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Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 84 (2006) 49–54 www.elsevier.com/locate/ypest
Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats Ismail Celik *, Yasin Tuluce Department of Biology, Faculty of Arts and Sciences, Yuzuncu Yil University, Van, Turkey Received 28 March 2005; accepted 18 May 2005 Available online 27 June 2005
Abstract This study aims to investigate the effects of indoleacetic acid (IAA) and kinetin (Kn), which are plant growth regulators (PGRs), on antioxidant defense systems [reduced glutathione (GSH), glutathione-S-transferase (GST), catalase (CAT)], and lipid peroxidation level (malondialdehyde, MDA) various tissues of rats. Rats (Sprague–Dawley albino) were exposed to 100 ppm IAA and Kn. One hundred parts per million of PGRs was administered orally to rats ad libitum for 21 days continuously. The PGRs treatments caused different effects on the content of MDA and antioxidant defense system in comparison to those of control rats. According to the results, the subchronic treatments of IAA caused significant decrease in the GSH concentration and CAT activity in erythrocyte. Kn decreased GSH concentration in erythrocyte too. While the MDA concentration in brain was increased significantly by IAA and Kn, Kn decreased significantly brain CAT and GST activity. The liver GST activity was decreased by IAA and Kn. But, liver CAT activity was increased by IAA. On the other hand, while IAA treatment caused a significant decrease kidney GST activity, Kn caused a significant decrease both kidney GST and CAT activity. Also, while heart CAT activity was decreased by IAA, heart GST activity was decreased by both IAA and Kn. Moreover, MDA concentration in heart was increased by Kn treatment. It was concluded that IAA might effect MDA and antioxidant defense on the animals at subchronic treatment. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Plant growth regulators; Malondialdehyde; Antioxidant defense system; Rats
1. Introduction Today, as the effects of plant growth regulators (PGRs) on plants are well known, they are used *
Corresponding author. E-mail address: [email protected] (I. Celik).
widely in agriculture. The toxic effects of these chemicals on animals are limited; therefore, this subject has attracted the interest of many researchers recently. Many chemicals are currently used in agriculture, and PGRs are among those widely used. The amounts of these substances placed into the
0048-3575/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.05.004
50
I. Celik, Y. Tuluce / Pesticide Biochemistry and Physiology 84 (2006) 49–54
environment may soon exceed those of insecticides [1]. The effects of different PGRs on insects have been investigated [2–7], but reports concerning vertebrates are very limited [8–16]. In the literature, it is reported that fecundity, longevity, and egg viability have been changed in different insects by PGRs treatment [2–7]. Furukawa et al. [8] findings indicate that IAA might induce the neuronal apoptosis in the S phase and lead to microencephaly. Hsiao et al. [9] resultsÕ suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Also, de Melo et al. [10] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of superoxide dismutase (SOD), CAT, and glutathione peroxidase. The addition of exogenous antioxidant enzymes (SOD and CAT) prevented the loss of cell membrane integrity induced by IAA. John et al. [11] observed that IAA possesses teratogenic effects in mice and rats. Ozmen et al. [12] observed that abscisic acid and gibberellic acid affect on sexual differentiation and some physiological parameters of laboratory mice. Also, it is reported that PGRs cause increase in the number of splenic plaque forming cells and circulating white blood cells, hematocrit values, and thymus weight in young deer mice [14]. El-Mofty and Sakr [15] found that gibberellin A3 (GA3) induced liver neoplasm in Egyptian toads, and they suggested that the tumors could be diagnosed as hepatocellular carcinomas. GA3 also induces microabscesses and hydropic degeneration in the liver and mononuclear inflammatory infiltration in the kidneys of laboratory mice, but not tumors [16]. On the other hand, some PGRs have been shown to affect the carbonic anhydrase isoenzymes of erythrocytes in humans and bovines [17]. The effects of IAA and Kn were also investigated on human serum enzymes in vitro. IAA was found to inhibit aspartate aminotransferase (AST) and activate amylase, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Kn inhibited muscle creatine kinase (CK-MB), while it activated AST and alanine aminotransferase (ALT) [18]. In the other hand, we have found that the level of IAA, AST, LDH, and CPK were increased significantly by IAA, and the level of AST, LDH, and
CPK were increased significantly by Kn in another study [19]. In addition, we determined that while Kn did not affect on the levels of MDA erythrocyte, muscle, heart, kidney, and liver tissues, IAA caused a significant increase in the MDA concentration in kidney and liver [20]. According to the U.S. Environmental Protection Agency (EPA), toxic xenobiotic chemicals are irritating to the eyes, skin, and mucous membrane and since it is easily absorbed dermally, orally, or by inhalation, can injure liver, kidney, muscle, and brain tissues. In spite of reason above, there is still great contradiction between results. Therefore, to achieve a more rational design of PGRs, it is necessary to clarify the mechanism of toxicity for PGRs and develop an understanding of structure–toxicity relationships. For this aim, the treatment of PGRs was done orally because the effect of chemicals represents a well characterized in vivo toxicity model system. The tissues were chosen due to its important role during detoxification in degradation and bioactivation of PGRs.
