SHORT REPORT UNEQUIVOCAL EVIDENCE OF GENOTOXIC POTENTIAL OF ARGEMONE OIL IN MICE Kausar M. ANSARI, Lalit K.S. CHAUHAN, Alok DHAWAN, Subhash K. KHANNA and Mukul DAS * Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India Consumption of mustard oil adulterated with argemone oil leads to a clinical condition, commonly referred to as “Epidemic Dropsy.” Since in vitro studies have shown that sanguinarine, an active benzophenanthridine alkaloid of arge- mone oil, intercalates DNA molecule, the in vivo clastogenic and DNA damaging potential of argemone oil was investi- gated in mice. Swiss albino mice were intraperitoneally ad- ministered 0.5, 1.0, 2.0 and 4.0 ml/kg body wt. of argemone oil to analyze chromosome aberrations and micronucleus test, while 0.25, 0.5, 1.0 and 2.0 ml/kg body wt. were given for alkaline comet assay. The frequencies of chromosomal aber- rations and micronucleated erythrocytes formation in mouse bone marrow cells increased in a dose-dependent manner following argemone oil treatment. However, significant in- duction in chromosomal aberrations (83%) and micronucle- ated erythrocytes formation (261%) were observed at a min- imum dose of 1.0 ml/kg. The results of comet assay revealed DNA damage in blood, bone marrow and liver cells following argemone oil treatment. Olive tail moment (OTM) and tail DNA showed significant increase in bone marrow (35– 44%) and blood cells (25– 40%) even at a dose of 0.25 ml/kg body wt. of argemone oil. In liver cells, OTM was significantly increased (20%) at a dose of 0.25 ml/kg, while all the comet parameters including OTM, tail length and tail DNA showed significant increase (31–101%) at a dose of 0.5 ml/kg. These results clearly suggest that single exposure of argemone oil even at low doses produces genotoxic effects in mice. © 2004 Wiley-Liss, Inc. Key words: argemone oil; genotoxicity; comet assay; chromosomal aberration; micronucleus assay Several incidences of adverse effects on human health have been reported due to consumption of mustard oil adulterated with arge- mone oil. 1 The clinical manifestations of the disease, commonly referred to as Epidemic Dropsy, include vomiting, diarrhea, pitting edema, erythema, glaucoma, breathlessness, etc. In extreme cases, cardiac arrest has also been reported. 1 The earliest incidence of argemone oil poisoning was observed in Kolkata in 1877. 2 Since then, several epidemics have been encountered in different states of India as well as in other countries such as Rangoon, Fiji, Mauritius and North West Cape districts of South Africa. 3,4 Recently, the largest epidemic occurred in New Delhi, during the months of August–September 1998, affecting over 3,000 persons with 65 deaths. 4 The toxic effects of argemone oil have been attributed to the presence of benzophenanthridine alkaloids, sanguinarine and di- hydrosanguinarine. 5 Argemone oil causes dilatation of the smaller arterioles and capillaries, leading to the leakage of serum albumin with a concomitant increase in globulin resulting in increased capillary permeability. 4,6 Experimental studies suggest that skin, liver, lungs, kidneys and heart are the target sites for argemone oil intoxication. 1,7 Studies indicate that argemone oil inactivates he- patic cytochrome P-450 with concomitant enhancement of mito- chondrial and microsomal lipid peroxidation. 8,9 The inactivation of cytochrome P-450 by argemone oil impairs the clearance of arge- mone alkaloids from the body. 10 while enhancement in lipid per- oxidation is due to production of reactive oxygen species, which may lead to over expression of heat shock protein (hsp-70). 11–13 In the recent past, it has been observed that the incidence of hepato- biliary/gall bladder cancers is statistically more in the states of the Gangetic basin than other states of India. 14,15 Among the usage pattern of fats and oils, mustard oil is one of the prime differences and preferred choice in the dietary habits of the population of the Gangetic basin. It was therefore argued whether consumption of argemone adulterated mustard oil has any role in hepatobiliary cancer. Since it is known that oxidative stress parameters may have a direct bearing on genotoxicity, the clastogenic and DNA dam- aging potential of argemone oil has been evaluated in the present study. MATERIAL AND METHODS Chemicals Agarose, Low melting agarose (LMA) was procured from BRL Life Technologies Inc. (Gaithersburg, MD). Ethidium bromide (EtBr), Trypan blue, Carboxyfluoresein, Ethylmethanesulfonate (EMS), Cyclophosphamide and Colchicine were obtained from Sigma Chemicals Co., Inc. (St. Louis, MO). All other chemicals and dyes used were of analytical reagent grade commercially available. Collection of seeds and extraction of oil Argemone mexicana seeds were procured from the outskirts of Lucknow city, Uttar Pradesh, India. The seeds were crushed and the oil was extracted with the help of a Soxlet apparatus using n-hexane. 9 The yield of argemone oil from the seeds was 35% (v/w) and the alkaloid, sanguinarine content was found to be 0.33% in oil. Animal treatment schedule Six- to 7-week-old male Swiss albino mice (202 g), obtained from an animal breeding colony of Industrial Toxicology Research Centre, Lucknow, were used in the present study. Five animals were kept per cage, having a bedding of rice husk and fed standard pellet diet (Ashirwad, Chandigarh, India) and water ad libitum. The animals were maintained in controlled atmosphere of 12 hr dark/light, 22  2°C and 70 – 80% humidity and sacrificed by cervical dislocation with minimal suffering following the rules laid down by the Animal Welfare Committee of ITRC. Argemone oil was diluted in pure and fresh mustard oil. Acute intraperitoneal LD 50 was determined 16 and found to be 8.4 ml/kg body wt. Seventy mice were divided into 7 groups of 10 each. Four groups of mice received argemone oil through the intraperitoneal (i.p.) route at doses of 0.5, 1.0, 2.0 and 4.0 ml/kg body wt. equivalent to 6, 12.5, 25 and 50% of LD 50 . Argemone oil was Grant sponsor: Technology Mission on Oil Seeds, Pulses and Maize (TMOP&M), New Delhi *Correspondence to: Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P.O. Box 80, Lucknow-226001, India. Fax: +91-522-2228227. E-mail: mditrc@rediffmail.com, mditrc@hotmail.com, mukuldas@itrc.res.in Received 25 January 2004; Accepted after revision 9 March 2004 DOI 10.1002/ijc.20319 Published online 4 August 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 112, 890 – 895 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer