Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry.

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150mm×4.6mm, 5µm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3-0.6ng/mL), the limits of quantitation (LOQs, 1.0-2.0ng/mL) and recoveries (89.1%-108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.

Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.

A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.

Joint action between binary mixtures of chlordimeform and insecticides.

Chlordimeform (CDF) was tested for its ability to act jointly with an organophosphorus insecticide (parathion) and a series of carbamate insecticides. Three arthropod species were used as test subjects: the two-spotted spider mite, Tetranychus urticae; the German cockroach, Blatella germanica; and the flour beetle, Tenebrio castaneum. However, CDF antagonized the toxicity of parathion toward B. germanica while it acted in a greater than additive fashion toward T. castaneum. No combination of CDF and insecticide tested acted jointly toward T. urticae. Species-specific differences in sensitivity, absorption, metabolism, and mode of delivery account for the varying results. Metabolic studies of pairs of compounds using two radiolabeled carbamates showed that CDF altered the metabolic detoxification of both carbaryl and aldicarb. The results suggest that CDF may inhibit MFOs as its mode of action.

XAMI and DCDM, agonists at cAMP-associated octopamine receptors in cockroach nerve cord, produce centrally mediated antinociception in mice.

The ability of XAMI (2,3-xylylaminomethyl-2'-imidazoline), the most potent agonist of cAMP-associated octopamine-sensitive adenylate cyclase in cockroach (Periplaneta americana) nerve cord yet reported, and DCDM (N-demethylchlordimeform), a partial octopamine agonist in this preparation, to produce centrally mediated antinociception in mice was evaluated. The antinociception produced by these compounds was compared to that previously reported for p-octopamine, a phenylethylamine and endogenous mammalian hydroxyphenolic analog of norepinephrine. Consonant with the reported greater agonistic activity of XAMI on octopamine-sensitive adenylate cyclase, XAMI was more potent than p-octopamine by spinal or supraspinal administration in the abdominal constriction test (E50 = 0.013 micrograms i.t., 1.45 micrograms i.c.v.) and in the 48 degrees C hot-plate test (ED50 = 0.06 micrograms i.t., 0.4 micrograms i.c.v.), but was inactive in the tail-flick test (up to 4.0 micrograms i.c.v. or i.t.). Unlike p-octopamine, both XAMI and DCDM were active by peripheral routes of administration. DCDM was orally active in the mouse acetylcholine-induced abdominal constriction test (ED50 = 9.98 mg/kg p.o.) and was active via the s.c. route in this test (ED50 = 2.36 mg/kg), the 48 degrees C hot-plate test (ED50 = 5.40 mg/kg) and the tail-flick test (ED50 between 15 and 30 mg/kg). It appeared to be a full agonist against these endpoints. XAMI produced dose-related antinociception in the abdominal constriction test (ED50 = 0.10 mg/kg s.c.) and in the 48 degrees C hot-plate test (ED50 = 3.71 mg/kg p.o. and 0.46 mg/kg s.c.), where the antinociceptive response persisted for at least 60 min following subcutaneous or oral administration. Both compounds were less potent via peripheral routes than clonidine (as reference) in these tests. Mechanistically, XAMI-induced antinociception was antagonized by yohimbine and idazoxan, but not the opiate antagonist naloxone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 2-adrenoceptors as a target for formamidine pesticides: in vitro and in vivo studies in mice.

While the toxicity in insects of formamidines such as chlordimeform (CDM), its demethylated metabolite DCDM, and amitraz (AMZ) appears to involve activation of an octopamine-sensitive adenylate cyclase, their mechanism of action in mammals remains elusive. There is increasing evidence, however, that alpha 2-adrenoceptors might mediate certain effects of CDM, DCDM, and AMZ. In the present study, we investigated whether formamidines can interact directly with adrenoceptors in mouse forebrain both in vitro and after in vivo administration. Formamidines were potent inhibitors of the binding of [3H]clonidine to alpha 2-adrenoceptors with IC50's of 13 microM, 29 nM, and 130 nM for CDM, DCDM, and AMZ, respectively. Binding of [3H]yohimbine was inhibited with similar potencies. All compounds also inhibited with equal (CDM) or lower potency the binding of [3H]spiperone to dopamine D2 receptors and were weak inhibitors or inactive toward alpha 1- and beta-adrenoceptors, cholinergic muscarinic, GABAA, opiate mu, benzodiazepine, and histamine 1 receptors. Administration of formamidines to mice caused a dose-dependent decrease of [3H]clonidine binding. [3H]Clonidine binding returned to control values within 5 hr following administration of CDM and DCDM, but was still significantly decreased up to 48 hr after AMZ. Among different brain regions, [3H]clonidine binding was decreased to a larger extent in cerebral cortex, hippocampus, and midbrain. In vitro and ex vivo kinetic binding studies indicated that the effect of formamidines on alpha 2-adrenoceptors was due to a decrease in affinity and not to an alteration of the density of [3H]clonidine binding sites. The results of these biochemical studies support the hypothesis that alpha 2-adrenoceptors represent an important target for formamidine neurotoxicity in mammals.

