Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis Daoud Ali a , N.S. Nagpure a, * , Sudhir Kumar b , Ravindra Kumar a , B. Kushwaha a , W.S. Lakra a a National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, India b Department of Zoology, University of Lucknow, Lucknow 226 007, India article info Article history: Received 24 August 2008 Accepted 17 December 2008 Keywords: DNA damage Chlorpyrifos Comet assay Micronucleus assay Genotoxic Channa punctatus abstract Chlorpyrifos (CPF) is the single largest selling agrochemical that has been widely detected in surface waters in India. The studies on long-term genotoxic effects of CPF in different tissues of fish using geno- toxic biomarkers are limited. Therefore, in the present study DNA damage by CPF in freshwater fish Chan- na punctatus using micronucleus (MN) and comet assays was investigated. The LC 50 – 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC 50 value sublethal and nonlethal concen- trations were determined. The DNA damage was measured in lymphocytes and gill cells as the percent- age of DNA in comet tails and micronuclei were scored in erythrocytes of fishes exposed to above concentrations of CPF. In general, significant effects for both the concentrations and time of exposure were observed in treated fish. It was found that MN induction in the blood was highest on day 14 at 203.0 lg/l of CPF. The highest DNA damage was observed on day 5, followed by a gradual non-linear decline in the lymphocytes and gill cells. The study indicated MN and comet assays to be sensitive and rapid methods to detect mutagenicity and genotoxicity of CPF and other pollutants in fishes. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Occupational exposure to pesticides is a common and alarming worldwide phenomenon. Approximately, 3 million cases of acute poisoning and 0.22 million deaths from pesticide exposure have been reported annually (Marrs, 1993; USDA, 1994; Yasmashita et al., 1997). Chlorpyrifos (O,O-diethylO-3,5,6-trichloro-2-pyridyl phosphorothioate) is a broad-spectrum organophosphate pesticide used heavily throughout the world for agriculture and domestic purposes. The main target of the pesticides is acetylcholinesterase, which hydrolyses acetylcholine in cholinergic synapses at neuro- muscular junctions (Ecobichon, 1991; Amitai et al., 1998). Chlor- pyrifos (CPF) elicits a number of other effects including hepatic dysfunction, immunological abnormalities, embryo toxicity, geno- toxicity, teratogenicity, neurochemical, and neurobehavioral changes (Muscarella et al., 1984; Muto et al., 1992; Thrasher et al., 1993; Bagchi et al., 1995; Song et al., 1998; Dam et al., 1999; Gomes et al., 1999). It is a widely used against fire ants, or- namental plant insects, cockroaches, mosquitoes, termites, horn flies, lice and fleas (EPA, 1986). Its persistence in sediments is hazardous to sea bottom dwellers (Schimmel et al., 1983). The high concentration of CPF immediately inhibits both DNA and protein syntheses without regional selectivity in 1-day old rats (Whitney et al., 1995). In India, CPF is classified as an extremely hazardous pesticide (ITRC, 1989); its residue has been found in scented roses and their products (Kumar et al., 2004). Its maximum concentration has been reported to be 88.6 lg/g in tissues of fishes Channa striata and Catla catla from Kolleru Lake, India (Amaraneni and Pillai, 2001) and 198.5 lg/g in sediment, prawn and water samples from prawn ponds near Kolleru Lake wetland (Amaraneni, 2006). Surprisingly, the soft drinks also contain CPF in a concentration of 4.8 ppb, which is 47 times higher than permissible limit (CSE, 2006). It has been reported to be genotoxic in C. punctatus and mice leukocytes (Porichha et al., 1998; Rahman et al., 2002) and root meristem cells of Crepis capillaris (Dimitrov and Gadeva, 1997). The exposure to 0.08 lg/l of CPF caused reproductive impairment in Daphnia magna (EPA, 1985). Since there is growing a concern over the presence of genotox- ins in the aquatic environment, the development of sensitive bio- markers for detection of genotoxic effects in aquatic organisms has gained importance (Hayashi et al., 1998). The micronucleus (MN) and comet assays are two sensitive, rapid and extensively used tools for detecting the mutagenic and genotoxic effects of chemicals in the environment (Tucker and Preston, 1996; Kassie 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.021 * Corresponding author. Tel.: +91 522 2442440/2442441; fax: +91 522 2442403. E-mail address: nagpurens@yahoo.co.in (N.S. Nagpure). Food and Chemical Toxicology 47 (2009) 650–656 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox