Both the naturally occurring pyrethrins (CAS 121-21-1 and CAS 121-29-9) and the synthetic pyrethroids (e.g., cyfluthrin, CAS 68359-37-5; cypermethrin, CAS 52315-07-8; deltamethrin, CAS 52918-63-5; and permethrin, CAS 52645-53-1) are widely used as potent insecticides and insect repellents in agricultural, public health, and domestic applications. They are also used as ectoparisiticides in veterinary and human medicine. Attractive features are their low environmental persistence and their rapid knockdown activity, whereby flying insects very quickly become uncoordinated and unable to fly before they are killed.

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target expression of the vesicular glutamate transporter 1 (VGLUT1) following injection into the dorsal hippocampus of adult mice, as partial reductions in VGLUT1 expression should attenuate glutamatergic signaling and similar reductions have been reported in schizophrenia. The VGLUT1-targeting vector attenuated tonic glutamate release in the dorsal hippocampus without affecting GABA, and selectively impaired novel object discrimination (NOD) and retention (but not acquisition) in the Morris water maze, without influencing contextual fear-motivated learning or causing any adverse locomotor or central immune effects. This pattern of cognitive impairment is consistent with the accumulating evidence for functional differentiation along the dorsoventral axis of the hippocampus, and supports the involvement of dorsal hippocampal glutamatergic neurotransmission in both spatial and nonspatial memory. Future use of this nonpharmacological VGLUT1 knockdown mouse model could improve our understanding of glutamatergic neurobiology and aid assessment of novel therapies for cognitive deficits such as those seen in schizophrenia.

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[3H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated 3H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of 3H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.

Objectives Progesterone has been shown to be neuroprotective in a number of preclinical central nervous system injury models including cerebral ischaemia. The aim of this study was to clarify differences in outcomes owing to different dosing regimens and the pharmacokinetic profile of progesterone, particularly in relation to brain levels. Methods Male C57 Bl/6 mice were injected intraperitoneally with progesterone (8 mg/kg in dimethylsulfoxide) or with a bolus injection followed by continuous subcutaneous infusion (1.0 mu l/h of a 50 mg/ml progesterone solution) via implanted osmotic minipumps. Plasma and brain samples were collected over 24 h from bolus-injected mice and 48 h from mice implanted with minipumps. Progesterone concentrations were measured by an enzyme-linked immunoassay and pharmacokinetic profiles were constructed. Key findings Intraperitoneally injected progesterone had a short half-life (fast component half-life of 0.2 h) in both plasma and brain. Minipump delivery resulted in higher concentrations of progesterone in plasma and particularly in brain over a longer period. The volume of distribution with intraperitoneal injection was 172.78 versus 1641.84 ng/h per g via minipump in the first 24 h. Conclusions A bolus intraperitoneal loading dose of progesterone followed by continuous delivery via osmotic minipump is an effective way of delivering progesterone to the brain.

Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited. The determination of individual OP protein binding targets has been hampered by the sensitivity of methods of detection and quantification of protein-pesticide adducts. We have overcome this limitation by the employment of a microchannel plate (MCP) autoradiographic detector to monitor a radiolabelled OP tracer compound. We preincubated rat thymus tissue in vitro with the OP pesticides, azamethiphos-oxon, chlorfenvinphos-oxon, chlorpyrifos-oxon, diazinon-oxon, and malaoxon, and then subsequently radiolabelled the free OP binding sites remaining with 3H-diisopropylfluorophosphate (3H-DFP). Proteins adducted by OP pesticides were detected as a reduction in 3H-DFP radiolabelling after protein separation by one dimensional polyacrylamide gel electrophoresis and quantitative digital autoradiography using the MCP imager. Thymus tissue proteins of molecular weights ~28 kDa, 59 kDa, 66 kDa, and 82 kDa displayed responsiveness to adduction by this panel of pesticides. The 59 kDa protein target (previously putatively identified as carboxylesterase I) was only significantly adducted by chlorfenvinphos-oxon (p < 0.001), chlorpyrifos-oxon (p < 0.0001), and diazinon-oxon (p < 0.01), the 66 kDa protein target (previously identified as serum albumin) similarly only adducted by the same three pesticides (p < 0.0001), (p < 0.001), and (p < 0.01), and the 82 kDa protein target (previously identified as acyl peptide hydrolase) only adducted by chlorpyrifos-oxon (p < 0.0001) and diazinon-oxon (p < 0.001), when the average values of tissue AChE inhibition were 30%, 35%, and 32% respectively. The ~28 kDa protein target was shown to be heterogeneous in nature and was resolved to reveal nineteen 3H-DFP radiolabelled protein spots by two dimensional polyacrylamide gel electrophoresis and MCP autoradiography. Some of these 3H-DFP proteins spots were responsive to adduction by preincubation with chlorfenvinphos-oxon. In addition, we exploited the useful spatial resolution of the MCP imager (~70 mm) to determine pesticide micolocalisation in vivo, after animal dosing and autoradiography of brain tissue sections. Collectively, MCP autoradiographic imaging provided a means to detect targets of OP pesticides, quantify their sensitivity of adduction relative to tissue AChE inhibition, and highlighted that these common pesticides exhibit specific binding character to protein targets, and therefore their toxicity will need to be evaluated on an individual compound basis. In addition, MCP autoradiography afforded a useful method of visualisation of the localisation of a small radiolabelled tracer within brain tissue.

