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Evaluation of the effects on the cerebellar cortex of CF-1 mice exposed to a single dose of cypermethrin/Evaluacion de los efectos de una dosis unica de cipermetrina en la corteza cerebelar de ratones CF-1/Avaliacao dos efeitos de uma dose unica de cipermetrina no cortex cerebelar de ratos CF-1.

Introduction

Cypermethrin it is a synthetic, photo stable pyrethroid with great insecticide power and a broad action spectrum, widely used for pest control in crops, with good effects on Lepidoptera, Coleoptera and Hemiptera. The inclusion of a a-cyano group classifies cypermethrin as a type II pyretroid (Soderlund et al., 2002). The a-cypermethrin makes up more of the 90% of the active form of the insecticide and corresponds with a rasemic mixture of the cis isomers (Lessenger, 1992). Cypermethrin it's considered as a class II insecticide by WHO (2010).

The pesticide has multiple ways of entrance to the body: dermal (Luty et al., 2000), oral (Manna et al., 2004), across the conjunctiva and through the respiratory tract epithelium (Martinez-Navarrete et al., 2008). This last one allows a high rate of absorption and is the best known (Martinez-Navarrete et al., 2008). Cypermethrin shows high affinity to lipids, its elimination kinetics from nervous tissue is 78% in 24h, 12% in 8 days and the remaining 10% between 18 and 24 days. The rate of excretion does not increase after repeated exposure. The half-life of cis-cypermethrin in fatty tissue of mice is 13 days, and the trans isomer has a half-life of one day (Soderlund et al., 2002; Wolansky and Harrill, 2008).

The principal mechanism of action is by alteration of the voltage-dependent sodium channels (VDSC) in the CNS, increasing the channel opening time, which in turn increases depolarization time (Ray and Fry, 2006). Cypermethrin also produces alterations on the gamma aminobutyric (GABA) receptor, altering the GABA dependent Cl- channel (GDCC). GABA is the principal neurotransmitter for Cl-transport in the CNS and the cerebellum has the highest concentration of GABA in the encephalon. Severe alterations of GABA concentration could lead to disruption of the cerebellar circuits, mainly affecting the Purkinje cells. Cypermethrin and deltamethrin produce decreases on the level of GABA in rats exposed with 1/10 of the LD50 (Manna et al., 2006). Permethrin (a Type I pyrethroid), decreases in the number of Purkinje cells in dermally exposed rats (Abdel-Rahman et al., 2001).

The objective of this study was to evaluate the effects of a low dose (1/5 DL50) of cypermethrin on the cerebellar cortex, focusing on the density of the Purkinje cell population and the expression of apoptotic (Apaf-1) and proliferation (Ki67) biomarkers, as regulators of cellular proliferation processes, through the route of caspases and cell cycle regulatory factors, respectively.

Materials and Methods

Materials

Thirty-three mice of the CF1 strain, 2.5-3 month old, weighing 25-30g, were kept under standard feeding conditions, with a 12h light and 12h darkness cycle in the animal house of the Faculty of Medicine, Universidad de Chile. Cypermethrin (CAS No. 52315-07-8) 92.5% p/p from Anasac was re-suspended in vegetable oil (Sunflower).

Experimental design

Animals were distributed in three groups: Group 1 (n= 3) were left untreated, Group 2 (n= 15) received 50pl vegetable oil i.p., and Group 3 (n = 15) received 50ml vegetable oil containing cypermethrin at 1/5 of the lethal i.p. dose ([LD.sub.50] of 485mg x [kg.sup.-1] (IPCS, 2008). For each group, the cerebellums of three mice were dissected at 24h and 8, 17, 25, and 34 days after cypermethrin treatment, following euthanasia according to NIH protocols (Mendez, 2009). These periods coincide with the time required for metabolism and elimination of cypermethrin, and its permanence in the fatty tissue with a half-life of 9-12 and 18 days, respectively. The cerebellum was fixed for 48h in 10% buffered formalin (phosphate buffer pH 7. 2) and thereafter subjected to standard histological techniques, such as paraffin embedding (56/58[degrees]C melting point). Sections of 5[micro]m were sliced and mounted in silanised slides (Star Frost).

Purkinje cell count

After hematoxylin eosine (H-E) staining, bright field photomicrographs were taken with a 1000 x objective. Cell counts were made by measuring a line in the monolayer of Purkinje cell, and an index was obtained considering number of Purkinje cells per lineal extent ([micro]m). A total of 30 fields were read per cerebellum.

Immunohystochemistry

Apoptosis was analyzed with antibody against Apaf-1 and cell proliferation was analyzed with antibody against Ki-67 (Clon SP6. Thermo Scientific Catalog RM-9106SO). Positive Purkinje cells were counted as the number of immune reactive cells in relation to the total of Purkinje cells. For Granular cells, positive cells were counted and tabulated as the number of immune reactive cell per unit area ([micro][m.sup.2]). A total of 30 fields were read per cerebellum (1000x).

