Abstract INTRODUCTION. WU Hai-hua 1, ZHU Kun-yan 2, GUO Ya-ping 1, ZHANG Xiao-min 1 and MA En-bo 1

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1 Agricultural Sciences in China 2008, 7(4): April 2008 Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S- Transferases in Six s of Oxya chinensis (Thunberg) (Orthoptera: Acrididae) WU Hai-hua 1, ZHU Kun-yan 2, GUO Ya-ping 1, ZHANG Xiao-min 1 and MA En-bo 1 1 Institute of Applied Biology, Shanxi University, Taiyuan , P.R.China 2 Department of Entomology, Kansas State University, Manhattan 66506, USA Abstract Specific activity, substrate specificity, and kinetic parameters (K m and ) of glutathione S-transferases (GSTs) towards three substrates, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzene chloride (pnbc) were investigated in six tissues (foregut, midgut, hindgut, fat body, hemolymph, and muscle) of Oxya chinensis. In addition, the inhibition in vitro (ethacrynic acid, and Cibacron Blue 3GA) of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body > midgut > hindgut > muscle > foregut > hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pnbc and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration (IC 50 ) values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues. Key words: Oxya chinensis, glutathione S-transferase (GST), tissue distribution, kinetic parameters, inhibition in vitro INTRODUCTION Oxya chinensis (Thunberg) (Orthoptera: Acrididae) is a common species of Oxya Serville. It is widely distributed in Africa, Australia, and Asia, including India, Malaysia, Japan, Philippines, and China (Zheng 1993). This widespread species mainly inhabits low-lying grasslands, rice fields, and their surrounding banks. It is a polyphagous insect and its food plants include a wide range of wild and uncultivated plants and cultivated crops such as rice, maize, sorghum, wheat, beans, cotton, sugarcane, and reeds (Chen 1999). O. chinensis is a major insect pest in rice. Traditionally, chemical Received 6 November, 2007 Accepted 30 February, 2008 WU Hai-hua, Ph D, wuhaihua04@163.com; Correspondence MA En-bo, Professor, Tel: , maenbo2003@sxu.edu.cn

2 Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six s of Oxya chinensis 463 insecticides are widely used to protect rice from the damage by this pest. Glutathione S-transferases (GSTs) are a group of multifunctional detoxification enzymes that catalyze the conjugation of reduced glutathione (GSH) with various xenobiotics and endogenous compounds possessing an electrophilic center (Armstrong 1991). These enzymes form an integral part of the phase-ii detoxification system (Kostaropoulos et al. 1996). Glutathione S- transferases play an important role in the detoxification of many substances including plant secondary metabolites. Many plant secondary metabolites have been shown to induce GST in phytophagous insects and similarly in predators that feed on these herbivores (Francis et al. 2000; Vanhaelen et al. 2001). Expression of GSTs is often tissue-specific (Mittapalli et al. 2007). distributions of GST have been studied in a number of organisms (Chien and Dauterman 1991; Francis et al. 2000; Tang et al. 2005; Tate et al. 1982). Thus, attempts were made to understand the tissue distribution of GST from O. chinensis. The objectives of this study were to: (1) compare the GST activities among the six tissues and between the sexes in O. chinensis; (2) characterize the GST substrate specificity and kinetic parameters in the six tissues, and (3) study the inhibitory properties of the GST in the six tissues of O. chinensis. MATERIALS AND METHODS Insects O. chinensis was reared for half a year in the laboratory in the College of Life Science and Technology of Shanxi University, China. Vigorous and uniform fifth-instar nymphs of F 1 generation were used for this study. Chemicals Bicinchoninic acid solution, 1,2-dichloro-4- nitrobenzene (DCNB), p-nitrobenzene chloride (pnbc), ethacrynic acid, and Cibacron Blue 3GA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Bio-Rad Laboratories (Hercules, CA). Reduced glutathione (GSH) was purchased from Bio. Basic Inc. (Calgary, Canada). 