Browsing by Author "Misra, Hara P."
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- The actions of gossypol on the physiologic antioxidant defense systemBender, Holly S. (Virginia Polytechnic Institute and State University, 1987)Gossypol , a yellow polyphenolic pigment found in cottonseeds, is known to promote the production of reactive species of oxygen in vitro, and has toxic actions on spermatogenic epithelium, hepatocytes and cardiac myocytes in vivo. Species vary in tissue sensitivity to the toxic effects of gossypol. The spermatogenic epithelium is the most sensitive tissue to gossypol in rats, followed by the liver. Toxic effects to the rat heart are found only after prolonged administration of gossypol. The antioxidant defense system that protects cells from injury by reactive species of oxygen was examined in the present study to determine a possible pathogenesis for gossypol associated tissue damage. The concentrations of several hepatic antioxidants including catalase, glutathione peroxidase, ascorbate and copper-zinc superoxide dismutase were decreased in gossypol treated rats. Catalase, glutathione peroxidase, ascorbate and glucose-6-phosphate dehydrogenase were decreased in the testis. In contrast, antioxidants including catalase and glutathione reductase were increased in the hearts of gossypol treated rats. The selective inhibition of testis and hepatic antioxidants may account for the greater sensitivity of these organs to reactive oxygen species generated by gossypol. The rat heart may adapt to oxidative insult by inducing the production of antioxidants. Glucose-6-phosphate dehydrogenase activity was decreased in the testis but not liver or heart of gossypol treated rats. This important enzyme is known to produce NADPH reducing equivalents for testosterone biosynthesis and the glutathione antioxidant system. In the present study, micromolar concentrations of gossypol inhibited glucose-6-phosphate dehydrogenase in a competitive manner with respect to glucose-6-phosphate. This may explain the degeneration of spermatogenic epithelium as well as decreases in serum testosterone concentrations in gossypol treated rats. Gossypol is known to cause infertility in women and female rats. The present study found irregularities in the estrous cycles and ultrastructural changes in endometrial macula adherentes of gossypol treated female rats.
- Amelioration of oxidative lung injury by antiarrhythmic agentsDas, Kumuda C. (Virginia Tech, 1992-10-15)Class I antiarrhythmic drugs, such as lidocaine, quinidine and procainamide, are known to be effective membrane stabilizers. However, the mechanism of such "membrane stabilization" has not been elucidated. In the present study we found that all three drugs are powerful scavengers of hydroxyl! radical. In addition, lidocaine was found to be a quencher of singlet oxygen. These drugs are also found to inhibit NADPH-dependent lipid peroxidation in bovine lung microsomes in a dose dependent manner. Since oxyradicals are implicated in the lipid peroxidation process and antiarrhythmic drugs were found to scavenge/quench reactive oxygen species, we proposed that the membrane Stabilizing effects of antiarrhythmic drugs may, in part, be due to their antioxidant properties. Ischemia-reperfusion injury has been studied in many organs. Despite the evidence of functional, metabolic and structural abnormalities during reperfusion, the precise mechanism of reperfusion lung injury remains obscure. Data from the organ models suggest that toxic oxygen metabolites play an important role in the mechanism of reperfusion tissue injury. Lidocaine has also been shown to be clinically valuable for the treatment and prevention of ventricular arrhythmia occurring after surgical correction of myocardial infraction. We found that the class I antiarrhythmic drugs are effective in ameliorating post-ischemic lung reperfusion injury in an ex vivo perfused rat lung model exposed to both normoxic and hyperoxic conditions. Since phagocytes are known to generate reactive oxygen species and play an important role in causing irreversible oxidative tissue injury during reperfusion of organs, we examined the role of antiarrhythmic agents on macrophage function. We found that these drugs inhibit superoxide and hydrogen peroxide production in stimulated macrophages in a dose dependent manner. The diminished production of superoxide was found to be not due to the inactivation of superoxide generating NADPH-oxidase enzyme but by inhibition of the phagocytosis process by these drugs The results of these studies indicate that the antiarrhythmic drugs, such as, lidocaine, quinidine and procainamide, are effective antioxidants and can protect biomembranes against lipid peroxidation injury and post-ischemic reperfusion injury of the lung. We have investigated the mechanism(s) of action of these drugs in ameliorating oxidative tissue injury and found that these drugs are not only effective in removing reactive oxygen species but also cause inactivation of pulmonary macrophage from inappropriately generating reactive species of oxygen. The fundamental knowledge derived from these Studies could lead to enhanced functional improvement of patients following cardiopulmonary bypass, pulmonary arterial embolectomy and acute respiratory distress syndrome, all of which undergo a period of elective/induced ischemia and reperfusion or oxidative stress.
- Antioxidant activity of Mn-salophen complex and its effects on antioxidant enzymes in Escherichia coliLiu, Zheng-Xian (Virginia Tech, 1994-11-05)Mn-salophen complex with superoxide-scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol. Visible absorption spectrum of the red-brown solution exhibited a broad absorption band at 430 - 450 nm with two shoulders between 500 and 600 nm which were absent with either salophen or manganic acetate alone. Titration of salophen with manganese(III) was consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity. The SOD-like activity of the complex in the xanthine-xanthine oxidase/cytochrome c assay was 1450 units/mg salophen. The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of E. coli QC779 sodA sodB (1 mg/ml). E. coli QC779 sodA sodB grew scantily after an 8 hour lag phase in aerobic M63 glucose minimal medium.
