Glutathione metabolism in activated human neutrophils: stimulation of glutathione synthesis and consumption of glutathione by reactive oxygen species

Since glutathione (GSH) is involved in the modulation of the function of polymorphonuclear leucocytes (PMN) such as phagocytosis and production of reactive oxygen species, the metabolism of GSH was studied in human PMN. The concentration of GSH in resting PMN amounted to 13.3 nmol 10(-7) PMN and remained stable over 100 min of incubation. Upon activation of PMN with phorbol myristate acetate intracellular GSH decreased to 50% of the resting concentration within 80 min. In the presence of buthionine sulfoximine, which inhibits the synthesis of GSH, the depletion of intracellular GSH was dramatically accelerated, indicating that activation of PMN is associated with a marked stimulation of GSH synthesis. Since a similar depletion of GSH was seen in the presence of propargylglycine, an inhibitor of the cystathionine pathway, most of the cysteine required for the resynthesis of GSH must originate from methionine and not from cysteine generated by the catabolism of GSH. Further studies showed that GSH is sequentially oxidized by O2-. and HOCl, first to GSSG and then to an unidentified compound, most likely a chloramine. In the presence of an adequate supply of GSH and NADPH which is required for the reduction of GSSG by glutathione reductase this further oxidation of GSSG was prevented. Thus, the highly toxic HOCl generated by PMN can be detoxified by the glutathione reducatase system. The capacity of PMN to re-synthesize GSH may be an important determinant of PMN function.     

Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth

The glutathione S-transferase (GST) superfamily is involved in detoxification of various xenobiotics. Using real-time PCR, mRNA encoding an omega-class GST of Bombyx mori (bmGSTO) was shown to be induced after exposure to various environmental stresses. A soluble form of recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells and purified to homogeneity. Cys 38 and Pro 39 were found to be highly conserved in omega-class GSTs, and their roles were investigated by site-directed mutagenesis/kinetic analysis. Mutations of Cys 38 and Pro 39 residues affected the catalytic efficiency of enzymes, indicating that the presence of Cys 38 and Pro 39 residues is important for bmGSTO activity. Thus, bmGSTO could contribute to increasing the environmental stress resistance of lepidopteran insects.     

达斡尔族谷胱甘肽S-转移酶GSTT1和GSTM1基因多态性

目的探讨内蒙古地区达斡尔族谷胱甘肽争转移酶（glutathione S-transferases，GSTs）GSTM1和GSTT1基因多态性分布特点，为内蒙古少数民族基因型研究提供相关数据。方法采用内对照聚合酶链反应技术（PCR）和凝胶成像分析方法，对220例内蒙古地区达斡尔族个体的GSTT1、GSTM1基因缺失型频率进行了分析。结果GSTM1基因缺失型、GSTT1缺失型在内蒙古地区达斡尔族人群中检出频率分别为50．8％和71．4％。同时具有GSTM1缺失型和TSTT1缺失型个体的检出频率为31．4％。结论中国达斡尔族人群GSTM1、GSTT1基因呈多态性分布，与汉旅相比存在一定差异，与蒙古族相比差异无统计学意义。     

Catalytic function of an Epsilon‐class glutathione S‐transferase of the silkworm

The glutathione S‐transferase (GST) superfamily is involved in the detoxification of various xenobiotics. A silkworm GST, belonging to a previously reported Epsilon‐class GST family, was identified, named bmGSTE, cloned, and produced in Escherichia coli. Investigation of this enzyme's properties showed that it was able to catalyse glutathione (GSH) with 1‐chloro‐2,4‐dinitrobenzene and ethacrynic acid, and also that it possessed GSH‐dependent peroxidase activity. The enzyme's highly conserved amino acid residues, including Ser11, His53, Val55, Ser68 and Arg112, were of interest regarding their possible involvement in its catalytic activity. These residues were replaced with alanine by site‐directed mutagenesis and subsequent kinetic analysis of bmGSTE mutants indicated that His53, Val55, and Ser68 were important for enzyme function.     

