The effects of some avermectins on bovine carbonic anhydrase enzyme

Abstract

Avermectins are effective agricultural pesticides and antiparasitic agents that are widely employed in the agricultural, veterinary and medical fields. The aim of this study was to investigate the inhibitory effects of selected avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin that are used as drugs against a wide variety of internal and external mammalian parasites, on the carbonic anhydrase enzyme (CA, EC 4.2.1.1.) purified from fresh bovine erythrocyte. CA catalyses the rapid interconversion of carbon dioxide (CO₂) and water (H₂O) to bicarbonate () and protons (H⁺) and regulate the acidity of the local tissues. Bovine erythrocyte CA (bCA) enzyme was purified by Sepharose-4B affinity chromatography with a yield of 21.96% and 262.7-fold purification. The inhibition results obtained from this study showed K_i values of 9.73, 17.39, 20.43, 13.39, 16.44 and 17.73 nM for abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin, respectively. However, acetazolamide, well-known clinically established CA inhibitor, possessed a K_i value of 27.68 nM.

• Avermectin
• bovine
• carbonic anhydrase
• enzyme inhibition
• enzyme purification

Introduction

Carbonic anhydrase (CA, carbonate hydrolases EC 4.2.1.1.) is a metalloenzyme involving Zn²⁺ that occurs in all organisms. CA is an enzyme that catalyses the reversible reactions of the hydration of CO₂ and dehydration of in living cells1–3. It has been detected primarily in vertebrates and mammals and secondarily in plants, algae and some bacteria4–6. It regulates pH in most tissues including erythrocytes. CA catalyses the reversible hydration of carbon dioxide (CO₂) in a two-step reaction. As a result of this reaction, and H⁺ are produced. CA allows the excretion from the body of CO₂ produced during respiration through the dissolving and moving of water. In addition, CA plays a very important role in many more physiological processes such as acid–base balance, ion exchange and providing the embodiment of the cardiovascular system7^,8. CA also catalyses the hydrolysis of carboxylic, sulphonic and phosphoric acid esters as well as hydration reactions from cyanate to carbamate from urea to cyanamide, and from aldehyde to geminal diol in addition to the hydration reaction of CO₂9–11.

The carbonic anhydrase enzyme, which generally provides transport of metabolic CO₂, plays a role in the accumulation of H⁺ and , in many tissues12^,13 such as the kidney, gastric mucosa and lens. The molecular mass of the enzyme in mammals has been found to be approximately 30 KDa5^,14^,15. Thus far, the CA enzyme in plants and animals has been different isoenzymes, which catalyses the same reaction but with having different physical and chemical properties16–18. Carbonic anhydrase isoenzymes are divided into different classes in terms of structure, tasks and localization. Five of these enzymes are cytosolic (CA I, II, III, VII and XIII), four are membrane bound (CA IV, IX, XII and XIV), two are mitochondrial (CA VA and VB) and one is located in the saliva (CA VI)19^,20. Three (CA VIII, X and XI) have been found to be non-catalytic21–23.

An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. As blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance. Many drugs are enzyme inhibitors. They are also used as herbicides and pesticides. The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalysing its reaction. Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology24.

Macrolide endectocides are active animal drugs against a wide spectrum of internal and external mammalian parasites such as intestinal worms nematodes, lungworms, heartworms, roundworms and arthropods, such as ticks, mites, lice and fleas25^,26. These animal drugs have very high potencies and consequently are convenient for the treatment of agricultural animals. These macrocyclic lactone compounds consist of two subgroups. The first group is the avermectins, which include abamectin, doramectin, emamectin and some others. Avermectins, as antiparasitic drugs and agricultural pesticides, are a class of neurotoxic macrocyclic lactone compounds27. They are widely used in the protection of animals and crops, such as peanut, corn, cattle and pigs, against mites and insects28. The second subgroup has saccharide substituents26^,29. Edible oils originating from plants and animals may contain avermectin residue, causing a potential public health risk. Avermectins are toxic to the nervous and growth systems, and their harm to the environment and humans has raised increasing concerns on the topic of residue analysis in food and agro-products30.

Many new avermectins have been synthesised both to improve the anti-parasitic efficacy and to change their pharmacokinetic profiles. Doramectin was biosynthesised from mutant Streptomyces avermitilis with a cyclohexyl group26^,31. S. avermitilis is responsible for the production of multiple avermectins including one of the most widely employed drugs against nematode and arthropod infestations. Ivermectin, as well as abamectin, widely used insecticide and anthelmintic32. The mechanism of action of these avermectins is the inhibition of glutamate-activated chloride channels, which occur in the muscles and nerves of arthropods. Avermectins residues degrade rapidly, thus forming a variety of products. The only residues of toxicological significance are avermectins as the presence of these residues in fruits can affect consumer health27.

