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Influence of Indole-3-butyric acid on antioxidant defense systems in various tissues of rats at subacute and subchronic exposure
Food and Chemical Toxicology 47 (2009) 2441–2444
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Influence of Indole-3-butyric acid on antioxidant defense systems in various tissues of rats at subacute and subchronic exposure Nuray Topalca a, Esref Yegin b, Ismail Celik a,* a b
Yuzuncu Yıl University, Faculty of Science and Letters, Department of Biology, 65080-Van, Turkey Dicle University, Faculty of Medicine, Department of Biochemistry, Diyarbakır, Turkey
a r t i c l e
i n f o
Article history: Received 15 May 2009 Accepted 26 June 2009
Keywords: Indole-3-butyric acid Antioxidant defense systems Rat
a b s t r a c t This study was carried out to investigate the effects of Indole-3-butyric acid (IBA), a plant growth regulator (PGR), on antioxidant defense systems (ADS) such as reduced glutathione (GSH) level and Glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px) superoxide dismutase (SOD) enzymes activity in various tissues of rats exposed to 25 and 50 ppm dosages of IBA for 20 and 45 days. Results showed that the administrations of IBA fluctuated GSH levels in some tissues of rats treated with both dosages and periods. With regard to the ADS enzymes, SOD and GST activities increased significantly in the most of the tissues in rats treated with both dosages and periods of IBA. Also, GSH-Px activity fluctuated after subacute and subchronic exposure with both dosages in some of the tissues in rats compared to that the control rats. The observations presented led us to conclude that the administrations of IBA at subacute and subchronic affected the ADS system in various tissues of rats. This may reflect the potential role of these parameters as useful biomarkers for toxicity of IBA. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction Indole-3-butyric acid (IBA) is a derivative of IAA (indole-3-acetic acid) naturally occurring plant growth regulators of the auxin class, affecting cell enlargement, division, and differentiation (Mickel, 1978). As a result of the industrial usage, this agrochemical is consumed by non-target organisms (Cokugras and Bodur, 2003). Although PGRs are used for pest control and giving rise product on a wide variety of crops, little is known about the biochemical or physiological effects in mammalian organisms. However, there are some studies about endogenous PGRs including IBA. de Melo et al. (2004) determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase in rat neutrophils and lymphocytes. Furukawa et al. (2004) indicated that IAA might induce the neuronal apoptosis in the S phase and lead to microencephaly. John et al. (1979) observed that IAA possesses teratogenic effects in gestation mice and rats at 500 mg/ kg/day. El-Mofty and Sakr (1988) found that GA3 induced liver neoplasm in Egyptian toads, and they suggested that the tumors could be diagnosed as hepatocellular carcinomas. GA3 also induces microabsceses and hydropic degeneration in the liver and mononuclear inflammatory infiltration in the kidneys of laboratory mice, but not tumors. Ozmen et al. (1995) observed that Abcisic acid * Corresponding author. Tel.: +90 432 2251707x2278; fax: +90 432 2251188. E-mail address: [email protected] (I. Celik).
(ABA) and Gibberellic acid (GA3) was affective on sexual differentiation and some physiological parameters of laboratory mice. In a study, IAA effect investigated on human serum enzymes in-vitro, it was found that IAA inhibited aspartate aminotransferase (AST) but activated amylase, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) (Celik and Kara, 1997). Also, it was reported that while the levels of LDH and CPK increased significantly by IBA, the levels of AST, LDH and CPK were increased significantly by IAA after subacute exposure with 100 ppm dosages (Celik et al., 2002). IAA was found to be linear-mixed type inhibitor for human serum BChE, and uncompetitive inhibitor for the horse serum BChE enzyme (Cokugras and Bodur, 2003). Further, PGRs may induce oxidative stress, leading to generation of free radicals and cause lipid peroxidation as one of the molecular mechanisms involved in PGRs-induced toxicity (Tuluce and Celik, 2006; Candeias et al., 1995; Celik et al., 2007, 2006a,b). Antioxidant defense systems, present in all aerobic organisms, include antioxidant enzymes and free radical scavengers whose function is to remove reactive oxygen species (ROS), thus protecting the functions of organisms from oxidative stress (Regoli and Principato, 1995). The sensitivity of cell to oxidants is attenuated by antioxidant defense system such as GSH, GST, catalase (CAT), SOD, glutathione peroxidase (GPx), GR and glucose-6-phosphate dehydrogenase (G6PD). The antioxidant defense system maintains a relatively low rate of the reactive and harmful OH. Oxidative stress occurs as a result of the effect of xenobiotics causing the disturbances in the antioxidant enzymes system (Oruc-Ozcan and Uner,
0278-6915/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.037
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2002). Among these, GPx, through reduction of both hydrogen peroxide and organic hydroperoxides, provides an efficient protection against oxidative damage and free radicals in the presence of reduced glutathione (GSH). Previously oxidized GSH is regenerated by GR. SOD catalyses dismutation of superoxide anion radicals, whereas CAT eliminates hydrogen peroxide. Antioxidant enzymes play a crucial role in maintaining cell homeostasis. Another group of enzymes, GST act as catalyst of a wide variety of conjugation reactions of glutathione with xenobiotic compounds containing electrophilic center. Additionally, there are glutathione-independent enzymes that act as part of the cellular defense system against toxicity originated by active oxygen forms (Halliwell and Gutteridge, 1989). Despite the reasons mentioned in above paragraphs, little is known regarding the IBA effects on antioxidant defense systems of vertebrata. In order to achieve a more rational design of IBA, it is necessary to clarify the mechanism of oxidative stress for IBA. To this end, the treatments of IBA were done orally because the effect of chemicals represents a well characterized in vivo toxicity model system. The enzymes were chosen due to their important role for antioxidant defense systems and important role during detoxification in degradation and bioactivation of IBA.
