Chromium(VI) induces oxidative stress in the mouse brain

Toxicology 150 (2000) 137 – 146 www.elsevier.com/locate/toxicol

Chromium(VI) induces oxidative stress in the mouse brain Marina Travacio *, Jose´ Marı´a Polo, Susana Llesuy Quı´mica General e Inorga´nica, Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, Facultad de Farmacia y Bioquı´mica, Uni6ersidad de Buenos Aires, Gu¨emes 4144 -170F, 1425 Buenos Aires, Argentina Received 6 January 2000; accepted 2 June 2000

Abstract Potassium dichromate was given to female Swiss mice (25 mg/kg per day) orally in water for 1 – 3 days. Brain homogenates were prepared to evaluate the occurrence of oxidative stress in this organ through the measurement of the antioxidant defense levels, and the extent of lipid peroxidation. In addition, mitochondrial fractions were isolated from brain homogenates to determine the production of reactive oxygen species in this subcellular fraction. The administration of potassium dichromate for 3 days caused increases of 72 and 74% in superoxide dismutase and catalase activities, respectively, in the homogenates. The treatment with this metal for 3 days increased brain homogenate chemiluminescence and thiobarbituric acid-reactive substances by 34 and 29%, respectively. The brain contents of the non-enzymatic antioxidants a-tocopherol and sulfhydryl groups decreased by 35 and 32%, respectively. Ascorbic acid levels were not modified by the administration of potassium dichromate. Finally, there was a significant increment in the mitochondrial production of oxidants in the brain of treated mice as compared with controls. These results suggest that chromium(VI) produces an increased formation of reactive oxygen species and brain lipid peroxidation. The increase in the antioxidant enzyme activities reflects an adaptive response against oxidative stress, while the reduction in the levels of non-enzymatic antioxidants might be due to their reaction with reactive oxygen species generated during the metabolism of chromium(VI). © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chromium; Brain; Oxidative stress; Lipid peroxidation; Antioxidants

1. Introduction Chromium is a metal that belongs to the first series of the transition elements and it can occur in different oxidation states. The trivalent form of chromium has a strong tendency to form coordi* Corresponding author. Tel: + 54-11-48323024; fax: +5411-43724786. E-mail address: [email protected] (M. Travacio).

nation compounds, while the hexavalent form is usually linked with oxygen (CrO24 − , chromate; Cr2O27 − , dichromate) and it is a strong oxidizing agent (Mertz, 1969). Chromium is a common constituent of organic matter. Mammalian tissues contain variable amounts of this metal and its levels in man are influenced by the diet consumed, age and endocrine disorders such as diabetes. This transition metal has been considered as an essential trace

0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 5 4 - 7

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element since it influences glucose, protein, and fat metabolism through a mechanism related to the action of insulin (Mertz, 1993). Trivalent chromium is poorly absorbed from the gastrointestinal tract while the absorption of chromate appears to be higher. Once absorbed, it appears in the plasma protein fraction and its excretion is mainly urinary (O’Flaherty, 1996). It has been suggested that this transition element has a predilection for proliferating tissues; however, high concentrations have also been found in the brain (Stearns et al., 1995a,b). This heavy metal is a widely used industrial chemical (Galva˜o and Corey, 1987) and it is known to cause cytotoxicity and mutagenesis in humans and animals (Von Burg and Liu, 1993; Bagchi et al., 1995). Chromium(VI) is more potent as a carcinogen in the form of CrO24 − and Cr2O27 − . Both chromium(VI) and chromium(III) are biologically active oxidation states since they are involved in redox cycling with the production of reactive oxygen species (Stohs and Bagchi, 1995). Free radical scavengers such as vitamin E protect cells from chromate-induced cytotoxicity, indicating that chromium-toxic effects may be due to the production of reactive oxygen species. Chromium(VI) enters many types of cells and under physiological conditions can be reduced intracellularly by hydrogen peroxide (H2O2), glutathione reductase, carbohydrates, ascorbic acid and reduced glutathione to produce reactive intermediates and, ultimately, chromium(III) (Stearns et al., 1995a,b). The most important mechanisms of oxygen activation by transition metals involve Fenton/Haber-Weiss chemistry and autoxidation to generate the powerful DNA-damaging hydroxyl radical (HO’) (Bagchi et al., 1995). Cr6 +

