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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract:  The aim of this study was to assess the toxic effects of chronic exposure to fluphenazine in liver and kidney of rats, as well as the possible protective effect of diphenyl diselenide on the fluphenazine-induced damage. Long-term treatment with fluphenazine caused an increase in lipid peroxidation levels in liver and kidney homogenates. Diphenyl diselenide treatment did not affect δ-aminolevulinate dehydratase (δ-ALA-D) activity, but fluphenazine alone or in combination with diphenyl diselenide showed an inhibitory effect on δ-ALA-D activity in liver. Diphenyl diselenide plus fluphenazine treatment increased the reactivation index of hepatic δ-ALA-D by approximately 80%. Superoxide dismutase activity decreased in liver of rats treated with fluphenazine alone. The combined treatment with fluphenazine and diphenyl diselenide was able to ameliorate superoxide dismutase activity in liver of rats. Catalase activity was augmented in liver from rats treated with fluphenazine, and this increase was prevented when diphenyl diselenide was co-administered. Taken together, these results indicate that the association of diphenyl diselenide with fluphenazine could protect the liver from lipid peroxidation and ameliorate superoxide dismutase and catalase activities. Moreover, our data point to the relationship between the oxidative stress and fluphenazine treatment in liver and kidney of rats.

Fluphenazine is one of the three anti-psychotic drugs enlisted in the recent (14th) World Health Organization Model List of Essential Medicines [1]. However, the use of this anti-psychotic medication can be associated with tardive dyskinesia, a debilitating involuntary hyperkinetic movement disorder, in 20–50% of individuals with a psychotic illness during chronic treatment [1–4]. Of particular importance, the use of neuroleptic drugs and the symptoms of tardive dyskinesia in humans or orofacial dyskinesia in rodents have been associated with oxidative stress [5–8].

The use of phenothiazines like fluphenazine has been associated with hepatic injury [9–11]. Indeed, agranulocytosis and the release of transaminase enzymes from liver cells are described as consequences of neuroleptic drug use [12]. Zimmerman [13] reported elevations of serum aspartate aminotransferase and serum alanine aminotransferase (ALT) on persons taking chlorpromazine, a phenothiazine derivative. The available evidence suggests that the release of AST and ALT can be due to the direct cytotoxic effect of phenothiazines on liver cells [14]. Isolated elevations of hepatic enzymes occur frequently with phenothiazine drugs (frequency evaluated to 20%) [15]. In this vein, literature has indicated that phenothiazines cause cytotoxicity in hepatocytes, which can be attributed to oxidative stress since it was prevented by antioxidants in vitro [16].

Seleno-organic compounds have been studied based on their potential antioxidant properties [17,18]. In fact, this class of compounds exhibits glutathione peroxidase-like activity and oxidizes sulfhydryl groups (-SH) during the reduction of H2O2[19–21]. Ebselen, a seleno-organic compound, has antioxidant properties in a variety of in vitro and in vivo models of neurotoxicity in rats [22–24]. Recent data from our laboratory indicated that ebselen plays a protective role against haloperidol-induced orofacial dyskinesia and reverses the increase in thiobarbituric acid-reactive species (TBARS) production caused by haloperidol administration [25]. Ebselen has also been demonstrated to protect the liver when injury was induced by paracetamol, CCl4, lipopolysaccharide and Propionibacterium acnes, alcohol and ischaemia-reperfusion injury [26].

The simplest of diaryl diselenides, diphenyl diselenide, has been shown to be even more active as a glutathione peroxidase mimic [27] and less toxic to rodents than ebselen [28]. Recently, data from our laboratory have indicated that diphenyl diselenide decreased the prevalence of vacuous chewing movements induced by long-term treatment with fluphenazine in rats [29]. Furthermore, diphenyl diselenide has a protective role in a variety of experimental models associated with the overproduction of free radicals in the brain, liver and kidney [26,30–32]. In addition, several researchers have demonstrated that liver is a therapeutic target of seleno-organic compounds, as well as the various clinical conditions in which hydroperoxides play a role [26].