1-chloro-2,4-dinitrobenzene (CDNB) was purchased from Sangon (Shanghai, China). Enzyme preparation 15 ml of hemolymph from the cuts of the thorax and abdomen of O. chinensis were collected using a micropipette. Hemolymph was blown into an ice-cold tube. Muscle was collected from the femur of hindleg. Foregut, midgut, hindgut, and fat body were dissected from groups of 90 fifth-instar nymphs with the aid of a dissecting microscope. In the case of foregut, midgut and hindgut, gut contents were removed. The foregut, midgut, hindgut and fat body tissues were then washed in 1.15% KCl. The dissected tissues were stored at -20 C. All weighed tissues were homogenized in ice-cold 0.1 M phosphate buffer (ph 7.5) at a ratio of 1:5 (w/v). All homogenates were centrifuged at g for 20 min at 4 C. Supernatants were transferred to fresh tubes and used as enzyme sources. Glutathione S-transferase activity assay The GST activity towards each of the three substrates (CDNB, DCNB and pnbc) in six different tissues were determined according to the methods of Zhu et al. (2000) and Habig et al. (1974) with slight modifications. Briefly, 10 µl of appropriately diluted enzyme preparation was mixed with 188 µl mm reduced glutathione in 0.1 M phosphate buffer (ph 7.5) and 2 µl 200 mm of each substrate was dissolved in acetone in a 96-well microplate. The change in absorbance was immediately recorded at 340 nm for CDNB, 345 nm for DCNB, and 310 nm for pnbc at 10-s intervals for 1 min using a SpectraMAX 190 microplate reader and SOFTmax software (Molecular Devices, Sunnyvale, CA, USA). All assays were corrected for nonenzymatic conjugation by using a negative control in which 10 µl of 0.1 M phosphate buffer (ph 7.5) replaced the enzyme preparation. The amount of glutathione conjugate formed was calculated using extinction coefficients of 9.6 mm -1 cm -1 for CDNB, 10.0 mm -1 cm -1 for DCNB, and 1.9 mm -1 cm -1 for pnbc.

3 464 WU Hai-hua et al. Kinetic studies of GST in different tissues in O. chinensis and IC 50 values between the sexes were subjected to Student s t-test. The Michaelis constant (K m ) and maximum velocity ( ) values of GST from different tissues were determined for each of the three substrates. This was done by recording the absorbance towards a range of concentrations of CDNB, DCNB, or pnbc from to µm and the GSH concentration was kept constant at 9.21 mm. Other assay conditions were the same as those described previously. Inhibitory studies in vitro of GST in different tissues in O. chinensis Two inhibitors (ethacrynic acid and Cibacron Blue 3GA) were used to characterize GST prepared from six different tissues of the grasshopper by using CDNB as a substrate. The stock solutions of ethacrynic acid and Cibacron Blue 3GA were prepared in acetone and water, respectively, and diluted by using 0.1 M phosphate buffer (ph 7.5). The highest final acetone concentration in the test solutions was 1%. The inhibitors were tested at concentrations ranging from to µm for ethacrynic acid and 7.81 to µm for Cibacron Blue 3GA. 10 ml of enzyme preparation and 10 µl inhibitor solutions were incubated for 10 min. The remaining GST activity was determined by adding the CDNB/GSH substrate mixture as previously described. Assay of protein Protein concentration of each enzyme preparation was determined according to Smith et al. (1985) using BSA as the standard. The measurements were carried out with the microplate reader at 560 nm. Data analysis Glutathione S-transferase activities, kinetic parameters, and median inhibition concentration (IC 50 ) values among different tissues were subjected to Fisher s least significant difference multiple comparisons using the Statistical Package for Social Science (SPSS) software. Glutathione S-transferase activities, kinetic parameters, RESULTS Comparison of GST activity among the six tissues and between the sexes in O. chinensis The present results showed that GSTs prepared from the six different tissues of the grasshopper can catalyze the conjugation reactions for all the three substrates examined. However, GST activities were different among the tissues (Table 1). In addition, GST activities conjugating CDNB were the highest, followed by pnbc, and DCNB. For CDNB as a substrate, the highest GST activity was found in the fat body, followed by the midgut, hindgut, muscle, and foregut. The lowest GST activity was found in the hemolymph. Glutathione S- transferase activities in the fat body were to fold and to 9.98-fold significantly higher than those in the other tissues in females and males. For DCNB as a substrate, the highest GST activity was found in the midgut, followed by the fat body, and the lowest GST activity was found in the hemolymph in both the sexes. Glutathione S-transferase activities in the midgut were to 9.81-fold and to 5.52-fold higher than those in the other tissues in females and males, respectively. For pnbc as a substrate, the highest GST activity was found in the fat body and in the muscle in females and males, respectively (Table 1). The lowest GST activity was found in the foregut. Table 1 also showed the comparison of the GST activity differences between the sexes. Glutathione S- transferase activities in the hemolymph and the muscle were significantly higher in males than those in females in conjugating-dcnb and conjugating-pnbc (P < 0.05). However, no significant differences in GST activities were obtained in other tissues between males and females (P > 0.05). Comparison of GST kinetic parameters among the six tissues and between the sexes in O. chinensis Table 2 showed the K m values of GSTs from the six

4 Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six s of Oxya chinensis 465 tissues towards the three substrates. The affinities of GSTs towards the three substrates were different in the six tissues. The kinetic parameter, K m values of GSTs conjugating-dcnb were the highest in the six tissues. Whereas K m values of GSTs conjugating-cdnb were the lowest, except in the midgut and the fat body where K m values of GSTs conjugating-pnbc were lower than those conjugating-cdnb. For CDNB as substrate, K m value in the fat body was the lowest, followed by the hindgut, the foregut, and the midgut. Kinetic parameter, K m values in the hemolymph was the highest, followed by the muscle. For DCNB as substrate, the rank order of K m values was fat body > midgut > muscle > hindgut > hemolymph > foregut in females. In males, the rank order was slightly changed to fat body > midgut > muscle > foregut > hindgut > hemolymph. For pnbc as substrate, K m values in the fat body were the lowest. The highest K m values were found in the hemolymph and in the muscle in females and males. When pnbc was used as a substrate, K m values in females were significantly higher than those in males in the midgut, hindgut, and fat body (P < 0.05) whereas K m value in males was significantly higher than those in females in the muscle (P < 0.05). Table 3 shows the values of GSTs from different tissues. For CDNB as substrate, value in the hemolymph was significantly lower than those in the other tissues in both the sexes (P < 0.05). In females, value in the hindgut was the highest, followed by the midgut and there was no significant difference between the two tissues (P > 0.05). In males, value Table 1 Comparisons of GST activities for conjugation CDNB, DCNB, and pnbc in the six tissues from O. chinensis Female CDNB DCNB pnbc CDNB DCNB pnbc Foregut ± cd 2.91 ± 0.27 b 2.39 ± 0.98 b ± 2.20 e 2.70 ± 0.83 b 3.94 ± 0.30 b Midgut ± b ± 6.86 a ± a ± b ± 2.72 a ± 3.04 a Hindgut ± b 2.90 ± 1.22 b ± 3.80 a ± c 2.96 ± 1.43 b ± 7.85 a Fat body ± a 9.20 ± 0.83 a ± 4.16 a ± a ± 0.42 a ± 3.37 a Hemolymph ± 2.02 d 1.34 ± 0.49 b 7.75 ± 4.95 ab ± 1.48 f 2.32 ± 0.26 b * ± 3.64 a Muscle ± 4.45 c 2.75 ± 0.51 b 8.88 ± 3.91 ab ± 4.13 d 3.22 ± 0.31 b ± 1.07 a * Results are the means ± SD of 3 replicates (n = 3), each with triplicate analyses. Means within columns followed by the same letter are not significantly different (P > 0.05, Fisher s LSD multiple comparison test). * indicates that there were significant differences between the sexes for the same substrate (P < 0.05, Student s t-test). The same as below. Male Table 2 K m values of GST conjugating three substrates in the six tissues of O. chinensis Female Male CDNB DCNB pnbc CDNB DCNB pnbc Foregut ± c ± a ± b ± c ± a ± a Midgut ± b ± c ± d * ± b ± b ± c Hindgut ± c ± a ± ab * ± 8.74 c ± a ± b Fat body ± c ± d ± 9.55 d * ± 9.69 c ± c ± 9.49 bc Hemolymph ± a ± a ± a ± a ± a ± a Muscle ± b ± b ± c ± a * ± b ± a * Table 3 values of GST conjugating three substrates in the six tissues of O. chinensis Female Male CDNB DCNB pnbc CDNB DCNB pnbc Foregut ± 4.21 d 6.54 ± 0.60 bc ± 0.73 abc * ± 1.88 c 7.41 ± 2.19 b ± 1.35 c Midgut ± 6.55 a ± 1.81 a * ± 1.05 ab ± a 6.55 ± 2.85 b ± 1.24 bc Hindgut ± a 8.63 ± 1.93 b ± 4.42 a ± a ± 0.86 a * ± 3.28 bc Fat body ± 1.74 b ± 1.20 a ± 2.64 c ± a * ± 2.22 a ± 3.35 a * Hemolymph ± 2.17 e 4.83 ± 0.47 cd ± 2.13 a * ± 0.31 d 4.79 ± 1.72 b ± 1.90 d Muscle ± 1.30 c 3.63 ± 0.31 d ± 2.16 bc ± 2.79 b * 4.06 ± 0.34 b ± 0.54 ab

5 466 WU Hai-hua et al. in the midgut was the highest, followed by the hindgut, and fat body. Although they have no significant differences among the three tissues (P > 0.05), their values were significantly higher than those in the other tissues (P < 0.05). Kinetic parameter, values in the hemolymph were to fold and to fold lower than those in the other tissues in females and males, respectively. For DCNB as substrate, the lowest value was found in the muscle, followed by the hemolymph and there was no significant differences between the two tissues in females and males (P > 0.05). In females, value in the fat body was the highest, followed by the midgut, whereas there were no significant differences between the two tissues (P > 0.05). In males, the highest value was found in the hindgut, followed by the fat body and no significant differences between the two tissues (P > 0.05) was found. For pnbc as substrate, in females, values in the hindgut and hemolymph were significantly higher than those in the other tissues (P < 0.05), whereas values in the fat body was significantly lower than those in other tissues (P < 0.05). In males, the highest value was found in the fat body whereas no significant difference between the fat body and the muscle (P > 0.05) was observed. The lowest value was found in the foregut. For CDNB as substrate, values in males were significantly higher than those in females in the fat body and the muscle (P < 0.05). For DCNB as substrate, value in females was significantly higher than those in males in the midgut (P < 0.05), whereas values in males were significantly higher than those in females in the hindgut (P < 0.05). The values conjugatingpnbc in females were significantly higher than those in males in the foregut and the hemolymph (P < 0.05), whereas values in females conjugating-pnbc was significantly lower than that of males in the fat body (P < 0.05). In vitro inhibition studies of GSTs The two inhibitors have different inhibitory potencies to GSTs from the six tissues (Table 4). The IC 50 values of the two inhibitors to GSTs from the six tissues were in the range of µm. In females, the IC 50 value in the hemolymph was the lowest, followed by the fat body whereas that in the foregut was the highest and followed by the hindgut and midgut when ethacrynic acid was used as the inhibitor. Cibacron Blue 3GA was effective to inhibit GST activity in the foregut and was less effective to that in the fat body. In males, GST in the muscle was sensitive to ethacrynic acid whereas that in the hindgut was less sensitive to ethacrynic acid. Cibacron Blue 3GA was a potent inhibitor to GST in the foregut whereas was a less potent inhibitor to GST in the fat body. The two inhibitors have various inhibitions to GST of different sexes in the same tissues. DISCUSSION The results showed that various tissues have different GST activities. This was related with the functions of each tissue and the physiological and toxicological implications of GST. Glutathione S-transferases are a family of detoxification enzymes that have essential roles in the survival of insects exposed to xenobiotics. The fat body of insect is where insects deposit toxins and detoxify, and the midgut is where insects digest food and take up chemical substances (Tang et al. 2005). Generally, it was thought that the fat body and midgut of insects were the metabolized regions to xenobiotics. The results in this study showed that GSTs were mainly distributed in the fat body and midgut. This suggested Table 4 IC 50 values for two inhibitors in vitro inhibition to GSTs of six tissues of O. chinensis Female Male Ethacrynic acid Cibacron Blue 3GA Ethacrynic acid Cibacron Blue 3GA Foregut ± a * ± 4.43 d ± b ± d Midgut ± a * ± a * ± 9.02 b ± b Hindgut ± 9.13 a ± 4.25 b * ± 8.00 a * ± 7.70 bc Fat body ± c ± a ± b * ± a Hemolymph ± 4.09 c ± c ± c ± 5.90 bcd Muscle ± b * ± c ± 3.14 d ± 8.41 cd

