e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1276–1282

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Neurotoxicity of neem commercial formulation (Azadirachta indica A. Juss) in adult zebrafish (Danio rerio) M.M. Bernardi a,∗ , S.G. Dias b , V.E. Barbosa b a

Post-Graduate Program of Environmental and Experimental Pathology and Post-Graduate Program of Dentistry, Paulista University, UNIP, Campus Indianapolis, Rua Dr. Bacelar, 1212, São Paulo cep: 04026-002, SP, Brazil b Graduate Biological Science Program, Paulista University, UNIP, Campus Marques, Av. Marquês de São Vicente, 3001, São Paulo cep: 05036-040, SP, Brazil

a r t i c l e

i n f o

a b s t r a c t

Article history:

The neurotoxic effects of a commercial formulation of Azadirachta indica A. Juss, also called

Received 18 June 2013

neem or nim, in adult zebrafish were determined using behavioral models. General activity,

Received in revised form

anxiety-like effects, and learning and memory in a passive avoidance task were assessed

2 October 2013

after exposure to 20 or 40 ␮l/L neem. The results showed that 20 ␮l/L neem reduced the

Accepted 5 October 2013

number of runs. Both neem concentrations increased the number of climbs to the water

Available online 18 October 2013

surface, and 40 ␮l/L increased the number of tremors. In the anxiety test, the 20 ␮l/L dose increased the number of entries in the light side compared with controls, but the latency

Keywords:

to enter the dark side and the freezing behavior in this side did not changed. In relation

Dark/light task

to controls, the 40 ␮l/L neem reduced the latency to enter in the light side, did not change

Fish

the number of entries in this side and increased freezing behavior in the light side. In the

General activity

passive avoidance test, pre-training and pre-test neem exposure to 40 ␮l/L decreased the

Anxiety

response to the learning task. Thus, no impairment was observed in this behavioral test.

Learning and memory

We conclude that neem reduced general activity and increased anxiety-like behavior but did not affect learning and memory. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

The neem tree, Azadirachta indica A. Juss, Meliaceae, neem, or nim, is indigenous to the Indian subcontinent and has several medicinal properties (Mbaya and Ogwiji, 2012). The bark and leaves of the tree are used as a bitter tonic and astringent. It is considered an antiseptic, a blood purifier, and useful in skin disorders. Its fruit is an emollient and purgative. A. indica is also considered beneficial for the treatment of a wide range of disorders, such as coughing, nausea, vomiting, fever, jaundice, gonorrhea, urinary tract infection, intestinal worm

infestation, and leprosy (Saxena and Hassan, 1999). Khillare and Shrivastav (2003) reported a potent espermicide property of A. indica aqueous extract. Various products based on the A. indica plant have been used, particularly as an antifeedant, antiattractant, repellent (Sharma et al., 1993), ecdysone inhibitor, oviposition deterrent, and sterilant in various genera of insects and pests (Schmutterer, 1990). Two types of botanical insecticides can be obtained from the seeds of A. indica, Meliaceae. Neem oil, obtained by cold-pressing seeds, can be effective against soft-bodied insects and mites, but it is also useful in the management of phytopathogens (Schmutterer, 2002). More

∗ Corresponding author at: Rua Dr. Bacelar, 1212, Vila Clementino, São Paulo, SP 04026-002, Brazil. Tel.: +55 11 5586 4000; fax: +55 11 2275 1541. E-mail addresses: [email protected], [email protected] (M.M. Bernardi). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.10.002