Statistical analysis

Data were analyzed statistically with non-parametric ANOVA (Kruskal-Wallis test) and post test (Dunn's multiple comparison test), considering always p<0,05.

Results

The three layers of the cerebellar cortex: Molecular, Purkinje and Granular (Figure 1) were differentiated in histological sections stained with hematoxylin eosin. Morphological alterations of the tissue were not observed.

Purkinje cell count

Purkinje cells were observed with their characteristic morphology forming a monolayer in the cerebellar cortex (Figure 1). The number of Purkinje cells was significant decreased (p<0.05) after the administration of cypermethrin. This was observed at all the times studied in comparison to the control group 1 (Figure 2). The decrease of the Purkinje cells was higher at days 17 and 25, which shows that the insecticide not only has an acute affect to the cerebellar tissue, but can also induce significant changes to the tissue in chronic stages (p [less than or equal to] 0.05).

[FIGURE 1 OMITTED]

Apoptosis

Immunoreactive cells showed a bright brown staining in both Purkinje cells and Granular cells. There was an increase of the expression of Apaf-1 in both cell populations. In Purkinje cells there was a higher expression of Apaf-1 in days 17 and 25, but this wasn't significant (p> 0.05). Granular cells showed a significant increase of the expression of Apaf-1 at day 25 (Figure 3), which concurs with the lowest count of Purkinje cells at days 17 and 25.

Ki-67

Both cell populations showed immunoreactive cells to the antibody. There was an increased of the expression of Ki-67 in the Purkinje cells at days 17 and 25, but this was not significant (p> 0.05). Granular cells showed a significant increase in the expression of Ki-67 at day 25 (Figure 4). Discussion

The effect of cypermethrin on the CNS is principally through an increase of the opening time of the voltage-dependent sodium channels, but also affects the GABA receptors and GABA encephalic levels. In the normal cerebellar circuits the Purkinje cells acts as their axis (Sarna and Hawkes, 2003), exerting an inhibitory function through GABA dependent Cl- channels. This implies that an alteration on the GABA receptors can affect the cerebellar circuits and the cerebellar cell population, which can induce a decrease in the Purkinje cell population, as observed in the present study. A cypermethrin effect on GABA levels was showed in rats exposed to the insecticide to a single dose of 145mg x [kg.sup.-1], producing a significant decrease in the concentrations of the neurotransmitter (Manna et al., 2006b).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

The main afferent pathway of the normal cerebellar circuit is through the mossy and the climbing fibers. The mossy fibers forms glutamatergic synapses on the Granular cells, and these cells in turn project their axons to the molecular layer, to synapse on the dendritic spines of Purkinje cells. This means that damage to Purkinje cells, as observed in this study, could alter the population of Granular cells. The climbing fibers come from the olivar complex and synapse directly on the Purkinje cells. Finally, the Purkinje cell axons synapse on neurons in deep cerebellar and vestibular nuclei that are constantly modulated by inhibition from the Purkinje cells, which are exclusively GABAergic (Sarna and Hawkes, 2003). This circuit can be altered by the presence of pyrethroids that reduce GABA levels (Manna et al., 2006b) and also reduce the number of Purkinje cells (Abdel-Rahman et al., 2001). In the present study a significant decrease in the number of Purkinje cells was observed after a single dose of cypermethrin.

The euthanasia of the animals was carried out at days 1, 8, 17, 25 and 34, which correspond with the biotransformation and excretion of cypermethrin. The trans isomer is almost completely eliminated in the first 24h, while the cis isomer is eliminated by day 8 from liver and kidney, and in days 12, 17, 24 and 28 from fatty tissue (Crawford et al., 1981; Rhodes et al., 1984; Soderlund et al., 2002).

There was a larger decrease in the Purkinje cell number at days 17 and 25; this concurs with previous results in cerebral cortex, where a significant decrease in the neuronal density was observed at day 25 in mice exposed to a 1/5 [DL.sub.50] of cypermethrin (Jimenez et al., 2008). This allows to determinate that cypermethrin has a significant effect in acute and chronic stages after exposure.

Other authors also described the chronic effect of cypermethrin in the CNS; this was shown in rats exposed to 1/10 and 1/20 of the LD50, where the animals had a significant decrease of the GFAP protein present in astrocytes at days 2 and 21 after exposure, the reduction being more pronounced in day 21 (Malkiewicz et al., 2006). This shows that cypermethrin not only leads to damage of neurons, but also affects the glial cells in the CNS.

An increase in the expression of Apaf-1 in the Purkinje and Granular cells was observed at all the times studied, with a significant increase in day 25, which agrees with the lowest numbers in the Purkinje cell population. This shows that the decrease in the cell number could be due top the apoptotic cell death induced by cypermethrin. Apoptosis induced by pyrethroids in Purkinje cells was also shown in rats exposed for 30 days to permethrin at daily doses of 0.14mg x [kg.sup.-1] (Abdel-Rahman et al., 2004). Also, cypermethrin-induced apoptosis was observed in telencephalon of tadpoles of Physalaemus biligonigerus, 96h after exposure to 193[micro]g x [l.sup.-1] of the insecticide (Izaguirre et al., 2000). The increase in the expression of Apaf-1 in Granular cells can be a direct effect of cypermethrin, or due to disruption of the cerebellar circuit after the death of the Purkinje cells, in turn inducing the death of Granular cells.

