Improved electrochemical analysis of neuropathy target esterase activity by a tyrosinase carbon paste electrode modified by 1-methoxyphenazine methosulfate.

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Bioelectrochemical analysis of neuropathy target esterase activity in blood.

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

A stable preparation of hen brain neuropathy target esterase for rapid biochemical assessment of neurotoxic potential of organophosphates.

Neuropathy target esterase (NTE) is a molecular target for organophosphate-induced delayed neurotoxicity (OPIDN). This enzyme has proved to be an excellent tool for the assessment of neuropathic potential of organophosphates (OP), in particular by comparison of an OP inhibitory activity in vitro against NTE and acetylcholinesterase. A large-scale OP screening for delayed neurotoxicity was largely prevented by the lack of an available stable preparation of NTE. To obtain a stable NTE preparation the influence of intensive freezing and subsequent lyophilization of paraoxon-preinhibited (P2 + P3) hen brain membrane fraction on NTE properties has been studied using two neuropathic OP: mipafox and O,O-dipropyldichlorovinyl phosphate (PrDChVP). It was shown that lyophilization preserved a high NTE specific activity and did not alter the inhibitor characteristics of the enzyme. A long-term storage study showed that lyophilized NTE preparation exhibited inhibitory features actually identical to those of the native enzyme during 1 year and retained rather high specific activity; in this case some loss of NTE specific activity has been observed. Comparative studies of inhibition of the native and lyophilized NTE preparations by a model series of phenyl phosphonates RO(C6H5)P(O)ON=CClCH3 (R = alkyl), demonstrated a good correlation between the values pI50 obtained with both enzyme preparations as well as identical structure-activity relationships for the lyophilized and native enzymes. The results allow the conclusion that the obtained NTE preparation can be used as a standard, stable and readily available source of NTE for assessing the anti-NTE activity of OP.

A new approach for determination of neuropathy target esterase activity.

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

[O,O-dialkyl-S-bromomethylthiophosphates--inhibitors of mammalian choline- and carboxyl esterases: structure-activity relationship]. / O,O-dialkil-S-brommetiltiofosfaty--ingibitory kholin- i karboksiésteraz mlekopitaiushchikh: sviaz' "Struktura-aktivnost'".

The interaction kinetics of potential pesticides, O,O-dialkyl S-bromomethylthiophosphates (RO)2P(O) SCH2Br (R = Et, i-Pr, n-Pr, n-Bu, or n-Am) with acetylcholinesterase, butyryl cholinesterase, and carboxyl esterase from warm-blooded animals was studied. All the compounds irreversibly inhibit these esterases, with k1 (M-1 min-1) being 1.8 x 10(4) - 1.9 x 10(6) for acetylcholinesterase, 2.0 x 10(6) - 4.1 x 10(7) for the more sensitive butyryl cholinesterase, and 2.3 x 10(7) - 2.3 x 10(8) and higher for the most sensitive carboxyl esterase. By using the Hansch and Kubinyi technique of multiple regression analysis, we quantitatively analyzed the relationship between the structure and inhibiting activity of these substances toward acetylcholinesterase and butyryl cholinesterase. Hydrophobic interactions were found to be important for the inhibition of both enzymes but are more pronounced in the case of butyryl cholinesterase. On the other hand, steric factors were much more significant in the inhibition of acetylcholinesterase. For both enzymes, the steric hindrances affect the phosphorylation stage of the enzyme.

[Antiesterase activity and toxicity of O,O-dialkyl-S-ethoxycarbonylbromomethylthiophosphates]. / Antiésteraznaia aktivnost' i toksichnost' O,O-dialkil-S-étoksikarbonilbrommetiltiofosfatov.

The interaction of potential pesticides, O,O-dialkyl S-ethoxycarbonylbromomethylthiophosphates (RO)2P(O)SCH(Br)COOC2H5 (R = Et, i-Pr, n-Pr, n-Bu, n-Am, or n-Hx) with the esterases of warm-blooded animals [acetylcholinesterase (ACE), butyryl cholinesterase (BCE), and carboxyl esterase (CE)] was studied. The acute toxicities of these compounds for mice were determined. All the compounds were non-hydrolyzable by CE and capable of irreversible inhibition of all these esterases with ki (M-1 min-1) of 1.2 x 10(5)-6 x 10(6), 2.0 x 10(6)-1.5 x 10(8), and 2.0 x 10(8), respectively. By using multiple regression analysis, we found that the steric factor plays a significant role in the inhibition of ACE, with the steric hindrances manifesting themselves even at the sorption stage. On the other hand, hydrophobic interactions predominate in the case of BCE, while steric properties of its substituents exert a markedly weaker effect and manifest themselves at the phosphorylation stage. We suggested the presence of an electrophilic region in the active site of ACE, which can interact with the ethoxycarbonyl group of the thiophosphates under study. The decrease in toxicities and the affinities to BCE and CE were found to correlate with an increase in the length of n-alkyl substituents of the compounds studied. This suggests that the unspecific esterases play a significant role as a buffer system in the exhibition of toxic effects by the thiophosphates under consideration.

