Influence of subacute and subchronic treatment of abcisic acid and gibberellic acid on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats

Pesticide Biochemistry and Physiology 86 (2006) 85–92 www.elsevier.com/locate/ypest

InXuence of subacute and subchronic treatment of abcisic acid and gibberellic acid on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats Yasin Tuluce, Ismail Celik ¤ Yuzuncu Yil University, Faculty of Arts and Sciences, Department of Biology, Van, Turkey Received 8 December 2005; accepted 25 January 2006 Available online 9 March 2006

Abstract This study describes the subacute and subchronic eVects of two plant growth regulators (PGRs) [abcisic acid (ABA) and gibberellic acid (GA3)] on serum marker enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), -glutamil transpeptidase (GGT)], antioxidant defense systems [reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] and lipid peroxidation level (Malondialdehyde D MDA) in various tissues of rats. Rats (Sprague–Dawley albino) were exposed to 75 ppm (parts per million) of ABA and GA3. Seventy-Wve parts per million of PGRs as drinking water was administered orally ad libitum for 25 and 50 days continuously. The PGRs treatments caused diVerent eVect on the serum marker enzymes, antioxidant defense systems and the content of MDA in comparison to those of control rats. Results show that ABA caused a signiWcant decrease in serum LDH and CPK activity with both periods. Also, GA3 signiWcantly decreased serum AST, CPK, and LDH activity with subacute and decreased serum ALT, CPK, LDH, and GGT treated with subchronic periods. The lipid peroxidation end product MDA signiWcantly increased in the erythrocyte, liver, brain, and muscle of rats treated with both the period of GA3 without signiWcantly change in the erythrocyte and muscle of rats treated with the subacute period of ABA. The GSH levels were signiWcantly depleted in the erythrocyte and brain of rats treated with both the period of GA3 without any change in the erythrocyte, liver, brain, and muscle of rats treated with both the period of ABA. Also GSH levels in the muscle signiWcantly depleted with the subchronic period of GA3. Antioxidant enzyme activities such as SOD signiWcantly decreased in the erythrocyte, liver and brain tissues but increased in the muscle tissue of rats treated with both the periods of GA3. Meanwhile, SOD signiWcantly decreased in liver and brain, and increased in muscle of rats treated with both the period of ABA. While CAT signiWcantly decreased in the all tissues of rats treated with both the period of GA3, decreased in the liver and muscle of rats treated with both the periods of ABA too. On the other hand, the ancillary enzyme GPx and GR activity in the erythrocytes, liver, brain and muscle were either signiWcantly depleted or not changed with two periods of PGRs. The drug metabolizing enzyme GST activity signiWcantly decreased in the brain of rats treated with subacute period of PGRs but increased in the erythrocytes of rats treated with subacute period of GA3. As a conclusion, ABA and GA3 had signiWcantly increased the activity of hepatic damage enzymes. Also the rats resisted to oxidative stress via antioxidant mechanism. However, the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat’s tissues. These data, along with changes, suggest that PGRs produced substantial systemic organ toxicity in the erythrocyte, liver, brain, and muscle during the period of a 25-day subacute and 50-day subchronic exposure. © 2006 Elsevier Inc. All rights reserved. Keywords: Plant growth regulators; Serum enzymes; Malondialdehyde; Antioxidant defense system; Rats

1. Introduction

*

Corresponding author. Fax: +90 432 2251114. E-mail address: [email protected] (I. Celik).

0048-3575/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.01.009

Antioxidant defenses, present in all aerobic organisms, include antioxidant enzymes and free-radical scavengers whose function is to remove reactive oxygen species, thus

86

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

protecting whose function organisms from oxidative stress [28]. The sensitivity of cell to oxidants is attenuated by antioxidant defense system such as GSH, GST, CAT, SOD, glutathione peroxidases (GPx),1 GR, and glucose-6-phosphate dehydrogenase (G6PD). Among these enzymes, the GPx, through reduction of both hydrogen peroxide and organic hydroperoxides, provide an eYcient protection against oxidative damage and free radicals in the presence of GSH. Oxidized glutathione (GSSG) is regenerated by GR. SOD catalyses dismutation of superoxide anion radicals, whereas CAT eliminates hydrogen peroxide. 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 defence system against toxicity originated by active oxygen forms [17]. Oxidative stress may produce DNA damage, enzymatic inactivation, and peroxidation of cell constituents, especially lipid peroxidation when antioxidant defences are impaired or overcome [25]. Many chemicals are currently used in agriculture, and PGRs are among those widely used. ABA plays important roles in many cellular processes including seed development, dormancy, germination, vegetative growth, and environmental stress responses. These diverse functions of ABA involve complex regulatory mechanisms that control its production, degradation, signal perception, and transduction. Because of the key role of ABA in plant stress responses, understanding these regulatory mechanisms will help devise rational strategies to breed or genetically engineer crop plants with increased tolerance to adverse environmental conditions [37]. GA3 plays also, important roles in many cellular processes including promotes stem elongation, overcomes dormancy in seed and buds, involved in parthenocarpic fruit development, Xowering, mobilization of food reserves in grass seed germination, juvenility, and sex expression [31]. The amounts of these substances placed into the environment may soon exceed those of insecticides [23]. 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 eVects in mammalian organisms. In the literature, it is reported that fecundity, longevity, and egg viability have been changed in diVerent insects by

