GSH (1 mM) to a reaction mixture containing 200 mM potassium phosphate buffer, pH 6.4, and sodium selenite (0–100 mM) at 39 C. Since no other sulfhydryl compound was added to the incubation medium, it can be considered that GSH was the only compound evaluated by Ellman’s reagent. 2.9. Protein quantification Protein was measured by the method of Bradford (1976) using bovine serum albumin as the standard.
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2.10. Statistical analysis Data on the in vivo effects of selenite and mercury were analyzed by two-way analysis of variance (ANOVA) (2 Se 2 Hg concentrations) followed by Duncan’s multiple-range test when necessary. Data on the in vitro effects of selenite and mercury were analyzed by threeway ANOVA (4 Hg 3 Se 2 tissues for TBARS production and 3 Hg 4 Se 2 tissues for ALA-D activity) followed by Duncan’s multiple range test when necessary. Data on the rate of GSH oxidation were analyzed by two-way ANOVA (4 Se 5 times), taking into account the time variable as a repeated measure. Results with Pp0:05 were considered significant.
3. Results Treatment of rats with a single dose of 17 mmol/kg mercuric chloride did not affect ALA-D activity, TBARS level, total thiol groups, or nonprotein thiol groups in liver, but significantly reduced (B15%) hepatic ascorbic acid content [F ð1; 25Þ ¼ 5:82; Po0:05; Table 1]. Additionally, mercury treatment
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significantly reduced ALA-D activity (B60%) [F ð1; 21Þ; 9.80; Po0:01] and ascorbic acid content (B35%) [F ð1; 21Þ; 10.68, Po0:01], but increased the TBARS level (B50%) in kidney [F ð1; 22Þ; 4.74, Po0:05; Table 2]. Pretreatment with 17 mmol/kg sodium selenite significantly increased the content of nonprotein thiol groups both in liver (35–60%) [F ð1; 21Þ; 9.19; Po0:01; Table 1] and in kidney (B50–160%) [F ð1; 21Þ; 5.74; Po0:03; Table 2]. Moreover, sodium selenite completely prevented the mercury-induced increase of the TBARS level [HgCl2 selenite interaction: F ð1; 22Þ; 10.23; Po0:01] and the decrease of ascorbic acid content in kidney [F ð1; 21Þ; 10.68; Po0:01; Table 2]. Pretreatment with sodium selenite partially prevented the mercury-induced ALA-D inhibition in kidney [F ð1; 21Þ; 5.37; Po0:05; Table 2]. The in vitro effect of sodium selenite and mercuric chloride on TBARS production and ALA-D activity in kidney and liver are shown in Figs. 1 and 2. Mercuric chloride significantly increased TBARS production by renal or hepatic tissue preparations in vitro [F ð3; 128Þ; 41.29; Po0:01; Fig. 1]. Sodium selenite did not affect basal TBARS production or the mercury-induced TBARS increase in vitro (Fig. 1).
Table 1 Effect of sodium selenite treatment on hepatic toxicity induced by mercuric chloride Na2SeO3
Control
ALA-D activity (nmol PBG/h/mg protein) TBARS level (nmol MDA/mg protein) Total thiol groups (nmol SH/mg protein) Non protein thiol groups (mmol SH/g tissue) Ascorbic acid (mg/g tissue)
Saline
HgCl2
Saline
HgCl2
11.9471.55 2.8970.37 113.3377.11 7.8071.17 145.1978.64
13.0971.61 2.3670.32 109.37717.01 6.6270.32 120.8978.13b
13.4472.47 2.4970.24 126.4079.13 10.5271.39a 137.5972.32
11.9471.83 3.1470.40 119.7977.55 10.7371.35a 117.09719.17b
Rats were subcutaneously injected with Na2SeO3 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg). After 6 h rats were injected with HgCl2 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg, sc). Twelve hours later, animals were killed and the hepatic toxicity was evaluated. Data are expressed as means7SEM (n ¼ 528). a Significantly different from the control groups (Po0:05). b Significantly different from the saline groups (Po0:05). Table 2 Effect of sodium selenite treatment on renal toxicity induced by mercuric chloride Na2SeO3
Control Saline ALA-D activity (nmol PBG/h/mg protein) TBARS (nmol MDA/mg protein) Total thiol groups (nmol SH/mg protein) Non protein thiol groups (mmol SH/g tissue) Ascorbic acid (mg/g tissue)
5.1270.46 1.9670.16 58.5677.35 0.9970.36 75.4777.03
HgCl2 a
1.9970.30 2.8970.19a 76.1779.37 0.8770.17 49.8974.80a
Saline
HgCl2
6.8771.43 2.0570.18 61.2976.41 1.4770.42c 75.7676.71
4.3970.57b 1.8770.14 64.6574.87 2.2870.59c 97.91711.20
Rats were subcutaneously injected with Na2SeO3 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg). After 6 h rats were injected with HgCl2 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg, sc). Twelve hours later, animals were killed and the renal toxicity was evaluated. Data are expressed as means7SEM (n ¼ 528). a Significantly different from other groups (Po0:05). b Significantly different from the Na2SeO3–saline group (Po0:05). c Significantly different from the control groups (Po0:05).
