Mira Škrinjarić-Špoljar

Response of hepatic microsomal mixed-function oxidases to various types of insecticide chemical synergists administered to mice

Abstract For several hours after a single intraperitoneal (i.p.) dose to mice, piperonyl butoxide, 2-methylpropyl 2-propynyl phenylphosphonate (NIA 16824), and 5,6-dichloro-l, 2,3-benzothiadiazole (WL 19255) inhibit hepatic microsomal mixed-function oxidase (mfo) activity and the apparent level of cytochrome P-450 but, after 24–72 hr, they induce increased mfo activity and P-450 content. Other types of insecticide chemical synergists studied are generally less active or inactive in this respect. The active compounds enhance the toxicity of several insecticide chemicals and markedly prolong hexobarbital sleeping time but not hexobarbital toxicity. The action of WL 19255 differs from that of piperonyl butoxide and NIA 16824 in that it is relatively non-specific in inhibiting enzymatic activity on several substrates, gives a more prolonged effect on P-450, and the magnitude of induction is greater.

Spontaneous reactivation and aging of dimethylphosphorylated acetylcholinesterase and cholinesterase

Abstract Spontaneous reactivation and aging of dimethylphosphorylated acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from human and bovine erythrocytes and rat brain, and cholinesterase (acylcholine acyl-hydrolase, EC 3.1.1.8) from human and rat plasma was studied (pH 7.4; 37°C), after inhibition by O,O- dimethyl-4-nitrophenyl phosphate or O,O- dimethyl-2,2-dichlorovinyl phosphate. The first-order rate constants obtained for spontaneous reactivation ( k r are 0.0136, 0.0092 and 0.00606 min−1 for acetylcholinesterase from human and bovine erythrocytes and rat brain, and 0.00504 min−1 for rat plasma cholinesterase. For human plasma cholinesterase, k r is ≥ 0.00014 min −1 . The rate of aging was calculated from the amount of enzyme which could not be reactivated with oximes. Aging of dimethylphosphorylated human erythrocyte and rat brain acetycholinesterase follows the kinetics of a first-order reaction with rate constants of aging ( k ag ) of 0.00297 and 0.00172 min−1, respectively. For rat plasma cholinesterase, k ag is about 0.00058 min−1, and this value was obtained from only two differeent times of aging. Due to aging, the kinetics of spontaneous reactivation deviates from first order. A theoretical equation is derived for calculating the time course of spontaneous reactivation. The agreement between theory and experiment was satisfactory when the time course was calculated using the experimentally obtained constants k r and ag .

Mechanism of inhibition in vitro of mammalian acetylcholinesterase and cholinesterase in solutions of O,O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate (trichlorphon)

Abstract The rate of decomposition of trichlorphon into DDVP was measured polarographically at pH 7.4. The first order rate constants of decomposition at 25° are 7.27 × 10 −4 and 6.05 × 10 −4 min −1 for trichlorphon concentrations of 0.150 and 15.0 mM respectively; at 37° the corresponding rate constants are 53.1 × 10 −4 and 37.1 × 10 −4 min −1 . The rate of decomposition of trichlorphon was also calculated from the kinetics of inhibition of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) in trichlorphon solutions at 25° and 37° (pH 7.4). The following enzyme sources were used: bovine erythrocytes and rat brain acetylcholinesterase, and human, horse and rat plasma cholinesterase. The rate of decomposition of trichlorphon was calculated by assuming that only DDVP formed from trichlorphon is the enzyme inhibitor, while trichlorphon itself does not act as an inhibitor. The calculated rate constants for the decomposition of trichlorphon are lower or just within the range of the rate constants obtained by the polarographic method. This agreement was taken as kinetic evidence that trichlorphon is not an inhibitor of mammalian cholinesterases. The effect of pH on enzyme inhibition supports this conclusion. The rate of inhibition of bovine erythrocyte acetylcholinesterase by DDVP is the same at pH 7.4 and pH 6.0 (37°). However, the rate of enzyme inhibition in trichlorphon solutions is 30 times faster at pH 7.4 than at pH 6.0, and this agrees with the greater stability of trichlorphon at the lower pH value. The rate of spontaneous reactivation of the enzyme was measured (37°, pH 7.4) after inhibition in trichlorphon solutions of acetylcholinesterase (human and bovine erythrocytes) and cholinesterase (human plasma). For all three enzyme preparations, the rate of spontaneous reactivation was the same as that obtained after inhibition by DDVP. All results point to the conclusion that trichlorphon in vitro is not an inhibitor of mammalian cholinesterases.

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.

Quinuclidinium-imidazolium compounds: synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice

Abstract Four compounds were prepared: 3-oxo-1- methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH2)3- (III) and -CH2-O-CH2- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD50) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD50 dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD50 doses of Soman.

3-Hydroxyquinuclidinium derivatives: synthesis of compounds and inhibition of acetylcholinesterase

Four compounds were prepared: 3-hydroxy-1-methylquinuclidinium iodide (I), 3-(N,N-dimethylcarbamoyloxy)-1-methylquinuclidinum iodide (II), and two conjugates of I and II with 2-hydroxyiminomethyl-3-methylimidazole in which two parts of the molecule were linked by -CH2-O-CH2- (III and IV). III and IV are new compounds and their synthesis and physical data were given. All compounds were tested as inhibitors of human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE). The enzyme activity was measured in 0.1 M phosphate buffer (pH 7.4) at 10 and 37 degrees C with acetylthiocholine (ATCh) as the substrate. The obtained enzyme/inhibitor dissociation constants were between 0.05 and 0.5 mM at 10 degrees C and between 0.2 and 0.6 mM at 37 degrees C. At both temperatures compounds III and IV had higher affinities for the enzyme than compounds I and II and this difference was more pronounced at 10 than at 37 degrees C. The carbamates II and IV were also progressive AChE inhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M(-1) min(-1) at 37 and 10 degrees C, respectively. Compound IV was a very weak carbamoylating agent with rate constants of inhibition of 100 and 63 M(-1) min(-1) at 37 and 10 degrees C, respectively. The oxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M(-1) min(-1) at 37 and 10 degrees C, respectively.

Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of radiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of 14C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) alpha-D- glucopyranosides and methyl 2-acetamido-2-deoxy-3,6- di-O-pivaloyl-(3,6-DPNAc) alpha-D-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCl2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgCl2 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (KM and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.