Uptake and glutathione conjugation of ethacrynic acid and efflux of the glutathione adduct by periportal and perivenous rat hepatocytes

We assessed the impact of zonal factors on the hepatic reduced glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by enriched periportal (PP) and perivenous (PV) rat hepatocytes was characterized by both saturable (V(max)(uptake) = 3.4 +/- 1.7 and 3. 2 +/- 0.8 nmol/min/mg protein and K(m)(uptake) = 51 +/- 13 and 44 +/- 15 microM) and nonsaturable (12 +/- 5 and 12 +/- 3 microl/min/mg protein) components. Values for the overall GSH conjugation rates of EA (200 microM) were similar among the zonal hepatocytes and resembled those for the influx transport rates. In the absence of the hepatocyte membrane, GSH conjugation in PV and PP hepatocyte cytosol was similar, but a higher perivenous GSH conjugation activity toward EA (PV/PP of 2.4) that mirrored the higher PV/PP ratios of immunodetectable GSTs Ya (1.7) and Yb2 (2.5) was found in cell lysates obtained by the dual-digitonin-pulse perfusion technique. The GSH conjugation rates in the subcellular fragments were, however, much greater than those observed for intact hepatocytes. Efflux rates of the glutathione conjugate EA-SG from zonal hepatocytes were similar, as were levels of the immunodetectable multidrug-resistance protein 2/canalicular multispecific organic anion transporter (Mrp2/cMoat) in the 100,000g pellets. The composite results suggest that the GSTs responsible for EA metabolism are more abundant in the PV region, albeit that the gradient of enzymatic activities is shallow. Despite the existence of zonal metabolic activity, the overall GSH conjugation rate of EA is homogeneous among cells because the reaction is rate limited by uptake, which occurs evenly. Results on EA-SG efflux suggest the acinar homogeneity in Mrp2/cMoat function for canalicular transport.     

脑缺血再灌注对大鼠不同脑区丙二醛、谷胱甘肽含量及谷胱甘肽过氧化物酶活性的影响

目的：探讨脑缺血再灌注时自由基代谢变化在迟发性神经元损伤中作用。方法：采用闭塞大鼠４条动脉全脑缺血模型，在全脑缺血３０分钟和缺血后再灌注不同时间，分别观察某些脑区丙二醛（ＭＤＡ）、谷胱甘肽（ＧＳＨ）含量以及谷胱甘肽过氧化物酶（ＧＳＨＰｘ）活性变化。结果：在大脑皮层和海马中，缺血３０分钟后，ＧＳＨ、ＧＳＨＰｘ显著下降，细胞膜ＭＤＡ有所增加，但无显著性差异。随再灌注时间延长，胞浆中ＧＳＨ逐渐回升，而ＧＳＨＰｘ进一步下降，并且细胞膜ＭＤＡ显著升高。在丘脑和下丘脑，各组ＧＳＨ、ＭＤＡ变化均无显著性差异，ＧＳＨＰｘ变化则与大脑皮层和海马中相似，但下降幅度较小。结论：脑缺血引发的自由基损伤主要发生在缺血再灌注期，且海马是脑缺血再灌注损伤中最易损伤区。     

