State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine

Abstract. Synthetic pyrethroids (SPs) are insecticides used in everyday life, in rural and forestry farms. The wide use of SPs is due to their ability to rapidly biodegradate, relatively low toxicity to mammals and high insecticidal activity. Due to the similarity of neurochemical processes, these compounds may also exhibit their neurotoxic properties in non-target objects, in addition to the effects on pests. An overview ofscientific literature has shown that the best biological model for investigating the potential neurotoxic effects of SPs and extrapolation of obtained experimental data to humans is the rat.
Objective — to investigate the effect of cypermethrin and zeta-cypermethrin in pre- and postnatal development; to conduct a comparative study of the neurotoxic properties of cypermethrin and zeta-cypermethrin.
Methods —the article presents the data of two conducted experiments studying the neurotoxic activity of representatives ofpyrethroid insecticides — cypermethrin and zeta-cypermethrin.
Results — it was found that cypermethrin and zeta-cypermethrin exhibit gender-related sensitivity: young male rats were more susceptible to cypermethrin and zeta-cypermethrin compared to females. The effect of cypermethrin and zeta-cypermethrin in the pre-and postnatal period is dose-dependent. Effect of cypermethrin in doses of 17,5 and 35 mg/kg and zeta-cypermethrin at a dose of 12,5 mg/kg in the pre- and postnatal period does not cause a neurotoxic effect in offspring. Cypermethrin exposure at a dose of 70 mg/ kg and zeta-cypermethrin exposure in doses of 35 and 70 mg/kg causes changes in behavioural reactions of offspring of both genders. Administration of cypermethrin at a dose of 70 mg/kg in the- pre and postnatal period affects motor activity and cognitive activity of young male rats (in terms of increasing the latent period of the first movement and decreasing the number of crossed squares). Administration of zeta-cypermethrin at a dose of 70 mg/kg causes in the offspring of both genders disorders in the psycho-emotional state (is a decrease in the parameters of the grooming), inhibition of motor and cognitive activity (is a decrease of the number of crossed squares and pointing,is a decrease of the number of crossed squares), and reduction of adaptive properties (is a increase of latent period of the first movement). Under the action of zeta-cypermethrin at a dose of 35 mg/kg in males, an increase in anxiety was observed (decrease in the total duration of the grooming). Zeta-cypermethrin showed a greater neurotoxicity on offspring compared with cypermethrin.
Key words: pesticides, synthetic pyrethroids, cypermethrin, zeta-cypermethrin, nervous system, prenatal period, postnatal period, behavioural reactions.

Introduction. Synthetic pyrethroids (SPs) are insecticides used in everyday life, in rural and forestry farms. Synthetic pyrethroids are the fourth generation of pesticides that have replaced carbamates, organophosphorus and organochlorine compounds. The wide use of pyrethroid insecticides is due to their ability to rapidly biodegradate, relatively low toxicity to mammals and high insecticidal activity. However, the use of these compounds is accompanied by the risk of new problems in the protection of the environment and public health [1–6].

Pyrethroid insecticides belong to preparations with pronounced neurotoxic effect for target objects — insect pests. Effect of SPs on insect pests is determined by neurophysiological action on the sensory, central and motor axons. SP molecules are absorbed through the insect cuticle, penetrating Na+ -ion channels of cell membranes and provoking their prolonged open state. Such prolongation enhances the passive flow of Na+ within the cell, which leads to a delay in returning Na+ -ion channels to the initial state. Depending on the dose, SPs acting on insects can provoke a “knockdown effect” or affect their motor functions that manifest in the form of changes in the ability to fly or crawl [7, 8].

Pyrethroid insecticides are highly toxic to fish [9, 10]. A neurotoxic effect was observed in adult Poecilia reticulata fish under the action of beta-cypermethrin, which manifested in the change in their behavioural reactions [11]. The neurotoxic action of SPs on the development of embryos of fish has been proved.  Under the effect of cypermethrin on Zebrafish embryos, concentration-dependent apoptosis was observed in the cells of the nervous system (NS) [12]. In similar studies, the action of cypermethrin in the same species of fish embryos resulted in a distortion of the body axis and a concentration-dependent death [13].

