Adam Vincze

Oxidative biodegradation of phosphorothiolates by fungal laccase

Organophosphorus (OP) insecticides and nerve agents that contain P‐S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl‐Amiton with specific activity: k sp=2200, 667 and 1833 nmol min−1 mg−1, respectively (pH 7.4, 37°C). A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX. The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52‐fold slower degradation of VX with k sp=42 nmol min−1 mg−1. The enzymatic biodegradation products were identified by 31P‐NMR and GC/MS analysis.

Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.

N1-(2-hydroxyethylthioethyl)-4-methyl imidazole (4-met-1-imid-thiodiglycol) in plasma and urine: a novel metabolite following dermal exposure to sulphur mustard

There is increasing interest in the mode of action and metabolism of alkylating agents and specifically sulphur mustard (HD). We report the detection of a new metabolite, 4-met-l-imid-thiodiglycol, formed following dermal exposure of pigs to HD. Alkylating agents are electrophilic in nature and react in vivo with N7 of guanine moieties in nucleic acids (Brookes et al. 1960). Proteins are predominantly alkylated on the cysteine, histidine and N-terminal amino acids (Bailey et al. 1987). Several alkylated metabolites of HD have been previously reported in the literature (Davison et al. 1960). The principal reaction product of DNA with HD, isolated from mice bearing Ehrlich ascites tumor, is 7-(2-hydroxyethylthioethyl) guanine (Brookes et al. 1960). The major metabolites in urine after intraperitoneal injection of (S35)-HD to rats are conjugates of 2,2-dicysteinyl ethyl sulphone (Roberts et al. 1963). There have been no case reports or pharmacological studies reporting the formation of alkylated histidine by HD. However, it is known that histidine in hemoglobin may undergo alkylation processes like 2-hydroxyalkylation following exposure to propylene oxide and ethylene oxide (Bailey et al. 1987). In the search of metabolites following dermal exposure of HD in pigs we have isolated a previously unknown compound. It was isolated from plasma and urine samples of pigs by extraction and purification on a C18 Sep-Pak column. Various modes of FAB/MS (fast atom bombardment/mass spectrometry) methods were used for identification. Low resolution measurements and linked scans were obtained on a VG 7035 MS instrument equipped with a Xenon gun. For high resolution measurement the VG 70 VSEQ with a Cesium gun was used.

Mass spectrometric investigation of the presence of 7-methyl ring-opened guanine derivatives in urine

The involvement of chemical alkylating agents in tumorigenesis and chemotherapy is well established and it was shown that one of the main sites of alkylation is the N-7 of guanine in DNA. Though excision of damaged bases is regarded as one of the repair mechanisms in damaged DNA there is a scarcity of information concerning the excised final metabolites in body fluids. This study attempts to demonstrate the usefulness of CAD MS/MS for the detection of the final metabolite-deformylated ring-opened 7 alkylguanine in urine. Such mass spectrometric methods can be used in biomedical studies.