J. Neurochem. (2011) 116, 996–1004.AbstractWe have previously shown that systemic administration of S(+)3‐chloropropanediol (3‐CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3‐CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20‐min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio. These challenge ammonia exposures evoked a stable response for at least 5 h when the probe was implanted in the rat inferior colliculus, but caused no behavioural response or morphological damage. Although 3‐CPD produced a rapid and sustained abolition of the ammonia response within 2 h, the field potential response of inferior collicular neurones to sound fell significantly to 75.0 ± 3.9% pre‐dose at up to 8 h but then fell markedly, reaching 20.5 ± 3.7% at 2 days. Blood flow in the inferior colliculus also showed only late changes, increasing substantially at 2 days. Astrocyte damage at the EM level was seen from 3 h, followed by loss of astrocytes from 18 h to a minimum of 7 ± 10% control at 3 days. The rapid abolition of the ammonia response suggests that in addition to selective astrocyte death, 3‐CPD also produces an earlier impairment of astrocyte function that precedes loss of neuronal function. This initial functional selectivity of 3‐CPD provides a potential investigative tool in neurochemical studies of astrocyte‐neuronal interactions.

Organophosphorus pesticides primarily elicit toxicity via their common covalent adduction of acetylcholinesterase (AChE), but pesticide binding to additional sensitive secondary targets may also compromise health. We have utilised tritiated-diisopropylfluorophosphate ((3)H-DFP) binding to quantify the levels of active immune and brain tissue serine hydrolases, and visualise them using autoradiography after protein separation by one-dimensional and two-dimensional techniques. Preincubation of protein extracts with pesticide in vitro or dosing of rats with pesticide in vivo was followed by (3)H-DFP radiolabelling. Pesticide targets were identified by a reduction in (3)H-DFP radiolabelling relative to controls, and characterised by their tissue presence, molecular weight, and isoelectric point. Conventional column chromatography was employed to enrich pesticide targets to enable their further characterisation, and/or identification by mass spectrometry. The major in vivo pesticide targets characterised were 66 kDa, serum albumin, and 60 kDa, likely carboxylesterase 1, both of which displayed differential pesticide binding character under conditions producing approximately 30% tissue AChE inhibition. The characterisation and identification of sensitive pesticide secondary targets will enable an evaluation of their potential contribution to the ill health that may arise from chronic low-dose pesticide exposures. Additionally, secondary targets may provide useful biomonitors and/or bioscavengers of pesticide exposures.

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase-III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress.

Developmental hypothyroidism causes brain retardation due to impairment of neuronal migration and oligodendroglial myelination. Brominated flame retardants (BFRs), potential environmental pollutants to cause bioaccumulation, usually exert low or no toxicity in conventional toxicity studies, but some of them are known to induce mild hypothyroidism. To search for target genes responsible for possible brain retardation due to developmental exposure to BFRs, we performed a global gene expression profiling specific to the cerebral white matter of rat pups exposed to decebromodiphenyl ether (DBDE) during development. Dams were given DBDE at 10, 100, or 1000 ppm in diet during the period from gestation day 10 to postnatal day 21 (weaning). As a positive control for hypothyroidism, methimazole (MMI) at 200 ppm or propylthiouracil (PTU) at 3 or 12 ppm was given via drinking water. At weaning, bilateral cerebral white matter and corpus callosum (CC) were selectively microdissected from male pups and subjected to microarray analysis. As a result, 10% of genes showing altered expression by DBDE were identical to those induced by anti-thyroid agents (MMI and PTU) including glial cell differentiation, axon guidance, myelination, or cellular migration. Also, DBDE-alone induced expression alteration of genes related to similar functions as with anti-thyroid agents. At 11 weeks of age, offspring were subjected to brain morphometry regarding white matter components. As a result, as well as anti-thyroid agents, DBDE reduced the CC area and the density of CNPase-positive oligodendrocytes suggestive of reduced white matter development. Results thus suggest that glial development-related genes obtained here with DBDE may be linked to or independent of developmental hypothyroidism that is shown here for the first time with DBDE as white matter hypoplasia.