1 Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ABA, abcisic acid; GA3, gibberellic acid; BHT, butylated hydroxytoluene; CAT, catalase; CDNB, 5,5⬘dithiobis-(2-nitrobenzoic acid); CPK, creatine phosphokinase; DTNB, 1-chloro-2,4-dinitrobenzene; EDTA, ethylenediaminetetraacetic acid; 2,4-D,2,4-dichlorofenoxyacetic acid; GST, glutathione-S-transferase; GPx, glutathione peroxidases; GSH, reduced glutathione; GR, glutathione reductase; G6PD, glucose-6-phosphate dehydrogenase; LDH, lactate dehydrogenase; NAA, naphthaleneacetic acid; NADPH, -nicotinamide adenine dinucleotide phosphate (reduced); MDA, Malondialdehyde; GSSG, oxidized glutathione; PGRs, plant growth regulators; ppm, parts per million; SOD, superoxide dismutase; TBA, thiobarbituric acid; TCA, trichloroacetic acid; TRIS, trihydroxymethyl aminomethane; TIBA, 2,3,5-triiodobenzoic acid.

PGRs treatment [16,33,34,10,1]. Furukawn et al. [15] Wndings indicate that IAA (indole acetic acid) might induce the neuronal apoptosis in the S phase and lead to microencephaly. Hsiao [18] results’ suggest that kinetin has eVective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Also, de Melo et al. [11] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of 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. [20] observed that IAA possesses teratogenic eVects in mice and rats. Ozmen et al. [27] observed that abscisic acid and gibberellic acid aVect on sexual diVerentiation and some physiological parameters of laboratory mice. Also, it is reported that PGRs causes increase in the number of splenic plaque forming cells and circulating white blood cells, hematocrit values, and thymus weight in young deer mice [24]. El-Mofty and Sakr [13] found that gibberellin A3 (GA3) induced liver neoplasm in Egyptian toads, and they suggested that the tumours could be diagnosed as hepatocellular carcinomas. GA3 also induces microabsceses and hydropic degeneration in the liver and mononuclear inXammatory inWltration in the kidneys of laboratory mice, but not tumours [32]. On the other hand, Some PGRs have been shown to aVect the antioxidant defense systems and MDA content [8]. The eVects of IAA and Kn (kinetin) 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) [6]. Also, we have found in another study that while the levels of LDH and CPK were increased signiWcantly by IBA (indole butiric acid), the levels of AST, LDH, and CPK were increased signiWcantly by IAA. In addition, the levels of AST, LDH, and CPK were increased signiWcantly by kinetin [7]. In addition, 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 [8,5]. 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. Oxidative stress occurs as a result of the eVect of xenobiotics causing the disturbances in the antioxidant enzymes system [25]. In spite of reason above, there is still great contradiction between results. Therefore, In order 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 eVect of chemicals represents a well characterized in vivo toxicity model system. The tissues were chosen due to its important role during detoxiWcation in degradation and bioactivation of PGRs.

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

2. Materials and methods 2.1. Materials PGRs, thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA), reduced glutathione (GSH), metaphosphoric acid, 5,5⬘dithiobis-(2-nitrobenzoic acid) (DTNB), trihydroxymethyl aminomethane (Tris), 1-chloro2,4-dinitrobenzene (CDNB), -nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), oxidized glutathione (GSSG), potassium chloride (KCl), hydrogen peroxide (H2O2), and sodium chloride (NaCl) 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 (Sprague–Dawley albino) 4 months of age with an average weighing 150–200 g were provided by the animal house of the Medical School of Yüzüncü YÂl University, and were housed in seven groups, each group containing six rats. The animals were housed at 20 § 2 °C an in daily light/ dark cycle. All animals were fed a group wheat–soybeanmeal-based 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 [35]. The ethic regulations have been followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments. 2.3. Treatment of rats This investigation was performed on male rats. The animals are housed for a minimum of Wve days to ‘acclimatize’ before being dosed with the substance. The animals are repeatedly dosed with a chemical over a period of 28–90 days. This is usually done orally (forcefeeding with a syringe or tube) but may also be administered dermally or inhaled. It is accepted 7–28 days for subacute and 28–90 days for subchronic. Therefore, the rats were exposed to 75 ppm ABA and GA3 ad libitum for 25 (subacute period) and 50 (subchronic period) days as drinking water. Seventy-Wve milligrams of the PGRs were dissolved in 1 ml of 1 N NaOH and then were diluted with tab water until 1000 ml to obtain a 75 ppm dose. For the control rats, only 1 ml of 1 N 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 § 3 ml during the tests. Consequently, the PGRs intake amount of each rat was about 2.2 § 0.3 mg per day. At the end of the treatments, the rats were anesthetized by inhalation of diethyl ether, and after blood and tissues