耐药人白血病细胞GSH含量、GST活力及其同工酶、MRP表达的研究

目的：了解多药耐药基因（ｍｄｒ１）机制以外导致人白血病细胞耐药的因素。方法：采用生化方法，对Ｋ５６２和ＨＬ６０敏感和耐药细胞株谷胱甘肽（ＧＳＨ）含量、谷胱甘肽Ｓ转移酶（ＧＳＴ）活力进行测定；用Ｎｏｒｔｈｅｒｎ杂交对ＧＳＴα、π、μ和多药耐药相关蛋白（ＭＲＰ）ｍＲＮＡ表达进行检测；用Ｗｅｓｔｅｒｎ杂交对ＧＳＴα、π、μ蛋白表达进行检测。结果：与敏感株相比，Ｋ５６２／Ｈ２０和Ｋ５６２／ＶＣＲ的ＧＳＨ含量、ＧＳＴ活力明显增高，ＨＬ６０／Ａｄｒ的ＧＳＨ含量和ＧＳＴ活力无明显增高，Ｎｏｒｔｈｅｒｎ和Ｗｅｓｔｅｒｎ杂交可见ＧＳＴα、π和ＧＳＴπ、μ在Ｋ５６２／Ｈ２０和Ｋ５６２／ＶＣＲ过度表达，ＨＬ６０／Ａｄｒ无ＧＳＴ同工酶过度表达，ＭＲＰ在三种耐药株均有过度表达。结论：ＧＳＨ、ＧＳＴ和ＭＲＰ参与了Ｋ５６２／Ｈ２０和Ｋ５６２／ＶＣＲ耐药，ＨＬ６０／Ａｄｒ耐药与ＧＳＨ、ＧＳＴ无关，与ＭＲＰ有关。在实际应用中，应对多种耐药指标同时进行检测     

深圳地区汉族急性白血病儿童GSTP1全编码区基因多态性分析

目的研究深圳地区汉族急性白血病(AL)患儿谷胱甘肽转移酶P1(GSTP1)基因全编码区内的单核苷酸多态性(SNPs)基因型和等位基因频率分布特征。方法用RT-PCR和变性梯度凝胶电泳(DGGE)技术对108例AL患儿和121例对照儿童的GSTP1全编码区内的SNPs进行筛查分析。结果在GSTP1全编码区内共筛查到3个SNPs位点,包括1个热点突变位点A313G(Ile105Val,rs1695)、1个错义突变位点G439T(Asp147Tyr,rs4986949)和1个同义突变位点T555C(Ser185Ser,rs4891),其在汉族儿童分布等位基因总频率分别为16.4%、1.3%和16.4%,且具有明显的种族差异性。GSTP1 A313G、G439T和T555C多态性各基因型和等位基因频率分布在AL患儿和对照组儿童中差异均无统计学意义(P分别为0.691和0.359;0.898和0.581、0.691和0.359)。结论对深圳地区汉族儿童GSTP1基因全编码区多态性进行筛查分析,确定了GSTP1 A313G,G439T和T555C 3个SNPs位点,其具有种族差异性且与儿童AL发病风险均无关。     

Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat.

Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.     

Analysis of glutathione: implication in redox and detoxification

BACKGROUND: Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. TESTS AVAILABLE: Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. CONCLUSION: Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects.     

血清谷胱甘肽过氧化物酶偶联连续监测法

应用偶联连续监测法测定血清谷胱甘肽过氧化物酶（ＧＳＨ－ＰＸ），特异性强，灵敏度高，快速、准确。本法批内ＣＶ为３．９％，批间ＣＶ４．９％。在最适条件下，反应的吸光度差（ΔＡ）４ｍｉｍ之内呈线性。胆红质高于正常１０倍不影响结果，避免溶血。测定２３９例年龄５８～８２岁健康中老年人，参考值为７．５５±１．４３μｋａｔａｌ／Ｌ。     