- Antioxidant Intervention With manganese(Iii)-Salophen in the Selenite Cataract Model: Implications for Cataract DiseaseDell, Kevin David (Virginia Tech, 1998-05-04)Cataract disease affects millions of people worldwide. It is characterized by the accumulation of light-scattering bodies within the lens that reduce visual acuity. Cataracts are effectively treated surgically, but at great expense, costing Medicare $3.4 billion in 1997. Development of an alternative therapy for this disease would provide medical and economic benefits. We have investigated a novel antioxidant, the superoxide scavenger Mn(III)-salophen, as a therapeutic agent in the selenite cataract model. Mn(III)-salophen has been shown to protect E. coli colonies against oxidative stress but was untested in a eukaryotic system. A total dose of 300 mmol/kg, given IP in four equal 75 mmol/kg doses spaced four hours apart, protects 75% of neonatal rats from nuclear cataract development five days after selenite injection. Selenite is toxic through its reaction with the endogenous antioxidant glutathione (GSH). The reduction of selenite to selenide through an intermediate, selenodiglutathione (GSSeSG) leads to generation of superoxide radical, one of several toxic oxygen species that can damage the lens. Mn(III)-salophen causes an in vitro preservation of the lifetime of GSSeSG by interrupting the reduction of selenite. We have established that the reduction of GSSeSG to selenide does not use GSH as a reducing agent, but rather depends upon electrons generated in the earlier reduction of selenite to selenodiglutathione. These electrons can be intercepted by known one-electron scavengers, arresting the metabolism of GSSeSG. Extensive proteolysis of lens crystallins and loss of calcium homeostasis occur in cataractous lenses from a rat treated with sodium selenite. The visual protection with Mn(III)-salophen is accompanied by a partial loss of the calcium homeostasis, a net increase in sodium, and calpain-mediated proteolysis of à -crystallins similar to lenses from animals treated with selenite alone. Although preservation of alpha-crystallins may contribute to the greater transparency in the protected lens, generalized à -crystallin proteolysis is insufficient for cataract formation. From these experiments we propose that Mn(III)-salophen minimizes the oxidative stress imposed upon the cell by interfering with the metabolism of selenodiglutathione. This allows the cell to compensate for the loss of cation homeostasis and prevents aggregation of proteolyzed crystallins into cataracts.
- Chemosensory Evaluation of Training and Oxidative Stress in Long Distance RunnersWhysong, Christan Yvonne (Virginia Tech, 2014-05-28)Athletes complete a balance of training loads and rest periods, risking overtraining when this balance favors excessive training. Diagnostic biomarkers have been suggested but a clear diagnostic method is not available. This preliminary study's objective was to use data standardization to improve an electronic nose's (enose) discrimination model for athletes' breathprints after cumulative and acute training loads. Collegiate long distance runners were observed throughout competitive training seasons. Prolonged training effects were observed through Profile of Mood States (POMS) surveys and blood and breath samples collected at the beginning (Pre-Study) and end of the training season (Post-Study). Immediate training effects were observed for one low (LI) and one high (HI) intensity acute training load. Subjects provided blood and breath samples before the LI (BSR) and HI (BLR), completed the training load, and provided blood and breath samples after each training load (ASR; ALR). Blood was analyzed for antioxidant enzymes (catalase, glutathione peroxidase, and glutathione reductase). Breath samples were analyzed with a Cyranose® 320 (C320) enose. Age, gender, and training loads affected oxidative states, with the HI having more effect than the LI. Mood profiles indicated healthy and successful athletes. Neither POMS nor blood parameters suggested overtrained athletes. The C320 successfully discriminated between breathprints of athletes correlating to the training loads. Direct data standardization through carbon dioxide as a baseline sensor purge correctly classified 100 percent of the data through linear discriminant analysis (LDA). Indirect data standardization by subtracting Pre-Study data from the subsequent data classes (e.g. BSR) correctly classified 96 percent of the data. An LDA on the combined blood parameters correctly classified 61.9 percent of the data. The blood analyses required invasive sample collections and involved procedures that took a long time (hours). In comparison, the best C320 model correctly classified 96 percent of the data and required less invasive sample collections, simple analysis, and short result times (minutes). Evidence suggested the C320 will provide a simple and noninvasive method for clinically diagnosing the onset of overtraining. The unit is small, handheld, rapid, and noninvasive so it could also be used on- site to provide immediate feedback for training optimization.
- Differential Prediction of Medical School Selection Factors for Rural and Non-Rural PopulationsPrice, Megan Rae (Virginia Tech, 2008-04-29)Differential predictive validity in assessing academic performance in institutions of higher education has been assessed for a number of years. Historically, this body of research focused on gender and ethnicity. This study extends that research to geographic region (e.g., rural and non-rural populations). Specifically, this study predicted relationships between preadmission variables of incoming grade point average (GPA) and medical college admissions test (MCAT) and output variables of medical school GPA and comprehensive osteopathic medical licensing exam (COMLEX). Results indicate incoming GPA and MCAT are good variables to use to predict academic performance in medical school and score on the licensing board exam. Further, rural populations presented similar scores on preadmission variables and, thus, are not at a disadvantage in the admission process. A second goal of this study was to explore differential prediction of medical school GPA and COMLEX Level 1 score for the MCAT for rural and non-rural populations. Results provide some evidence of differential prediction of COMLEX score for the physical and biological sciences MCAT sub-tests such that rural populations' performance on the COMLEX Level 1 exam was underpredicted. Hence, when rural and non-rural populations present the same physical sciences and biological sciences MCAT sub-test score, the rural sub-group is predicted to obtain a lower COMLEX score and non-rural sub-group is predicted to obtain a higher COMLEX score. Further, when the two sub-groups present different MCAT scores for the physical and biological sciences sub-test, they are likely to obtain similar scores on the COMLEX. Implications and recommendations for future research are discussed.
- Effect of oxidative stress on Escherichia coli sodA-sodB-: protection by the mimic of superoxide dismutase, Mn(III)-salophenKittiponkul, Vipavadee (Virginia Tech, 1996-12-19)The effect of Mn(III)-salophen, a superoxide scavenger, against oxidative stress was evaluated in Escherichia coli sodA- sodB-. Oxidative stress was imposed by exposure of the cells to paraquat or hyperoxia. Cells were grown in LB medium overnight, washed and resuspended in the indicated glucose/salts medium supplemented with casamino acids. The effect of Mn(III)-salophen in the oxidative stress model in vivo was measured in terms of the cell growth. Mn(III)-salophen ( 60 nM) completely protected E. coliJI132sodA- sodB-against 1.0 μM paraquat. Equivalent amounts of Mn(III) acetate, a Mn(III)-salophen component, also protected against paraquat toxicity in aerobic E. coli JI132sodA- sodB-. Fe(III)-salophen which has no superoxide scavenging activity, did not protect the cells against paraquat toxicity. The protective effect of Mn(III)-salophen against the paraquat toxicity was proposed to come from the intracellular superoxide scavenging activity of either the complex itself, its component Mn(III), or both, but not by inhibiting the uptake of paraquat. The protective effect of Mn(III)-salophen and Mn(III) in the glucose/salts medium containing casamino acids was also observed in E. coli sodA- sodB- in 100% and 50% oxygen. Hyperoxia increases intracellular levels of superoxide radicals that are intercepted by Mn(III)salophen and Mn(III).
- Effects of taurine and hypotaurine on oxidative lung injuryGarland, Carroll Moses (Virginia Tech, 1995-08-05)The present studies are based on the premise that pulmonary injury, during periods of hypoxia, ischemia, and reperfusion, may be due to increased production of reactive oxygen species, including the superoxide anion (O₂-), hydroxyl radical (-OH), and hydrogen peroxide (H₂O₂), and on the premise that this injury can be ameliorated by antioxidant pre-treatment. The sulfur-containing (λ²-amino acids, taurine and its precursor, hypotaurine, have been shown indirectly to possess antioxidant properties by several investigators. The mechanism(s) by which taurine and hypolaurine exert their antioxidant effects has(have) remained unclear despite many years of intensive study, as does the precise physiological role for these two β-amino acids. The goals of the present study were: 1) to evaluate the effects of taurine and hypolaurine in experiments that model biochemical events which are believed to be important components of oxidative pulmonary injury; 2) to assess the potential antioxiodant ability of the amino acids by determining their capacity to scavenge the free radicals, -OH and O₂-., directly; and 3) to investigate the effect of these amino acids on reperfusion injury of rat lungs in an ex vivo ischemia-reperfusion injury model. The results of this study indicate that taurine and hypotaurine are not effective in detoxifying H₂O₂ and, in fact, taurine was found to augment H₂O₂ production in phorbol myristate acetate-stimulated macrophages. At 26, 78, and 104 mM, taurine was found to elevate H₂O₂ production 13%, 28%, and 43%, respectively, above the positive control. Taurine (5-120 mM) and hypotaurine (2-10 mM) were also ineffective (p > 0.05) in protecting biomembranes against free radical-induced lipid peroxidation. However, taurine (10-300 mM) and hypotaurine (2-30 mM) were found to possess the ability to scavenge hydroxyl radicals. Taurine (148 and 193 mM) and hypotaurine (19 mM) were found to possess the ability to scavenge superoxide at the high end of the concentration range tested. This was demonstrated by the ability of these amino acids to compete with both ferricytochrome c for available O₂- and deoxyribose for available -OH, within the rdespective systems designed to produce these two reactive species. Additionally, in an EPR study using 5,5-dimethyl-l-pyrroline-Noxide (DMPO) as a spin trap, both taurine and hypotaurine caused dose-dependent inhibition of DMPO-OH and DMPO-OOH adduct formation. In the ex vivo rat lung model, the addition of 5 and 10 mM taurine to the perfusion medium 20 minutes prior to the induction of ischemia appeared not to provide significant protection (p > 0.05) against reperfusion injury to isolated rat lungs exposed to 60 minutes of ischemia followed by 30 minutes of reperfusion. However, the data obtained from the ex vivo lung experiments was variable and must be interpreted with caution. Furthermore, in preliminary studies it was found that 50 mM taurine may be toxic to the isolated, perfused, rat lung. In conclusion, the antioxidant properties of taurine and hypotaurine are due to their capability to scavenge some of the reactive species of oxygen. The apparent inability of low concentrations of taurine to ameliorate post-ischemic reperfusion injury of lungs is consistent with the fact that relatively high concentrations of taurine were needed for the amino acid to demonstrate significant scavenging of O₂- and -OH.
- Endovascular trophoblast cell behavior in normal and abnormal pregnancyEndo, Yasuhiro (Virginia Tech, 1995-04-05)Preeclampsia is an important disease during pregnancy and causes significant maternal and fetal mortality and morbidity. Despite intense research efforts, the etiology and pathogenesis of the disease remain largely unknown. Since placentas from preeclamptic patients are smaller than normal, and cytokine growth factors are suggested to be important in placental growth, the effects of macrophage-colony stimulating factor (M-CSF) on human trophoblast cells were examined. While term trophoblast cells did not respond to M-CSF, those from early trimester and choriocarcinoma cells showed enhanced growth after treatment. In addition, the serum level of M-CSF in hypertensive pregnant women at the second trimester were significantly lower than those of normal pregnant women. These data suggest possible roles of M-CSF in preeclampsia. When M-CSF was administered to pregnant rats on days 8-11, rats had smaller placentas at day 12 and increased fetal resorption rate at day 20. The effects of interleukin-12 (IL-12) was also examined on days 8-11. While placental development was normal at both days 12 and 20, fetuses were significantly smaller at day 20. To remedy the difficulties and dangers associated with obtaining human placentas, I characterized endovascular trophoblast cell behavior in pregnant rats. In normal pregnancy, rat trophoblast cells simulated all features of human endovascular trophoblast behavior including selective invasion into the spiral arteries, retrograde migration, embedding, and secretion of PAS-positive materials as well as IIphysiological changes," In pregnancy terminated with a certain type of spontaneous fetal resorption, defective endovascular trophoblast cell behavior was observed, which was similar to that reported in preeclamptic pregnancy. Finally, the roles of cytoskeleton on trophoblast cell locomotion were investigated in vivo with a cytoskeleton-disrupting agent, cytochalasin B. This treatment impaired trophoblast cell invasion at day 12 and induced smaller fetuses at day 20, suggesting the importance of cytoskeleton in trophoblast movement. In conclusion, the results suggest the importance of the use of appropriate specimens and endpoints in the study of pregnancy, and rats may serve as a suitable animal model for the study of endovascular trophoblast cell behavior with clinical relevance to preeclampsia.
- Genistein Induces Pancreatic beta-Cell Proliferation through Activation of Multiple Signaling Pathways and Prevents Insulin-Deficient Diabetes in MiceFu, Zhuo; Zhang, Wen; Zhen, Wei; Lum, Hazel; Nadler, Jerry; Bassaganya-Riera, Josep; Jia, Zhenquan; Wang, Yanwen; Misra, Hara P.; Liu, Dongmin (Endocrine Society, 2010-07)Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. However, studies on whether genistein has an effect on pancreatic beta-cell function are very limited. In the present study, we investigated the effect of genistein on beta-cell proliferation and cellular signaling related to this effect and further determined its antidiabetic potential in insulin-deficient diabetic mice. Genistein induced both INS1 and human islet beta-cell proliferation after 24 h of incubation, with 5 mu M genistein inducing a maximal 27% increase. The effect of genistein on beta-cell proliferation was neither dependent on estrogen receptors nor shared by 17 beta-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of protein kinase A (PKA) or ERK1/2 completely abolished genistein-stimulated beta-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for beta-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in streptozotocin-induced diabetic mice, concomitant with improved islet beta-cell proliferation, survival, and mass. These results demonstrate that genistein may be a natural antidiabetic agent by directly modulating pancreatic beta-cell function via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway. (Endocrinology 151: 3026-3037, 2010)
- Immune mechanisms in murine brucellosis: studies with strain RB51, a rough mutant of Brucella abortusBagchi, Tamishraha (Virginia Tech, 1990-05-05)The roles of humoral and cell mediated immune responses in murine brucellosis were investigated in this study.B. abortus strain 19, the current vaccine strain, is known to induce an antibody as well as cell mediated immune responses, both of which protect mice against smooth strain 2308. B. abortus rough strain RB51 does not induce an o-side chain specific antibody response and yet protects mice against smooth strain 2308. Passive transfer experiments using serum and nylon wool enriched T cells obtained from micevaccinated with strain 19 and strain RB51 were carried out. Immune senum from strain 19 vaccinated mice protected against challenge with strain 2308 but not strain RB51. Nylon wool enriched T cells from strain 19 vaccinated mice protected recipient mice against challenge with both strains RB51 or 2308. Serum obtained from RB51 vaccinated mice did not protect recipient mice against challenge with either strain RB51 or strain 2308. Nylon wool enriched T cells from the same vaccinated mice, however, protected mice against challenge with both strains. Thioglycollate elicited mouse peritoneal macrophages could be activated by recombinant gamma-interferon to kill ingested B. abortus. This was true for both the rough strain RB51 and smooth strain 2308, although RB51 exhibited greater susceptibility to killing. Macrophages already invaded by either strain RB51 or strain 2308 retained their responsiveness to gamma-interferon activation am could kill either strain of B. abortus following activation by gamma-interferon. Results obtained in this investigation indicate that strain RB51 protects mice against strain 2308 by probably inducing a cell mediated immune response.
- Immunotoxic and Oxidative Effects of Endosulfan and Permethrin on Murine SPlenocytes, in vitroVemireddi, Vimala (Virginia Tech, 2004-05-06)Indiscriminate use of pesticides appears to alter immune response in non-target organisms such as humans and other animals. Thus, immune modulation is considered as one of the potential risks and consequences following exposure to these chemicals. Because of the widespread usage, exposure to mixtures of pesticides during the lifetime of individuals is unavoidable and can result in potentiation of the toxic effects. Because immune cells are more susceptible to toxic insults at a lower dose than most other cell types, the effects of pesticides and their mixtures on murine splenocytes were evaluated. C57BL/6 male mouse splenocytes, in vitro, were exposed to permethrin and endosulfan, individually and in-combination (25-200 µM). The immunotoxic potential of these pesticides was monitored using a flow cytometric technique in combination with 7-Amino Actinomycin D (7-AAD) staining. Endosulfan exposures (25-150 µM) resulted in time- and dose-dependent increase in apoptotic and necrotic cell death in murine splenocytes, in vitro. Permethrin exposure (50-200 µM) resulted in neither a time-dependent/dose-dependent loss of splenocyte viability nor induction of apoptosis in splenocytes. With mixtures of permethrin and endosulfan, depressed viability and enhanced early apoptosis and late apoptosis/necrosis were observed. Exposure to mixtures of 50 µM endosulfan with 50 or 100 µM permethrin increased late apoptosis/necrosis compared to exposure to either chemical alone. DNA fragmentation, a hall mark of apoptosis was observed by DNA ladder technique, confirming the occurrence of apoptosis. Morphological observation using cytospun slides was also carried out to further confirm the presence of apoptosis and necrosis. These findings suggest that the immunotoxicity of endosulfan both individually and in mixtures with permethrin is associated with the occurrence of apoptotic and necrotic processes. Further, the ability of these pesticides to alter the oxidative status of the cells, via reactive oxygen species (ROS) generation and modulation of intracellular antioxidant enzymes levels, was investigated. We monitored the generation of ROS such as hydrogen peroxide (H₂O₂) with 2´, 7´- dichlorofluorescin diacetate (DCFH-DA) assay and superoxide anion (O₂⁻) with hydroethidine (HE) assay in combination with flow cytometry. Spectrophotometric techniques were used to measure antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPX). Results of the analyses revealed that individual pesticides increased the production of H₂O₂ in a time and dose-dependent manner. Both time and dose-dependent increases in O₂⁻ production were caused by permethrin; whereas endosulfan exposure resulted in only a dose-dependent increase. However, exposure to mixtures of these pesticides had little or no effect on the generation of H₂O₂ and O₂⁻ radicals as compared to individual pesticides. The levels of SOD and GPX in pesticide-treated splenocytes were found to be not different from solvent control. An increase in GR and CAT levels in cells was noticed with permethrin (100 µM) exposure. These findings suggest that permethrin and endosulfan have the ability to affect the cellular oxidative status and can cause toxicity in immune cells, in vitro.