87

samples were obtained, they were sacriWced. The blood samples were obtained from a cardiac puncture using syringe for the determination of serum enzyme levels and biochemical analysis. For serum enzyme levels, blood samples were put immediately into ice-chilled siliconized disposable glass tubes. The serum samples were obtained by centrifuging blood samples at 4000g for 15 min. at 4 °C, and enzyme levels were measured in these serum samples. 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 three times with physiological saline (0.9% NaCl). The GSH concentration in erythrocytes and tissues were measured just after the animals were sacriWced because of tremendous loss of GSH. The concentration of GSH and MDA and the activity of SOD, GPx, GR, CAT, and GST in erythrocytes were measured in the pellets. The tissues were dissected and put in Petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples were taken and kept at ¡78 °C until analysis. The tissues were homogenized for 5 min in 0.115 M ice-cold KCl solution (1:5 w/v) using a glass–porcelain homogenizer (20 KHz frequency ultrasonic, Jencons ScientiWc Co.) and then centrifuged at 7000g for 15 min. All processes were carried out at 4 °C. Supernatants and homogenates were used to determine antioxidant defense systems and MDA. 2.4. Biochemical analysis The erythrocyte and tissues MDA concentration was determined using the method described by Jain et al. [19], based on TBA reactivity. BrieXy, 0.2 ml erythrocyte pellets or supernatant obtained from tissues, 0.8 ml phosphate buVer (pH 7.4), 0.025 ml BHT, and 0.5 ml of 30% 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 TeXon-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 PharmaciaBiotech, Biochrom Ltd., UK). The erythrocyte and tissues GSH concentration was measured using the method described by Beutler et al. [4]. BrieXy, 0.2 ml fresh erythrocyte pellet or supernatant was added to 1.8 ml distilled water. Three milliliter 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 Wltered (Whatman No. 42). Two milliliter of Wltrate was taken and added into another tube, and then 8 ml of the phosphate solution (0.3 M disodium

88

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

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 the spectrophotometer. CAT (EC 1.11.1.6) activity was determined using the method described by Beutler [3]. BrieXy, 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 37 °C was used for quantitative determination of CAT activity. The decrease in optical density of the system is measured against that of the blank for 10 min (20 l of 95% ethanol was added in 1 ml of hemolysate to break down complex of catalase and H2O2). One EU was deWned as the enzyme breaking 1 mol H2O2 per min at 25 °C and optimal pH (pH 8.0). GST (EC 2.5.1.18) 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 Mannervik and Guthenberg [21]. One EU was deWned as the enzyme conjugation of 1 mol of glutathione with CDNB per min at 25 °C and optimal pH (pH 7.8). GR (EC 1.6.4.2) activity was assayed at 37 °C and 340 nm by following the oxidation of NADPH by GSSG [3]. One EU was deWned as the enzyme oxidation 1 mol of NADPH per min at 25 °C and optimal pH (pH 8.0). SOD (EC 1.15.1.1) activity was measured at 505 nm and 37 °C and calculated using inhibition percentage of formazon formation. One EU was deWned as the enzyme inhibition percentage of formazon formation 1 mol per min at 25 °C and optimal pH (pH 7.8) [22]. To determine the activity of GPx (EC 1.11.1.9), tbutyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GPx and NADPH. The activity of GPx was assayed at 37 °C and 340 nm by calculating the diVerence in absorbance values during the oxidation of NADPH [3]. One EU was deWned as the enzyme oxidation 1 mol of NADPH per min at 25 °C and optimal pH (pH 8.0). 2.5. Measurement of enzyme levels Serum enzyme activities [AST (EC 2.6.1.1), ALT (E.C 2.6.1.2), CPK (EC 2.7.3.2), -glutamil transpeptidase (EC