− H2O2 + O− + HO’ 2 “ O2 + OH

Hydroxyl radical is genotoxic by modifying DNA bases and cause DNA-protein crosslinks, depurination, and strand scission (Sunderman, 1984). On the other hand, transition metals promote lipid peroxidation in living cells through the generation of reactive oxygen species (Bagchi et al., 1997). The brain is particularly vulnerable to oxygen free radical attack since this organ consumes 20%

of the body’s oxygen. Furthermore, the brain has large amounts of polyunsaturated fatty acids which can be attacked by reactive oxygen species produced during the cellular reduction of chromium(VI) and this process would cause membrane damage (Coyle and Puttfarcken, 1993). Although it has been suggested that the treatment of rodents with chromium(VI) can lead to lipid peroxidation in the brain (Bagchi et al., 1997), a complete characterization of oxidative stress parameters has not yet been carried out in this organ. The purpose of this study was to evaluate the occurrence of oxidative stress in the brain of mice treated with potassium dichromate. Enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic (tissue sulfhydryl groups, a-tocopherol and ascorbic acid) antioxidant defenses were evaluated in mouse brain homogenates. The measurements of spontaneous chemiluminescence and thiobarbituric acid-reactive substances were used to assess brain lipid peroxidation. Since mitochondria is a well known source of reactive oxygen species, the production of oxidants in brain mitochondrial fractions was evaluated by measuring the oxidation of 2%,7%dichlorofluorescein diacetate and luminol-amplified chemiluminescence.

2. Methods

2.1. Animals and treatment Female Swiss mice weighing 25–30 g were used in accordance with institutional guidelines and the Guiding Principles in the Use of Animals in Toxicology. The animals were housed two per cage and allowed to acclimate 5 days prior to experimental use. The mice were maintained at a temperature of 21°C with a 12-h light/dark cycle, and free access to tap water and standard food. Treated mice received 25 mg potassium dichromate (K2Cr2O7) per kg of body weight per day, orally in drinking water for 1–3 days (Bagchi et al., 1995). To ensure that the administration of K2Cr2O7 did not modify K+ serum content, K+ and Na+ were measured in mice serum by using

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an ion selective electrode. None of these ions concentrations was modified in treated animals as compared with control values (K+, 5.9 9 0.3 mEq/l; Na+, 1459 1 mEq/l).

2.2. Preparation of homogenates Mice were sacrificed by cervical dislocation after 1 and 3 days of chromium treatment. The brain was excised, immediately cooled in ice, and homogenized in 120-mM KCl, 30-mM phosphate buffer (pH 7.4). Mouse brain homogenates were centrifuged at 700 ×g for 10 min to discard nuclei and cell debris. The supernatant fraction obtained was called ‘homogenate’.

2.3. Preparation of mitochondrial fractions Brains were obtained as described before and homogenized in MSTE buffer (0.23 M mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM Tris – HCl; pH 7.3). Mouse brain homogenates were centrifuged at 700×g for 10 min and the supernatant fractions were centrifuged at 5000 ×g for 10 min. The pellets were washed twice and re-suspended in 0.2 ml of MSTE buffer. It was considered to consist mainly of heavy intact mitochondria and was termed the ‘mitochondrial fraction’. These procedures were carried out at 0 – 2°C (Boveris et al., 1972).

2.4. Chromium content Total chromium content was measured in brain homogenates of control and treated animals. The samples were digested using a PROLABO MICRODIGEST 301 (France) and total chromium levels were determined by atomic absorption spectrophotometry (atomic absorption spectrophotometer, Varian AA-575, with Carbon rod atomizer CRA-90, Varian Associates, Palo Alto, CA, USA) as described by Ueno et al., 1995. Chromium content was expressed as ppm.

2.5. Enzymatic antioxidants 2.5.1. Superoxide dismutase Superoxide dismutase activity was determined

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in brain homogenates by following the inhibition of the rate of autocatalytic adrenochrome formation at 480 nm with a Hitachi U-2000 (Hitachi, Tokyo, Japan). The reaction medium contained 1 mM epinephrine and 50 mM glycine–NaOH (pH 10.2) (Misra and Fridovich, 1972). The enzyme activity was expressed as U/g brain. One unit corresponds to the volume of homogenate that reduces the adrenaline autoxidation rate to 50% of its value.

