Assessment of Avermectins-Induced Toxicity in Animals

Macrocyclic lactones, particularly the avermectins, have completely revolutionized the approaches aimed at control of parasites. These avermectins are the most widely used anti-parasitic drugs in veterinary field with sales exceeding one billion US dollars annually. However, before clinical usage, their safety evaluation in the animals is a major critical factor that must be considered. Many studies have reported the negative effects of avermectins like ivermectin, abamectin, doramectin, and eprinomectin on the host animals. These harmful effects arise from avermectins targeting GABA and glutamate-gated chloride channels present both in the parasites and the host animals. In this review, various modes of avermectins action along with the negative effects on the host like nephrotoxicity, hepatotoxicity, neurotoxicity, reproductive toxicity, and endocrine disruption were discussed in detail. Furthermore, other important issues like ecotoxicity, drug resistance, and drug residues in milk associated with avermectins usage were also discussed, which need special attention.


Introduction
Parasitic organisms like helminths, insects, and arachnids pose a serious threat to the welfare of both humans and animals [1][2][3][4][5]. There exists a significant host-parasite specificity with the parasites getting their metabolic and physiological needs from their hosts [3]. They not only cause diseases, but also result in huge economic losses in terms of reduced productivity and retarded growth in their hosts, thus emphasizing the need for control of these parasites using novel approaches [6,7]. Basically, there are two approaches aimed at control of parasites including either the use of synthetic molecules or the use of natural products [3].
The chemical and structural diversity of natural products has always revolutionized the biological field with the discovery of new drugs [8]. For example, the discovery of avermectins, a group of macrocyclic lactones, in 1976 in Japan changed the concept of parasite control [9,10]. These avermectins are produced by fermentation of Streptomyces avermitilis,

Avermectins
Avermectins are the group of 16 distinct chemical compounds that belong to the drug category of macrocyclic lactones and which have well established insecticidal, acaricidal, and nematicidal activities [11][12][13][14]. These are originally produced by soil dwelling grampositive bacterium of group actinomycetes, Streptomyces avermitilis, through the process of fermentation [35][36][37][38]. Macrocyclic backbone is the main component of avermectins to which a hexahydrobenzofuran and a spiroketal agent are attached [11]. At the C-13 position, the avermectins possess a bisoleandrosyloxy group as the main identification point [11,13,36]. There are eight different components classified into two main groups A and B (A1 a , A2 a , A1 b , A2 b , B1 a , B2 a , B1 b , and B2 b ) which are yielded by bacterium Streptomyces avermitilis. The A and B components vary only with respect to the presence of either methoxy or hydroxy groups at the C-5 position respectively. The general structure of avermectins described by various scientists is elaborated by Figure 1 [12,35,39].

Mode of Avermectins Action against Parasites
Avermectins, regarded as potential neurotoxins, target the gamma aminobutyric acid (GABA) receptors and the glutamate gated chloride ion channels (GluCl) which are concerned with neurotransmission in parasites [10,17,41]. GABA, a neurotransmitter, causes opening of the chloride ion channels of the organism resulting in the influx of chloride ions. Avermectins act as agonists of chloride channels and cause changes in permeability of these channels, thus resulting in neurotransmission disturbance [13,36,[42][43][44]. This disturbance induces neuronal membrane hyperpolarization, paralysis, and ultimately the death of the parasite. The GABA receptors in the mammals are found only in the brain, which is shielded by the blood-brain barrier. Avermectins cannot cross this protective barrier, thus making the avermectins relatively safe in mammals [10,13,17,45]. This mode of action is briefly summarized in Figure 3.

Mode of Avermectins Action against Parasites
Avermectins, regarded as potential neurotoxins, target the gamma aminobutyric acid (GABA) receptors and the glutamate gated chloride ion channels (GluCl) which are concerned with neurotransmission in parasites [10,17,41]. GABA, a neurotransmitter, causes opening of the chloride ion channels of the organism resulting in the influx of chloride ions. Avermectins act as agonists of chloride channels and cause changes in permeability of these channels, thus resulting in neurotransmission disturbance [13,36,[42][43][44]. This disturbance induces neuronal membrane hyperpolarization, paralysis, and ultimately the death of the parasite. The GABA receptors in the mammals are found only in the brain, which is shielded by the blood-brain barrier. Avermectins cannot cross this protective barrier, thus making the avermectins relatively safe in mammals [10,13,17,45]. This mode of action is briefly summarized in Figure 3.