2.3.2.2) and LDH (EC 1.1.1.27)], were measured by an auto analyzer (BM/HITACHI-911), using the kits. 2.6. Analysis of data The data were expressed as means § 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 examine diVerences between means of the treatments and the control rats. The signiWcance level was accepted at p 6 0.05 for all tests. 3. Results The results of experiment showed that the treatment of rats with ABA and GA3 caused changes in the activities of serum enzymes (Table 1), in the concentration of MDA and GSH, and antioxidant enzymes such as CAT, GST, GR, GPx, and SOD in erythrocyte, liver, brain, and muscle in comparison to control rats (Tables 2–5). To Wnd out the signiWcance of biochemical changes in diVerent tissues exposed to ABA and GA3 for 25 and 50 days, the data have been subjected to Mann–Whitney U test. According to the results, ABA caused a signiWcant decrease in serum LDH and CPK activity with both periods. Also, while GA3 signiWcantly decreased serum AST, CPK, and LDH activity with subacute periods, decreased serum ALT, CPK, LDH, and GGT treated with subchronic periods. The lipid peroxidation end product MDA signiWcantly increased in the erythrocyte, liver, brain, and muscle of rats treated with both the period of GA3 without signiWcantly change in the erythrocyte and muscle of rats treated with the subacute period of ABA. The GSH levels were signiWcantly depleted in the erythrocyte and brain of rats treated with both the period of GA3 without any change in the erythrocyte, liver, brain, and muscle of rats treated with both the period of ABA. Also GSH levels in the muscle signiWcantly depleted with the subchronic period of GA3. Antioxidant enzyme activities such as SOD signiWcantly decreased in the erythrocyte, liver, and brain tissues but increased in the muscle

Table 1 EVects of 2.2 § 0.3 mg daily ABA and GA3 on serum marker enzyme levels of rats Period

Parameters

Control X § SD

ABA X § SD

GA3 X § SD

Subacute

AST(U/L) ALT (U/L) LDH (U/L) CPK (U/L) GGT

150.33 § 12.31 56.50 § 9.14 1555 § 153.2 1209.3 § 126.5 0.67 § 0.52

132.50 § 19.87 63.00 § 8.79 1144 § 79¤ 640.5 § 99.3¤ 1.17 § 0.41

130.33 § 13.09¤ 63.50 § 5.13 1035 § 38¤ 804.5 § 222.2¤ 0.67 § 0.52

Subchronic

AST(U/L) ALT (U/L) LDH (U/L) CPK (U/L) GGT

163.7 § 33.4 69.50 § 5.89 1375.3 § 230.6 1106.7 § 39.5 2.00 § 0.63

Each value represents the mean § SD. ¤ p < 0.05.

156.3 § 31.9 64.83 § 3.13 971.7 § 81.7¤ 730.7 § 159.2¤ 1.83 § 0.41

136.2 § 17.8 54.83 § 1.17¤ 896.7 § 99.8¤ 750.3 § 114.2¤ 0.83 § 0.41¤

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

89

Table 2 EVects of 2.2 § 0.3 mg daily ABA and GA3 on antioxidant defense and MDA in erythrocyte of rats Period

Parameters

Subacute

GSH (mg/dl) MDA (nmol/ml) CAT (U/ml) GST (U/ml) GR (U/ml) SOD (U/ml) GPx (U/ml)

Control X § SD 39.59 § 1.27 1.02 § 0.26 68.02 § 9.98 27.83 § 1.21 2.55 § 0.33 485.50 § 167.21 95.26 § 12.55

ABA X § SD 35.18 § 1.66¤ 1.37 § 0.31 52.25 § 9.06¤ 28.92 § 3.01 2.22 § 0.26 397.14 § 123.29 120.92 § 24.98

GA3 X § SD 32.29 § 1.56¤ 2.04 § 0.16¤ 30.12 § 5.93¤ 30.67 § 2.92¤ 2.80 § 0.41 267.93 § 67.97¤ 93.73 § 21.80

Subchronic

GSH (mg/dl) MDA (nmol/ml) CAT (U/ml) GST (U/ml) GR (U/ml) SOD (U/ml) GPx (U/ml)

39.51 § 3.43 1.01 § 0.15 65.99 § 8.50 37.38 § 3.80 2.76 § 0.36 464.70 § 124.10 94.24 § 10.63

36.66 § 2.03 1.44 § 0.15¤ 63.76 § 9.69 34.42 § 3.28 2.50 § 0.37 416.86 § 159.33 108.01 § 30.97

34.85 § 2.85¤ 1.71 § 0.18¤ 48.80 § 5.40¤ 36.37 § 3.08 3.74 § 0.35¤ 253.43 § 80.31¤ 112.08 § 20.61

Each value represents the mean § SD. ¤ p < 0.05.

