Impact of Some Avermectins on Lactoperoxidase in Bovine Milk

Abstract

Many macrocyclic lactones, including avermectins, are known to be used as a veterinary drug, agricultural pesticides, and insecticides. Lactoperoxidase (EC 1.11.1.7) is one of the peroxidases found in milk. Lactoperoxidase has a natural host defense system against micro-organisms and a natural antimicrobial system. In this study, some macrocyclic lactones, including emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin were investigated for in vitro inhibitory effects on the bovine lactoperoxidase enzyme, which was purified using amberlite CG-50 H⁺ resin and sepharose 4B-L-tyrosine-sulphanamide affinity chromatography 344.6-fold, with a yield of 61.1% and a specific activity of 39.11 EU/mg protein. Emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin are also known strong antiparasitary properties. In this study, we demonstrated that avermectins have strong lactoperoxidase inhibitory effects. Of these, the emamectin-benzoate was shown to have the most inhibiting effect against lactoperoxidase with Ki value of 6.82 ± 2.60 µM.

Keywords:

• Lactoperoxidase
• Enzyme purification
• Enzyme inhibition
• Avermectins
• Affinity chromatography

Introduction

A subfamily of macrocyclic lactones that are known avermectins include: abamectin, doramectin, eprinomectin, ivermectin, emamectin, and the milbemycin moxidectin used as a drug for veterinary applications.^[1] Many macrocyclic lactones are used in practice as veterinary drugs, agricultural pesticides, and insecticides. Ivermectin and abamectin are the most known, and more recently moxidectin, doramectin, eprinomectin, and emamectin benzoate have begun to be used, showing impressive results at low doses.^[2,3] These lactone compounds consist of two subgroups: The first group is the avermectins^[4] and the second subgroup has saccharide substituents.^[5] Avermectins are toxic to the nervous and growth systems in many animals and plants.^[6]

Gamma-aminobutyric acid (GABA) is found in most invertebrates and vertebrates. Avermectins inhibit the GABA neurotransmission in nematodes. Among the effects, they are shown to block nerve signals by interfering with the glutamate-gated chlorid channel receptors.^[7] Avermectins interact with vertebrate and invertebrate GABA receptors^[8] effecting stability in their glutamate-gated chloride channels.^[9,10] The soil nematode, Caenorhabditis elegans, is a useful model for studying the avermectins mechanism.^[11,12] Abamectin is a mixture of avermectins: avermectin B1a and B1b. Abamectin, derived from the natural fermentation product of soil bacterium Streptomyces avermitilis, has been found to be toxic to different species of insects and mites. Abamectin is used as insecticide in a range of agronomic fruits, vegetables, and ornamental crops by interfering with neural and neuromuscular systems.^[13,14] Avermectin residues degrade rapidly, thus forming a variety of products. These compounds are neurotoxins. When their residues are present in fruits, they have shown to threatens the the health of consumers, and also high doses have caused death from respiratory failure.^[13] Avermectins were isolated from cultures of Streptomyces avermitilis and the milbemycins were isolated from S. Cyanogrise or S. hygroscopicus. These groups have been showed insecticidal features.^[5]

Abamectin’s cishydrogenated form is called ivermectin, consisting of the major isomer, B1a that contains a 2-butyl group, and the minor isomer (B1b), an isopropyl group.^[15] Ivermectin is an antiparasitic agent used against many intestinal worms; mostly mites, ticks, and some lice. The dose of ivermectin must be controlled; overdoses are very toxic. Ivermectin effects the nervous system and muscle function, intervening to inhibit neurotransmission.^[5] abamectin’s 4”-deoxy-4”-methylamino form is known as emamectin, which is produced by the fermentation soil actinomycete Streptomyces avermitilis.^[16] Emamectin benzoate is generally in salt form.^[17] Emamectin is widely used as an insecticide because of its chloride channel activation properties.^[18]

Doramectin is a derivative of ivermectin. It can be used to control and treat internal parasites, such as roundworms, lungworms, and some external parasites. Moxidectin is produced by Streptomyces cyanogriseus and is used as a drug to control parasites in many animals.^[19] This compound has shown strong effects against nematodes in cattle and sheep.^[20] In adult non-pregnant sheep, Moxidectin is administered subcutaneously, and its^[21] tissue distribution^[22] was characterized as displaying long-term residence in plasma and tissues, a large distribution volume, and a linear pharmacokinetic pattern.^[23]