Toxicity Studies
The avermectins are potent antiparasitic drugs which may pose a serious toxicity threat to animals. By damaging the various organs of the body, these may even lead to death of animals, as seen in various cases [27,[46][47][48]. These damaging effects are seen in dose-dependent and dose-time dependent manners [49,50]. The reason is that avermectins are lipophilic and, hence, tend to accumulate in fatty tissues and the liver where they induce oxidative stress leading to tissue damage through lipid peroxidation [51]. The other important factor is the breed susceptibility. In different animals, the same doses may be damaging. Additionally, some animals like dogs and rats are deficient in P-glycoprotein, which acts as an efflux pump against avermectins, thus increasing their vulnerability to the negative effects of avermectins [30,52]. In toxicity analysis, a combined evaluation of various biochemical parameters provides better identification of the organ being damaged by the drug under investigation. A detailed review of various toxicity studies is described below.

Toxicity Studies
The avermectins are potent antiparasitic drugs which may pose a serious toxicity threat to animals. By damaging the various organs of the body, these may even lead to death of animals, as seen in various cases [27,[46][47][48]. These damaging effects are seen in dose-dependent and dose-time dependent manners [49,50]. The reason is that avermectins are lipophilic and, hence, tend to accumulate in fatty tissues and the liver where they induce oxidative stress leading to tissue damage through lipid peroxidation [51]. The other important factor is the breed susceptibility. In different animals, the same doses may be damaging. Additionally, some animals like dogs and rats are deficient in Pglycoprotein, which acts as an efflux pump against avermectins, thus increasing their vulnerability to the negative effects of avermectins [30,52]. In toxicity analysis, a combined evaluation of various biochemical parameters provides better identification of the organ being damaged by the drug under investigation. A detailed review of various toxicity studies is described below.

Nephrotoxicity
The evaluation of serum creatinine and the blood urea levels post-administration of drugs provides a good overview for nephrotoxicity analysis [46]. Various studies conducted on avermectins (mainly the ivermectin and the abamectin) have proven these drugs to induce nephrotoxicity in many animals like mice, bats, rabbits, and rats [28,34,[53][54][55][56]. The main molecular mechanism through which avermectins exert their nephrotoxic effect is the lipid peroxidation which results from the action of reactive oxygen species [51]. This oxidative damage results in histopathological changes like interstitial nephritis, glomerular damage, interstitial infiltration areas of round cells, and tubular necrosis as well as elevated levels of serum creatinine, urea, and the uric acid in the blood [46,57,58]. For example, ivermectin causes nephrotoxicity through elevated levels of creatinine and decreased protein synthesis and glucose levels in pigs and dogs [59,60]. Various other reports of avermectins causing nephrotoxicity are summarized in the Table 1.

Hepatotoxicity
The liver is the main organ that catabolizes and neutralizes most toxins and drugs present in the body [66]. These drugs or toxins may induce hepatic injury, which can escalate into complete hepatic failure and even death of the animal may also occur [67]. Avermectins are highly lipophilic with the highest concentration found in the liver of animals post-administration regardless of the administration route [68][69][70]. They cause hepatotoxicity by affecting the liver enzymes, altering the mitochondrial bioenergetics of hepatocytes, inducing oxidative stress, and enhancing the autophagy in the liver tissues of the treated animals [65,66,[71][72][73][74]. Various histopathological changes are also observed like dilated blood vessels, infiltration of leucocytes, and hepatocytes degeneration [75]. As a repairing process, autophagy clears the damaged organelles and proteins produced as a result of hepatocytes damage. For example, avermectins administration in pigeons at different concentrations induced hepatotoxicity and increased the apoptosis in a dose dependent manner [74]. Similarly, other hepatotoxic reports of avermectins are compiled in Table 2.

Neurotoxicity
Generally, the use of avermectins in animals has a wide safety margin as far as the nervous system is concerned [77,78]. This is due to the P-glycoprotein associated with the blood-brain barrier that prevents the avermectins from exerting their neurotoxic effects by inhibiting their penetration in the brain [78,79]. P-glycoprotein is responsible for multi-drug resistance and uses avermectins as substrate [77,80]. Thus, the P-glycoprotein expression is linked with the neurotoxicity of avermectins in animals like rats and dogs, which are somewhat deficient in P-glycoprotein [30,81]. The avermectins exhibit nervous effects by damaging the cerebral cortex and inducing diffused cerebellar dysfunction [79]. They do so by modulating the binding of GABA and benzodiazepine receptors, affecting the chloride channels and targeting the Cys-loop receptors of the mammalian brain [39,82,83]. Various signs observed in neurotoxicity studies are depression, tremors, salivation, ataxia, convulsions, mydriasis, coma, and ultimately death in animals like dogs, cattle, and lions, etc. [77][78][79]84]. Neurotoxic effects produced by various avermectins are described in Table 3.

Reproductive Toxicity
There are reports of avermectins negatively affecting the reproductive system of the animals [34,46]. As discussed earlier, avermectins induce neurotoxicity by damaging the brain, which is responsible for production of reproductive hormones; therefore, they indirectly affect the reproductive system of animals as well [63,89]. In males, avermectins cause testicular damage, thereby affecting the sperm count as well as the sperm motility [63,90]. The avermectins, by crossing the blood-testes barrier, reach the germ cells of the seminiferous tubules, causing a reduced meiotic index that ultimately results in decreased sperm count [63,91]. The targeting of the brain and the testes by avermectins results in sexual behavioural changes as well as impaired hormones production [92,93]. Likewise, avermectins also target the female reproductive organs, producing deleterious effects like degenerated and hemorrhagic reproductive organs, degenerated ova, and atritic follicles [62]. They have the potential of crossing the placental barrier and are also passed in milk during lactation and produce developmental anomalies in offspring at high doses [94,95]. Various reports of avermectins causing reproductive toxicity are summarized in Table 4.

Endocrine Disruption
The organisms administered sub-lethal doses of a drug may experience different side effects including the endocrine disruption [96]. These are the endocrine glands which produce hormones responsible for metabolism, growth, and development of cells. Thus, any disruption in the endocrine system will have a negative impact on the organism [97]. At present, there is limited research available describing the role of avermectins as endocrine disruptors. Additionally, there are different gaps regarding mammalian endocrinal toxicology which are not properly addressed [98]. Among the avermectins, ivermectin and abamectin are investigated as endocrine disruptors. Sexual maturation in Holstein heifers was advanced by 3.5 months following continuous ivermectin administration from birth till puberty. This early maturity may be associated with the elevated levels of insulin-like growth factor and luteinizing hormone [31]. In Baladi cows, the injection of a therapeutic dose (0.2 mg/Kg) of ivermectin one day post-parturition caused 3 months delay in estrous. It caused disturbances in the levels of luteinizing hormone, follicle-stimulating hormone, cortisol, estradiol, progesterone, and prolactin [32]. Similarly, ivermectin was also shown to suppress the sexual behavior in estradiol treated female rats at therapeutic dose [99]. In another study where the male albino rats were exposed to sublethal dose of abamectin, significant alterations in sex hormones as well as the thyroid hormones were observed [97,100,101]. Likewise in humans, abamectin is placed under the category which is more likely to cause endocrine disruption according to the joint UK-German document [102].
The toxic effects of avermectins in animals discussed above can be briefly described, as in Figure 4.

Milk Residues
The lipophilic drugs in plasma easily cross the epithelial barrier of mammary glands and concentrate in the milk. The same is true with the avermectins which are highly lipophilic in nature and, hence, diffuse readily in milk [23,103]. This lipophilicity can be estimated from concentration ratio of drugs in milk vs. plasma. The greater the value, the higher the lipophilicity of the drug. In various reports, ivermectin and abamectin were shown to have concentration values close to 1 and 0.2 respectively in the experimental animals, showing abamectin to be relatively safe in view of milk residues [104,105]. The milk with drug residues is fit neither for human consumption nor for animals' offspring. So, there is a need for either the discovery of new, safe drugs or the development of a new administration technique that minimizes the milk residues and the milk-rejection period.