The tissues were dissected and put in Petri dishes. After washing the tissues with 0.9% NaCl, samples were taken and kept at 78 °C until the analysis. The tissues were homogenized for 5 min in 50 mM ice-cold KH2PO4 solution (1:5 w/v) using a glass–porcelain homogenizer (20 kHz frequency ultrasonic, Jencons Scientific Co.) and then centrifuged at 7000g for 15 min. All processes were carried out at 4 °C. Supernatants were used to determine antioxidant defense system enzymes. 2.4. Biochemical analysis The erythrocyte and tissues GSH concentration was measured using the method described by Beutler et al. (1963). Briefly, 0.2 mL fresh erythrocyte pellets or the tissue supernatants were added to 1.8 mL distilled water. Three mL of the precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 mL distilled water) was mixed with haemolysate. The mixture was allowed to stand for approximately 5 min and then filtered (whatman N 42). Two millilitre of filtrate was taken and added into another tube, and then 8 mL of the phosphate solution (0.3 M disodium hydrogen phosphate) and 1 mL DTNB were added. A blank was prepared with 8 mL of phosphate solution, 2 mL diluted precipitating solution (three parts to two parts distilled water), and 1 mL DTNB reagent. A standard solution of the glutathione was prepared (40 mg/100 ml). The optical density was measured at 412 nm in a spectrophotometer (Novaspec II Pharmacia-Biotech, Biochrom Ltd., UK). GST (EC 2.5.1.18) was assayed at 25 oC spectrophotometrically by following the conjugation of glutathione with CDNB at 340 nm as described by Mannervik and Guthenberg (1981). To determine the activity of GPx (EC 1.11.1.9), t-butyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GPx and NADPH (Beutler, 1984). SOD (EC 1.15.1.1) activity was measured at 505 nm and 37 °C and calculated using inhibition percentage of formazon formation (McCord and Fridovich, 1969).
2. Materials and methods
2.5. Analysis of data
2.1. Chemicals
All data were expressed as mean ± standard deviation (SD). The statistical analyses were made using the Minitab 13 for windows packet program. Means and Standard deviations were calculated according to the standard methods for all parameters. One way ANOVA statistical test was used to determine the differences between means of the treatments and the control group accepting the significance level at p 6 0.05 (See Tables 1 and 2).
Trichloroacetic acid (TCA) ethylenediaminetetraacetic acid (EDTA), GSH, metaphosphoric acid, 5,50 dithiobis-(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced b-Nicotinamide adenine dinucleotide phosphate (NADPH), oxidized glutathione (GSSG), potassium dihydrogenephosphate (KH2PO4), sodium chloride (NaCl) and Indole-3-butyric acid (IBA) of technical grade used in this study were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Kits for antioxidant enzymes analysis were supplied by Randox Laboratories Ltd. 2.2. Animals Rats (Wistar) 4 months of age with and average weighing approximately 200– 250 g were provided by the animal house of the Sciences Faculty of Yüzüncü Yıl University, and were housed in 3 groups. The animals were housed at 20 ± 2 °C an in daily light/dark cycle. All animals were fed a group wheat–soybean-mealbased diet and water ad libitum in stainless cages, and received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and published by the National Institutes of Health. The ethic regulations have been followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments. This study was approved by The Ethic Committee of Yüzüncü Yıl University. 2.3. Treatment of rats This investigation was performed on male rats. The animals ere housed for a minimum of two weeks to ‘acclimatize’ before being dosed with the substance. The rats were exposed to 25 and 50 ppm IBA ad libitum 20 days for subacute and 45 days for subchronic applications as drinking water. Twenty-five and fifty milligrams of the IBA were dissolved in 1 mL of 1 N NaOH, and then were diluted with tap water until 1000 mL to obtain a 25 and 50 ppm dosages. For the control rats, only 1 mL of 1 N NaOH was added to 1000 mL of tap water. Because the PGRs are photoactive compounds the drinking water containing IBA was prepared and refreshed every day in amber bottle. Since all rats have the same physiologic characters, daily water consumption of all groups of rats was approximately 25 ± 3 mL during the tests. Consequently, the IBA intake amount of each rat was about 2.1 ± 0.3 mg per day. At the end of the treatments, after the rats were anesthetized blood and tissues samples were obtained. The blood samples were obtained from a cardiac puncture using syringe for the determination of biochemical analysis. For biochemical analysis, blood samples were put immediately into silicon disposable glass tubes with EDTA as an anticoagulant. Blood samples were centrifuged at 4000g for 15 min at 4 °C and erythrocyte pellets were obtained. Then the pellets were washed tree times with physiological saline (0.9% NaCl). The GSH concentration in erythrocytes and tissues were measured just after the animals were sacrificed because of tremendous loss of GSH. Also, SOD, GSH-Px and GST activities in the erythrocytes were measured in the pellets.
3. Results Following the exposure to 25 and 50 ppm dosages of IBA, the effects of IBA administration on the erythrocytes and brain, kidney, hearth, muscle, liver, lungs and spleen tissues oxidative stress damages index were evaluated as the level of GSH and the activity of antioxidant enzymes such as SOD, GR and GST in the tissues samples from control and treated rats. It was observed that GSH level and GSH-Px activity were fluctuated in the some tissues of rats treated with 25 and 50 ppm dosage of IBA at subacute and subchronic exposure. The GST increasing in the all of tissues of rats treated with 25 ppm and 50 ppm IBA were significant at subacute and subchronic periods. Also, SOD activity increased significantly with both dosages of IBA except for muscle tissue at subacute, and brain and spleen tissues at subchronic period compared to control rats (Tables 3 and 4). 4. Discussion In recent years, a significant increase in the use of PGRs against harmful agricultural pests and giving rise to losing product have been observed in Turkey and the rest of the world. One of the major reasons for the increase is the ease of using PGRs and ensuring an absolute result. In this study, IBA was preferred because information on its negative effects on ADS of higher animals is very limited for in vivo, oral exposures. Also, IBA is a derivative of IAA found in plants as endogen hormones and wide variety of biologically active compounds. In this study, experimental group was exposed to two dosages of chemical substance. Antioxidant defense system constituent were used as important biomarkers for detection of toxic nature of IBA. Four antioxidant markers (GSH, SOD, GSH-Px and GST) were evaluated for oxidative stress.