- Immunotoxic Effects of Mixtures of Endosulfan and Permethrin Via Caspase Dependent Thymocyte ApoptosisKeenan, James John (Virginia Tech, 2003-03-21)Altered immune responses have been observed following occupational, inadvertent, or therapeutic exposure to xenobiotics. Many pesticides are known to cause immunotoxicity. Exposure to mixtures of pesticides, either concurrently or sequentially, may result in potentiating this effect partly because one can effect the metabolism of the other. The objective of this study was to determine the effect of the insecticides endosulfan, permethrin and their mixtures on C57/BL6 male mice thymocytes in vitro and to ascertain the mechanism by which these effects take place. Permethrin, a broad-spectrum synthetic pyrethroid, is a widely used insecticide in agriculture and public health. Endosulfan is a highly toxic chlorinated hydrocarbon insecticide used worldwide. We examined the immunotoxic potential of these pesticides using a flow cytometric technique in combination with 7-Amino Actinomycin D (7AAD) to distinguish live, early apoptotic, and late apoptotic/necrotic cells. DNA ladder assay, a hallmark of apoptosis, was also used to determine the occurrence of apoptosis. Both endosulfan and permethrin were found to cause significant apoptotic death of thymocytes in a dose- and time- dependent manner. Thus, permethrin at 50, 100 or 300 µM was found to cause 5.5, 11.5 and 26.1% increases in early apoptotic cell death relative to control, respectively. Endosulfan at 25, 50 or 250 µM was found to cause 11.9, 15.7 and 68.0% early apoptotic cell death, respectively. For the mixture study, concentrations of 100 µM permethrin and 50 µM endosulfan were selected and found to cause 27.1% apoptosis. Thus, these pesticides in mixture have an additive immunotoxic effect. Increases in late-apoptotic/necrotic cells were found at these concentrations for either pesticide when exposed for 12 hours. DNA ladder assay confirmed the presence of DNA fragments and therefore the presence of significant apoptotic cell death. Apoptosis is a morphologically distinct form of cell death that can be mediated by a variety of pathological and physiological stimuli. Because permethrin and endosulfan were found to induce apoptosis in C57/BL6 mice thymocytes in vitro, the objective of the second half of this study was to elucidate the potential mechanism by which these pesticides regulate apoptosis in immune cells. Caspases are a family of cystine-dependent, aspartate-directed proteases that have an integral role in apoptotic cell death. Caspases, which are normally inactive in healthy cells, are activated during apoptosis and form an irreversible cascade. There are two subsets of caspases, initiator caspases (i.e. caspase 8 and 9) and effector caspases (i.e. caspases 3 and 6). Caspase 3, a downstream effector of apoptosis, is activated by many different pathways and is an apoptotic marker in cells. Caspase 8 is the apical caspase in the extrinsic pathway. Caspase 9 is the apical caspase in the intrinsic pathway, therefore we investigated mechanisms of pesticide induced apoptosis involving the thymocyte caspase system. Thymocytes from C57/BL6 mice were incubated with varying concentrations of pesticides for varying amounts of time. Active caspase 3 was then measured using EnzCheck Caspase 3 Assay Kit. Relative fluorescence for permethrin exposed cells after 12 hours incubation in the presence of pesticides at 150, 100, and 50 µM and 40 minutes in the presence of AFC-substrate was found to be 387, 386, and 297, respectively. Relative fluorescence for endosulfan exposed cells at 150, 100 and 50 µM was 188, 177, and 294. Caspase 3 activity increased as permethrin concentrations increased and decreased as endosulfan concentrations were increased. Then the extrinsic and intrinsic pathways of apoptosis were further investigated. Active caspase 8 was measured using the ApoAlert Caspase Fluorescent Assay Kit. Relative fluorescence for permethrin exposed cells after 7 hours incubation in the presence of pesticides at 100, 150, and 200 µM was found to be 35.5, 10.5, and 0, respectively. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 32.8, 63.8, 69.5, and 55.5, respectively. A mixture study was then performed using endosulfan (50, 100, 150 µM) combined with permethrin (100 µM). All combinations were found to have more than an additive effect, therefore the extrinsic pathway seems to be involved. Caspase 9 activity was measured using Caspase 9/Mch6 Fluorometric Protease Assay Kit. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 43, 73, 78.9, and 5.12, respectively. Relative Fluorescence for permethrin exposed cells at 100, 150 and 200 µM was found to be 34.5, 39, and 55.5, respectively. A mixture study was then performed using endosulfan (25, 50 µM) combined with permethrin (100 µM). Both combinations were found to have less than an additive effect. These results suggest that apoptosis caused by both endosulfan and permethrin exert their effects via the caspase pathway. The results also show that mixtures of pesticides have a less than additive effect on caspase 9 activation and more than an additive effect on caspase 8 activation, therefore the extrinsic pathway is predominantly involved in thymocyte apoptosis caused by mixtures of permethrin and endosulfan.
- Immunotoxicity of Pesticide Mixtures and the Role of Oxidative StressOlgun, Selen (Virginia Tech, 1998-08-04)The immunotoxic effects of multiple pesticide exposure were evaluated. C57BL/6 mouse thymocytes were exposed to lindane, malathion, and permethrin, either separately or in mixtures of two pesticides, in concentrations ranging from 37.5 uM to 1mM. These exposures caused both apoptotic and necrotic cell death in thymocytes as evaluated by 7-aminoactinomycin-D, Annexin-V/PI, and lactate dehydrogenase release assays. When cells were exposed to lindane+malathion, or lindane+permethrin, a significantly greater-than-additive cytotoxicity was observed. The pesticide exposure caused DNA ladder formation with increased laddering in mixtures. Further, the effect of these pesticides on thymocyte oxidative stress was investigated. Thymocytes treated with any of these pesticides generated superoxide and H2O2. The lindane + malathion caused more-than-additive increase in superoxide production compared to single treatments of these pesticides. However, the effect of the lindane + permethrin was not significantly different from individual components of this mixture. The effects of pesticides on antioxidant enzymes were also investigated and only mixtures were found to have significant effects. Alteration in transcription factor NFkB level was measured as an indicator of oxidative stress in thymocytes following 12 h pesticide exposure, in vitro. Only lindane + malathion was found to increase the protein level. Furthermore, the effects of pesticides and their mixtures on immune functions of mice were studied in vivo. Animals (8-12 week old, male mice) were randomly divided into groups of six and injected intraperitoneally with three different doses (one-half, one-third, one-fourth, or one-eight of LD50) of individual pesticides. Exposure to individual pesticides did not alter the thymus/body or spleen/body weight ratios, thymic or splenic cell counts, or CD4/CD8 or CD45/CD90 ratios. However, anti-sRBC plaque forming cell (PFC) counts were significantly lowered with all treatments. Two other groups of animals were injected with lindane + malathion or lindane + permethrin at one-third of the LD50 of each pesticide. Exposure to pesticide mixtures did not alter the CD4/CD8 or CD45/CD90 ratios. However, the thymus/ and spleen/body weight ratios, thymic and splenic cell counts, and PFC counts were significantly lowered. These data indicate that lindane, malathion, and permethrin are immunotoxic and their mixtures can cause higher toxicity compared to individual exposures. In addition, these data support the hypothesis that oxidative stress were induced in thymocytes by exposure to these pesticides in vitro.
