Molecular Mechanisms of Amitraz Mammalian Toxicity - American

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Review

Molecular mechanisms of amitraz mammalian toxicity: A comprehensive review of existing data Javier Del Pino, Paula Viviana Moyano-Cires, Maria Jose Anadon, Maria Jesus Diaz, Margarita Lobo, Miguel Andres Capo, and Maria Teresa Frejo Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500534x • Publication Date (Web): 14 May 2015 Downloaded from http://pubs.acs.org on May 22, 2015

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Chemical Research in Toxicology

Molecular mechanisms of amitraz mammalian toxicity: A comprehensive review of existing data.

Javier del Pino1,*, Paula Viviana Moyano-Cires2, Maria Jose Anadon2, María Jesús Díaz1, Margarita Lobo1, and Miguel Andrés Capo1, María Teresa Frejo1

1

Departament of Toxicology and Pharmacology, Veterinary School, Complutense University of

Madrid, 28040 Madrid, Spain. 2

Department of Toxicology and Legal Medicine, Medicine School, Complutense University of

Madrid, 28040 Madrid, Spain.

Running Title: Molecular mechanisms of amitraz mammalian toxicity.

*Corresponding author:

Javier del Pino PharmD, PhD Departament of Toxicology and Pharmacology Veterinary School Complutense University of Madrid Avda. Puerta de Hierro s/n 28040 Madrid. Spain Phone: +34-916628170 E-mail: [email protected]

ACS Paragon Plus Environment

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TABLE OF CONTENTS (TOC) GRAPHIC


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ABSTRACT Amitraz is a formamidine pesticide widely used as an insecticide and acaricide. Amitraz poisoning cases in humans and animals are still being described up to date, which is a cause of concer for health authorities. Amitraz was reported not to pose unreasonable risks or adverse effects to humans or the environment as the other commercialized member of the formamidine family, chlordimeform, which was removed from the market because of carcinogenic effects in animal studies. Amitraz was classified as a non-quantifiable “Suggestive Evidence of Carcinogenicity” and not genotoxic, but recently it has been reported that it could induce genotoxic effects. Moreover, since the previous published evaluations made by EPA and JMPR there have been new reported data on amitraz toxicity related to genotoxicity, oxidative stress, cell death, immunotoxicty, endocrine disruption and developmental toxicity which indicate that the risk of this compound could be underestimated. Furthermore, there is missing information about the dose-response relationship for some mechanisms and toxic effects described for amitraz and its metabolites, the mechanism of action by which several toxic effects are produced, and the amitraz pharmacokinetic on different species. According to this, the new information reported should be taken into account and more studies should be performed to fill gaps of missing information for a complete hazard identification and therefore an exhaustive risk assessment of amitraz. This review is aimed at updating the current knowledge on molecular mechanisms of amitraz mammalian toxicity, pointing out the missing information, providing some possible explanation of the mechanism by which some toxic effects observed are produced, and a future direction on its research. To our knowledge this is the first review on molecular mechanisms of amitraz toxicity.

KEY WORDS: Neurotoxicity, reproductive and developmental toxicity, immunotoxicity, genotoxicity, cancer, hazard identification.


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CONTENTS Introduction Mechanisms of amitraz toxicity Agonist of alpha-adrenergic receptors Inhibition of histamine H1 receptor Prostaglandin synthetase inhibition Inhibition of monomanino oxidase Adenylyl cyclase inhibition Voltage dependent calcium ion channels activation Generation of reactive oxygen species Cell death Endocrine disruption Neurotoxicity Reproductive and developmental toxicity Reproductive toxicity Developmental toxicity Neurodevelopmental toxicty Immunotoxicity Genotoxicity Cancer Conclusions Funding Sources Acknowledgments Abbreviations References

INTRODUCTION

Formamidine pesticides were developed in the late 1950’s and early 1960’s due to the development of resistances to conventional insecticides. The two formamidines primarily marketed and most widely used are chlordimeform and amitraz. Amitraz (1,5 di-(2,4-dimethylphenyl)-3methyl-1,3,5-triaza-penta-1,4-diene) (Figure 1) was first patented in 1971, registered as a pesticide technical grade in 1975,1 and marketed in 1981. Different agencies have evaluated the amitraz toxicity and the lethal dose 50 (LD50) or lethal concentration 50 (LC50) values of amitraz via inhalation, dermal and oral (Table 1) or its metabolites via oral on different species (Tables 2) based on acute toxicity studies.1,2 The EPA (Environmental Protection Agency), according to acute toxicity studies, classifies amitraz as Class


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III-slightly toxic by the oral and inhalation routes, as Class II-moderately toxic by the dermal route, as Class IV-not a dermal irritant and only slight irritant to the eyes and not a dermal sensitizer.1 Amitraz is rapidly absorbed, distributed, metabolized, and eliminated primarily via urine when administered orally to mammals.1,2 No differences have been described between species and genders in the rates and routes of excretion. In all species studied, 55-74% of the dose was excreted in the urine within the first 24 hours after dosing.1,2 The degradation products present in the urine include N'-[2,4-dimethylphenyl]-N-methylformamidine (BTS-27271), 2,4-dimethylformanilide (BTS-27919), 2,4-dimethylaniline (BTS-24868), 4-formamido-3-methylbenzoic acid (BTS-39098), 4-amino-3-methylbenzoic acid (BTS-28369), and several unknown metabolites.3 Moreover, the spectrum of metabolites observed was similar in all species studied. BTS-27271 and BTS-27919 are the main metabolites of amitraz and a cause of concern due to the content of 2,4-dimethylaniline moiety, which could lead to developmental and genotoxic effects.1,2 Furthermore, BTS-27271 has been found to be more potent than amitraz with regard to its miticidal activity4 and mammalian toxicity.5,6 The action of these metabolites has been described only for some of the mechanisms and effects of amitraz, thus further studies are needed to determine their participation on the rest of mechanisms and effects. On the other hand, there is a lack of information about the levels of amitraz and its metabolites that reach the different tissues from different species and genders in the range of toxic doses. In this regard, there are only two studies on amitraz plasma levels, one in ponies and sheeps after intravenously administration of amitraz,7 and another one on dogs orally, where the plasma toxic doses observed were between 5 and 25 mg/L,8 but no data are available in other species. With only this information, an extrapolation of the in vitro results to the in vivo situation is very difficult if not impossible, thus a wide range of studies is necessary to cover this missing information for hazard identification and therefore future risk assessment of this compound.


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MECHANISMS OF AMITRAZ TOXICITY

Different mechanisms of amitraz toxicity have been described (Figure 2) as presented below.

Agonist of alpha-adrenergic receptors.

Activation of α2-adrenergic receptors has been reported as the main mechanism of amitraz toxicity, since it plays a role in the induction of hypotension, bradycardia, miosis and mydriasis, altered mental status, hypothermia, convulsion, polyuria, gastrointestinal hypomotility and hyperglycaemia.9-16 Initially, a hypothesis was established about the role of the α2 receptor in neuroendocrine toxicity of this insecticide in mammals, based on the similarities observed between this receptor and octopaminergic receptors, which are the targets of formamidine pesticides in insects.17 This hypothesis was subsequently reinforced by experimental studies, which revealed that amitraz causes similar effects to those observed with clonidine, a selective α2 receptor agonist, and the simultaneous administration of yohimbine, an α2, but not α1, receptor antagonist, reversed the effects of amitraz on cardiac function, intestinal motility, pupillary aperture, and blood pressure among others and reduced lethality.11-15 This hypothesis was proved through binding studies by Costa et al.,9,10,18 which shown that amitraz, in vivo and in vitro, is able to strongly inhibit [3H] clonidine binding to α2 receptor in mouse and rat brain. In this sense, Costa et al.,18 showed that amitraz inhibitory concentration 50 (IC50) of α2 in mouse forebrain in vitro was 130 nM and Costa et al.,9 described inhibition of α2 receptor at dose of 7.5 mg/kg in mouse brain in vivo. Moreover, Costa et al.,10 reported that amitraz IC50 was 95-110 nM in rat brain in vitro, and the inhbition of α2 receptor was from 25 mg/kg doses in rat brain in vivo. These in vivo results show that rats are less sensitive to amitraz inhibition of α2 receptor than mice, which is not in agreement with the higher sensitivity to amitraz toxicity seen in rats in vivo as shown by the oral LD50 (Table 1). However, in vitro results are in accordance with sensitivity differences presented to amitraz toxicity. This could


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be due to the fact that in vivo, amitraz is metabolized to BTS-27271, which is more active than amitraz itself, presenting higher LD50 values in rats than mice. Later, Abu-Basha et al.,19 reported that amitraz and its metabolite BTS-27271 (0.01, 0.1, 1, and 10 µM) actions on insulin and glucagon secretion are mediated by α2D-adrenergic receptors as showed with idazoxan, an α2A/2Dadrenergic receptor antagonist, treatment in rat pancreas in vitro and Altobelli et al.,20 showed that amitraz (0.1-10 mM) acts as a partial agonist of presynaptic α2D-adrenergic receptors in the rat hypothalamus synaptosomes. These results suggest that α2D-adrenergic receptors subtype may be the subtype amitraz acts on but further studies are needed to confirm this hypothesis. On the other hand, Costa et al.,18 described that amitraz also presents a low agonist action on α1 adrenergic receptor in mouse forebrain in vitro with an IC50 of 10 nM. Moreover, Cullen and Reynoldson21 showed that selective α1 and α2 adrenergic receptor antagonists prazosine (20 µg/kg) and yohimbine (30 µg/kg) respectively blocked bradycardia produced by intracisterna magna injections of amitraz (5-25 µg/kg) in dogs, suggesting that α1 receptor may also participate in the toxic effects induced by amitraz. These results are in contradiction with the studies comented above where α1 receptor antagonist prazosin at high doses did not reverse toxic effects induced by amitraz. Taking all these data into account, some effects mediated by amitraz could be due to an action only on α2 receptor in some cases, and in other cases on both receptors, being the action on α1 adrenergic receptor masked by α2 agonist actions due to the reported low agonist action of α1. Moreover, other mechanisms besides α2 agonist action could contribute to some signs reported above, and this issue will be discussed in the following sections.

Inhibition of histamine H1 receptor

Amitraz causes toxicity in horses characterized by drowsiness and stasis of intestine,22 which are also side effects of many antihistamines,23 suggesting that these signs may be due to an antihistaminergic action of amitraz. Based on these signs, the effects of amitraz on guinea


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pig ileum in vitro were investigated.24 The results indicated that amitraz (3.4 µM) inhibits contraction of intestines which was stimulated by H1 histamine agonists 2-methylhistamine (0.32 µM) and 2-pyridylethylamine (1.64 µM) in a 52% and 47% respectively, but not the stimulated by cholinergic agonists such as acetylcholine and methacholine. Pass and Seawright24 concluded that amitraz is a H1 antagonist in guinea pig ileum and that this action could be relevant in cases of gastrointestinal toxicity. After that, Costa et al.,18 described that amitraz is able to inhibit [3H] mepyramine (3.5 nM) binding to H1 receptor in rat brain in vitro with an IC50 of 10-5M, concluding that it is a week inhibitor of H1 receptor. Finally, Pass and Mogg5 reported that Amitraz (10-5M) and BTS-27271 (10-5M) had significant H1 antagonist activity on isolated guinea pig ileum, increasing effective concentration 50 (EC50) of 2-pyridylethylamine H1 agonist 2.5 ± 0.35 and 8.3 ± 1.75 times respectively from control. However, amitraz (1 and 2 mg/kg, iv) and BTS-27271 (0.68 and 1.35 mg/kg, iv) caecal stasis induced in sheeps was reversed partially by intravenous administration of 2pyridylethylamine (10 mg/kg) but it was completely reversed by intravenous administration of yohimbine (0.5 mg/kg), which confirms that amitraz is a weak inhibitor of H1 receptor and indicates that this action in vivo is secondary to α2 adrenergic receptor agonist activity. The discrepancies on H1 antagonist activity in vitro between Costa et al.,18 and Pass and Mogg5 could be related to the differences on species and tissues tested or to differences on vehicule used. In this regard, the discrepancies between species could be due to the higher toxicity presented by amitraz in guinea pigs than rats, as represented by LD50 orally (Table 1), which could reflect a higher antagonist potency of amitraz on H1 receptor in guinea pigs. Moreover, it has been suggested that H1 receptors in rat may not be structurally identical with those in guinea-pig and it may present different afinity for histamine and antagonists as was observed with the selective H1 antagonist mepyramine25 which could also explain the differences observed between species. Besides, H1 receptors have been reported to present polimorfism26-28 and the variants expressed in the intestine and brain could be different, thus having different sensitivity to amitraz and its metabolites. Further studies are required to confirm the differences on antagonist potency between species and tissues in vivo,


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determine the dose-response relationship and survey the possible toxic effects mediated by this mechanism. In this regard, the inhibition of H1 receptor, besides the delay of intestinal motility, could contribute other effects like cell death29, behavioural30,31 and reproductive32-35 alterations, seizures36,37, and anti-inflammatory38 action among others which will be discussed in the following sections.

Prostaglandin synthetase inhibition

Formamidines were proposed to be prostaglandin synthetase inhibitors based on studies in isolated guinea pig ileum, which show aspirin-like actions with depression of electrically induced responses, reversible by prostaglandin E1 but not by naloxone.39,40 According to this, Yim et al.,41 evaluated the antipyretic and anti-inflammatory activities of amitraz given intraperitoneally to rats at 5 to 80 mg/kg doses, compared with two known inhibitors of prostaglandin synthesis, aspirin (20 to 320 mg/kg) and indomethacin (0.25 to 2 mg/kg). The results show that amitraz behaves as an antipiretic and anti-inflammatory agent. Moreover, Yim et al.,41 found that amitraz caused inhibition of prostaglandin E2 (PGE2) synthesis with an IC50 of 880 µM. This action could contribute, besides to the antipiretic and anti-inflamatory activity described, to induction of hyperthermia42-50, aggressiveness,51 developmental52-56 and reproductive toxicity57-60 among others which will be discussed in the following sections. There are no studies about amitraz metabolites activity on prostaglandin synthetase or on weather this action is mediated only by amitraz, its metaboloites or both. Thus, it is important to develop more studies to clarify this point. Moreover, the mechanisms and kinetics of inhibition by amitraz and the toxic effects mediated through this mechansim remain to be established and further studies are needed to elucidate them.


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Inhibition of monoamine oxidase (MAO).

The most important derivative pathway for numerous bioactive amines is represented by the oxidative deamination performed by MAO type A and B, two isoenzymes coded by two related but separate genes.61 Among amitraz effects that could be mediated by MAO inhibition are motor activitiy alterations,62 aggressiveness,63,64 reproductive65,66 and neurodevelopmental toxicity67-71 among others, which could also be mediated by other mechanisms as disccused in the following sections. Initially, Knowles and Roulston72 hypothetized that formamidines are inhibitors of MAO based on tick poisoning effects and on the known ability of amidine compound to inhibit MAO on mamals. This hipothesis was confirmed by Aziz and Knowles73 and Benezet and Knowles74 which studied the inhibition of rat liver´s and brain´s MAO in vitro by amitraz, BTS-27271 and BTS27919 with an IC50 in liver of 6.6 x 10-7 M and 2.7 x 10-5 M for amitraz and BTS-27271, and in brain of 5 x 10-5 M, 3.8 x 10-5 M and 2.3 x 10-5 M for amitraz BTS-27271 and BTS-27919 respectively. According to these data, amitraz was a more potent inhibitor of MAO on liver than its metabolite BTS-27271, but this difference was erased in brain studies. This could be due to the fact that amitraz and its metabolite present different selectivity for the MAO isforms and the isoform for which amitraz is selective was more expressed in the brain, thus the IC50 is increased. Later, in vitro and in vivo experiments conducted by Moser and MacPhail75 showed that amitraz inhibited both MAO-A and MAO-B in a dose-dependent way at dose ≥100 mg/kg. Amitraz appeared to be more selective for MAO-B when given in vivo, although MAO-A was also inhibited at dose ≥ 300 mg/kg, but this selectivity was not observed in vitro where only amitraz was tested.75 The discrepancies observed on MAO A and B inhibition between in vitro and in vivo experiments were suggested to be due to the fact that on in vivo studies amitraz is metabolized and thus one of its metabolites could present selectivity on MAO B. Althoug, Moser and MacPhail75 did not observed selectivity in vitro by amitraz, the results of Aziz and Knowles73 and Benezet and Knowles74 suggest that amitraz


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presents selectivity for one isoform. This lack of selectivity observed by Moser and MacPhail75 could be due to the fact that the MAO isoenzyme for which amitraz is selective is more expressed in the brain. In this regard, MAO A has been reported to present higher concentrations than MAO B in rat brain, thus if amitraz would present more selectivity for MAO A this would be masked by its higher expression,76 explaining the differences observed. These data suggest that amitraz could present higher selectivity on MAO A and some of its metabolites on MAO B. On the other hand, insulin has been reported to increase enzimatic activities of MAO-A and MAO-B,77 thus a decrease of insulin levels, such as that produced by amitraz, could reduce these activities, contributing indirectely to amitraz inhibition of MAO-A and MAO-B activities. According to all exposed above, further studies are requiered to determine the mechanisms of MAO inhibition by amitraz and its metabolites, their selectivity on MAO isoenzymes and the toxic effects mediated through this inhibition.