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1276–1282

highly valued than neem oil are the medium-polarity extracts of the seed residue after removal of the oil. These extracts contain the triterpene azadirachtin complex. Neem seeds contain more than a dozen azadirachtin analogs, but the major form is azadirachtin; the remaining minor analogs likely contribute little to the overall efficacy of the extract (Schmutterer, 2002). Although neem extract is considered to have low toxicity in non-target aquatic life (Martinez, 2002), it was reportedly associated with respiratory problems and a delay in growth (Omoregie and Okpanachi, 1992). It was also shown to interfere with the antioxidant defense system, decrease liver catalase activity, and activate the detoxifying enzyme glutathioneS-transferase in several fish species (Winkaler et al., 2007). Exposure to neem extract was also shown to damage gill and kidney tissue (Winkaler et al., 2007). We previously compared the acute toxicity of neem oil and a commercial formulation in Artemia sp. The commercial formulation was significantly more toxic than the pure oil. We concluded that the formulated product used as an agricultural pesticide represents a greater risk to animals in the aquatic environment than the pure oil of the plant (Bevilacqua et al., 2008). Because of the scarcity of data on the toxic effects of this bipesticide, we investigated the acute neurotoxicity of a commercial neem product in Danio rerio (zebrafish). As zebrafish are easy to obtain, maintain, and reproduce in the laboratory—decisive characteristics when choosing a species to be used for testing—this fish species is the most widely used in scientific research. Zebrafish have also been used to study developmental (Bailey et al., 2013; Overman and Hertog, 2013) and biomedical (McGrath and Seng, 2013; Scalzo and Levin, 2004) and evaluate the aquatic exposure risk to toxicants (Froehlicher et al., 2009; Zhou et al., 2010). Also, valuable information has been evaluated about zebrafish with regard to neurotoxicological studies of chemicals (de Esch et al., 2012a,b; Levin, 2011; Levin and Tanguay, 2011). The Levin group especially focused on behavioral models of anxiety and learning/memory processes in zebrafish (Eddins et al., 2009; Levin, 2011; Levin et al., 2007). In the present study we investigated the neurotoxic effects of different dosages of a neem product on zebrafish behavior as an expression of nervous system function. Behavioral tests were conducted focusing in particular on activity of the zebrafish, on anxiety and cognitive aspects.

2.

Materials and methods

2.1.

Animals

The water hardness was 42 mg L−1 CaCO3 , pH 7.0 ± 0.2. The luminous intensity was 600 lux, with a 12 h/12 h light/dark photoperiod. With the exception of during the experiments, the aquaria were aerated using air compressors and connected to water filtration systems with acrylic wool and active charcoal to improve water quality. Every 7 days, 25% of the total water volume was changed. To feed the fish, Tetramin, a supplied as recommended by the manufacturer and in accordance with CETESB 1990 guidelines, was used. During testing, the fish were not fed, and pH, dissolved oxygen, and conductivity were analyzed at the beginning and end of each test.

2.2.

Commercial neem formulation

The experiments were performed with leaf extracts of A. indica. Each 100 ml of the extract (Dal neem, Dalquim Com Ind. Ltda) contained 8.5 ml of the leaf extract, 4.5 ml of emulsifier, 4.5 ml of anhydrous alcohol and q.s.p. (quod satis para or that sufficiently prepared), and 100 ml of vegetable oil. The neem concentrations were approximately 7- and 14-times the lethal dose in zebrafish that are exposed for 96 h, which was calculated in our laboratory as 2.80 ␮l/L.

2.3.

Behavioral tests

2.3.1.

General activity

This method was established in our laboratory based in previous observations of adult zebrafish behaviors. An aquarium with 15 cm length × 10 cm height × 15 cm width was used to observe general activity. The front wall of the aquarium was divided into six equal 5 cm2 parts. Because adult zebrafish had maximum size of 5 cm, the counting of area crossed was of sufficient sensitivity to detect zebrafish movements. The fish were individually introduced into the aquarium that contained 20 or 40 ␮l/L of the neem extract. General activity was observed 0–5, 10–15, and 20–25 min after exposure to the bioinsecticide. The following parameters were recorded: (1) run frequency, i.e., the number of times that the fish ran in any direction, except when they going to climb; a unit was counted each time the fish started and stopped a run; (2) the number of times the fish climbed to the water surface, and (3) the number of times the fish presented tremors, i.e., a unit was counted each time the fish started and stopped rapids and progressive contractions from whole body from head to tail. The procedure was filmed for later observation by visual inspection from an observer.

2.3.2.