By the nature of toxic effects on mammals, SPs are distributed to insecticides of type 1 and type 2. They differ in their chemical structure, namely the presence of cyano groups. It can be predicted that the toxic properties of SPs depend on the presence of cyano groups. Representatives of type 1, which do not contain cyano group in their structure (e. g. cysmethrin, permethrin, allethrin, etc.), cause hyperactivity, aggressive behaviour, tremor and muscle contracture upon the effect on animals. At the same time, the peculiarities of the toxic effects of cyano-containing pyrethroids of type 2 (e. g. deltamethrin, cypermethrin, etc.) are salivation, seizures and choreoathetosis. Type 2 SPs surpass the insecticidal activity of type 1 hundreds of times [4,14,15].

SP neurotoxic effect in mammals is due to a violation of the process of excitation of the nerve cell, followed by the depolarisation of its membrane. Such a depolarisation results in a significant deceleration in the opening or closure of the Na+ ion channel. This results in the activation of the incoming sodium flow during the generation of the potential of an action of the neurons, and as a consequence, the refracterity phase of the nerve fibre increases, impulse generation is disturbed in the interception of Ranvier node, the rate of propagation of excitation along the nerve cell is reduced. There is an opinion that high-affinity SP binding to the a-subunit of Na+ -ion channel in the area of Ranvier node is the basis of their neurotoxic effect [8, 16].

Violations of the normal functioning of Na⁺ ion channels on mammalian nervous cell membranes leads to the inhibition of Na⁺, K⁺, ATPase, and monoamine oxidase (MAO) activity [8, 16, 17]. This is confirmed by studies performed on rats. When animals were exposed to cypermethrin and fenvalerate, there had a decrease in Na⁺, K⁺, ATPase and MAO in the brain [18].

In addition to their effect on Na+ -ion channels of nerve cells, there is an additional mechanism for cyano-containing pyrethroids, which includes the effect of SPs on the ionophore channels of gamma-aminobutyric acid (GABA) receptors in cerebral synapses [4, 16, 17]. The study describes the reduction of GABA in the brain of the rats just after a single oral administration of alpha-cypermethrin at a dose of 145 mg/kg [19]. The ability of pyrethroid insecticides to interact with GABA receptors leads to disturbances in the balance of inhibitory and excitatory processes both in the peripheral and in the central nervous system [4, 16, 20].

The scientific literature sufficiently well describes the neurotoxic action of SPs in experiments in adult laboratory animals (rats and mice). Studying the effect of pyrethroid insecticides on rats noted functional changes in the nervous system (NS). For example, when animals were exposed to cypermethrin and alpha-cypermethrin, they presented a weakening of muscle strength and a decrease in motor coordination [5, 19]. In the studies of behavioural reactions under the action of various representatives of SPs (fenvalerate, deltamethrin, cypermethrin, cyhalothrin, etc.) in rats, the motor activity in various behavioural tests (“open field”, “cross-like labyrinth”) was reduced [5]. In studying the effect of fenvalerate on mice, changes in behavioural reactions were also noted. For example, when mice were exposed to this compound, there was an increase in the latent period of the first movement and a decrease in motor-searching activity in the “open field” conditions [21].

Together with studies on the effects of SPs on adult animals, there are experimental data indicating the neurotoxic effect in young animals exposed to their effect in the postnatal period [22]. For example, a single administration of cypermethrin at a dose of 0.5 mg/kg at Day 10 after birth (DAB), young male rats showed neuro-behavioural abnormalities in various behavioural tests. Authors attribute this disorders with the decrease in the level of neuroproteins as a result of cypermethrin exposure. At repeated testing in the post-lactation period, change in behavioural reactions continued in young mice. The results obtained indicate long-term or even irreversible processes through the effect of cypermethrin [23].

In similar experiments, under the effect of cypermethrin and permethrin from DAB 8 to 15, young rats showed changes in behavioural reactions under conditions of “open field” [22]. Comparing the effect of cypermethrin on rats of different ages, the following was concluded: degeneration of dopaminergic neurons in animals occurs only under exposures during the lactation period (from DAB 5 to 19) [24]. This can be explained by the fact that NS completes its formation in the postnatal period [25].

Since data are available on the effects of pyrethroid insecticides on animals of different ages, its effect on the development of offspring cannot be ignored [26, 27]. Studies of the previous years have shown that most SPs are characterised by penetration of transplacental barrier [28, 29], which may affect the development of foetal NS in the prenatal period [30]. It is known that the brain at the stage of embryonic development is particularly vulnerable to the adverse effects of chemical substances. The main phase of neurogenesis takes place in the prenatal period [31–33]. This is the period when the brain develops from the ectodermal cells of the complex organism of the embryo, which consists of billions of precisely located, highly specialised and interrelated cells. In a normally developing brain, the neurons must precisely move along certain paths from the points of origin to their destination, by establishing a connection to other cells. Influence at this stage of brain development can lead to irreversible violations in later life [26, 32, 34]. It is therefore assumed that the brain at the development stage may act as a target for the effects of pesticides, in particular, SPs.

Experimental studies of some authors describe the intrauterine effect of SPs (permethrin, fenvalerate, cypermethrin) and related morphological, biochemical and functional changes in NS of mice in the postnatal period.  For example, morphological (decrease in the thickness of the hippocampus in the middle brain) and biochemical changes (increased levels of norepinephrine and dopamine) occurred in mice exposed to permethrin during the period of active organogenesis (10.5 days of pregnancy) [33]. In similar studies, there was an increase in the level of dopamine and a decrease in the activity of monoamine oxidase in the brain during exposure to fenvalerate and cypermethrin in the prenatal period in mice [31]. In addition to morphological and biochemical changes, animals also showed functional disorders of the NS. In the experiment under conditions of intrauterine exposure to permethrin in mice, motor activity in the “open field” test decreased [33]. Under exposure to cypermethrin from day 1 of pregnancy to day 15 of lactation, mice showed a violation of adaptive capacity in various behavioural tests [35].

As can be seen from the scientific literature, different biological models (fish, mice, rats) were used to evaluate the neurotoxic properties of SPs. However, for extrapolation of obtained experimental data to humans, rats are one of the best biological models in studies of neurotoxic properties of pesticides. Recently, the OECD (Organisation for Economic Co-operation and Development) has developed guidelines for the study of adverse effects at the developmental stage and initiation of NS functioning in the postnatal period depending on the dose and exposure time of chemicals. These methodological approaches are reflected in guideline 426 (Developmental Neurotoxicity Study) [36].

Currently, a group of cypermethrins (cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin) is widely used among SPs. Cypermethrin and its isomers are well-studied in toxicological terms in various biological models. In particular, studies were conducted to study their neurotoxic properties in adults and young laboratory animals. Experiments to study the effects of cypermethrins in the pre- and postnatal period in accordance with the requirements of the OECD Guideline 426 were not carried out.

Objective of this work was to investigate the effect of cypermethrin and zeta-cypermethrin in pre- and postnatal development; to conduct a comparative study of the neurotoxic properties of cypermethrin and zeta-cypermethrin.

Materials and methods. The study of the neurotoxic action of SPs was carried out on the basis of two experiments [37,38]. The first study explored the effect of cypermethrin and the second — zeta-cypermethrin. In two experiments one biological model, Wistar Hannover rats was used.

Rats were obtained from SPF nursery of the SE “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine and allocated according to cypermethrin doses: group 1 — control, group 2 — 17.5 mg/kg body weight, group 3 — 35 mg/kg body weight, group 4 — 70 mg/kg body weight; and zeta-cypermethrin: group 1 — control, group 2 — 12.5 mg/kg body weight, group 3 — 35 mg/kg body weight, group 4 — 70 mg/kg body weight. In both control groups, distilled water was administered in animals with an emulsifier OP-10 in equivalent amounts. Husbandry of the two experiments was the same, the rats had free access to water and feed. The exposition in two studies lasted from the day 6 of pregnancy to the day 21 of lactation. The test substances were administered orally with a metal probe in the morning at the same time.