[Mechanism of action of 2-aryloxy-2-thio-1,3,2-oxazaphosphorinane pesticides]. / Issledovanie mekhanizma deistviia pestitsidov riada 2-ariloksi-2-tio-1,3,2-oksazafosforinana.

The interaction of 2-aryloxy-2-thio-1,3,2-oxazaphosphorinanes exhibiting nematocide, insecticide/acaricide, and synergetic activities with monoamine oxidases and the interaction of the corresponding oxones, 2-aryloxy-2-oxo-1,3,2-oxazaphosphorinanes, with various cholinesterases, carboxyl esterases, and monoamine oxidases were studied. We showed that the thioderivatives inhibited monoamine oxidases, whereas oxones, which are, as a rule, weak cholinesterase inhibitors, strongly inhibited carboxyl esterases of the American cockroach and were transformed with monoamine oxidases into the strong cholinesterase inhibitors, acyclic phosphamidates. This allowed us to explain the low toxicity of the thioderivatives, the high toxicity of the oxoderivatives, and the great difference in toxicities of thio- and oxocompounds in the 1,3,2-oxazaphosphorinane series. The capacity of thioderivatives to inhibit monoamine oxidases and of oxoderivatives and their further activation products to inhibit carboxyl esterases, i.e., both enzymes responsible for pyrethroid detoxication in insects, explains the synergetic activity of the 1,3,2-oxazaphosphorinane series.

Comparative studies of O,O-dialkyl-O-chloromethylchloroformimino phosphates: interaction with neuropathy target esterase and acetylcholinesterase.

Acetylcholinesterase (AChE) and neuropathy target esterase (neurotoxic esterase, NTE) are two major target enzymes for organophosphorus (OP) esters. The relative potency of an OP ester to react with AChE or with NTE in vitro correlates with its relative potency in vivo to cause acute toxicity (death) or organopohosphate-induced delayed neurotoxicity (OPIDN). On this basis extrapolation from in vitro to in vivo data now seems justifiable to predict risk of OPIDN. The kinetics of NTE and AChE inhibition by experimental pesticides of the general formula (RO)2P(O)ON=CClCH2Cl, where R = methyl, ethyl, isopropyl, propyl, isobutyl, butyl, pentyl, has been studied. Compounds with short R (methyl, ethyl) were shown to be far more potent inhibitors of AChE than NTE. Both anti-NTE activity, selectivity for NTE and, correspondingly, the propensity of compounds to cause OPIDN rise with increasing their hydrophobicity. A high value of ki(NTE)/ki(AChE) for R = pentyl suggests that this compound would have the potential to cause OPIDN at doses lower than the LD50. A quantitative structure-activity relationships (QSAR) analysis indicated that NTE and AChE have different structural and electronic requirements for their respective OP inhibitors.

[The inhibition of synaptic acetylcholinesterase by postsynaptic muscle relaxants]. / Tormozhnie sinapticheskoi atsetilkholinésterazy postsinapticheskimi myshechnymi relaksantami.

In experiments on narcotized cats records of contractions of the anterior tibial muscle in response to indirect stimulation showed that the mean dose of acetylcholine (Ach) blocking neuromuscular conduction and that of carbachole (Cch) for their injection into the corresponding femoral artery was 18.9 and 0.16 mumole/kg, respectively. In the period of partial blockade induced by depolarizing myorelaxants, the blocking dose of Ach reduced to a greater measure than that of Cch. Most nondepolarizing myorelaxants used in the experiment increased the blocking Ach dose less than the Cch dose. Benzoquinone possessing the highest antiacetylcholinesterase activity in vitro even reduced the blocking dose of Ach. This difference is apparently due to inhibition of the activity of synaptic acetylcholinesterase by the muscle relaxants.

[A comparative study of the interaction of phosphoric acid dichlorovinyl esters with a neurotoxic esterase from the brain of hens and rats]. / Sravnitel'noe issledovanie vzaimodeistviia dikhlorvinilovykh éfirov kislot fosfora s neirotoksichnoi ésterazoi mozga kur i krys.