Adenylyl cyclase inhibition

Cyclic adenosine monophosphate (cAMP) is a second messenger which regulates physiological processes, such as cell adhesion and growth, cell survival, energy homeostasis, neuronal signaling, and muscle relaxation. Alterations in cAMP signaling have been observed in a number of pathophysiological conditions such as cell death, neurotoxicity and developmental toxicity among others.78-80 Chen and Hsu6 hypothesized that amitraz and its metabolites (0.1-10 µM) activate α2 receptor which inhibits insulin release by decreasing intracellular cyclic AMP levels. This hypothesis was confirmed because amitraz and its metabolite BTS-27271 inhibited insulin release stimulated by forskolin (1 µM), an adenylyl cyclase activator, showing a cyclic AMP insulin-dependent release in a rat beta-cell line (RINm5F). Moreover, 3-isobutyl-1-methylxanthine (IBMX; 100 µM), a non-specific inhibitor of cyclic AMP phosphodiesterases, elevated cyclic AMP concentrations increasing insulin release, effects that were both reversed by amitraz, BTS-27271


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and medetomidine (0.1 µM), an specific α2 receptor agonist. Furthermore, yohimbine (0.01, 0.1 and 1 µM) also prevented the effect of amitraz and BTS-27271 on IBMX-stimulated accumulation of cyclic AMP but prazosin (1 µM), did not. Thus, it was proved that amitraz and its metabolite BTS27271 decrease cyclic AMP by inhibiting adenylyl cyclase through its action on α2 receptor in vitro. Further studies are needed to confirm this effect in vivo, the amitraz actions mediated by this mechanism and the dose-response relationship.

Voltage-dependent calcium ion (Ca2+) channels activation.

Ca2+ is a second messenger which regulates physiological processes, like mitochondrial metabolism, cell cycle entry, and cell survival. Alteration of its signal leads to pathological effects such as neurotoxicity,81 cell death82,83

and neurodevelopmental toxicity.84,85 Shin and Hsu,86

reported that amitraz and its active metabolite BTS-27271 (10-8-10-5 M) caused a dose-dependent increase in myometrial contractility in the luteal phase strips in vitro. The alpha 2-adrenoceptor antagonist, yohimbine (10-8-3x10-7 M), blocked this effect of amitraz and BTS-27271 in a dosedependent manner. When uterine strips were pretreated with the Ca2+ free Tyrode's solution or the voltage-dependent Ca2+ channel blocker verapamil (3x10-5 M), the contractile effects were completely abolished. These results suggested that amitraz and its metabolite are able to induce an increase in extracellular Ca2+ influx through voltage-dependent Ca2+ channels activation, mediated by its alpha 2-adrenoceptors activity which produces myometrial contractions, however subsequent studies are needed to confirm these data both in vitro and in vivo, and posible effects mediated by this mechanism.


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Generation of reactive oxygen species

Oxidative stress can be the result of either an increase of ROS production and/or a reduction in the mechanisms of ROS elimination. ROS can directly modify cellular macromolecules leading to a variety of toxic effects including lipid peroxidation, protein dysfunction, nucleic acid oxidation, and cell death. Kruk and Bounias87 reported that amitraz (50 µM to 2 mM) oxidation leads to the generation of OH˙ and O2˙. Recently, Radakovic et al.,88 evaluated the influence of oxidative stress on DNA damage and cell death induced by amitraz in human lymphocytes. This study showed that the co-treatment of amitraz (3.5 µg/mL) and antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced DNA damage and the percentage of apoptotic and necrotic cells in comparison with amitraz alone treatment. These results indicated that amitraz treatment induces free radicals, generating DNA damage and cell death. Furthermore, it was reported that amitraz (5 mg/kg intraperitoneally) decreases hepatic glutathione and non protein sulfydryls (NPSH) (to a maximum of 40%) due to its α2-agonist action causing weakness of the antioxidant defense mechanism,89 which could result in oxidative stress. Further estudies are requiered to confirm these data in vivo and if this effect is mediated by amitraz, its metabolites or both. The generation of ROS could lead to genotoxicity,90-92 cell death,93,94 carcinogenesis90 and developmental toxicity95,96 among others which will be discussed in the following sections. The mechanism through which amitraz could induce oxidative stress is unknown. It is becoming increasingly clear that various xenobiotic-inducible CYPs are associated with different signaling pathways contributing to ROS (superoxide, H2O2, and OH˙) production and causing cell/tissue injury.97 Moreover, it was reported that estrogen metabolism by CYP generates reactive quinones and semiquinones which leads to the production of ROS and oxidative stress.97 Furthermore, an increase of blood glucose levels results in mitochondrial injury and endothelial dysfunction by generating reactive oxygen species and inhibiting nitric oxide production, respectively.98 In this way, amitraz was reported to induce cytochrome P450 1A1, 2B1, 2E1 and


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nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase,99,100 which were related with ROS generation,97,101-105 increased glucose levels and estradiol metabolism, being these the most probable mechanisms of oxidative stress generation by amitraz. Thus, further studies are requiered to determine the mechansims through which amitraz, its metabolites or both may induce oxidative stress and the effects that could be mediated through this action.

Cell death

Multiple authors have described amitraz potential ability to induce cell death. Gregorc and Bowen106 were the first ones to study amitraz (0.174 µg/larvae) induced cell death in honeybee larvae. They observed necrotic and apoptotic cell death in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after amitraz treatment. Cell death was reduced to 36% in the epithelial cells, 48 h after treatment. This study showed that amitraz is able to induce apoptotic and necrotic cell death. Afterwards, Ueng et al.,99 evaluated cell viability in amitraz treated MCF-7 human breast cancer cells using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method. They found that treatment with 0.1, 1, and 10 µM amitraz for 3 h, 1 day, and 2 days had no effects on cell viability, but treatment with 100 µM amitraz produced 22%, 27%, and 69% decreases in cell viability, while 500 µM amitraz resulted in 40%, 69%, and 70% decreases. Moreover, Young et al.,107 also tested cell viability on human lymphoblastoid WIL2NS cells treated with amitraz (11.9 µM, 119 µM, 1.19 mM, and 11.9 mM) using MTT assay. The concentrations studied, except 11.9 µM and 119 µM amitraz concentrations, caused a reduction in cell viability. Young et al.,108 also studied amitraz effects on luteinized granulosa treated cells viability by MTT assay, finding that exposure for periods of 2-72 h to 3.4 µM and 34.1 µM amitraz did not cause cell death. However, after 24 h exposure to 341 µM amitraz concentration it induced citotoxicty and after 72 h it caused cell death.108 Recently, Padula et al.,109 analyzed cell death by Annexin Vpropidium iodide (AnnV-PI) staining apoptosis test in CHO cells, showing an increase of apoptotic


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cell death frequency, at concentrations of 8.5 µM and 12.8 µM. Finally, Radakovic et al.,88 also studied cell death using AnnV-PI staining apoptosis test, showing apoptotic and necrotic cells on human lymphocytes treated with 11.9 µM amitraz. According to all exposed above there is a discrepancy on the concentration that induces cell death in vitro by amitraz which could be related to the use of different cell lines, solvents and cell culture protocols. Moreover, Olegsbya et al.,110 reported that amitraz poisoning (78 mg/kg) induced in Scottish terrier dog necrosis in liver and kidney. All these studies show the amitraz ability to induce cell death, probably being this either apoptotic and/or necrotic cell death, but weather this effect is mediated by amitraz, its metabolites or both and the mechanisms through which they could produce this effect are unknown. Amitraz was reported to induce CYP2E1,99 which induction was described to generate oxidative stress and apoptotic cell death.103,111 Moreover, amitraz is able to block H1 receptors,22 being shown that H1 antagonists induce apoptotic cell death in malignant melanoma cells involving DNA damage, caspase-2 activation and the mitochondrial pathway.29 Furthermore, amitraz could induce voltagedependent Ca2+ channels activation, increasing Ca2+ cellular levels,86 and it has been stated that abnormal Ca2+ elevation leads to apoptosis induction.82,83 Amitraz also inhibits adenylyl cyclase, decreasing cyclic AMP levels,6 which reduction may induce apoptotic cell death.112 Finally, amitraz was described to generate oxidative stress,87,88 which is able to induce cell death. The mechanism that leads to cell death could be any of the above or a combination of them. On the other hand, no studies have been developed to research whether amitraz is able to induce the last type of cell death, autophagy, which may be induced by oxidative stress.93,94 Further studies are required to determine dose-response relationship in vivo for cell death, the types of cell death produced and the mechanisms through which amitraz, its metabolites or both could induce them. Amitraz induction of cell death could lead to endocrine disruption, neurotoxicity, immunotoxicity, reproductive and developmental toxicity being necessary more studies to clarify this.