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Fig. 1. The in vitro effect of sodium selenite (Na2SeO3) and mercuric chloride (HgCl2) on hepatic (A) and renal (B) TBARS production. The tissue preparation was preincubated with Na2SeO3 and HgCl2 (added simultaneously) at 39 C for 60 min before TBARS measurement. TBARS production is expressed as a percentage of control (absence of Na2SeO3 and HgCl2) (mean7SEM, n ¼ 426). The control TBARS production for hepatic and renal preparation was 6.21 and 6.73 nmol MDA formed/mg of protein, respectively.
Fig. 2. The in vitro effect of sodium selenite (Na2SeO3) and mercuric chloride (HgCl2) on hepatic (A) and renal (B) ALA-D activity. The enzyme preparation was preincubated with Na2SeO3 and HgCl2 (added simultaneously) at 39 C for 10 min before the enzyme reaction was started. The ALA-D activity is expressed as a percentage of control activity (absence of Na2SeO3 and HgCl2) (mean7SEM, n ¼ 729). The control activities of hepatic and renal ALA-D were 10.40 and 4.81 nmol porphobilinogen formed/h/mg of protein, respectively.
Both renal and hepatic ALA-D activities were significantly inhibited by mercuric chloride [100 mM; F ð2; 132Þ; 33.26; Po0:01] and sodium selenite [12 mM onwards, F ð3; 132Þ; 43.98; Po0:01; Figs. 2A and B] in vitro. Three-way ANOVA (2 tissue 4 selenite concentrations 3HgCl2 concentrations) also revealed a significant selenite HgCl2 interaction [F ð6; 132Þ; 4.22; Po0:01], indicating a synergistic effect of these compounds on ALA-D activity (Fig. 2). In order to clarify the interaction of sodium selenite with the thiol groups in assay conditions similar to those used in ALA-D assays, we determined the effect of sodium selenite on the disappearance of reduced glutathione sulfhydryl groups (Fig. 3). Two-way ANOVA (4 selenium concentrations 5 times) revealed a significant selenium time [F ð4; 48Þ; 6.42; Po0:01] interaction, indicating that the oxidative effect of sodium selenite (10 mM onwards) on glutathione increased with time.
4. Discussion Inorganic mercury has a nonuniform distribution after absorption, being accumulated mainly in kidneys (Klaassen, 1996; Emanuelli et al., 1996). Accordingly, the results obtained indicated that subcutaneous exposure to a single dose of mercuric chloride significantly increased the TBARS level and decreased the ALA-D activity and ascorbic acid level in kidneys, while there was only a marginal decrease in the ascorbic acid level in liver (Tables 1 and 2). We have previously demonstrated that mercuric chloride exposure leads to ALA-D inhibition (Emanuelli et al., 1996; Rocha et al., 1995, 2001). The high bonding affinity between mercury and thiol groups is probably responsible for this inhibition and has been implicated in the toxicity of mercuric ions (Clarkson, 1997). Although the mercury-induced decrease of both total and nonprotein thiol groups have been reported previously
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Fig. 3. The effect of sodium selenite (Na2SeO3) on the rate of glutathione (1 mM) oxidation. Sulfhydryl groups of glutathione were evaluated at 412 nm with 5,50 -dithio-bis-(2-nitrobenzoic acid). Results are the means of four independent experiments. SEM were less than 10% of respective means.
(Stohs and Bagchi, 1995), in the present study we could not observe any alteration in the level of renal thiol groups (Table 2). The result obtained may be related to the dose of mercury used, since previous studies revealed that low toxic or nontoxic doses increase renal content of the nonprotein thiol compound GSH, while higher doses decrease the renal content of GSH (Zalups, 2000). The mercury-induced reduction of the ascorbic acid level and the increase of the TBARS level are in agreement with literature data suggesting that mercury treatment may induce oxidative damage (Lund et al., 1993; Stohs and Bagchi, 1995). Ascorbic acid administration has been proved to reduce mercuric chloride toxicity (Chatterjee and Rudra Pal, 1975), suggesting a relationship between these compounds. Besides, mercuric ions lead to the oxidation of ascorbic acid in vitro (Sanehi et al., 1975). Since Hg2+ does not react with appreciable affinity with ascorbic acid, it is plausible that the mercury-induced reduction of ascorbic acid levels may be related to the reported mercury-induced increase of reactive oxygen species (Lund et al., 1993). Despite the essentiality of selenium compounds, toxicity may be manifested after the ingestion of levels only slightly higher than those nutritionally required (Spallholz, 1994). Selenium toxicity has been related to the oxidation of endogenous thiol compounds and/or to oxidative stress (Tsen and Tappel, 1958; Spallholz, 1994). In the present study, sodium selenite exposure per se did not induce lipid or thiol oxidation, suggesting that a nontoxic dose of selenite was used. In fact, sodium selenite induced an increase of nonprotein thiol groups content both in liver and in kidney (Tables 1 and 2). This is not the first report on a sodium seleniteinduced increase of thiol group content, since Zia and
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Islam (2000) reported that sodium selenite might induce both a reduction and an increase of thiol group content in different brain regions. Rao et al. (1998) demonstrated that mercury-induced damage in mammalian ascorbic acid metabolism could be prevented by simultaneous administration of sodium selenite. Moreover, El-Demerdash (2001) reported that selenite could prevent mercury-induced oxidative damage (TBARS) when both compounds were simultaneously administered. Although some previous reports did not find protection when mercury-exposed animals were pretreated with selenite (Naganuma et al., 1984; Magos, 1991), in the present study pretreatment with sodium selenite 6 h before HgCl2 exposure prevented mercury effects in kidney (ALA-D activity, TBARS, and ascorbic acid level), but not in liver (ascorbic acid level). In line with this result, Frisk et al. (2001) observed that pretreatment of human K-562 cells with sodium selenite protected against HgCl2 toxicity. Additionally, Wang et al. (2001) observed that sodium selenite pre-treatment of cultured renal cells increased the protective effects of selenium administered later in