谷胱甘肽-S-转移酶P1基因多态性与晚期胃癌患者对顺铂化疗疗效的相关性研究

目的 基因多态性通过影响药物的代谢、转运和作用靶点从而导致药物疗效和毒性的个体差异,寻找明确的生物学标记来识别获益人群已成为最大的挑战.本研究旨在观察谷胱甘肽-S-转移酶P1(GSTP1)基因多态性与以顺铂(DDP)为基础的化疗方案治疗晚期胃癌疗效间的关系.方法 收集经病理学确诊的晚期胃癌患者59 例.所有病例化疗前抽取外周静脉血,提取脱氧核糖核酸(DNA),用连接酶检测反应技术(PCR-LDR)检测研究对象的GSTP1 基因型.所有患者经多西他赛(DOCETAXEL)/DDP/5-氟尿嘧啶(5-FU) 联合方案化疗,化疗结束后观察疗效及其与GSTP1 基因多态性的关系.结果 59 例晚期胃癌患者中,15 例(25.4%)为GSTP1 G/G 基因型,21 例(35.6%)为GSTP1 G/A 基因型,23 例(39.0%)为GSTP1 A/A 基因型.其中4 例完全缓解,14 例部分缓解,19 例稳定,22 例进展,总有效率为30.5%(18/59).GSTP1 G/G 基因型患者的化疗有效率(73.3%)明显高于G/A 基因型患者(19.0%)(χ2 ＝10.616,P ＝0.005),同样明显高于A/A 基因型患者(13.0%)(χ2 ＝14.202,P ＝0.001).G/A 基因型患者的化疗有效率与A/A 基因型患者之间差异无统计学意义(χ2 ＝0.31,P＝0.856).突变基因型(G/G +G/A)对化疗较敏感,其化疗有效率是野生基因型(A/A)的3.2 倍(95% CI 1.442 ～7.302,P ＝0.004).结论 GSTP1 基因型对预测以DDP 为基础化疗方案治疗晚期胃癌的疗效具有较好的临床意义.     

Age and gender dependent alterations in the activities of glutathione related enzymes in healthy subjects

Objectives: Oxidative stress as a result of increased free radical production is implicated in the pathogenesis of several diseases. Specific antioxidant enzymes have a crucial role in the prevention of these deleterious effects. Since the activities of these enzymes differ significantly in different populations and seem to be affected by various environmental factors, in this study we aimed to determine the reference values of glutathione related antioxidant enzyme activities in the erythrocytes of healthy subjects and to investigate the possible variations as a function of age and gender in a healthy Turkish Mediterranean population.

Design and methods: 130 healthy subjects (12–90 yr, 82 females, 48 males) were divided into six different age groups. Erythrocyte glutathione peroxidase (GSH-PX), glutathione reductase (GR) and glutathione-s-transferase (GST) activities were measured on a Hitachi 704 autoanalyser by the modification of previously described manual UV spectrophotometric methods.

Results: No significant differences were observed in erythrocyte GSH-PX, GR and GST activities between different age groups. Overall, GST activities were significantly higher in females compared with males (8.08 ± 1.39, 6.88 ± 1.51 U/g Hb respectively, mean ± SD, p < 0.001). A significant positive correlation between GSH-PX and GR activities was observed (r = 0.49, p < 0.001).

Conclusion: The results of this study suggested that the activities of GSH-PX, GR and GST did not depend. GST activities overall were higher in females. The reference values that we obtained were different than the previous reports. This situation implies that each population should determine its own reference values and should investigate the influence of environmental factors and life style habits on the activities of these enzymes that constitute a major part of the antioxidant defense system in the human organism.     

Age-dependent excretion of 3-hydroxy-3-methylglutaric acid (HMG) and ketone bodies in the urine of full-term and pre-term newborns

Total ketone bodies and 3-hydroxy-3-methylglutaric acid (HMG) were determined in urines of full-term and pre-term newborns from the first day after birth to an age of 17. Significantly higher levels of the two catabolites were observed in the first two weeks of life of the pre-term newborns. A peak of excretion of both ketone bodies and HMG was found between the 7th and the 10th day after birth in both groups of newborns. After the 3rd month there is no significant difference between full-term and pre-term children as far as the excretion of the two analytes is concerned.     

