Animal hygiene assessment by spontaneous reactivation and aging kinetics of pesticide compounds

The main aims of this paper were to investigate the rate of spontaneous reactivation of butyrylcholinesterase from plasma and serum of farm animals inhibited by the organophosphorus, chlorfenvinphos and trichlorfon. Organophosphorus are among the most toxic of all substances that cause poisoning in farm animals and are the most frequently encountered insecticides, commonly detected in agricultural products, animalderived foodstuffs, environmental samples and home use and represent a significant potential health risk.The first-order rate constants obtained for spontaneous reactivation (ks) for chlorfenvinphosand trichlorfon for plasma and serum was ranged between 0.012 to 0.811h -1 , while aging of phosphorylated butyrylcholinesterase follows the kinetics of a firstorder reaction with rate constants aging (ka) ranged between 0.004to 0.653 h -1 . Half-time (t1/2) for spontaneous reactivation and aging are higher in trichlorfon compared to chlorfenvinphos and ranged from 1.1 to 28.9h (sheep), 2.1 to 63 h (cattle) and 2.8 to 53.3 h (goat), respectively.

This phenomena spontaneous reactivation, which proceeds very slowly and depends on the leaving group of the original organophosphorus inhibitor, but on the residual substituted groups on the phosphorus atom and the source of butyrylcholinesterase enzyme [1,7].
However, organophosphorus-inhibited butyrylcholinesterase changes gradually into a nonreactivatable form on storage, then this phenomena is called aging. It was assumed that the aging might be caused by a migration of the phosphoryl group from an initial position to form more stable bond or by the elimination of serine phosphate to lose serine hydroxyl group.
Generally is accepted that spontaneous reactivation and aging mechanism for alkoxyl group of organophosphorus residue bound to butyrylcholinesterase [8,9]. Recently, the standard treatment for organophosphorus poisoning comprises a mixture of atropine, an oxime, e.g.
obidoxime. The oxime compounds that can reactivate organophosphorus-inhibited butyrylcholinesterase by attaching to the phosphorus atom of the organophosphorus compounds forming an oxime-phosphonate, which ruptures away from the butyrylcholinesterase molecule [1,10].The main aims of this paper were to investigate the rate of spontaneous reactivation of butyrylcholinesterase from plasma and serum of farm animals inhibited by the chlorfenvinphos and trichlorfon. As well as, to investigate the time course of aging of organophosphorus-inhibited butyrylcholinesterase from plasma and serum of farm animals.

MATERIALS AND METHODS
Samples were collected from healthy farm animals (sheep, cattle and goats) from local abattoirs were used in this paper. The samples (plasma and serum) were transported on ice to the laboratory for immediate processing. To obtain serum blood samples were allowed to clot for at least 2 h at 30 °C, after which they were centrifuged at 6000 g for 15 min. To obtain plasma 4 ml blood samples were added to anticoagulant heparin, 3.7 mg, 0.1% in 10 ml centrifuge tubes. Plasma was separated by centrifugation at 6000 g for 10 min [11][12][13].Butyrylcholinesterase activity was determined by the Ellman method [14], adapted for use with microtitre plates as described by Haigh et al. [15] using butyrylthiocholine iodideas substrate (2 mM final concentration) for measuring butyrylcholinesterase activity.

Statistical analysis
The method was used to calculate the means and standard errors (SE). Differences with P<0.05 were regarded to have statistical significance. The pseudo-first-order rate for constants k s and k a were calculated by a non-linear regression analysis [1,17].

RESULTS AND DISCUSSION
This is the first study, which compares the ks and ka of organophosphorus inhibited butyrylcholinesterase in the plasma and serums of farm animals. Phosphorylated butyrylcholinesterase is susceptible to spontaneous hydrolysis of an alkyl-ester bond, resulting in a negatively charged residue which is resistant towards nucleophilic attack [18,19]. The rate of ksto inhibit erythrocyte butyrylcholinesterase by chlorfenvinphos has been observed to be 0.92 min-1 for cattle [17,20], 0.347 h-1 for rat [21], while ks values to trichlorfon was 0.012 h-1 for human [21]. The ks and ka kinetics of butyrylcholinesterase inhibited by chlorfenvinphos and trichlorfonwere determined in plasma and serumfor sheep, cattle and goat using Ellman method as described in the section of Materials and Methods (Tables1 and 3) . Plasma ks and ka kinetic parameters for reaction between butyrylcholinesterase and two organophosphorus (chlorfenvinphos and trichlorfon) are shown in Table 1. The ks for the three animals was decreased according to the rank order of sheep >cattle >goat for chlorfenvinphos andtrichlorfon.

