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The effect of ingested sulfite on visual evoked potentials, lipid peroxidation, and antioxidant status of brain in normal and sulfite oxidase-deficient aged rats Ozlem Ozsoy, Sinem Aras, Ayse Ozkan, Hande Parlak, Mutay Aslan, Piraye Yargicoglu and Aysel Agar Toxicol Ind Health published online 23 October 2014 DOI: 10.1177/0748233714552688 The online version of this article can be found at: http://tih.sagepub.com/content/early/2014/10/22/0748233714552688

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Article

The effect of ingested sulfite on visual evoked potentials, lipid peroxidation, and antioxidant status of brain in normal and sulfite oxidase-deficient aged rats

Toxicology and Industrial Health 1–11 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714552688 tih.sagepub.com

Ozlem Ozsoy1, Sinem Aras1, Ayse Ozkan1, Hande Parlak1, Mutay Aslan2, Piraye Yargicoglu3 and Aysel Agar1 Abstract Sulfite, commonly used as a preservative in foods, beverages, and pharmaceuticals, is a very reactive and potentially toxic molecule which is detoxified by sulfite oxidase (SOX). Changes induced by aging may be exacerbated by exogenous chemicals like sulfite. The aim of this study was to investigate the effects of ingested sulfite on visual evoked potentials (VEPs) and brain antioxidant statuses by measuring superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities. Brain lipid oxidation status was also determined via thiobarbituric acid reactive substances (TBARS) in normal- and SOX-deficient aged rats. Rats do not mimic the sulfite responses seen in humans because of their relatively high SOX activity level. Therefore this study used SOX-deficient rats since they are more appropriate models for studying sulfite toxicity. Forty male Wistar rats aged 24 months were randomly assigned to four groups: control (C), sulfite (S), SOX-deficient (D) and SOX-deficient þ sulfite (DS). SOX deficiency was established by feeding rats with low molybdenum (Mo) diet and adding 200 ppm tungsten (W) to their drinking water. Sulfite in the form of sodium metabisulfite (25 mg kg1 day1) was given by gavage. Treatment continued for 6 weeks. At the end of the experimental period, flash VEPs were recorded. Hepatic SOX activity was measured to confirm SOX deficiency. SOX-deficient rats had an approximately 10-fold decrease in hepatic SOX activity compared with the normal rats. The activity of SOX in deficient rats was thus in the range of humans. There was no significant difference between control and treated groups in either latence or amplitude of VEP components. Brain SOD, CAT, and GPx activities and brain TBARS levels were similar in all experimental groups compared with the control group. Our results indicate that exogenous administration of sulfite does not affect VEP components and the antioxidant/oxidant status of aged rat brains. Keywords Sodium metabisulfite, visual evoked potentials, lipid peroxidation, antioxidant enzymes, aged rats, sulfite oxidase deficiency 1

Introduction The aging process is associated with certain biochemical, morphological, and electrophysiological alterations in the central nervous system (CNS) (Curti and Benzi, 1990; Gupta et al., 1991). The nature of the mechanisms underlying the aging process is presently not well understood. The aging process involves increased production of reactive oxygen species

Department of Physiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey 2 Department of Biochemistry, Faculty of Medicine, Akdeniz University, Antalya, Turkey 3 Department of Biophysics, Faculty of Medicine, Akdeniz University, Antalya, Turkey Corresponding author: Aysel Agar, Department of Physiology, Faculty of Medicine, Akdeniz University, Arapsuyu, Antalya 07070, Turkey. Email: [email protected]