D. rerio, with a maximum size of 5 cm and 8–9 months of age, were obtained from a commercial breeder (Izael Ba Hi, Indaiatuba, São Paulo, Brazil) and brought to the laboratory within 30 min in plastic bags with sufficient air. The plastic bags were placed in a maintenance aquarium with 80 L for approximately 30–35 min for acclimation. The bags were then opened, and the fish were allowed to swim into the aquarium water. The animals were maintained in the laboratory for 15 days for acclimation. Dechlorinated water from São Paulo was used and maintained at a temperature of 23 ± 2 ◦ C by heaters.

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Light/dark preference test

This procedure was based on the black-and-white model proposed by Blaser et al. (2010) to evaluate anxiety-like behavior in zebrafish. To assess anxiety-like behavior, a 40 cm aquarium was used, divided into three compartments: black called as dark side with 15 cm, clear side, the light side with 15 cm, illuminated by an 835.66 lux incandescent bulb, and intermediate side with 10 cm, separated from the other two compartment by two glass walls. The black side was previously determined to be the side that the fish preferred, and the light side was the anxiogenic side. To study anxiety-like effects, 30 fish were divided into two experimental groups and one control group (n = 10/group). The experimental fish were exposed to 20 or

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Fig. 1 – Scheme of the anxiety test.

40 ␮l/L neem for 5 min in a 2 L aquarium and then placed in the intermediate compartment of the test aquarium. The control group was subjected to the same procedure but was not exposed to the bioinsecticide. Five minutes later, i.e., the habituation period, the glass walls were removed. The latency in sec to enter the light side, the number of entries in the light side, and the freezing behavior (total time in which the fish remained without movements) in each side were measured for 5 min (Fig. 1). The procedure was filmed for later evaluation by visual inspection from an observer.

2.3.3.

Passive avoidance test

To assess passive avoidance, a 40 cm aquarium (aquarium B) was used, divided into three compartments: black with 15 cm, clear with 15 cm, illuminated by an 835.66 lux incandescent bulb, and intermediate with 10 cm, separated from the other two compartments by two glass walls. We found that the fish preferred the black compartment 80% of the time. An iron Lshaped device to which a wire was connected with a weight at its tip was attached over the dark side of the tank. When the fish penetrated the dark side, i.e., the preferred side of the aquarium, the weight was released into the water. Two sessions were performed. First the fish was introduced in the intermediated compartment to habituation. Five minutes later the glass walls were removed. Each time the fish entered the dark side, the weight was released into the water. This procedure was performed 10 times. The test session was performed similarly to the training session, but the weight was not released. The procedure was filmed for later evaluation by visual inspection from an observer. Two treatments were performed. In the first experiment the zebrafish received the bioinsecticide pre-training to study the neem effects on learning and memory. Then, 20 fish were divided into two equal groups were exposed to 40 ␮l/L neem for 5 min in a 2 L aquarium called (aquarium A). The control group was trained and tested as the experimental group but not is exposed to neem. Both groups were trained and the tests were performed 24 h later in aquarium B, which contained only normal water. The experimental design is shown in Fig. 2. In the second experiment the zebrafish was exposed to neem pretest to evaluate the evocation of the learned task. Thus, another 20 fish were trained in aquarium B with normal water and divided 24 h later into two equal groups. The experimental group was exposed to aquarium A containing 40 ␮l/L neem, for 5 min and then placed in the intermediated compartment of the aquarium B, which contained only normal water, and 5 min later the test was performed. The control group was trained and tested as the experimental group but not is exposed to neem. The experimental design is shown in Fig. 3.

Fig. 2 – Scheme of passive avoidance of pre-training treatment.

Fig. 3 – Scheme of passive avoidance of pretest treatment.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1276–1282

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The number of attempts to enter the dark compartment, i.e., punished compartment, number of crossings into the dark compartment, and duration and frequency of immobility were assessed in the test session.

2.4.

Statistical analysis

The results are expressed as mean ± SEM. Homoscedasticity was verified using the F or Bartlett’s test. Normality was verified using the Kolmogorov–Smirnov test. Two-way analysis of variance (ANOVA) was used to analyze the general activity data, followed by the Bonferroni post hoc test, with treatment and session as factors. The anxiety data were compared using one-way ANOVA followed by Tukey’s multiple comparison test. The passive avoidance test data were compared using Student’s t-test. The level of statistical significance was p < 0.05.