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N. Topalca et al. / Food and Chemical Toxicology 47 (2009) 2441–2444 Table 1 Effects of subacute and subchronic treatment of IBA on GSH level in tissues of rats. Period
Tissue
GSH Control
25 ppm
50 ppm
X ± SD
X ± SD
X ± SD
Subacute
Erythrocytes (mg/dL) Brain (mg/g) Kidney (mg/g) Hearth (mg/g) Muscle (mg/g) Liver (mg/g) Lungs (mg/g)
3.21 ± 0.37 12.12 ± 1.17 28.58 ± 2.66 19.57 ± 1.55 17.50 ± 1.05 45.29 ± 4.99 26.69 ± 2.28
2.87 ± 0.31 42.80 ± 2.21* 89.00 ± 5.34* 14.40 ± 1.77* 10.65 ± 1.37* 32.46 ± 2.66* 21.04 ± 1.73
2.66 ± 0.14* 57.40 ± 1.28* 77.80 ± 3.78* 30.21 ± 1.39* 20.87 ± 1.76 76.66 ± 2.65* 40.11 ± 3.19*
Subchronic
Erythrocytes (mg/dL) Brain (mg/g) Kidney (mg/g) Hearth (mg/g) Muscle (mg/g) Liver (mg/g) Lungs (mg/g) Spleen (mg/g)
3.21 ± 0.37 12.12 ± 1.17 28.58 ± 2.66 19.57 ± 1.55 17.50 ± 1.05 45.29 ± 4.99 26.69 ± 2.28 33.88 ± 2.67
2.92 ± 0.23 7.23 ± 0.25* 18.08 ± 0.31* 9.70 ± 0.46* 8.90 ± 0.43* 33.19 ± 1.61* 15.49 ± 1.00* 28.53 ± 0.53
2.76 ± 0.16 11.30 ± 1.11 25.91 ± 1.97 15.19 ± 1.02* 14.60 ± 0.45* 39.42 ± 2.61 26.34 ± 1.03 29.95 ± 1.88
Each value represents the Mean ± SD. Significantly different from control rats (One way ANOVA).
*
The results of the present study have demonstrated that the applied dosages of IBA could have affected the antioxidant defense systems in the rats. This is evidenced from our observation that, upon IBA treatment in vivo, the activities of GSH-Px, GST and SOD activities and GSH level differ from those of controls in the tissues of rats exposed to 25 and 50 ppm dosages of IBA for 20 and 45 days. So far, no study examining the effects of IBA on ADS in vivo have been made on rat. Therefore, we could not have the chance to compare our results with the previous results. The results in the present study showed that the GSH depletion in the all tissues except for lungs tissue of rats treated with both dosages of MP were significant. Reduced GSH and its metabolizing enzymes provide the major defense against ROS-induced cellular damage (Avellini et al., 1993). GSH depletion might enhance the risk of the oxidative stress (Regoli and Principato, 1995). A considerable decline in GSH content in the tissue under the present experimental model may be due to its utilization to challenge
the prevailing oxidative stress under the influence of ROS generated from IBA. The antioxidative enzymes such as GST and SOD were also seriously affected by IBA; SOD significantly increased in the almost tissues of rats treated with both doses of IBA whereas GSH-Px activity fluctuated with two doses of IBA. Although the reasons for such effect of IBA are not understood at the present, it is conceivable that IBA might be interacting primarily with the tissues, resulting in fluctuated enzymes activities by the way of increased reactive oxygen radicals as result of stress condition in the rats. Doyotte et al. (1997) pointed out that a decreased enzyme activity’s response may accompany a first exposure to pollutants, which can be followed by an induction of antioxidant systems. Thus, the existence of an inducible antioxidant system may reflect an adaptation of organisms. In contrast, Dimitrova et al. (1994) suggested that the superoxide radicals by themselves or after their transformation to H2O2 cause an oxidation of the cysteine in the enzyme and
Table 2 Effects of subacute and subchronic treatment of IBA on GST enzyme in tissues of rats.
Table 3 Effects of subacute and subchronic treatment of IBA on GSH-Px enzyme in tissues of rats.
