Saudi Journal of Biological Sciences (2009) 16, 69–76

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Effect of azadirachtin of neemix-4.5 on SWR/J mice

F.M. Abou-Tarboush a,*, H.M. El-Ashmaoui b, H.I. Hussein c, D. Al-Rajhy c,

M. Al-Assiry c

a King Saud University, College of Science, Department of Zoology, Saudi Arabiab National Research Center, Cell Biology Dept., Giza, Egyptc King Saud University, College of Sciences of Foods and Agriculture, Plant Protection Department, 2460, Riyadh 11451,Saudi Arabia

Available online 27 October 2009

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KEYWORDS

Neemix-4.5;

Azadirachtin;

Toxicity;

SWR/J mice;

Toxic effect;

Neem-based insecticide

Corresponding author.mail address: ftarbush@ksu.

19-562X ª 2009 King Saud

view under responsibility of

i:10.1016/j.sjbs.2009.10.003

Production and h

edu.sa (F

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Abstract Inbred normal SWR/J male and female mice, 8–10 weeks old and weighing 22.55–

26.72 g, were used throughout the study. A total of 100 males and 100 females were used and were

divided into 20 groups, 10 animals in each group.

Azadirachtin of neemix-4.5, a commercial botanical pesticide derived from the neem tree, orally

administered to male and female SWR/J mice at a dose level 9.0 mg/kg (1/10 LD50) for different

treatment periods (2, 4, 6, 8 or 11.5 weeks) has produced signs of toxicity, mortality and changes

in body and tissue weights of both sexes at almost all treated periods used in the present study.

Moreover the oral administration of this dose level for 11.5 weeks has also resulted in some histo-

pathological changes in the livers, kidneys and testes of treated animals compared with the control

group, and the degree of these changes ranged from mild to severe in these organs of treated males.

However, conflicting results have been reported concerning the toxicity of azadirachtin in mamma-

lian species using different formulations of neem-based pesticides. It appears, therefore, that the

toxicity produced by neemix-4.5 in the present study may be due to factors other than azadirachtin

in this formulation.ª 2009 King Saud University. All rights reserved.

.M. Abou-Tarboush).

ity. All rights reserved. Peer-

d University.

lsevier

1. Introduction

Increasing concern about pesticide accumulation in the envi-ronment stimulates search for natural compounds that couldreplace synthetic insecticides in insect pest control (Adel and

Sehnal, 2000). Neem is the most promising potential sourceof biopesticide of botanical origin (Schmutterer, 1995; Raizadaet al., 2001). During the past two decades, neem seeds

(Azadirachta indica, A. Juss) has gained increasing attentionas a natural insecticide, and its activity has been evaluatedagainst many economically important insect species (Schmut-

terer, 1990; Hashem et al., 1998; Kreutweiser et al., 2002; Lianget al., 2003; Charleston et al., 2005).

Nomenclature

S spermatozoa

A arteryK Kupffer cellsD damaged spermatocytesI interstitial space

H hepatocyteN nucleus

Pr proximal convoluted tubule

Si sinusoidSp spermatocytesIc interstitial cellsL lumen

G glomerulusDi distal convoluted tubule

70 F.M. Abou-Tarboush et al.

Neem-based insecticides have deterrent, antiovipositional,antifeedant, growth-regulating, fecundity- and fertility-reduc-

ing properties on insects (Mordue and Blackwell, 1993;Hashem et al., 1998). The principle insecticidal component ofneem extracts is the limonoid, azadirachtin. Azadirachtin has

been effectively used against more than 400 species of insectsand has proved to be one of the most promising plant ingredi-ents for integrated pest management at the present time (Isam,

1999; Walter, 1999; Saber et al., 2004).Although neem has shown every indication of being safe to

mammals in normal use, the possibility of future hazardsshould not be ignored (Anon, 1992; Raizada et al., 2001). Its

residual persistence on foods is also unknown. Moreover, thereis very little information on physiology (Jacobson, 1986) andtoxicity of azadirachtin (Raizada et al., 2001). Furthermore,

most toxicity studies on azadirachtin have been done on in-sects which show rapid loss of mobility and reduced fitness(Akudugu et al., 2001).