Eprinomectin is a member of the avermectin class of compounds, and the only anthelmintic registered for a zero milk withdrawal period, making it suitable for lactating animals.^[24] Eprinomectin is a novel avermectin developed from abamectin. Like ivermectins, eprinomectin provides against activity for nematodes and arthopodes. It exhibits higher potency against endoparasites than other avermectins. Furthermore, it exhibits a very favorable milk to plasma ratio resulting in low residue.^[25] Against insecticides chemical control, it is known as the main method.^[26] The use of these chemicals in drugs for veterinary applications poses important questions. Because of its extensive use and the slow elimination from the bovine body, there may be residues in the milk, liver, and muscle tissues. In a Brazilian study, only eprinomectin is indicated for use in cattle producing milk for human consumption.^[27]

Milk is known to have the best defense system because it contains lactoferrin, lysozyme, immunoglobulins, and lactoperoxidase (LPO), all known as essential antimicrobial factors. The LPO system has the ability to converts thiocyanate (SCN^–) to hipotiyosiyanate (OSCN^–), dependent on its reaction with hydrogen peroxide (H₂O₂).^[26,28] These are both present in biological fluids and, together with LPO, they are called the lactoperoxidase system (LPS). The LPO system shows the effect on bacteria, destroying its cell walls.^[29,30] LPO is a very important enzyme for the immune system, and during lactation the use of these drugs should be noted, particularly as these chemicals can pass into the milk and may show adverse effects. Bovine milk has been shown to have similar properties to (human) breast milk.^[31,32]

In this study we used purified bovine LPO enzyme on some antiparasitary properties lactones, including emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin. We investigated their in vitro effects on LPO, in which secreted in milk plays an important role in protecting the mammary glands and intestinal tract of the newborn, against pathogenic microorganisms.

Materials And Methods

Chemicals and Materials

Fresh bovine milk was obtained from the Veterinary Faculty of Atatürk University (Erzurum, Turkey). CNBr-activated-sepharose 4B, L-tyrosine, sulphanilamide, amberlite CG-50- NH₄⁺ resin protein assay reagents and chemicals for electrophoresis were purchased from Sigma-Aldrich Co. (Sigma-Aldrich Chemie GmbH Export Department Eschenstrasse 5, 82024 Taufkirchen, Germany). Standard protein markers for electrophoresis were obtained from Thermo Scientific. Emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin and all other chemicals were of analytical grade and obtained from Merck and Sigma-Aldrich Co.

Determination of LPO Activity

LPO activities were determined according to the procedure of Shindler and Bardsley^[33] with a minor modification.^[34] One unit of enzyme is defined as the amount of enzyme catalyzing the oxidation of l µmol of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) min^–1 at 298 K (Molar absorption coefficient, 32,600 M^–1cm^–1). This activity method is based on the oxidation of ABTS as a chromogenic substrate by H₂O₂, resulting in a product that absorbs at 412 nm.^[35,36] Km and Vmax values were calculated from Lineweaver-Burk graphs^[37] as previously described.^[38–41]

Protein Determination

The quantitity of protein was determined according to the Bradford method^[42] as described in our previous studies.^[43–45] Bovine serum albumin was used as a standard protein.^[46–48]

Purification of LPO

LPO was purified using Sepharose 4B-L-tyrosine-sulphanamide affinity column chromatoghraphy.^[49–51] The eluate was applied to the column and equilibrated with phosphate buffer (10 mM, pH 6.8). The affinity gel was washed with phosphate buffer (400 mL, 25 mM, pH 6.8) then, LPO enzyme was eluted with a solution of NaCl/Na₂HPO₄ (1 M/0.25 M, pH 6.8), measuring the Rz (A₄₁₂/A₂₈₀) of the fractions. The enzyme solution was dialyzed overnight against a sodium phosphate buffer (0.5 M, pH 6.8).

SDS–PAGE

For enzyme purity control, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed after LPO purification according to the procedure of Laemmli^[52] as previously described.^[53–56] The stacking and running gels contained 3% (w/v) and 10% (w/v) acrylamide, respectively, and 0.1% (w/v) SDS. The electrode buffer was Tris/Glycine (0.025 M/0.2 M, pH 8.3). The sample buffer was prepared by mixing 0.65 mL of Tris–HCl (1 M, pH 6.8), 3 mL of 10% (w/v) SDS, 1 mL of neat glycerol, 1 mL of 0.1% (w/v) bromophenol blue, 0.5 mL of β-mercaptoethanol and 3.85 mL of water. A 20 mg aliquot of enzyme (50 mL) was added to 50 mL of sample buffer, and the mixture was heated in a boiling waterbath for 3 min. The cooled LPO sample was loaded into each lane of the stacking gel. LPO was analyzed separately by polyacrylamide gel electrophoresis. Initially, an electrical potential of 80 V (Hoefer Scientific Instruments SE 600) was applied until the bromophenol blue dye reached the running gel. Then, the electrical potential was increased to 200 V for 3–4 h. Gels were stained for 1.5 h in 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol and 10% (v/v) acetic acid, and destained with methanol/acetic acid.^[57–59]