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Endocrine disruption

Endocrine disruption is an important mechanism for reproductive and developmental toxicities of pesticides and xenobiotics.100,112,113 Endocrine disruptors may elicit their effects by interfering with the synthesis, transport, metabolism, binding, action, or elimination of the endogenous hormones critical in homeostasis, reproduction, and development.114 Several reports have described the ability of amitraz to disrupt hormones by different mechanisms. First of all, amitraz has been reported to disrupt hormones through activation of α2adrenergic receptor. In this way, it has been shown that amitraz (30 mg/kg/day) inhibits hypothalamic gonadotropin-releasing hormone (GnRH) release and the ovulatory luteinizing hormone (LH) surge in rats likely through activation of α2-adrenergic receptor.115 Moreover, Young et al.,108 studied estradiol and progesterone steroidogenesis on human luteinized granulosa cells treated with 3.4, 34.1, 170.4 and 340 µM amitraz for periods of 2-72 h. They observed that exposure from 34.1 to 340 µM amitraz inhibited progesterone production after a 4 h exposure, whilst having no effect upon estrogen production. Co-administration of amitraz with the α2adrenergic receptor antagonist yohimbine prevented amitraz-mediated inhibition of progesterone production, proposing that this effect is mediated by α2-adrenergic receptor. Amitraz also inhibited human chorionic gonadotropin (HCG) stimulated progesterone production, but not estrogen production. These authors suggested that this differential inhibition of progesterone but not estrogen production may be due to an amitraz action on the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase (3βHSD), which converts pregnenolone to progesterone, because pregnenolone is also a precursor for estrogen synthesis, and the amount of pregnenolone converted to progesterone depends upon the activity of this enzyme only. Subsequent studies are needed to confirm the disruption of progesterone in vivo and the implication of amitraz metabolites on it. Furthermore, it was reported that amitraz (1.85 mg/kg, topically) suppresses insulin release and increases plasma glucose in dogs,116 and this effect was shown to be mediated by α2 receptors.117 Furthermore, Chen


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and Hsu6 found that amitraz and its metabolite BTS-27271 (0.1-10 µM) inhibit insulin release in the rat β-cell line RINm5F in a dose-dependent way through activation of α2 receptors. Finally, AbuBasha et al.,19 showed that amitraz and BTS-27271 (0.01, 0.1, 1, and 10 µM) inhibit insulin secretion in a concentration-dependent manner, but stimulate glucagon secretion (10 µM and 1 µM respectively) in a perfused rat pancreas model mediated by α2D receptors. On the other hand, induction of P450 may reduce endogenous serum hormone concentrations and consequently decrease hormonal activity.118 Chou et al.,119 studied the effect of amitraz (25 and 50 mg/kg/day) on estradiol (E2) and testosterone serum levels and its respective metabolites. Amitraz was administered intraperitoneally to male rats for 4 days and to intact female rats or ovariectomized (OVX) and 0.5 mg/kg/day E2-supplemented female rats for 7 days. It was found that in OVX E2-supplemented rats, 50 mg/kg/day amitraz caused an 85% decrease of serum E2 concentration and a marked increase of 2-OH-E2 concentration. Amitraz at 25 and 50 mg/kg/day produced 9.0-fold or greater increases of serum testosterone and 2β-OH-testosterone levels in males. Amitraz at 25 mg/kg/day resulted in no or minimal increases of liver microsomal formation of E2 or testosterone metabolites. Amitraz at 50 mg/kg/day produced an increase of E2 and testosterone metabolites formation in males and intact females. According to these data the authors suggested that amitraz induces hepatic E2 and testosterone metabolism in male and female rats, which is supported by a previous report which shows that amitraz is an inductor of CYP1A, 2B, and 3A proteins in immature female rat liver.99 Hepatic P450 enzyme modulation may alter metabolism and elimination of E2 or testosterone which may consequently lead to changes of circulating steroid level118,120 as shown by this study. On the other hand, Ueng et al.,99 investigated the effect of amitraz on estrogenic activity in MCF-7 cells, performing estrogen receptor binding tests and DNA synthesis studies. Treatment of MCF-7 human breast cancer cells with amitraz (0.1, 1, and 10 µM) antagonized E2 binding to the estrogen receptor (no effect, 8% and 17% respectively) and treatment with E2 (10 nM) unlabelled displaced [3H] E2 from the receptor by 72%. DNA synthesis in cells treated with amitraz (0.1, and 1


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µM) alone was decreased (15% and 24% respectively) and in co-treated cells with amitraz (0.01, 0.1, and 1 µM) and E2 (10 nM) it was similar to cells treated with E2 alone. These data show that amitraz is a weak antiestrogen, but in vivo confirmation of this result is needed. Additional studies using more sensitive assays such as reporter gene activation in vitro may assist in identifying the amitraz degradation products that show antiestrogenic and endocrine disruption effects. The relationship between amitraz-elicited P450 induction and antiestrogenicity remains to be investigated. Future studies are needed to define the roles of these P450 enzymes in the antiestrogenicity, the dose-response relationship between amitraz exposure and edocrine disruption and the possible neuro, immuno, reproductive and developmental toxicity effects of amitraz mediated by endocrine disruption.

NEUROTOXICITY

Amitraz induces toxic effects on peripheral nervous system and is able to cross the bloodbrain barrier (BBB)2 inducing also toxicity on central nervous system (CNS). The neurotoxic effects observed by amitraz exposure were, mydriasis, vomiting, CNS depression, sedation, loss of righting reflex, motor incoordination, coma as well as behavioral effects such as hyperreactivity to external stimuli, aggressiveness and neurochemical effects among others.62,67,75,116,122-132 Amitraz, as commented above, disrupts sex hormones, insulin, glucagon and PGE2. In this sense, sex steroids (estrogen, progesterone and testosterone) are major modulators of mammalian brain function, affecting behavior and modulating neuronal activity.133-137 In addition, PGE2 has been shown to present multiple functions in the nervous system, including its role in fever, pain, inflammation, sleep, regulation of membrane excitability, sexual behavior, and synaptic transmission, integration and plasticity.42-50 Recent studies confirm that endogenous basal levels of PGE2 are necessary for synaptic plasticity and memory acquisition.46,138 Moreover, insulin can modulate neuronal firing via actions at KATP channels, alter synaptic plasticity and modulate


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cognitive processes, thus, insulin lack or resistance is associated with cognitive deficits.139,140 Furthermore, estradiol and progesterone have been shown to improve outcomes in animal models of a number of neurologic diseases, including traumatic brain injury, ischemia, spinal cord injury, peripheral nerve injury, demyelinating disease, neuromuscular disorders and seizures, through reduction of edema, improvement of neuronal survival and modulation of inflammation and apoptosis.141-143 The endocrine disruption, induced by amitraz, of these hormones could mediate some of the behavioural and cognitive alterations observed. According to literature, amitraz induces several of its neurotoxic effects mainly through activation of alpha-adrenergic receptors, specifically α2-adrenergic receptors.11-16 Moreover, sex hormones regulate the expression and activity of these receptors. In this way, testosterone has been reported to up regulate the expression of the α1144 and α2 adrenergic receptors.145-147 In addittion, estrogens have been shown to increase the α1-adrenoceptor density but decrease the affinity, while progesterone decreased the density, but the available binding sites increased their affinity.148,149 Estradiol has also been described to increase α2 adrenergic receptor expression and progesterone decreases it,150 and desensitizes presynaptic α2A/D adrenergic receptors.151,152 The endocrine disruption induced by amitraz on these hormones could affect the expresion and activity of these receptors, contributing to the neurotoxic effects reported. Besides, hormones also regulate the expression of other neurotransmitter receptors which also could contribute to the neurotoxic effects. In this regard, estradiol treatment in combination with progesterone has been described to increase 5-HT2A receptor mRNA in the ventral hippocampus in female rats153, and decrease the expression of serotonin reuptake transporter154 and 5-HT1A autoreceptor mRNAs155 in the dorsal raphe nucleus (DRN) of ovariectomized macaques. Moreover, estradiol alone decreases 5-HT2C receptor gene expression in the ventral hippocampus in female rats.153 In addition, progesterone alone was shown to increase serotonin transporter, 5-HT1A and 5-HT2A receptor expression,156,157 and it (or its metabolites) can act directly and rapidly on neurotransmitter receptors such as the GABA-A