2. Materials and methods 2.1. Materials Plant growth regulators (PGRs) of technical grade were supplied by the Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals Rats (Sprague–Dawley albino) three months of age with an average weighing 150–200 g were provided by the animal house of the Medical School of Yu¨zu¨ncu¨ Yı´l University, and were housed in three groups, each group containing six rats. 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 ÔGuide for the Care and Use of Laboratory AnimalsÕ prepared by the National Academy of Science and published by the National Institutes of Health [21]. The animals were housed at 20 ± 2 °C an in daily light/dark cycle.
I. Celik, Y. Tuluce / Pesticide Biochemistry and Physiology 84 (2006) 49–54
2.3. Treatment of rats This investigation was performed on male rats. A dosage of IAA and Kn was used. The rats were exposed to 100 ppm of IAA and Kn ad libitum for 21 days. Hundred milligrams of PGRs was dissolved in 1 ml of 1 N NaOH, and then were diluted with tap water to obtain a 100 ppm dose. For the control rats, only 1 ml of NaOH was added to 1000 ml of tap water. The control rats were given only this drinking tap water. Since all rats have the same physiologic characters, daily water consumption of all groups rat was approximately 30 ± 5 ml during the tests. Consequently, the PGRs intake amount of each rat was about 3 ± 0.5 mg per day. At the end of the treatments, the rats were anesthetized by inhalation of diethyl ether, and after blood and tissues samples were obtained, they were sacrificed. The blood samples were obtained from a cardiac puncture using syringe for the determination of MDA levels. Blood samples were put immediately into silicon disposable glass tubes with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The erythrocyte packet was obtained by centrifuging blood samples at 4000g for 15 min at 4 °C, and washing with physiological saline (0.9% NaCl) three times. The concentration of GSH and MDA in erythrocytes, and the erythrocyte CAT and GST activity were measured in these erythrocyte pellets. The tissues were dissected and put in petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples taken and kept at 78 °C until analysis. The tissues were homogenized for 5 min in 0.115 M ice-cold potassium chloride (KCl) 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. Supernatant were used to determine biochemical analysis. 2.4. Biochemical analysis The erythrocyte and tissues MDA concentration was determined using the method described by Jain et al., [22], based on thiobarbituric acid
51
(TBA) reactivity. Briefly, 0.2 ml of erythrocyte packets or supernatant obtained from tissues, 0.8 ml of phosphate buffer (pH 7.4), 0.025 ml of butylated hydroxytoluene (BHT) and 0.5 ml of 30% trichloroacetic acid (TCA) were added to the tubes and mixed. After 2 h incubation at 20 °C, the mixture was centrifuged (400g) for 15 min. After this, 1 ml supernatant was taken and added to each tube, and then 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% TBA were added. These tubes with Teflon-lined screw caps were incubated at 90 °C in a water bath for 15 min and cooled to room temperature. The optical density was measured at 532 and 600 nm in a spectrophotometer for erythrocyte MDA, and the optical density was measured at 532 for tissues MDA concentration (Novaspec II Pharmacia-Biotech, Biochrom Ltd., UK). The erythrocyte and tissue GSH concentration was measured using the method described by Beutler et al. [23]. Briefly, 0.2 ml of erythrocyte pellet or supernatant was added to 1.8 ml of distilled water. Three milliliters 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. Two milliliters of the filtrate was taken and added into another tube, and then 8 ml of the phosphate solution and 1 ml of 5,5 0 -dithiobis-(2-nitrobenzoic acid) (DTNB) were added. A blank was prepared with 8 ml of phosphate solution, 2 ml of diluted precipitating solution (three parts to two parts distilled water), and 1 ml of DTNB reagent. A standard solution of the glutathione was prepared (40 mg/100 ml). The optical density was measured at 412 nm in the spectrophotometer. CAT activity was determined using the method described by Beutler [24], Briefly, 1 M Tris–HCl, 5 mM EDTA (pH 8), 10 mM H2O2, and H2O were mixed and the rate of H2O2 consumption at 240 nm and the 37 °C was used for quantitative determination of CAT activity. GST was assayed at 25 °C spectrophotometrically by following the conjugation of glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm as described by Mannervic and Guthenberg [25].
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2.5. Analysis of data The data were expressed as mean ± standard deviation (SD). For statistical analysis the SPSS/ PC+ package (SPSS/PC+, Chicago, IL, USA) was used. For all parameters, means and SD were calculated according to the standard methods. The Mann–Whitney U test was employed to test differences between means of the treatments and the control rats. The significance level was accepted at p 6 0.05 for all tests.