Results and Discussion

Drugs to ensure animal health and growth are widely used in many fields.^[60] In a veterinary application, the development of some compounds show strong activity against both ectoparasites and endoparasites.^[1,61] The avermectins are some of these compounds showing potent anthelmintic and other insecticidal activities. However, avermectins are very potent drugs, so effects can be seen on non-target organisms.^[7] Some of the residues of these drugs are not desirable, because they pose a threat to human health, promoting the proliferation of multi-antibiotic resistant bacteria strains.^[60]

Avermectins are macrocyclic lactones and pentacyclic polyketide-derived compounds, linked to a disaccharide of the methylated deoxysugar oleandrose.^[62] They are used to treat food animals, for example; cattle, swine, sheep, bison, deer, and reindeer against nematodes and arthropods, and also for mastitis. Emamectin is used to control sea lice in fish farms and selamectin is used for the treatment of pets against heart and round worms. Attention is drawn to the use avermectin drugs, as while the use of these antiparasitic compounds may ensure benefits to the mammalian, their indiscriminate use might result in the presence of residues in milk and dairy products. Among this group of drugs, only eprinomectin and moxidectin are permitted for use in dairy cattle. Eprinomectin was designed to exhibit a low milk/plasma ratio,^[5] and moxidectin is less toxic for a larger acceptable daily intake. Avermectins are hydrophobic 16-membered macrocyclic lactone^[6] and avermectins’ 4”-position has been the most common studied because of easy access.^[7] Emamectin differs from avermectins B_1a and B_1b by the presence of a hydroxyl group at the 4”-epimethylamino group rather than the 4-position (Fig. 1). Emamectin play an activatory role in chloride channel and binding gamma aminobutyric acid receptor and alloying nerve signals within arthropods.^[63] This molecule induces the release of GABA from the synapses between nerve cells in insects and arthropods, increasing GABA’s affinity for its receptor on the post-junction membrane of muscle cells, increasing their permeability to chloride ions, due to the hypotonic concentration gradient.^[64] The resistance of gastrointestinal nematodes has drawn attention from the goat and sheep industry.^[65,66] Some studies have shown avermectin resistance in goats in Switzerland, and a further study, focusing on Boer goats and Dorper sheep, has shown that avermectin resistance seems to be wide spread in the gastrointestinal nematodes of these breeds.^[67]

FIGURE 1 Chemical structures of some avermectins, including emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin.

FIGURE 2 Sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) band of LPO. Column “c” is purified LPO from bovine milk by Sepharose 4B-L-Tyrosine sulphanilamide affinity column chromatography. Column “a” is standard proteins (Line 1: 250 kDa, Line 2: 150 kDa, Line 3: 100 kDa, Line 4: 70 kDA, Line 5: 50 kDA, Line 6: 40 kDA, Line 7: 30 kDA, Line 8: 20 kDa, Line 9: 15 kDa).

As can be seen in Table 1, LPO was purified 355.6-fold with a yield of 61.1% with Amberlite CG- 50-NH₄⁺ resin and CNBr-activated-sepharose 4B affinity chromatography from skimmed bovine milk and when the purity of this enzyme was checked by SDS-PAGE, a single band was detected, approximately corresponding to 80 kDA (Figure 2). The purified LPO migrated to a similar distance on the SDS-PAGE gel as LPO purified from other sources.^[68,69] The Rz (A₄₁₂/A₂₈₀) value for LPO was 0.9. Km and Vmax were calculated by a Lineweaver-Burk graph using ABTS substrate. The optimum pH was found to be 6.0. Km and Vmax values of the purified enzyme were determined to be 0.14 mM and 0.55 μmol/min.mL, respectively, for pH: 6.0 at 20^oC.