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receptor.158 Insulin has also been shown to modulate N-methyl-D-aspartate (NMDA), α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels159,160 and GABA receptors.161-163 On the other hand, there is controversy concerning the mechanisms by which amitraz exerts neurotoxic effects such as reduction of motor activity, aggressiveness and seizure induction. In rats, low to intermediate doses of amitraz (6.25-25 mg/kg/day) showed a decrease in motor activity.129 High doses of amitraz (50-100 mg/kg/day) affected the amplitude of evoked potentials and decreased the body weight and body temperature of the treated rats.128 In addition, at doses higher than 100 mg/kg/day it produced agressiveness and inhibition of MAO. Thus, MAO inhibition would not be the main mechanism involved in alterations of motor activity caused by amitraz.75 According to Moser and MacPhail,75,164 motor effects might be caused by the action of amitraz on the α2-adrenergic receptor, because the dose range over which it produces MAO inhibition is much higher than that which suppressed motor activity. However, Florio et al.,62 described that amitraz effects on motor activity were a consequence of its MAO inhibition within the CNS, an action that could be responsible for the enhancement of catecholamine levels, although these effects were not observed in a dose-dependent way. In this sense, Del Pino et al.,132 evaluated the effects of amitraz oral exposure (20, 50 and 80 mg/kg/day, 5 days) on monoamine levels in male rats’ brain region at 30 and 60 days of age. Amitraz caused changes in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) and their metabolite levels in a brain regional-, dose- and age-related manner. In the brain regions (hypothalamus, midbrain, hippocampus, striatum and prefrontal cortex) studied, amitraz induced a statistically significant increase in 5-HT, NE and DA content and a statistically significant decrease in their metabolites 5-hydroxy-3-indolacetic acid (5-HIAA), 3-metoxy-4 hydroxyphenyl-

ethyleneglycol

(MHPG),

3,4-hydroxyphenylacetic

acid

(DOPAC)

and

homovanillic acid (HVA) levels respectively with age interaction, except for the NE increase in prefrontal cortex and hippocampus, the 5-HIAA decrease in midbrain and the DOPAC decrease in hypothalamus and striatum which were without age interaction. Furthermore, amitraz evoked a statistically significant decrease in 5-HT, NE and DA turnover in the brain regions studied.


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These findings were in accordance with those reported by Florio et al.,62 which observed that amitraz at dose of 100 mg/kg increased the NE and DA levels in hypothalamus and striatum respectively and decreased the HVA levels in striatum, suggesting the effects on motor activity could be related to an increase of monoamines levels probably by MAO inhibition. In this sense, it has been suggested that MAO-B activity increases in the whole brain of aging rats, whereas MAOA is unchanged,76,165,166 thus the increased effect observed may be related to the increase with age of the MAO-B activity. However, in relation to the animals age, amitraz did not produced its effect on all regions studied for all neurotransmitters, suggesting that other mechanisms of action for amitraz, different from the inhibitory effect of MAO, could be present. Alternatively, the changes in the NE, DA and 5-HT and its metabolites levels in a brain regional-, dose- and age-related manner observed in rats could be also attributed to a possible amitraz effect on sex steroid hormones that modulate the expression of enzymes such as tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), tryptophan hydroxylase (TRH), MAO, catechol-O-metyltransferase (COMT), aldehyde dehydrogenase (AD) and aldehyde reductase (AR) required for synthesis and metabolism of these neurotransmitters.167-174 In this context, amitraz, possibly through disruption of T hormone, could mediate this effect. However, testosterone in CNS could be metabolized to E2 by aromatase or to dihydrotestosterone (DHT) by reductase enzymes in a region- and receptor-specific manner175-177 for this reason the final effect of amitraz could be mediated not only via T changes but also via E2 or DHT. Supporting this hypothesis, Del Pino et al.,67 showed that maternal exposure to amitraz (20 mg/kg/day, orally on days 6–21 of pregnancy and 1–10 of lactation) affected monoamine levels in brain regions of male and female offspring rats at 60 days of age, displaying a sex interaction which effect could be mediated by an alteration in sex steroids levels. Otherwise, the aggressiveness induced by amitraz could be related to its MAO inhibition activity. In this way, low levels of MAO in brain have been shown to result in a higher predisposition to aggressiveness. It was observed that MAO-A knockout (KO) mice exhibited a


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number of aberrant phenotypes, including high brain concentrations of 5-HT and norepinephrine, dysmorphic barrel fields in the sensorimotor cortex, marked reactive aggression towards intruder conspecifics, maladaptive reactivity to environmental cues and autism-related responses.63,64 Besides, it has been reported that mice deficient in prostaglandin E receptor subtype EP1 manifest behavioral disinhibition, including impulsive aggression with defective social interaction, impaired cliff avoidance, and an exaggerated acoustic startle response.51 Thus, the inhibition of PGE2 synthesis could impair the activity of this receptor contributing to the aggressive behavior. Moreover, Al-Thani et al.,178 showed a decrease in the weight of the preputial glands in adult male Swiss mice exposed to amitraz at a dose level of 40 ppm (equivalent to about 5.4 mg/kg/day) for 12 weeks. Preputial glands produce behavior-modulating pheromones that alter fighting and other behaviors in rodents.179,180 This would also be a possible mechanism through which amitraz alters behavior, being necessary more studies to elucidate amitraz effects on pheromone production and its effects on behavior. Finally, Gilbert181 reported that amitraz (50 mg/kg/day) was able to induce seizure in Male Long-Evans rats which could be mediated through alpha-2 adrenergic agonist and/or local anesthetic-like properties of these compounds. Subsequently, Gilbert and Mack182 showed that both mechanisms are implicated on seizures induction. Moreover, other mechanisms besides the last ones could be implicated on amitraz induced seizures. In this way, the inhibition of histamine H1 receptor has also been implicated in seizure induction,36,37 and it is known that seizure induction is related to dysfunctions on gabaergic and glutamatergic systems, voltage-gated sodium, potassium and calcium channels.81 Further studies are needed to clarify amitraz implication on seizures induction through an alteration of these neurotransmitter systems and voltage ion channels. Taken together, amitraz and its metabolites may exert neurotoxicity mainly through a combination of inhibition of MAO, alpha-adrenergic agonist activity, inhibition of histamine H1 receptor, inhibition of PGE2 synthesis and sex steroid actions among others. Further studies are required to elucidate the relative contributions of these mechanisms and the metabolites


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participation on amitraz neurotoxic effects. Information regarding sex steroid hormone levels and expression of the enzymes involved in the synthesis and metabolism of monoamine neurotransmitters in the different CNS regions of male and female rats after amitraz exposure would be necessary to evaluate the significance of an endocrine-disrupting activity of amitraz on its neurotoxic effects. Moreover, although acute and chronic neurotoxic effects have been reported in animals, there is only information about acute neurotoxic effects in humans. This point is important due to the fact that the Reference Dose (RD) and the Adjusted Population Dose (PAD) have been calculated from NOAEL values of acute neurotoxicity data in humans,183 but as it has been observed in animals studies, the doses from which chronic neurotoxic effects take place could be lower than those from which acute neurotoxic effects are produced. Thus, these levels of safety exposure could not cover the risk of chronic neurotoxic effect in the population. This is especially important in producers, handlers and applicators as well as in populations living in areas close to its use, because the safety values could be exceeded if the security measures suggested are not follow. Therefore, further studies are required to confirm this point, to make a complete hazard identification to re-evaluate, if necessary, the safe levels of exposure for all population and thus make a an exhaustive risk assessment.

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

Reproductive toxicity

Several unpublished studies were used by JMPR2 and USEPA1 to evaluate the reproductive effect of amitraz in rat and mouse. In rat, amitraz exposure was associated with an increase in the percentage of deaths and a prolonged estrus. In mouse, amitraz exposure was associated with a decrease in food consumption, a decrease in body weight, a prolongation of pro-estrus, and a shortening of diestrus. The no-observed-adverse-effect levels (NOAEL) proposed for reproductive