In view of a lack of or little information on its toxicity pro-file, an attempt has been made in the present study to evaluatethe short-and long-term toxicity of azadirachtin of neemix-4.5(a commercial botanical pesticide derived from the neem tree)

in male and female of SWR/J mice.

2. Material and methods

Inbred normal SWR/J male and female mice, 8–10 weeks oldand weighing 22.55–26.72 g, were used throughout the study.The animals were kept and bred in an environmentally con-

trolled room with a temperature of 22 ± 1 �C, a relativehumidity of 45 ± 5% and a light/dark cycle of 10/14 h. Mouse

Table 1 Effects of the dose level 9 mg/kg of azadirachtin of neemix-4.5 applied at different durat

Treatment

duration

No. of

males

used

Body wt. in

g at the start

of exp.

(Mean ± SE)

Body wt. in

g at the end

of exp.

(Mean ± SE)

Liver wt.

in g

(Mean ± SE)

Kidney wt. in g

(Mean ± SE)

He

wt

(M

R-kidney L-kidney

Control 10 25.23 ± 0.44 32.67 ± 0.59 1.94 ± 0.11 0.29 ± 0.02 0.28 ± 0.02 0.1

2 weeks 10 24.32 ± 1.44 30.63 ± 1.44 2.03 ± 0.12 0.23 ± 0.02* 0.23 ± 0.02* 0.1

Control 10 24.43 ± 0.84 32.85 ± 0.63 2.14 ± 0.09 0.28 ± 0.01 0.28 ± 0.01 0.1

4 weeks 10 25.65 ± 0.86 31.15 ± 1.41 1.82 ± 0.05** 0.22 ± 0.01* 0.21 ± 0.01* 0.1

Control 10 26.38 ± 0.14 33.98 ± 1.03 2.22 ± 0.09 0.30 ± 0.02 0.29 ± 0.02 0.1

6 weeks 10 25.64 ± 0.71 27.10 ± 1.29** 1.85 ± 0.13** 0.22 ± 0.02* 0.21 ± 0.02* 0.1

Control 10 26.51 ± 1.47 35.07 ± 2.03 2.25 ± 0.09 0.29 ± 0.02 0.29 ± 0.02 0.1

8 weeks 10 24.86 ± 0.46 26.86 ± 0.69** 1.74 ± 0.07** 0.20 ± 0.01** 0.20 ± 0.01** 0.1

Control 10 26.72 ± 0.71 41.32 ± 1.22 2.34 ± 0.07 0.32 ± 0.01 0.31 ± 0.01 0.1

80 days 10 25.29 ± 0.38 32.63 ± 1.85** 1.86 ± 0.6** 0.25 ± 0.01** 0.25 ± 0.01** 0.1

* Differences are statistically significant from the control at p< 0.05.

** Differences are statistically significant from the control at p < 0.01.

food (commercially available in Saudi Arabia) and water wereoffered ad libitum.

A total of 100 males and 100 females were used and weredivided into 20 groups, 10 animals in each group. Groups1–5 (males) and 6–10 (females) were orally treated once daily

with the dose level 9.0 mg/kg body weight (1/10 LD50) ofazadirachtin of neemix-4.5 (Thermo Trilogy Corp., USA) dis-solved in sterile distilled water for 2, 4, 6, 8 or 11.5 (80 days)

weeks. Control mice (groups 11–15 and 16–20) were similarlytreated with the corresponding volumes of the vehicle alone.At the end of each duration period, animals were weighedand then killed by cervical dislocation. They were then dis-

sected and the weights of their livers, kidneys, spleens, hearts,lungs, testes or ovaries were recorded. Those organs from the80-day treated groups and their controls were immediately

fixed in Bouins fixative, processed for the usual paraffinembedding and 7.0 lm thick paraffin sections were cut accord-ing to the methods of Drury and Wallington (1967) and

Humason (1979), stained with haematoxylin and eosin andthen examined for histopathological changes. Moreover, thenumbers of spermatozoa in the testes of males of controland azadirachtin of neemix-4.5-treated groups were deter-

mined (Bhunya and Behera, 1987).The data obtained were analyzed statistically using the stu-

dent’s t-test and a 2 · 2 contingency table (X2) (Sokal and

Rohlf, 1981).