2.5.2. Catalase Catalase activity was measured in the homogenates treated with Triton X-100 by following the decrease in absorption at 240 nm. The reaction medium consisted of 50 mM phosphate buffer (pH 7.2) and 10 mM H2O2 (Chance et al., 1979). The results were expressed in mU/g brain. One unit corresponds to 1 nmol catalase per g tissue. 2.5.3. Glutathione peroxidase Glutathione peroxidase activity was measured in the homogenates by following NADPH oxidation at 340 nm as described by Flohe´ and Gunzler (1984). The reaction medium consisted of 30 mM phosphate buffer (pH 7.0), 0.17 mM reduced glutathione, 0.2 U/ml glutathione reductase, and 0.5 mM tert-butyl hydroperoxide. The activity of glutathione peroxidase was expressed as mU/g brain. One unit corresponds to 1 mmol NADPH per min per g tissue. 2.6. Non-enzymatic antioxidants 2.6.1. Tissue sulfhydryl groups Tissue sulfhydryl groups were measured spectrophotometrically at 412 nm in brain homogenates after reaction with 5,5%-dithiobis-(2nitrobenzoic acid). Proteins were eliminated through the addition of 0.5 M HClO4 (Ellman, 1959). Thiol content was expressed in mmol/g tissue. 2.6.2. a-Tocopherol content Brain homogenates were extracted with 4 ml of hexane and centrifuged at 1000× g for 5 min. The hexane layer was removed and evaporated to

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dryness under nitrogen. The residue was dissolved in methanol/ethanol 1/1 v/v. Measurements of a-tocopherol were made by high performance liquid chromatography (HPLC) on an 8-C reverse phase column (Supelco, Bellefonte, PA, USA) with in line electrochemical detection (Bioanalytical System, West Lafayette, IN, USA). a-Tocopherol concentrations were referred to commercial standards and expressed as nmol/g brain (Lucesoli and Fraga, 1995).

2.6.3. Ascorbic acid content Ascorbic acid content was quantified by HPLC with electrochemical detection (Bioanalytical System). Homogenates were treated with 7% (w/v) metaphosphoric acid. After centrifugation at 1500× g for 10 min, 0.3 ml of 0.8% (w/v) metaphosphoric acid were added to 90 ml of the supernatant. Results were expressed in mmol/g tissue (Kutnik et al., 1987). 2.7. Lipid peroxidation 2.7.1. Spontaneous chemiluminescence The chemiluminescence of homogenates was measured using a liquid scintillation counter in the out-of-coincidence mode. The protein content was adjusted to 1 mg/ml of reaction medium consisting of 120 mM KCl, 30 mM phosphate buffer (pH 7.4), in a final volume of 2 ml. Samples were incubated at 30°C for 30 min. The emission was expressed as cpm/mg of protein (Adamo et al., 1989). 2.7.2. Thiobarbituric acid-reacti6e substances (TBARS) TBARS were measured fluorometrically. The calibration curve was carried out with a malondialdehyde (1,1,3,3-tetramethoxipropane) standard. Brain homogenates were treated with 4% w/v butylated hydroxytoluene to prevent the autoxidation of the samples. Samples were treated with 12 mM sodium dodecylsulfate, 50 mM HCl, 0.7% w/v phosphotungstic acid, 0.15% w/v thiobarbituric acid, and heated at 100°C for 60 min. Butanol was used as extraction solvent and fluorescence intensity of the extracts was measured at 515 – 553 nm. Results were expressed in terms of nmol/g of tissue (Yagi, 1976).

2.8. Oxidant species production 2.8.1. Mitochondrial oxidation of 2 %,7 %dichlorofluorescein diacetate (DCFH-dAc) Mitochondrial fraction samples were incubated in 2 ml of 40 mM Tris–HCl buffer (pH 7.0) in the presence of 100 mM DCFH-dAc at 37°C for 1 h. The resulting fluorescent compound was measured after 15, 30, 45, and 60 min of incubation in a fluorescence spectrophotometer (Hitachi F-3000, Hitachi Ltd., Tokyo, Japan) at 488-nm excitation and 525-nm emission wavelengths (Le Bel et al., 1992). Protein content was adjusted to 0.25 mg/ml of reaction medium and corrections for autofluorescence were made by inclusion of parallel blanks (assay mixture without the sample) in each experiment. The results were expressed in arbitrary units of fluorescence intensity per milligram of protein (I per mg protein). 2.8.2. Mitochondrial luminol-amplified chemiluminescence Protein content of mitochondrial fractions was adjusted to 1 mg/ml with 50 mM glycine–NaOH buffer (pH 9.0) and these samples were incubated at 37°C for 30 min in darkness. After the addition of 4 mM luminol, the luminescence was measured in a luminometer (Labsystem Luminoskan, EL, Finland) for 60 min. Results correspond to the maximal value of photoemission and were expressed in arbitrary units of luminescent intensity per milligram of protein (I per mg protein) (Samuni et al., 1991). 2.9. Protein measurement Protein content was assayed by the method of Lowry et al. (1951), using bovine serum albumin as standard.