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together with alterations in calcium homeostasis and mitochondrial dysfunction (Lores-Arnaiz and Bustamante, 2011). Recently accumulated evidence has suggested that alterations in the status of the antioxidant defense system play a pivotal role in the development of oxidative stress in aging (Cand and Verdetti, 1989). Human beings are exposed to both endogenous and exogenous sulfites. Endogenous sulfites are generated in mammalian tissues from sulfur-containing amino acids, such as cysteine and methionine. Sulfites are widely used in foods, beverages, and drug industries as antioxidants and antimicrobials. Sodium metabisulfite (Na2O5S2), potassium metabisulfite, sodium bisulfite, and potassium sulfite have been listed as sulfite compounds (Taylor et al., 1986). Alongside its antioxidative properties, sulfite also has an effect like an oxidant. It is known that Na2O5S2 can react with acids and water, releasing toxic sulfur dioxide gas. So, it is a double-edged sword with antioxidant as well as prooxidant properties (Lavoie et al., 1994). The toxicity of sulfite to humans apparently depends on the physical conditions of the individual and on other factors including its dose. Ingested sulfite enters the systemic circulation by gastrointestinal absorption and is distributed essentially to all body tissues including the brain (Gunnison and Benton, 1971; Gunnison and Jacobsen, 1987). Both endogenously- and exogenously derived sulfite must be detoxified in mammalian tissues. Sulfite oxidase (SOX), which is a molybdenum (Mo)-containing enzyme located in the mitochondria, oxidizes sulfite into sulfate in a two-electron oxidation, and protects the cells from sulfite toxicity (Cohen and Fridovich, 1971). This reaction is the terminal step in the oxidative degradation of sulfur-containing amino acids (cysteine and methionine) and membrane components such as the sulfatides (Woo et al., 2003). A high activity of SOX is seen in liver, kidney, and heart tissues, whereas the brain, spleen, and testis exhibit very low activities (Cabre et al., 1990). The physiological importance of this enzyme is seen in individuals with SOX deficiency, which is a rare genetically transmitted disease (Mudd et al., 1967). Deficiency of SOX in humans leads to progressive cerebral degeneration, major neurological abnormalities, dislocated ocular lenses, mental retardation, severe seizures, and early death, usually between 2 years and 6 years of age (Johnson, 1985). Rats have been used for the evaluation of sulfite toxicity. However, rats may not be the most appropriate model for prediction of sulfite toxicity in humans.

Rats do not mimic the responses observed in humans, since a rat liver has about a 10- to 20-fold greater SOX activity than a human liver (Johnson and Rajagopalan, 1976). Therefore, it has been suggested that SOXdeficient rats might be a more useful model to predict sulfite toxicity in humans (Dulak et al., 1984). Besides the many kinds of biological and toxicological effects in multiple organs, earlier studies by our group have determined the adverse effects of sulfite on the visual system assessed by visual evoked potentials (VEPs) (Agar et al., 2000; Aydin et al., 2005; Derin et al., 2009; Kucukatay et al., 2006a; Ozturk et al., 2011; Savcioglu et al., 2011), which consist of several components arising from the retina, optic pathway, subcortex, and cortex. Thus, VEPs offer an important method for evaluating the toxic effects of sulfite on the visual system after occupational, environmental, and ingested sulfite exposures. As shown in our earlier studies (Agar et al., 2000; Aydin et al., 2005; Derin et al., 2009; Kucukatay et al., 2006b; Ozturk et al., 2011; Savcioglu et al., 2011), Na2O5S2 and sulfur dioxide caused an increase in lipid peroxidation process, which was accompanied by changes in VEPs. These results clearly indicated that sulfite markedly affects the visual system. However, the effect of sulfite on VEPs of both normal and SOX-deficient aged animals has not yet been examined and information on biochemical aspects of toxicology is limited. Therefore, this study was designed to investigate the effects of sulfite ingestion on the brain and retina of normal and SOX-deficient aged rats by means of electrophysiological and biochemical parameters.

Materials and methods Animals Forty 24-month-old male Wistar albino rats weighing 500–550 g, used in this experimental study, were obtained from Animal Care Unit, Akdeniz University, Turkey. The experimental protocols conducted on animals were approved by the Akdeniz University’s Animal Care and Use Committee. The animals were housed in groups of four to five rats in stainless steel cages under standard conditions (24 + 2 C and 50 + 5% humidity) with a 12-h light–dark cycle.