3. Results

Data in Table 1 show that azadirachtin of neemix-4.5 at thedose level 9.0 mg/kg body weight has significantly (p < 0.05)

ions on the body weight and other parameters of treated SWR/J male mice.

art

. in g

ean ± SE)

Spleen

wt. in g

(Mean ± SE)

The two-lung

wt. in g

(Mean ± SE)

Testis wt. in g (Mean ± SE) No. of

sperm/ml (·106)(Mean ± SE)

R-testis L-testis

8 ± 0.01 0.27 ± 0.03 0.25 ± 0.01 0.131 ± 0.003 0.131 ± 0.03 43.10 ± 2.04

9 ± 0.01 0.24 ± 0.01 0.26 ± 0.01 0.126 ± 0.007 0.127 ± 0.002 39.21 ± 1.84

6 ± 0.01 0.25 ± 0.01 0.24 ± 0.01 0.128 ± 0.003 0.126 ± 0.003 38.00 ± 2.29

7 ± 0.01 0.23 ± 0.04 0.23 ± 0.01 0.118 ± 0.003* 0.117 ± 0.003* 29.67 ± 2.26*

7 ± 0.01 0.26 ± 0.03 0.25 ± 0.01 0.135 ± 0.009 0.132 ± 0.009 40.67 ± 1.46

7 ± 0.01 0.21 ± 0.01* 0.25 ± 0.02 0.108 ± 0.009** 0.107 ± 0.009* 28.65 ± 1.47**

8 ± 0.01 0.24 ± 0.01 0.27 ± 0.01 0.141 ± 0.005 0.143 ± 0.003 39.89 ± 0.75

5 ± 0.01* 0.14 ± 0.01* 0.25 ± 0.01 0.093 ± 0.003** 0.095 ± 0.002** 28.18 ± 1.08**

8 ± 0.01 0.26 ± 0.01 0.26 ± 0.01 0.136 ± 0.003 0.134 ± 0.005 42.10 ± 2.32

5 ± 0.01* 0.16 ± 0.01* 0.26 ± 0.01 0.115 ± 0.005** 0.116 ± 0.004** 32.50 ± 2.36**

Table 2 Effects of the dose level 9 mg/kg of azadirachtin of neemix-4.5 applied at different durations on the body weight and other parameters of treated SWR/J female mice.

Treatment

duration

No. of

females

used

Body wt.

in g at the

start of exp.

(Mean ± SE)

Body wt.

in g at the

end of exp.

(Mean ± SE)

Liver

wt. in g

(Mean ± SE)

Kidney wt. in g (Mean ± SE) Heart

wt. in g

(Mean ± SE)

Spleen

wt. in g

(Mean ± SE)

The two-lung

wt. in g

(Mean ± SE)

Ovary wt. in g (Mean ± SE)

R-kidney L-kidney R-ovary L-ovary

Control 10 24.36 ± 0.62 30.71 ± 1.81 2.32 ± 0.15 0.23 ± 0.01 0.23 ± 0.01 0.17 ± 0.01 0.24 ± 0.04 0.30 ± 0.02 0.019 ± 0.002 0.019 ± 0.002

2 weeks 10 25.10 ± 0.52 28.37 ± 1.31 2.02 ± 0.06 0.21 ± 0.01 0.21 ± 0.01 0.16 ± 0.01 0.20 ± 0.19 0.26 ± 0.02 0.017 ± 0.007 0.018 ± 0.001

Control 10 25.39 ± 0.41 29.82 ± 0.92 1.84 ± 0.06 0.22 ± 0.01 0.21 ± 0.01 0.16 ± 0.01 0.23 ± 0.02 0.27 ± 0.01 0.018 ± 0.002 0.018 ± 0.002

4 weeks 10 24.53 ± 0.28 25.48 ± 1.61** 1.78 ± 0.03 0.20 ± 0.01 0.20 ± 0.01 0.15 ± 0.01 0.20 ± 0.02 0.26 ± 0.01 0.015 ± 0.003 0.015 ± 0.002