2.10. Chemicals tert-Butyl hydroperoxide and Trolox were purchased from Aldrich Chemicals (Milwaukee, WI, USA). 2,2%-Azobis (2-amidinopropane) was obtained from Polysciences (Warrington, PA, USA). 1,1,3,3-Tetramethoxypropane and other chemicals were purchased from Sigma (St. Louis, MO, USA).

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2.11. Statistics

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The administration of potassium dichromate to mice produced a significant increase of 60% in the brain content of chromium, after 3 days of treatment (control value (cv), 0.3190.02 ppm). Superoxide dismutase, catalase, and glutathione peroxidase activities were determined in brain homogenates of control and potassium dichromatetreated mice. Superoxide dismutase activity increased significantly by 65% after the first day of chromium(VI) administration as compared with controls (cv, 79 93 U/g brain). This increment remained constant after 3 days of treatment (Fig. 1). Catalase activity showed a different profile. A 1-day treatment did not cause a significant difference in the activity of this enzyme as compared with control values. Notwithstanding, the administration of potassium dichromate for 3 days produced a significant increase of 74% in catalase activity (cv, 1.8490.07 mU/g brain) (Fig. 1). On

the other hand, glutathione peroxidase was not affected by the administration of chromium (cv, 2269 20 mU/g brain) (Fig. 1). The content of sulfhydryl groups decreased progressively in brain homogenates of chromiumtreated mice as compared with controls. The levels of thiol groups reached a significant decrement of 35% after 3 days of potassium dichromate administration (cv, 1.099 0.04 mmol/g brain) (Fig. 2). a-Tocopherol content decreased in chromiumtreated mice. The levels of vitamin E slightly decreased since the first day of treatment (cv, 0.789 0.03 nmol/g brain). After 3 days of chromium administration, the content of a-tocopherol reached a significant reduction of 32% as compared with controls (Fig. 2), paralleling the decrease seen for sulfhydryl groups. Ascorbic acid levels were measured in brain homogenates of control and chromium-treated mice, after 1 and 3 days of treatment. As seen in Fig. 2, the content of ascorbic acid in the brains of treated animals was not modified by the administration of the chromium(VI) salt (cv, 0.739 0.02 mmol/g tissue). Spontaneous chemiluminescence was measured in brain homogenates. The photoemission was not modified after the administration of chromium(VI) for 1 day. However, this parameter increased sig-

Fig. 1. Effect of the administration of potassium dichromate on superoxide dismutase ( ), catalase (), and glutathione peroxidase () activities in brain homogenates. Symbols indicate mean values from six to eight animals and bars indicate S.E.M. *, P B 0.05.

Fig. 2. Time course of the effect of potassium dichromate on sulfhydryl groups ( ), a-tocopherol content (), and ascorbic acid content () of brain homogenates. Symbols indicate mean values from six to animals and bars indicate S.E.M. *, PB 0.05.

Results were expressed as mean values 9S.E.M. ANOVA followed by Student – Newman – Keuls tests were used to analyze the differences between mean values.

3. Results

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Table 1 Lipid peroxidation and oxidant species productiona Oxidative parameters

Homogenates Chemiluminescence (cpm/mg protein) TBARS (nmol/g tissue)

Time of treatment (days) 0

1

3

28 800 91200 4.1 90.1

26 000 9 1500 3.7 9 0.1

38 600 9 800b 5.3 9 0.2b

2.9 90.1 21.2 90.6

2.9 90.1 24.5 9 0.6

Mitochondria Chemiluminescence (I per mg protein)c DCFH-dAc oxidation (I per mg protein)c

4.0 90.3b 30.0 92.0b

a Homogenates brain lipid peroxidation estimated through the measurement of spontaneous chemiluminescence and TBARS. Mitochondria production of brain mitochondrial oxidant species assessed through the measurement of the oxidation of 2%,7%-dichlorofluorescein diacetate (DCFH-dAc) and luminol-amplified chemiluminescence. The values represent mean 9S.E.M. from six to eight animals. b PB0.05. c The results of both DHFH-dAc oxidation and mitochondrial chemiluminescence are expressed in arbitrary units per milligram of protein (I per mg protein).

