Conjugation Of 1-chloro-2,4-dinitrobenzene Research Articles

A novel theta-class glutathione S-transferase gene in Rhopalosiphum padi (L.) (Hemiptera: Aphididae): Identification, recombinant protein expression and function analyses of RpGSTT1

Glutathione S-transferases (GSTs) play a major role in the detoxification of insecticides and the protection against oxidative damage caused by ROS (reactive oxygen species). The bird cherry-oat aphid, Rhopalosiphum padi (L.), is a major crop pest worldwide. Chemical insecticides are currently used to control aphids, but insecticide resistance is an evolutionary issue for aphid control. In this research, we identified and cloned the full-length cDNA of a theta-class GST gene (RpGSTT1) from R. padi, which contains 702 bp open reading frame encoding 233 amino acids. RpGSTT1 was clustered together with theta classe of insect GSTs in the phylogenetic tree. Effect of insecticide exposure on the GST enzyme activity was investigated in vivo. After affinity purification, the recombinant RpGSTT1 protein was able to catalyze the conjugation of 1-chloro-2, 4-dinitrobenzene (CDNB). A disc diffusion assay showed that the RpGSTT1 can increase resistance to cumene hydroperoxide-induced oxidative stress. In addition, the purified recombinant RpGSTT1 protected super-coiled DNA from oxidative damage. By quantitative PCR, The relative expression level of RpGSTT1 in the aphid was highly up-regulated after 12 h exposure to β-cypermethrin, isoprocarb, malathion and sulfoxaflor, respectively. To our knowledge, RpGSTT1 is the first theta class of GST gene reported in bird cherry-oat aphid. Our findings indicate that RpGSTT1 may be involved in insecticide detoxification in R. padi.

CpGSTd3 is a lambda-Cyhalothrin Metabolizing Glutathione S-Transferase from Cydia pomonella (L.)

Little is known about the role of specific delta GST genes in the detoxification of lambda-cyhalothrin in the global quarantine fruit pest codling moth, Cydia pomonella (L.). Real-time quantitative PCR shows that CpGSTd3 was ubiquitously expressed at all developmental stages and is most abundant in the larval stage and lowest in the egg stage; the mRNA level of CpGSTd3 is higher in the midgut and Malpighian tubules of fourth-instar larvae and abdomens of adults than in other tissues. Exposure of fourth-instar larvae to an LD10 dosage of lambda-cyhalothrin significantly induced the transcript of CpGSTd3 at 3 h, but the mRNA level was down-regulated after 12 h of treatment. Recombinant CpGSTd3 expressed in Escherichia coli was able to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and with an IC50 value of 0.65 mM for lambda-cyhalothrin. Metabolism assays indicate that recombinant CpGSTd3 could metabolize lambda-cyhalothrin. These results suggest that CpGSTd3 is probably a lambda-cyhalothrin metabolizing GST in C. pomonella.

8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1.

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

GST isoenzymes in matched normal and neoplastic breast tissue

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore we investigated GST activity and the protein expression of glutathione S-transferases (GSTs) isoenzymes known to be involved in the metabolism of endogenous and exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. The interindividual variation in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and of 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) with glutathione (GSH) by cytosolic glutathione S-transferases (GSTs) were investigated in human breast matched normal and tumor samples. The GSTA, GSTM, GSTP and GSTT isoenzymes from the crude extracts of matched breast normal and tumor tissues in terms of their immunological properties using western blotting were compared. In most of the samples, the GST activities were higher in the tumor than in the normal cytosolic fractions against both CDNB and EPNP. In the western blotting analysis, it was proved statistically that in normal and tumor epithelial cells, there was difference between GST pi and theta isoenzymes expressions (p0.05). In normal epithelium there was a stronger GST theta expression than in invasive tumor tissues (p=0.013). However, the stronger GST pi expression was observed in tumor epithelium than in normal epithelium in human breast cancers (p=0.000). We found the GSTP protein level and GST activities were higher in the breast tumor than in the normal cytosolic fractions against both CDNB and EPNP, thus implicating a certain biological importance.

