Assessing the effect of ß-glucan diets on innate immune response of tilapia macrophages against trichlorfon exposure: an in vitro study.

The widespread use of pesticides in some areas where fish species such as tilapia are farmed may cause damage to the environment and affect commercial fish and therefore, human health. Water leaching with the pesticide trichlorfon, during the fumigation season in the field, can affect water quality in fish farms and consequently affect fish health. At the same time, the use of immunomodulatory compounds such as ß-glucan supplied in the diet has become widespread in fish farms as it has been shown that improves the overall immune response. The present research examines the immunomodulatory impacts observed in macrophages of Nile tilapia (Oreochromis niloticus) after being fed a diet supplemented with ß-glucan for 15 days, followed by their in vitro exposure to trichlorfon, an organophosphate pesticide, at concentrations of 100 and 500 µg mL-1 for 24 h. The results showed that ß-glucan diet improved the viability of cells exposed to trichlorfon and their antioxidant capacity. However, ß-glucan did not counteract the effects of the pesticide as for the ability to protect against bacterial infection. From the present results, it can be concluded that ß-glucan feeding exerted a protective role against oxidative damage in cells, but it was not enough to reduce the deleterious effects of trichlorfon on the microbicidal capacity of macrophages exposed to this pesticide.

Dynamics of dominant rhizospheric microbial communities responsible for trichlorfon absorption and translocation in maize seedlings.

In this study, the available phosphorus (AP) and TCF concentrations in soils and maize (Zea mays) seedling tissues were measured in response to escalating TCF concentrations during 216 hr of culture. Maize seedlings growth considerably enhanced soil TCF degradation, reaching the highest of 73.2% and 87.4% at 216 hr in 50 and 200 mg/kg TCF treatments, respectively, and increased AP contents in all the seedling tissues. Soil TCF was majorly accumulated in seedling roots, reaching maximum concentration of 0.017 and 0.076 mg/kg in TCF-50 and TCF-200, respectively. The hydrophilicity of TCF might hinder its translocation to the aboveground shoot and leaf. Using bacterial 16 S rRNA gene sequencing, we found that TCF addition drastically lessened bacterial community interactions and hindered the complexity of their biotic networks in rhizosphere than in bulk soils, leading to the homogeneity of bacterial communities that were resistant or prone to TCF biodegradation. Mantel test and redundancy analysis suggested a significant enrichment of dominant species Massilia belonging to Proteobacteria phyla, which in turn affecting TCF translocation and accumulation in maize seedling tissues. This study provided new insight into the biogeochemical fate of TCF in maize seedling and the responsible rhizobacterial community in soil TCF absorption and translocation.

Colorimetric assay based on peroxidase-like activity of dodecyl trimethylammonium bromide-tetramethyl zinc (4-pyridinyl) porphyrin for detection of organophosphorus pesticides.

A simple and sensitive colorimetric assay for detecting organophosphorus pesticides (OPs) was developed based on 3,3',5,5'-tetramethylbenzidine (TMB)/hydrogen peroxide (H2O2)/dodecyl trimethylammonium bromide (DTAB)-tetramethyl zinc (4-pyridinyl) porphyrin (ZnTPyP). In this system, based on the peroxidase-like activity of DTAB-ZnTPyP, H2O2 decomposes to produce hydroxyl radicals, which oxidize TMB, resulting in blue oxidation products. The OPs (trichlorfon, dichlorvos, and thimet) were first combined with DTAB-ZnTPyP through electrostatic interactions. The OPs caused a decrease in the peroxidase-like activity of DTAB-ZnTPyP due to spatial site blocking. At the same time, π-interactions occurred between them, and these interactions also inhibited the oxidation of TMB (652 nm), thus making the detection of OPs possible. The limits of detection for trichlorfon, dichlorvos, and thimet were 0.25, 1.02, and 0.66 µg/L, respectively, and the corresponding linear ranges were 1-35, 5-45, and 1-40 µg/L, respectively. Moreover, the assay was successfully used to determine OPs in cabbage, apple, soil, and traditional Chinese medicine samples (the recovery ratios were 91.8-109.8%), showing a great promising potential for detecting OPs also in other complex samples.

Correlation of bacterial community with phosphorus fraction drives discovery of Actinobacteria involved soil phosphorus transformation during the trichlorfon degradation.