- Investigations of Insulin-Like Growth Factor I Cell Surface Binding: Regulation by Insulin-Like Growth Factor Binding Protein-3 and Heparan Sulfate ProteoglycanBalderson, Stephanie D. (Virginia Tech, 1997-05-22)The primary aim of this text is to gain insight on how cellular activation by a insulin-like growth factor (IGF-I), in the presence of insulin-like growth factor binding protein-3 (IGFBP-3), is influenced by heparan sulfate proteoglycans (HSPG). Initial research will be presented, assumptions and hypotheses that were included in the development of mathematical models will be discussed, and the future enhancements of the models will be explored. There are many potential scenarios for how each component might influence the others. Mathematical modeling techniques will highlight the contributions made by numerous extracellular parameters on IGF-I cell surface binding. Tentative assumptions can be applied to modeling techniques and predictions may aid in the direction of future experiments. Experimentally, it was found that IGFBP-3 inhibited IGF-I Bovine Aortic Endothelial (BAE) cell surface binding while p9 HS slightly increased IGF-I BAE cell surface binding. IGFBP-3 has a higher binding affinity for IGF-I (3 x 10-9 M) than p9 HS has for IGF-I (1.5 x 10-8 M) as determined with cell-free binding assays. The presence of p9 HS countered the inhibiting effect of IGFBP-3 on IGF-I BAE cell surface binding. Although preliminary experiments with labeled p9 HS and IGFBP-3 indicated little to no cell surface binding, later experiments indicated that both IGFBP-3 and p9 HS do bind to the BAE cell surface. Pre-incubation of BAE cells with either IGFBP-3 or p9 HS resulted in an increase of IGF-I BAE cell surface binding . There was a more substantial increase of IGF-I surface binding when cells were pre-incubated with IGFBP- 3 than p9 HS. There was a larger increase of IGF-I BAE cell surface binding when cells were pre-incubated with p9 HS than when p9 HS and IGF-I were added simultaneously. This suggests that IGFBP-3 and p9 HS surface binding plays key role in IGF-I surface binding, however, p9 HS surface binding does not alter IGF-I surface binding as much as IGFBP-3 surface binding seems to. Experimental work helps further the understanding of IGF-I cellular activation as regulated by IGFBP-3 and p9 HS. Developing mathematical models allows the researcher to focus on individual elements in a complex systems and gain insight on how the real system will respond to individual changes. Discrepancies between the model results and the experimental data presented indicate that soluble receptor inhibition is not sufficient to account for experimental results. The alliance of engineering analysis and molecular biology helps to clarify significant principles relevant to the conveyance of growth factors into tissue. Awareness of the effects of individual parameters in the delivery system, made possible with mathematical models, will provide guidance and save time in the design of future therapeutics involving growth factors.
- Mechanism of Action of Antipsychotics, Haloperidol and Olanzapine in vitroMahapatra, Vijaylaxmi (Virginia Tech, 2001-01-31)Schizophrenia affects 1-1.5% of people in the United States alone. Haloperidol (HP), a butyrophenone and a typical antipsychotic, has been used as an antipsychotic drug in human. Unfortunately, the therapeutic effects of HP also come with severe extrapyramidal side effects, resulting in movement disorders in patients. Olanzapine, a new atypical neuroleptic, seems to have better efficacy, with less severe adverse effects. There has been increasing evidence of the role of reactive oxygen species (ROS) and oxidative stress in the pathogenesis of Schizophrenia. We therefore hypothesized that the differences between HP and Olz could be partly because of the differences in the oxidative stress they cause. We studied the pro-oxidant and antioxidant effects of these two drugs in vitro and examined the mechanism of their cytotoxicity in a neuronal cell model using PC-12 cells. HP was found to be ineffective as a superoxide radical scavenger but appeared to be a potent scavenger of hydroxyl radicals with a rate constant of ~6.78 X 109 M-1s-1. Olz on the other hand was found to scavenge hydroxyl radical at a rate of 34.1 X 109 M-1s-1. This was shown using the hydroxyl radical dependent deoxyribose degradation assay and EPR spin trapping methods. HP was also found to quench singlet oxygen in a dose-dependent manner. HP was found to enhance the microsomal lipid peroxidation in a dose-dependent manner and at 10 µM it augmented the lipid peroxide accumulation by 100% whereas Olz, at the same concentrations had trivial effects. Light microscopy and two cytometric apoptotic/viability probes (7-aminoactinomycin D and Annexin-V) were employed to evaluate mechanisms of drug-induced cell death in PC-12 pheochromocytoma cells exposed to HP or Olz. At low dose (50 µM), HP was more cytotoxic than Olz. At high concentrations (150 mM) each of these antipsychotic drugs caused a significant increase in cell death that was readily detectable by all the techniques. Light microscopy with trypan blue staining indicated that necrosis was the predominate form of cell death with both drugs. Apoptotic cells were rarely observed by microscopy in vehicle or drug-exposed cells. Further, no increase in early cellular apoptosis was observed using the Annexin-V probe. 7AAD and Annexin-V both showed drug-related increases in the late apoptotic/necrotic cell death window. These data, along with the cytologic evaluations suggest that cell death in PC-12 pheochromocytoma cells exposed to HP or Olz may primarily be necrotic in nature, rather than apoptotic. Because Olz at a low dose was less cytotoxic and was found to have lower pro-oxidant action than HP the secondary effects manifested in patients with chronic treatment with HP may, at least in part, be attributed to the pro-oxidant effects of the drug.