Abamectin is a type of large-ring lactone disaccharide compound and a natural fermentation product of the soil-dwelling actinomycete S. avermitilis33^,34. Abamectin is the commercialised major pair of isomers of avermectin itself, and ivermectin is the cis-hydrogenated product of abamectin29. It is highly lipophilic and used both as a biocide and as an anthelminthic drug. The toxicological mechanism of abamectin is believed to affect the γ-aminobutyric acid (GABA) system and Cl⁻ channels of animal cells35, in which the GABA receptor is responsible for regulating the neural basal tone36.

Doramectin is an endectocide compound with exceptional potency and a broad antiparasitic spectrum of activity against nematodes and arthropods31. The Doramectin belongs to the avermectins and has a high antiparasitic activity, and thus it has been widely used in food-producing animals37. This compound is extensively used worldwide to control endo- and eczoparasites in livestock animals38.

Emamectin is a semi-synthetic avermectin that kills invertebrates by acting as a neuroblocker, increasing the permeability of chloride ions at synapses39, which results in the paralysis and ultimately death of the parasite. Emamectin benzoate is approved as a treatment for sea lice (Lepeophtheirus salmonis) infestations in many regions of the world where marine salmonid aquaculture exists and has been the preferred therapeutic for sea lice (L. salmonis)40.

Eprinomectin is another member of the avermectin class of anthelmintic compounds, commercially launched in the late 1990s as a novel avermectin. It is marketed as a pour-on formulation for use in cattle41. In addition to broad-spectrum activity, it exhibits interesting low elimination in milk and was licensed, in lactating cows only, as a pour-on formulation42. It has been used intensively to control parasites on dairy animals as it has minimal disposition rates into the milk suggesting that it can be used safely in lactating animals with zero milk-withdrawal time43.

Ivermectin is a semi-synthetic product obtained from avermectin, naturally synthesized by the microorganism S. avermitilis. It is used as an active ingredient with broad-ranging medical applications for the treatment of rashes, worms and lice, acting on the nervous system and muscles function, resulting in paralysis and death of the parasites44.

Moxidectin, belonging to the milbemycin family, is a highly efficacious veterinary anthelmintic and a potential alternative to ivermectin for preventive chemotherapy and elimination of human onchocerciasis. It exhibits broad-spectrum activity against nematodes and arthropods45.

The objective of this research was to purify the bovine carbonic anhydrase (bCA) enzyme from bovine erythrocyte by Sepharose-4B affinity chromatography chromatography to determine the in vitro effects of the above-mentioned avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin on the bCA enzyme. Another main goal of this research was to compare the inhibition effects of these avermectins to acetazolamide, a clinical carbonic anhydrase inhibitor (Figure 1).

Figure 1. Chemical structures of used avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin.

Materials and methods

Chemicals

Avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin, Sepharose 4B, protein assay reagents, p-nitrophenylacetate (NPA) and l-tyrosine were obtained from Sigma-Aldrich Co. (St. Louis, MO). All other chemicals were highly pure and obtained from Merck (Darmstadt, Germany).

Purification of CA from bovine erythrocytes by affinity chromatography

Bovine blood was sampled from the jugular vein using a 250-mL heparinized blood bag and transferred to centrifuge tubes. Whole blood samples were centrifuged at 1500×g for 15 min, and the plasma was removed by dropper. Erythrocytes were washed with NaCl solution (0.9%) three times, always were centrifuged at 1500×g, and the supernatants were removed. Erythrocytes and pure water at zero degrees (1/5) were gently mixed for 30 min and subjected to haemolysis and the hemolysate (4 °C, 13.500× g) was centrifuged46^,47. The pH value of bovine erythrocyte hemolysate was adjusted to 8.7 using Tris powder. A Sepharose-4B-l-tyrosine-sulphanilamide affinity column was used for purification48 and equilibrated with 25 mM Tris–HCl/0.1 M Na₂SO₄ (pH 8.7). Hemolysate was loaded onto the column at pH 8.7, and the Sepharose-4B-l-tyrosine-sulphanilamide gel was washed with 25 mM Tris–HCl/22 mM Na₂SO₄ (pH: 8.7). The bCA was eluted with 0.1 M CH₃COONa/0.5 M NaClO₄ (pH: 5.6). All procedures were performed at 4 °C (Table 1)49^,50.