Experimental design and treatments Four experimental groups, each consisting of 10 rats, were formed, namely the control (C) group, the group treated with sulfite (S), the SOX-deficient (D) group,

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Table 1. Daily diet with normal and SOX-deficent status in groups. Groups

SOX status

Diet

Supplementation

C S D DS

Normal Normal Deficient Deficient

Standart rat chow Standart rat chow Low Mo diet Low Mo diet

Vehicle Sulphite (25 mg kg1 day1) W (200 ppm) vehicle W (200 ppm) vehicle þ sulphite (25 mg kg1 day1)

SOX: sulfite oxidase; C: control; S: sulfite; D: SOX-deficient; DS: SOX-deficient þ sulfite.

and the SOX-deficient þ sulfite group (DS). Sulfite in the form of Na2O5S2 (25 mg kg1 day1) was given by gavage to the S and DS groups for 6 weeks (Kucukatay et al., 2005; Kucukatay et al., 2006a). Since the given dose of sulfite was dissolved in distilled water, the C and D groups received distilled water for the same period. Rats in the C and S groups were fed with standard rat chow and tap water ad libitum. SOX deficiency was produced in rats by maintaining low-Mo diets (AIN 76a, Research Dyets Inc, Bethlehem, Pennsylvania, USA) with concurrent addition of tungsten (W) to their drinking water at 200 ppm in the form of sodium tungstate for 6 weeks (Table 1). All groups exhibited similar weight gains and survival rates with no signs of toxicity.

VEP recordings VEPs from experimental groups were recorded with stainless steel subdermal electrodes (Nihon Kohden NE 223 S, Nihon Kohden Corporation, Tokyo, Japan) under light diethylether anesthesia. The reference and active electrodes were placed 0.5 cm in front of and behind the bregma, respectively. The active electrode was also placed 0.4 cm lateral to the midline over area 17 of the visual cortex. A ground electrode was placed on the tail. After 5 min of dark adaptation, a photic stimulator (Nova-Strobe AB Biopac System, Santa Barbara, California, USA) at the lowest intensity setting was used to provide the flash stimulus at a distance of 15 cm, which allowed lighting of the entire pupilla from the temporal visual field. The repetition rate of the flash stimulus was 1 Hz, and the flash energy was 0.1 J. VEP recordings from both right and left eyes were obtained, and throughout the experiments, the eye not under investigation was closed with appropriate black carbon paper and cotton. Body temperature was maintained between 37.5 C and 38 C using a heating pad (Hetzler et al., 1988). A total of 100 responses were averaged with the averager of Biopac MP100 data acquisition equipment (Biopac System).

The analysis time was 300 ms. The frequency bandwidth of the amplifier was 1–100 Hz. The gain was selected as 20 V div1. The microprocessor was programmed to reject any sweeps contaminated with larger artifacts, and at least two averages were obtained to ensure response reproducibility. Peak latencies of the components were measured from the stimulus artifact to the peak in milliseconds. Amplitudes were measured as the voltage between successive peaks.

Tissue collection and preparation At the end of the experimental period, the rats were anesthetized with urethane (1 g kg1, intraperitoneally) and their abdomens were opened by a midline incision. The rats were perfused transcardially with 100 ml heparinized saline. Brains and livers were quickly removed and stored at 80 C until analyzed. Livers were used to determine SOX activity, while brains were analyzed for thiobarbituric acid reactive substances (TBARS) and antioxidant enzymes. A portion of each rat’s liver (1 g) was weighed and homogenized for 1 min in 4 ml, 50 mmol phosphate buffer (pH 7.4). The homogenate was centrifuged at 2100 g for 10 min at 4 C. Supernatants were collected for SOX activity assay. Brain tissues were sonicated (Bandelin Sonopuls, HD 2070, Bandelin Electronic GmbH & Co.KG, Berlin, Germany) on ice in 3 ml of phosphate buffer (pH 7.2), including 50 mM di-potassium hydrogen phosphate for 1 min. After centrifugation of brain homogenates for the assay of TBARS (15,000 g for 10 min at 4 C), catalase (CAT)/glutathione peroxidase (GPx; 10,000 g for 15 min at 4 C), and superoxide dismutase (SOD; 1500 g for 5 min at 4 C) activities; supernatants were collected and stored at 80 C until biochemical analysis.

Hepatic SOX activity assay Hepatic SOX actitivity was determined as described in a previous paper (Cohen and Fridovich, 1971).