Period
Period
Tissue
GST
Tissue
GSH-Px
Control
25 ppm
50 ppm
Control
25 ppm
X ± SD
X ± SD
X ± SD
X ± SD
X ± SD
X ± SD
50 ppm
Subacute
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
5.75 ± 0.46 2.26 ± 0.42 3.14 ± 0.37 1.39 ± 0.19 1.55 ± 0.28 6.98 ± 0.69 3.25 ± 0.24 1.43 ± 0.31
9.46 ± 0.63* 13.25 ± 0.59* 8.98 ± 0.47* 1.83 ± 0.38* 3.00 ± 0.47* 22.45 ± 0.65* 4.64 ± 0.61* 2.58 ± 0.27*
10.13 ± 0.95* 7.63 ± 0.71* 9.11 ± 0.69* 1.97 ± 0.38* 7.93 ± 0.69* 13.24 ± 1.59* 4.40 ± 0.43* 2.77 ± 0.24*
Subacute
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
12.93 ± 1.47 6.85 ± 0.19 12.11 ± 1.44 17.73 ± 1.59 10.03 ± 0.69 15.38 ± 1.65 14.91 ± 1.22 19.21 ± 1.01
15.67 ± 1.88 9.15 ± 0.64* 9.25 ± 0.69* 12.45 ± 1.60* 8.77 ± 0.29 12.11 ± 1.53 9.02 ± 0.57* 10.90 ± 0.45*
14.17 ± 1.60 7.69 ± 0.63 8.93 ± 0.63* 6.71 ± 0.43* 13.39 ± 1.25* 10.09 ± 1.25* 25.07 ± 0.78* 22.64 ± 0.77
Subchronic
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
5.75 ± 0.46 2.26 ± 0.42 3.14 ± 0.37 1.39 ± 0.19 1.55 ± 0.28 6.98 ± 0.69 3.25 ± 0.24 1.43 ± 0.31
9.79 ± 0.71* 7.63 ± 0.46* 6.50 ± 0.46* 2.75 ± 0.44* 4.90 ± 0.70* 10.15 ± 0.61* 4.14 ± 0.22* 3.38 ± 0.10*
11.25 ± 0.94* 6.50 ± 0.48* 10.20 ± 1.11* 5.43 ± 0.45* 5.26 ± 0.41* 17.38 ± 1.81* 6.85 ± 0.49* 6.49 ± 0.41*
Subchronic
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
12.93 ± 1.47 6.85 ± 0.19 12.11 ± 1.44 17.73 ± 1.59 10.03 ± 0.69 15.38 ± 1.65 14.91 ± 1.22 19.21 ± 1.01
16.90 ± 2.16* 11.84 ± 1.56* 12.06 ± 1.59 10.85 ± 1.65* 13.50 ± 1.63* 11.34 ± 1.26* 9.44 ± 0.80* 7.41 ± 0.26*
16.01 ± 2.14* 7.77 ± 0.88 7.87 ± 0.73*
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
8.56 ± 0.95* 15.46 ± 1.43* 8.11 ± 0.37* 9.84 ± 0.88* 9.42 ± 0.84*
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Table 4 Effects of subacute and subchronic treatment of IBA on SOD enzyme in tissues of rats.
Acknowledgments
Period
The authors are grateful to the University Grant Commission (The University of Yuzuncu Yil, No. 2007-FBE-YL77) for providing financial assistance during the tenure of Research Associate ship.
Subacute
Tissue
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
Subchronic Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
SOD Control
25 ppm
50 ppm
X ± SD
X ± SD
X ± SD
974.21 ± 53.89 1088.37 ± 60.23* 1205.18 ± 54.40* 1167.12 ± 24.55 1144.22 ± 17.68 1253.12 ± 17.43 1278.95 ± 12.71 1155.98 ± 30.19 1261.34 ± 24.36 1288.91 ± 8.69
1265.87 ± 15.86* 1314.49 ± 9.59* 1315.92 ± 2.83* 1271.57 ± 12.38 1252.60 ± 33.98* 1333.20 ± 12.30* 1265.61 ± 10.20*
1288.37 ± 20.92* 1299.77 ± 15.28* 1315.18 ± 8.31* 1312.14 ± 14.55* 1302.07 ± 22.69* 1331.69 ± 4.44* 1326.92 ± 6.16*
974.21 ± 53.89 1224.80 ± 44.57* 1323.56 ± 36.30* 1167.12 ± 24.55 1144.22 ± 17.68 1253.12 ± 17.43 1278.95 ± 12.71 1155.98 ± 30.19 1261.34 ± 24.36 1288.91 ± 8.69
1224.14 ± 11.62 1271.39 ± 14.23* 1337.25 ± 8.03* 1322.06 ± 10.87* 1260.40 ± 19.47* 1336.08 ± 3.51* 1298.40 ± 8.74
2636.59 ± 7.84* 1276.68 ± 10.99* 2656.24 ± 8.48* 2650.18 ± 8.95* 2395.77 ± 38.80* 2513.83 ± 33.59* 2595.25 ± 18.62*
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
decrease SOD activity. Consequently, the decreased and increased ADS enzymes activities might have reflected a cellular oxidative stress due to IBA exposure. Meanwhile, GST activity significantly increased in the all tissues at subacute and subchronic treatment with both doses of IBA. The enzymatic antioxidants such as SOD, GR and GST have been shown to be sensitive indicators of increased oxidative stress in Mugil sp obtained from a polluted area containing high concentrations of polyaromatic hydrocarbons, polychlorinated biphenyls, and pesticides (Rodriguez-Arizaet et al., 1993). However, the increased activities of GST are known to serve as protective responses to eliminate xenobiotics (Smith and Litwack, 1980). In addition, findings of this study suggest that the fluctuated levels in the constituents of antioxidant defense systems in the tissues of rats exposed to IBA may be dependent on the differences between interstitial concentrations. Namely, the systems might have to be exposed to different xenobiotic concentration due to blood volume differences in the tissues. There have been reported that the long-term intoxication with plant growth regulators leads to a gradual exhaustion of SOD, GR, GSH-Px and GST or cause increase of antioxidative defense systems (Tuluce and Celik, 2006; Celik and Tuluce, 2006; Candeias et al., 1995; Celik et al., 2007, 2006a,b). The results are partly in accordance with our findings although the treatment time and manner, the setting of studies and concentrations of chemical are different. Our observations led us to conclude that the administrations of subacute and subchronic IBA cause changes in the antioxidant defense systems in the various tissues of rats. Thus, any external stressor such as IBA, even at nonlethal concentration can have a toxic effect on the organism. From the foregoing observations it may also be postulated that antioxidative constituents might offer a certain result of choice for monitoring biotoxicity of direct acting compounds such as IBA. Such a test will also be of value in pollution studies, and also be of interest to understand molecular basis of refractoriness of IBA toxicity. 5. Conflict of interest This study was approved by the ethic committee of Yüzüncü Yıl University.