δ-Aminolevulinate dehydratase (δ-ALA-D) is a sulfhydryl-containing enzyme highly susceptible to oxidizing agents and is inhibited in different pro-oxidant situations [33–36]. The inhibition of δ-ALA-D may impair the haem biosynthesis and may result in the accumulation of aminolevulinic acid (ALA) that has been demonstrated to be a pro-oxidant molecule under significant physiological conditions [37–39]. Based on this, δ-ALA-D can be suggested as a marker of oxidative stress.

The hepatotoxicity mechanism of phenothiazines is not completely understood [10,14,15]. In spite of the several reports of hepatic injury by phenothiazinic drugs, few data are available about their effects on kidney and the role of oxidative stress on these effects. In this way, the rationale for this study was to evaluate the oxidative stress in the liver and kidney of rats chronically treated with fluphenazine, a phenothiazine, as well as to assess the potential protective effect of diphenyl diselenide on the fluphenazine-induced damage.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Chemicals.  Fluphenazine enantate (Flufenan®) was kindly donated by Cristália (Brazil). Diphenyl diselenide was synthesized by the method previously described [40]. Thiobarbituric acid, aminolevulinic acid and dl-dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO, USA). HgCl2, NaCl, K2HPO4, KH2PO4, trichloroacetic acid (TCA), para-dimethylaminobenzaldehyde, and glacial acetic acid were purchased from Reagen (Rio de Janeiro, RJ, Brazil). All other chemicals were purchased from Merck (Darmstadt, Germany).

Animals.  Male Wistar rats weighing 270–320 g and aged 3–3.5 months from our own breeding colony were kept in cages of three or four animals each. They were placed in a room with controlled temperature (22 ± 3°) on a 12-hr light/dark cycle with lights on at 7:00 a.m. and had continuous access to food and water. The animals were maintained and used in accordance with the guidelines of the Brazilian Association for Laboratory Animal Science.

Treatment.  For chronic treatment, 36 animals were used in total. Rats were divided into control, diphenyl diselenide, fluphenazine and fluphenazine plus diphenyl diselenide groups with nine animals in each group:

  • Control: received soy oil (1 ml/kg) every 21 days intramuscularly (i.m.) and three times a week in alternating days subcutaneously (s.c.);

  • Diphenyl diselenide: received diphenyl diselenide 3 times a week in alternating days (1 mg/kg, s.c.), and the vehicle (1 ml/kg soy oil) was administered at each 21 days (i.m.);

  • Fluphenazine: received fluphenazine enantate at each 21 days (25 mg/kg, i.m.), and the vehicle (1 ml/kg soy oil) was administered three times a week in alternating days (s.c.);

  • Combined treatment: received fluphenazine enantate at each 21 days (25 mg/kg, i.m.), and diphenyl diselenide three times a week in alternating days (1 mg/kg, s.c.).

The treatment was carried out over the course of 6 months and was based on previous studies [4,29,41–43].

Tissue preparation.  Animals were killed by decapitation. Liver and kidney were quickly removed, placed on ice and homogenized at seven and five volumes of 0.9% NaCl, respectively. The homogenates were centrifuged at 4000 ×g for 10 min. to yield a low-speed supernatant fraction (S1) that was used for the biochemical and enzymatic assays. To perform superoxide dismutase and catalase assays, S1 was diluted as described in the respective sections.

Lipid peroxidation assay.  Thiobarbituric acid reactive species (TBARS) were determined as described by Ohkawa et al. [44]. In brief, samples were incubated at 100° for 1 hr in a medium containing 8.1% sodium dodecyl sulfate, 1.4 M acetic acid pH 3.4 and 0.6% thiobarbituric acid. The pink chromogen produced in the reaction was measured spectrophotometrically at 532 nm. Results were expressed as nmol of TBARS/gram of tissue.

Enzyme assays.