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An aliquot quantity of the supernatant was mixed with 5% Triton X-100 and further diluted (1:10) with 50 mmol phosphate buffer (pH 7.4). This diluted mixture was used to measure SOX activity at room temperature by monitoring the reduction of cytochrome c at 550 nm. The mixture was added to the cuvettecontaining 10 mmol sodiumsulfite, 0.2 mmol cytochrome c, Triton X-100 (% 5), 100 mmol tris(hydroxymethyl)aminomethane (Tris)–hydrochloric acid (HCl; pH 8.5), and potassium cyanide (10 mmol in Tris–HCl) in a final volume of 2.5 ml. The slow nonenzymatic rate of reduction of cytochrome c was first recorded and its rate was subtracted from the recorded total rate. One unit of SOX activity was defined as the amount of enzyme that caused an absorbance change of 0.1 min1 under these conditions. The results were expressed as units per milligram protein.

Measurement of SOD activity The activity of SOD was estimated using an SOD activity assay kit (Cayman Chemical, Ann Arbor, Michigan, USA, no. 706002). The activity of SOD was assessed by measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine in a convenient 96-well format. One unit of SOD activity was defined as the amount of enzyme needed to exhibit 50% dismutation of superoxide radical. The standard curve generated using this enzyme provides a means to accurately quantify the activity of all three types of SODs (Cu/Zn-, Mn-, and Fe-SOD).

that caused the oxidation of 1 mol NADPH to NADP per minute at 25 C.

TBARS assay Brain TBARS levels were measured by a fluorometric method as described in a previous paper using 1,1,3,3tetraethoxypropane as a standard (Wasowicz et al., 1993). Tissue samples (50 l) were introduced into a tube containing 1 ml of distilled water. After the addition of 1 ml of a solution containing 29 mmol L1 2-thiobarbituric acid in acetic acid (8.75 mol L1), samples were placed in a water bath and heated for 1 h at 95– 100 C. After the samples had cooled, 25 l, 5 mol L1 HCl was added, and the reaction mixture was extracted by agitation for 5 min with 3.5 ml of n-butanol. After centrifugation (3000 g, 10 min), the butanol phase was separated, and the fluorescence of the butanol extract was measured in a spectrofluoremeter (Perkin Elmer Luminescence spectrometer, LS50B, Waltham, Massachusetts, USA) using wavelengths of 525 nm for excitation and 547 nm for emission. The results were expressed as nanomoles per milligram protein.

Protein determinations The protein concentrations were determined spectrophotometrically (Shimadzu RF-5500, Kyoto, Japan) by a protein assay reagent kit (Pierce, Rockford, Illinois, USA), which is based on a modified Bradford method using bovine serine albumin as a standard.

Measurement of CAT activity

Statistical analysis

The activity of CAT was determined using a commercially available kit (Cayman Chemical, no. 707002) according to the manufacturer’s instructions. One unit of enzyme activity was defined as the amount of enzyme that caused the formation of 1 mmol formaldehyde per minute at 25 C.

An analysis of variance (ANOVA) was performed on all parameters of VEPs for the factors of side (right and left) and groups. Prior to completion of ANOVA, the homogeneity of variance was calculated by the Statistical Package for Social Sciences Version 20 statistic software (SPSS Inc., Chicago, Illinois, USA). Post hoc comparisons of the means were carried out using Tukey’s test. Differences of other data were also analyzed by Kruskal–Wallis test, followed by Dunn’s multiple comparison test. Significance levels were set at p < 0.05.

Measurement of GPx activity The activity of GPx was determined using the GPx assay kit (Sigma–Aldrich Chemie, Steinheim, Germany, no. CGP-1) according to the manufacturer’s instructions. Oxidized glutathione is recycled to its reduced state by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). The oxidation of NADPH to NADP is accompanied by a decrease in absorbance at 340 nm. One unit of enzyme activity was defined as the amount of enzyme

Results Animal health and survival No changes were observed in the general appearance and behavior of animals during the experimental

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Figure 1. Hepatic SOX activity in experimental groups. Values are expressed as means + SEM, n ¼ 10, *p < 0.05 compared with control group. SOX: sulfite oxidase; SEM: standard error of mean. Table 2. The means and SD of peak latencies (in milliseconds) for each VEP component.a Groups C S D DS