Volume 9, Issue 2, June 2014 , p.p(29-41) ISSN 1992 -0849
Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com 34 However, ka values in plasma for animals was reduced in order of cattle >goat >sheep for chlorfenvinphos and trichlorfon.The relative activity between chlorfenvinphos and trichlorfon in cattle found highest ks(4) and lowest in ka (0.5). Kinetic ks and ka of all tested animals in plasma gave no correlation between ka and ks (R2 < 0.24) ( Table 1).  The half time (t1/2) from plasma for ks and ka kinetic parameters for reaction among butyrylcholinesterase and (chlorfenvinphos and trichlorfon) are shown in Table 2.
All farm animals studied in this research, the half time for trichlorfon was higher than chlorfenvinphos for spontaneous reactivation, in contrast with the values of aging ( The letters between the chlorfenvinphos and trichlorfon is significantly different (ANOVA, P< 0.05).
Serum ks and ka kinetics for reaction among butyrylcholinesterase and two organ phosphorus (chlorfenvinphos and trichlorfon). The values of ks for animals were decreased according to the rank order of goat >sheep > cattle for chlorfenvinphos while in range sheep >goat >cattle for trichlorfon. Instead, first-order rate constants for ka values was decreased according to the rank order of goat >cattle >sheep for chlorfenvinphos while decreased: sheep >goat> cattle for trichlorfon. Chlorfenvinphos/trichlorfonwere1.9, 8.9 and 18.9, respectively for ks and 0.8, 3 and 50.8, respectively for ka. The comparison between the ks and ka kinetics in the serums of all tested animals gave also poor correlation between ka and ks (R2 < 0.22) ( Table 3). Half time (t1/2) from serum for ks and ka kinetic parameters for reaction among butyrylcholinesterase and two organophosphorus (chlorfenvinphos and trichlorfon) are shown in Table 4. Again the half time in all farm animals, the trichlorfon was higher than chlorfenvinphos for ks, while chlorfenvinphos for aging was much higher (5-times) than that of trichlorfon (Table4). Other than t1/2for aging was nearly similar in trichlorfon than chlorfenvinphos in goat (Table4). The letters between the chlorfenvinphos and trichlorfon is significantly different (ANOVA, P< 0.05).
Comparable to our work the chlorfenvinphos and trichlorfon was higher than above results.However, ks in the plasma proceeded substantially faster with goat chlorfenvinphos and trichlorfon compared with the sheep and cattle. While serum ks was faster with cattle exposed to chlorfenvinphos and trichlorfon compared with sheep and goat (Tables1 and 3). Literature values of the half time (t1/2) of ks for butyrylcholinesterase are about 2 and 58 h for organophosphorus in human erythrocyte [21,22]. The recovery of rate ka of reserved erythrocyte butyrylcholinesterase by organophosphorus was 2.62 x 104 min-1 and 7.77 x 104 min-1 for cattle and horse, respectively [16]. The serum trichlorfon-inhibited butyrylcholinesterase ka proceeded markedly later than ka of sheep, cattle and goats. In addition, t1/2 of ka for human erythrocyte butyrylcholinesterase is about 41 h for organophosphorus [21], concurring with our results which ranged between 21to 231 h in serum and ranged between 4.62 to 24.75 in plasma (

CONCLUSIONS
This research is provided original data concerning an enzymological characterization of these inhibitors in these farm animals. As well as in this research, we surveyed recent developments in our understanding of the kinetic properties of k s and k a for sheep, cattle and goat butyrylcholinesterase is comparable in view of interactions with chlorfenvinphos and trichlorfon.In plasma from all three animals studied (sheep, cattle and goats), the rate of the aging process is much slower than spontaneous reactivation of butyrylcholinesterase inhibited with both of them (chlorfenvinphos and trichlorfon). This was also true in serum with chlorfenvinphos and trichlorfon, although the difference was less. Hence, in the case of t 1/2 butyrylcholinesterase inhibited with trichlorfon the enzyme will tends to become much higher than chlorfenvinphos. The determination of reactivation and aging constants of chlorfenvinphos and trichlorfon organophosphorus pesticides with farm animals butyrylcholinesterase indicates that a structure activity relationship can be derived for inhibition as well as for spontaneous reactivation but not for aging and oxime-induced reactivation.