- Mechanism of TNF-α cytotoxicity in a leukemia virus transformation modelMishra, Shrikant (Virginia Tech, 1991)Abelson murine leukemia virus (A-MuLV)-induced transformation was investigated to determine whether cells not sensitive to TNF-α could be made sensitive to the cytolytic action of TNF-α when infected with this retrovirus. Mouse embryonic fibroblast cell line CL.7 was found to be relatively insensitive to TNF-α. Upon transformation with A-MuLV, these cells gave rise to a clone (3R.1) which was found to be insensitive to TNF-α and another clone (6R.1) which had an increased sensitivity to TNF-α. The differential cytotoxicity was observed when cells were treated with TNF-α, for 18 hr, at 0 to 100 units/ml, at 37°C. The mechanism of this differential cytotoxicity was further investigated. Thus, TNF-R levels on the cell surface were found to be not correlated with the differential TNF-α response. The A-MuLV transformation suppressed the epidermal growth factor-receptor (EGF-R) in 3R.1 clone and induced its levels significantly in the 6R.1 clone (p<0.05). Cell surface EGF-receptor (EGF-R) levels in CL.7 and 3R.1 clones were lower than the 6R.1 clone (p<0.05). Although the EGF-R levels in all the clones were induced with TNF-α, the expression of EGF-R correlated with the susceptibility to TNF-α. The role of antioxidants, such as α-tocopherol and β-carotene, (known anti-cancer agents) in modulating TNF-α-induced EGF-R expression was investigated. In both the untransformed and the transformed clones, f-carotene suppressed the constitutive and the TNF-α induced EGF-R levels whereas α-tocopherol was found to have an enhancing effects. Studies with metabolic inhibitors on TNF-R and EGF-R expression indicate that inhibitors of the arachidonic acid cascade and modulators of protein kinase-C (PK-C), could influence the binding and internalization of TNF-α and thereby controlling the physiologic future of the cells. The A-MuLV specific V-abl protein, p120, tyrosine phosphorylation was determined by a radio-labelled anti-phosphotyrosine antibody in an antigen capture assay. TNF-α had little effect on p120 phosphotyrosine levels of TNF-α insensitive CL.7 and 3R.1 clones. The, TNF-α sensitive, 6R.1 clone, however, was found to induce its p120 specific phosphotyrosine upon exposure to TNF-α for 8 hr. Thus, TNF-α modulated the tyrosine phosphorylation of p120 only in the TNF-α-sensitive cell line. The mitochondrial toxicity of TNF-α was determined by monitoring the rate of quenching of a cationic spin probe CAT 16. Mitochondrial preparation from CL.7 and 3R.1 clones had higher ability to quench CAT 16 signal with TNF-α incubation time than mitochondria from the 6R.1 cells. This indicates that the differential TNF-α cytotoxicity manifested in A-MuLV transformed clones may, in part, be due to the differential mitochondrial toxicity of this cytokine. The hypothesis that TNF-α cytotoxicity was mediated via an oxidative process was tested on the TNF-α sensitive L929 cells. Using a flow cytometric detection system it was determined that TNF-α produced intracellular hydrogen peroxide in these cells which was sensitive to concentration and incubation time of TNF-α. Superoxide radicals were also generated during TNF-α action on L929 cells, as determined by the use of the spin trap PBN in conjunction with EPR spectroscopic techniques. The PBN-OOH spin adduct spectrum peaked at 9 hr of TNF-α incubation and was inhibitable upto 30 % with 10 µM of desferral-Mn complex (a known SOD mimic). These data indicate that superoxide and hydrogen peroxide are common events in TNF-α dependent cell killing process. The differential TNF-α cytotoxicity was found to depend on differences in the antioxidant status of the target clones. Thus, it was found that Cu/Zn-SOD, Mn-SOD, GSH-Peroxidase and GSH-Reductase enzymes were all induced significantly in the CL.7 clone (p<0.05) upon incubation with 100 units/ml of TNF-α for 18 hrs. TNF-α had little effect on the antioxidant enzymes of both 3R.1 and 6R.1 cells. However, the constitutive levels of most antioxidant enzymes were found to be higher in 3R.1 cells than in the 6R.1 cells. Therefore, the susceptibility of 6R.1 to TNF-α may, in part, be due to a low level of antioxidant enzymes present in this clone. In conclusion we found that the differential cytotoxicity of TNF-a may, in part, due to: (1) differential EGF-R expression, (2) differential mitochondrial cytotoxicity, and (3) differential ability to modulate the tyrosine phosphorylation in untransformed and A-MuLV transformed cells and (4) differential antioxidant status of these cells to handle oxidative stress imposed by TNF-α.
- Mechanisms by which hypoxia augments Leydig cell viability and differentiated cell function in vitroKukucka, Mark A. (Virginia Tech, 1993-04-18)The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture.
- Mechanisms of Cytotoxicity and Intracellular Trafficking for Gene Delivery PolymersGrandinetti, Giovanna (Virginia Tech, 2011-06-30)Herein, different polymer libraries were examined to determine the effect polymer structure has on intracellular events. The effect of different polyamine lengths in copolymers on cellular uptake, the effect of modifying end groups of trehalose-containing polymers on transfection efficiency, and the effect of different linker lengths between galactose and a hepatocyte-targeted polymer on transfection efficiency were studied. Furthermore, it was demonstrated that polymers with terbium chelated in their repeat units could potentially be used for Förster resonance energy transfer (FRET) studies to monitor pDNA release from the polymer. Much of the work in this dissertation focuses on elucidating the intracellular mechanisms of linear poly(ethylenimine) (PEI) and how it compares to poly(L-tartaramidopentaethylenetetramine) (T4) and poly(galactaramidopentaethylenetetramine) (G4), two poly(glycoamidoamine)s synthesized by our group. The long-term goal of this project is to develop structure-function relationships between polymers and pDNA delivery efficacy that will result in the rational design of safe, efficient vehicles for therapeutic nucleic acid delivery. Many polymers used as DNA delivery vehicles display high cytotoxicity. Often, the polymers with the highest transfection efficiency are the most toxic, as demonstrated herein by PEI and T4 with varying polymer lengths. Therefore, it was of interest to study how polymer structure influences mechanisms of cytotoxicity. To this end, studies on several mechanisms of cytotoxicity, including nuclear envelope permeabilization, were conducted. Longer polymers induced more cytotoxic responses than shorter ones, and it appears that hydroxyl groups in the repeat unit of polymers play a role in polyplex formation. This research has also led us to a potential link between transfection efficiency and cytotoxicity; the polymers with the highest transfection efficiency were also the most toxic, and were also able to induce the most nuclear envelope permeability. It is possible that these polymers' ability to permeabilize the nuclear envelope is what causes their high transfection efficiency and high toxicity. In addition, flow cytometry and confocal microscopy studies revealed that polymer structure plays a role in nuclear trafficking; poly(glycoamidoamine)s G4 and T4 more dependent on intracellular machinery than PEI. This research demonstrates the impact that changes in polymer structure have on intracellular mechanisms.