conjunction with mercury. The in vivo protection of selenium against mercury effects has been attributed to the formation of a Hg–Se– S complex, reducing the availability of mercury (Suzuki et al., 1998; Gailer et al., 2000). The formation of this complex depends on the previous reduction of selenite (Se4+) to selenide (Se2) (Gailer et al., 2000; Farina et al., 2003a), which can be held in red blood cells in vivo (Sasakura and Suzuki, 1998). The reduced form of selenium has been demonstrated to form an equimolar complex with Hg in the plasma, which subsequently binds to selenoprotein P (Yoneda and Suzuki, 1997). In vitro, sodium selenite per se significantly inhibited ALA-D in agreement with previous reports (Barbosa et al., 1998; Jacques-Silva et al., 2001). We have also observed that sodium selenite in vitro is a more potent inhibitor of the sulfhydryl-containing enzyme ALA-D than mercuric chloride (Fig. 1), suggesting that selenite could promptly react with thiol groups in this assay condition. This proposal was confirmed by the rapid depletion of the GSH thiol groups when this molecule was incubated with sodium selenite (10 mM onwards) in assay conditions similar to those used in the ALA-D assay. This result is in agreement with the proposal that an excess of sodium selenite may be toxic due to the oxidation of thiol groups (Tsen and Tappel, 1958; Seko et al., 1989). ALA-D inhibition by selenite is probably related to the catalytic oxidation of sulfhydryl groups of enzyme with a concomitant reduction of Se4+ to Se2 (Farina et al., 2003a) similar to that reported for organic forms of selenium (Maciel et al., 2000; Farina et al., 2002). Contrary to the results obtained in vivo (Table 2), sodium selenite did not prevent mercury effects in vitro
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(ALA-D activity and TBARS production), but potentiated ALA-D inhibition by this metal (Figs. 1 and 2). The S1 fraction used in the in vitro experiments did not contain intact erythrocytes. Therefore, results obtained in vitro could be related to the inability of the S1 fraction to substantially reduce selenite to selenide, which may have prevented the formation of the Hg–Se–S complex. Nevertheless, this hypothesis seems hardly probable because the S1 fraction contains high amounts of glutathione, which was suggested as the reductant agent responsible for selenite reduction in erythrocytes (Gailer et al., 2000). An alternative explanation is that in vivo the inhibitory effect of selenite was not observed because after subcutaneous administration it would be reduced in erythrocytes before an interaction with renal proteins (e.g., ALA-D), while in vitro it would be in direct contact with ALA-D. The results of the present study indicate that although both mercury and selenium are potent inhibitors of ALA-D activity in vitro, pretreatment with a nontoxic dose of sodium selenite partially or totally prevented in vivo mercury effects in kidney, including prevention of ALA-D inhibition. This finding is of particular importance, since it points to a possible preventive use of sodium selenite in populations at risk of mercury exposure, such as mining workers and riverside people in mining areas (Boischio and Henshel, 1996).
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Environmental Research 95 (2004) 166–173
Effects of mercury and selenite on d-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats$ Juliano Perottoni,a Luciana P. Lobato,b Aline Silveira,a Joa˜o Batista Teixeira Rocha,a and Tatiana Emanuellia,b, a
Master Science Course in Biochemical Toxicology, Department of Chemistry, Center of Nature and Exacts Sciences, Federal University of Santa Maria, 97105-900 Santa Maria, RS, Brazil b Department of Alimentary Technology and Sciences, Center of Rural Science, Federal University of Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 10 April 2003; received in revised form 14 August 2003; accepted 29 August 2003
Abstract The present study evaluates the effects of Na2SeO3 and HgCl2 on kidney and liver of adult rats. In vivo, HgCl2 (17 mmol/kg, sc) reduced ascorbic acid levels in liver (B15%), whereas in kidney it reduced ALA-D activity (B60%) and ascorbic acid levels (B35%) and increased TBARS content (B50%). Na2SeO3 (17 mmol/kg, sc) exposure increased the content of nonprotein thiol groups in liver (35–60%) and kidney (B50–160%), partially prevented mercury-induced ALA-D inhibition in kidney, and completely prevented a mercury-induced increase of TBARS content and decrease of ascorbic acid levels in kidney. In vitro, HgCl2 and Na2SeO3 inhibited renal and hepatic ALA-D, while HgCl2 increased TBARS in renal and hepatic tissue preparations. Na2SeO3 increased the rate of glutathione oxidation in vitro. Results indicated that Na2SeO3 protected against HgCl2 effects in vivo (prevention of mercury interaction with thiol groups and of mercury-induced oxidative damage). In vitro, Na2SeO3 did not prevent mercury effects, but potentiated ALA-D inhibition by mercury, probably due to its ability to oxidize thiol groups. r 2003 Elsevier Inc. All rights reserved. Keywords: Sodium selenite; Mercuric chloride; 5-Aminolevulinate dehydratase; Thiobarbituric acid-reactive substances; Oxidative stress
1. Introduction Although mercury has long been considered a toxic metal, it possesses a number of important industrial uses, and poisoning from occupational exposure and environmental pollution continues to be a concern (Boischio and Henshel, 1996; Klaassen, 1996). Inorganic
$ This study was supported by CNPq (Grants 420010/01-7 to T.E. and 470539/01-1 to J.B.T.R.), CAPES, and FAPERGS. J.P. was the recipient of the CAPES Fellowship. L.P.L. was the recipient of a FAPERGS Scientific Initiation Fellowship. T.E. and J.B.T.R. are the recipients of the CNPq Research Fellowship (Procs. 550691/02-2 and 523761/95-3). Studies involving animals were conducted according to the guidelines of the Committee on the Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sa˜o Paulo, Brazil. Corresponding author. Departamento de Tecnologia e Cieˆncia dos Alimentos, Centro de Cieˆncias Rurais, Universidade Federal de Santa Maria, Camobi, Santa Maria-RS 97105-900, Brazil. Fax: +55-55-2208353. E-mail address: [email protected] (T. Emanuelli).