P1 (ms) 26.72 28.06 27.94 28.94

N1 (ms)

+ 0.92 + 0.7 +0.9 + 1.38

41.66 44.88 40.33 42.05

P2 (ms)

+ 1.35 + 1.96 + 1.5 + 2.84

54.33 58.55 52.61 51.89

N2 (ms)

+ 1.96 + 2.6 + 2.3 + 2.63

67.27 72.05 63.39 66.39

+ 2.03 + 2.5 + 2.22 + 2.97

P3 (ms) 99.77 + 103.33 + 98.16 + 101.33 +

1.61 2.8 1.68 2.64

VEP: visual evoked potential; SOX: sulfite oxidase; C: control; S: sulfite; D: SOX-deficient; DS: SOX-deficient þ sulfite. a Values are expressed as means + SD for 10 rats in each group. The mean value of each component was determined by averaging the data of both eyes.

Table 3. The means and standard errors of peak-to-peak amplitudes of each groups.a Groups C S D DS

P1N1 (V) 6.5 + 4.77 + 7.06 + 5.52 +

0.74 0.45 0.98 0.63

N1P2 (V) 5.22 3.98 4.22 3.1

+ 1.18 + 0.44 + 0.46 + 0.39

P2N2 (V) 4.28 4.2 4.31 2.88

+ 0.52 + 0.34 + 0.67 + 0.45

N2P3 (V) 6.22 5.28 8.13 6.12

+ + + +

2.66 0.55 0.57 0.54

SOX: sulfite oxidase; C: control; S: sulfite; D: SOX-deficient; DS: SOX-deficient þ sulfite. a Values are expressed as means þ SE for 10 rats in each group. The mean value of each component was determined by averaging the data of both eyes.

period. There was no significant difference between the control and treated groups in either food consumption or body weight (data not shown).

very effective. This diet resulted a decrease in SOX activity of D groups compared with SOX normal groups (p < 0.05). Sulfite administration did not cause an alteration in SOX activity in either SOX normal or SOX-deficient animals (S and DS groups).

Hepatic SOX Activity The hepatic SOX activity level of each group at the end of 6 weeks is presented in Figure 1. Our results clearly demonstrate that induction of SOX deficiency by maintaining a low-Mo diet with W supplementation was

Visual evoked potentials The mean and standard deviation of peak latencies and peak-to-peak amplitudes of VEP components are

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Figure 2. Effect of sulfite on brain SOD activity in experimental groups. Values are expressed as means + SEM, n ¼ 10. SOD: superoxide dismutase; SEM: standard error of mean.

Figure 3. Effect of sulfite on brain CAT activity in experimental groups. Values are expressed as means + SEM, n ¼ 10. CAT: catalase; SEM: standard error of mean.

shown in Tables 2 and 3, respectively. Measurements were made on three positive and two negative potentials, which were seen in all the groups. We did not find significant differences in latencies between right and left eyes. Data from stimulation of both eyes were averaged. No significant differences were observed in the recorded latencies and amplitudes among the different experimental groups.

difference was observed in brain antioxidant enzyme levels among different experimental groups.

Brain levels of TBARS Lipid peroxidation was measured as the amount of brain TBARS levels of the rats. Brain TBARS values for the studied groups are summarized in Figure 5. No significant difference was noted in brain TBARS levels among different experimental groups.

Brain levels of antioxidant enzyme activities Measured brain SOD, CAT, and GPx enzyme activity values for the studied groups are summarized in Figures 2, 3, and 4, respectively. No significant

Discussion In this study, we examined the effects of sulfite on VEPs in normal and SOX-deficient aged rats. In this

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Figure 4. Effect of sulfite on brain GPx activity in experimental groups. Values are expressed as means + SEM, n ¼ 10. GPx: glutathione peroxidase; SEM: standard error of mean.

