- In vitro effects of pesticide exposure on the activity of the paraoxonase-1 enzyme from sheep liver microsomes. - They are also used as nerve gases, such as sarin and soman. - Therefore, the effects of these compounds on enzymatic activity are well known. - In the present study, we investigated the in vitro effects of fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, and dichlorvos pesticides on sheep liver microsomal PON1. - The pesticides inhibited sheep liver PON1 in vitro, and the IC 50 values for these compounds were and 0.2 µ M, respectively.. - Key words: Paraoxonase, fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, dichlorvos, inhibition. - These materials have been shown to affect the growth of productivity and the specialization of cultures. - 1 It is well known that pesticides have neurotoxic effects. - and microorganisms such as bacteria. - These effects can range from simple irritation of the skin and eyes to more severe effects, such as affecting the nervous system and also causing cancer, Alzheimer’s disease, Parkinson’s disease, and other metabolic disorders. - 1 − 3 Recently, it has been reported that environmental factors are critical in the regulation of the immune system. - 4 The cause of Alzheimer’s disease is not known, although it is theorized to be caused by a genetic predisposition and/or certain environmental factors. - It is known that a toxicological mechanism is associated with enzymatic reactions in metabolism. - In particular, some enzymes, such as carbonic anhydrase, glucose 6-phosphate dehydrogenase, and other enzymes involved in glucose metabolism, including paraoxonase, are known drug targets. - PON1 and PON3 are expressed primarily in the liver, while PON2 is expressed in various tissues, including the brain, liver, and kidneys. - PON1 was the first member of this family to be identified, and it is the most thoroughly studied. - 6,7 PON1 is a liver and plasma enzyme that catalyzes the hydrolysis and inactivation of various organophosphates, including paraoxon and the insecticides parathion and chlorpyriphos as well as the nerve agents sarin and soman. - 8 In our study, the effects of the pesticides fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, and dichlorvos on PON1 enzyme activity were studied. - It is one of the members of the aryloxyphenoxypropionate herbicide family, which is used to control certain annual and perennial grass weeds in cereals, certain pulse crops, vegetables, and certain feed and forage crops. - It is a mixture of highly active isomers of cyhalothrin and is used to control a wide range of pests in a variety of applications. - It is used on a wide array. - 13 − 15 For instance, our laboratory group studied the in vitro effects of heavy metals on the activity of PON1 from human serum. - Consequently, this study was conducted to purify PON1 from sheep liver microsomes and investigate the in vitro inhibitory effects of some commonly used pesticides on this enzyme.. - It is involved in the detoxification of several organophosphorus insecticides. - PON1 is known to play a critical role in the protection of HDL and LDL particles from oxidation, the antioxidant effects against lipid peroxidation on cellular membranes, and the anti-inflammatory process. - In addition, it is reported to be associated with protective properties against cardiovascular diseases. - The PON1 enzyme has a significant role in the benign nature of HDL. - With a decrease in the amount of HDL, the amount and activity of the PON1 enzyme drop off. - Thus, an increase in the number of atherosclerotic lesions is observed due to oxidation of LDL. - In particular, free radicals formed by oxidative stress cause the expression of the PON1 enzyme to be reduced.. - Therefore, HDL synthesis will be insufficient and, consequently, the level of PON1 gene expression and the PON1 concentration in the serum will be reduced. - Some of these enzymes are known drug targets in the literature. - (2008) reported that intravenous anesthetics, such as propofol, ketamine, and etomidate, significantly inhibit human PON1 activity both in vitro and in vivo. - The authors determined the rank order based on the effects of drugs to be as follows: etomidate >. - One study examined the effects of EDTA, Mg 2. - p-hydroxymercuribenzoate (p-OH-MB), and phenyl mercuric acetate (PMA) on paraoxonase activity from human liver microsomes. - competitive inhibitors, Zn was noncompetitive, and the results were mixed for Hg. - They reported that variations in the expression and catalytic activity of the PON1 enzyme are associated with polymorphisms of the PON1 gene. - 28 It is well known that environmental conditions have important effects on all living things, including humans. - These unsuitable factors lead to genetic disorders and thus mutations and lack of expression of the enzymes, and they result in more than 100 diseases, including Alzheimer’s disease, cancer, and Parkinson’s diseases, that are specifically associated with oxidative stress (Figure 1). - 29,30 In this respect, studies on the structures and activities of the affected enzymes are important. - 31 − 34 In the present study, PON1 was purified with a specific activity of 1822.22 EU/mg proteins at an approximately 141-fold concentration from sheep liver microsomes (Table 1). - In the purification steps, we included the preparation of a microsomal pellet, ammonium sulfate precipitation, DEAE Sephadex A-50 anion exchange chromatography, and Sephadex G-100 gel filtration chromatography. - Finally, the impact of the pesticides fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, and dichlorvos on PON1 activity was determined. - Purification steps of sheep liver microsomes PON1.. - Although there are benefits associated with the use of pesticides, they are also potentially hazardous to humans, animals, other organisms, and the environment. - The inhibitory effects of fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, and dichlorvos on the activity of PON1 from sheep liver microsomes were determined in this study. - However, it is important to consider their role in the balance of nature. - This process can be with occurrence of all reactions of the metabolism, quickly. - It is well known that enzymes catalyze almost all chemical reactions in the metabolism of the living systems. - This could result in some changes in the metabolisms of living organisms, and cause detrimental effects for both the life kingdoms and the environment.. - Purification of the paraoxonase-1 enzyme was confirmed by SDS-PAGE analysis. - Paraoxone was used as a substrate in the studies. - A blind tube contained all of the reaction mixtures except for the enzyme solution.. - IC 50 values for inhibition of sheep liver microsomal PON1 by pesticides.. - DEAE-Sephadex A50, 1-naphthylamine, paraoxon, the protein assay reagents, and the chemicals for elec- trophoresis were obtained from Sigma-Aldrich (Germany). - The PON1 activity assay was performed according to our previous study 14 at 25 ◦ C, and diethyl p-nitrophenyl phosphate (paraoxon) was used as a substrate. - Preparation of the microsomal samples. - Briefly, sheep livers (20 g) were removed, placed in beakers on ice, rinsed with an ice-cold homogenization buffer (including 0.25 M sucrose, 5 mM Tris/HCl pH 7.4), minced with scissors, and then placed in 4-fold of the same ice-cold buffer. - The homogenate was centrifuged at 460 × g for 10 min, and the precipitate was discarded. - The supernatant was centrifuged at 12,500 × g for 60 min, and the precipitate was discarded. - The microsomal pellet derived from the sheep liver tissue was suspended in a 5 mM Tris/HCl buffer (pH 7.4).. - Purification of sheep liver microsomes PON1. - The eluted fractions were collected, and the enzyme activity was checked at 412 nm. - 40,41 Protein standards and PON1 samples from sheep liver microsomes were loaded into each slot of the stacking gel (slab gel dimensions: 16 × 18 cm). - The gels were stained for 1.5 h in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid and destained with methanol/acetic acid. - We examined the inhibitory effects of several commonly used pesticides: fenoxaprop-p-ethyl, lambda cy- halothrin, imidacloprid, and dichlorvos. - The PON1 activities were measured in the presence of different concentrations of pesticides. - The control ac- tivity was assumed to be 100% in the absence of any inhibitor. - For each pesticide, a graph of the percent of activity versus the drug concentration graph was drawn, and the IC 50 values were calculated.. - in vitro
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