3. Results The results of experiment showed that the treatment of rats with IAA and Kn caused changes in the levels of MDA and GSH, and in the activity of CAT and GST in erythrocyte, muscle, liver, heart, and kidney in comparison to control rats (Table 1). To find out the significance of increases in different tissues, MDA on exposure to IAA and Kn for 21 days, the data obtained have been subjected to Mann–Whitney U test. According to the results, the subchronic treatment of IAA caused significant decrease in the GSH concentration and CAT activity in erythrocyte, and Kn decreased erythrocyte GSH activity too. While the level of brain MDA was increased significantly by IAA and Kn, Kn decreased significantly brain CAT and GST activity. While liver GST activity was decreased by IAA and Kn, IAA increased liver CAT activity. On the other hand, while IAA treatment caused a significant decrease kidney GST activity, Kn caused a significant decrease both kidney GST and CAT activity. Also, while IAA decreased heart CAT and GST activity, Kn and MDA concentration increased, and decreased GST activity.
4. Discussion In this study, IAA and Kn were preferred because information on its negative effects on higher animals is very limited for in vivo, oral exposures. Also, IAA and Kn are found in a wide variety of biologically active compounds. The data collected
in this study were all from one time-point of the experiment. We found that the treatment to IAA and Kn increased the production of lipid peroxides, and inhibited antioxidant defense in various rat tissues. So far, no study examining the effect of IAA and Kn in vivo has been made on rat erythrocyte and that of tissues MDA content and antioxidant enzymes activities. Therefore, we could not have the chance to compare our results with the previous results. However, Candeias et al. [26] investigated the peroxidation of liposomes by a haem peroxidase and hydrogen in the presence of IAA and derivates. They found that these compounds can accelerate lipid peroxidation up to 65-fold, and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. Also, we determined that while Kn did not affect on the levels of MDA erythrocyte, muscle, heart, kidney, and liver tissues, IAA caused a significant increase in the MDA concentration in kidney and liver [20]. Consequently, these studies agree with our results although the settings of studies are different. In the other hand, de Melo et al. [10] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of CAT. This report is in contradiction with our findings. In addition, because of high variability in analyzing MDA content and antioxidant enzymes–chemicals interaction in vitro and in vivo, and inconsistent factors like treatment time and manner, the setting of studies, purity of chemicals, and species tissue differences etc., it is difficult to compare the present data to different studies regarding the toxicological effect. The present study indicates that exposure IAA and Kn can result toxicological effects in vertebrates. This is evidenced from our observation that, upon PGRs treatment in vivo, the concentration of MDA in brain and heart was higher than control rats. In addition, the PGRs exerted significant decrease erythrocytes GSH. Also, the PGRs caused significant change some tissues GST and CAT activity. Therefore, an increase or decrease in the antioxidant defense may result in an increase of superoxide radicals. The reasons for such affect of PGRs are not understood at the present. But,
I. Celik, Y. Tuluce / Pesticide Biochemistry and Physiology 84 (2006) 49–54
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Table 1 Effects of 100 ppm IAA and Kn on antioxidant defense and MDA in various tissues of rats (mean ± SD). Tissue
Parameters
Erythrocyte
GSH (mg/dl) MDA (nmol/ml) CAT (U/ml) GST (U/ml)
22.73 ± 3.49 1.315 ± 0.279 190.5 ± 42.7 7.688 ± 1.229
14.73 ± 0.62a 1.322 ± 0.175 107.6 ± 44.2b 6.919 ± 0.513
13.18 ± 0.46a 1.567 ± 0.316 124.7 ± 51.5 6.099 ± 0.525
Brain
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
64.08 ± 6.52 56.09 ± 13.76 29.87 ± 3.92 5.750 ± 0.537
57.45 ± 10.14 95.33 ± 11.31c 21.30 ± 5.40 4.275 ± 1.011
61.05 ± 9.36 104.7 ± 23.7a 19.26 ± 4.27c 3.331 ± 0.648a
Liver
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
85.81 ± 14.90 61.91 ± 14.60 16.53 ± 3.02 28.469 ± 1.217
87.07 ± 3.89 46.24 ± 14.27 22.96 ± 5.08b 26.744 ± 0.844b
89.83 ± 17.06 66.58 ± 10.24 14.48 ± 1.75 25.031 ± 1.511c
Kidney
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
62.06 ± 4.59 37.82 ± 6.69 26.19 ± 6.49 6.200 ± 0.944
61.58 ± 2.77 33.29 ± 10.90 18.09 ± 6.01 4.906 ± 0.427b
58.79 ± 4.77 40.93 ± 12.87 13.60 ± 5.32c 4.438 ± 0.409a
Heart
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
65.13 ± 5.71 31.86 ± 6.88 19.70 ± 3.65 1.9750 ± 0.2018
66.21 ± 1.52 23.44 ± 7.76 11.29 ± 3.01d 1.5063 ± 0.184a
61.10 ± 4.27 49.35 ± 8.18b 18.46 ± 3.98 1.5938 ± 0.2037e
a b c d e
Control X ± SD
IAA X ± SD
Kn X ± SD
a = 0.012. b = 0.013. c = 0.021. d = 0.016. e = 0.047.