Nutritional and racial determinants of the increase in plasma homocysteine levels after methionine loading

Background: Low circulating plasma levels of total homocysteine (tHcy) are associated with a lower prevalence of coronary heart disease among black people than among white people living in Burkina Faso.Objective: The purpose of this study was to provide a rationale for a possible mechanism for the decrease in plasma tHcy levels among black people compared with white people living in Burkina Faso.Methods: Healthy, black, adult, lifelong inhabitants of Burkina Faso and healthy, white adults born in Italy but living in Burkina Faso ≥5 years were eligible for enrollment. Controlled diets were assigned to all subjects for 2 weeks before the study. After an overnight (12-hour) fast, a methionine-loading test was performed in all subjects. Plasma levels of tHcy, cysteine, glutathione, and cysteinylglycine were measured simultaneously using high-performance liquid chromatography after fasting (baseline) and at either 4 and 8 hours (n = 30) or 2, 4, 6, and 8 hours (n = 4) after methionine loading. During the 12 hours after loading, the clinical conditions and adverse events of subjects were monitored. Results were analyzed using the Student t test and Mann-Whitney U test.Results: Seventeen black adults (9 males, 8 females; median age, 21 years) and 17 white adults (8 males, 9 females; median age, 35 years) were enrolled. Mean plasma levels of tHcy, cysteine, and glutathione increased from mean baseline levels more slowly in the black group than in the white group and peaked 8 hours after methionine loading (16.8 ± 3.0 μmol/L, 130.4 ± 25.7 μmol/L, and 68.3 ± 21.2 μmol/L, respectively). In the white group, these levels peaked 4 hours after loading (16.1 ± 4.0 μmol/L, 215.8 ± 18.6 μmol/L, and 38.6 ± 12.4 μmol/L, respectively). Only the mean plasma cysteinylglycine level decreased significantly (from 35.7 ± 11.4 μmol/L to 19.0 ± 6.1 μmol/L; P < 0.01) in the black group after 4 hours. This decrease was followed by an increase after 8 hours (29.6 ± 12.0 μmol/L). In the white group, a less remarkable change in mean cysteinylglycine level was observed, with a peak after 4 hours (16.3 ± 4.3 μmol/L).Conclusions: The findings of this study suggest that, in addition to lower plasma tHcy levels, the metabolism of plasma tHcy is different in black people than in white people after methionine loading. This difference may be due to different alimentary habits associated with a reduced dietary availability of methionine. Moreover, the higher plasma levels of glutathione before and after methionine loading appear to occur exclusively in black people compared with whites and correspond with the variation of cysteinylglycine, suggesting that, in addition to nutritional factors, a racial component may contribute to the difference in plasma levels of tHcy. This difference also might explain, in part, the lower prevalence of coronary heart disease in black people living in Burkina Faso compared with that in other populations.     

铜绿假单胞菌MexAB-OprM、MexXY-OprM主动外排系统的耐药作用

目的探讨MexAB-OprM、MexXY-OprM主动外排系统(外排泵)在多重耐药铜绿假单胞菌耐药机制中的作用。方法选取36株临床分离的多重耐药铜绿假单胞菌,采用琼脂二倍稀释法,测定环丙沙星对铜绿假单胞菌的MIC及进行泵抑制剂碳酰氰基-对-氯苯腙(CCCP)存在情况下的干预试验;用实时定量RT-PCR测定结构基因mexA、mexX的mRNA表达水平来判断MexAB-OprM、MexXY-OprM外排泵表达情况;用PCR法分别扩增其调控基因mexR、mexZ,并对其产物测序,用Blast软件在GenBank与已知序列比较,研究其过度表达的机制。结果在CCCP作用下,36株铜绿假单胞菌中24株菌对环丙沙星的敏感性(MIC)由20～320 mg/L提高到2.5～40 mg/L,主动外排表型阳性率为66.7%(24/36),15株菌高表达Mex-AB-OprM外排系统(41.7%),21株高表达MexXY-OprM外排系统(58.3%)。15株高表达MexAB-OprM外排系统铜绿假单胞菌同时表达MexXY-OprM外排系统;12株干预试验阴性的铜绿假单胞菌均无mexA、mexX高表达;随机选择mexA、mexX高表达的4株细菌均发生mexR、mexZ基因突变,出现氨基酸替代。结论主动外排系统MexAB-OprM、MexXY-OprM在多重耐药铜绿假单胞菌中过度表达是铜绿假单胞菌多重耐药的机制之一;外排泵MexAB-OprM、MexXY-OprM高表达分别与调控基因mexR、mexZ发生变异有关。     