Figure 5. Effect of sulfite on brain TBARS levels in experimental groups. Values are expressed as means + SEM, n ¼ 10. TBARS: thiobarbituric acid reactive substances; SEM: standard error of mean.

regard, we investigated the oxidant/antioxidant status of the brain by measuring lipid peroxidation and antioxidant enzyme activities. Our study indicates that there is no detrimental effect of sulfite on VEPs in normal and SOX-deficient aged rats. Additionally, no effect of sulfite was observed on brain TBARS and antioxidant enzyme activities in either normal or SOX-deficient aged rats. It can be concluded that the given dose of sulfite does not have an effect on VEPs and on brain oxidant status in aged rats. The acceptable daily intake (ADI) for sulfites was established as 0–0.7 mg kg1 body weight by The Joint Food and Agriculture Organisation (FAO)/World

Health Orgqanisation (WHO) Expert Committee on Food Additives (FAO/WHO, 1975). The ADI value was based on long-term studies in rats, including a threegeneration work of reproductive toxicity, with a noobserved-effect level of 0.25% Na2S2O5 in the diet, equivalent to 70 mg kg1 body weight day1 of sulfur dioxide equivalents (Til et al., 1972). By applying the typical 100-fold safety factor, the ADI value was determined for humans as 0–0.7 mg kg1. However, the daily intake of sulfite may not be in agreement with this value in many cases. Studies have shown that it is possible to consume 180–200 mg of sulfite from foods and beverages in a single day or meal (Gunnison and Jacobsen,

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1987; Taylor et al., 1986). This dosage of sulfite was also used in our previous studies (Kucukatay et al., 2005, 2006a; Ozsoy et al., 2012; Savcioglu et al., 2011). Due to the generation of very reactive ionic species and potentially toxic interactions with molecules of biological importance, sulfite levels must be strictly regulated, via mechanisms such as SOX-mediated detoxification. Studies have shown an inverse correlation between SOX levels in liver and sulfite toxicity in several species of laboratory animals (Tejnorova, 1978). In this study, hepatic SOX activity was measured as an indicator of SOX status in the body because of its high level of SOX activity compared to other organs. The results of this study clearly demonstrate that feeding rats with a high-W/low-Mo diet effectively reduced SOX activity. By competing with Mo for the active site of newly formed SOX molecules, W causes the production of nonfunctional molecules instead, which, in turn, cancel out SOX enzyme activity (Gunnison et al., 1987). There are significant differences among species in their SOX activity. The most notable is the difference between rats and humans. It was shown that the liver of a rat has about a 10- to 20-fold greater SOX activity than a human liver (Johnson and Rajagopalan, 1976). It was shown that the normal level of SOX activity in a human liver tissue is 0.009 U mg1 protein, whereas liver tissue of rats exhibited 0.06 U mg1 protein activity of SOX (Beck-Speier et al., 1985). Figure 1 shows that SOX-deficient rats have a decrease in SOX activity compared with those rats in the C group. For this reason, SOX-deficient rats were used in this experiment as a more appropriate model of sulfite toxicity in humans due to their reduced SOX activity. VEPs represent relevant information on determining the integrity of the visual system and have an accepted clinical utility regarding visual and other neuropathologies (Chiappa and Ropper, 1982; Halliday et al., 1972; Ozsoy et al., 2011; Wright et al., 1986). In addition to this, it is well known that VEPs are a sensitive method for detecting early alterations of optic pathway in experimental toxicology studies (Agar et al., 2000; Aydin et al., 2005; Kucukatay et al., 2006a; Otto et al., 1988; Savcioglu et al., 2011). As is known, VEP components are P1, N1, P2, N2, and P3. Components of VEP found in our laboratory are in considerable agreement with those found in other laboratories regarding general trends in normative data (Dyer et al., 1987; Sisson and Siegel, 1989). In this study, sulfite exposure did not affect the latencies and amplitudes of VEP components. Previous studies of our laboratory demonstrated that sulfite in