nificantly by 34% in brain homogenates after the third day of treatment (Table 1). The content of TBARS in brain homogenates of treated mice was not modified by the administration of potassium dichromate to mice for 1 day. However, after 3 days of treatment, the values of TBARS had increased by 29% in brain homogenates (Table 1). The production of reactive oxygen species was determined in mitochondrial fractions isolated from brain homogenates of control and chromium(VI)-treated mice. After 3 days of treatment, the oxidation of DCFH-dAc had increased significantly by 47% as compared with control values (Table 1). The generation of oxidants in brain mitochondria was also estimated through the measurement of luminol-enhanced luminescence. After the third day of chromium administration, the photoemission of mitochondrial fractions had increased by 38% in treated animals as compared with controls (Table 1).

4. Discussion Several authors have suggested that reactive oxygen species are implicated in the toxicity of chromium(VI) (Sugiyama, 1992; Bagchi et al.,

1997). Dı´az-Mayans et al. (1986) have shown that the oral administration of chromium(VI) caused neurotoxicity, described as a significant decrease in the motor activity of animals. Common molecular mechanisms have been suggested to be involved in the toxicity of different xenobiotics, including heavy metals. These mechanisms are related to the production of reactive oxygen species. According to this hypothesis, chromium(VI) itself is not the cytotoxic agent, but rather oxygen free radicals generated through the cellular reduction of chromium(VI) (Miesel et al., 1995). Chromium reduction intermediates are believed to react with hydrogen peroxide to form the hydroxyl radical (Kadiiska et al., 1994), which may finally attack proteins, DNA, and membranes lipids, thereby disrupting cellular functions and integrity (Bagchi et al., 1997). In the present study, the activities of the antioxidant enzymes superoxide dismutase and catalase were increased by chromium-treatment. This response seems to follow a temporal sequence. When chromium was administrated to mice for 1 day, the only parameter significantly modified was the activity of superoxide dismutase, which increased by 65% as compared with control values. After 3 days of treatment, both superoxide dismutase and catalase activities increased by 72 and 74%, respectively. Since superoxide dismutase catalyzes the

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dismutation of superoxide anion to hydrogen peroxide, which is in turn the substrate of catalase, this fact could explain the temporal sequenced response observed in the increment of the two enzyme activities (Fig. 3). It has been reported that catalase and superoxide dismutase inhibited chromate-induced DNA strand breaks in cultured cells (Sugiyama, 1992). As these enzymes have a protective role against oxygen free radical-induced damage, their induction can be understood as an adaptive response to oxidative stress. It has been reported that different models of oxidative stress involve a biphasic response of the antioxidant enzyme activities. First, the enzymatic activities are markedly decreased but at longer times, the activity levels are increased probably as a consequence of de novo synthesis and/or enzymatic activation (Travacio and Llesuy, 1996). In our study, we did not detect this decrement in antioxidant enzyme activities, which might have occurred within the first 24 h of treatment.

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The administration of potassium dichromate to mice caused a progressive decrease of sulfhydryl group content in brain homogenates. This parameter has been related to lipid peroxidation. Isolated hepatocytes supplemented with glutathione suppressed heavy metal-induced lipid peroxidation (Sugiyama, 1992; Fig. 3). Glutathione is the major low molecular weight hydrosoluble thiol antioxidant inside the cells. In our experimental model, an increase in the production of reactive oxygen species is believed to consume glutathione during its metabolism leading to reduced levels of total sulfhydryl groups in brain homogenates. In addition, it has been proposed that heavy metals may be reduced by glutathione to form different complexes (Sugiyama, 1992). Vitamin E content was also decreased as a consequence of the treatment with chromium(VI). Several authors have suggested previously that a-tocopherol has a protective role against chromium-induced damage (Sugiyama, 1991;

Fig. 3. Proposed mechanism of chromium-generated brain oxidative stress, antioxidant defenses against reactive oxygen species, and lipid peroxidation generating light emission (hy) and TBARS. O− 2 , superoxide anion; HO,·hydroxyl radical; H2O2, hydrogen peroxide; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH/GSSG, glutathione; aTOH/aTO’, a-tocopherol; A’−/AH−, ascorbic acid; RH, polyunsaturated fatty acids; R’, carbon-centered radical; ROO’, peroxyl radical; ROOOOR, tetroxide; 1O2, singlet oxygen; CO*, excited carbonyl.