The study of the functional state of the central nervous system was conducted under an “open field” conditions. The open field is a uniformly illuminated square area 44 x 44 cm in size made of opaque black plastic, divided into 36 equal squares with walls 30 cm high. Experiments were conducted at day 13, 17 and 21 (DAB). The rats were placed in the centre of the field, and their behaviour was evaluated for 3 minutes and recorded: latent period of the first movement (time to the first step), the number of crossed squares (horizontal activity of animals), pointing (vertical activity of animals) and grooming (washing) during the observation period. Testing of animals took place at the same time.

Experiments in animal were conducted in accordance with OECD 426 (Guideline for Testing of Chemicals. Developmental Neurotoxicity Study) and requirements of the Bioethics Committee of the SE “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine.

Results and discussion. Analysis of changes in behavioural reactions of young female and male rats over time in terms of “open field” allowed distinguishing the following characteristic features.

Studies conducted at DAB 13 showed that the effects of cypermethrin and zeta-cypermethrin in experimental groups of offspring of both genders with lower and moderate doses, behavioural reactions compared to the controls were unchanged. Both test substances caused changes in behavioural reactions only in young male rates at maximum doses. Males of these groups showed the following: under exposure to cypermethrin — an increase in the latent period of the first movement (control: 25 % young rats; group 70 mg/kg: 63 %) and decrease in the number of crossed squares (control: 31.44 ± 2.13; group 70 mg/kg: 23.00 ± 3.49); under exposure to zeta-cypermethrin: decrease in the number of crossed squares (control: 30.46 ± 2.73; group 70 mg/kg: 22.19 ± 2.59). The latent period of the first movement characteriіes the rate of adaptation of animals to the new environment. The parameters of the number of crossed squares (motor activity of animals) is associated with the state of emotional comfort of the young rats [39, 40].  The table schematically shows changes in the behavioural reactions of the offspring.

Therefore, cypermethrin and zeta-cypermethrin at DAB 13 did not affect the behavioural responses of young female rats in all studied doses according to the studied parameters (latent period of the first movement, crossed squares, pointing, grooming). Both test substances caused a decrease in the number of crossed squares only in young male rats at a dose of 70 mg/kg (under the action of cypermethrin in the group 70 mg/kg, the parameter decreased by 26.85 % compared to the control group; under the action of zeta-cypermethrin — by 27.15 % respectively).

Table. Comparative characteristics of cypermethrin and zeta-cypermethrin according to “open field” test parameters

Studies conducted at DAB 21 showed that under the effects of cypermethrin and zeta-cypermethrin in experimental groups, changes in behavioural reactions were not observed compared to the control only for lower doses. Under the effect of cypermethrin at a dose of 7  mg/kg in young male rats, also there was an increase in latent period of the first movement, like at DAB 13 (control: 25 %; group 70 mg/kg: 75 % of young rats) and decrease in the number of crossed squares (control: 37.44 ± 3.06; group 70 mg/kg: 28.88 ± 3.14). While under the effects of zeta-cypermethrin in males at the same maximum dose, in addition to reducing the number of crossed squares (control: 41.15 ± 2.31; group 70 mg/kg: 29.92 ± 2.59), there was also an increase in the latent period of the first movement (control: 12 %; group 70 mg/kg: 31 % of young rats), decrease in the number of pointing that characterise the searching activity of young rats [40] (control: 6.88 ± 1.15; group 70 mg/kg: 3.69 ± 0.40) and total duration of grooming (control: 18.08 ± 2.39; group 70 mg/kg: 10.27 ± 2.00). Grooming is a marker of the anxiety of animals. Changes in the parameters of the grooming are characterised by the increased emotional stress of the young rats [42]. It should be noted that the decrease in the total amount of grooming (control: 2.19 ± 0.19; group 70 mg/kg: 1.54 ± 0.23) and number of crossed squares (control: 43.35 ± 3.42; group 70 mg/kg: 29.73 ± 3.63) were also registered in the females of group 70 mg/kg. In addition, young male rats of group 35 mg/kg also showed a decrease in the total duration of grooming (control: 18.08 ± 2.39; group 35 mg/kg: 12.54 ± 0.93).