The Use of Fungal Laccase for Oxidation of Phosphorothiolates

Enzymatic degradation of organophosphorus (OP) insecticides and nerve agents involves the hydrolytic breakdown of the bond between the phosphorus atom and either O-alkyl, 0-aryl (P-OR) or halogen (P-X) moieties. These moieties also serve as leaving groups during irreversible inhibition of acetylcholinesterase (AChE) by these OP’s. However, the P-S bond in phosphorothiolates is more resistant to enzymatic hydrolysis. So far, phosphorothiolates were decomposed rapidly only by chemical oxidation. In the current work we have successfully employed enzymatic oxidation for the rapid degradation of P-S containing OP’s. Degradation kinetics was followed by measuring the residual inhibitory activiy of AChE by the OP compound at specified time intervals. Purified laccase isolated from the white rot fungus Pleurotus ostreatus (Po) together with as 2,2’ azinobis (3-ethylbenzthiazoline - 6-sulfonate) (ABTS) as a mediator caused rapid degradation of the persistent nerve agent 0-ethyl S-[N,N-diisopropylaminoethyl] methylphosphonothiolate (VX) (t½=10 min with 6µg/ml enzyme, ksp = 2200 nmole min-1mg-1) and its structural analog 0,0-diethyl S-[N,N-diisopropylaminoethyl] phosphorothiolate (DiPr-Amiton) (ksp =1833 nmole min-1mg-1). The optimal pH for VX degradation by Pleurotus laccase and ABTS was 7.4 whereas DiPr-Amiton was decomposed at a higher rate at pH 8. The maximal rate of VX and DiPr-Amiton degradation by Po laccase and ABTS was obtained with ABTS:OP molar ratio of 20:1 and 10:1, respectively. The complete degradation of both optical isomers of VX and identification of oxidative biodegradation products were displayed by 31P NMR and GC/MS analysis. A thermostable laccase purified from the fungus Chaetomium thermophylium (Ct) in the presence of the mediator ABTS (ABTS:VX, 20:1) caused a 52 fold slower degradation of VX than by Po laccase (t1,2 =16 min, with 1 mg/ml enzyme in phosphate buffer 0.05M, pH 7.4, 37°C). The specific activity (ksp) of laccase from Ct for the degradation of VX was 42 nmole min-1mg-1. Thus, fungal

Sequestration of Toxic Phosphorylated Oximes by Stoichiometric and Catalytic Scavengers

Several quaternary oxime reactivators of phosphorylated cholinesterases (ChE) are effective antidotes in organophosphates (OP) toxicity. Reactivation of OP-inhibited ChEs probably proceeds via nucleophilic displacement which causes the accumulation of a phosphorylated oxime (POX) intermediate. Several POXs that were generated in situ over the last 4 decades from a variety of oxime antidotes and OPs, have been demonstrated to be more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs (1, 2). Their rapid removal is expected to improve therapeutic management of OP toxicity by oxime antidotes (1). In order to examine the possibility of augmenting the endogenous detoxification of POXs by exogenous stoichoimetric (e. g., ChE) or a catalytic (e. g., phosphotriesterase; PTE) scavenger, the diethylphosphoric acid esters of 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) and of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) were synthesized and their structure and homogeneity was determined by 1H, 31P, MS, and UV spectroscopies. DEP-4PAM was fully characterized as inhibitor of butyrylcholinesterases (BChE) and AChEs, and as a substrate of a PTE purified from Pseudomonas sp. In addition, the stability of the POXs and their decomposition pathways in aqueous solutions were studied by product identification. DEP-4PAM has t1/2 of 11 min in phosphate buffer pH 8.0, and was found to inhibit ChEs with the following ranking order: human BChE (7.1×108 M−1 min−1) »equine BChE > mouse AChE > fetal bovine serum AChE. PTE-induced hydrolysis of DEP-4PAM was recorded at kcat/Km = 3.6×107 M−1 min−1 with Km = 0.78 mM.

Chemical and Biochemical Studies on the Conversion of Alkylating Agents to Phosphorothiolates and their Subsequent Sequestration by Cholinesterases

Abstract In endeavor to develop a universal, mild decontamination solution of vesicants and nerve agents, we have examined the possibility of transforming bis(β-chloroethyl)sulfide (sulfur mustard; HD) to an organophosphorus (OP) compound that will have a significant preference for inhibition of human butyrylcholinesterase (HuBChE) compared with acetylcholinesterase (AChE).

Gas-chromatographic quantitation of lindane in household insecticide spray cans

ZusammenfassungDas Treibmittel wird durch Ausfrieren abgetrennt und die Insecticidlösung gas-chromatographisch mit Electron-capture Detektor analysiert. Das reine α-Isomere dient als innerer Standard. Der Fehler beträgt ± 5%.SummaryA method was developed for the determination of the lindane content of commercial household insecticide spray aerosols. After the propellant is removed from the chilled contents of the spray packages the hydrocarbon solution is analysed for its lindane content by electron-capture gas chromatography. Pure α-isomer of hexachlorocyclohexane serves as the internal standard. Experimental error is ± 5%.