Cytogenetic toxicity of neem P.K. Khan a, *, K.S. Awasthy b a Department of Zoology, Patna Women’s College, Patna 800 001, India b Department of Zoology, K.K.M. College, Pakur 816 107, India Accepted 11 April 2003 Abstract The cytogenetic toxicity of the leaf extract of neem was evaluated in murine germ cells. The extract was found to induce struc- tural and numerical changes in the spermatocyte chromosomes as well as synaptic disturbances in them at their first metaphase. A significant increase in the frequency of sperms with abnormal head morphology and the decrease in mean sperm count were also observed. This spermatotoxic effect of the neem extract corroborates its germ cell mutagenicity. The possible role of azadirachtin, the most active principle present in the neem extract, in producing the observed genotoxic effect is discussed. # 2003 Elsevier Ltd. All rights reserved. 1. Introduction The promising properties of neem (Azadirachta Indica Juss) have been realized world-wide (National Research Council, 1992; Stone, 1992). As a contraceptive, various neem formulations are reported to adversely affect the reproductive performance in males (Riar et al., 1993; Sinha et al., 1984; Upadhyay and Talwar, 1993). It is therefore imperative to assess the cytogenetic toxicity, if any, of neem in male germ cells. Azadirachtin (C 35 H 44 O 16 ), a steroid akin to tetra- nortriterpenoid (limonoid) is the most active principle present in the extract of neem (Singh et al., 1993). Structural analyses suggest genotoxic and carcinogenic potentials of this limonoid (Rosenkranz and Klopman, 1995a,b). Moreover, the leaf extract of neem has been shown to induce chromosomal aberrations in murine bone marrow cells (Awasthy et al., 1995, 1999; Khan and Awasthy, 2001). Rojanapo and Tepsuwan (1992) has also reported mutagenicity of flower extract of neem towards Salmonella typhimurium TA 98. In the present work, the differentiating spermatogo- nial cells of mice were exposed in vivo during their last 2–3 generations of proliferation to the leaf extract of neem. Screening of spermatocyte chromosomes at their first metaphase and analysis of sperm head morphology as well as sperm count were done to evaluate the geno- toxic potentials of the extract in male germ line cells. 2. Materials and methods Mature leaves of neem, collected during the flowering season (late March), were washed in running tap water, oven dried (at 60  C), coarsely powdered in a glass mortar and then soxhlated with 80% ethanol (v/v). The extract so obtained was made ethanol free under vac- corotatory evaporator and finally obtained in the form of a latex. A homogeneous suspension of the latex was prepared by using a 10% aqueous solution of gum-aca- cia as its vehicle. Experiments were performed in 6–8-week-old labora- tory bred male Swiss albino mice, Mus musculus (S-cdri, seed colony obtained from Central Drug Research Institute, Lucknow, India). The animals were segregated into five experimental groups (two controls and three treated), each comprising 10 mice. A description of the experimental groups and the treatment protocol is given in Table 1. The treatment were given once daily through oral gavaging for seven consecutive days (multiple treatment regimen) to affect a larger proportion of stem cell population (Salvadori et al., 1988). Separate sets of animals were used from meiotic chromosome test as well as for sperm toxicity test. All animals were maintained 0278-6915/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0278-6915(03)00123-6 Food and Chemical Toxicology 41 (2003) 1325–1328 www.elsevier.com/locate/foodchemtox * Corresponding author. E-mail address: parimal_khan@yahoo.co.in (P.K. Khan).