ACTIVITY OF PROPYL p-BENZOYLOXYBENZOATE AS A GLUTATHIONE S-TRANSFERASE(S) INHIBITOR: THE COMPARISON BETWEEN COMPUTATIONAL CHEMISTRY APPROACH AND EMPIRICAL OBSERVATION

The activity of propyl p-benzoyloxybenzoate as a glutathione S-transferase(s) (GSTs) inhibitor has been examined through computational chemistry based theoretical approach and laboratory experiment. This research was related to the nature of GSTs as multifunctional enzymes, which play an important role in the detoxification of electrophilic compounds, the process of inflammation and the effectivities of anticancer compounds. Quantitative Structure-Activity Relationship (QSAR) study, which was established on curcumin and its derivatives using computational chemistry approach, was used to examine the theoretical activity of p-benzoyloxybenzoate as a GSTs inhibitor. Empirical observation on GSTs inhibition was examined using formation reaction model of GS-CNB conjugate through conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) with GSTs (prepared from rat's liver) as catalysts. The result showed that the difference between the activities of propyl p-benzoyloxybenzoate as a GSTs inhibitor obtained from the computational chemistry approach and the empirical observation were not statistically significant at 95% level of confidence. Keywords: Propyl p-benzoyloxybenzoate, inhibitor, glutathione S-transferase (GSTs), QSAR

Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake

Brown bullheads ( Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxification by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent K m and V max for CDNB at fixed nucleophile (glutathione, GSH) concentrations of 1.8–1.9 mM and 12.1–14.6 μmol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), Δ5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were significantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodruff). No site differences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads efficiently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems.

Quantification of individual glutathione S-transferase isozymes in hepatic and pulmonary tissues of naphthalene-tolerant mice.

Acute exposure to naphthalene produces severe bronchiolar epithelial cell necrosis in mice, whereas subchronic exposure to naphthalene (200 mg/kg/7 days) fails to produce epithelial necrosis and renders the animals tolerant to subsequent challenge doses of naphthalene. Mechanisms responsible for the development of tolerance have not been delineated. The few studies exploring naphthalene tolerance focus on expression of microsomal enzymes and have yet to delve into expression of the hepatic detoxification enzymes such as glutathione S-transferases (GSTs; EC 2.5.1.18). Glutathione conjugation catalyzed by GSTs accounts for one of the two primary routes of naphthalene detoxification. In this study, we rigorously quantify levels of individual GST isozymes expressed within the livers and lungs of mice with acquired tolerance to naphthalene. Subchronic exposure to naphthalene increases the abundance of some hepatic GSTs to levels as much as 68% greater than controls. Naphthalene-tolerant mice displayed increases in mGSTM1 (51%), mGSTM2 (58%), and mGSTP1 (66%), whereas no significant difference in mGSTA3 was observed between exposed and control mice. Extracts of pulmonary tissues from naphthalene-tolerant mice showed minor increases in levels of mGSTP1 (7%) and Peak 8 isozyme (27%) and decreases in levels of mGSTM1 (31%), mGSTM2 (17%), and mGSTA3 (8%). The total enzymatic activity for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) was 22% lower in lung extracts from naphthalene-tolerant animals than in controls. These results indicate that induction of hepatic GSTs is substantial and may be an important factor in the development of tolerance to naphthalene.

Biochemical Basis for the Extreme Sensitivity of Turkeys to Aflatoxin B1

Poultry are the most susceptible food animal species to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). Feed contaminated with even small amounts of AFB1 results in significant adverse health effects in poultry. The purpose of this study was to explain the biochemical mechanism(s) for this extreme sensitivity. We measured microsomal activation of AFB1 to the AFB1-8,9-epoxide (AFBO), the putative toxic intermediate, as well as cytosolic glutathione S-transferase (GST)-mediated detoxification of AFBO, in addition to other hepatic phase I and phase II enzyme activities, in 3-week-old male Oorlop strain turkeys. Liver microsomes prepared from these turkeys activated AFB1in vitro with an apparent Km of 109 μM and a Vmax of 1.25 nmol/mg/min. Preliminary evidence for the involvement of cytochromes P450 (CYP) 1A2 and, to a lesser extent, 3A4 for AFB1 activation was assessed by the use of specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was supported by activity toward ethoxyresorufin and nifedipine, as well as by Western immunoblotting using antibodies to human CYPs. Cytosol prepared from turkey livers exhibited GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), but at a much lower rate than that observed in other species. Western immunoblotting indicated the presence of α and ς class GSTs and another AFB1-detoxifying enzyme, AFB1-aldehyde reductase (AFAR). Turkey liver cytosol also had quinone oxidoreductase (QOR) activity. Importantly, cytosol exhibited no measurable GST-mediated detoxification of microsomally activated AFB1, indicating that turkeys are deficient in the most crucial AFB1-detoxification pathway. In total, our data indicate that the extreme sensitivity of turkeys to AFB1 may be attributed to a combination of efficient AFB1 activation and deficient detoxification by phase II enzymes, such as GSTs.

