Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (
An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis [
CbE substrate, S-phenyl thioacetate (PSA), 98% purity; ChE substrate, acetylthiocholine iodide (AcTChI), 98% purity; and 5 5′-dithiobis(2-nitrobenzoic acid) (DTNB) were supplied by the Sigma Chemical Company. Malathion [S-1,2-bis(ethoxycarbonyl) ethyl O,O-dimethyl phosphorodithioate] was obtained from G.L. Industries (E) Ltd., Guwahati, India. All other reagents and solvents used in this paper were of analytical grade.
Blood samples from healthy quail, duck, and chick from local markets in Kirkuk were used in this paper. They were maintained in batches of 3–15 birds in cages with dimensions of 75
To obtain plasma, 1 mL blood samples were added to anticoagulant (EDTA, 7.2 mg, final concentration 0.18%) in 5 mL centrifuge tubes. Plasma was separated by centrifugation at 5000 g for 10 min [
Esterase enzyme activity was determined at room temperature 25°C by the Ellman method [
For the measurement of maximal inhibitory concentrations (IC20, IC50, and IC80) blood samples were inhibited for 30 min at room temperature 25°C with appropriate concentration malathion compound, depending on preliminary range finding tests [
The statistical analysis was performed using SigmaPlot version 11. Differences among groups were determined by one-way analysis of variance (ANOVA) followed by Fisher’s LSD test. All results are presented as mean
The effect of dilutions on CbE and ChE activities was determined in serum, plasma, and erythrocyte for quail, duck, and chick as described in above section of Materials and Methods (Figures
Esterase enzyme activities in plasma diluted for quail (a), duck (b), and chick (c). A, B denote significantly different between mean ChE with different dilutions (analysis of variance (ANOVA),
Esterase enzyme activities in erythrocyte diluted for quail (a), duck (b), and chick (c). Key to the figures is listed under Figure
Esterase enzyme activities in serum for quail (a), duck (b), and chick (c). Key to the figures is listed under Figure
Blood from erythrocyte was seen significant (
Pearson correlation coefficient and
Dilution | Plasma | Erythrocyte | Serum | ||||||
---|---|---|---|---|---|---|---|---|---|
Quail | Duck | Chick | Quail | Duck | Chick | Quail | Duck | Chick | |
1 : 5 | 0.219 | −0.111 | −0.402 | −0.648 | −0.063 | 0.112 | 0.411 | 0.102 | 0.071 |
(0.22) | (0.52) | (0.22) | (0.21) | (0.82) | (0.21) | (0.22) | (0.71) | (0.10) | |
1 : 10 | −0.337 | 0.081 | 0.123 | 0.253 | −0.580 | −0.326 | 0.026 | 0.209 | 0.138 |
(0.10) | (0.90) | (0.04) | (0.13) | (0.13) | (0.02) | (0.93) | (0.52) | (0. 06) | |
1 : 15 | 0.311 | −0.179 | −0.083 | 0.152 | −0.451 | −0.224 | 0.544 | −0.091 | 0.417 |
(0.31) | (0.32) | (0.21) | (0.54) | (0.12) | (0.74) | (0.04) | (0.92) | (0.09) | |
1 : 20 | −0.245 | 0.162 | 0.242 | 0.352 | −0.324 | 0.157 | 0.446 | −0.216 | 0.429 |
(0.74) | (0.13) | (0.12) | (0.43) | (0.24) | (0.55) | ( 0.04) | (0.02) | (0.18) | |
1 : 25 | 0.221 | 0.205 | 0.134 | −0.078 | −0.121 | −0.251 | 0.559 | −0.075 | 0.317 |
(0.11) | (0.89) | (0.43) | (0.13) | (0.42) | (0.65) | (0.32) | (0.08) | (0.12) |
Values in the table are the
Distribution of the individual values of CbE and ChE activities in all tested birds (quail, duck, and chick) and blood samples (plasma, serum, and erythrocyte) in dilutions (1 : 5, 1 : 10, 1 : 15, 1 : 20, and 1 : 25).
Regression analyses between CbE and ChE activities in all tested blood contents from quail, duck, and chick across different dilutions (1 : 5, 1 : 10, 1 : 15, 1 : 20, and 1 : 25).
Regression analyses between CbE and ChE activities in all tested birds for plasma, serum, and erythrocyte across different dilutions (1 : 5, 1 : 10, 1 : 15, 1 : 20, and 1 : 25).
Box plots of the range esterase enzyme activity reported to be taken in different dilutions (1 : 5, 1 : 10, 1 : 15, 1 : 20, and 1 : 25).