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Kadiiska et al., 1994; Bagchi et al., 1997). This lipid-soluble antioxidant inhibits lipid peroxidation and protects cell membranes against oxidative damage by scavenging peroxyl radicals (Niki, 1996). A reduction in the levels of a-tocopherol in the brain of chromium-treated mice could be related to its reaction with free radicals generated during the physiological reduction of chromium(VI) (Fig. 3). Ascorbate reduces free radicals with the formation of dehydroascorbate, thus acting as an antioxidant (Choi, 1993; Fig. 3). According to this, ascorbic acid is expected to be consumed in a situation of oxidative stress. However, the ascorbic acid content in brain homogenates was not modified by the oral administration of potassium dichromate to mice. Due to the large content of this compound in the brain, its consumption would not cause a significant change in the levels of this antioxidant in the mouse brain. Free radical scavengers such as a-tocopherol and glutathione would inhibit the propagation steps of the free radical chain reaction by interaction with carbon- or oxygen-free radicals. The reduced levels of these non-enzymatic anti-oxidants is in accordance with the results obtained when spontaneous chemiluminescence and TBARS were measured in brain homogenates. Since the brain contains large amounts of polyunsaturated fatty acids, it is particularly susceptible to free radical attack and therefore lipid peroxidation (Coyle and Puttfarcken, 1993). In the present study, lipid peroxidation was assessed by the determination of spontaneous chemiluminescence and TBARS in brain homogenates of control and treated mice. Both parameters increased significantly after 3 days of potassium dichromate administration. These results are in accordance with those obtained by Bagchi et al. (1997) who detected oxidative lipid metabolites in the urine of chromium(VI)-treated rats. The increase observed in lipid peroxidation may be due to the formation of hydroxyl radical (HO’) through a Haber-Weiss reaction, catalyzed by chromium (Fig. 3). This radical is capable of abstracting a hydrogen atom from a methylene group of polyunsaturated fatty acids (RH) enhancing lipid peroxidation. The lu-

minescence produced by lipid peroxidation may be used to monitor cellular oxidative stress (Lissi et al., 1989). The increased light emission detected in our study may be due to excited carbonyls (CO*) or singlet oxygen (1O2), generated during later stages of lipid peroxidation. These excited species return to their ground state by emitting light (Cadenas et al., 1994; Fig. 3). There are several intracellular sources of oxygen free radicals. Since mitochondria is a well known site where these species might be produced, we studied the generation of reactive oxygen species in mitochondrial fractions by two different methods — oxidation of DCFH-dAc and luminol-amplified chemiluminescence (Cadenas, 1985; Le Bel et al., 1992). The oxidation of DCFH, considered as a direct method to estimate the reactive oxygen species formation in mitochondria, significantly increased in brain mitochondria fractions isolated from mice treated with potassium dichromate for 3 days, as compared with controls. Luminol-amplified chemiluminescence is another parameter used to estimate the production of oxidant species in mitochondria and it had also increased after 3 days of chromium administration. These results indicate that chromium(VI) can promote the formation of oxidant species in brain mitochondria leading to a situation of oxidative stress in this organ, particularly taking into account that antioxidant defenses are decreased by the treatment. Therefore, one of the intracellular sites where chromium can catalyze the oxygen free radicals formation is the mitochondrial fraction. In summary, potassium dichromate administration to mice produced brain lipid peroxidation through a mechanism that involves reactive oxygen species. Tissue sulfhydryl groups and a-tocopherol levels were decreased in correlation with the increase in spontaneous chemiluminescence and TBARS. On the other hand, superoxide dismutase and catalase activities were significantly increased after 3 days of chromium(VI) administration reflecting an adaptive response against the oxidative challenge. According to these findings, the biochemical mechanism of cellular damage due to chromium(VI) exposure is believed to be an increase in the steady state concentration of

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oxygen free radicals through a Fenton/Haber – Weiss cycle. Moreover, one of the intracellular sources of reactive oxygen species production increased may be mitochondria according to the results obtained from the measurements of DCFH oxidation and luminol-amplified chemiluminescence.

Acknowledgements The authors wish to express their gratitude to Dr Patin˜o (Departamento de Toxicologı´a del Poder Judicial de la Nacio´n) for the measurements of chromium levels in brain homogenates. This research was supported by grants from Universidad de Buenos Aires (Buenos Aires, Argentina).

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