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effects (1.47 mg/kg/day/male and 1.69 mg/kg/day/female) were lower than the systemic toxicity NOAEL (4.84 mg/kg/day/male and 5.22 mg/kg/day/female). These studies did not satisfy the data requirements for EPA Guideline 83-4 (Reproductive Toxicity) due to technical deficiencies in their conduction and were unacceptable for regulatory purposes to EPA,1,183,184 which required as confirmatory of the present data a study of the developmental neurotoxicity and reproductive toxicity potential of amitraz in rat. Recently, Al-Thani et al.,178 studied amitraz effect on reproduction and fertility in adult male Swiss mice exposed via drinking water at doses of 0, 1.5, 3 and 6 mg/kg/day amitraz for 12 weeks. They reported that amitraz at 1.5 mg/kg/day showed decreases in fertility index, sperm production, the number of viable fetuses and an increase in post-implantation loss in mice. They also showed a decrease in the weight of the preputial glands, the testes and the epididymis, which could be related to the significant decreases in the sperm counts. The pregnancy rate was significantly reduced in females impregnated by the tested males, which could be related to the significant reduction in sperm count and/or to poor quality of the semen. Moreover, the significant decrease described in the average body weight gain in the tested males indicates general toxicity. This magnitude of toxicity might have affected the animals indirectly rather than having any specific effect on fertility. Lately, Lim et al.,185 evaluated the effects of amitraz on development and reproductive parameters in rats following the Organisation for Economic Co-operation and Development (OECD) Test Guideline 421.186 Amitraz was administered via drinking water at doses of 0, 1.5, 4.5 and 13 mg/kg/day to male rats from 2 weeks before mating to the end of 14-day mating period and to females from 2 weeks before mating, throughout mating, gestation and up to lactational day 4. They observed a decrease in food consumption at 4.5 mg/kg/day, maternal toxicity in rats at 13 mg/kg/day and decreases in the seminal vesicle weight, sperm motility, and the number of live pups and an increase in the post-implantation loss. However, no adverse effects of amitraz on mating, fertility, and pregnancy indices and pre-coital time were observed. Under these experimental conditions, the NOAEL for general and reproduction/developmental toxicity was believed to be 4.5


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mg/kg/day. There are discrepancies between the studies of Al-Thani et al.,178 and Lim et al.,185 showing that mice are more sensitive to amitraz toxicity that rats. According to the LD50 values, rats are more sensitive to amitraz toxicity than mice but with respect to its metabolite BTS-27271 this sensitivity is reversed, suggesting that this metabolite could be more implicated in reproductive toxic effects than amitraz. Moreover, these discrepancies may be explained by the differences in animal species, metabolism and mechanisms that control reproduction, dosing form and protocol test used to evaluate the reproductive toxicity. According to these studies amitraz may have serious effects on fertility and reproduction. The mechanism through which amitraz induces these effects could be mediated by its action on monoamine systems, endocrine disruption and genotoxicty among others. Monoamine systems participate in the regulation of sexual behavior, ovulation and implatantion among other reproductive functions and an alteration on the levels of monoamine neurotransmitters could disrupt reproduction. In this sense, administration of MAO inhibitors to female rats has been reported to increase occurrence of degenerated or low developmental-stage embryos, showing that catecholamines and serotonin can have negative impact on preimplantation development.65,66 Moreover, treatment with selective serotonin reuptake inhibitors (SSRIs) during pregnancy indicates potentially hazardous effects on fertility and prenatal development.187-189 Embryo exposure to serotonin or the specific 5-HT1D receptor agonist sumatriptan has been described to impair preimplantation development.190,191 Furthermore, mouse treatment of preimplantation embryos in vitro with α2-adrenergic agonists reduced significantly the proportion of embryos which reached higher developmental stages and the mean embryo cell number was significantly lower.192,193 Moreover, steroid hormones like progesterone and estradiol as well as gonadotropins are fundamentally involved in the regulation of the female sexual behavior, menstrual cycle and in the establishment and maintenance of pregnancy.194-196 Estradiol and progesterone are both needed to support implantation.197,198 Irritations in this fine regulated balance may result in implantation


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failure and undesired pregnancy outcome. Furthermore, as was commented above, sex steroids affect the expression and activity of α1-adrenoceptors which are reported to coordinate the expression of female reproductive behaviors and LH surge,199 or control male fertility, spermatogenesis, and steroidogenic capacity of Leydig cells.200 Thus, its alteration by endocrine disruption could affect these actions. Furthermore, Prostaglandins (PGs) are considered to be very important mediators involved in female reproductive processes.57-60 They have been implicated in the coordination of ovarian function, particularly in ovulation and implantation. In this way, PGE2 seems to be one of several local signals within the follicle that acts to coordinate oocyte maturation with the time of follicle rupture, ensuring the release of an optimally-mature oocyte at ovulation,201 stimulates progesterone synthesis to regulate ovulation,202-204 and is required for embryo implantation.205,206 In addition, PGE2 participates in male fertility206 regulating sperm concentration207 and motility of spermatozoa.208,209 According to these studies prostaglandin inhibition induced by amitraz could be one of the mechanisms implicated in reproductive toxicity. Additionally, histamine was reported to exert effects on female reproductive functions playing a role in steroidogenesis, stimulating progesterone and estradiol secretion in granulose cells, in follicular development, ovulatory and implantation processes.32,33,211-213 H1 receptor signaling has been shown to mediate the stimulatory effect of histamine on implantation, being blockaded by H1 antagonists.33 Moreover, histamine has been reported to have a role in penile erection,34 stimulate steroidogenesis, and potentiate the effects of LH in testicular parenchyma.35 Amitraz antagonist effect on H1 receptor could also contribute to the reproductive toxicity effects observed. Besides, insulin has been reported to be implicated on reproduction process and a decrease of its levels, like that induced by amitraz, has been associated with reproductive neuroendocrinopathy in both humans and animal models for diabetes, which is the result of multilevel dysfunction within the hypothalamus hypophyseal-gonadal axis.35,214


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Taking all these data together, reproductive toxicicty may take place at levels of exposure lower than those from which systemic toxicity occurs, and since the RD has been calculated from data of acute neurotoxicity in humans, this safe exposure levels may not cover the effects over reproductive toxicty. Further studies to confirm that the doses from which reproductive toxicity is produced may not be covered by the RD, to determine the mechanisms through which reproductive toxicity effects are produced and the contribution to these effects of amitraz metabolites are needed to perform an exhaustive risk assessment of amitraz.

Developmental toxicity

The possible adverse effects of amitraz on developmental outcomes have also been examined using experimental animals. JMPR2 and EPA1 evaluated the potential developmental effects of amitraz in rats and rabbits using unpublished studies submitted to them. According to these studies, amitraz had a teratogenic potential in laboratory animals. These studies had a number of deficiencies under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) guidelines, which included the absence of test article characterization, insufficient number of pregnant animals or animals tested, lack of clinical observations, of an analysis of dosing solution, of dosage levels justification, and inadequate soft tissue examinations; for this reason EPA considered them unacceptable for regulatory purposes, requiring a study of the developmental neurotoxicity and reproductive toxicity potential of amitraz in rats.1,183,184,215 On the other hand, Palermo-Neto et al.,130 studied the developmental and behavioral effects of prenatal amitraz exposure. Pregnant rats were orally gavaged with amitraz (20 mg/kg/day) on days 1, 4, 7, 10, 13, 16, and 19 of pregnancy, resulting in the opening of the vagina at an earlier age, as well as in earlier fur development, delays in incisor eruption in the offspring, a higher locomotor activity and rearing frequency and shorter immobility time when observed in an open-field 30 days after birth but not at 60 and 90 days of age. A subsequent study also showed that the postnatal


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exposure of dams treated with amitraz (10 mg/kg/day) caused transient developmental and behavioral changes in the exposed offspring.131 The results showed that the median effective time (ET50) for fur development, eye opening, testis descent, onset of the startle response were increased and it took more time (in seconds) to perform the surface righting reflex on postnatal days (PND) 3, 4 and 5 in rats postnatally exposed to amitraz. Postnatal exposure to amitraz did not change locomotion and rearing frequencies or immobility time in the open-field on PND 30, 60 and 90. Later, Osano et al.,216 evaluated developmental effects of amitraz (0.34, 0.68, 1.7, 3.4, 6.8, 17, 34 µM) and its metabolite 2,4-dimethylaniline (12.9, 26.8, 51.6, 103.1, 206.3, 412.5, 825.1 µM) on embryos of Xenopus laevis. They reported that 2,4-dimethylaniline did not induce lethality to the embryos of Xenopus laevis at tested concentrations. In this study, axial flexures and a prominent effect of generalized edema induced by amitraz were observed which may have been due to a disruption of osmoregulation resulting from cell membrane lipid bilayer disruption. Also, it was observed that the surviving embryos at 17 µM amitraz were edematous. The significant effect of 2,4-dimethylaniline was a progressive loss of pigment together with encephalomegaly, that was observable from 206.3 µM dose test media. At 825.1 µM there was total loss of the pigment, leading to loss of color contrast between the eyes and the rest of the body, and the bifurcation at the forebrain was indistinguishable as a result of swelling of the brain. The amitraz teratogenicity index (TI) was 2.7, indicating a teratogenic effect of the compound, which is increased after its degradation into 2,4-dimethylaniline (TI > 5). Recently, Kim et al.,217 studied amitraz (3, 10, and 30 mg/kg/day) effects on the initiation and maintenance of pregnancy and embryo-fetal development in Sprague-Dawley rats given amitraz from days 1 through 19 of gestation. The results showed that a 19-day oral repeated dose of amitraz during pregnancy is embryotoxic and teratogenic in rats at the maternally toxic dose. Significant maternal toxicity of amitraz was observed in the 10 and 30 mg/kg/day groups, which was evidenced by the suppression or decrease in body weight, body weight gain, corrected body weight, and food consumption. The embryo-fetal developmental toxicity was observed in the 10