Control 10 25.05 ± 0.84 28.37 ± 0.85 1.98 ± 0.12 0.22 ± 0.02 0.22 ± 0.02 0.17 ± 0.01 0.22 ± 0.02 0.24 ± 0.01 0.020 ± 0.001 0.021 ± 0.002

6 weeks 10 24.58 ± 1.01 25.78 ± 0.95* 1.72 ± 0.12* 0.19 ± 0.01 0.19 ± 0.01 0.16 ± 0.01 0.21 ± 0.02 0.25 ± 0.02 0.014 ± 0.001** 0.014 ± 0.001**

Control 10 24.68 ± 0.55 29.72 ± 0.42 2.09 ± 0.05 0.23 ± 0.02 0.23 ± 0.02 0.19 ± 0.02 0.21 ± 0.02 0.29 ± 0.02 0.018 ± 0.001 0.019 ± 0.001

8 weeks 10 25.85 ± 0.75 24.53 ± 0.92** 1.65 ± 0.07** 0.17 ± 0.01** 0.17 ± 0.01** 0.15 ± 0.01* 0.16 ± 0.01* 0.24 ± 0.01** 0.013 ± 0.002* 0.013 ± 0.002**

Control 10 23.29 ± 0.37 32.71 ± 0.26 1.86 ± 0.06 0.22 ± 0.01 0.22 ± 0.01 0.18 ± 0.01 0.22 ± 0.02 0.25 ± 0.01 0.025 ± 0.002 0.024 ± 0.002

80 days 10 22.55 ± 0.43 29.28 ± 0.85** 1.54 ± 0.07* 0.17 ± 0.01** 0.16 ± 0.01** 0.15 ± 0.01* 0.17 ± 0.13* 0.21 ± 0.01* 0.014 ± 0.001** 0.014 ± 0.001**

* Differences are statistically significant from the control at p < 0.05.

** Differences are statistically significant from the control at p < 0.01.

Plate 1 Photomicrograph of cross-section of liver obtained from the control group. H and E stain.

Plate 2 Photomicrograph of cross-section of liver obtained from the 80-day treated group. H and E stain.

Effect of azadirachtin of neemix-4.5 on SWR/J mice 71

Plate 3 Photomicrograph of cross-section of liver obtained from the 80-day treated group. H and E stain.

Plate 4 Photomicrograph of cross-section of kidney cortex obtained from the control group. H and E stain.

72 F.M. Abou-Tarboush et al.

reduced the means of the liver and testis weights, and sperm

count when applied for 4 weeks and on. The means of bodyand spleen weights were also significantly (p < 0.05) reducedwhen azadirachtin applied for 6 weeks and on. Moreover,

the mean heart weight was significantly (p < 0.05) reducedwhen azadirachtin applied for 8 weeks and on. On the otherhand, the mean kidney weight was significantly (p < 0.05) re-duced at all duration periods used in the present study. How-

ever, no significant (p< 0.05) effect was seen on the mean lungweight of such treated males.

Data in Table 2 show that azadirachtin of neemix-4.5 at the

dose level 9.0 mg/kg has significantly (p< 0.05) reduced themean body weight of treated females when applied for 4 weeksand on. The means of liver and ovary weights were signifi-

cantly (p< 0.05) reduced when azadirachtin applied for

6 weeks and on. Furthermore, the means of kidney, heart,

spleen and lung weights were significantly (p < 0.05) reducedin such treated females when azadirachtin applied for 8 weeksand on. Moreover, 1 male and 1 female, 2 males and 2 females

and 3 males and 3 females (out of 10 in each) were died duringthe duration periods of the treatment with azadirachtin at 6, 8and 11.5 weeks, respectively.

The microscopic examination of heart, lung, spleen and

ovaries of mice treated with the dose level 9.0 mg/kg of azadi-rachtin for a period of 80 days did not show any significant tis-sue damage and were comparable with those of control

groups. However, the microscopic examination of livers, kid-neys and testes showed some significant changes includingmultiple vacuolations, disturbed normal arrangement of hepa-

tic cells, increase in Kuffer cell number, more condensed nuclei

Plate 5 Photomicrograph of cross-section of kidney cortex obtained from the 80-day treated group. H and E stain.

Plate 6 Photomicrograph of cross-section of testis obtained from the control group. H and E stain.