Table 3 EVects of 2.2 § 0.3 mg daily ABA and GA3 on antioxidant defense and MDA in liver of rats Period

Parameters

Subacute

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

Control X § SD 1.82 § 0.08 8.93 § 2.98 38.58 § 5.63 3.67 § 0.10 2.30 § 0.56 1615.66 § 170.05 21.48 § 1.02

ABA X § SD 1.88 § 0.045 16.77 § 3.03¤ 27.75 § 2.19¤ 3.80 § 0.44 2.08 § 0.35 1026.63 § 142.18¤ 18.20 § 2.24¤

GA3 X § SD 1.77 § 0.08 18.39 § 3.15¤ 19.63 § 3.83¤ 3.55 § 0.11 2.33 § 0.28 1496.06 § 225.59 21.15 § 1.86

Subchronic

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

1.88 § 0.03 11.12 § 1.68 36.04 § 3.73 3.87 § 0.12 2.84 § 0.26 1587.22 § 207.03 25.04 § 2.14

1.84 § 0.05 23.15 § 4.76¤ 19.29 § 1.57¤ 3.97 § 0.10 2.35 § 0.29¤ 1078.06 § 203.83¤ 23.29 § 1.01

1.90 § 0.052 26.28 § 3.51¤ 15.57 § 2.54¤ 3.91 § 0.11 2.72 § 0.26 1161.87 § 239.67¤ 24.40 § 1.30

Each value represents the mean § SD. ¤ p < 0.05.

Table 4 EVects of 2.2 § 0.3 mg daily ABA and GA3 on antioxidant defense and MDA in brain of rats Period

Parameters

Subacute

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

11.06 § 1.13 25.18 § 1.83 6.77 § 0.76 17.24 § 0.21 2.63 § 0.20 960.11 § 71.86 1.53 § 0.73

9.51 § 1.07 28.68 § 5.54¤ 5.78 § 0.64 15.84 § 0.36¤ 1.72 § 0.20¤ 725.29 § 179.73¤ 0.68 § 0.04¤

7.68 § 0.77¤ 33.54 § 1.82¤ 4.94 § 0.64¤ 16.35 § 0.16¤ 1.47 § 0.26¤ 574.13 § 115.15¤ 0.71 § 0.10¤

Subchronic

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

9.35 § 0.22 21.40 § 2.09 7.22 § 0.55 17.50 § 0.22 2.61 § 0.33 970.38 § 220.61 1.47 § 0.29

8.88 § 0.36 22.84 § 2.09 5.75 § 0.71¤ 17.14 § 0.26 2.57 § 0.46 709.49 § 185.66 1.36 § 0.12

6.09 § 0.11¤ 24.82 § 2.55¤ 4.96 § 0.55¤ 17.24 § 0.53 1.554 § 0.330¤ 549.64 § 136¤ 1.78 § 0.39

Each value represents the mean § SD. ¤ p < 0.05.

Control X § SD

ABA X § SD

GA3 X § SD

90

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

Table 5 EVects of 2.2 § 0.3 mg daily ABA and GA3 on antioxidant defense and MDA in muscle of rats Period

Parameters

Subacute

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

Control X § SD 1.62 § 0.06 11.31 § 2.02 8.04 § 0.87 3.22 § 0.09 1.02 § 0.17 448.29 § 75.89 2.16 § 1.12

ABA X § SD 1.63 § 0.07 14.19 § 2.52 6.68 § 0.81¤ 3.21 § 0.07 1.13 § 0.14 663.51 § 118.36¤ 1.95 § 0.51

GA3 X § SD 1.65 § 0.16 17.38 § 2.31¤ 3.05 § 0.45¤ 3.20 § 0.09 1.29 § 0.14¤ 821.56 § 92.21¤ 1.49 § 0.39

Subchronic

GSH (mg/g) MDA (nmol/g) CAT (U/g) GST (U/g) GR (U/g) SOD (U/g) GPx (U/g)

1.58 § 0.04 12.09 § 1.13 9.14 § 0.79 3.45 § 0.16 0.83 § 0.12 467.99 § 116.18 2.41 § 0.60

1.62 § 0.04 16.35 § 1.66¤ 5.67 § 0.93¤ 3.44 § 0.15 1.10 § 0.12¤ 751.29 § 195.25¤ 1.39 § 0.14¤

1.92 § 0.07¤ 14.73 § 0.89¤ 3.30 § 0.53¤ 3.380 § 0.12 1.29 § 0.20¤ 846.23 § 131.27¤ 3.11 § 0.92

Each value represents the mean § SD. ¤ p < 0.05.