0013-9351/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2003.08.007
mercury in the Hg2+ form has a great affinity for SH groups of endogenous biomolecules (Clarkson, 1997). Thus, it is invariably found in cells and tissues attached to thiol-containing proteins and small-molecular-weight thiols such as cysteine and glutathione (GSH). At relatively low toxic or nontoxic doses, mercury increases the renal content of GSH, probably due to the induction of GSH synthesis (Zalups, 2000). However, higher doses of inorganic mercury decrease the renal content of GSH (Zalups, 2000). Thiol-containing enzymes, such as daminolevulinate dehydratase (ALA-D), are inhibited by mercury (Rocha et al., 1995, 2001; Emanuelli et al., 1996). Additionally, mercury can also give rise to free radicals that induce lipid, protein, and DNA oxidation (Lund et al., 1993; Clarkson, 1997). The exact mechanisms underlying free-radical production are still not completely understood, but Woods et al. (1990a, b) have shown that in vitro Hg2+ both hinders the antioxidant potential of glutathione and yields reactive species via thiol complexation. Lipid oxidation yields hydroperoxides and aldehydes, such as malondialdehyde. The
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latter may be measured by reactions with thiobarbituric acid (TBARS) and constitutes a useful index of lipid peroxidation (Ohkawa et al., 1979). Sulfhydryl compounds are considered the most effective agents in the chelation therapy of mercury intoxication (Nielsen and Andersen, 1991). The recommended treatment for intoxication with inorganic or elemental mercury includes dimercaprol or d-penicillamine (Klaassen, 1996). Nevertheless, dimercaprol that possesses a low therapeutic index (Andersen, 1989; Kojima et al., 1989) has been suggested to be neurotoxic (Nogueira et al., 2000) and may occasionally potentiate mercury toxicity (Emanuelli et al., 1996, 1998). Additionally, complexes of Hg2+ with meso-2,3-dimercaptosuccinic acid (Endo and Sakata, 1995; Flora, 1999; Frumkim et al., 2001) and 2,3-dimercaptopropane-1sulfonic acid (Pingree et al., 2001), sulfhydryl compounds that are becoming the drugs of choice for the treatment of heavy metal intoxication, are more potent in vitro inhibitors of hepatic ALA-D than Hg2+ alone (Nogueira et al., unpublished results). Selenium is an essential dietary trace element that plays a crucial role in glutathione peroxidase (Rotruck et al., 1973), thioredoxin reductase (Xia et al., 2003), and other enzymes. It is worth noting that the reduced form of selenium (selenide) presents an affinity constant for mercury even higher than that of sulfhydryl compounds (Clarkson, 1997; Yoneda and Suzuki, 1997). Accordingly, Hg–selenium interactions have been experimentally reported to counteract mercury toxicity (Whanger, 1981; Hoffman and Heinz, 1998; El-Demerdash, 2001). Simultaneous administration of sodium selenite with mercuric chloride usually protects against mercury toxicity in vivo (Naganuma et al., 1984; Rao et al., 1998; El-Demerdash, 2001). Nevertheless, there is some controversy concerning the protection afforded by previous administration of sodium selenite. Some authors have reported an absence of protection (Naganuma et al., 1984) and even an increase of mercury toxicity when selenite has been given 1–2 h before HgCl2 (Magos, 1991). Most studies on Hg–selenium interactions have evaluated selenite protection against mercury-induced glutathione depletion and oxidative damage (Chung et al., 1982; Urano et al., 1997; Yoneda and Suzuki, 1997; Gailer et al., 2000), and, to the best of our knowledge, few studies have evaluated the protective effects of selenite against mercury-induced inhibition of thiol-containing enzymes (Farina et al., 2003a, b). The sulfhydryl-containing enzyme ALA-D, which is susceptible to mercury inhibition (Rocha et al., 1995, 2001; Emanuelli et al., 1996), has recently been demonstrated to be a potential molecular target for selenium intoxication (Maciel et al., 2000; Farina et al., 2001; Jacques-Silva et al., 2001). Therefore, it is of interest to evaluate the combined effect of mercury and selenium on ALA-D activity.
