Simultaneous characterization of multiple Psoraleae Fructus bioactive compounds in rat plasma by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry for application in sex-related differences in pharmacokinetics.

A method for the simultaneous quantification of 13 bioactive compounds (psoralen, isopsoralen, isobavachin, bakuchalcone, neobabaisoflavone, bavachin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone, and bakuchiol) by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry has been developed and validated in rat plasma. Osthol was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. These analytes were separated using a gradient mobile phase system of water and acetonitrile at a flow rate of 0.2 mL/min on a reverse-phase C18 column and analyzed in the selected multiple reactions monitoring mode. All calibration curves were linear (r > 0.9952) over the tested ranges. The intra- and interday accuracy and precisions of these analytes at three different concentration levels were within the acceptable limits of <15% at all concentrations. The mean recoveries of these analytes at three concentrations were more than 60.2% and the matrix effects were in the range of 85-115%. Stability studies proved that the analytes were stable under the tested conditions. The developed method was applied to evaluating the pharmacokinetic study of 13 bioactive compounds after oral administration of Psoraleae Fructus in rat of different genders. Some active compounds in Psoraleae Fructus had sex-related pharmacokinetics.

Unique interactions between scorpion toxins and small conductance Ca(2+)-activated potassium channels.

Small conductance Ca(2+)-activated potassium channels (SK channels) distributing in the nervous system play an important role in learning, memory and synaptic plasticity. Most pharmacological properties of them are determined by short-chain scorpion toxins. Different from most voltage-gated potassium channels and large-conductance Ca(2+)-activated potassium channels, SK channels are only inhibited by a small quantity of scorpion toxins. Recently, a novel peptide screener in the extracellular pore entryway of SK channels was considered as the structural basis of toxin selective recognition. In this review, we summarized the unique interactions between scorpion toxins and SK channels, which is crucial not only in deep-researching for physiological function of SK channels, but also in developing drugs for SK channel-related diseases.

COH-203, a novel microtubule inhibitor, exhibits potent anti-tumor activity via p53-dependent senescence in hepatocellular carcinoma.

5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one (COH-203) is a novel synthesized analogue of combretastatin A-4 that can be classified as a microtubule inhibitor. In this study, we evaluated the anti-hepatoma effect of COH-203 in vitro and in vivo and explored the underlying molecular mechanisms. COH-203 was shown to be more effective in inhibiting the proliferation of liver cancer cells compared with normal liver cells. COH-203 also displayed potent anti-tumor activity in a hepatocellular carcinoma xenograft model without significant toxicity. Mechanistic studies demonstrated that treatment with COH-203 induced mitotic arrest by inhibiting tubulin polymerization in BEL-7402 liver cancer cells. Long-term COH-203 treatment in BEL-7402 cells led to mitotic slippage followed by senescence via the p14(Arf)-p53-p21 and p16(INK4α)-Rb pathways. Furthermore, suppression of p53 via pifithrin-α (p53 inhibitor) and p53-siRNA attenuated COH-203-induced senescence in BEL-7402 cells, suggesting that COH-203 induced senescence p53-dependently. In conclusion, we report for the first time that COH-203, one compound in the combretastatin family, promotes anti-proliferative activity through the induction of p-53 dependent senescence. Our findings will provide a molecular rationale for the development of COH-203 as a promising anti-tumor agent.

Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

Selective inhibition of CCR7(-) effector memory T cell activation by a novel peptide targeting Kv1.3 channel in a rat experimental autoimmune encephalomyelitis model.