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and 30 mg/kg/day groups and included increased fetal death and postimplantation loss, decreased litter size and fetal body weight, increased fetal external, visceral and skeletal abnormalities, and reduced fetal ossifications in many skeletal districts. Therefore, it was unclear that the developmental toxic effect of amitraz observed in this study was either a secondary effect to maternal toxicity or a direct effect on the rat embryo-fetuses themselves. They estimated that the NOAEL of amitraz is 3 mg/kg/day for pregnant dams and embryo-fetal development. However, these toxicology reports on amitraz did not totally comply with specific testing guidelines. In this way, Lim et al.,185 tested amitraz developmental toxicity in rats following the Test Guideline 421.186 The results of this study showed that amitraz caused maternal and developmental toxicity to rats at 13 mg/kg/day. The developmental toxicity by amitraz exposure included decreased number of alive pups at birth and PND 4 and increased post-implantation loss. The significant increase in the post-implantation loss suggests that an interruption of pregnancy occurred during the embryo-fetal developmental stage, but not in the germinal developmental stage. According to these studies amitraz induces embryotoxicity and teratogenicity on animals exposed throughout pregnancy and lactation. The recent works provide some information on developmental toxicity of amitraz that may constitute a significant contribution in the process of hazard identification within the risk assessment process. There are discrepancies between the studies of Palermo-Neto et al.,130,131 and the studies of Kim et al.,217 and Lim et al.,185 about the dose that causes maternal toxicity, being for the last ones lower than 20 mg/kg/day. Moreover, there is also disagreement about whether developmental toxicity takes place at the same time than maternal toxicity as described by Kim et al.,217 and Lim et al.,185 being important to clarify these discrepancies because if maternal toxicity takes place at the same time that developmental toxicity, the effects observed could be due to maternal toxicity more than to a direct effect of amitraz. The mechanism through which amitraz induces developmental toxicity could be either direct or through maternal toxicity. Among the direct mechanisms of developmental toxicity caused by amitraz, one could be through genotoxic action, but there is controversy about the genotoxic


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potential of amitraz. Other possible mechanisms are oxidative stress or endocrine disruption induced by amitraz. Developing embryos seem to be very sensitive to ROS, especially during early organogenesis, apparently due to their reduced antioxidant capacity, leading to the production of oxidative stress and embryonic damage.95,96 Moreover, sex steroids control sexual differentiation of the brain and the body219 and their disruption during the critical periods of time could induce deep changes on sexual differentiation. Moreover, it has been reported that disruption of sex steroids during development produces teratogenic effects.219,220 Accumulating evidence indicates that monoaminergic neurotransmitters play an important role in basic developmental processes such as embryogenesis

and

morphogenesis,

controlling

cell

proliferation,

differentiation

and

migration.68,221-228 Further studies will be needed to clarify the effects of amitraz and its metabolites during development, whether the effects produced are transient or permanent, to determine the mechanisms through which amitraz and its metabolites induce developmental toxicity and if these are independent from maternal toxicity mechanisms. Finally, according to animal studies, the doses seen to produce developmental toxicity may be lower than the ones that produce systemic toxicity, so the RD may not cover the developmental toxicity effects. Thus, to avoid underestimation of the risk that this compound poses to human health all these studies are required in order to have an exhaustive risk assessment.

Neurodevelopmental toxicity

Developmental

neurotoxicity

involves

alterations

in

behavior,

neurochemistry,

neurohistology and/or gross dysmorphology of central nervous system occurring in the offspring, as a result of chemical exposure of the mother during pregnancy or lactation.229 Several studies have researched the neurodevelopment toxicity of amitraz in rats.130,131 They observed that treatment of pregnant female rats with amitraz during gestation (20 mg/kg/day) or during lactation (10


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mg/kg/day) caused transient developmental and behavioral changes in the exposed offspring as commented above. Recently, Del Pino et al.,67 evaluated the effect of maternal exposure to amitraz (20 mg/kg/day, orally on days 6 to 21 of pregnancy and 1 to 10 of lactation) on brain region monoamine levels and the neurotransmitter utilization rate (turnover) in male and female offspring rats at 60 days of age. The results of this study showed that all these neurotransmitter systems were altered in a brain regional-related manner. In male and female offspring, amitraz induced a significant decrease in prefrontal cortex of 5-HT (higher decrease in females) and its metabolite 5-HIAA (higher decrease in males) and DA and its metabolites DOPAC and HVA levels (higher decrease in females). Nevertheless, the authors reported that striatum DA, 5-HT and their corresponding metabolite contents decreased in male and female offspring without statistical distinction of sex. In contrast, amitraz did not modify 5-HT content, but caused an increase in 5-HIAA content in the medulla oblongata and hippocampus in male and female offspring. Alterations in the hippocampus DA, DOPAC and HVA levels after amitraz exposure were also observed displaying a sex interaction (higher effect in females). NE levels also showed a decrease after amitraz treatment in the prefrontal cortex and striatum without statistical sex interaction, but MHPG levels decreased in both regions with sex interaction (higher decrease in females’ striatum and higher decrease in males’ prefrontal cortex). Amitraz evoked increases in 5-HT turnover in the prefrontal cortex as well as in DA turnover in the striatum and hippocampus and decreases in NE turnover in the hypothalamus, prefrontal cortex and striatum. The maternal toxicity after exposure to amitraz was not observerd. The present findings indicated that maternal exposure to amitraz induced long-term alteration in the noradrenergic, serotonergic and dopaminergic systems in their offspring in the prefrontal cortex, striatum and hippocampus, and those variations could be related to several alterations in the functions in which these brain regions are involved. The mechanism of the effects of prenatal and postnatal exposure to amitraz is unknown. Amitraz could affect the neuronal cell replication, differentiation, synaptogenesis and axonogenesis,


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steroid metabolism, and functional development of neurotransmitter systems, effects that could result in the behavioral and neurochemical alterations observed in these studies. In this sense, it is assumed that the monoaminergic neurotransmitters play a role during development, defined as "morphogenetic" prior to assuming their roles as transmitters in the mature brain.67-71 Any changes in the levels of catecholamines during development or any agonist action on its receptors could have a profound effect on brain structural and functional development.223,230 Since the endogenous levels of these transmitters are highly regulated by MAO, any change in this enzyme can profoundly affect the developing brain. As amitraz is classified as a MAO inhibitor-like agent, this mechanism could support the alterations in the noradrenergic, serotonergic and dopaminergic systems in adulthood of rats exposed during development observed in the study of Del Pino et al.67 Besides, the agonist action of amitraz on α2 adrenergic receptor could also contribute to these alterations. Further studies determining MAO activity in treated animals should clarify if the activity of MAO was permanently altered after developmental exposure to amitraz. But also, the possibility of a direct effect of amitraz in the brain on the functional development of monoaminergic systems cannot be omitted. The loss of these projections is likely to play an important role in the alterations reported. In this regard, previous works performed in rats, that studied the gestational effects of MAO inhibitors exposure on serotonin and dopamine innervation density, showed that the administration of clorgyline and deprenyl throughout gestation as well as several days after birth, produced in offspring at 30 days of age, a significant reduction of serotonergic innervation particularly in the cerebral cortex, without observed changes in the development of the dopaminergic system.231 Moreover, conditions that increase DA turnover, as were observed by Del Pino et al.,67 could potentially increase the formation of reactive metabolites especially under conditions in which the ratio of available dopamine to antioxidant capacity is high,232 conducing to neuronal death. On the other hand, amitraz showed region-dependent effects that could be explained by regional kinetic differences which increase amitraz levels in the affected regions, but the gender-