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Considering the unsatisfactory performance of the chelation therapy commonly used in mercury intoxication, it is important to clarify the therapeutic effects of alternative drugs such as sodium selenite. In the present study, we examined whether pretreatment with sodium selenite could provide protection against the effects of HgCl2. ALA-D activity, TBARS production, thiol groups, and ascorbic acid levels were measured in liver and kidney of rats after exposure to sodium selenite and mercuric chloride. The interaction between sodium selenite and mercuric chloride was also investigated in vitro by measuring ALA-D activity and TBARS production in liver and kidney. The effect of sodium selenite on the rate of oxidation of reduced glutathione in vitro has been evaluated as well.
2. Materials and methods 2.1. Animals Adult male Wistar rats from our own breeding colony (250–350 g) were maintained in an air-conditioned room (22–25 C) under natural lighting conditions, with water and food (Guabi, RS, Brasil) ad libitum. Animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sa˜o Paulo, Brazil. 2.2. Chemicals Glacial acetic acid, ortho-phosphoric acid, hydrochloric acid, sodium selenite, sulfuric acid, perchloric acid, ethanol, HgCl2, NaCl, K2HPO4, KH2PO4, 5,50 -dithiobis-(2-nitrobenzoic acid), hydrogen peroxide, and ascorbic acid were obtained from Merck (Rio de Janeiro, RJ, Brazil), and butylated hydroxytoluene, sodium dodecyl sulfate, dimethyl sulfoxide, 2,4-dinitrophenylhydrazine, tris(hydroxymethyl)aminomethane, cysteine, thiobarbituric acid, d-aminolevulinic acid, bovine serum albumin, reduced glutathione, and Coomassie brilliant blue G were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 1,1,3,3-Tetramethoxypropane was obtained from Aldrich (Milwaukee, WI, USA). Tricholoroacetic acid was obtained from Reagen (Rio de Janeiro, RJ, Brazil). p-Dimethylaminobenzaldehyde was obtained from Riedel (Hae¨n, Germany). 2.3. Exposure A nephrotoxic dose of mercuric chloride was chosen based on the studies of Fukino et al. (1984) and Emanuelli et al. (1996), in which acute exposure to a similar dose increased lipid peroxidation and decreased ALA-D activity in renal tissue. Considering that the
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molar ratio of Hg2+ to Se is 1:1 (Sasakura and Suzuki, 1998), the dose of sodium selenite was equimolar to that of mercuric chloride. Sodium selenite and HgCl2 solutions were prepared in 120 mM NaCl and 10 mM phosphate buffer, pH 7.4. For the in vivo studies, rats (25) received a subcutaneous injection (1 mL/kg body wt) of vehicle (120 mM NaCl, 10 mM phosphate buffer, pH 7.4) or sodium selenite (17 mmol/kg). After 6 hours, animals were subcutaneously injected (1 mL/kg body wt) with vehicle or HgCl2 (17 mmol/kg). Rats were anaesthetized with ether and killed by decapitation 12 h after the last injection to determine ALA-D activity, TBARS, thiol groups, and ascorbic acid content. 2.4. ALA-D activity Kidneys and livers of treated animals were quickly removed, placed on ice, and homogenized in 150 mM NaCl of 7 and 10 vol, respectively. The homogenate was centrifuged at 4000g at 4 C for 10 min to yield a lowspeed supernatant fraction (S1) that was used for enzyme assay. The enzyme assay was carried out as described by Sassa (1982) by measuring the rate of product (porphobilinogen) formation, except that 200 mM potassium phosphate buffer, pH 6.4, and 2.5 mM d-aminolevulinic acid were used. The reaction was started 10 min after the addition of the enzyme preparation (0.8–1.1 mg of protein) by the addition of the substrate and carried out for 90 min at 39 C. The reaction product was determined using a modified Ehrlich’s reagent at 555 nm with a molar absorption coefficient of 6.1 104 for the Ehrlich-porphobilinogen salt. In vitro ALA-D assays were carried out as described above, except that untreated animals were used and HgCl2 (0–100 mM) and/or sodium selenite (0–40 mM) was included in the incubation assay. 2.5. TBARS determination Kidneys and livers of treated animals were homogenized in 7 vol of a medium containing 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4, and 7.2 mM butylated hydroxytoluene to prevent further oxidation. The homogenate was centrifuged at 4000g at 4 C for 10 min and immediately used to determine thiobarbituric acid-reactive substances. TBARS were assayed as described by Ohkawa et al. (1979). Briefly, samples were incubated at 100 C for 120 min in a medium containing 0.45% sodium dodecyl sulfate, 100 mM hydrochloric acid, 1.4 M acetic acid, pH 3.4, and 0.3% thiobarbituric acid. After centrifugation, the reaction product was determined at 532 nm using 1,1,3,3-tetramethoxypropane as a standard. In vitro TBARS determination was carried out as described above, except that untreated animals were used, butylated hydroxytoluene was omitted, and tissue