Effect of azadirachtin of neemix-4.5 on SWR/J mice 73

in hepatic and renal cells, decrease in sperm number and/or ab-

sence of such sperms in some of the seminiferous tubules andreduction in intertubular spaces in the testes of treated animalsPlates 1–9.

4. Discussion

The present results have clearly demonstrated that the oral

administration of the dose level 9.0 mg/kg (1/10 LD50) bodyweight of azadirachtin of neemix-4.5 to SWR/J mice for varioustreatment periods has resulted in significant reduction in bodyweight and in the weights of most organs tested of treated ani-

mals especially at the 4-, 6-, 8- and 11.5-week periods.Moreover,such treatment has also resulted in some histological changes inthe livers, kidneys and testes of the 11.5-week treated group and

these changes ranged frommild to severe in these organs of those

treated animals compared with their control group.Similar observations have been made by Vijjan and Parihar

(1983) using neem seed cake feeding for 90 days in rats. Neem

seed cake has also been reported to exhibit toxic effect in sheep(Vijjan et al., 1982) and cattle (Ketkar, 1976). The major toxicsymptom observed in those studies was the depression in thegrowth rate of treated animals. Furthermore, Narayanan

et al. (1993) reported that the chronic administration of neemoil to adult albino rats caused microscopic lesions in the liversand kidneys. Moreover, gastric intubation with 100 mg/kg of

neem leaf extract for 20 consecutive days has significantly re-duced the percent increase in body weight in mice (Pandaand Kar, 2000). In addition, the subacute effect of neem oil

(25 and 50 mg/kg) administrated daily for 8 days to adult male

Plate 7 Photomicrograph of cross-section of testis obtained from the 80-day treated group. H and E stain.

Plate 8 Photomicrograph of cross-section of testis obtained from the control group. H and E stain.

74 F.M. Abou-Tarboush et al.

rats caused significant decrease in sperm counts, epididymalweight and marked structural changes in testes (Manoranji-tham et al., 1993; Sampathraj et al., 1993). However, azadi-

rachtin technical 12% orally administered to male andfemale rats at the dose levels of 500, 1000 or 1500 mg/kg/dayfor 90 days did not produce any signs of toxicity, mortality,

changes in tissue weight, pathology and serum and bloodparameters (Raizada et al., 2001). Moreover, Pillai and Sant-hakumari (1984) have reported that nimbidin, a commercial

formulation containing azadirachtin, has failed to produceany signs of toxicity in mice, rats and dogs.

The discrepancy between the present results and those of

Raizada et al. (2001) and Pillai and Santhakumari (1984) couldwell be due to the nature of formulations used. Jacobson

(1995) has reviewed and concluded that neem products maybe toxic to some vertebrates but, however, attributed theseto be a result of mycotoxin and/or other toxins (Raizada

et al., 2001). Neem-Rich 100 is reported to have little influenceon biochemical parameters of serum in rats (Quadari et al.,1984). Neem oil, however, has been reported to produce great-

er general toxicity (Narayanan et al., 1993), particularly to themale reproductive tissues (Manoranjitham et al., 1993; Sampa-thraj et al., 1993). Furthermore, Mahboob et al.(1998) have

shown that low-level exposure of vapocide (isolated from neemoil) has a significant effect on the xenobiotic detoxificationmechanism of different tissues of rats. However, Raizada

et al. (2001), as mentioned, reported that azadirachtin techni-cal 12% did not produce any adverse effects in rats. In the light

Plate 9 Photomicrograph of cross-section of testis obtained from the 80-day treated group. H and E stain.

Effect of azadirachtin of neemix-4.5 on SWR/J mice 75

of those studies mentioned, it appears that the toxicity pro-duced by neemix-4.5 in the present study may be due to factorsother than azadirachtin.

Such factors could cause the adverse effects observed in thepresent study through their cytotoxic effects on the rapidlyproliferating cells, their depletive effect on the energy sources

and/or their disturbing effect on the hormonal homeostasis.

Acknowledgements

This work was supported by the General Directorate ResearchGrants Programs, King Abdulaziz City for Science and Tech-nology, Riyadh, Saudi Arabia. Research Project No. LGP-4-38.

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