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dependent effects suggest a sex hormone interaction on amitraz effects. In this sense, amitraz was reported to modulate serum concentrations of E2 and testosterone in rats,100 which play a role in the development of catecholamines systems233-236 and mammalian sex differentiation.237 Moreover, it was described that sex steroid hormones modulated the expression of enzymes which regulate the metabolism and synthesis of monoaminergic neurotransmitters in a gender and region-dependent way.167-175 This could possibly be another mechanism by which developmental exposure to amitraz may alter permanently the monoamine levels in prefrontal cortex, striatum and hippocampus, and not only by MAO inhibition, suggesting that the sex differences observed on these neurotransmitter systems could be related to the endocrine disruption induced by amitraz. Besides endocrine disruption and MAO inhibition, other mechanisms could induce developmental neurotoxicity by amitraz. In this manner, amitraz is able to inhibit adenylate cyclase and activate calcium voltage channels increasing cyclic AMP levels6 and intracellular calcium concentrations.86 In this way, cyclic AMP levels have been shown to control intracellular processes underlying synaptic plasticity and memory formation, guide axonal elongation, and support neuronal survival in the developing brain80 and together with Ca2+ plays a pivotal role in neuronal differentiation and maturation.84,85 Moreover, amitraz inhibits prostaglandin synthetase, decreasing the prostaglandin E2 production,41 mechanisms that could be also related to neurodevelopmental impairment. Furthermore, it has been described that prostaglandin E2 participates during development, together with sex hormones, in the organization of brain regional sex differences, as shown in differences on dendritic spines number and shape, which are necessary to develop sexual behavior. Also, PGE2 and sex hormones regulate synaptic plasticity, neuronal cell death and apoptosis, neural differentiation, proliferation, and migration,52-56 and in adulthood they regulate sleep, sexual behavior, and synaptic transmission, integration and plasticity.47,48 Prenatal exposure to amitraz may result in either direct damage or enhanced vulnerability of the neurotransmitter systems to future toxic insult. These effects may represent a large number of actions involved in the development of synaptic dysfunction in these neurotransmitter systems that


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ultimately contribute to behavioral anomalies. Nevertheless further behavioral testing is needed to confirm this suggestion. Moreover, it is important to confirm that these effects of amitraz are not transient and the implication of amitraz metabolites on them. Lastly, as indicated above the doses from which developmental toxicity is produced may be lower than those that produce systemic toxicity, so they may not be covered by the RD. Therefore, further studies are required to determine if the doses that produce neurodevelopmental toxicity are covered or not by the RD. These data will help to perform an exhaustive risk assessment of amitraz.

IMMUNOTOXICITY

The knowledge about amitraz effects on immune system is very limited. Amitraz has been reported to inhibit prostaglandin synthetase (5 to 80 mg/kg) showing anti-inflammatory activity.41 Besides, it blocks H1 receptor (1 and 2 mg/kg, iv)5 which could reinforce this anti-inflammatory effect and may have an anti-allergic effect due to histamine mediated allergic and inflammatory responses through histamine H1-receptors.38 Moreover, JMPR8 tested the amitraz potential to cause delayed contact hypersensitivity using two unpublished studies. The first study developed a guinea pig maximization test injecting to animals 5% w/w (17 mg/kg/day) amitraz in Alembicol D (a coconut oil) into a 4x6 cm clipped area of the dorsal skin on the scapular region. A week after animals were retreated with a topical application of 15 or 30% w/w (50 or 100 mg/kg/day) amitraz in Alembicol D and left for 48 h. Two weeks later the animals were challenged topically with 15 or 30% (50 or 100 mg/kg/day) amitraz in Alembicol D reporting a dermal reaction more marked and persistent than those seen in the control group 24, 48 and 72 h after challenge. The second study performed a buehler test, administrating 500 mg (417 mg/kg/day) of amitraz to guinea pig on the anterior right flank for 6 h under a dry compress on days 1, 8 and 15. After two weeks rest period, the animals received an amitraz 500 mg challenge. The cutaneous reactions were evaluated 12 and 48 h after the challenge, describing no cutaneous reactions. These differences may be related to the


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different type of test used, vehicule and time of exposure, being needed more studies to confirm the potential causing delayed contact hypersensitivity of amitraz. Recently, Institoris et al.,238 evaluated the effects of amitraz (5.29, 10.6, 21.1, 26.5 mg/kg/day for 4 weeks, orally) on humoral immune response and cell-mediated immunity performing a plaque forming cell (PFC) assay and a delayed type hypersensitivity (DTH) test respectively. At all doses tested the number of plaques formed did not change, but at the highest dose the spleen cell number of the animals immunized with Sheep red blood cells (SRBC) was significantly reduced. As a consequence, the PFC content of the spleen was also decreased at this dose. These data suggest a lack of effects on humoral immune response by amitraz. On the other hand, the two higher doses decreased the maximum and shortened the decay of DTH reaction, but the difference was statistically significant only at the highest dose, which suggests that amitraz affects cell-mediated immunity. Based on these findings, the Lower Observed Effects Level (LOEL) dose for the immune function was 26.5mg/kg/day. On the other hand, the endocrine and nervous system participate in the regulation of the immune system and their alterations may lead to immunotoxicity.194,239-241 Amitraz is able to alter monoamine neurotransmitters,62,67,132 which regulate several functions of the immune system activities, such as proliferation, cytolytic activity, cytokine and antibody release, and chemotaxis. Thus, an increase of their levels could inhibit cytotoxic activity of T cells, increase mobility and proliferation of lymphocytes, phagocytosis, cytolytic properties and synthesis of chemokines and cytokines.231,242-244 Moreover, as commented above, amitraz presents endocrine disruptor effects such as inhibition of GnRH release and LH surge which modulate immune system and their disruption may play a role in immune mediated diseases.245 Moreover, it disrupts sex steroids progesterone, E2 and testosterone, which influence the different effector cells of the immune system, modulating their coordinated response. In this way, it has been described that estrogen enhances immune responses, while progesterone and androgens decrease it.246 Exposure to testosterone reduces natural killer


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(NK) cell activity and increases the synthesis of pro-inflammatory products and anti-inflammatory cytokines.247-249 E2 can have bipotential effects on inflammatory responses, with low doses enhancing pro-inflammatory cytokine production and Th1 responses and with high or sustained concentrations reducing production of pro-inflammatory cytokines and augmenting Th2 responses and humoral immunity.249-253 Progesterone suppresses innate immune responses, including macrophage and NK cell activity as well as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signal transduction.249 Also, amitraz suppresses insulin release but stimulates glucagon secretion which are involved oppositely with the inflammatory response.254 Insulin is a proinflammatory hormone which lack of action has been related with immunosupression in diabetes. The alterations observed in diabetes disease include: decreases of leukocyte-endothelial interactions and reduced accumulation of leukocytes in inflammatory lesions;255-259 reduced mast cell degranulation;260 lowered airway inflammation to antigen challenge,261,262 and reduction in lymph node retention capacity.263 Disruption of these hormones may also induce immunological abnormalities and/or alterations in the lymphatic organs.98,264 According to these endocrine disruptor effects commented above, amitraz could induce immunosuppression, being important to evaluate its possible effects on cellular and humoral immunity. In summary, amitraz has anti-inflammatory activity and appears to have cell mediated inmunotoxicity and may cause delayed contact hypersensitivity, but more studies are necessary to confirm these results, explore if the endocrine disruption and neurotoxic effects could affect immune response and elucidate the possible mechanisms by which amitraz, its metabolites or both induced immunotoxicity, in order to perform a complete hazard identification and therefore an exhaustive risk assessment of this compound.


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GENOTOXICITY

The possible genotoxic effects of amitraz and its metabolites have been examined using unpublished in vitro and in vivo tests. JMPR2 and EPA1 evaluated the genotoxic potential of amitraz and its metabolites using Salmonella gene mutation assay (Ames test), the host mediated assay, the L5178Y mouse lymphoma assay, tests for in vitro and in vivo chromosomal aberrations, a mouse dominant lethal assay, tests for DNA damage, and tests for morphological cell transformation (Tables 3 and 4). According to these studies amitraz did not show an induction of structural chromosomal aberrations or DNA damage, thus, it was considered negative for genotoxic potential while 2,4-dimethylaniline was positive. The results analyzed to conclude that amitraz is not directly mutagenic are contradictory, being both positive and negative mutagenic effects observed. Moreover, some of these studies have deficiencies, being considered unacceptable under FIFRA guidelines for regulatory purposes.215 In this way, some gene mutation assays showed insufficient documentation to support findings, and inadequate documentation and/or supporting data. Moreover, some of the in vivo cromosomic aberration studies presented no justification for dose selection, lack of female data, appropriate sampling times, scoring criteria, test article purity, dosing solution analysis, and inadequate number of females per group. Furthermore, some tests for DNA damage had incomplete documentation of procedures and no indication of test article purity. According to these deficiencies, more studies are required to clarify the amitraz genotoxic potential, not being discardable a direct mutagenic effect. Similarly to these evaluations, Kimmel et al.,265 showed mutagenic effects only for 2,4-dimethylaniline in the Ames test, but not for amitraz, and Grilli et al.,266 reported amitraz was not able to induce DNA strand breaks on rat hepatocytes. Lately, Osano et al., 267 used the Mutatox genotoxicity test, which determines substances that damage or intercalate DNA, inhibit synthesis of new DNA, are direct mutagens causing base substitution or frame shifts, or are SOS inducing agents, to test the genotoxicity of amitraz and its metabolite 2,4-dimethylaniline, showing that both were genotoxic at very low concentrations (

Molecular Mechanisms of Amitraz Mammalian Toxicity - American

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