δ-ALA-D activity.  δ-ALA-D activity was assayed according to the method of Sassa [45] by measuring the rate of product (porphobilinogen/PBG) formation. The reaction product was determined using modified Ehrlich's reagent at 555 nm with a molar absorption coefficient of 6.1 × 10M−1 for the Ehrlich-PBG salt. The incubation medium contained an aliquot of tissue supernatants and potassium phosphate buffer (pH 6.8) 0.084 M. The reaction was initiated by the addition of δ-ALA 2.4 mM and the incubations were carried out for 90 and 150 min., for liver and kidney respectively, at 37°. Afterwards, the reaction was stopped by the addition of TCA 10% containing HgCl2 0.01 M. The activity of δ-ALA-D was expressed as nmol of PBG/mg of protein/hr. Simultaneously, a set of tubes was assayed using the same protocol, except that 2 mM dl-dithiothreitol was added in order to obtain the reactivation index. dl-dithiothreitol is a –SH reducing agent that has been used in vitro to prevent and/or revert δ-ALA-D inhibition by oxidizing agents [33–36]. The reactivation index indicates the extent of the reactivation of δ-ALA-D activity. The reactivation index of δ-ALA-D activity was calculated as follows:

  • image

Superoxide dismutase activity.  To verify superoxide dismutase activity, S1 of kidney and liver were adequately diluted to 40 and 60 volumes with 0.9% NaCl, respectively, and the assay was performed according to the method of Misra and Fridovich [46]. Briefly, epinephrine rapidly autooxidizes at pH 10.5 producing adrenochrome, a pink-coloured product that can be detected at 480 nm. The addition of samples (10, 20 and 30 µl) containing superoxide dismutase inhibits the autooxidation of epinephrine. The rate of inhibition was monitored during 180 sec. at intervals of 30 sec. The amount of enzyme required to produce 50% inhibition at 25° was defined as one unit of enzyme activity (UI).

Catalase activity.  Catalase activity was measured by the method of Aebi [47]. An aliquot of liver and kidney supernatants (10 µl) diluted with 60 and 40 volumes of 0.9% NaCl, respectively, was added to a quartz cuvette and the reaction was started by the addition of freshly prepared H2O2 (30 mM) in phosphate buffer (50 mM, pH 7). The rate of H2O2 decomposition was measured spectrophotometrically at 240 nm during 120 sec. at intervals of 15 sec. Catalase activity was expressed as percentage of control.

Protein measurement.  Protein was assayed by the method of Lowry et al. [48] with bovine serum albumin as standard.

Statistical analysis.  Data were analysed statistically by one-way anova, followed by Duncan's post hoc tests. The results were considered statistically significant when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Lipid peroxidation.

Chronic treatment with fluphenazine increased TBARS production in the liver when compared to control and diphenyl diselenide groups (P < 0.05). Diphenyl diselenide administration did not modify hepatic TBARS levels. However, in the combined treatment, diphenyl diselenide caused a decrease in hepatic TBARS levels observed after fluphenazine treatment, returning TBARS levels to control values (fig. 1A, P < 0.05).

image

Figure 1. Effect of diphenyl diselenide and/or fluphenazine treatments on TBARS production in liver (A) and kidney (B) homogenates. Data are expressed as mean ± S.E.M. for nine rats per group. Experiments were performed in duplicates. *P < 0.05 as compared to control group by Duncan's multiple range test. P < 0.05 as compared to fluphenazine group by Duncan's multiple range test.

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Long-term treatment with fluphenazine caused an increase in TBARS levels of about 50% in kidney homogenates when compared to the control group (P < 0.05). Treatment with diphenyl diselenide did not change the TBARS levels in kidney. However, when the combined treatment was used, diphenyl diselenide protected from the increase in renal TBARS levels (fig. 1B, P < 0.05).

δ-ALA-D activity.

Diphenyl diselenide treatment did not change hepatic δ-ALA-D activity (fig. 2A). However, fluphenazine inhibited δ-ALA-D activity in liver (fig. 2A, P < 0.05) and the combined treatment did not restore the enzyme activity. In vitro, dl-dithiothreitol, a classical agent that restores oxidized thiol groups, caused an increase in hepatic δ-ALA-D activity of all groups (fig. 2B). In fact, the combined treatment increased the hepatic δ-ALA-D reactivation index and this increase was the highest among the groups (fig. 2C, P < 0.05).

image

Figure 2. Effect of diphenyl diselenide and/or fluphenazine treatments on δ-ALA-D activity in liver homogenates. (A) Enzyme determined without dl-dithiothreitol (DTT), (B) in the presence of 2 mM DTT and, (C) on the enzyme reactivation index. Data are expressed as mean ± S.E.M. for nine rats per group. Experiments were performed in duplicates. *P < 0.05 as compared to control group by Duncan's multiple range test.