the increased content of MDA may result from an increase of hydroxyl radicals (OH) increase. However, it is conceivable that IAA and Kn might be interacting primarily with the liver, brain, and kidney tissues, resulting in lipid peroxidation synthesis by the way of increase superoxide radicals as a result of stressed condition in the rats, leading to an increase in lipid peroxidation. Because, it knows that OH can initiate lipid peroxidation in tissues [27] and MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation [28]. The decreased activity of GST may lead to decreased protection against oxidants [29]. It is not a general rule that increases in pollutant concentrations induce antioxidant activity. Doyotte et al. [30] pointed out that a decreased 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. Nevertheless, the physiological the role of a single antioxidant enzyme in the cell is poorly understood because of complex interactions and interrelationships among individual components. Findings of this study suggest that further experiments should be performed to elicit what is responsible for the elevation of MDA content in tissues, and for the decreasing or increasing level of antioxidant enzymes. In addition, the different values of antioxidant marker enzymes and MDA content in the tissues of rats exposed to PGRs may depend on differences of interstitial. Namely, the systems might have to be exposed to different xenobiotic concentrations due to blood volume differences in the tissues. In conclusion, these results suggest that the tissues MDA content and antioxidant markers
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may be offer with means for monitoring toxicity of compounds such as PGRs. This test results may be used in oncoming investigations if more studies confirm our findings. Such a test will be of value in pollution studies, and also be of interest to understand molecular basis of refractoriness PGRs toxicity.
References [1] L.G. Mickel, ‘‘Plant Growth Regulators’’ Controlling biological behavior with chemicals, Chem. Eng. News. 56 (1978) 18. [2] A.A. Guarra, Effect of biological active substance in the diet on development and reproduction of Heliothis spp, J. Econ. Entomol. 63 (1970) 1518–1521. [3] N.S. Visscher, Plant growth hormones affect Grasshopper growth and reproduction. Proc. Sch. Int. Symp. IncectPlant Relationship. Wageningen, (1982) 57–62. [4] W. De Man, A. De Loof, T. Briers, R. Huybrechts, Effect of abscisic acid on vitellogenesis in Sarcophaga bullata, Entomol. Exp. Appl. 29 (1991) 259–267. [5] C. Alanso, The Effects of gibberellic acid upon developmental processes in Drosophila hydlei, Entomol. Exp. Appl. 14 (1971) 73–82. [6] N.S. Visscher, Regulation of Grasshopper fecundity, longevity and egg viability by plant growth hormones, Experimentia 36 (1980) 130–131. [7] N.S. Visscher, Special report dietary plant growth hormones affects insect growth and reproduction, Bull. Plant Growth Reg. Soc. Am. 11 (1983) 4–6. [8] S. Furukawa, M. Abe, K. Usuda, I. Ogawa, Indole-3acetic acid induces microencephaly in rat fetuses, Toxicol. Pathol. 32 (6) (2004) 659–667. [9] G. Hsiao, M.Y. Shen, K.H. Lin, C.Y. Chou, N.H. Tzu, C.H. Lin, D.S. Chou, T.F. Chen, J.R. Sheu, Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo, Eur. J. Pharmacol. 465 (3) (2004) 281–287. [10] M.P. de Melo, T.M. de Lima, T.C. Pithon-Curi, R. Curi, The mechanism of indole acetic acid cytotoxicity. Toxicol. Lett. 14;148(1–2) (2004) 103–111. [11] J.A. John, C.D. Blogg, F.J. Murray, B.A. Schwetz, P.J. Gehring, Teratogenic effects of the plant hormone indole3-acetic acid in mice and rats, Teratology 19 (3) (1979) 321–324. [12] M. Ozmen, S.F. Topcuoglu, S. Bozcuk, N.A. Bozcuk, Effects of abscisic acid and gibberellic acid on sexual differentiation and some physiological parameters of laboratory mice, Turk. J. Biol. 19 (1995) 357–364. [13] N.S. Visscher, Effects of Abscisic Acid in Animal Growth and Reproduction, Praeger Science Publisher, New York, 1983, pp. 553–579.