supernatants were preincubated at 37 C for 60 min in a medium containing HgCl2 (0–400 mM) and/or sodium selenite (0–40 mM) before the TBARS assay was carried out. 2.6. Thiol group determination Thiol groups were determined as described by Ellman (1959) at 412 nm, after reaction with 5,50 -dithio-bis-(2nitrobenzoic) acid. Tissues of treated animals were homogenized in 7 vol of 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4, and centrifuged at 4000g at 4 C for 10 min, and supernatants were used to determine the content of total thiol groups. Nonprotein thiol groups were determined in the fraction obtained after dilution of the supernatants with 1 vol of 4% trichloroacetic acid followed by centrifugation and neutralization with 1 M tris(hydroxymethyl)aminomethane. A standard curve using cysteine was used to calculate the content of total and nonprotein thiol groups in the tissue samples. 2.7. Ascorbic acid determination Ascorbic acid determination was performed as described by Benderitter et al. (1998) and Jacques-Silva et al. (2001). Briefly, tissues of treated animals were homogenized in 7 vol of 10 mM tris(hydroxymethyl)aminomethane/HCl, pH 7.4 and centrifuged at 4000g at 4 C for 10 min, and protein was removed by dilution with 1 vol of 4% trichloroacetic acid followed by centrifugation. Part of the sample was incubated at 37 C in a medium containing 4.5 mg/mL dinitrophenylhydrazine, 0.6 mg/mL thiourea, 0.075 mg/mL CuSO4, and 0.675 mol/L H2SO4 (final volume of 1 mL). After 3 h, 1 mL 65% H2SO4 was added and the samples were read at 520 nm. A standard curve was constructed using ascorbic acid. 2.8. Determination of the rate of GSH oxidation The rate of GSH oxidation was followed by the disappearance of –SH groups as described by Ellman (1959). Reduced –SH groups of GSH were quantified at 0, 10, 20, 30, and 60 min after the addition of GSH (1 mM) to a reaction mixture containing 200 mM potassium phosphate buffer, pH 6.4, and sodium selenite (0–100 mM) at 39 C. Since no other sulfhydryl compound was added to the incubation medium, it can be considered that GSH was the only compound evaluated by Ellman’s reagent. 2.9. Protein quantification Protein was measured by the method of Bradford (1976) using bovine serum albumin as the standard.
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2.10. Statistical analysis Data on the in vivo effects of selenite and mercury were analyzed by two-way analysis of variance (ANOVA) (2 Se 2 Hg concentrations) followed by Duncan’s multiple-range test when necessary. Data on the in vitro effects of selenite and mercury were analyzed by threeway ANOVA (4 Hg 3 Se 2 tissues for TBARS production and 3 Hg 4 Se 2 tissues for ALA-D activity) followed by Duncan’s multiple range test when necessary. Data on the rate of GSH oxidation were analyzed by two-way ANOVA (4 Se 5 times), taking into account the time variable as a repeated measure. Results with Pp0:05 were considered significant.
3. Results Treatment of rats with a single dose of 17 mmol/kg mercuric chloride did not affect ALA-D activity, TBARS level, total thiol groups, or nonprotein thiol groups in liver, but significantly reduced (B15%) hepatic ascorbic acid content [F ð1; 25Þ ¼ 5:82; Po0:05; Table 1]. Additionally, mercury treatment
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significantly reduced ALA-D activity (B60%) [F ð1; 21Þ; 9.80; Po0:01] and ascorbic acid content (B35%) [F ð1; 21Þ; 10.68, Po0:01], but increased the TBARS level (B50%) in kidney [F ð1; 22Þ; 4.74, Po0:05; Table 2]. Pretreatment with 17 mmol/kg sodium selenite significantly increased the content of nonprotein thiol groups both in liver (35–60%) [F ð1; 21Þ; 9.19; Po0:01; Table 1] and in kidney (B50–160%) [F ð1; 21Þ; 5.74; Po0:03; Table 2]. Moreover, sodium selenite completely prevented the mercury-induced increase of the TBARS level [HgCl2 selenite interaction: F ð1; 22Þ; 10.23; Po0:01] and the decrease of ascorbic acid content in kidney [F ð1; 21Þ; 10.68; Po0:01; Table 2]. Pretreatment with sodium selenite partially prevented the mercury-induced ALA-D inhibition in kidney [F ð1; 21Þ; 5.37; Po0:05; Table 2]. The in vitro effect of sodium selenite and mercuric chloride on TBARS production and ALA-D activity in kidney and liver are shown in Figs. 1 and 2. Mercuric chloride significantly increased TBARS production by renal or hepatic tissue preparations in vitro [F ð3; 128Þ; 41.29; Po0:01; Fig. 1]. Sodium selenite did not affect basal TBARS production or the mercury-induced TBARS increase in vitro (Fig. 1).