tissue of rats treated with both the periods of GA3. Also, SOD signiWcantly decreased in liver and brain, and increased in muscle of rats treated with both the period of ABA. While CAT signiWcantly decreased in the all tissues of rats treated with both the period of GA3, decreased in the liver and muscle of rats treated with both the periods of ABA too. On the other hand, the ancillary enzyme GPx and GR activity in the erythrocytes, liver, brain, and muscle were either signiWcantly depleted or not changed with two periods of PGRs. The drug metabolizing enzyme GST activity signiWcantly decreased in the brain of rats treated with both the periods of ABA and subacute period of GA3 but increased in the erythrocytes of rats treated with subacute period of GA3. 4. Discussion In recent years, a signiWcant 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, ABA and GA3 were preferred because information on its negative eVects on higher animals is very limited for in vivo, oral exposures. Also, ABA and GA3 are found in plants as endogen hormones and 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 ABA and GA3 increased some the activities of serum marker enzymes, the production of lipid peroxides, and aVected antioxidant defense in various rat tissues. So far, no study examining the eVect of ABA and GA3 in vivo have 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. 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 diVerences etc., it is diYcult to compare the present data to diVerent studies regarding the for toxicological eVect. Too the extent that chemical aVect, little is known about the biochemical or physiological eVects in vertebrates. Ozmen et al. [27] observed that abscisic acid and gibberellic acid aVect on sexual diVerentiation and some physiological parameters of laboratory mice. Furthermore, El-Mofty and Sakr [13] found that Gibberellin A3 (GA3) induced liver neoplasm in Egyptian toads, and they suggested that the tumours could be diagnosed as hepatocellular carcinomas. In this study, PGRs caused a signiWcant alteration in the serum enzyme activities in rats treated with both periods of PGRs in comparison to those of controls (Table 1). Namely, ABA caused a signiWcant decrease in serum LDH and CPK activity with both periods. Also, while GA3 signiWcantly decreased serum AST, CPK, and LDH activity with subacute periods, decreased serum ALT, CPK, LDH, and GGT treated with subchronic periods. The reasons for such aVect of PGRs are not understood at present certainly, but it may be doe to the ranking of test chemical eVect on tissues. However, it is conceivable that the PGRs, as toxicological agent like other pesticides, might be interacting primarily with the liver and muscle tissue cells’ membrane, resulting in structural damage, changes in enzyme leakage and in metabolism of the constituents. PGRs may lead to pressurize of this enzyme into plasma as a result of enzyme leakage breakdown. These enzymes are mainly localized in the cytoplasm. Any damage in hepatic cells may result in alteration in the serum levels. The decrease in the activities of these enzymes in the serum may result only consequent to impairment of the synthesis in cell of tissues with subsequent liberation of the enzymes into the circulation from the damaged tissue. Transaminases are intracellular enzymes which exist in only a small amount of the serum. Therefore, damage to liver cell may

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

result in leakage of the enzymes into the plasma due to a large concentration gradient [36]. Conversely, in this study, ABA caused a signiWcant decrease in serum LDH and CPK activity with both periods. Also, while GA3 signiWcantly decreased serum AST, CPK, and LDH activity with subacute periods, decreased serum ALT, CPK, LDH, and GGT treated with subchronic periods. Similarly, another researcher had determined that have decreased AST and ALT activities in the serum of Channa striatus following exposure to xenobiotics [30]. Oruc and Uner [26] also found an increase in the serum LDH activity in Cyprinus carpio following exposure to 2,4-diamin. On the other hand, Celik et al. [7] determined that while the levels of LDH and CPK were increased signiWcantly by IBA, the levels of AST, LDH, and CPK were increased signiWcantly by IAA. In addition, the levels of AST, LDH, and CPK were increased signiWcantly by kinetin. Although the treatment, materials of studies and the setting of studies are diVerent, this result is in accordance with our result partly. In addition to decreased the serum marker enzymes, the results of the present study have also demonstrated that the rats treated with both doses of PGRs could have aVected the antioxidant defense systems in vertebrates. This is evidenced from our observation that, upon PGRs treatment in vivo, the concentration of MDA and the antioxidant defense markers in erythrocytes, liver, brain, and muscle diVer from that of control rats. The present study showed that The lipid peroxidation end product MDA signiWcantly increased in the erythrocyte, liver, brain and muscle of rats treated with both the period of GA3 without signiWcantly change in the erythrocyte and muscle of rats treated with the subacute period of ABA. The reasons for such aVect of PGRs are not understood at the present. But, the increased content of MDA may result from an increase of hydroxyl radicals («OH). However, it is conceivable that ABA and GA3 might be interacting primarily with the erythrocytes, liver, brain, and muscle tissues, resulting in lipid peroxidation processes by way of increase superoxide radicals as result of stressed condition in the rats, leading to an increase in lipid peroxidation. MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation [14]. It is known that «OH can initiate lipid peroxidation in tissues [17]. Results also showed that the GSH levels were signiWcantly depleted in the erythrocyte and brain of rats treated with both the period of GA3 without any change in the erythrocyte, liver, brain, and muscle of rats treated with both the period of ABA. Also GSH levels in the muscle signiWcantly depleted with the subchronic period of GA3. However, antioxidant enzyme activities such as SOD signiWcantly decreased in the erythrocyte, liver, and brain tissues but increased in the muscle tissue of rats treated with both the periods of GA3. On the other hand, SOD signiWcantly decreased in liver and brain, and increased in muscle of rats treated with both the period of ABA. While CAT signiWcantly decreased in the all tissues of rats treated with both the period of GA3, decreased in the liver and muscle of rats

