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Biomedical subjects

J E Casida

Publications and source records attributed to J E Casida.

At least 19 recordsLinked to original sources
PubMed2002

Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil).

1. Hyssop oil is an important food additive and herbal medicine and the principal active ingredients are (-)-cis- and (-)-trans-3-pinanones. No information is available on their metabolism or specific mode of action. 2. The metabolites of cis- and trans-3-pinanones were examined from mouse and human liver microsomes and human recombinant P4503A4 with NADPH and on administration to mouse by gas chromatography/chemical ionization mass spectrometry comparison with standards from synthesis. 3. The major metabolite of cis-3-pinanone in each P450 system and in brain of the i.p.-treated mouse in quantitative studies was 2-hydroxy-cis-3-pinanone, and two minor metabolites were hydroxypinanones other than 2-hydroxy-trans-3-pinanone and 4S-hydroxy-cis-3-pinanone. The urine from oral cis-3-pinanone treatment examined on a qualitative basis contained conjugates of metabolites observed in the microsomal systems plus 2,10-dehydro-3-pinanone. 4. Trans-3-pinanone was metabolized more slowly than the cis-isomer in each system to give hydroxy derivatives different than those derived from cis-3-pinanone. 5. Cis- and trans-3-pinanones and hyssop oil act as gamma-aminobutyric acid type A (GABAA) receptor antagonists based on inhibition of 4'-ethynyl-4-n-[2,3-(3)H(2)]propylbicycloorthobenzoate ([(3)H]EBOB) binding in mouse brain membranes (IC(50) of 35-64 microM) and supported by tonic/clonic convulsions in mouse (i.p. LD(50) 175 to >250 mg kg(-1)) alleviated by diazepam. The cis-3-pinanone metabolites 2-hydroxy-cis-3-pinanone and 2,10-dehydro-3-pinanone exhibit reduced toxicity and potency for inhibition of [(3)H]EBOB binding.

Animals↗
PubMed2001

Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.

Animals↗
PubMed2001

Analgesic and toxic effects of neonicotinoid insecticides in mice.

Several nicotinic agonists with the 6-chloro-3-pyridinyl moiety are potent insecticides (e.g., the neonicotinoids imidacloprid and thiacloprid) while others are candidate nonopioid and nonantiinflammatory analgesics (i.e., epibatidine and several heterocyclic analogs). This study examines the hypothesis for the first time that the neonicotinoid insecticides and their imine metabolites and analogs display analgesic (antinociceptive) activity or adverse toxic effects associated with their action on binding to the alpha 4 beta 2 nicotinic acetylcholine receptor (AChR) subtype. Seven 6-chloro-3-pyridinyl compounds were studied, i.e., imidacloprid and thiacloprid, the corresponding imines and an olefin derivative, a nitromethylene analog, and (+/-)-epibatidine. Like (-)-nicotine and carbachol, they all act as full agonists in the (86)rubidium ion efflux experiment with intact mouse fibroblast M10 cells stably expressing the alpha 4 beta 2 nicotinic AChR. Their agonist action is correlated with binding affinity to the alpha 4 beta 2 receptor from M10 cells. Imidacloprid, thiacloprid, and their imine analogs are not antinociceptive agents in mice by abdominal constriction and hot plate analgesic tests. Their agonist actions at the alpha 4 beta 2 receptor correlate instead with their toxicity. Surprisingly, the nitromethylene analog, a weak agonist, is as potent as (-)-nicotine in inducing antinociception, and the effect persists longer than that caused by (-)-nicotine. However, mecamylamine (1 mg/kg) prevents antinociception induced by (-)-nicotine but not by the nitromethylene analog. Interestingly, this nitromethylene neonicotinoid insecticide gives 80-100% mortality within 15 min at 3 mg/kg with mecamylamine pretreatment at 2 mg/kg, doses at which each agent alone gives no lethality. Therefore, analgesic and toxic effects of the nitromethylene analog differ in their mechanism of action from (-)-nicotine and (+/-)-epibatidine.