There was an increase in the expression of Ki-67 in the Purkinje cells at days 17, 25 and 34. This was not statistical significant in comparison with the control groups, althoug it was significant at day 25 in comparison with days 1 and 8. Granular cells also showed a significant increase in the expression of Ki-67 in day 25. This change can be a response to the decreased cellular population resulting from the expose to cypermethrin, causing activation of the cell cycle leading to an increase in the expression of Ki-67. However, the cells do not complete the cycle; it has been shown that cells can return to a quiescent state even after having synthesized DNA (Scholzen and Gerdes, 2000). Cells can undergo cellular arrest and apoptosis after entering the cell cycle if they do not successfully past the checkpoints of the cycle (Liu and Greene, 2001). Neurons are highly differentiated cells, and they do not have the adequate cellular machinery to successfully complete a cell cycle, so that after they start it they can end in cellular arrest and apoptosis.

Cypermethrin has a significant effect on the cerebellar cortex, altering both Purkinje and Granular cells. This is established by the reduction in density of Purkinje cell population and the increases in the expression of Apaf-1 and Ki-67 in both Purkinje and Granular cells. Ki-67 is a unique peptide that appears in the G1 and G2 phases of the cell cycle, and has a short half life. Meanwhile, Apaf-1 brings together, through the caspase pathway, the intrinsic and extrinsic apoptosis pathways.

Cypermethrin produces acute and chronic effects in the cerebellar cortex. The insecticide can remain in fatty tissue even 28 days after the exposure (Rhodes et al., 1984) and can cause a chronic effect to the cerebellar neurons. Such chronic effect of cypermethrin can be produced by two processes: 1) as mentioned, the insecticide can remain in fatty tissue after 28 days of exposure, and 2) because of the damage to cerebellar circuits the intact neurons activate the cellular machinery trying to compensate this lost circuits, ending in cellular arrest (Liu and Greene, 2001; Migliorini et al., 2002). It is difficult to determine which one of the proposed processes has more importance in the damage produced by cypermethrin. Also, this damage has been observed in other tissues under the similar experimental conditions (Jimenez et al., 2008; Rodriguez et al., 2009).

Conclusion

In adult mice, cypermethrin at a 1/5 LD50 produces acute and chronic effects on the cerebellar cortex, causing a decrease in the number of Purkinje cells. It also increases the expression of apoptotic biomarkers such as Apaf-1 in Purkinje and Granular cells, and increases the expression of Ki-67 in these cells. Cypermethrin can cause subclinic damage to the cerebellar cortex due to direct damage and induction of apoptosis.

Received: 03/31/2014. Modified: 10/28/2014. Accepted: 11/03/2014.

German Venegas F. Veterinarian, Master in Veterinary Sciences, Doctoral Student in Veterinary Medicine, Universidad Nacional Andres Bello (UNAB), Chile. Professor, UNAB, Chile.

Cristian Donoso. Veterinarian, Universidad de Chile (UChile). Reasearch Asistant, Universidad de Chile (UChile).

Camilo Arriaza. Veterinarian and Master of Science, Professor, UChile.

Omar Espinoza-Navarro. Doctor in Celullar and Genetic Biology, Universidad Autonoma of Madrid, Espana. Professor, Universidad de Tarapaca, Chile. Address: Department of Biology, Faculty of Science, Universidad de Tarapaca. General Velasquez No. 1775. Arica, Chile. e-mail: oespinoz@uta.cl

Maria Eugenia Castro. Psychologist, Master of Science and Doctor in Psychology, Universite de Paris, France. Professor, Playa Ancha University, Chile.

Cristian Torres. Veterinarian and Doctor in Veterinary Sciences, UNAB, Chile. Professor, UChile.

Felipe Lillo. Veterinarian, Master in Veterinary Sciences, Doctoral Student in Veterinary Medicine, UNAB, Chile. Professor, UNAB, Chile.

Hector Rodriguez B. Veterinarian and Master of Science, UChile. Doctor in Biomedicine, Universidad Alcala de Henares, Spain. Professor UChile. Address: Hector Rodriguez. Morphology Laboratory. Faculty of Medicine, Universidad de Chile. Avenida Independencia 1027. Santiago, Chile. POBox 8389100. e-mail: hrodrigu@ med.uchile.cl

ACKNOWLEDGMENTS

This work was supported by the Major Research Project UTA No. 4712-13 from the Universidad de Tarapaca, Chile and Fondecyt No. 1101046.

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Title Annotation:articulo en ingles
Author:Venegas F., German; Donoso, Cristian; Arriaza, Camilo; Espinoza-Navarro, Omar; Castro, Maria Eugenia
Publication:Interciencia
Date:Nov 1, 2014
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