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Influence of Indole-3-butyric acid on antioxidant defense systems in various tissues of rats at subacute and subchronic exposure Nuray Topalca a, Esref Yegin b, Ismail Celik a,* a b
Yuzuncu Yıl University, Faculty of Science and Letters, Department of Biology, 65080-Van, Turkey Dicle University, Faculty of Medicine, Department of Biochemistry, Diyarbakır, Turkey
a r t i c l e
i n f o
Article history: Received 15 May 2009 Accepted 26 June 2009
Keywords: Indole-3-butyric acid Antioxidant defense systems Rat
a b s t r a c t This study was carried out to investigate the effects of Indole-3-butyric acid (IBA), a plant growth regulator (PGR), on antioxidant defense systems (ADS) such as reduced glutathione (GSH) level and Glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px) superoxide dismutase (SOD) enzymes activity in various tissues of rats exposed to 25 and 50 ppm dosages of IBA for 20 and 45 days. Results showed that the administrations of IBA fluctuated GSH levels in some tissues of rats treated with both dosages and periods. With regard to the ADS enzymes, SOD and GST activities increased significantly in the most of the tissues in rats treated with both dosages and periods of IBA. Also, GSH-Px activity fluctuated after subacute and subchronic exposure with both dosages in some of the tissues in rats compared to that the control rats. The observations presented led us to conclude that the administrations of IBA at subacute and subchronic affected the ADS system in various tissues of rats. This may reflect the potential role of these parameters as useful biomarkers for toxicity of IBA. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction Indole-3-butyric acid (IBA) is a derivative of IAA (indole-3-acetic acid) naturally occurring plant growth regulators of the auxin class, affecting cell enlargement, division, and differentiation (Mickel, 1978). As a result of the industrial usage, this agrochemical is consumed by non-target organisms (Cokugras and Bodur, 2003). Although PGRs are used for pest control and giving rise product on a wide variety of crops, little is known about the biochemical or physiological effects in mammalian organisms. However, there are some studies about endogenous PGRs including IBA. de Melo et al. (2004) determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase in rat neutrophils and lymphocytes. Furukawa et al. (2004) indicated that IAA might induce the neuronal apoptosis in the S phase and lead to microencephaly. John et al. (1979) observed that IAA possesses teratogenic effects in gestation mice and rats at 500 mg/ kg/day. El-Mofty and Sakr (1988) found that GA3 induced liver neoplasm in Egyptian toads, and they suggested that the tumors could be diagnosed as hepatocellular carcinomas. GA3 also induces microabsceses and hydropic degeneration in the liver and mononuclear inflammatory infiltration in the kidneys of laboratory mice, but not tumors. Ozmen et al. (1995) observed that Abcisic acid * Corresponding author. Tel.: +90 432 2251707x2278; fax: +90 432 2251188. E-mail address: [email protected] (I. Celik).
(ABA) and Gibberellic acid (GA3) was affective on sexual differentiation and some physiological parameters of laboratory mice. In a study, IAA effect investigated on human serum enzymes in-vitro, it was found that IAA inhibited aspartate aminotransferase (AST) but activated amylase, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) (Celik and Kara, 1997). Also, it was reported that while the levels of LDH and CPK increased significantly by IBA, the levels of AST, LDH and CPK were increased significantly by IAA after subacute exposure with 100 ppm dosages (Celik et al., 2002). IAA was found to be linear-mixed type inhibitor for human serum BChE, and uncompetitive inhibitor for the horse serum BChE enzyme (Cokugras and Bodur, 2003). Further, PGRs may induce oxidative stress, leading to generation of free radicals and cause lipid peroxidation as one of the molecular mechanisms involved in PGRs-induced toxicity (Tuluce and Celik, 2006; Candeias et al., 1995; Celik et al., 2007, 2006a,b). Antioxidant defense systems, present in all aerobic organisms, include antioxidant enzymes and free radical scavengers whose function is to remove reactive oxygen species (ROS), thus protecting the functions of organisms from oxidative stress (Regoli and Principato, 1995). The sensitivity of cell to oxidants is attenuated by antioxidant defense system such as GSH, GST, catalase (CAT), SOD, glutathione peroxidase (GPx), GR and glucose-6-phosphate dehydrogenase (G6PD). The antioxidant defense system maintains a relatively low rate of the reactive and harmful OH. Oxidative stress occurs as a result of the effect of xenobiotics causing the disturbances in the antioxidant enzymes system (Oruc-Ozcan and Uner,
0278-6915/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.037
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2002). Among these, GPx, through reduction of both hydrogen peroxide and organic hydroperoxides, provides an efficient protection against oxidative damage and free radicals in the presence of reduced glutathione (GSH). Previously oxidized GSH is regenerated by GR. SOD catalyses dismutation of superoxide anion radicals, whereas CAT eliminates hydrogen peroxide. Antioxidant enzymes play a crucial role in maintaining cell homeostasis. Another group of enzymes, GST act as catalyst of a wide variety of conjugation reactions of glutathione with xenobiotic compounds containing electrophilic center. Additionally, there are glutathione-independent enzymes that act as part of the cellular defense system against toxicity originated by active oxygen forms (Halliwell and Gutteridge, 1989). Despite the reasons mentioned in above paragraphs, little is known regarding the IBA effects on antioxidant defense systems of vertebrata. In order to achieve a more rational design of IBA, it is necessary to clarify the mechanism of oxidative stress for IBA. To this end, the treatments of IBA were done orally because the effect of chemicals represents a well characterized in vivo toxicity model system. The enzymes were chosen due to their important role for antioxidant defense systems and important role during detoxification in degradation and bioactivation of IBA.