Analgesics, Non-Narcotic↗
PubMed2001

GABA receptor subunit composition relative to insecticide potency and selectivity.

Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

Animals↗
PubMed2001

Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling.

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.

1-Methyl-4-phenylpyridinium↗
PubMed2001

Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides.

Organophosphorus (OP) compound-induced inhibition of acetylcholinesterase (AChE) and neuropathy target esterase explains the rapid onset and delayed neurotoxic effects, respectively, for OP insecticides and related compounds but apparently not a third or intermediate syndrome with delayed onset and reduced limb mobility. This investigation tests the hypothesis that fatty acid amide hydrolase (FAAH), a modulator of endogenous signaling compounds affecting sleep (oleamide) and analgesia (anandamide), is a sensitive target for OP pesticides with possible secondary neurotoxicity. Chlorpyrifos oxon inhibits 50% of the FAAH activity (IC50 at 15 min, 25 degrees C, pH 9.0) in vitro at 40--56 nM for mouse brain and liver, whereas methyl arachidonyl phosphonofluoridate, ethyl octylphosphonofluoridate (EOPF), oleyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (oleyl-BDPO), and dodecyl-BDPO give IC50s of 0.08--1.1 nM. These BDPOs and EOPF inhibit mouse brain FAAH in vitro with > or =200-fold higher potency than for AChE. Five OP pesticides inhibit 50% of the brain FAAH activity (ED50) at <30 mg/kg 4 h after ip administration to mice; while inhibition by chlorpyrifos, diazinon, and methamidophos occurs near acutely toxic levels, profenofos and tribufos are effective at asymptomatic doses. Two BDPOs (dodecyl and phenyl) and EOPF are potent inhibitors of FAAH in vivo (ED50 0.5--6 mg/kg). FAAH inhibition of > or =76% in brain depresses movement of mice administered anandamide at 30 mg/kg ip, often leading to limb recumbency. Thus, OP pesticides and related inhibitors of FAAH potentiate the cannabinoid activity of anandamide in mice. More generally, OP compound-induced FAAH inhibition and the associated anandamide accumulation may lead to reduced limb mobility as a secondary neurotoxic effect.

Amidohydrolases↗
PubMed2001

Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

Binding Sites↗
PubMed2001

Diethylphosphorylation of rat cardiac M2 muscarinic receptor by chlorpyrifos oxon in vitro.

The acute toxicity of chlorpyrifos oxon (CPO), the metabolically-activated form of the major organophosphorus insecticide chlorpyrifos, is attributable to diethylphosphorylation of acetylcholinesterase at its esteratic site. As a secondary effect, CPO is known to compete with agonist binding to the M2 muscarinic acetylcholine receptor (mAChR). This study tests the hypothesis that [ethyl-1,2-(3)H]CPO labels the M2 mAChR in rat cardiac membrane proteins. Of four labeled protein regions observed, only one had an apparent molecular mass (70-75 kDa) consistent with that of glycosylated M2 mAChR. It was identified as M2 muscarinic receptor by Western blotting and immunoprecipitation using a cardiac-specific M2 mAChR monoclonal antibody, providing the first direct evidence for diethylphosphorylation of a muscarinic receptor. This may be a functionally important M2 mAChR site, but the toxicological relevance and species and organ specificity of diethylphosphorylation are unknown.

Animals↗
PubMed2001

Structure and diversity of insect nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is an agonist-regulated ion-channel complex responsible for rapid neurotransmission. The vertebrate nAChR, assembled from five homologous subunits, penetrates the synaptic membrane. Different subunit combinations lead to receptor subtypes with distinctive pharmacological profiles. In comparison with mammalian nAChRs, the insect receptor is poorly understood relative to functional architecture and diversity. Several genes for Drosophila, Locusta and Myzus encoding insect nAChR subunits have been identified, although the functional assembly and presence of different subtypes of these receptors are not defined. The insect nAChR is the primary target site for the neonicotinoid insecticides, thereby providing an incentive to explore its functional architecture with neonicotinoid radioligands, photoaffinity probes and affinity chromatography matrices. This review considers the current understanding of the structure and diversity of insect nAChRs based mainly on recent studies in molecular biology and protein biochemistry.