different doses (Aydin et al., 2005; Derin et al., 2009; Kucukatay et al., 2006a; Ozturk et al., 2011; Savcioglu et al., 2011) and sulfur dioxide (Agar et al., 2000) have significant detrimental effects on the latencies of VEP components. In one of our previous studies, we concluded that this detrimental effect of sulfite on VEP latencies was increased in a dose-dependent (100 and 260 mg kg1 day1) manner. We did not detect any effect of sulfite (25 mg kg1 day1) on VEPs in this study, while we did observe prolonged VEP latencies of adult rats with the same dose of sulfite in our previous studies (Kucukatay et al., 2006a; Savcioglu et al., 2011). This discrepancy might be related to the age of the rats. Our result is in agreement with an earlier study which shows that only the P3 component of VEP was prolonged in 24-month-old rats that were exposed to sulfur dioxide (Kilic, 2003). According to this study, the number of VEP components affected by sulfur dioxide decreased with age in rats aged 3, 12, and 24 months (Kilic, 2003). Therefore, it seems that sulfite does not affect older animals as it does in adult animals in terms of visual system. The CNS is protected against destructive effects of free radicals by antioxidant enzymes. In this study, brain SOD, CAT, and GPx activities were unchanged in all sulfite-exposed groups compared with their respective C groups. Previous studies indicated that SOD, CAT, and GPx activities were significantly increased in hippocampus with sulfite exposure in young groups. This significant increment of hippocampus antioxidant capacity in the sulfite group may be an adaptive response to sulfite-dependent oxidative stress (Elmas et al., 2005; Kucukatay et al., 2007). However, no change in brain antioxidant enzyme activities was observed in aged rats that were inhaling sulfur dioxide (Kilic, 2003). Therefore, our results are in accordance with this study. Although the biochemical basis of brain aging is still unclear, the early investigations (Benzi, 1979; Benzi et al., 1980) on age-linked changes in cerebral enzymes related to energy transduction showed that aging causes a decrease in the enzyme activities of synaptosomes, mitochondria, gliosomes, and microsomes. These decreases could be due to the modified turnover of specific enzymatic protein, increase in the content of unspecific proteins, changed permeability of the compartmentalizing membranes to the enzymes’ substrates, or age-related changes in the physicochemical status of the parenchymal tissue (Benzi, 1979; Benzi et al., 1980). Therefore, it seems that sulfite does not have an additive effect on mechanisms in the aged brains that were mentioned above.

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Although little information about the mechanism of sulfite toxicity on the CNS exists, sulfur- and oxygencentered free radicals may play an important role in the development of this phenomenon (Abedinzadeh, 2001). There have been several reports on lipid peroxidation status affected by sulfite exposure (Derin et al., 2009; Kucukatay et al., 2005; Kucukatay et al., 2006a; Ozturk et al., 2011). Despite these reports, we have found that there were no significant effects of sulfite treatment on brain TBARS levels of normal and SOX-deficient aged rats. The two previous studies carried out in our laboratory found that elevated TBARS levels and prolonged VEP latencies were positively correlated (Aydin et al., 2005; Kucukatay et al., 2006a). These changes of VEPs seem to result from lipid peroxidation caused by sulfite. In this study, the latencies of VEP components were not affected by sulfite. Therefore, our VEPs and TBARS data from this study are in a positive corelation, as were in our previous studies (Aydin et al., 2005; Kucukatay et al., 2006a). In our previous study, following sulfur dioxide exposure, there was an increase in TBARS production in all age-groups; however, the extent of this increase was greater in young rats compared to middle- and old-aged rats (Yargicoglu et al., 1999). Therefore, from the results of this study as well as our previous study (Yargicoglu et al., 1999), it could be suggested that the lipid peroxidative effect of sulfite might be masked by increased lipid peroxidation with age. Aging is an important variable that influences VEP latencies (Kilic, 2003). The age-related increase in the latencies of VEP components has therefore been related to changes in optic pathways or cortex. Also demyelination, axonal swelling, apoptosis, and fiber loss have been described in the optic pathway (Wright et al., 1985). Another possible mechanism is the changing of ion channels. In the light of previous articles and this research, it could be concluded that sulfite exposure does not affect the visual system or oxidant/antioxidant balance of aged brains as it does in young brains. For this reason, this study raises the necessity of evaluating young people with increased risk of adverse health consequences. Conflict of interest The authors declared no conflicts of interest.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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The effect of ingested sulfite on visual evoked potentials, lipid peroxidation, and antioxidant status of brain in normal and sulfite oxidase-deficient aged rats.

Sulfite, commonly used as a preservative in foods, beverages, and pharmaceuticals, is a very reactive and potentially toxic molecule which is detoxifi...
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