The tissues were dissected and put in Petri dishes. After washing the tissues with 0.9% NaCl, samples were taken and kept at 78 °C until the analysis. The tissues were homogenized for 5 min in 50 mM ice-cold KH2PO4 solution (1:5 w/v) using a glass–porcelain homogenizer (20 kHz frequency ultrasonic, Jencons Scientific Co.) and then centrifuged at 7000g for 15 min. All processes were carried out at 4 °C. Supernatants were used to determine antioxidant defense system enzymes. 2.4. Biochemical analysis The erythrocyte and tissues GSH concentration was measured using the method described by Beutler et al. (1963). Briefly, 0.2 mL fresh erythrocyte pellets or the tissue supernatants were added to 1.8 mL distilled water. Three mL of the precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 mL distilled water) was mixed with haemolysate. The mixture was allowed to stand for approximately 5 min and then filtered (whatman N 42). Two millilitre of filtrate was taken and added into another tube, and then 8 mL of the phosphate solution (0.3 M disodium hydrogen phosphate) and 1 mL DTNB were added. A blank was prepared with 8 mL of phosphate solution, 2 mL diluted precipitating solution (three parts to two parts distilled water), and 1 mL DTNB reagent. A standard solution of the glutathione was prepared (40 mg/100 ml). The optical density was measured at 412 nm in a spectrophotometer (Novaspec II Pharmacia-Biotech, Biochrom Ltd., UK). GST (EC 2.5.1.18) was assayed at 25 oC spectrophotometrically by following the conjugation of glutathione with CDNB at 340 nm as described by Mannervik and Guthenberg (1981). To determine the activity of GPx (EC 1.11.1.9), t-butyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GPx and NADPH (Beutler, 1984). SOD (EC 1.15.1.1) activity was measured at 505 nm and 37 °C and calculated using inhibition percentage of formazon formation (McCord and Fridovich, 1969).
2. Materials and methods
2.5. Analysis of data
2.1. Chemicals
All data were expressed as mean ± standard deviation (SD). The statistical analyses were made using the Minitab 13 for windows packet program. Means and Standard deviations were calculated according to the standard methods for all parameters. One way ANOVA statistical test was used to determine the differences between means of the treatments and the control group accepting the significance level at p 6 0.05 (See Tables 1 and 2).
Trichloroacetic acid (TCA) ethylenediaminetetraacetic acid (EDTA), GSH, metaphosphoric acid, 5,50 dithiobis-(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced b-Nicotinamide adenine dinucleotide phosphate (NADPH), oxidized glutathione (GSSG), potassium dihydrogenephosphate (KH2PO4), sodium chloride (NaCl) and Indole-3-butyric acid (IBA) of technical grade used in this study were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Kits for antioxidant enzymes analysis were supplied by Randox Laboratories Ltd. 2.2. Animals Rats (Wistar) 4 months of age with and average weighing approximately 200– 250 g were provided by the animal house of the Sciences Faculty of Yüzüncü Yıl University, and were housed in 3 groups. The animals were housed at 20 ± 2 °C an in daily light/dark cycle. All animals were fed a group wheat–soybean-mealbased diet and water ad libitum in stainless cages, and received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and published by the National Institutes of Health. The ethic regulations have been followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments. This study was approved by The Ethic Committee of Yüzüncü Yıl University. 2.3. Treatment of rats This investigation was performed on male rats. The animals ere housed for a minimum of two weeks to ‘acclimatize’ before being dosed with the substance. The rats were exposed to 25 and 50 ppm IBA ad libitum 20 days for subacute and 45 days for subchronic applications as drinking water. Twenty-five and fifty milligrams of the IBA were dissolved in 1 mL of 1 N NaOH, and then were diluted with tap water until 1000 mL to obtain a 25 and 50 ppm dosages. For the control rats, only 1 mL of 1 N NaOH was added to 1000 mL of tap water. Because the PGRs are photoactive compounds the drinking water containing IBA was prepared and refreshed every day in amber bottle. Since all rats have the same physiologic characters, daily water consumption of all groups of rats was approximately 25 ± 3 mL during the tests. Consequently, the IBA intake amount of each rat was about 2.1 ± 0.3 mg per day. At the end of the treatments, after the rats were anesthetized blood and tissues samples were obtained. The blood samples were obtained from a cardiac puncture using syringe for the determination of biochemical analysis. For biochemical analysis, blood samples were put immediately into silicon disposable glass tubes with EDTA as an anticoagulant. Blood samples were centrifuged at 4000g for 15 min at 4 °C and erythrocyte pellets were obtained. Then the pellets were washed tree times with physiological saline (0.9% NaCl). The GSH concentration in erythrocytes and tissues were measured just after the animals were sacrificed because of tremendous loss of GSH. Also, SOD, GSH-Px and GST activities in the erythrocytes were measured in the pellets.
3. Results Following the exposure to 25 and 50 ppm dosages of IBA, the effects of IBA administration on the erythrocytes and brain, kidney, hearth, muscle, liver, lungs and spleen tissues oxidative stress damages index were evaluated as the level of GSH and the activity of antioxidant enzymes such as SOD, GR and GST in the tissues samples from control and treated rats. It was observed that GSH level and GSH-Px activity were fluctuated in the some tissues of rats treated with 25 and 50 ppm dosage of IBA at subacute and subchronic exposure. The GST increasing in the all of tissues of rats treated with 25 ppm and 50 ppm IBA were significant at subacute and subchronic periods. Also, SOD activity increased significantly with both dosages of IBA except for muscle tissue at subacute, and brain and spleen tissues at subchronic period compared to control rats (Tables 3 and 4). 4. Discussion In recent years, a significant increase in the use of PGRs against harmful agricultural pests and giving rise to losing product have been observed in Turkey and the rest of the world. One of the major reasons for the increase is the ease of using PGRs and ensuring an absolute result. In this study, IBA was preferred because information on its negative effects on ADS of higher animals is very limited for in vivo, oral exposures. Also, IBA is a derivative of IAA found in plants as endogen hormones and wide variety of biologically active compounds. In this study, experimental group was exposed to two dosages of chemical substance. Antioxidant defense system constituent were used as important biomarkers for detection of toxic nature of IBA. Four antioxidant markers (GSH, SOD, GSH-Px and GST) were evaluated for oxidative stress.