Glutathione transferase activities toward herbicides used selectively in soybean

Using extracts from suspension-cultured cells of soybean (Glycine max cv. Mandarin) as a source of active enzymes, the activities of glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and selective herbicides were determined to be in the order CDNB fomesafen > metolachlor = acifluorfen > chlorimuron-ethyl. GST activities showed a thiol dependence in a substrate-specific manner. Thus, GST activities toward acifluorfen and fomesafen were greater when homoglutathione (hGSH), the endogenously occurring thiol in soybean, was used as the co-substrate rather than glutathione (GSH). Compared with GSH, hGSH addition either reduced or had no effect on GST activities toward other substrates. In the absence of enzyme, the rates of hGSH conjugation with acifluorfen, chlorimuron-ethyl and fomesafen were negligible, suggesting that rapid hGSH conjugation in soybean must be catalysed by GSTs. GST activities were subsequently determined in 14-day-old plants of soybean and a number of annual grass and broadleaf weeds. GST activities of the plants were then related to observed sensitivities to post-emergence applications of the four herbicides. When enzyme activity was expressed on a mg -1 protein basis, all grass weeds and Abutilon theophrasti contained considerably higher GST activity toward CDNB than soybean. With fomesafen as the substrate, GST activities were determined to be in the order soybean Echinochloa crus-galli > Digitaria sanguinalis > Sorghum halepense = Setaria faberi with none of the broadleaf weeds showing any activity. This order related well to the observed selectivity of fomesafen, with the exception of A. theophrasti, which was partially tolerant to the herbicide. Using metolachlor as the substrate the order of the GST activities was soybean > A. theophrasti S. halepense > Amaranthus retroflexus > Ipomoea hederacea, with the remaining species showing no activity. GST activities toward metolachlor correlated well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds. Acifluorfen and chlorimuron-ethyl were selectively active on these species, but GST activities toward these herbicides could not be detected in crude extracts from whole plants.

Induction of rat liver microsomal epoxide hydrolase by its endogenous substrate 16 alpha, 17 alpha-epoxyestra-1,3,5-trien-3-ol.

1. The influence of the endogenous steroid epoxides 16 alpha, 17 alpha-epoxyestra-1,3,5(10)-trien-3-ol (estroxide) and 16 alpha, 17 alpha-expoxiandrost-4-en-3-one (androstene oxide) and their metabolic precursors estra-1,3,5(10), 16-tetraen-3-ol (estratetraenol) and androsta-4, 16-dien-3-one (androstadienone) on the specific activities of hepatic microsomal and soluble epoxide hydrolase, glutathione S-transferase, dihydrodiol dehydrogenase, and 7-ethoxycoumarin deethylase was investigated in the male Sprague-Dawley rat. 2. Both estroxide and estratetraenol induced microsomal epoxide hydrolase activity towards styrene oxide and estroxide itself 2-2.5-fold and glutathione conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) 1.6-fold after intraperitoneal administration of a high dose of compound (300 mg per kg of body weight). 3. In addition, estroxide decreased 7-ethoxycoumarin deethylation down to 20% of the activity observed in the untreated rat, whereas estratetraenol enhanced the activity of soluble epoxide hydrolase towards trans-stilbene oxide by a factor of 1.7. 4. In contrast, neither androstene oxide nor androstadienone showed a significant influence on any of the parameters under investigation. Dihydrodiol dehydrogenase was not significantly changed by any of the treatments.