TABLE 1 Purification scheme for LPO obtained after the Amberlite CG-50 H⁺ resin and Sepharose 4B-L-tyrosine-sulphanamide affinity chromatography purification steps (LPO: Lactoperoxidase enzyme purified from bovine milk)

Purification steps	Total volume (mL)	Enzyme activity (EU/mL.min)	Total enzyme activity (EU/mL.min)	Protein (mg/mL)	Total protein (mg)	Specific activity (EU/mg)	Yield (%)	Purification fold
Crude homogenate	50	1.21	60.5	11.1	555.0	0.11	100	1.0
Sepharose 4B-L-tyrosine-sulphanamide affinity chromatography	7	5.28	36.96	0.135	0.945	39.11	61.1	355.6

To show the inhibition effects of the lactons, the most suitable parameters are the K_i and IC₅₀ values. In this study both K_i and IC₅₀ parameters for these compounds on bovine LPO were first determined (Table 2). Among these compounds, Eprinomectin is shown to have the most successful inhibitory effect. The K_i value was determined to be 4.80 ± 1.95 µM. The obtained IC₅₀ value was 16.90 µM (Table 2).

TABLE 2 The half maximal inhibitory concentration (IC₅₀), inhibition constant (K_i), and inhibition type of avermectins against LPO purified from bovine milk by Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography (LPO: Lactoperoxidase)

Compounds	IC50 (µM)	R^2	Ki (µM)	Inhibition type
Emamectin-benzoate	4.33	0.9500	6.82 ± 2.60	Competitive
Eprinomectin	16.90	0.9458	4.80 ± 1.95	Competitive
Moxidectin-vetranal	99.00	0.9272	61.31 ± 9.89	Competitive
Abamectin	138.60	0.9068	103.73 ± 34.03	Competitive
Doramectin	173.20	0.9601	80.14 ± 29.38	Competitive
Ivermectin	231.00	0.9096	519.97 ± 47.62	Non-competitive

The IC₅₀ values obtained were, respectively: emamectin-benzoate (4.33 µM), eprinomectin (16.90 µM), doramectin (173.2 µM), abamectin (138.6 µM), moxidectin-vetranal (99.00 µM), and ivermectin (231.0 µM). On the other hand, K_i values of these compounds described in following order emamectin-benzoate (6.82 ± 2.60 µM), eprinomectin (4.80 ± 1.95 µM), doramectin (80.14 ± 29.38 µM), abamectin (103.73 ± 34.03 µM), moxidectin-vetranal (61.31 ± 9.89 µM), and ivermectin (519.97 ± 47.6278 µM). As shown in Table 2, the results of this study clearly demonstrate the kinetic properties of chemicals (K_i and IC₅₀ values) on LPO and their inhibition type.

The uses of these chemicals in veterinary application poses important questions because of their slow elimination from the bovine system.^[2] Their extensive use may result in the presence of their residues in the milk, liver, and muscle tissue. Milk contains lactoferrin, lysozyme, immunoglobulins, and LPO that are known to be essential antimicrobial factors. The LPO system has the ability to convert SCN^– to OSCN^– dependent on reaction with hydrogen peroxide (H₂O₂). These are both present in biological fluids, and together with LPO, they are called the LPO system.^[29,30] LPO is crucial, and a prominent enzyme in milk, with oxidoreductase activity that was found in milk, together with adequate amounts of SCN^– and peroxide. Biological systems can produce hydrogen peroxide.^[70–72]

Conclusion

As a result, in this study avermectins are known as a pesticides and antiparasitic agents, which are widely employed in the agricultural, veterinary, and medical fields. This study investigated the effect of some avermectins against LPO activity. On this basis the inhibitory effect of these antiparasitics on the LPO enzyme has been studied. IC₅₀ and K_i values have been calculated for each of the avermectins. The results showed that the avermectins effectively inhibited LPO with micromolar level with K_i values ranging from 4.80 to 519.97 µM. Furthermore, it was demonstrated that that emamaectin-benzoat has strong inhibitory effects on LPO with K_i values of 6.82 ± 2.60 µM. Because of the chemicals slow elimination from the bovine system, the use of these chemicals in drugs for veterinary application poses important questions; especially during the lactation period, when use of these drugs is crucial to the regulation of their metabolism. As a result it is essential that dose adjustments should be made. Also, the results obtained from this study demostrated that some avermectines molecules showed effective inhibiton effects against LPO. These macrocyclic lactones compounds are used as a veterinary drugs for food producing animals. Their residues are investigated in foods such as milk.

Funding

İlhami Gülçin extends his sincere appreciation to the Research Chairs Program at King Saud University for funding this research.

Additional information

Funding

İlhami Gülçin extends his sincere appreciation to the Research Chairs Program at King Saud University for funding this research.