3.

Results

3.1.

General activity

Fig. 4 shows the general activity of the zebrafish exposed to 20 or 40 ␮l/L neem. The two-way ANOVA of the number of runs (Fig. 4A) revealed a significant effect of treatment (F2,81 = 3.68, p = 0.029) but not session (F2,81 = 0.15, p = 0.861) and no treatment × session interaction (F4,81 = 0.10, p = 0.081). The Bonferroni post hoc test indicated fewer runs in the 20 ␮l/L group in the first and second sessions compared with controls. With regard to the number of climbs to the water surface (Fig. 4B), the two-way ANOVA revealed a significant effect of treatment (F2,81 = 8.23, p = 0.0006) but not session (F2,81 = 1.01, p = 0.37) and no treatment × session interaction (F4,81 = 0.61, p = 0.66). Compared with the control group, an increased number of climbs to the water surface was observed in all of the sessions after exposure to 20 ␮l/L neem. This parameter was also increased in the 10–15 min and 20–25 min sessions in fish treated with 40 ␮l/L neem. With regard to the number of tremors (Fig. 4C), the two-way ANOVA revealed a significant effect of treatment (F2,81 = 14.12, p < 0.0001) but not session (F2,81 = 1.24, p = 0.295) and no treatment × session interaction (F4,81 = 0.30, p = 0.875). Compared with the control group, the post hoc test revealed an increased number of tremors after exposure to the higher 40 ␮l/L neem dose in all of the sessions.

3.2.

Light/dark preference test

Fig. 5 shows the results of the light/dark preference test in fish exposed to 20 or 40 ␮l/L neem. The one-way ANOVA revealed differences in the latency to enter the light compartment between groups (F2,27 = 3.74, p = 0.03; Fig. 4A). The post hoc test indicated that the latency to enter the light side was decreased in fish treated with 40 ␮l/L neem compared with the control group. With regard to the number of entries into the light side, the one-way ANOVA revealed differences between groups (F2,27 = 6.53, p = 0.005; Fig. 4B). The post hoc test indicated an increase in the number of entries into the light side in the 20 ␮l/L neem group compared with the control group

Fig. 4 – Number of (A) runs, (B) climbs to the water surface, and (C) tremors after zebrafish were exposed to 20 and 40 ␮l/L neem. The data are expressed as mean ± SEM. *p < 0.05 (two-way ANOVA followed by Bonferroni post hoc test; n = 10/group).

(p < 0.05). No difference was found between the control and 40 ␮l/L neem groups (p > 0.05). The two-way ANOVA revealed significant differences in freezing behavior between the sides of the aquarium (F1,36 = 22.87, p = 0.002) but not between treatments (F1,36 = 0.11, p = 0.8), with no interaction between factors (F1,36 = 6.54, p = 0.08). The Bonferroni post hoc test revealed an increased percentage of freezing in the light side compared with the dark side.

3.3.

Passive avoidance test

The number of crossings into the dark compartment, number of attempts to enter the same side, and frequency of immobility were decreased by pre-training exposure to 40 ␮l/L neem in the test session compared with the training session. No difference was observed in immobility time between training and

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Table 1 – Pre-training and pre-test exposure of zebrafish to 40 ␮l/L neem during 5 min. Training Treatment Pre-training CDC NADC IMF IMT Pre-test CDC NADC IMF IMT

Testing

p

18.40 5.30 2.40 93.70

± ± ± ±

4.84 1.43 0.93 9.10

6.00 0.10 0.90 90.70

± ± ± ±

1.49 0.10 0.32 33.41

Neurotoxicity of neem commercial formulation (Azadirachta indica A. Juss) in adult zebrafish (Danio rerio).

The neurotoxic effects of a commercial formulation of Azadirachta indica A. Juss, also called neem or nim, in adult zebrafish were determined using be...
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