[14] L.J. Olson, R.D. Hinsdill, Influence of feeding chlorocholine chloride and glyposine on selected immune parameters in deer mice peromiseus moniculatus, Toxicology 30 (1984) 103–114. [15] M.M. El-Mofty, S.A. Sakr, Induction of neoplasms in the Egyptian Toad by gibberellin A3, Oncology 45 (1988) 61– 64. [16] H. Ustun, T. Tecimer, M. Ozmen, S.F. Topcuoglu, N.A. Bozcuk, Effects of gibberellic acid and benzoprenin on mice. Histopatalogic review, Ank. Patol. Bu¨lt. 9 (1992) 36–40. [17] I. Celik, V. Turkoglu, H. Camas, In vitro activation human and bovine erythrocyte carbonic anhydrase ´ısozymes by some plant growth regulators, Biosci. Res. Bull. 13 (1997) 99–104. [18] I. Celik, M. Kara, The effects of plant growth regulators on activity of eight serum enzymes in vitro, J. Environ. Sci. Health A 32 (1997) 1755–1761. [19] I. Celik, H. Ozbek, Y. Tuluce, Effects of subchronic treatment of some plant growth regulators on serum enzyme levels in rats, Turk. J. Biol. 26 (2002) 73–76. [20] I. Celik, Y. Tuluce, N. Ozok, Effects of indoleacetic acid and kinetin on lipid peroxidation in various rats tissues, Turk. J. Biol. 26 (2002) 73–76. [21] World Medical Association Declaration of Helsinki, 2000, 52nd WMA General Assembly, Edinburgh, Scotland. [22] S.K. Jain, R. McVie, J. Duett, J.J. Herbst, Erythrocyte membrane lipid peroxidation and glycolylated hemoglobin in diabetes, Diabetes 38 (1989) 1539–1543. [23] E. Beutler, O.B. Dubon, M. Kelly, Improved method for the determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [24] E. Beutler, Red Cell Metabolism: A manual of Biochemical methods, third ed., Grune and Startton, New York, 1984, pp. 105–106. [25] B. Mannervik, C. Guthenberg, Glutathione S-transferase (Human Plasenta). Methods Enzymol. 77 (1981) 231–235. [26] L.P. Candeias, L.K. Folkes, M. Porssa, J. Parrick, P. Wardman, Enhancement of lipid peroxidation by indole-3acetic acid and derivates: substituent effects, Free Radic. Res. 23 (1995) 403–418. [27] B. Halliwell, J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease, Biochem. J. 219 (1984) 1–14. [28] B.A. Freeman, J.D. Crapo, Hyperoxia increases oxygen radical production in rat lung and lung mitochondria, J. Biol. Chem. 256 (1981) 10986–10992. [29] P. Amstad, A. Peskin, A.G. Shah, M.E. Mirault, R. Moret, I. Zbinden, P. Cerutti, The balance between Cu, Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress, Biochemist 30 (1991) 9305–9313. [30] A. Doyotte, C. Cossu, M.C. Jacquin, M. Babut, P. Vasseur, Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus, Aquat. Toxicol. 39 (1997) 93–110.
Pesticide Biochemistry and Physiology 84 (2006) 49–54 www.elsevier.com/locate/ypest
Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats Ismail Celik *, Yasin Tuluce Department of Biology, Faculty of Arts and Sciences, Yuzuncu Yil University, Van, Turkey Received 28 March 2005; accepted 18 May 2005 Available online 27 June 2005
Abstract This study aims to investigate the effects of indoleacetic acid (IAA) and kinetin (Kn), which are plant growth regulators (PGRs), on antioxidant defense systems [reduced glutathione (GSH), glutathione-S-transferase (GST), catalase (CAT)], and lipid peroxidation level (malondialdehyde, MDA) various tissues of rats. Rats (Sprague–Dawley albino) were exposed to 100 ppm IAA and Kn. One hundred parts per million of PGRs was administered orally to rats ad libitum for 21 days continuously. The PGRs treatments caused different effects on the content of MDA and antioxidant defense system in comparison to those of control rats. According to the results, the subchronic treatments of IAA caused significant decrease in the GSH concentration and CAT activity in erythrocyte. Kn decreased GSH concentration in erythrocyte too. While the MDA concentration in brain was increased significantly by IAA and Kn, Kn decreased significantly brain CAT and GST activity. The liver GST activity was decreased by IAA and Kn. But, liver CAT activity was increased by IAA. On the other hand, while IAA treatment caused a significant decrease kidney GST activity, Kn caused a significant decrease both kidney GST and CAT activity. Also, while heart CAT activity was decreased by IAA, heart GST activity was decreased by both IAA and Kn. Moreover, MDA concentration in heart was increased by Kn treatment. It was concluded that IAA might effect MDA and antioxidant defense on the animals at subchronic treatment. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Plant growth regulators; Malondialdehyde; Antioxidant defense system; Rats
1. Introduction Today, as the effects of plant growth regulators (PGRs) on plants are well known, they are used *
Corresponding author. E-mail address: [email protected] (I. Celik).