Animals↗
PubMed2001

The insecticide target in the PSST subunit of complex I.

Current insecticides have been selected by sifting and winnowing hundreds of thousands of synthetic chemicals and natural products to obtain commercial preparations of optimal effectiveness and safety. This process has often ended up with compounds of high potency as inhibitors of the electron transport chain and more specifically of complex I (NADH:ubiquinone oxidoreductase). Many classes of chemicals are involved and the enzyme is one of the most complicated known, with 43 subunits catalyzing electron transfer from NADH to ubiquinone through flavin mononucleotide and up to eight iron-sulfur clusters. We used a potent photoaffinity ligand, (trifluoromethyl)diazirinyl[3H]pyridaben, to localize the insecticide target to a single high-affinity site in the PSST subunit that couples electron transfer from iron-sulfur cluster N2 to ubiquinone. Most importantly, all of the potent complex I-inhibiting pesticides, despite their great structural diversity, compete for this same specific binding domain in PSST. Finding their common mode of action and target provides insight into shared toxicological features and potential selection for resistant pests.

Animals↗
PubMed2001

alpha- and beta-Thujones (herbal medicines and food additives): synthesis and analysis of hydroxy and dehydro metabolites.

Essential oils containing alpha- and beta-thujones are important herbal medicines and food additives. The thujone diastereomers are rapidly metabolized convulsants acting as noncompetitive blockers of the gamma-aminobutyric acid-gated chloride channel. Synthesis and analysis of the metabolites are essential steps in understanding their health effects. Oxidation of alpha- and beta-thujones as their 2,3-enolates with oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) gave the corresponding (2R)-2-hydroxythujones assigned by (1)H and (13)C NMR and X-ray crystallography. alpha-Thujone was converted to 4-hydroxy-alpha- and -beta-thujones via the 3,4-enol acetate on oxidation with peracid and osmium tetroxide, respectively. Ozonation provided 7-hydroxy-alpha- and -beta-thujones, and by dehydration provided the 7,8-dehydro compounds. 4,10-Dehydrothujone was prepared from sabinene via sabinol. The hydroxy and dehydro derivatives are readily identified and analyzed by GC/MS as the parent compounds and trimethylsilyl and methyloxime derivatives. A separate study established that all of these compounds are metabolites of alpha- and beta-thujones.

Bicyclic Monoterpenes↗
PubMed2001

Detoxification of alpha- and beta-Thujones (the active ingredients of absinthe): site specificity and species differences in cytochrome P450 oxidation in vitro and in vivo.

Alpha- and beta-Thujones are active ingredients in the liqueur absinthe and in herbal medicines and seasonings for food and drinks. Our earlier study established that they are convulsants and have insecticidal activity, acting as noncompetitive blockers of the gamma-aminobutyric acid (GABA)-gated chloride channel, and identified 7-hydroxy-alpha-thujone as the major metabolite and 4-hydroxy-alpha- and -beta-thujones and 7,8-dehydro-alpha-thujone as minor metabolites in the mouse liver microsome-NADPH system. We report here unexpected site specificity and species differences in the metabolism of the thujone diastereomers in mouse, rat, and human liver microsomes and human recombinant P450 (P450 3A4), in orally treated mice and rats, and in Drosophila melanogaster. Major differences are apparent on comparing in vitro microsome-NADPH systems and in vivo urinary metabolites. Hydroxylation at the 2-position is observed only in mice where conjugated 2R-hydroxy-alpha-thujone is the major urinary metabolite of alpha-thujone. Hydroxylation at the 4-position gives one or both of 4-hydroxy-alpha- and -beta-thujones depending on the diastereomer and species studied with conjugated 4-hydroxy-alpha-thujone as the major urinary metabolite of alpha- and beta-thujones in rats. Hydroxylation at the 7-position of alpha- and beta-thujones is always a major pathway, but the conjugated urinary metabolite is minor except with beta-thujone in the mouse. Site specificity in glucuronidation favors excretion of 2R-hydroxy- and 4-hydroxy-alpha-thujone glucuronides rather than those of three other hydroxythujones. Two dehydro metabolites are observed from both alpha- and beta-thujones, the 7,8 in the P450 systems and the 4,10 in urine. Two types of evidence establish that P450-dependent oxidations of alpha- and beta-thujones are detoxification reactions: three P450 inhibitors block the metabolism of alpha- and beta-thujones and strongly synergize their toxicity in Drosophila; six metabolites assayed are less potent than alpha- and beta-thujones as inhibitors of [(3)H]ethynylbicycloorthobenzoate binding to the GABA(A) receptor in mouse brain membranes and as toxicants to Drosophila.