Intracellular glutathione and lipid peroxide availability and the secretion of vasoactive substances by human umbilical vein endothelial cells after incubation with TNF-alpha

BACKGROUND: The major pathophysiologic changes observed in preeclampsia suggest that endothelial cell dysfunction plays an important role in this disorder. The pathway mediating to endothelial cell dysfunction is unknown, however, the pathogenesis of preeclampsia is thought to be related to increased oxidative stress and increased vasoconstriction. The concentration of tumor necrosis factor alpha (TNF-alpha), a cytokine produced by macrophages and many other cell types, has been observed to be significantly increased in preeclampsia. It has been hypothesized that TNF-alpha overproduction by the placenta may then may produce an increase in plasma levels and subsequent endothelial dysfunction in preeclampsia. This study investigated the effect of TNF-alpha on glutathione and lipid peroxide levels and on the secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs). METHODS: Human umbilical vein endothelial cells were incubated for 24 h in the presence of different concentrations of TNF-alpha (0-1000 pg mL-1) that were shown in an earlier experiment to have no effects on the vitality and proliferation rate of HUVECs. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs), assessed by malondialdehyde and 4-hydroxyalkenal, were measured in endothelial cell lysates. For the measurement of vasoactive substances, levels of prostacyclin (PGI2), determined by 6-keto-prostaglandin F1a, thromboxane A2 (TXA2), measured by thromboxane B2, endothelin-1 (ET-1), and nitric oxide (NO), measured by total nitrite, were assessed in endothelial cell supernatants. RESULTS: At lower concentrations (10-100 pg mL-1), TNF-alpha increases the intracellular content of LPO and GSH, stimulates the secretion of ET-1 and TXA2, but inhibits the secretion of PGI2 in endothelial cells compared with control cells. At concentration of 1000 pg mL-1, TNF-alpha increases the secretion of PGI2 and TXA2, but it decreases the ET-1 concentration. TNF-alpha has no effect on NO secretion. CONCLUSION: These findings demonstrate that at concentrations corresponding to values in plasma from preeclamptic women, TNF-alpha induces oxidative stress and results in altered secretion of vasoactive substances in favour of vasoconstrictors in human endothelial cells. We conclude that TNF-alpha may participate in the pathway leading to endothelial cell dysfunction seen in preeclampsia.     

Functional diversification of three delta‐class glutathione S‐transferases involved in development and detoxification in Tribolium castaneum

Glutathione S‐transferases (GSTs) are members of a multifunctional enzyme superfamily. Forty‐one GSTs have been identified in Tribolium castaneum; however, none of the 41 GSTs has been functionally characterized. Here, three delta‐class GSTs, TcGSTd1, TcGSTd2 and TcGSTd3, of T. castaneum were successfully cloned and expressed in Escherichia coli. All of the studied GSTs catalysed the conjugation of reduced glutathione with 1‐chloro‐2,4‐dinitrobenzene. Insecticide treatment showed that the expression levels of TcGSTd3 and TcGSTd2 were significantly increased after exposure to phoxim and lambda‐cyhalothrin, whereas TcGSTd1 was slightly upregulated only in response to phoxim. A disc diffusion assay showed that overexpression of TcGSTD3, but not TcGSTD1 or TcGSTD2, in E. coli increased resistance to paraquat‐induced oxidative stress. RNA interference knockdown of TcGSTd1 caused metamorphosis deficiencies and reduced fecundity by regulating insulin/target‐of‐rapamycin signalling pathway‐mediated ecdysteroid biosynthesis, and knockdown of TcGSTd3 led to reduced fertility and a decreased hatch rate of the offspring, probably caused by the reduced antioxidative activity in the reproductive organs. These results indicate that TcGSTd3 and TcGSTd2 may play vital roles in cellular detoxification, whereas TcGSTd1 may play essential roles in normal development of T. castaneum. These delta‐class GSTs in T. castaneum have obtained different functions during the evolution.