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N. Topalca et al. / Food and Chemical Toxicology 47 (2009) 2441–2444 Table 1 Effects of subacute and subchronic treatment of IBA on GSH level in tissues of rats. Period
Tissue
GSH Control
25 ppm
50 ppm
X ± SD
X ± SD
X ± SD
Subacute
Erythrocytes (mg/dL) Brain (mg/g) Kidney (mg/g) Hearth (mg/g) Muscle (mg/g) Liver (mg/g) Lungs (mg/g)
3.21 ± 0.37 12.12 ± 1.17 28.58 ± 2.66 19.57 ± 1.55 17.50 ± 1.05 45.29 ± 4.99 26.69 ± 2.28
2.87 ± 0.31 42.80 ± 2.21* 89.00 ± 5.34* 14.40 ± 1.77* 10.65 ± 1.37* 32.46 ± 2.66* 21.04 ± 1.73
2.66 ± 0.14* 57.40 ± 1.28* 77.80 ± 3.78* 30.21 ± 1.39* 20.87 ± 1.76 76.66 ± 2.65* 40.11 ± 3.19*
Subchronic
Erythrocytes (mg/dL) Brain (mg/g) Kidney (mg/g) Hearth (mg/g) Muscle (mg/g) Liver (mg/g) Lungs (mg/g) Spleen (mg/g)
3.21 ± 0.37 12.12 ± 1.17 28.58 ± 2.66 19.57 ± 1.55 17.50 ± 1.05 45.29 ± 4.99 26.69 ± 2.28 33.88 ± 2.67
2.92 ± 0.23 7.23 ± 0.25* 18.08 ± 0.31* 9.70 ± 0.46* 8.90 ± 0.43* 33.19 ± 1.61* 15.49 ± 1.00* 28.53 ± 0.53
2.76 ± 0.16 11.30 ± 1.11 25.91 ± 1.97 15.19 ± 1.02* 14.60 ± 0.45* 39.42 ± 2.61 26.34 ± 1.03 29.95 ± 1.88
Each value represents the Mean ± SD. Significantly different from control rats (One way ANOVA).
*
The results of the present study have demonstrated that the applied dosages of IBA could have affected the antioxidant defense systems in the rats. This is evidenced from our observation that, upon IBA treatment in vivo, the activities of GSH-Px, GST and SOD activities and GSH level differ from those of controls in the tissues of rats exposed to 25 and 50 ppm dosages of IBA for 20 and 45 days. So far, no study examining the effects of IBA on ADS in vivo have been made on rat. Therefore, we could not have the chance to compare our results with the previous results. The results in the present study showed that the GSH depletion in the all tissues except for lungs tissue of rats treated with both dosages of MP were significant. Reduced GSH and its metabolizing enzymes provide the major defense against ROS-induced cellular damage (Avellini et al., 1993). GSH depletion might enhance the risk of the oxidative stress (Regoli and Principato, 1995). A considerable decline in GSH content in the tissue under the present experimental model may be due to its utilization to challenge
the prevailing oxidative stress under the influence of ROS generated from IBA. The antioxidative enzymes such as GST and SOD were also seriously affected by IBA; SOD significantly increased in the almost tissues of rats treated with both doses of IBA whereas GSH-Px activity fluctuated with two doses of IBA. Although the reasons for such effect of IBA are not understood at the present, it is conceivable that IBA might be interacting primarily with the tissues, resulting in fluctuated enzymes activities by the way of increased reactive oxygen radicals as result of stress condition in the rats. Doyotte et al. (1997) pointed out that a decreased enzyme activity’s response may accompany a first exposure to pollutants, which can be followed by an induction of antioxidant systems. Thus, the existence of an inducible antioxidant system may reflect an adaptation of organisms. In contrast, Dimitrova et al. (1994) suggested that the superoxide radicals by themselves or after their transformation to H2O2 cause an oxidation of the cysteine in the enzyme and
Table 2 Effects of subacute and subchronic treatment of IBA on GST enzyme in tissues of rats.
Table 3 Effects of subacute and subchronic treatment of IBA on GSH-Px enzyme in tissues of rats.