Table 1 Effect of sodium selenite treatment on hepatic toxicity induced by mercuric chloride Na2SeO3
Control
ALA-D activity (nmol PBG/h/mg protein) TBARS level (nmol MDA/mg protein) Total thiol groups (nmol SH/mg protein) Non protein thiol groups (mmol SH/g tissue) Ascorbic acid (mg/g tissue)
Saline
HgCl2
Saline
HgCl2
11.9471.55 2.8970.37 113.3377.11 7.8071.17 145.1978.64
13.0971.61 2.3670.32 109.37717.01 6.6270.32 120.8978.13b
13.4472.47 2.4970.24 126.4079.13 10.5271.39a 137.5972.32
11.9471.83 3.1470.40 119.7977.55 10.7371.35a 117.09719.17b
Rats were subcutaneously injected with Na2SeO3 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg). After 6 h rats were injected with HgCl2 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg, sc). Twelve hours later, animals were killed and the hepatic toxicity was evaluated. Data are expressed as means7SEM (n ¼ 528). a Significantly different from the control groups (Po0:05). b Significantly different from the saline groups (Po0:05). Table 2 Effect of sodium selenite treatment on renal toxicity induced by mercuric chloride Na2SeO3
Control Saline ALA-D activity (nmol PBG/h/mg protein) TBARS (nmol MDA/mg protein) Total thiol groups (nmol SH/mg protein) Non protein thiol groups (mmol SH/g tissue) Ascorbic acid (mg/g tissue)
5.1270.46 1.9670.16 58.5677.35 0.9970.36 75.4777.03
HgCl2 a
1.9970.30 2.8970.19a 76.1779.37 0.8770.17 49.8974.80a
Saline
HgCl2
6.8771.43 2.0570.18 61.2976.41 1.4770.42c 75.7676.71
4.3970.57b 1.8770.14 64.6574.87 2.2870.59c 97.91711.20
Rats were subcutaneously injected with Na2SeO3 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg). After 6 h rats were injected with HgCl2 (17 mmol/kg) or 120 mM NaCl, 10 mM potassium phosphate buffer, pH 7.4 (1 mL/kg, sc). Twelve hours later, animals were killed and the renal toxicity was evaluated. Data are expressed as means7SEM (n ¼ 528). a Significantly different from other groups (Po0:05). b Significantly different from the Na2SeO3–saline group (Po0:05). c Significantly different from the control groups (Po0:05).
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Fig. 1. The in vitro effect of sodium selenite (Na2SeO3) and mercuric chloride (HgCl2) on hepatic (A) and renal (B) TBARS production. The tissue preparation was preincubated with Na2SeO3 and HgCl2 (added simultaneously) at 39 C for 60 min before TBARS measurement. TBARS production is expressed as a percentage of control (absence of Na2SeO3 and HgCl2) (mean7SEM, n ¼ 426). The control TBARS production for hepatic and renal preparation was 6.21 and 6.73 nmol MDA formed/mg of protein, respectively.
Fig. 2. The in vitro effect of sodium selenite (Na2SeO3) and mercuric chloride (HgCl2) on hepatic (A) and renal (B) ALA-D activity. The enzyme preparation was preincubated with Na2SeO3 and HgCl2 (added simultaneously) at 39 C for 10 min before the enzyme reaction was started. The ALA-D activity is expressed as a percentage of control activity (absence of Na2SeO3 and HgCl2) (mean7SEM, n ¼ 729). The control activities of hepatic and renal ALA-D were 10.40 and 4.81 nmol porphobilinogen formed/h/mg of protein, respectively.
Both renal and hepatic ALA-D activities were significantly inhibited by mercuric chloride [100 mM; F ð2; 132Þ; 33.26; Po0:01] and sodium selenite [12 mM onwards, F ð3; 132Þ; 43.98; Po0:01; Figs. 2A and B] in vitro. Three-way ANOVA (2 tissue 4 selenite concentrations 3HgCl2 concentrations) also revealed a significant selenite HgCl2 interaction [F ð6; 132Þ; 4.22; Po0:01], indicating a synergistic effect of these compounds on ALA-D activity (Fig. 2). In order to clarify the interaction of sodium selenite with the thiol groups in assay conditions similar to those used in ALA-D assays, we determined the effect of sodium selenite on the disappearance of reduced glutathione sulfhydryl groups (Fig. 3). Two-way ANOVA (4 selenium concentrations 5 times) revealed a significant selenium time [F ð4; 48Þ; 6.42; Po0:01] interaction, indicating that the oxidative effect of sodium selenite (10 mM onwards) on glutathione increased with time.
4. Discussion Inorganic mercury has a nonuniform distribution after absorption, being accumulated mainly in kidneys (Klaassen, 1996; Emanuelli et al., 1996). Accordingly, the results obtained indicated that subcutaneous exposure to a single dose of mercuric chloride significantly increased the TBARS level and decreased the ALA-D activity and ascorbic acid level in kidneys, while there was only a marginal decrease in the ascorbic acid level in liver (Tables 1 and 2). We have previously demonstrated that mercuric chloride exposure leads to ALA-D inhibition (Emanuelli et al., 1996; Rocha et al., 1995, 2001). The high bonding affinity between mercury and thiol groups is probably responsible for this inhibition and has been implicated in the toxicity of mercuric ions (Clarkson, 1997). Although the mercury-induced decrease of both total and nonprotein thiol groups have been reported previously
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Fig. 3. The effect of sodium selenite (Na2SeO3) on the rate of glutathione (1 mM) oxidation. Sulfhydryl groups of glutathione were evaluated at 412 nm with 5,50 -dithio-bis-(2-nitrobenzoic acid). Results are the means of four independent experiments. SEM were less than 10% of respective means.