Trichlorfon (TCF) is a broad-spectrum phosphorus (P)-containing pesticide, yet its effects on soil P fraction transformation and bacterial communities during the TCF degradation in soils is unknown. In this study, we investigated soil TCF degradation behavior at different contents of 50, 100 and 200 mg/kg, and analyzed residual TCF contents and metabolites by gas chromatography mass spectrometry after 216-h incubation. Our results suggested that TCF was gradually degraded in soils and was be initially hydrolyzed to dichlorvos via P-C bond cleavage and then other P-containing metabolites. By analyzing different P fractions and soil microbial community composition, we found significant increases of soil available phosphorus contents from 2.76 mg/kg (control) to 3.23 mg/kg (TCF-50), 5.12 mg/kg (TCF-100) and 5.72 mg/kg (TCF-200), respectively. Inorganic CaCl2-P was easily and instantly transformed to primary mineral inorganic P (Pi) forms of HCl-P and citrate-P, while the proportion of enzyme-P (a labile organic P) fluctuated throughout TCF degradation process. Soil available P contents and Pi fractions were significantly correlated with the relative abundance of Actinobacteria. These results highlighted that Actinobacteria is the dominant soil species utilizing TCF as P sources to increase its community richness, and subsequently affect the transformation of P fractions to regulate soil P cycle. Our study gives new understanding on the microorganisms can involve soil P transformation during organophosphorus pesticides degradation in soils, highlighting the importance of bacteria in P transformation and pesticides soil decontamination.

Protective role of rutin dietary supplementation mediated by purinergic signaling in spleen of silver catfish Rhamdia quelen exposed to organophosphate pesticide trichlorfon.

Evidence suggests the involvement of purinergic signaling, a mechanism mediated by extracellular nucleotides and nucleosides, with the impairment of immune and inflammatory responses in silver catfish (Rhamdia quelen) exposed to trichlorfon. Plant-derived substances have been considered potent anti-inflammatory agents due to effects on the purinergic system, such as the use of the flavonoid rutin. The aim of this study was to determine whether a diet containing rutin is able to prevent or reduce trichlorfon-induced impairment of immune responses through alteration of the purinergic pathway. Spleen adenosine triphosphate (ATP) levels were significantly higher in silver catfish exposed to 11 mg/L trichlorfon for 48 h compared to the control group, while adenosine (Ado) levels were significantly lower. Spleen ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity was significantly lower in silver catfish exposed to trichlorfon compared to control group, while adenosine deaminase activity was significantly higher. Spleen metabolites of nitric oxide, interleukin-1, and IL-6 were significantly higher in silver catfish exposed to trichlorfon compared to control group. Diet with 3 mg rutin/kg diet was able to prevent all the alterations elicited by trichlorfon, except restoring spleen ATP levels. The purinergic exposure signaling is involved in impairment of immune and inflammatory responses in fish exposed to trichlorfon due to reduction in ATP hydrolyses and by an increase in Ado deamination, leading to release of pro-inflammatory mediators. Use of rutin-added diet exerted an essential role in protecting the silver catfish spleen from trichlorfon-induced impairment on immune and inflammatory responses, preventing all alterations on splenic purinergic signaling.

Protective effects of diet containing rutin against trichlorfon-induced muscle bioenergetics disruption and impairment on fatty acid profile of silver catfish Rhamdia quelen.

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage.

Integrated biomarker parameters response to the toxic effects of high stocking density, CuSO4, and trichlorfon on fish and protective role mediated by Angelica sinensis extract.

The present study explored the protective role of dietary the extract of Angelica sinensis (EAs) on high density, CuSO4, or trichlorfon-treated Crucian carp (Carassius auratus auratus). Firstly, the study showed that the optimum density for growth and growth inhibition was 0.49 and 0.98 fish L-1 water, respectively. Dietary EAs relieved the high density-induced growth inhibition in Crucian carp. The appropriate concentration of EAs for recovery of growth was estimated to be 4.30 g kg-1 diet in high-density fish. Moreover, high density decreased both digestive and absorptive enzyme activities and increased lipid oxidation in digestive organs, suggesting the ability of high density to induce oxidative damage. However, dietary EAs inhibited the oxidative damage through elevating ROS scavenging ability and enzymatic antioxidant activity in digestive organs. Secondly, our data demonstrated that the appropriate concentration of CuSO4 to induce the decrease in feed intake (FI) was 0.8 mg Cu L-1 water. Dietary EAs returned to FI of Crucian carp treated with CuSO4. The appropriate concentration of EAs for recovery of FI was estimated to be 4.25 g kg-1 diet. Moreover, dietary EAs suppressed the CuSO4-induced decrease in digestion and absorption capacity and increase in protein metabolism in digestive organs of Crucian carp. Finally, the present results suggested that dietary EAs inhibited the trichlorfon-induced rollover (loss of equilibrium) in Crucian carp. The appropriate concentration of EAs for inhibition of rollover was estimated to be 4.18 g kg-1 diet. Moreover, trichlorfon stimulated not only the decrease in energy metabolism but also lipid and protein oxidation, suggesting that trichlorfon caused loss of function and oxidative damage in muscles of fish. However, dietary EAs improved muscular function and inhibited oxidative damage via quenching ROS and elevating non-enzymatic and enzymatic antioxidant activity in muscles of trichlorfon-induced fish. So, EAs could be used as an inhibitor of high density, CuSO4, and trichlorfon stress in fish.

Fluorescence assay for three organophosphorus pesticides in agricultural products based on Magnetic-Assisted fluorescence labeling aptamer probe.

There has been increasing recent concern about the agricultural use of organophosphorus pesticides. A rapid and sensitive fluorescence assay for the detection of three organophosphorus pesticides has therefore been developed using 6-carboxy-fluorescein labeling aptamer as the probe and functionalized magnetic nanoparticles as the separation carrier. The aptamer hybridized with complementary DNA conjugated on the surface of the magnetic nanoparticles to form a magnetic aptamer-complementary DNA complex. Upon introducing the target organophosphorus pesticide, the aptamer departed from the complementary DNA, resulting in the fluorescence signal. Under optimized conditions, the limits of detection (LODs, S/N = 3) for trichlorfon, glyphosate, and malathion were 72.20 ng L-1, 88.80 ng L-1, and 195.37 ng L-1, respectively. The method was applied for the detection of trichlorfon, glyphosate, and malathion in spiked lettuce and carrot samples. The recoveries were in the range of 79.4%-118.7%, which were in good agreement with those obtained by gas chromatography, and the relative standard deviations were also acceptable. The method therefore has high sensitivity, so provides a means for the detection of multiple organophosphorus pesticides.

A study on biomimetic immunoassay-capillary electrophoresis method based on molecularly imprinted polymer for determination of trace trichlorfon residue in vegetables.

Pesticide residue in vegetables is a serious problem that has adverse effects on human health. In our study, we designed and synthesized a molecularly imprinted polymer that can selectively recognize trichlorfon. Using the polymer material as biomimetic antibody, we developed a biomimetic immunoassay-capillary electrophoresis method with improved sensitivity for the detection of trichlorfon. We evaluated the competitive reactions between HRP labeled trichlorfon hapten and free trichlorfon with the biomimetic antibody. Factors that affected the sensitivity of our method were tested in detail. Under optimal conditions, the limit of detection (LOD, IC15) and the sensitivity (IC50) of this method were 0.16mgL-1 and 0.13µgL-1 for trichlorfon. We used this method to determine the trichlorfon spiked in the kidney bean and cucumber samples with recoveries ranging from 78.8% to 103%. We also detected residual trichlorfons in the leek samples, and these results were verified by gas chromatography method.

Performance of trichlorfon degradation by a novel Bacillus tequilensis strain PA F-3 and its proposed biodegradation pathway.

The novel trichlorfon (TCF)-degrading bacterium PA F-3, identified as Bacillus tequilensis, was isolated from pesticide-polluted soils by using an effective screening and domesticating procedure. The TCF biodegradation pathways of PA F-3 were also systematically elucidated. As revealed by high-performance liquid chromatography, the TCF residues in the mineral salt medium demonstrated that PA F-3 can utilize TCF as its sole carbon source and reach the highest degradation of 71.1 % at an initial TCF concentration of 200 mg/L within 5 days. The TCF degradation conditions were optimized using response surface methodology as follows: temperature, 28 °C; inoculum amount, 4 %; and initial TCF concentration, 125 mg/L. Biodegradation treatments supplemented with exogenous carbon sources and yeast extract markedly increased the microbial dry weights and TCF-degrading performance of PA F-3, respectively. Meanwhile, five metabolic products of TCF were identified through gas chromatography/mass spectrometry, and a biodegradation pathway was proposed. Results indicated that deoxidation and dehydration (including the cleavage of the P-C phosphonate bond and the C-O bond) were the preferred metabolic reactions of TCF in this TCF-degrading bacterium.

Biodegradation of the Organophosphate Trichlorfon and Its Major Degradation Products by a Novel Aspergillus sydowii PA F-2.

Trichlorfon (TCF) is an important organophosphate pesticide in agriculture. However, limited information is known about the biodegradation behaviors and kinetics of this pesticide. In this study, a newly isolated fungus (PA F-2) from pesticide-polluted soils was identified as Aspergillus sydowii on the basis of the sequencing of internal transcribed spacer rDNA. This fungus degraded TCF as sole carbon, sole phosphorus, and sole carbon-phosphorus sources in a mineral salt medium (MSM). Optimal TCF degradation conditions were determined through response surface methodology, and results also revealed that 75.31% of 100 mg/L TCF was metabolized within 7 days. The degradation of TCF was accelerated, and the mycelial dry weight of PA F-2 was remarkably increased in MSM supplemented with exogenous sucrose and yeast extract. Five TCF metabolic products were identified through gas chromatography-mass spectrometry. TCF could be initially hydrolyzed to dichlorvos and then be degraded through the cleavage of the P-C bond to produce dimethyl hydrogen phosphate and chloral hydrate. These two compounds were subsequently deoxidized to produce dimethyl phosphite and trichloroethanal. These results demonstrate the biodegradation pathways of TCF and promote the potential use of PA F-2 to bioremediate TCF-contaminated environments.

Trichlorfon-induced haematological and biochemical changes in Cyprinus carpio: ameliorative effect of propolis.

Trichlorfon is among the most commonly used products to treat fish parasites in aquaculture. We investigated the effectiveness of propolis in alleviating the toxicity of trichlorfon on haematological and oxidant/antioxidant parameters in carp Cyprinus carpio. Fish were exposed to sublethal concentrations (11 and 22 mg l-1) of trichlorfon, and propolis (10 mg kg-1 of fish weight) was simultaneously administered. At the end of 14 d administration, blood and tissue (liver, kidney, gill) samples were collected. Haematological changes (red and white blood cell count, haemoglobin concentration, haematocrit level and erythrocyte indices: mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration) were determined in the blood samples, while antioxidant parameters (malondialdehyde and reduced glutathione levels and superoxide dismutase, catalase and glutathione peroxidase activities) were evaluated in the liver, kidney and gill samples. Trichlorfon led to negative alterations in the haematological and antioxidant parameters investigated. The administration of propolis alleviated this effect and suggests that fish treated with trichlorfon improve their physiological status when fed a propolis-supplemented diet.

Study of a molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography for simultaneous determination of trace trichlorfon and monocrotophos residues in vegetables.

BACKGROUND: Organophosphate pesticide residues are harmful to human health because of their potential mutagenic and carcinogenic properties. Therefore, it is of great importance to development an accurate and reliable analytical method to prevent their uncontrolled effects on environmental pollution and human health. RESULTS: This study reports a new method of molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography (MISPE-HPLC) for simultaneous determination of two organophosphate pesticides residues. Two types of molecularly imprinted polymers (MIPs) were prepared using the trichlorfon and monocrotophos as the template molecule, respectively, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The recognition ability and adsorption-desorption dynamic of each imprinted polymer toward the trichlorfon or monocrotophos were characterised. Using the mixture of trichlorfon-MIP and monocrotophos-MIP (20:80, wt/wt) as solid-phase extraction sorbent, the factors affecting the pre-concentration on the analytes and the sensitivity of the MISPE-HPLC method were optimised. Under optimal condition, the linear range was 0.005-1.0 mg L⁻¹. The limit of detection was 4.2 µg g⁻¹ for trichlorfon, and 1.2 ng g⁻¹ for monocrotophos. The peak area precision [Relative standard deviation (RSD)] for three replicates was 2.9-4.5%. The blank rape and cauliflower samples spiked with trichlorfon and monocrotophos at 0.05 and 0.005 µg g⁻¹ levels were extracted and determined by this method with recoveries ranging from 88.5% to 94.2%. Moreover, this method was successfully applied to the quantitative detection of the trichlorfon and monocrotophos residues in leek samples. CONCLUSION: With good properties of high sensitivity, simple pre-treatment and low cost, this MISPE-HPLC method could provide a new tool for the rapid determination of multi-pesticide residues in the complicated food samples.

Effect of glabridin from Glycyrrhiza glabra on learning and memory in mice.

Glabridin was isolated from the roots of Glycyrrhiza glabra and its effects on cognitive functions and cholinesterase activity were investigated in mice. Glabridin (1, 2 and 4 mg kg (-1), P. O.) and piracetam (400 mg kg (-1), i. p.), a clinically used nootropic agent, were administered daily for 3 successive days to different groups of mice. The higher doses (2 and 4 mg kg (-1), P. O.) of glabridin and piracetam significantly antagonized the amnesia induced by scopolamine (0.5 mg kg (-1), I. P.) in both the elevated plus maze test and passive avoidance test. Furthermore, glabridin (2 and 4 mg kg (-1), P. O.) and metrifonate (50 mg kg (-1), I. P.), used as a standard drug, both remarkably reduced the brain cholinesterase activity in mice compared to the control group. Therefore, glabridin appears to be a promising candidate for memory improvement and it will be worthwhile to explore the potential of glabridin in the management of Alzheimer patients.

Impact of the botanical insecticide Neem Azal on survival and reproduction of the biting louse Damalinia limbata on angora goats.

Secondary metabolites present in the neem tree (Azadirachta indica A. Juss, Meliaceae), exhibit a wide range of biological activities in insects. However, few studies have been undertaken to assess the potential of neem products as insecticides for the control of ectoparasites of domestic animals. This study was undertaken to estimate the efficacy of Neem Azal, an azadirachtin-rich extract of neem seeds, in controlling Damalinia limbata (Phthiraptera) louse infestation of angora goats. The study was conducted on a fibre animal farm situated in Central Italy. Groups of 11-12 goats were treated with Neem Azal at an azadirachtin concentration of 650ppm or 125ppm, with Neguvon or were left untreated. Their louse burden was assessed fortnightly to monthly for 22 weeks. A reduction in louse densities of 76-96% was observed from week 2 to week 18 after treatment with the neem solution containing azadirachtin at a concentration of 650ppm. At the lower test concentration (125ppm) a reduction of 60-92% could be recorded from week 2 to week 14. Neem Azal was found to reduce the survival of both adult and nymph stages of D. limbata and to interfere with oviposition and oogenesis of female lice. A decrease in oviposition was observed in neem exposed female lice and the examination of their ovaries revealed morphological alterations in both vitellogenic and previtellogenic ovarioles at the follicular and germinal level. Since neem compounds target different life stages and physiological processes of D. limbata, the development of insecticide resistance by biting lice exposed to neem-based insecticides appears unlikely. For this reason and for its prolonged activity, which in principle allows angora goats to be protected for a large part of the mohair production cycle, neem-based insecticides may have a potential interest for mohair producing breeders.

Peroxinectin gene transcription of the giant freshwater prawn Macrobrachium rosenbergii under intrinsic, immunostimulant, and chemotherapeutant influences.

Peroxinectin (PE) gene expressions were determined using real-time PCR in the giant freshwater prawn Macrobrachium rosenbergii based on moulting; prawns were fed diets containing different concentrations of sodium alginate, and were exposed to different concentrations of copper sulphate, benzalkonium chloride (BKC), and trichlorfon. Results showed that PE mRNA expression of prawns was the highest in stage A, significantly decreased in stage B, and reached the lowest level in stages D0/D1. The PE transcript was significantly higher in prawns fed the 1.0 gkg(-1) sodium alginate-containing diet than those fed the 2.0 gkg(-1) sodium alginate-containing diet and those fed the control diet. PE transcripts significantly decreased in prawns exposed to 0.1-0.4 mgL(-1) copper sulphate after 96 h, 0.3-1.0 mgL(-1) BKC after 96 h, and 0.2-0.4 mgL(-1) trichlorfon after 48 h. It was concluded that the status of PE gene expression was seriously affected by the moult cycle, immunostimulant, and chemotherapeutants.

Comparative brain cholinesterase-inhibiting activity of Glycyrrhiza glabra, Myristica fragrans, ascorbic acid, and metrifonate in mice.

The central cholinergic pathways play a prominent role in the learning and memory processes. Acetylcholinesterase is an enzyme that inactivates acetylcholine. The present study was undertaken to estimate the acetylcholinesterase- inhibiting activity of extracts of Glycyrrhiza glabra, Myristica fragrans seeds, and ascorbic acid and compare these values with a standard acetylcholinesterase-inhibiting drug, metrifonate. Aqueous extract of G. glabra (150 mg/kg p.o. for 7 successive days), n-hexane extract of M. fragrans seeds (5 mg/kg p.o. for 3 successive days), ascorbic acid (60 mg/kg i.p. for 3 successive days), and metrifonate (50 mg/kg i.p.) were administered to young male Swiss albino mice. Acetylcholinesterase enzyme was estimated in brains of mice. G. glabra, M. fragrans, ascorbic acid, and metrifonate significantly decreased acetylcholinesterase activity as compared with their respective vehicle-treated control groups.

Trafficking of the muscarinic m2 autoreceptor in cholinergic basalocortical neurons in vivo: differential regulation of plasma membrane receptor availability and intraneuronal localization in acetylcholinesterase-deficient and -inhibited mice.

In vivo, the abundance of receptors at the neuronal plasma membrane may be critical in the mediation of pre- and postsynaptic responses. Thus, we have studied the membrane availability and intraneuronal distribution of the m2 muscarinic autoreceptor (m2R) in cholinergic neurons of the nucleus basalis magnocellularis (NBM) projecting to the frontal cortex (FC). We have studied the subcellular compartmentalization of m2R at somatodendritic postsynaptic and axonal presynaptic sites in control animals (AChE +/+) and in two animal models: mice displaying acute acetylcholinesterase (AChE) inhibition by treatment with metrifonate, and AChE-deficient mice (AChE -/-). In control animals, m2R was mainly located at the plasma membrane in the somatodendritic field of NBM and in cortical varicosities. Acute AChE inhibition and chronic AChE deficiency induced a dramatic decrease of cell surface m2R in the somatodendritic compartment. This finding was associated with two different intracytoplasmic events: (1). internalization of m2R in endosomes after acute AChE inhibition, (2). exaggerated storage of m2R in the endoplasmic reticulum and Golgi complex in AChE -/- mice. In contrast, the m2R density was higher at the membrane of cortical varicosities in AChE -/- mice but unchanged in acutely AChE-inhibited mice. Our data demonstrate that acute and chronic stimulation provoke, in vivo, depletion of the membrane store of somatodendritic m2R through different intracellular mechanisms: endocytosis of receptors from the plasma membrane to the cytoplasm (acute) or regulation of their delivery from intracytoplasmic stores to the plasma membrane (chronic). The increase of m2R at the membrane of axonal varicosities after chronic stimulation suggest modulation of presynaptic cholinergic activity, including neurotransmitter release.

The effects of long-term treatment with metrifonate, a cholinesterase inhibitor, on cholinergic activity, amyloid pathology, and cognitive function in APP and PS1 doubly transgenic mice.

Recent studies in cell cultures have shown that modulating the cholinergic activity can influence the processing and metabolism of amyloid precursor protein (APP). To investigate whether acetylcholinesterase inhibitors (ChEIs) could decrease production of amyloid beta-peptide (A(beta)) and slow down the accumulation of A(beta) also in vivo, we chronically administered metrifonate (100 mg/kg, po), a second-generation ChEI, to 7-month-old doubly transgenic APP+PS1 mice and their nontransgenic littermate controls for 7 months. Behavioral studies, including open field test, T maze, and water maze, were conducted after 6 months treatment with metrifonate, and the mice were sacrificed at the age of 14 months for biochemical and histological analyses. The long-term treatment with metrifonate failed to inhibit the marked overproduction and deposition of A(beta) in the APP+PS1 mice; in contrast, it increased both A(beta)40 and A(beta)42 levels in the hippocampus. However, the A(beta)42 to 40 ratio was significantly reduced by the treatment. In addition, the number of amyloid plaques in the hippocampus did not differ between the treatment and the control groups. Tolerance to cholinesterase inhibition might be induced in the mouse brain because the inhibition rate of AChE was attenuated from about 80 to 50% during the experiment in both APP+PS1 and nontransgenic mice. The metrifonate treatment did not affect cognitive testing parameters but reduced swimming speed and locomotor activity in both genotypes. Our results do not support the idea that ChEIs would slow down the progression of amyloid pathology in Alzheimer's disease.