Inhibitor Report for: O-hexyl-O-2,5-dichlorophenyl-phosphoramidate

This study shows the EDTA-resistant, Ca(2+) and Cu(2+)-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 microM HDCP in presence of 100 or 300 microM Cu(2+), or 2.5 mM Ca(2+) or 5 mM EDTA at 37 degreesC for 30-60 min. The results shown an activator effect of Cu(2+) on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (-37 microM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed -50% less HDCPase activity (13-17 microM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7-13 microM DCP) with both copper concentrations. These Cu(2+)-dependent hydrolysis were stereospecific to R(+)-HDCP (p0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca(2+) did not show a significant activating effect on the HDCPase activity (1-8 microM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu(2+)-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.

The Ca(2+)-dependent and EDTA-resistant hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and paraoxon was studied in serum and subcellular fractions of liver, kidney and brain of hen, rat and rabbit. HDCP was the best substrate among all the tissues studied, except that of rabbit serum which showed the highest Ca(2+)-dependent paraoxon hydrolysing activity (paraoxonase). High HDCP hydrolysing activity (HDCPase) was detected in the brain tissue of the three species studied, whereas low or no paraoxonase was found. The HDCPase/paraoxonase ratio of Ca(2+)-dependent hydrolysing activities ranged from 0.5 to 83 for tissues of the same species. EDTA-resistant HDCPase activity was more than 50% of the total activities in hen tissues, with an almost undetectable Ca(2+)-dependent paraoxonase activity in most organs. The same response was observed in rat tissues, except for serum where the Ca(2+)-dependent HDCPase and paraoxonase activities were higher (70 and 25% of total activities, respectively). EDTA-resistant HDCPase and paraoxonase activities represented less than 25% of all activities in rabbit tissues. Paraoxon has traditionally been the substrate for measuring organophosphorus hydrolysing activities. However, HDCP could be a good substrate in addition to paraoxon for monitoring other phosphotriesterases in biological tissues.

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.

This study shows the EDTA-resistant, Ca(2+) and Cu(2+)-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 microM HDCP in presence of 100 or 300 microM Cu(2+), or 2.5 mM Ca(2+) or 5 mM EDTA at 37 degreesC for 30-60 min. The results shown an activator effect of Cu(2+) on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (-37 microM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed -50% less HDCPase activity (13-17 microM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7-13 microM DCP) with both copper concentrations. These Cu(2+)-dependent hydrolysis were stereospecific to R(+)-HDCP (p0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca(2+) did not show a significant activating effect on the HDCPase activity (1-8 microM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu(2+)-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.

O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) is a racemic organophosphate compound (OP) that induces delayed neuropathy in vivo. The O-hexyl 2,5-dichlorophenyl phosphoramidate R (R-HDCP) isomer inhibits and ages neuropathic target esterase (NTE) in hen brain. Moreover, human serum paraoxonase-1 (PON1) is a Ca(2+)-dependent enzyme capable of hydrolyzing OPs. The enzymatic activity of PON1 against OPs depends on the genetic polymorphisms present at position 192 (glutamine or arginine). The catalytic efficiency of PON1 is an important factor that determines neurotoxic susceptibility to some OPs. In the present study, we characterized the stereospecific hydrolysis of HDCP by alloforms PON1 Q192R human serum by chiral chromatography. Forty-seven human samples were characterized for the PON1 192 polymorphism. The hydrolysis data demonstrate that the three alloforms of PON1 show an exclusive and significant stereospecific Ca(2+)-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate S isomer (S-HDCP) at 19-127 microM at the concentrations that remain in all the samples. This stereoselective Ca(2+)-dependent hydrolysis of S-HDCP is inhibited by EDTA and is independent of the PON1 Q192R alloform. The present research reinforces the hypothesis that R-HDCP (an isomer that inhibits and causes NTE aging) is the enantiomer that induces delayed neuropathy by this chiral phosphoramidate due to the low hydrolysis level of the R-HDCP observed in this study.

The Ca(2+)-dependent and EDTA-resistant hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and paraoxon was studied in serum and subcellular fractions of liver, kidney and brain of hen, rat and rabbit. HDCP was the best substrate among all the tissues studied, except that of rabbit serum which showed the highest Ca(2+)-dependent paraoxon hydrolysing activity (paraoxonase). High HDCP hydrolysing activity (HDCPase) was detected in the brain tissue of the three species studied, whereas low or no paraoxonase was found. The HDCPase/paraoxonase ratio of Ca(2+)-dependent hydrolysing activities ranged from 0.5 to 83 for tissues of the same species. EDTA-resistant HDCPase activity was more than 50% of the total activities in hen tissues, with an almost undetectable Ca(2+)-dependent paraoxonase activity in most organs. The same response was observed in rat tissues, except for serum where the Ca(2+)-dependent HDCPase and paraoxonase activities were higher (70 and 25% of total activities, respectively). EDTA-resistant HDCPase and paraoxonase activities represented less than 25% of all activities in rabbit tissues. Paraoxon has traditionally been the substrate for measuring organophosphorus hydrolysing activities. However, HDCP could be a good substrate in addition to paraoxon for monitoring other phosphotriesterases in biological tissues.

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.