In order to validate a rodent biochemical model of delayed neurotoxicity of organophosphates (OP) inhibition of rat and hen brain neurotoxic esterase (NTE) by some dichlorovinyl phosphates and phosphonates was studied in vitro and in vivo. It was shown that compounds investigated exhibited the similar inhibitory potency to NTE from both species in vitro, in addition rat and hen NTE showed the same sensitivity to variation of the structure of OP inhibitors. A good correlation was found between pI50 estimated with enzymes from rat and hen trains: r2 = 0.951, n = 18, p < or = 0.05. NTE activities were also measured in rat and hen brains after acute administration of various dosages of potent axonopathic compound dipropyldichlorovinyl phosphate. The results obtained indicate that difference in species susceptibility to neurotoxic action of OP, in particular the absence of ataxia in rats, is not caused by difference in target enzyme sensitivity to axonopathic organophosphates.

[Mechanism of action of thiophosphoroorganic insectoacaricides containing fragments of N-carbaalkoxylated amino acids]. / Issledovanie mekhanizma deistviia tiofosfororganicheskikh insektoakaritsidov, soderzhashchikh fragmenty N-karbalkoksilirovannykh aminokislot.

Toxicity and insecticide and acaricide activity of compounds (1) is significantly dependent on the nature of amino acid (n = 1 or 2) and substituents in carbamate and amino acid ester groups (R and R1). Investigation of interaction of these compounds with mammalian carboxylesterases, and the appropriate "oxones" with choline esterases of mammal and arthropoda revealed that the lower toxicity and activity of beta-alanine derivatives (n = 2) compared with glycine derivatives (n = 1) are due to the more rapid hydrolysis by carboxylesterases (detoxication). The low toxicity of dithiophosphonate with R = Me, R1 = Bu(i), n = 1 and the high toxicity of its isomer with R = Bu(i), R1 = Me, n = 1 are associated with the more rapid oxidative cleavage of isobutyl group in comparison with the other substituents, because detoxication occurs by the cleavage of R1 and activation--by that of R respectively.

A new non-depolarizing myorelaxant with high activity and specificity.

The curare-like activity and mode of action of tercuronium (p,p"-bistriethylammonium-p-terphenyl dibenzen-sulphonate) have been investigated in experiments with cats, rabbits, mice and pigeons as well as with rat phrenic diaphragm preparations. The curare-like activity of tercuronium was higher than that of tubocurarine and was close to that of pancuronium bromide. The curare-like activity of tercuronium was higher than that of tubocurarine and was close to that of pancuronium bromide. The neuromuscular blocking doses (ED95) of tercuronium, (+)-tubocurarine and pancuronium in cats, for example, were 0.08 microM/kg, 0.4 microM/kg and 0.04 microM/kg respectively. The time of development and duration of action were similar to those of (+)-tubocurarine and pancuronium. Tercuronium is a nondepolarizing myorelaxant. In experiments with cats the antagonism of neostigmine (0.1 mg/kg) to tercuronium was more pronounced than in the case of (+)-tubocurarine, pancuronium or gallamine. Tercuronium affected neither the arterial pressure nor the heart rate when given in neuromuscular blocking doses. Tercuronium did not block transmission through autonomic ganglia and had no atropine-like action. Only a 10-fold increase in the dose of tercuronium produced the ganglion blocking effect in cats. Under artificial respiration, cats and rabbits tolerated tercuronium in a dose 200 times exceeding its myoparalytic dose. It is concluded that tercuronium is distinguishable from (+)-tubocurarine by its high neuromuscular blocking activity as well as by its specificity and more pronounced neostigmine antagonism.

[Tercuronium, a new nondepolarizing myorelaxant with high activity and selective action]. / Terkuronii--novyi nedepoliarizuiushchii miorelaksant s vysokoi aktivnost'iu i izbiratel'nost'iu deistviia.

Tercuronium is p',p"-bis-triethylammonium-p-terphenyl dibenzosulfonate. As a curarelike agent tercuronium is 4--8 times as potent as (+)-tubocurarine. The time of the development and lasting of the blocking effect of tercuronium is approximately the same as that of (+)-tubocurarine. In a blocking dose tercuronium does not exert any effect either on the vegetative ganglia or arterial blood pressure. Partial blocking of the transmission through the vegetative ganglia as well as an insignificant and short-term drop of arterial blood pressure are recorded after intravenous injection of 10 myoparalytic doses of tercuronium. The antagonism of neostigmine against tercuronium was more pronounced than against (+)-tubocurarine, pancuronium and gallamine.