(Stohs and Bagchi, 1995), in the present study we could not observe any alteration in the level of renal thiol groups (Table 2). The result obtained may be related to the dose of mercury used, since previous studies revealed that low toxic or nontoxic doses increase renal content of the nonprotein thiol compound GSH, while higher doses decrease the renal content of GSH (Zalups, 2000). The mercury-induced reduction of the ascorbic acid level and the increase of the TBARS level are in agreement with literature data suggesting that mercury treatment may induce oxidative damage (Lund et al., 1993; Stohs and Bagchi, 1995). Ascorbic acid administration has been proved to reduce mercuric chloride toxicity (Chatterjee and Rudra Pal, 1975), suggesting a relationship between these compounds. Besides, mercuric ions lead to the oxidation of ascorbic acid in vitro (Sanehi et al., 1975). Since Hg2+ does not react with appreciable affinity with ascorbic acid, it is plausible that the mercury-induced reduction of ascorbic acid levels may be related to the reported mercury-induced increase of reactive oxygen species (Lund et al., 1993). Despite the essentiality of selenium compounds, toxicity may be manifested after the ingestion of levels only slightly higher than those nutritionally required (Spallholz, 1994). Selenium toxicity has been related to the oxidation of endogenous thiol compounds and/or to oxidative stress (Tsen and Tappel, 1958; Spallholz, 1994). In the present study, sodium selenite exposure per se did not induce lipid or thiol oxidation, suggesting that a nontoxic dose of selenite was used. In fact, sodium selenite induced an increase of nonprotein thiol groups content both in liver and in kidney (Tables 1 and 2). This is not the first report on a sodium seleniteinduced increase of thiol group content, since Zia and
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Islam (2000) reported that sodium selenite might induce both a reduction and an increase of thiol group content in different brain regions. Rao et al. (1998) demonstrated that mercury-induced damage in mammalian ascorbic acid metabolism could be prevented by simultaneous administration of sodium selenite. Moreover, El-Demerdash (2001) reported that selenite could prevent mercury-induced oxidative damage (TBARS) when both compounds were simultaneously administered. Although some previous reports did not find protection when mercury-exposed animals were pretreated with selenite (Naganuma et al., 1984; Magos, 1991), in the present study pretreatment with sodium selenite 6 h before HgCl2 exposure prevented mercury effects in kidney (ALA-D activity, TBARS, and ascorbic acid level), but not in liver (ascorbic acid level). In line with this result, Frisk et al. (2001) observed that pretreatment of human K-562 cells with sodium selenite protected against HgCl2 toxicity. Additionally, Wang et al. (2001) observed that sodium selenite pre-treatment of cultured renal cells increased the protective effects of selenium administered later in conjunction with mercury. The in vivo protection of selenium against mercury effects has been attributed to the formation of a Hg–Se– S complex, reducing the availability of mercury (Suzuki et al., 1998; Gailer et al., 2000). The formation of this complex depends on the previous reduction of selenite (Se4+) to selenide (Se2) (Gailer et al., 2000; Farina et al., 2003a), which can be held in red blood cells in vivo (Sasakura and Suzuki, 1998). The reduced form of selenium has been demonstrated to form an equimolar complex with Hg in the plasma, which subsequently binds to selenoprotein P (Yoneda and Suzuki, 1997). In vitro, sodium selenite per se significantly inhibited ALA-D in agreement with previous reports (Barbosa et al., 1998; Jacques-Silva et al., 2001). We have also observed that sodium selenite in vitro is a more potent inhibitor of the sulfhydryl-containing enzyme ALA-D than mercuric chloride (Fig. 1), suggesting that selenite could promptly react with thiol groups in this assay condition. This proposal was confirmed by the rapid depletion of the GSH thiol groups when this molecule was incubated with sodium selenite (10 mM onwards) in assay conditions similar to those used in the ALA-D assay. This result is in agreement with the proposal that an excess of sodium selenite may be toxic due to the oxidation of thiol groups (Tsen and Tappel, 1958; Seko et al., 1989). ALA-D inhibition by selenite is probably related to the catalytic oxidation of sulfhydryl groups of enzyme with a concomitant reduction of Se4+ to Se2 (Farina et al., 2003a) similar to that reported for organic forms of selenium (Maciel et al., 2000; Farina et al., 2002). Contrary to the results obtained in vivo (Table 2), sodium selenite did not prevent mercury effects in vitro
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(ALA-D activity and TBARS production), but potentiated ALA-D inhibition by this metal (Figs. 1 and 2). The S1 fraction used in the in vitro experiments did not contain intact erythrocytes. Therefore, results obtained in vitro could be related to the inability of the S1 fraction to substantially reduce selenite to selenide, which may have prevented the formation of the Hg–Se–S complex. Nevertheless, this hypothesis seems hardly probable because the S1 fraction contains high amounts of glutathione, which was suggested as the reductant agent responsible for selenite reduction in erythrocytes (Gailer et al., 2000). An alternative explanation is that in vivo the inhibitory effect of selenite was not observed because after subcutaneous administration it would be reduced in erythrocytes before an interaction with renal proteins (e.g., ALA-D), while in vitro it would be in direct contact with ALA-D. The results of the present study indicate that although both mercury and selenium are potent inhibitors of ALA-D activity in vitro, pretreatment with a nontoxic dose of sodium selenite partially or totally prevented in vivo mercury effects in kidney, including prevention of ALA-D inhibition. This finding is of particular importance, since it points to a possible preventive use of sodium selenite in populations at risk of mercury exposure, such as mining workers and riverside people in mining areas (Boischio and Henshel, 1996).
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