widely in agriculture. The toxic effects of these chemicals on animals are limited; therefore, this subject has attracted the interest of many researchers recently. Many chemicals are currently used in agriculture, and PGRs are among those widely used. The amounts of these substances placed into the
0048-3575/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.05.004
50
I. Celik, Y. Tuluce / Pesticide Biochemistry and Physiology 84 (2006) 49–54
environment may soon exceed those of insecticides [1]. The effects of different PGRs on insects have been investigated [2–7], but reports concerning vertebrates are very limited [8–16]. In the literature, it is reported that fecundity, longevity, and egg viability have been changed in different insects by PGRs treatment [2–7]. Furukawa et al. [8] findings indicate that IAA might induce the neuronal apoptosis in the S phase and lead to microencephaly. Hsiao et al. [9] resultsÕ suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Also, de Melo et al. [10] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of superoxide dismutase (SOD), CAT, and glutathione peroxidase. The addition of exogenous antioxidant enzymes (SOD and CAT) prevented the loss of cell membrane integrity induced by IAA. John et al. [11] observed that IAA possesses teratogenic effects in mice and rats. Ozmen et al. [12] observed that abscisic acid and gibberellic acid affect on sexual differentiation and some physiological parameters of laboratory mice. Also, it is reported that PGRs cause increase in the number of splenic plaque forming cells and circulating white blood cells, hematocrit values, and thymus weight in young deer mice [14]. El-Mofty and Sakr [15] found that gibberellin A3 (GA3) induced liver neoplasm in Egyptian toads, and they suggested that the tumors could be diagnosed as hepatocellular carcinomas. GA3 also induces microabscesses and hydropic degeneration in the liver and mononuclear inflammatory infiltration in the kidneys of laboratory mice, but not tumors [16]. On the other hand, some PGRs have been shown to affect the carbonic anhydrase isoenzymes of erythrocytes in humans and bovines [17]. The effects of IAA and Kn were also investigated on human serum enzymes in vitro. IAA was found to inhibit aspartate aminotransferase (AST) and activate amylase, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Kn inhibited muscle creatine kinase (CK-MB), while it activated AST and alanine aminotransferase (ALT) [18]. In the other hand, we have found that the level of IAA, AST, LDH, and CPK were increased significantly by IAA, and the level of AST, LDH, and
CPK were increased significantly by Kn in another study [19]. In addition, we determined that while Kn did not affect on the levels of MDA erythrocyte, muscle, heart, kidney, and liver tissues, IAA caused a significant increase in the MDA concentration in kidney and liver [20]. According to the U.S. Environmental Protection Agency (EPA), toxic xenobiotic chemicals are irritating to the eyes, skin, and mucous membrane and since it is easily absorbed dermally, orally, or by inhalation, can injure liver, kidney, muscle, and brain tissues. In spite of reason above, there is still great contradiction between results. Therefore, to achieve a more rational design of PGRs, it is necessary to clarify the mechanism of toxicity for PGRs and develop an understanding of structure–toxicity relationships. For this aim, the treatment of PGRs was done orally because the effect of chemicals represents a well characterized in vivo toxicity model system. The tissues were chosen due to its important role during detoxification in degradation and bioactivation of PGRs.
2. Materials and methods 2.1. Materials Plant growth regulators (PGRs) of technical grade were supplied by the Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals Rats (Sprague–Dawley albino) three months of age with an average weighing 150–200 g were provided by the animal house of the Medical School of Yu¨zu¨ncu¨ Yı´l University, and were housed in three groups, each group containing six rats. 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 ÔGuide for the Care and Use of Laboratory AnimalsÕ prepared by the National Academy of Science and published by the National Institutes of Health [21]. The animals were housed at 20 ± 2 °C an in daily light/dark cycle.