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In kidney, fluphenazine alone did not alter δ-ALA-D activity (fig. 3A). However, when the combined treatment was used, the δ-ALA-D activity decreased when compared to the control and fluphenazine groups (fig. 3A, P < 0.05). In vitro, dl-dithiothreitol restored δ-ALA-D activity in kidney homogenates (fig. 3B) as well as in the liver. Renal δ-ALA-D reactivation index values were not modified by diphenyl diselenide, fluphenazine or combined treatment (fig. 3C).

image

Figure 3. Effect of diphenyl diselenide and/or fluphenazine treatments on δ-ALA-D activity in kidney homogenates. (A) Enzyme determined without dl-dithiothreitol (DTT), (B) in the presence of 2 mM DTT and, (C) on the enzyme reactivation index. Data are expressed as mean ± S.E.M. for nine rats per group. Experiments were performed in duplicates. *P < 0.05 as compared to control group by Duncan's multiple range test. P < 0.05 as compared to fluphenazine group by Duncan's multiple range test.

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Superoxide dismutase activity.

Long-term treatment with fluphenazine caused a significant decrease (about 50%) in hepatic superoxide dismutase activity (P < 0.05). Treatment with diphenyl diselenide did not modify hepatic superoxide dismutase activity. However, in the combined treatment, diphenyl diselenide compound recovered hepatic superoxide dismutase activity inhibited by fluphenazine. In fact, the activity of the combined treatment was not significantly different from control or diphenyl diselenide groups (fig. 4A). Isolated treatment with either diphenyl diselenide or fluphenazine did not change renal superoxide dismutase activity. However, the combined treatment caused a reduction in renal superoxide dismutase activity (fig. 4B, P < 0.05).

image

Figure 4. Effect of diphenyl diselenide and/or fluphenazine treatments on superoxide dismutase (SOD) activity in liver (A) and kidney (B) homogenates. Data are expressed as mean ± S.E.M. for nine rats per group. Experiments were performed in duplicates. *P < 0.05 as compared to control group by Duncan's multiple range test.

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Catalase activity.

Treatment with fluphenazine caused a significant increase in catalase activity of rat liver homogenates (P < 0.05). Isolated treatment with diphenyl diselenide did not change hepatic catalase activity. However, combined treatment partially prevented the increase in catalase activity caused by fluphenazine (fig. 5A). Renal catalase activity was not modified by diphenyl diselenide, fluphenazine or the combined treatment (fig. 5B).

image

Figure 5. Effect of diphenyl diselenide and/or fluphenazine treatments on catalase (CAT) activity in liver (A) and kidney (B) homogenates. Data are expressed as mean ± S.E.M. for nine rats per group. Experiments were performed in duplicates. *P < 0.05 as compared to control group by Duncan's multiple range test. The catalase activity in control groups was 291.41 and 173.25 µmol H2O2/mg protein/min for liver and kidney enzyme, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present investigation was carried out with the purpose to evaluate oxidative stress in liver and kidney of rats chronically treated with fluphenazine. We demonstrated here that the long-term treatment with fluphenazine caused an increase in lipid peroxidation (TBARS), a reduction in superoxide dismutase activity and an increase in catalase activity in liver, showing a relationship of fluphenazine administration and oxidative stress. Fluphenazine also induced lipid peroxidation in kidney, although catalase and superoxide dismutase activities were not altered in this organ.

Phenothiazines are extensively oxidized in the body to form cation radicals [49], which are believed to be sulphur-centred cation radicals [50,51]. It has been hypothesized that peroxidase-catalyzed drug oxidation causes protein binding and oxidative stress, which can contribute with cell death [52]. Furthermore, hepatocyte cytotoxicity induced by phenothiazines was markedly enhanced by non-toxic concentrations of extracellular H2O2/peroxidase, and also caused ascorbate, GSH and NADH cooxidation and reactive oxygen species formation [16]. In this way, we could suggest that the fluphenazine-induced lipid peroxidation could result from the production of fluphenazine radical metabolites catalyzed by peroxidases.