Table 1. Summary of purification procedure for bovine erythrocyte carbonic anhydrase (bCA) by a Sepharose-4B-Thyrosine-sulphanilamide affinity column chromatography.

Purification steps	Activity (EU/mL)	Total volume (mL)	Protein (mg/mL)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification fold
Haemolysate	2321	28.5	67.5	1923.75	66 148.5	34.39	100.0	1.0
Sepharose-4B-Thyrosine-sulphanilamide  affinity column chromatography	4843.0	3.0	0.536	1.608	14 529.0	9035.45	21.96	262.7

Esterase activity assay

This method is based on the esterase activity of CA. The esterase activity was tested by following the alteration in the absorbance of NPA to 4-nitrophenylate ions at 348 nm over a period of 3 min at 25 °C using a spectrophotometer (Beckman Coulter UV-VIS, Krefeld, Germany) according to the method described by Verpoorte et al.51. The enzymatic reaction was performed using 0.4 mL of 0.05 M Tris–H₂SO₄ buffer (pH 7.4), 0.36 mL of 3 mM NPA, 0.23 mL H₂O and 0.01 mL of enzyme solution in the presence in a 1-mL cuvette. The blank measurement value was obtained without the enzyme content but using the same cuvette52.

Protein determination

During each purification step, the protein assay was determined spectrophotometrically using the standard protein of bovine serum albumin by the Bradford method at 595 nm, as described previously53–58. Bovine serum albumin was used as the positive control59–63.

SDS polyacrylamide gel electrophoresis

Purification was followed by SDS polyacrylamide gel electrophoresis to check the enzyme purity64–66. It was conducted in 10% and 3% acrylamide for the running and the stacking gel, respectively; including 0.1% SDS according to the Laemmli procedure67 as described previously68–71.

Inhibition effect of avermectins on CA enzyme activity

In our study, bCA was purified from bovine erythrocyte, and NPA, the substrate of bCA, was used to examine the esterase activity. The effects of increasing concentrations of avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin on bCA were determined colorimetrically using CO₂ hydration7. This method was described previously20^,72. Briefly, bCA samples were added to 4.2 mL (final volume) of incubation mixture containing 1 mL of Veronal buffer (0.025 M, pH 8.2), bromothymol blue (0.1 mL, w/v: 0.04%) and saturated CO₂ solution in water (2.5 mL). The bCA activity was measured by a colorimetric method considering the required time for the pH to change from 8.2 to 6.3. One enzyme unit (EU) for CO₂-hydratase activity of bCA was calculated by using the equation (t_o − t_c/t_o), where t_o and t_c are the times for the pH change of the nonenzymatic and enzymatic reactions, respectively. Each concentration of each drug studied was tested three times73^,74. For the IC₅₀ values, five different inhibitor concentrations were processed. Table 2 reports by of K_i values obtained using the esterase method at three different inhibitor concentrations.

Table 2. Bovine carbonic anhydrase (bCA) inhibition data with certain avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin by an esterase assay with 4-nitrophenylacetate as substrate.

Avermectin	IC50 (nM)	r^2	Ki (nM)
Abamectin	14.39	0.9670	9.74 ± 1.91
Doramectin	14.64	0.9886	17.39 ± 4.89
Emamectin	18.68	0.9815	20.43 ± 9.49
Eprinomectin	14.63	0.9989	13.39 ± 2.47
Ivermectin	14.48	0.9898	16.44 ± 5.26
Moxidectin	20.84	0.9902	17.73 ± 3.75
Acetazolamide*	23.94	0.9827	27.68 ± 5.65

*Acetazolamide was used as a positive standard inhibitor for bCA.

Results and discussion

Avermectins are members of a group of pentacyclic 16-membered lactone compounds with endectocide activity. Avermectins are antiparasitic drugs and agricultural pesticides, a class of neurotoxics macrocyclic lactone compounds27. They are widely used in animal and crop protection, such as peanut, cattle, corn and pigs, against mites and insects75. Accordingly, edible oils originating from plants and animals may contain avermectins residues, causing potential public health risk. Avermectin is toxic to nervous and growth systems; their harm to the environment and humans has raised increasing concerns on the topic of residue analysis in food and agro-products76. The cytosolic bCA enzyme was effectively inhibited with inhibition constants in the nanomolar range by various avermectins including abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin, which demonstrated a K_i values of 9.74 ± 1.91 nM (r²: 0.9670), 17.39 ± 4.89 nM (r²: 0.9886), 20.43 ± 9.49 nM (r²: 0.9815), 13.39 ± 2.47 nM (r²: 0.9989), 16.44 ± 5.26 nM (r²: 0.9898) and 17.73 ± 3.75 nM (r²: 0.9902), respectively. Based on these results, as can be seen in Table 2, abamectin, possessing no carbonyl group at the part of the molecule, was found to be the best bCA inhibitor with K_i value of 9.74 ± 1.91 nM. However, acetazolamide is a well-known example of a clinically established carbonic anhydrase inhibitor9^,77 and was found to have a K_i of 27.68 ± 5.65 nM (r²: 0.9827) against bCA.

It has been reported that extensive use of avermectins can result in environmental pollution, and it is important to evaluate the potential impact of these antibiotics on ecological and living systems. These antibiotics have been shown to increase the rate of apoptosis and the expression levels of caspase and mRNA in the liver of pigeons. Some ultra-structural alterations, including mitochondrial damage and chromatin aggregation, become severe with increased exposure dose. Exposure to avermectins induced significant changes in antioxidant enzyme including glutathione peroxidase and superoxide dismutase activities and malondialdehyde levels, and augmented protein carbonyl content and DNA-protein crosslinking, in a concentration-dependent manner in the liver of pigeons78. Glutathione peroxidase and superoxide dismutase are major enzymes in the defence system, and they play important roles in protecting the living organisms from environmental stresses79^,80. In addition, residues of avermectin drugs or their metabolites in livestock faeces have toxic effects on non-target aquatic and terrestrial organisms. In addition, it has been reported that avermectins could induce oxidative damage to the brain tissue and serum of pigeons. Recently, it was reported that animals usually present poisoning reactions indicative of central nervous system depression, such as incoordination, tremors, salivation, pupil dilation and coma in animals when exposed to excessive doses of avermectins81. On the other hand, CA, which is strongly involved in the transport of metabolic CO₂ has great importance in different tissues5^,82. In some studies, the similarities in the crystal structures of CA isoenzymes have been identified. These results were achieved several times with CA purified from different organisms83^,84. CA activity can be enhanced or reduced by many chemicals, pharmaceuticals and pesticides85. Structures such as toxic chemicals and drugs released to the atmosphere have the effect of distorting the natural balance. This study examined the effects of certain pesticides on the esterase activity of bCA. Currently, all creatures are exposed to the effects of these pollutants by the ongoing contamination of the environment. The waste materials of factories in many countries are buried or released into the air. Naturally, this pollution may then be transferred to resources and to living creatures in nature, which is a great threat to future generations. Therefore, enzyme activity studies regarding the use of pesticides are of great importance85.

Thus far, our group have reported the CA inhibitory effects of a wide spectrum of biological active compounds including melatonin14, CAPE77, vitamin E4, capsaicin9, antioxidant phenols49, phenolic acids52, natural product polyphenols and phenolic acids86, (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives73, novel phenolic sulphamides83, novel sulphonamide derivatives of aminoindanes and aminotetralins84, novel phenolic benzylamine derivatives85, novel sulphamide analogues of dopamine related compounds87, morphine88, bromophenols derivatives89, spirobisnaphthalenes90 new benzotropone derivatives6 and novel sulphamides and sulphonamides incorporating tetralin scaffold21 have been reported by our groups. These extensive studies indicate the importance of CA enzyme inhibitors.

Carbonic anhydrase enzyme has a Zn²⁺ ion in the active site. Avermectins also include electronegative atoms that interact with the active site. For this reason avermectins can inhibit the bCA enzyme even at very low concentrations. Avermectins such as abamectin are pentacyclic polyketide-derived compounds linked to a disaccharide of the methylated deoxysugar oleandrose. The IC₅₀ values of abamectin and doramectin are similar and slightly lower than emamectin benzoate. The available chemical structures of emamectin show a pyranose ring and the quaternary ammonium salt including benzoic acid, unlike abamectin and doramectin.

Conclusion

As discussed in this study, the CA enzyme is virtually ubiquitous in all living systems and play important roles in pH regulation, carboxylation reactions, fluid balance, bone resorption, tumorigenicity, calcification, the synthesis of bicarbonate and many other pathological and physiological processes. Avermectins are effective common pesticides and antiparasitic agents that are widely employed in the agricultural, veterinary and medical fields. The results of this study clearly showed that the tested avermectins inhibited bCA with nanomolar K_i and IC₅₀ values. Even at very low concentrations, there are significant amounts of inhibition.

Acknowledgements

I.G. and S.H.A. would like to extend his sincere appreciation to the Research Chairs Program at King Saud University for funding this research.

Declaration of interest

The authors declare no competing of interests.