91

treated with both the periods of ABA too. On the other hand, the ancillary enzyme GPx and GR activity in the erythrocytes, liver, brain, and muscle were either signiWcantly depleted or not changed with two periods of PGRs. An increase in the constituent of antioxidant defense systems may result an increase of superoxide radicals. The drug metabolizing enzyme GST activity signiWcantly decreased in the brain of rats treated with both the periods of ABA and subacute period of GA3 but increased in the erythrocytes of rats treated with subacute period of GA3. The decreased activity of GST may lead to decreased protection against oxidants [2]. It is not a general rule that increases in pollutant concentrations induce antioxidant activity. Doyotte et al. [12] pointed out that a decreased response may accompany a Wrst exposure to pollutants, which can be followed by an induction of antioxidant systems. Thus, the existence of an inducible antioxidant system may reXect 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 diVerent values of antioxidants marker enzymes and MDA content in the tissues of rats exposed to PGRs may dependent on diVerences of interstitial. Namely, the systems might have to be exposed to diVerent xenobiotic concentration due to blood volume diVerences in the tissues. The enzymatic antioxidants such as SOD, GR, GPx, GST, and CAT 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 [29]. The increased activities of SOD, CAT, GPx, GR, and GST are known to serve as protective responses to eliminate reactive free radicals [9]. However, Candeias et al. [5] investigated the peroxidation of liposomes by a haem peroxidase and hydrogen in the presence of IAA and derivatives. 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 aVect on the levels of MDA erythrocyte, muscle, heart, kidney, and liver tissues, IAA caused a signiWcant increase in the MDA concentration in kidney and liver [8]. Consequently, these studies agree with our results although the chemicals of studies are diVerent. In conclusion, these results suggest that the serum marker enzymes, tissues antioxidant markers and MDA content may be oVer with means for monitoring toxicity of compounds such as ABA and GA3. The test results may be used in oncoming investigations if more studies conWrm our Wndings. Such a test will be of value in pollution studies, and also be of interest to understand molecular basis of refractoriness ABA and GA3 toxicity.

92

Y. Tuluce, I. Celik / Pesticide Biochemistry and Physiology 86 (2006) 85–92

Acknowledgments The authors are grateful to the University Grant Commission (The University of Yuzuncu Yil, No. 2003-FED052) for providing Wnancial assistance during the tenure of Research Associateship. References [1] C. Alanso, The EVects of gibberellic acid upon developmental processes in Drosophila hydlei, Entomol. Exp. Appl. 14 (1971) 73–82. [2] 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 aVects the sensitivity of mouse epidermal cells to oxidative stress, Biochemistry 30 (1991) 9305–9313. [3] E. Beutler, Red Cell Metabolism, A manual of Biochemical methods, third ed., Grune and Startton, New York, 1984, 105–106. [4] E. Beutler, O.B. Dubon, M. Kelly, Improved method for the determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [5] L.P. Candeias, L.K. Folkes, M. Porssa, J. Parrick, P. Wardman, Enhancement of lipid peroxidation by indole-3-acetic acids and derivates, substituent eVects, Free Radic. Res. 23 (1995) 403–418. [6] I. Celik, M. Kara, The eVects of plant growth regulators on activity of eight serum enzymes in vitro, J. Environ. Sci. Health A. 32 (1997) 1755–1761. [7] I. Celik, H. Ozbek, Y. Tuluce, EVects of subchronic treatment of some plant growth regulators on serum enzyme levels in rats, Turk. J. Biol. 26 (2002) 73–76. [8] I. Celik, Y. Tuluce, EVects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats, Pestic. Biochem. Physiol. 84 (2006) 49–54. [9] C.C. Cheung, G.J. Zheng, A.M. Li, B.J. Richardson, P.K.S. Lam, Relationship between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis, Aquat. Toxicol. 52 (2001) 189–203. [10] W. De Man, A. De Loof, T. Briers, R. Huybrechts, EVect of abscisic acid on vitellogenesis in Sarcophaga bullata, Entomol. Exp. Appl. 29 (1991) 259–267. [11] M.P. de Melo, T.M. de Lima, T.C. Pithon-Curi, R. Curi, The mechanism of indole acetic acid cytotoxicity, Toxicol. Lett. 148 (1-2) (2004) 103–111, 14. [12] A. Doyotte, C. Cossu, M.C. Jacquin, M. Babut, P. Vasseur, Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or Weld exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus, Aquat. Toxicol. 39 (1997) 93–110. [13] M.M. El-Mofty, S.A. Sakr, Induction of neoplasms in the Egyptian Toad by gibberellin A3, Oncology 45 (1988) 61–64. [14] B.A. Freeman, J.D. Crapo, Hyperoxia increases oxygen radical production in rat lung and lung mitochondria, J. Biol. Chem. 256 (1981) 10986–10992. [15] S. Furukawa, M. Abe, K. Usuda, I. Ogawa, Indole-3-acetic acid induces microencephaly in rat fetuses, Toxicol. Pathol. 32 (6) (2004) 659–667. [16] A.A. Guarra, EVect of biological active substance in the diet on development and reproduction of Heliothis spp., J. Econ. Entomol. 63 (1970) 1518–1521. [17] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, second reprint, Clarendon Press, Oxford, 1989, p. 543.