6 Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six s of Oxya chinensis 467 that, like many other insects, GSTs in the two tissues played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. Some studies showed that GST activities were the highest in the fat body and midgut (Snyder et al. 1995; Tang et al. 2005; Tate et al. 1982). Furthermore, the relatively high GST activities in the hindgut might be related with the Malpighian tubes in the hindgut where the highest GST activity was found in other studies (Konno and Shishido 1992). The Malpighian tubes in O. chinensis have to be dissected to study their GST characteristics in the future work. In this study relatively high GST activity was found in the femur muscles of hindleg, which are responsible for hopping. This phenomenon can be interpreted by two possibilities. One is that they may have toxic substances that need to be detoxified by GSTs in the muscles of O. chinensis; the other is that GSTs have other roles besides detoxification. The roles of GSTs in the muscles of O. chinensis need to be further studied. Similarly, the general GST activity pattern of Episyrphus balteatus corresponded to the localization of the GST two isoforms in Musca domestica and Drosophila melanogaster that appear mainly in the wing muscles (Clayton et al. 1998; Franciosa and Bergé 1995). Foregut of insect plays an important role in receiving, grinding, and storing foods and has partial metabolizing ability. The results in our study suggested that the GST of foregut in O. chinensis can metabolize substances. Hemolymph was known to be involved in detoxification (Patton 1961). In Musca domestica, it was found that one of the dipteran GST isozymes, GST-1, was only distributed in hemolymph cells and played a role in the detoxification of insecticides (Franciosa and Bergé 1995). Thus, low GST activity in the hemolymph of O. chinensis suggested that GST in hemolymph played a weak role in detoxifying xenobiotics. In the present study it was found that GST isoenzymes, with overlapping substrate specificities were expressed in the different tissues of O. chinensis, and they show altered substrate specificities, in vitro inhibition potency and kinetic constants. The observed changes in the kinetic parameters among the different tissues of the insect might be explained by differential expression patterns of multiple GSTs so that the insect GSTs have different affinities and maximum velocities towards the same substrate. Most organisms possess multiple GSTs belonging to two or more classes with differing catalytic activities to accommodate the wide range of substrate specificities. Yu (2002) found that fall armyworm larval midgut has six cytosolic GST isozymes, whereas the fat body contained three isozymes. The results showed that the pattern of activity toward various substrates was different among isozymes. For example, midgut (MG) GST-3 was not active toward p-nitrophenyl acetate, whereas MG GST-4 was not active toward trans-4-phenyl-3-buten-2-one (TPBO). Furthermore, conjugating-dcnb activity was not detected in any of these isozymes (Yu 2002). Multiple forms of GST were also observed in other insects, including Diptera (Snyder et al. 1995), Coleoptera (Kostaropoulos et al. 1996), Hymenoptera (Valles et al. 2003), and Lepidoptera (Kirby and Ottea 1995). From present studies it can be inferred that the changes of substrate specificity, kinetic parameters and inhibitory properties of GST might be because of the presence of different and multiple isozymes among the six tissues of O. chinensis. The information contained in this study might be useful in understanding the distribution, substrate specificity, kinetic parameters, and inhibition properties of the isoenzymes in the six tissues of the grasshopper. Specifically, our study could serve as an initial step in elucidating the characteristics of GST. Further studies will be focused on the purification of different isozymes and the molecular analysis of GST activity in these tissues of O. chinensis. Acknowledgements This work was supported by the National Natural Science Foundation of China ( , ), and Science and Technology Commission of Shanxi Province, China (041005, ). References Armstrong R N Glutathione S-transferases: reaction mechanism, structure, and function. Chemical Research in Toxicology, 4, Chen Y L The Locust and Grasshopper Pests of China. China Forestry Publishing House, Beijing. (in Chinese) Chien C, Dauterman W C Studies on glutathione S- transferase in Helicoverpa (= Heliothis) zea. Insect Biochemistry, 21, Clayton J D, Cripps R M, Sparrow J C, Bullard B

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