- Molecular basis of MPTP-induced Parkinson's diseaseZang, Lun-Yi (Virginia Tech, 1993)Self-administration of 1-methyl-4-pheny]-1,2,3,6-tetrahydropyridine (MPTP) has resulted in irreversible symptoms of Parkinson's disease in several young drug abusers. It was found that this neurotoxicant selectively destroys neuronal cells in the substantia nigra of humans and other primates. Although the mechanism of action of MPTP is not fully understood, it is now generally believed that the crucial species for MPTP neurotoxicity is not MPTP itself, but rather some of its metabolites. MPDP⁺, an intermediate in the metabolism of the neurotoxin MPTP, was found to generate superoxide radical (⋅O₂⁻) during its autoxidation process. The generation of ⋅O₂⁻ was detected by their ability to reduce ferricytochrome c. Superoxide dismutase (SOD) inhibited this reduction in a dosedependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP⁺, but also on the pH of the system. Thus, the rate of autoxidation of MPDP⁺ and the sensitivity of this autoxidation to superoxide dismutase inhibitable ferricytochrome c reduction were both augmented as the pH was raised from 7.0 to 10.5. The rate constant (kc) for the reaction of superoxide radical with ferricytochrome c to form ferrocytochrome c was found to be 3.48 x 10⁵ M⁻¹S⁻¹. The rate constant (kMPDP⁺) for the reaction of MPDP⁺ with ferricytochrome c was found to be 4.86 M⁻¹S⁻¹. The generation of ⋅O₂⁻ was further confirmed by spin-trapping in combination with EPR techniques using 5, 5-dimethyl-1-pyrrolonine-N-oxide (DMPO) as the spin trapping agent. The rate of formation of spin adduct (DMPO-O₂⁻) was dependent not only on the concentrations of MPDP⁺ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP⁺, and of SOD to effectively compete with this reaction for the available ⋅O₂⁻, was used as a convenient competition reaction to quantitatively determine various kinetic parameters. Using this technique, the rate constant for scavenging of superoxide radicals by superoxide dismutase was found to be 7.56 x 10⁹ M⁻¹S⁻¹. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP⁺ was found to be 4.48 x 10⁻¹⁰ M⋅S⁻¹. The rate constant (k₁) for MPDP⁺ making superoxide radical was found to be 3.97 x 10⁻⁶ Sec⁻¹. The second order rate constant (kDMPO) for DMPO trapping superoxide radicals was found to be 10.2 M⁻¹S⁻¹. The life time of superoxide radical at pH 10.0 was calculated to be 1.25 seconds. These data indicate that superoxide radicals are produced during spontaneous oxidation of MPDP⁺ and that EPR spin trapping techniques can be used to determine the rate constants and life time of free radicals generated in aqueous solution. Monoamine oxidase type B (MAO-B), an enzyme present in mitochondrial membranes, is known to metabolize MPTP to MPDP⁺, which then spontaneously oxidizes to MPP⁺. In the studies of MAO-B catalyzed oxidation of MPTP, the neurotoxicant was found to generate reactive oxygen species during its interaction with the enzyme. The kinetic parameters, Km and Vmax, for MAO-B catalyzed oxidation of MPTP to the corresponding species MPDP⁺ were found to be 0.194 mM and 0.335 µM/min, respectively. The generation of ⋅O₂⁻ and hydroxyl (⋅OH) radicals was detected as the DMPO spin adduct by spin trapping in combination with EPR techniques. Addition of Fe²⁺ (10 µM) to this system caused a 5-fold enhancement in EPR signal intensity of the DMPO-OH adduct. Catalase, a scavenger of hydrogen peroxide (H₂O₂), inhibited the DMPO-OH spin adduct formation in a dose-dependent manner, indicating that H₂O₂ is produced in the MAO-B catalyzed oxidation of MPTP. Ethanol, a well known scavenger of hydroxy] radical, rapidly produced an alpha-hydroxyethyl radical signal. SOD inhibited the formation of DMPO-O₂⁻ and DMPO-OH spin adducts in a dose-dependent fashion. These data suggest that ⋅O₂⁻ are produced during the oxidation of MPTP by MAO-B and that the generation of H₂O₂ and ⋅OH was secondary to the production of ⋅O₂⁻. MPTP and its metabolites, MPDP⁺ and MPP⁺, were found to inhibit the activity of acetylcholinesterase (AChE). The kinetic parameter, Km for the substrate (acetylthiocholine), was found to be 0.216 mM and Ki values for MPTP, MPDP⁺ and MPP⁺ to inactivate AChE were found to be 2.14, 0.265 and 0.197 mM, respectively. The inactivation of AChE by these neurotoxicants was found to be dose-dependent. It was found that MPTP, MPDP⁺ and MPP⁺ are neither substrates of AChE nor the time-dependent inactivators. The studies of reaction kinetics indicate that the inactivation of ACHE by these inactivators is via a mixed-type inhibition. The dilution of the enzyme-inhibitor complex completely reversed the MPTP inhibition but only partially reversed the MPDP+ and MPP+ inhibition. These data indicate that MPTP and its metabolites can inactivate AChE and thereby increase ACh level in the basal ganglia of the brain, leading to potential cell dysfunction. These results suggest that once MPTP enters the basal ganglia of the brain, it can be catalyzed by MAO-B to generate a series of reactive species, including ⋅O₂⁻, H₂O₂ and ⋅OH, which are known to destroy cell membranes, enzymes and other important biological molecules. The nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may largely be due to the reactivity of these reactive oxygen species in combination with the inactivation of the AChE enzyme in the brain, leading to potential cell dysfunction.