Human serum (HS) and human serum albumin (HSA) were able to hydrolyse the carbamate carbaryl. Carbarylase activity found in HSA was slightly activated by 1 mM Zn2+, Mn2+, Cd2+, Ni2+ and Na+ and by 0.01 mM Pb2+. The organophosphorus compounds paraoxon and O-hexyl O-2,5-dichlorophenyl phosphoramidate, caprylic acid, palmitic acid and the carboxyl ester p-nitrophenyl butyrate inhibited the hydrolysis of carbaryl by HSA, being in the last case a competitive inhibition. Using selective amino acid reagents, we concluded that Cys, Trp, Arg and Tyr seem to play important roles in the carbarylase activity of HSA. In addition, Tyr and Arg seem to be located in the active centre of the enzyme since carbaryl protected the activity from the inhibition. It was concluded that HSA hydrolyses carbaryl by a mechanism similar to that described for rabbit serum albumin based in transient carbamylation of a Tyr residue. The extrapolation of the hydrolysis rate to physiological albumin concentrations suggests that albumin might be playing a critical role in the detoxication of carbaryl.

Neuropathy target esterase (NTE), thought to be the target for organophosphate polyneuropathy, is operationally defined as that neural phenyl valerate esterase resistant to paraoxon (40 microM) and sensitive to mipafox (50 microM; 20 min, pH 8.0, 37 degrees C). The time course of inhibition of particulate paraoxon pretreated esterases by mipafox showed that the lines indicating the rate of inhibition did not pass through the log 100% activity when extrapolated at zero time. Slopes of inhibition of NTE were not linearly related to the concentration of mipafox. Kinetic parameters derived from Wilkinson type plots were: Ka = 49-199 microM, k(+2) = 0.24-0.64 min(-1) and k(a) = 3.1-5.0 mM(-1) m(-1). When mipafox was removed (either by dilution or centrifugation) before the addition of phenyl valerate intercepts below 100% disappeared. We confirm that the formation of Michaelis complex between NTE and mipafox is not prevented by phenyl valerate and that inhibition proceeds after addition of phenyl valerate. We compared inhibitions obtained with experiments by using the traditional method (sequential incubation with inhibitors and phenyl valerate) to those obtained with a method where mipafox is removed before the addition of substrate. When calculating fixed-time 50% inhibitory concentrations (IC50s) of some inhibitors for NTE, the longer the hydrolysis time, the lower were the IC50s. Therefore, the inhibitory potency of certain NTE inhibitors, is accurately assessed only when calculating second-order rate constants (k(a)).

O-Hexyl O-2,5, dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. The chicken has very low activity of Ca-dependent organophosphorus-hydrolases (OP-hydrolases) such as paraoxonase. HDCP is degraded at a similar rate in rat and hen plasma (16 and 21 nmol/min/microliters plasma, respectively) when measured by the loss of its anti-cholinesterase potency (Diaz-Alejo et al., 1990). The time course of the HDCP hydrolysis was not significantly affected by the following treatments: (a) 0.5-1 mM Ca2+ or 1-10 mM EDTA added at 30 min before starting the reaction at 37 degrees C; (b) preincubation with a carboxylesterase inhibitor 100 microM diisopropyl phosphorosfluoridated (DFP) for 60 min at 37 degrees C; (c) preincubation with 100 microM HDCP for 60 min at 37 degrees C; and (d) the presence of 50 microM DCP. However, the hydrolysis of HDCP was slightly modified by the other product of its hydrolysis. There is no contribution to the HDCP hydrolysis by covalent binding to carboxylesterase proteins. The course of the hydrolysis of HDCP was similar when measured by either the loss of anti-cholinesterase potency or the DCP liberated. HDCP is hydrolysed by an OP-hydrolase which is not Ca-dependent and is present in hen in contrast to the best known OP-hydrolases which are Ca-dependent and are undetectable in birds.

One of the main detoxification mechanisms of organophosphorus (OP) compounds is hydrolysis by OP hydrolysing enzymes (OP-hydrolases) or phosphoric triester hydrolases. We previously reported an OP-hydrolase from hen plasma which hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP). In this study, a total of 18 cations, as well as several thiol blocking reagents, ethylenediaminetetraacetic acid (EDTA) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) were assayed as activators or inhibitors of the HDCP hydrolysing activity of hen plasma in vitro. Of the 18 inorganic cations only 1 M Na+ caused any inhibition. Most of the cations, including Ca2+, exerted no detectable effect; however, 1 mM Cu2+ was found to produce an activation of up to 263%, with a lesser activation of up to 168% for 1 mM Zn2+. The thiol blocking reagents methyl vinyl ketone (MVK) and N-ethylmaleimide (NEM) inhibited the enzyme in a time-dependent manner, the maximum effect depending upon concentration in the case of NEM, but not in the case of MVK; however, 5,5'-dithiobis (2-nitrobenzoic acid) caused inhibition that was concentration dependent but which was independent of time. Other thiol blocking reagents such as p-hydroxymercuribenzoic acid (sodium salt), phenylmercuric acetate, iodoacetic acid (sodium salt) and iodoacetamide produced only slight inhibition, as did EDTA. Finally, the OP compound mipafox exerted no detectable effect.

The organophosphorus (OP) compound O-hexyl-O-2,5-dichlorophenyl phosphoramidate (H-DCP) is hydrolysed in the plasma, liver and brain of hens and rats. We study in hen plasma the effect of tissue and substrate concentrations and of pH on the hydrolysing activity of H-DCP. The data on each tissue concentration were fitted to a double exponential mathematical model. The kinetics of this activity was not linear; in a first exponential component or fast initial phase (k1 = (1.603 +/- 0.248) x 10(-3) min-1/microliter plasma (n = 4, S.E.)) about 15% of the total compound was hydrolysed, followed by a slow second phase (k2 = (0.144 +/- 0.026) x 10(-3) min-1/microliter plasma (n = 4, S.E.)) in which the remaining 85% was hydrolysed. Both constants increased in value with pH. The hydrolytic activity and rate constants increased with the amount of plasma, but no change in kinetics was observed. The kinetic data are discussed in terms to lend support to the hypothesis of a stereospecific degradation of H-DCP.