The voltage-gated Kv1.3 K(+) channel in effector memory T cells serves as a new therapeutic target for multiple sclerosis. In our previous studies, the novel peptide ADWX-1 was designed and synthesized as a specific Kv1.3 blocker. However, it is unclear if and how ADWX-1 alleviates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. In this study, the administration of ADWX-1 significantly ameliorated the rat experimental autoimmune encephalomyelitis model by selectively inhibiting CD4(+)CCR7(-) phenotype effector memory T cell activation. In contrast, the Kv1.3-specific peptide had little effect on CD4(+)CCR7(+) cells, thereby limiting side effects. Furthermore, we determined that ADWX-1 is involved in the regulation of NF-κB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2 pathway of CD4(+)CCR7(-) cells. The elevated expression of Kv1.3 mRNA and protein in activated CD4(+)CCR7(-) cells was reduced by ADWX-1 engagement; however, an apparent alteration in CD4(+)CCR7(+) cells was not observed. Moreover, the selective regulation of the Kv1.3 channel gene expression pattern by ADWX-1 provided a further and sustained inhibition of the CD4(+)CCR7(-) phenotype, which depends on the activity of Kv1.3 to modulate its activation signal. In addition, ADWX-1 mediated the activation of differentiated Th17 cells through the CCR7(-) phenotype. The efficacy of ADWX-1 is supported by multiple functions, which are based on a Kv1.3(high) CD4(+)CCR7(-) T cell selectivity through two different pathways, including the classic channel activity-associated IL-2 pathway and the new Kv1.3 channel gene expression pathway.

Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family.

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 µM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

[Primary co-culture of cortical neurons and astrocytes of new-born SD rats].

This study is to establish a simple and practical co-culture method of cortical neurons and astrocytes of rats. The cortex of the new-born SD rats was digested by 0.125% pancreatic enzyme, and the differential adherence was applied to obtain the mixed cell suspension of neurons and astrocytes. A low concentration of cytarabine was used to inhibit the astrocytes in a moderate way to get neuronal and astrocyte co-culture. The morphological characteristics of the cells in different times were observed under the inverted microscope. The cells began to adhere the wall 2 h after the inoculation. Neurons and astrocytes grew in a good condition under the inverted microscope 9 days after the inoculation. The results of the immunofluorescence staining and Rosenfeld's staining indicated that the co-culture of neurons and astrocytes was successful and the ratio of neurons and astrocytes was close to 1:1. A new neurons and astrocytes co-culture method, which is simple and convenient, was successfully established. It will be an efficient method for the related researches about neuronal and astrocyte co-culture in vitro.

Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells.

AIM: To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells. METHODS: The cell viability was measured using MTT assay. Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining, respectively. The mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye rhodamine 123. DCF-induced fluorescence was used to measure the intracellular ROS level. Protein expression was examined using Western blot. RESULTS: Treatment of HeLa cells with oridonin (20-160 µmol/L) inhibited the cell growth in time- and concentration-dependent manners. The cells treated with oridonin (80 µmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes. In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L), the oridonin-induced apoptosis was significantly enhanced. Treatment of HeLa cells with oridonin (20-120 µmol/L) induced intracellular ROS generation in a concentration-dependent manner. In the presence of the ROS scavenger NAC (5 mmol/L), the oridinin-induced ROS generation was markedly reduced. NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis. Furthermore, oridonin significantly reduced ΔΨm, which was blocked by NAC. Oridonin markedly increased Bax expression in mitochondria, and decreased Bcl-2 expression in both the cytosol and mitochondria. Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol. All the effects were blocked by NAC. Oridonin increased the levels of caspase-3 and caspase-8, and decreased the expression of pro-caspase 3 and pro-caspase 9, which were blocked by NAC. CONCLUSION: ROS plays a critical role in oridonin-induced apoptosis and autophagy.

Effects of acute and chronic administration of MK-801 on c-Fos protein expression in mice brain regions implicated in schizophrenia with or without clozapine.

This study investigated the effects of acute and chronic administration of the non-competitive NMDA receptor antagonists MK-801 on c-Fos protein expression in different brain regions of mice with or without clozapine. MK-801 (0.6 mg/kg) acute administration produced a significant increase in the expression of c-Fos protein in the layers III-IV of posterior cingulate and retrosplenial (PC/RS) cortex, which was consistent with the previous reports. Moreover, we presented a new finding that MK-801 (0.6 mg/kg) chronic administration for 8 days produced a significant increase of c-Fos protein expression in the PC/RS cortex, prefrontal cortex (PFC) and hypothalamus of mice. Among that, c-Fos protein expression in the PC/RS cortex of mice was most significant. Compared to acute administration, we found that MK-801 chronic administration significantly increased the expression of c-Fos protein in the PC/RS cortex, PFC and hypothalamus. Furthermore, pretreatment of mice with clozapine significantly decreased the expression of c-Fos protein induced by MK-801 acute and chronic administration. These results suggest that c-Fos protein, the marker of neuronal activation, might play an important role in the chronic pathophysiological process of schizophrenic model induced by NMDA receptor antagonist.

Chlorpyrifos induces delayed cytotoxicity after withdrawal in primary hippocampal neurons through extracellular signal-regulated kinase inhibition.

In this study, the delayed effect and related mechanism after chlorpyrifos (CPF) withdrawal was studied in primary rat hippocampal neurons. The results showed that 10 muM CPF induced no detectable cytotoxicity during 96 h continuous exposure while its withdrawal after 48 h exposure induced evident cytotoxicity, as indexed by decreased methyl thiazolyl tetrazolium (MTT) metabolism, increased loss of neurons immunostained by neuron-specific enolase (NSE) antibody, and the increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cell rate in the following 24 h and 48 h incubation in the absence of CPF. Extracellular signal-related kinase (ERK)1/2 activation by phosphorylation was observed and persisted during CPF exposure. However, CPF withdrawal after 48 h exposure led to inhibition of ERK1/2 phosphorylation. Carbacol and nerve growth factor (NGF), which are ERK1/2 activators, protected the neurons after CPF withdrawal, while atropine and PD98059, which are ERK1/2 inhibitors, exacerbated the cytotoxicity, indicating the involvement of inhibition of ERK1/2 phosphorylation in CPF-induced delayed cytotoxicity. In conclusion, CPF withdrawal after exposure induced delayed cytotoxicity in cultured neurons. Inhibition of ERK1/2 phosphorylation was found to be related to the delayed cytotoxicity. This finding may provide a new insight into the toxicological mechanism of organophosphorus pesticides, especially chronic organophosphate-induced neuropsychiatric disorder characterized by delayed occurrence.

Chronic organophosphate (OP)-induced neuropsychiatric disorder is a withdrawal syndrome.

Chronic organophosphate-induced neuropsychiatric disorder is a less well-characterized syndrome, which is usually delay-occurred, persists long and is similar to the symptom of cholinergic deficit, its mechanism is unclear. The characteristics of chronic organophosphate-induced neuropsychiatric disorder are somewhat opposite to the direct action of OP pesticide, since withdrawal effect is usually opposite to the original effect of a drug, hypothesis that chronic organophosphate-induced neuropsychiatric disorder is a kind of withdrawal syndrome is suggested.

Synergistic antitumor effects of anthracenylmethyl homospermidine and alpha-difluoromethylornithine on promyelocytic leukemia HL60 cells.

The present study was designed to assess the synergistic antitumor effects of anthracenylmethyl homospermidine (ANTMHspd), a novel polyamine conjugate, with alpha-difluoromethylornithine (DFMO) and to elucidate the mechanism of these effects on human leukemia HL60 cells. Cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis and mitochondria membrane potential (MMP) were evaluated by flow cytometry. Caspases and cytochrome c were detected by Western Blot analysis. The combination treatment strongly inhibited cell proliferation, induced cell apoptosis and caused an accumulation in the G1 phase with an accompaniment decrease in S phase. Moreover, reduction of MMP, release of cytochrome c and activation of caspase-3 and caspase-9 but not caspase-8 were observed during the combination-mediated apoptosis. All these findings demonstrated that the combination treatment with DFMO and ANTMHspd resulted in synergistic antitumor effects on HL60 cells.

[Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism].

OBJECTIVE: To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism. METHODS: Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot. RESULTS: NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner. CONCLUSION: NNAMB induces apoptosis via mitochondrial pathway in K562 cells.

[Process of HIV-1 reverse transcription and its detection by using PCR].

Human immunodeficiency virus (HIV) is a retrovirus, belongs to Lentiviridae family. As long as viral genetic material entering into host cytoplasm, double-strand DNAs synthesis occurs which is catalyzed by reverse transcriptase (RT) with viral plus-strand RNA as template. This reverse transcription is a key link of HIV-1 life cycle and an important target for anti-HIV drug development. The process of reverse transcription can be divided into several steps: formation of minus-strand strong-stop DNA; the first translocation; initiation of plus-strand DNA synthesis; and, the second translocation and the completion of both strands. These steps can be detected individually by using polymerase chain reaction (PCR) according to the amplified products on the region of R/U5, U3, U5/PBS and the sequence between LTR and Gag. In this review, we summarize the principle for detecting stages of HIV-1 reverse transcription by using PCR.

VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

The majority of molecular targets of anticancer agents are limited to a subset of patients, and therefore identification of more specific biomarkers that can be used to improve clinical outcomes is of increasing interest. The present study showed that von Hippel­Lindau tumor suppressor (VHL) tumor­suppressor activity may influence the therapeutic response to Aurora kinase A (AURKA) inhibitors in human renal cell carcinoma (RCC). VHL protein (pVHL) expression was evaluated by immunoblotting in the human RCC cell lines CAKI, ACHN, 786­O, 769­P and A498. The anti­tumor activities of alisertib, an AURKA­specific chemical inhibitor, were detected by Cell Counting Kit­8 assay in vitro and mouse xenograft model in vivo. Additionally, the VHL­mediated anti­tumor activity was assessed in 769­P and CAKI cells via the loss or gain of VHL. The results revealed that VHL­deficient 786­O, 769­P and A498 cells were sensitive to alisertib. By contrast, alisertib­resistant CAKI and ACHN cells expressed the wild type VHL gene. In addition, rescue or knockdown of VHL was observed to increase or decrease alisertib anti­proliferation activity, respectively, in RCC cells. The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenografts models. Taken together, these results suggested that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors.

Never-in-mitosis A-related kinase 8, a novel target of von-Hippel-Lindau tumor suppressor protein, promotes gastric cancer cell proliferation.

Previous research has revealed that the von-Hippel-Lindau tumor suppressor protein (pVHL) may downregulate never-in-mitosis A-related kinase 8 (NEK8) via hypoxia-inducible factor-α (HIF-α). The HIF-independent functions of pVHL also serve an important role in its tumor-suppressor action. In the present study, the association between pVHL and NEK8 was demonstrated in the human gastric cancer cell line, SGC-7901, indicating a direct interaction of pVHL with NEK8. Subsequently, it was reported that MG-132, a specific proteasome inhibitor, may attenuate pVHL overexpression-induced reductions in NEK8 protein expression levels. In addition, the present study revealed that pVHL may stimulate the rapid degradation of NEK8 protein and promote its ubiquitination. The association between the expression profile of NEK8 and the survival status of patients with gastric cancer was analyzed from an online database. Kaplan-Meier survival plots indicated that higher expression levels of NEK8 may lead to poor survival, as suggested by the transcriptomic data of 1,065 patients with gastric cancer. It was found that NEK8-knockdown mediated by RNA interference inhibited SGC-7901 and SNU-1 proliferation, colony formation and migration in vitro, and tumor growth in vivo. Collectively, the present study proposed that NEK8 may be a novel target of pVHL as a ubiquitin E3 ligase, and may serve a role as a potential oncoprotein in human gastric cancer.

Molecular cloning and electrophysiological studies on the first K(+) channel toxin (LmKTx8) derived from scorpion Lychas mucronatus.

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.

Effect of MK-801 and ketamine on hydroxyl radical generation in the posterior cingulate and retrosplenial cortex of free-moving mice, as determined by in vivo microdialysis.

This study investigated the effect of MK-801 and ketamine, N-methyl-D-aspartate (NMDA) receptor antagonists which can induce schizophrenic symptoms and have neurotoxicity in human and animals, on hydroxyl radical (*OH) generation in the posterior cingulate and retrosplenial (PC/RS) cortex of free-moving mice using the salicylic acid trapping technique. MK-801 (0.6 mg/kg) or ketamine (50 mg/kg) acute administration significantly increased *OH levels in mouse PC/RS cortex. The basal *OH levels after MK-801 and ketamine administrations for 7 consecutive days were significantly increased compared with the naive basal levels. MK-801 (0.6 mg/kg) or ketamine (50 mg/kg) challenge after chronic administration further significantly increased dialysate levels of *OH. Our study also found that the release of *OH was secondary to stereotyped behavior, and the intensity of stereotyped behavior induced by MK-801 was more than that induced by ketamine. The results suggested that NMDA receptor antagonists participate in the generation of *OH in the PC/RS cortex of mouse, and oxidative stress, derived from the formation of free radicals, might play an important role in the pathophysiology of these two models of schizophrenia.

Effect of acute and chronic MK-801 administration on extracellular glutamate and ascorbic acid release in the prefrontal cortex of freely moving mice on line with open-field behavior.

The present study was designed to investigate the effects of acute and chronic administration of MK-801 (0.6 mg/kg), a noncompetitive NMDA-receptor antagonist on extracellular glutamate (Glu) and ascorbic acid (AA) release in the prefrontal cortex (PFC) of freely moving mice using in vivo microdialysis with open-field behavior. In line with earlier studies, acute administration of MK-801 induced an increase of Glu in the PFC. We also observed single MK-801 treatment increased AA release in the PFC. In addition, our results indicated that the basal AA levels in the PFC after MK-801 administration for 7 consecutive days were significantly decreased, and basal Glu levels also had a decreased tendency. After chronic administration (0.6 mg/kg, 7 days), MK-801 (0.6 mg/kg) challenge significantly decreased dialysate levels of AA and Glu. Our study also found that both acute and chronic administration of MK-801 induced hyperactivity in mice, but the intensity of acute administration was more than that of chronic administration. Furthermore, in all acute treatment mice, individual changes in Glu dialysate concentrations and the numbers of locomotion were positively correlated. In conclusion, this study may provide new evidence that a single MK-801 administration induces increases of dialysate AA and Glu concentrations in the PFC of freely moving mice, which are opposite to those induced by repeated MK-801 administration, with an unknown mechanism. Our results suggested that redox-response might play an important role in the model of schizophrenic symptoms induced by MK-801.

Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice.

BACKGROUND: Paraquat (PQ; 1, 1'-dimethyl-4, 4'-bipyridinium), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPTP (1-methyl-1, 2, 3, 6-tetrahydropyridine), has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). Aging is an accepted risk factor for idiopathic Parkinson's disease. The aim of this study was to test the hypothesis that paraquat could induce PD-like nigrostriatal dopaminergic degeneration in aging C57BL/6 mice. METHODS: Senile male C57BL/6 mice were intraperitoneally injected with either saline or PQ at 2-day intervals for a total of 10 doses. Locomotor activity and performance on the pole test were measured 7 days after the last injection and animals were sacrificed one day later. Level of dopamine (DA) and its metabolites levels in the striatum were measured by high-performance liquid chromatography with an electrochemical detector (HPLC-ECD), and numbers of tyrosine hydroxylase (TH) positive neurons were estimated using immunohistochemistry. RESULTS: Locomotor activities were significantly decreased and the behavioral performance on the pole test were significantly impaired in the PQ treated group. Level of DA and its metabolites levels in the striatum were declined by 8 days after the last injection. Immunohistochemical analyses showed that PQ was associated with a reduction in numbers of tyrosine hydroxylase positive neurons. CONCLUSIONS: Long-term repeated exposes to PQ can selectively impair the nigrostriatal dopaminergic system of senile mice, suggesting that PQ could play an important role in the pathogenesis of Parkinson's disease (PD). Our results also validate a novel model of PD induced by exposure to a toxic environmental agent.