[18] 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. [19] S.K. Jain, R. McVie, J. Duett, J.J. Herbst, Erythrocyte membrane lipid peroxidation and glycolylated hemoglobin in diabetes, Diabetes 38 (1989) 1539–1543. [20] J.A. John, C.D. Blogg, F.J. Murray, B.A. Schwetz, P.J. Gehring, Teratogenic eVects of the plant hormone indole-3-acetic acid in mice and rats, Teratology 19 (3) (1979) 321–324. [21] B. Mannervik, C. Guthenberg, Glutathione S-transferase (Human Plasenta), Methods Enzymol. 77 (1981) 231–235. [22] J.M. McCord, I. Fridovich, Superoxide dismutase, Anenzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6053. [23] L.G. Mickel, “Plant Growth Regulators” Controlling biological behavior with chemicals, Chem. Eng. News. 56 (1978) 18. [24] L.J. Olson, R.D. Hinsdill, InXuence of feeding chlorocholine chloride and glyposine on selected immune parameters in deer mice peromiseus moniculatus, Toxicology 30 (1984) 103–114. [25] O.E. Oruc, N. Uner, Marker enzyme assesment in the liver of Cyprinus carpio (L.) exposed to 2,4-D and azinphosmethyl, J. Biochem. Mol. Toxicol. 16 (2002) 183–188. [26] E.O. Oruc, N. Uner, EVects of 2,4-diamin on some parameters of protein and carbohydrate metabolisms in the serum, muscle and liver of Cyprinus carpio, Environ. Pollut. 105 (1999) 267–272. [27] M. Ozmen, S.F. Topcuoglu, S. Bozcuk, N.A. Bozcuk, EVects of abscisic acid and gibberellic acid on sexual diVerentiation and some physiological parameters of laboratory mice, Turk. J. Biol. 19 (1995) 357–364. [28] F. Regoli, G. Principato, Glutathione, glutathione dependent and antioxidant enzymesin mussel, Mytilus galloprovincialis, exposed to metals under Weld and laboratory conditions, Implications for the use of biochemical biomarkers, Aquat. Toxicol. 31 (1995) 143–164. [29] A. Rodriguez-Ariza, J. Peinado, C. Pueyo, J. Lopez-Barea, Bichemical indicators of oxidative stress in Wsh from polluted littoral areas, Can. J. Fish Aquat. Sci. 50 (1993) 2568–2573. [30] K.A. Sadhu, D.K. Chowdhury, P.K. Mukhopadhyay, Relationship between serum enzymes, histological features and enzymes in hepatopancreas after sublethal exposure to malathion and phosphamidon in the murrel Channa striatus (BL.), Int. J. Environ. Studies 24 (1985) 35–45. [31] F.B. Salisbury, C.W. Ross, Plant Physiology, Wadsworth, Belmont, CA, 1992, pp. 357–407, 531–548. [32] H. Ustun, T. Tecimer, M. Ozmen, S.F. Topcuoglu, N.A. Bozcuk, EVects of gibberellic acid and benzoprenin on mice, Histopatalogic Rev. Ank. Patol. Bült. 9 (1992) 36–40. [33] N.S. Visscher, Regulation of Grasshopper fecundity, longevity and egg viability by plant growth hormones, Experimentia. 36 (1980) 130–131. [34] N.S. Visscher, Special report dietary plant growth hormones aVects insect growth and reproduction, Bull. Plant Growth Reg. Soc. Am. 11 (1983) 4–6. [35] WMA., World Medical Association Declaration of Helsinki, 52nd WMA General Assembly, Edinburgh, Scotland, 2000. [36] F. Wroblewski, J.S. la Due, Serum glutamic oxaloacetic transaminase activity as index of liver cell injury. A preliminary report, Ann. Int. Med. 43 (1955) 345–360. [37] L. Xiong, J.K. Zhu, Regulation of abscisic acid biosynthesis, Plant Physiol. 133 (2003) 29–36.