Biochemical effects of vepacide (from Azadirachta indica) on Wistar rats during subchronic exposure

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Abstract

We investigated the effect of Vepacide (from Azadirachta indica), a neem-based pesticide, on acid (AcP) and alkaline (AkP) phosphatase in different tissues of male and female albino Wistar rats. Subchronic doses of Vepacide in coconut oil (80, 160, and 320 mg/kg; maximum volume of 0.2 mL) were administered orally for 45 or 90 days. The administration of Vepacide resulted in a significant increase in AcP and AkP in serum, kidney, lung, and liver tissue (AkP only in liver), whereas a significant decrease of AcP in liver was observed in male and female rats after 45 and 90 days of treatment with moderate and high doses. The alterations in serum, liver, kidney, and lung tissues of both male and female rats caused by this compound were statistically significant, and the changes were also dose and time dependent. The alterations in male rats were not statistically significant when compared with female rats, indicating that there were no sexual differences. The withdrawal study (28 days post-treatment) revealed significant recovery, indicating reversal of the toxic symptoms once the toxicant was removed. There was a high degree of positive correlation between results for serum as compared to those for kidney, lung, and liver (AkP only for liver). However, there was a high negative correlation between AcP results for serum as compared with those for liver. The alterations in these enzymes indicated that lung tissue was the most susceptible, followed by liver and kidney. AcP and AkP are marker enzymes, and their increase in serum, with parallel increases in different tissues, might be due to the increased permeability of plasma membranes. The decrease in liver AcP may be due to the necrosis of cellular tissues. The changes observed in these enzyme activities could be useful as biomarkers of exposure to Vepacide.

Introduction

The indiscriminate use of synthetic pesticides has resulted in environmental pollution and a toxicity risk to nontarget organisms. While searching for alternatives to commercially available synthetic pesticides, the Indian Institute of Chemical Technology (IICT) in Hyderabad isolated a free-flowing solid from seed kernels of the neem tree (Azadirachta indica) and has designated it as Vepacide (US Patent 5856256). The main active ingredient of neem is azadirachtin, a well-known biopesticide with good insecticidal activity against a broad range of insect pests (Kreutzweiser et al., 1999). It is selective, less resistant, eco-friendly, and minimally toxic for mammals (Kataria et al., 1998). In addition to its insecticidal activity, the antifeedant effect of neem-containing azadirachtin against Culex tarsalis and Culex quinquefasciatus has been reported at 5 and 10 ppm (Su and Mulla, 1998). It has also been reported that A. indica leaves, at a dose of 200 mg/kg, exerted significant anti-inflammatory activity in cotton pellet granuloma assays in rats (Chattopadhyay, 1998). A 50% ethanol extract of neem flower and bark showed contraceptive activity in male rats (Purohit et al., 1990).

In earlier studies we found that Vepacide, when administered orally to rats in subchronic doses of 80, 160 and 320 mg/kg, inhibited brain acetylcholinesterases (AChE), Na+-K+-, Mg2+-, and Ca2+-ATPases (Rahman et al., 1999). It also altered feed intake, body weight gain, and hematological and biochemical parameters in rats (Rahman et al., 1996). Similarly, Vepacide caused an increase of aspartate and alanine aminotransferase in serum, kidney, and lung, whereas these enzymes decreased in rat liver (Rahman et al., 2001). The half-maximal lethal dose (LD50) value of Vepacide was found to be 1566.85±134.06 mg/kg in rat by the oral route (Mahboob et al., 1998). It has been reported that petroleum ether extracts of neem seed kernel at a dose of 1 g/kg caused a significant decrease in hemoglobin, MCHC, and lipid peroxidation, as well as a significant inhibition of erythrocyte catalase. They also caused an increase in white blood cell count and reduced glutathione and blood urea nitrogen in rat blood (Kataria et al., 2000). However, evidence is very scanty on the long-term effect of neem, especially on nontarget biochemical parameters. Therefore, in this study we investigated the subchronic effect of Vepacide on the enzymes acid phosphatase (AcP) and alkaline phosphatase (AkP) in serum and different tissues of male and female Wistar rats. AcP is a marker enzyme for lysosomes and is thus related to general cell metabolism, whereas AkP is a sensitive membrane-bound enzyme biomarker related to the transport of various metabolites (EL-Demerdash, 2001). We also studied sexual differences and reversal of toxicity. Furthermore, we investigated the correlation between serum effects and those in various tissues such as liver, kidney, and lung, caused by the treatment of Vepacide.

Section snippets

Test material

The test compound Vepacide was extracted from neem seed kernels using methanol after treatment with hexane and then ethyl acetate or dichloromethane. Vepacide contains 12% azadirachtin as active ingredient; the remaining 88% consists of nimbinal, nimbin, azadiradione, epoxydiradione, gedunin, 14,15-dioxy-17,β-gedunin, 40-acetyl nimbanediol, 17β-hydroxy-azadridione, and 17,α-hydro-azadriodione (Rahman et al., 1999).

Treatment of rats

Male and female albino Wistar rats weighing 100–120 g were obtained from NIN

Results

Vepacide, when administered in three different subchronic doses to male and female rats by the oral route for a period of 90 days, caused a 10% mortality among female rats at a high dose (320 mg/kg). The rats showed a loss in body weight and feed intake. They showed such symptoms as dullness, irritation, lacrimation, and diarrhea. The rats treated with a moderate dose (160 mg/kg) also showed these symptoms, but they were less marked. However, neither the rats treated with the low dose (80 mg/kg)

Discussion

The aim of this study was to evaluate the biochemical alterations in rats after subchronic administration of Vepacide. The investigation assessed AcP and AkP activity in various tissues of male and female rats. The study showed that subchronic treatment with Vepacide led to the suppression of body weight gain and feed intake, along with clinical signs and symptoms in the moderate- and high-dose groups related to stress and growth retardation. Similar loss of body weight was reported in

Acknowledgements

The authors express their sincere thanks to K.V. Raghavan (Director, IICT, Hyderabad, India) for his keen interest and encouragement during this study. This study is IICT Communication No. 02.415.

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