Absinthe↗
PubMed2001

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.

Animals↗
PubMed2000

5-Azidoimidacloprid and an acyclic analogue as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors.

The 5-azido analogue of the major insecticide imidacloprid, 1-(5-azido-6-chloropyridin-3-ylmethyl)-2-nitroiminoimidaz olidine (1), and an acyclic analogue, N-(5-azido-6-chloropyridin-3-ylmethyl)-N'-methyl-N' '-nitroguanidine (2), were prepared in good yields as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors (nAChRs). The essential intermediate was 5-azido-6-chloropyridin-3-ylmethyl chloride (3) prepared in two ways: from 6-chloro-5-nitronicotinic acid by selective reduction and then diazotization, and from N-(6-chloropyridin-3-ylmethyl)morpholine by an electrophilic azide introduction with lithium diisopropylamide followed by chlorine substitution of morpholine with ethyl chloroformate. Coupling of 3 with 2-nitroiminoimidazolidine gave 1. Conversion of 3 to 2 was achieved in good yields via the hexahydrotriazine intermediate 14. Fortuitously, the azido substituent in 1 and 2 increases the affinity 7-79-fold for rat brain and recombinant alpha4beta2 nAChRs (K(i)s 4.4-60 nM competing with [(3)H](-)-nicotine) while maintaining high potency on both insect nAChRs (Drosophila and Myzus) (K(i)s 1-15 nM competing with [(3)H]imidacloprid). Azidopyridinyl compounds 1 and 2 are therefore candidate photoaffinity probes for characterization of both mammalian and insect receptors.

Animals↗
PubMed2000

Imidacloprid, thiacloprid, and their imine derivatives up-regulate the alpha 4 beta 2 nicotinic acetylcholine receptor in M10 cells.

Neonicotinoids are the most important new class of insecticides of the last decade. They act as nicotinic acetylcholine receptor (AChR) agonists. This investigation tests the hypothesis for the first time that neonicotinoid insecticides and their imine derivatives up-regulate the alpha 4 beta 2 nicotinic AChR subtype, which represents >90% of the high-affinity [(3)H]nicotine binding sites in mammalian brain. The alpha 4 beta 2 receptor stably expressed in mouse fibroblast M10 cells was assayed after 3 days' exposure to the test compound, as [(3)H]nicotine binding following immunoisolation by monoclonal antibody (mAb 299) or as [(125)I]mAb 299 labeling for cell surface receptors. We found that imidacloprid (IMI) (one of the most important insecticides) and thiacloprid (THIA) increased [(3)H]nicotine binding levels (up-regulation of the alpha 4 beta 2 AChRs) by five- to eightfold with EC50s of approximately 70,000 and 19,000 nM, respectively, compared with 760 nM for (-)-nicotine. In contrast, two imine analogs [the desnitro metabolite of IMI (DNIMI) and the descyano derivative of THIA] gave up-regulation by eightfold and EC50s of 870 and 500 nM, respectively. The potency order for up-regulation by the five aforementioned compounds was correlated with their in vitro IC50s for inhibiting [(3)H]nicotine binding (r(2) = 0.99, n = 5), indicating that binding to the alpha 4 beta 2 receptor initiates the up-regulation. A potent olefin derivative of the THIA imine up-regulated with an EC50 of 22 nM. DNIMI-induced up-regulation mainly occurred intracellularly rather than at the cell surface. These findings in alpha 4 beta 2-expressing M10 cells indicate the possibility that some neonicotinoid insecticides or their metabolites, on accidental human exposure or when used for flea control on dogs, may also up-regulate the receptor in mammals.

Animals↗
PubMed2000

Alpha-thujone (the active component of absinthe): gamma-aminobutyric acid type A receptor modulation and metabolic detoxification.

Alpha-thujone is the toxic agent in absinthe, a liqueur popular in the 19th and early 20th centuries that has adverse health effects. It is also the active ingredient of wormwood oil and some other herbal medicines and is reported to have antinociceptive, insecticidal, and anthelmintic activity. This study elucidates the mechanism of alpha-thujone neurotoxicity and identifies its major metabolites and their role in the poisoning process. Four observations establish that alpha-thujone is a modulator of the gamma-aminobutyric acid (GABA) type A receptor. First, the poisoning signs (and their alleviation by diazepam and phenobarbital) in mice are similar to those of the classical antagonist picrotoxinin. Second, a strain of Drosophila specifically resistant to chloride channel blockers is also tolerant to alpha-thujone. Third, alpha-thujone is a competitive inhibitor of [(3)H]ethynylbicycloorthobenzoate binding to mouse brain membranes. Most definitively, GABA-induced peak currents in rat dorsal root ganglion neurons are suppressed by alpha-thujone with complete reversal after washout. alpha-Thujone is quickly metabolized in vitro by mouse liver microsomes with NADPH (cytochrome P450) forming 7-hydroxy-alpha-thujone as the major product plus five minor ones (4-hydroxy-alpha-thujone, 4-hydroxy-beta-thujone, two other hydroxythujones, and 7,8-dehydro-alpha-thujone), several of which also are detected in the brain of mice treated i.p. with alpha-thujone. The major 7-hydroxy metabolite attains much higher brain levels than alpha-thujone but is less toxic to mice and Drosophila and less potent in the binding assay. The other metabolites assayed are also detoxification products. Thus, alpha-thujone in absinthe and herbal medicines is a rapid-acting and readily detoxified modulator of the GABA-gated chloride channel.

Absinthe↗
PubMed2000

Regional modification of [(3)H]Ethynylbicycloorthobenzoate binding in mouse brain GABA(A) receptor by endosulfan, fipronil, and avermectin B(1a).

[(3)H]Ethynylbicycloorthobenzoate ([(3)H]EBOB), a high affinity radioligand for the noncompetitive blocker site of the GABA(A) receptor, is used here for quantitative autoradiography to determine regional binding in mouse brain and the effects on this binding of administering toxic doses of endosulfan, fipronil, and avermectin B(1a) (AVM). Animals were euthanized 4-8 min after 1 LD50 or 2 LD50 doses of the two channel blockers and 32 min after 1 LD50 or 4 LD50 doses of the channel activator AVM. Specific binding of [(3)H]EBOB was determined for 20-microm brain sections as the difference in labeling on incubation with 2 nM [(3)H]EBOB either alone (total binding) or with 5 microM alpha-endosulfan (nonspecific binding). The highest specific labeling was observed for layers I and IV of the cerebral cortex, the globus pallidus, and the medial septal nucleus/nucleus of the vertical limb of the diagonal band. Dose-dependent inhibition by endosulfan was highest in the nucleus accumbens and least in the cerebellum and periaqueductal gray matter. Fipronil had much less effect on binding even at severely toxic doses. AVM increased [(3)H]EBOB binding in most regions and was the only one of the three agents inhibiting in vitro [(3)H]strychnine binding to the glycine receptor. In summary, the noncompetitive blocker site was strongly inhibited with dose dependence and regional selectively by alpha-endosulfan but was generally poorly inhibited or activated by fipronil and was activated by avermectin.

Animals↗