Resistance
Avermectins have been used in animals as mass drug administration strategy (MDA) with the purposes of treatment and prophylaxis from parasitic diseases. However, this

Milk Residues
The lipophilic drugs in plasma easily cross the epithelial barrier of mammary glands and concentrate in the milk. The same is true with the avermectins which are highly lipophilic in nature and, hence, diffuse readily in milk [23,103]. This lipophilicity can be estimated from concentration ratio of drugs in milk vs. plasma. The greater the value, the higher the lipophilicity of the drug. In various reports, ivermectin and abamectin were shown to have concentration values close to 1 and 0.2 respectively in the experimental animals, showing abamectin to be relatively safe in view of milk residues [104,105]. The milk with drug residues is fit neither for human consumption nor for animals' offspring.
So, there is a need for either the discovery of new, safe drugs or the development of a new administration technique that minimizes the milk residues and the milk-rejection period.

Resistance
Avermectins have been used in animals as mass drug administration strategy (MDA) with the purposes of treatment and prophylaxis from parasitic diseases. However, this extensive application of the drugs has led to rapid selection and resistance in the parasites which can survive the drug administration [37]. Various studies have shown the resistance to be associated with genetic mutations but the main mechanism of development of resistance is yet not known [37,106]. Some of the techniques like genetic crossing and genome-wide sequencing may help us better understand the mechanism of resistance development towards avermectins [107][108][109]. Apart from understanding the resistance development mechanism, there is need to develop integrated approaches using various options for control of parasites [37,106].

Ecotoxicity
Administration of avermectins in animals results in their excretion of faeces, thus posing an impact on the biological processes of nature [110]. The non-specificity of avermectins in their action leads them to exert their deleterious effect not only on the parasites but also on the non-parasitic species found in the ecosystem like dung beetles. These non-parasitic environmental organisms may have an important role in food webs like nutrient recycling in the environment and organic matter decomposition [110]. The sustained slow-release strategy for avermectins poses an even greater threat to the ecosystem [111]. The avermectins are somewhat resistant to environmental degradation and, thus, tend to accumulate in the environment [112]. For example, there are reports of a direct relationship between the dung beetle population in the field and the concentration of avermectins in dung [13,113]. It is important to highlight the potential effects of un-controlled and over the label use of Avermectins in animals and humans. The magnitude of resistance may further be amplified due to the residues of avermectin drugs in the environment. Therefore, there is a need to adopt some comprehensive strategy for the use of avermectins in animals to protect the environment from their toxic effects.

Materials and Methods
We performed a systematic review of the scientific literature using the Web of Science, PubMed, and Google Scholar databases through multiple combinations of Avermectins terms with "Toxicity", "Animals", "Parasite", and "Safety". We limited the search to studies in various types of toxicity (Nephrotoxicity, Hepatotoxicity, Neurotoxicity, Reproductive Toxicity). We obtained over 500 articles that were analyzed and subsequently those that corresponded to the objectives of this study were selected. Following this criterion, we chose and used 113 articles as a reference for this review.

Conclusions
The avermectins, owing to their broad spectrum of action, find a crucial role in parasitic control practices with huge sales of these products throughout the world. However, the side effects linked with their usage puts emphasis on a comprehensive approach towards understanding all the aspects associated with them. Due to their non-target specificity, these act both on the parasites as well as the host animals to which they are administered. Nephrotoxicity, hepatotoxicity, neurotoxicity, and reproductive toxicity are the main side-effects produced by them in the host animals. Apart from these effects, these compounds also pose a major threat to the ecosystem and the food web with additional risk of resistance development in the parasites. So, there is need for research focusing on the above-mentioned issues to overcome these major constraints of avermectins usage in animals.

Conflicts of Interest:
The authors declare no conflict of interest.