Period
Period
Tissue
GST
Tissue
GSH-Px
Control
25 ppm
50 ppm
Control
25 ppm
X ± SD
X ± SD
X ± SD
X ± SD
X ± SD
X ± SD
50 ppm
Subacute
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
5.75 ± 0.46 2.26 ± 0.42 3.14 ± 0.37 1.39 ± 0.19 1.55 ± 0.28 6.98 ± 0.69 3.25 ± 0.24 1.43 ± 0.31
9.46 ± 0.63* 13.25 ± 0.59* 8.98 ± 0.47* 1.83 ± 0.38* 3.00 ± 0.47* 22.45 ± 0.65* 4.64 ± 0.61* 2.58 ± 0.27*
10.13 ± 0.95* 7.63 ± 0.71* 9.11 ± 0.69* 1.97 ± 0.38* 7.93 ± 0.69* 13.24 ± 1.59* 4.40 ± 0.43* 2.77 ± 0.24*
Subacute
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
12.93 ± 1.47 6.85 ± 0.19 12.11 ± 1.44 17.73 ± 1.59 10.03 ± 0.69 15.38 ± 1.65 14.91 ± 1.22 19.21 ± 1.01
15.67 ± 1.88 9.15 ± 0.64* 9.25 ± 0.69* 12.45 ± 1.60* 8.77 ± 0.29 12.11 ± 1.53 9.02 ± 0.57* 10.90 ± 0.45*
14.17 ± 1.60 7.69 ± 0.63 8.93 ± 0.63* 6.71 ± 0.43* 13.39 ± 1.25* 10.09 ± 1.25* 25.07 ± 0.78* 22.64 ± 0.77
Subchronic
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
5.75 ± 0.46 2.26 ± 0.42 3.14 ± 0.37 1.39 ± 0.19 1.55 ± 0.28 6.98 ± 0.69 3.25 ± 0.24 1.43 ± 0.31
9.79 ± 0.71* 7.63 ± 0.46* 6.50 ± 0.46* 2.75 ± 0.44* 4.90 ± 0.70* 10.15 ± 0.61* 4.14 ± 0.22* 3.38 ± 0.10*
11.25 ± 0.94* 6.50 ± 0.48* 10.20 ± 1.11* 5.43 ± 0.45* 5.26 ± 0.41* 17.38 ± 1.81* 6.85 ± 0.49* 6.49 ± 0.41*
Subchronic
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
12.93 ± 1.47 6.85 ± 0.19 12.11 ± 1.44 17.73 ± 1.59 10.03 ± 0.69 15.38 ± 1.65 14.91 ± 1.22 19.21 ± 1.01
16.90 ± 2.16* 11.84 ± 1.56* 12.06 ± 1.59 10.85 ± 1.65* 13.50 ± 1.63* 11.34 ± 1.26* 9.44 ± 0.80* 7.41 ± 0.26*
16.01 ± 2.14* 7.77 ± 0.88 7.87 ± 0.73*
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
8.56 ± 0.95* 15.46 ± 1.43* 8.11 ± 0.37* 9.84 ± 0.88* 9.42 ± 0.84*
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Table 4 Effects of subacute and subchronic treatment of IBA on SOD enzyme in tissues of rats.
Acknowledgments
Period
The authors are grateful to the University Grant Commission (The University of Yuzuncu Yil, No. 2007-FBE-YL77) for providing financial assistance during the tenure of Research Associate ship.
Subacute
Tissue
Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
Subchronic Erythrocytes (U/mL) Brain (U/g) Kidney (U/g) Hearth (U/g) Muscle (U/g) Liver (U/g) Lungs (U/g) Spleen (U/g)
SOD Control
25 ppm
50 ppm
X ± SD
X ± SD
X ± SD
974.21 ± 53.89 1088.37 ± 60.23* 1205.18 ± 54.40* 1167.12 ± 24.55 1144.22 ± 17.68 1253.12 ± 17.43 1278.95 ± 12.71 1155.98 ± 30.19 1261.34 ± 24.36 1288.91 ± 8.69
1265.87 ± 15.86* 1314.49 ± 9.59* 1315.92 ± 2.83* 1271.57 ± 12.38 1252.60 ± 33.98* 1333.20 ± 12.30* 1265.61 ± 10.20*
1288.37 ± 20.92* 1299.77 ± 15.28* 1315.18 ± 8.31* 1312.14 ± 14.55* 1302.07 ± 22.69* 1331.69 ± 4.44* 1326.92 ± 6.16*
974.21 ± 53.89 1224.80 ± 44.57* 1323.56 ± 36.30* 1167.12 ± 24.55 1144.22 ± 17.68 1253.12 ± 17.43 1278.95 ± 12.71 1155.98 ± 30.19 1261.34 ± 24.36 1288.91 ± 8.69
1224.14 ± 11.62 1271.39 ± 14.23* 1337.25 ± 8.03* 1322.06 ± 10.87* 1260.40 ± 19.47* 1336.08 ± 3.51* 1298.40 ± 8.74
2636.59 ± 7.84* 1276.68 ± 10.99* 2656.24 ± 8.48* 2650.18 ± 8.95* 2395.77 ± 38.80* 2513.83 ± 33.59* 2595.25 ± 18.62*
Each value represents the Mean ± SD. * Significantly different from control rats (One way ANOVA).
decrease SOD activity. Consequently, the decreased and increased ADS enzymes activities might have reflected a cellular oxidative stress due to IBA exposure. Meanwhile, GST activity significantly increased in the all tissues at subacute and subchronic treatment with both doses of IBA. The enzymatic antioxidants such as SOD, GR and GST have been shown to be sensitive indicators of increased oxidative stress in Mugil sp obtained from a polluted area containing high concentrations of polyaromatic hydrocarbons, polychlorinated biphenyls, and pesticides (Rodriguez-Arizaet et al., 1993). However, the increased activities of GST are known to serve as protective responses to eliminate xenobiotics (Smith and Litwack, 1980). In addition, findings of this study suggest that the fluctuated levels in the constituents of antioxidant defense systems in the tissues of rats exposed to IBA may be dependent on the differences between interstitial concentrations. Namely, the systems might have to be exposed to different xenobiotic concentration due to blood volume differences in the tissues. There have been reported that the long-term intoxication with plant growth regulators leads to a gradual exhaustion of SOD, GR, GSH-Px and GST or cause increase of antioxidative defense systems (Tuluce and Celik, 2006; Celik and Tuluce, 2006; Candeias et al., 1995; Celik et al., 2007, 2006a,b). The results are partly in accordance with our findings although the treatment time and manner, the setting of studies and concentrations of chemical are different. Our observations led us to conclude that the administrations of subacute and subchronic IBA cause changes in the antioxidant defense systems in the various tissues of rats. Thus, any external stressor such as IBA, even at nonlethal concentration can have a toxic effect on the organism. From the foregoing observations it may also be postulated that antioxidative constituents might offer a certain result of choice for monitoring biotoxicity of direct acting compounds such as IBA. Such a test will also be of value in pollution studies, and also be of interest to understand molecular basis of refractoriness of IBA toxicity. 5. Conflict of interest This study was approved by the ethic committee of Yüzüncü Yıl University.
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