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2.3. Treatment of rats This investigation was performed on male rats. A dosage of IAA and Kn was used. The rats were exposed to 100 ppm of IAA and Kn ad libitum for 21 days. Hundred milligrams of PGRs was dissolved in 1 ml of 1 N NaOH, and then were diluted with tap water to obtain a 100 ppm dose. For the control rats, only 1 ml of NaOH was added to 1000 ml of tap water. The control rats were given only this drinking tap water. Since all rats have the same physiologic characters, daily water consumption of all groups rat was approximately 30 ± 5 ml during the tests. Consequently, the PGRs intake amount of each rat was about 3 ± 0.5 mg per day. At the end of the treatments, the rats were anesthetized by inhalation of diethyl ether, and after blood and tissues samples were obtained, they were sacrificed. The blood samples were obtained from a cardiac puncture using syringe for the determination of MDA levels. Blood samples were put immediately into silicon disposable glass tubes with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The erythrocyte packet was obtained by centrifuging blood samples at 4000g for 15 min at 4 °C, and washing with physiological saline (0.9% NaCl) three times. The concentration of GSH and MDA in erythrocytes, and the erythrocyte CAT and GST activity were measured in these erythrocyte pellets. The tissues were dissected and put in petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples taken and kept at 78 °C until analysis. The tissues were homogenized for 5 min in 0.115 M ice-cold potassium chloride (KCl) 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. Supernatant were used to determine biochemical analysis. 2.4. Biochemical analysis The erythrocyte and tissues MDA concentration was determined using the method described by Jain et al., [22], based on thiobarbituric acid
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(TBA) reactivity. Briefly, 0.2 ml of erythrocyte packets or supernatant obtained from tissues, 0.8 ml of phosphate buffer (pH 7.4), 0.025 ml of butylated hydroxytoluene (BHT) and 0.5 ml of 30% trichloroacetic acid (TCA) were added to the tubes and mixed. After 2 h incubation at 20 °C, the mixture was centrifuged (400g) for 15 min. After this, 1 ml supernatant was taken and added to each tube, and then 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% TBA were added. These tubes with Teflon-lined screw caps were incubated at 90 °C in a water bath for 15 min and cooled to room temperature. The optical density was measured at 532 and 600 nm in a spectrophotometer for erythrocyte MDA, and the optical density was measured at 532 for tissues MDA concentration (Novaspec II Pharmacia-Biotech, Biochrom Ltd., UK). The erythrocyte and tissue GSH concentration was measured using the method described by Beutler et al. [23]. Briefly, 0.2 ml of erythrocyte pellet or supernatant was added to 1.8 ml of distilled water. Three milliliters 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. Two milliliters of the filtrate was taken and added into another tube, and then 8 ml of the phosphate solution and 1 ml of 5,5 0 -dithiobis-(2-nitrobenzoic acid) (DTNB) were added. A blank was prepared with 8 ml of phosphate solution, 2 ml of diluted precipitating solution (three parts to two parts distilled water), and 1 ml of DTNB reagent. A standard solution of the glutathione was prepared (40 mg/100 ml). The optical density was measured at 412 nm in the spectrophotometer. CAT activity was determined using the method described by Beutler [24], Briefly, 1 M Tris–HCl, 5 mM EDTA (pH 8), 10 mM H2O2, and H2O were mixed and the rate of H2O2 consumption at 240 nm and the 37 °C was used for quantitative determination of CAT activity. GST was assayed at 25 °C spectrophotometrically by following the conjugation of glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm as described by Mannervic and Guthenberg [25].
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2.5. Analysis of data The data were expressed as mean ± standard deviation (SD). For statistical analysis the SPSS/ PC+ package (SPSS/PC+, Chicago, IL, USA) was used. For all parameters, means and SD were calculated according to the standard methods. The Mann–Whitney U test was employed to test differences between means of the treatments and the control rats. The significance level was accepted at p 6 0.05 for all tests.
3. Results The results of experiment showed that the treatment of rats with IAA and Kn caused changes in the levels of MDA and GSH, and in the activity of CAT and GST in erythrocyte, muscle, liver, heart, and kidney in comparison to control rats (Table 1). To find out the significance of increases in different tissues, MDA on exposure to IAA and Kn for 21 days, the data obtained have been subjected to Mann–Whitney U test. According to the results, the subchronic treatment of IAA caused significant decrease in the GSH concentration and CAT activity in erythrocyte, and Kn decreased erythrocyte GSH activity too. While the level of brain MDA was increased significantly by IAA and Kn, Kn decreased significantly brain CAT and GST activity. While liver GST activity was decreased by IAA and Kn, IAA increased liver CAT activity. On the other hand, while IAA treatment caused a significant decrease kidney GST activity, Kn caused a significant decrease both kidney GST and CAT activity. Also, while IAA decreased heart CAT and GST activity, Kn and MDA concentration increased, and decreased GST activity.