Concentration-inhibition curves for malathion in the serum for quail in presence of dilution 1 : 5, dilution 1 : 10, and dilution 1 : 15 were found to be the highest reaction rates in dilution 1 : 10 for long time (4 h), whereas reducing reaction 10 rates in dilution 1 : 10 for short time (1 h) (Figure
Malathion point estimates (IC20, IC50, and IC80;
Preincubation condition | Slop | IC20 ( |
Slop | IC50 ( |
Slop | IC80 ( |
---|---|---|---|---|---|---|
Long (4 h) | ||||||
Dilution (1 : 5) | 0.22 ± 0.655a | 3.15 ± 0.991a | 0.67 ± 0.126 | 1.1 ± 0.432a | 0.66 ± 0.718 | 1.1 ± 0.121 |
Dilution (1 : 10) | 1.4 ± 0.122 | 0.50 ± 0.654 | 0.89 ± 0.117 | 0.78 ± 0.121 | 0.64 ± 0.611 | 1.2 ± 0.211 |
Dilution (1 : 15) | 0.11 ± 0.418b | 6.3 ± 1.2b | 0.71 ± 0.112 | 0.98 ± 0.212 | 0.84 ± 0.801 | 0.83 ± 0.221 |
Short (1 h) | ||||||
Dilution (1 : 5) | 0.34 ± 0.372a | 2.1 ± 0.876a | 0.29 ± 0.501a | 2.4 ± 0.311a | 0.37 ± 0.48a | 1.9 ± 0.421 |
Dilution (1 : 10) | 0.66 ± 0.931 | 1.1 ± 0.765 | 0.27 ± 0.145 | 2.6 ± 0.911 | 0.29 ± 0.18 | 2.4 ± 0.987 |
Dilution (1 : 15) | 0.12 ± 0.583b | 5.8 ± 1.1b | 0.53 ± 0.196b | 1.31 ± 0.876 | 0.24 ± 0.41b | 2.9 ± 0.766 |
Values in the table are the mean ± SE of three individual serum esterase (CbE) for quail. For the measurement of IC20, IC50, and IC80, esterase enzyme was inhibited for 30 min at room temperature 25°C with appropriate concentrations for malathion compound. Then results were fitted with an exponential decay using SigmaPlot 11. a,bdenotes significant difference (analysis of variance (ANOVA),
Kinetic parameters for hydrolysis of esterase in various dilutions.
Preincubation condition | CbE | ChE | ||||
---|---|---|---|---|---|---|
|
|
|
|
|
|
|
Dilution (1 : 5) | 176 ± 15.3a | 16,765 ± 865a | 95.3a | 265 ± 23.4a | 2023 ± 232a | 7.6a |
Dilution (1 : 10) | 543 ± 21.3 | 12,765 ± 932 | 23.5 | 234 ± 32.4 | 17,165 ± 654 | 73.4 |
Dilution (1 : 15) | 753 ± 28.4 | 7653 ± 121b | 10.2 | 943 ± 71.2 | 1154 ± 111 | 1.2 |
Values in the table are the mean ± SE of three individual serum esterase (CbE and ChE) for quail. For the measurement of
Concentration-inhibition curves for malathion in serum for quail in the presence of dilution 1 : 5, dilution 1 : 10, and dilution 1 : 15. Three preincubation conditions were used: long (4 h) (a) and short (1 h) (b). Experimental data (symbols) and model output (lines) are fitted by single rectangular equation of a hyperbola using a SigmaPlot 11.
Two forms of esterase enzyme activities have been identified in mammalian blood. These are distinguished according to their substrate specificity [
The results of kinetic studies using thiol side esters for the esterase enzyme show that PSA for the CbE activity and AcTChI for the ChE activity (dilution 1 : 10) are the most suitable substrate for the enzyme kinetic. The somewhat smaller ratios at the higher concentrations (less than half at the IC80 compared to IC20) with dilution 1 : 10, but not dilution 1 : 15, suggest some saturation of the CbE in that range of the curve (Table
In conclusion, this is the first paper that provided original data concerning an enzymological dilution characterization in the blood samples from birds used for human consumption. This paper succeed in establishing a gentle dilution in all three tested birds (quail, duck, and chick) and blood (plasma, serum, and erythrocyte); the dilution 1 : 10 recorded higher esterase enzyme activity than other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25). Furthermore, our results also pointed at the importance of estimating different kinetic dilutions effects prior to using in birds as biomarker tools of environmental exposure to anti-CbE or anti-ChE pesticides intoxication. Finally, in spite of this paper, further studies are required under different laboratories and different OP pesticide compounds in order to improve and to increase our knowledge about this very interesting enzyme as a potential biochemical marker for pesticide compounds.
The author declares that there is no conflict of interests and he has no financial or personal relationships with other people or organizations that can inappropriately influence his work; there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in this paper.
The author wishes to thank the farmers who participated in this study and the animal health assistants and laboratory technicians for their expert work in collecting the samples and for their useful assistance. The author is also thankful to management and principal of College of Nursing, Kirkuk, for the providing facilities for present work.