HPLC determination of the metabolite 4-chloro-o-toluidine in the urine of workers occupationally exposed to chlordimeform.

A reversed phase HPLC method (UV detection) is described or the analysis of 4-chloro-o-toluidine in the urine of workers occupationally exposed to chlordimeform. The procedure involves extracting an alkaline hydrolysate of urine with hexane, evaporating off the solvent, and reconstituting the residue with an aqueous acetonitrile mobile phase.

Formamidine-mediated inhibition of rat platelet aggregation.

The formamidine pesticide chlordimeform (CDF) was a strong inhibitor of aggregation of rat platelets induced by collagen and arachidonic acid and was a weak inhibitor of that induced by ADP. With the exception of 4-chloro-o-formotoluidide which was an inhibitor of arachidonic acid-induced aggregation only, the CDF metabolites were without discernible effect on aggregation induced by these agents. Amitraz, another formamidine pesticide, inhibited arachidonic acid-induced aggregation but was without effect on that induced by collagen or ADP. Inhibition of collagen- and/or arachidonic acid-induced aggregation by formamidines was concentration-dependent. Although platelets underwent shape change, primary aggregation was markedly inhibited, and secondary aggregation was abolished in some cases. CDF, its two N-desmethyl metabolites, and octopamine, but not amitraz, caused significantly elevated levels of cyclic AMP in platelet rich plasma as compared to controls; however, this effect did not fully account for the action of formamidines on aggregation.

Interaction of formamidines with the platelet 5-hydroxytryptamine uptake system.

1. The activity of the insecticide/acaricide N'-(4-chloro-o-tolyl)-N,N-dimethylformamidine (chlordimeform), its two formamidine metabolites, N'-(4-chloro-o-tolyl)-N-methylformamidine (demethylchlordimeform) and N'(4-chloro-o-tolyl) formamidine (didemethylchlordimeform), and 116 other formamidines and related compounds as inhibitors of rat platelet 5-hydroxytryptamine (5-HT) uptake was studied. Though several formamidines were more active than chlordimeform (pI50 3.9), none was as potent as imipramine. Didemethylchlordimeform (pI50 4.4) was the most potent formamidine examined. 2. Inhibition of 5-HT uptake by chlordimeform was mixed. Moreover, chlordimeform inhibition of 5-HT uptake by reserpinized platelets was not significantly different from uptake by non-reserpinized platelets. 3. Chlordimeform and its two formamidine metabolites caused release of platelet 5-HT, and their potency as releasers paralleled their activity as uptake inhibitors. 4. Electron microscopy indicated that chlordimeform treatment changed platelet shape and size but apparently did not alter physical integrity of the membrane. 5. It was suggested that platelet 5-HT storage vesicles were the most probable site of formamidine action.

Uptake and persistence of pesticides in cultured human cells.

The uptake and persistence of 14C-labeled pesticides in cultur-s of human embryonic lung diploid cells were studied. Chlordimeform, DDT, parathion, aldrin and dieldrin were selected as test compounds. Results obtained from these experiments divided the chemical into two groups. The first group consisted of three chemical DDT, aldrin and dieldrin which are generally classified as persistent insecticides while the second group was composed of chlordimeform and parathion, generally considered to be non-persistent insecticides. The rate of initial cellular incorporation of first group was approximately 20 to 70 times greater than the non-persistent insecticides. During the course of an incubation, the amounts of pesticide taken up by the cells decreased gradually.