We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9+/-1%, 67+/-2%, 56+/-2%, 54+/-2% and 8+/-1%, respectively. After 1 h of incubation with 19 microM (3)H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of (3)H-DFP, increasing the (3)H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.

Oxidative stress has been implicated in the pathogenesis of several neurodegenerative diseases and may result from excessive free radical production due to increased local metabolism. Non-competitive N-methyl-d-aspartate (NMDA) antagonists (MK-801 and phencyclidine) increase glucose metabolism in many brain areas and induce cytoplasmic vacuoles, heat shock protein and necrotic cell death in neurones of the rodent posterior cingulate and retrosplenial cortex. We have investigated the effect of several antioxidants with differing properties on MK-801-induced neuronal loss. Free radical scavengers (dimethyl sulfoxide (DMSO) and α-tocopherol) and spin traps (N-tert-butyl-α-(2-sulfophenyl)-nitrone (S-PBN) and 5-(diethoxyphosphoryl)-5-methyl-1-pyrrole N-oxide (DEPMPO)), produced marked attenuation of MK-801-induced neuronal necrosis in the rat posterior cingulate and retrosplenial cortex. Further, administration of DMSO could be delayed by up to 4h after MK-801 dosing and still achieve between 80 and 86% reduction in neuronal loss. We also show that MK-801 administration rapidly induced a four-fold and prolonged increase in cerebral blood flow in the posterior cingulate. This elevated regional blood flow was only transiently reduced by DMSO administration. The anterior cingulate, a region which undergoes no neuronal loss, showed only a two-fold increase in regional blood flow following MK-801 administration. These results support a hypothesis that oxidative stress plays a role in MK-801-induced neuronal necrosis since pathological changes can be attenuated by several antioxidants.

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.

There is a need for mechanistic understanding of the lasting ill health reported in several studies of workers exposed to organophosphorus (OP) pesticide. Although the acute toxicity is largely explicable by acetylcholinesterase inhibition and the lasting effects of frank poisoning by direct excitotoxicity or indirect consequences of the cholinergic syndrome, effects at lower levels of exposure would not be predicted from these mechanisms. Similarly, reversible interactions with nicotinic and muscarinic receptors in adults would not predict continuing ill health. Many OP pesticides produce protein adduction, and the lasting nature of this makes it a candidate mechanism for the production of continuing ill health. We found significant adduction of partially characterized protein targets in both rat brain and thymus by azamethiphos, chlorfenvinphos, chlorpyrifos—oxon, diazinon—oxon, dichlorvos and malaoxon, in vitro and pirimiphos-methyl in vivo. The diversity in the adduction pattern seen across these agents at low dose levels means that any longer term effects of adduction would be specific to specific organophosphates, rather than generic. This presents a challenge to epidemiology, as most exposures are to different agents over time. However, some adducted proteins are also expressed in blood, notably albumin, and so may provide exposure measures to increase the power of future epidemiological studies. Human & Experimental Toxicology (2007) 26, 347—353

Our recent report that fructose supported the metabolism of some, but not all axons, in the adult mouse optic nerve prompted us to investigate in detail fructose metabolism in this tissue, a typical central white matter tract, as these data imply efficient fructose metabolism in the central nervous system (CNS). In artificial cerebrospinal fluid containing 10 mmol/L glucose or 20 mmol/L fructose, the stimulus-evoked compound action potential (CAP) recorded from the optic nerve consisted of three stable peaks. Replacing 10mmol/L glucose with 10 mmol/L fructose, however, caused delayed loss of the 1st CAP peak (the 2nd and 3rd CAP peaks were unaffected). Glycogen-derived metabolic substrate(s) temporarily sustained the 1st CAP peak in 10mmol/L fructose, as depletion of tissue glycogen by a prior period of aglycaemia or high-frequency CAP discharge rendered fructose incapable of supporting the 1st CAP peak. Enzyme assays showed the presence of both hexokinase and fructokinase ( both of which can phosphorylate fructose) in the optic nerve. In contrast, only hexokinase was expressed in cerebral cortex. Hexokinase in optic nerve had low affinity and low capacity with fructose as substrate, whereas fructokinase displayed high affinity and high capacity for fructose. These findings suggest an explanation for the curious fact that the fast conducting axons comprising the 1st peak of the CAP are not supported in 10 mmol/L fructose medium; these axons probably do not express fructokinase, a requirement for efficient fructose metabolism.