4. Discussion In this study, IAA and Kn were preferred because information on its negative effects on higher animals is very limited for in vivo, oral exposures. Also, IAA and Kn are found in a wide variety of biologically active compounds. The data collected
in this study were all from one time-point of the experiment. We found that the treatment to IAA and Kn increased the production of lipid peroxides, and inhibited antioxidant defense in various rat tissues. So far, no study examining the effect of IAA and Kn in vivo has been made on rat erythrocyte and that of tissues MDA content and antioxidant enzymes activities. Therefore, we could not have the chance to compare our results with the previous results. However, Candeias et al. [26] investigated the peroxidation of liposomes by a haem peroxidase and hydrogen in the presence of IAA and derivates. They found that these compounds can accelerate lipid peroxidation up to 65-fold, and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. Also, we determined that while Kn did not affect on the levels of MDA erythrocyte, muscle, heart, kidney, and liver tissues, IAA caused a significant increase in the MDA concentration in kidney and liver [20]. Consequently, these studies agree with our results although the settings of studies are different. In the other hand, de Melo et al. [10] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of CAT. This report is in contradiction with our findings. In addition, because of high variability in analyzing MDA content and antioxidant enzymes–chemicals interaction in vitro and in vivo, and inconsistent factors like treatment time and manner, the setting of studies, purity of chemicals, and species tissue differences etc., it is difficult to compare the present data to different studies regarding the toxicological effect. The present study indicates that exposure IAA and Kn can result toxicological effects in vertebrates. This is evidenced from our observation that, upon PGRs treatment in vivo, the concentration of MDA in brain and heart was higher than control rats. In addition, the PGRs exerted significant decrease erythrocytes GSH. Also, the PGRs caused significant change some tissues GST and CAT activity. Therefore, an increase or decrease in the antioxidant defense may result in an increase of superoxide radicals. The reasons for such affect of PGRs are not understood at the present. But,
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Table 1 Effects of 100 ppm IAA and Kn on antioxidant defense and MDA in various tissues of rats (mean ± SD). Tissue
Parameters
Erythrocyte
GSH (mg/dl) MDA (nmol/ml) CAT (U/ml) GST (U/ml)
22.73 ± 3.49 1.315 ± 0.279 190.5 ± 42.7 7.688 ± 1.229
14.73 ± 0.62a 1.322 ± 0.175 107.6 ± 44.2b 6.919 ± 0.513
13.18 ± 0.46a 1.567 ± 0.316 124.7 ± 51.5 6.099 ± 0.525
Brain
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
64.08 ± 6.52 56.09 ± 13.76 29.87 ± 3.92 5.750 ± 0.537
57.45 ± 10.14 95.33 ± 11.31c 21.30 ± 5.40 4.275 ± 1.011
61.05 ± 9.36 104.7 ± 23.7a 19.26 ± 4.27c 3.331 ± 0.648a
Liver
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
85.81 ± 14.90 61.91 ± 14.60 16.53 ± 3.02 28.469 ± 1.217
87.07 ± 3.89 46.24 ± 14.27 22.96 ± 5.08b 26.744 ± 0.844b
89.83 ± 17.06 66.58 ± 10.24 14.48 ± 1.75 25.031 ± 1.511c
Kidney
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
62.06 ± 4.59 37.82 ± 6.69 26.19 ± 6.49 6.200 ± 0.944
61.58 ± 2.77 33.29 ± 10.90 18.09 ± 6.01 4.906 ± 0.427b
58.79 ± 4.77 40.93 ± 12.87 13.60 ± 5.32c 4.438 ± 0.409a
Heart
GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g)
65.13 ± 5.71 31.86 ± 6.88 19.70 ± 3.65 1.9750 ± 0.2018
66.21 ± 1.52 23.44 ± 7.76 11.29 ± 3.01d 1.5063 ± 0.184a
61.10 ± 4.27 49.35 ± 8.18b 18.46 ± 3.98 1.5938 ± 0.2037e
a b c d e
Control X ± SD
IAA X ± SD
Kn X ± SD
a = 0.012. b = 0.013. c = 0.021. d = 0.016. e = 0.047.
the increased content of MDA may result from an increase of hydroxyl radicals (OH) increase. However, it is conceivable that IAA and Kn might be interacting primarily with the liver, brain, and kidney tissues, resulting in lipid peroxidation synthesis by the way of increase superoxide radicals as a result of stressed condition in the rats, leading to an increase in lipid peroxidation. Because, it knows that OH can initiate lipid peroxidation in tissues [27] and MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation [28]. The decreased activity of GST may lead to decreased protection against oxidants [29]. It is not a general rule that increases in pollutant concentrations induce antioxidant activity. Doyotte et al. [30] pointed out that a decreased 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. Nevertheless, the physiological the role of a single antioxidant enzyme in the cell is poorly understood because of complex interactions and interrelationships among individual components. Findings of this study suggest that further experiments should be performed to elicit what is responsible for the elevation of MDA content in tissues, and for the decreasing or increasing level of antioxidant enzymes. In addition, the different values of antioxidant marker enzymes and MDA content in the tissues of rats exposed to PGRs may depend on differences of interstitial. Namely, the systems might have to be exposed to different xenobiotic concentrations due to blood volume differences in the tissues. In conclusion, these results suggest that the tissues MDA content and antioxidant markers
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may be offer with means for monitoring toxicity of compounds such as PGRs. This test results may be used in oncoming investigations if more studies confirm our findings. Such a test will be of value in pollution studies, and also be of interest to understand molecular basis of refractoriness PGRs toxicity.
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