A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase

Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.

Mixed-function oxidase activity and conjugating enzymes in two species of antarctic fish

Enzyme activities of phase I and phase II of the hepatic detoxication system were investigated in two species of fish from the Ross Sea in Antarctica. The fish had extremely low phase I enzyme (BPMO and aldrin epoxidase). The reductase regenerating enzyme activities were comparable to those found in fish of temperate seas. Transferases of cytosol showed highest activity in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and little or no conjugation of other substrates. The GSSG reductase and the glutathione transferase with peroxidase activity (CuOOH substrate) were low; the Sedependent glutathione peroxidase activity was not detectable. Interspecific differences were found for most of the enzymes tested.

Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats

The activities of three drug conjugation reactions, glutathione, glucuronic acid and sulphate conjugation and the synthesis of glutathione, have been measured in hepatocytes isolated from strept-ozotocin-induced male diabetic rats. The intracellular content of reduced glutathione (GSH) was decreased in diabetic rat hepatocytes compared with controls. Following depletion of the intracellular GSH stores with diethylmaleate, the resynthesis of GSH in the presence of 0.5 mM l-methionine, occurred faster in diabetic rat hepatocytes than in those from control rats indicating that the cystathione pathway may be more efficient in the diabetic animals. In contrast, there was no significant difference in the resynthesis of GSH between control and diabetic rat hepatocytes in the presence of l-cysteine. The GSH conjugation of 1-chloro-2, 4-dinitrobenzene (CDNB) and 3, 4-dichloronitrobenzene (DCNB) was deficient in diabetic rat hepatocytes, although only the effect on the former reaction was statistically significant (P < 0.05). The V max for CDNB conjugation was significantly lower (P < 0.05) in cytosolic fractions prepared from diabetic rat liver than in control rat liver fractions. This was accompanied by an increase in the affinity of the enzyme for CDNB. In contrast, the V max and K m for the conjugation of DCNB in cytosolic fractions were unaffected by the induced-diabetes. Glucuronic acid conjugation of both 1-naphthol and phenolphthalein was markedly deficient in diabetic rat hepatocytes. The intracellular concentrations of the cofactor for glucuronidation, UDP-glucuronic acid, were decreased in diabetic rat liver and this was thought to contribute to the defect in glucuronidation. The sulphation of 1-naphthol was not significantly altered by the induced diabetes. Deficiencies in glutathione and glucuronic acid conjugation in streptozotocin-induced diabetic rats may result in an increased susceptibility to xenobiotic induced cytotoxicity.

Tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] an atrazine synergist: Enzymatic conversion to a potent glutathione S-transferase inhibitor

Glutathione S-transferases (GST) from corn, giant foxtail, onion, pea, house fly, and equine liver catalyzed conjugation of tridiphane with glutathione (GSH). The conjugate was characterized by soft ionization mass spectral methods. Tridiphane and the GSH conjugate of tridiphane both inhibited GSH conjugation of atrazine in vitro (corn and giant foxtail). Tridiphane did not inhibit GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) in corn or giant foxtail; however, the GSH conjugate of tridiphane was a competitive inhibitor with respect to GSH and was four times more effective with extracts from giant foxtail ( K i = 2 μM) than from corn ( K i = 8 μM). The GSH conjugate of tridiphane inhibited a variety of GST enzymes with several different substrates. When compared to other inhibitors of GST, only triphenyl tin chloride was more effective than the GSH conjugate of tridiphane in inhibition of GST from giant foxtail. Both GST and GSH decreased in corn and increased in giant foxtail as tissues matured. The catabolism of the GSH conjugate of tridiphane was compared in crude enzyme systems from corn, giant foxtail, and onion. The rate of catabolism was much greater in extracts from corn leaves than from giant foxtail leaves. Inhibition of GSH conjugation of CDNB was reversed as the GSH conjugate of tridiphane was catabolized. The possibility that synergism of atrazine toxicity by tridiphane is mediated by conversion of tridiphane to a GSH conjugate is discussed in relationship to the relative rates of GSH conjugation of tridiphane and atrazine, concentrations of GSH, K i values, tissue age, and stability of the conjugate in different tissues.