Fluphenazine chronic treatment induced alterations in hepatic superoxide dismutase and catalase enzymes activities. These findings are consistent with those of Cadet and Perumal [53], who reported alterations of superoxide dismutase and catalase activity in the brain of rats after chronic fluphenazine treatment. The decrease in hepatic superoxide dismutase activity caused by fluphenazine treatment also could contribute to the increase in TBARS levels in the liver.

Diphenyl diselenide was used in this study based on its hepatoprotective and antioxidant properties [31,32]. Indeed, diphenyl diselenide was effective in protecting liver and kidney against lipid peroxidation induced by fluphenazine. This protective effect on TBARS was accompanied by a partial restoration of catalase activity in liver. Diphenyl diselenide was able to ameliorate superoxide dismutase activity in liver of rats treated with fluphenazine. In this way, the protective effects of diphenyl diselenide could be attributed to the thiol peroxidase-like activity that has been described for organoselenium compounds and to other antioxidant properties of diphenyl diselenide [26,54]. In contrast, the renal superoxide dismutase activity was diminished by the treatment with fluphenazine and diphenyl diselenide. The decrease in superoxide dismutase activity in this group was unexpected and may indicate an interaction between the antioxidant properties of the selenium compound and fluphenazine metabolites. Nevertheless, the administration of diphenyl diselenide was not accompanied by any sign of lipid peroxidation (TBARS) in the kidney.

Fluphenazine treatment caused an inhibition of hepatic δ-ALA-D activity, and the combination of fluphenazine and diphenyl diselenide was unable to restore the enzyme activity. In fact, this combination increased the partial inhibition caused by fluphenazine alone. Diphenyl diselenide could oxidize the cysteinyl residues in the active site of δ-ALA-D [33,34]. However, in this case diphenyl diselenide alone did not affect δ-ALA-D activity, only when associated to fluphenazine. In this way, the interaction of fluphenazine and diphenyl diselenide could provoke the oxidation of δ-ALA-D sulfhydryl groups in a more pronounced way than fluphenazine alone. This is supported by the reactivation index, which was higher in rats treated with the combined treatment than other treated groups.

Quite the opposite of liver, fluphenazine alone did not cause any effect on renal δ-ALA-D activity, although the combination of fluphenazine and diphenyl diselenide resulted in inhibition of δ-ALA-D activity. dl-dithiothreitol was able to restore δ-ALA-D activity in kidney. However, we did not observe differences in the reactivation index for δ-ALA-D among the groups. In this way, we could suggest that the mechanism underlying the inhibitory effect of these compounds on renal δ-ALA-D was not related to the oxidation of -SH groups. The decreased activity of the renal δ-ALA-D in the combined treatment could be attributed to an additive inhibitory effect of fluphenazine and diphenyl diselenide on the enzyme activity.

Drug-induced liver and kidney toxicity is common and can lead to acute liver and kidney failure [55,56]. Neuroleptic drugs have been implicated in biological and/or clinical hepatotoxicity although the precise mechanisms remain unclear [15]. Since anti-psychotics are going to be the drugs of choice for the treatment of psychotic disorders, the understanding of the effects of their action on oxidative stress and oxidative cellular injury may be very important [57]. This study demonstrated for the first time an association between oxidative stress and fluphenazine chronic treatment in liver and kidney of rats, indicating a possible mechanism for fluphenazine hepatotoxicity. Moreover, these data may provide useful indications about the benefits of diphenyl diselenide administration to protect liver from a variety of hepatotoxicants since diphenyl diselenide protected from oxidative damage caused by fluphenazine in the liver of rats. However, we believe that further studies are necessary to test the hypothesis of whether oxidative stress could contribute to the fluphenazine-induced hepatotoxicity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Supported by FINEP research grant ‘Rede Instituto Brasileiro de Neurociência (IBN-Net)’# 01.06.0842–00. Additional support given by CNPq, FAPERGS, CAPES and Cristália-SP. C.W. receives fellowship from CAPES. C.L.D.C., R.F., C.W.N. and J.B.T.R. are the recipients of CNPq fellowships.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References