Therefore, both test substances at DAB 21 induced the same changes in behavioural reactions in the males of group 70 mg/kg only in terms of crossed squares and the latent period of the first movement. When comparing the number of crossed squares under the effect of cypermethrin and zeta-cypermethrin, there was no significant difference (under the action of cypermethrin in the group 70 mg/kg — the parameter decreased by 32.12 % relative to control; under the action of zeta-cypermethrin — by 27.29 %, respectively). The increase in the latent period of the first movement was recorded in the overwhelming number of experimental males precisely under the action of cypermethrin compared to the young rats exposed to zeta-cypermethrin (under the action of cypermethrin in the group 70 mg/kg, the parameter increased by 317.05 % compared to the control, under the action of zeta-cypermethrin in the group 70 mg/kg — by 284.85 %).

Thus, summing up the experimental data obtained, it can be stated that cypermethrin at lower and middle doses (17.5 mg/kg and 35 mg/kg) did not cause behavioural abnormalities in both genders during postnatal development. Administration of cypermethrin at a dose of 70 mg/kg in the- pre and postnatal period affects motor activity and cognitive activity of young male rats (in terms of increasing the latent period of the first movement and decreasing the number of crossed squares). Cypermethrin did not affect the searching and emotional activity of animals at all studied doses. Under the action of zeta-cypermethrin at a lower dose (12.5 mg/kg), no behavioural reactions were reported in young male and female rats. Under the action of zeta-cypermethrin in males of the group 35 mg/kg, an increase in anxiety was observed (decrease in the total duration of the grooming). Zeta-cypermethrin at a dose of 70 mg/kg causes in the offspring of both genders disorders in the psycho-emotional state (♂♀ — decrease in the parameters of the grooming), inhibition of motor and cognitive activity (♂ — decrease of the number of crossed squares and pointing, ♀ — decrease of the number of crossed squares), and reduction of adaptive properties (♂ — increase of latent period of the first movement).

Conclusion

Based on the comparison of the results obtained in the study of behavioural reactions, the following conclusion can be drawn from the effects of the same dose level of cypermethrin and zeta-cypermethrin:

1. Cypermethrin and zeta-cypermethrin exhibit gender-related sensitivity: young male rats were more susceptible to cypermethrin and zeta-cypermethrin compared to females.

2. The effect of cypermethrin and zeta-cypermethrin in the pre- and postnatal period is dose-dependent.

3. Effect of cypermethrin at doses of 17.5 and 35 mg/kg and zeta-cypermethrin at a dose of 12.5 mg/kg in the pre- and postnatal period does not cause a neurotoxic effect in the offspring.

4. Cypermethrin exposure at a dose of 70 mg/ kg and zeta-cypermethrin exposure in doses of 35 and 70 mg/kg causes changes in behavioural reactions of offspring of both genders. Administration of cypermethrin at a dose of 70 mg/kg in the- pre and postnatal period affects motor activity and cognitive activity of young male rats (in terms of increasing the latent period of the first movement and decreasing the number of crossed squares). Administration of zeta-cypermethrin at a dose of 70 mg/kg causes in the offspring of both genders disorders in the psycho-emotional state (♂♀ — decrease in the parameters of the grooming), inhibition of motor and cognitive activity (♂ — decrease of the number of crossed squares and pointing, ♀ — decrease of the number of crossed squares), and reduction of adaptive properties (♂ — increase of latent period of the first movement). Under the action of zeta-cypermethrin at a dose of 35 mg/kg in males, an increase in anxiety was observed (decrease in the total duration of the grooming).

5. Zeta-cypermethrin showed a greater neurotoxicity on offspring compared with cypermethrin.

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