Assessment of Carcinogenic Potential of Repeated Fish Fried Oil in Mice Manoj K. Pandey and Mukul Das* Food Toxicology Division, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India Our prior studies have shown that single topical treatment of repeated fish fried oil extract (RFFE), containing various polycyclic aromatic hydrocarbons (PAHs), to the dorsal epidermis of mice caused enhancement of DNA damage along with higher expression of p53 and p21WAF1 proteins and cell-cycle arrest. In the present study carcinogenic potential of repeated fish fried oil (RFFO) and RFFE was assessed. Single topical application of RFFO (100 mL/animal) and RFFE (100–500 mg/animal) to Swiss albino female mice resulted in significant induction (1.8- to 7.4-fold) of ornithine decarboxylase activity. Twice weekly topical application of methylcholanthrene (MCA) for 24 wk or single topical application of 7,12-dimethylbenzanthracene (DMBA) or RFFO or RFFE, as initiator followed by twice weekly application of 12-O-tetradecanoyl phorbol myristate acetate (TPA) as promoter for 24 wk, resulted in development of skin papillomas after 6, 7, 18, and 9 wk, respectively. The cumulative number of tumors in MCA, DMBA/TPA, RFFE (200 mg)/TPA, and RFFE (500 mg)/TPA groups were 276, 168, 34, and 58 after 24 wk while negligible or minimal initiating activity was noticed in RFFO/TPA group. No tumors were found in animals either given twice weekly topical application of RFFO or a single initiating dose of DMBA followed by twice weekly application of RFFO. Histopathology of skin of animals treated with RFFE/TPA showed marked proliferation of epidermal layers along with abnormal mitosis and multinucleated tumor appearance. Skin of animals in groups RFFO/TPA and DMBA/RFFO showed sloughing and regeneration of epidermal layers, oedema along with proliferation of fibroblasts. Histochemical localization of g- glutamyl transpeptidase was found to be substantially higher in skin of mice treated with RFFO/TPA and RFFE/TPA. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA resulted in significant induction of cutaneous aryl hydrocarbon hydroxylase (AHH) (421–432%), ethoxyresorufin-O-deethylase (252–316%), and glutathione S- transferase (133–245%) activities. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA led to significant reduction in glutathione content (39–44%) with a concomitant increase in lipid peroxidation (254–492%). Animals treated with RFFO/TPA and RFFE/TPA led a significant decrease in catalase (43–69%) and superoxide dismutase (20– 31%) activities while glutathione reductase activity was found to be diminished (23–51%) in RFFO, RFFO/TPA, DMBA/ RFFO, and RFFE/TPA treated groups. These results suggest that RFFE possess skin tumor initiating activity and that it may have weak promoting activity as well, which may involve free radicals. ß 2006 Wiley-Liss, Inc. Key words: repeated fish fried oil; PAHs; ODC; tumor INTRODUCTION Skin, a major body interface with the environ- ment, is exposed to numerous physical and chemical agents, some of which exhibit toxic manifestation in cutaneous as well as in extracutaneous tissues following precutaneous absorption [1]. The skin is one of the common sites of human cancer. Both benign and malignant tumors may be derived from viable keratinocytes and melanocytes of the epider- mis and rarely from skin appendages, blood vessels, peripheral nerves, and lymphoid tissue of dermis [2,3]. Experimental studies have shown that poly- cyclic aromatic hydrocarbons (PAHs), found to be present in soot, coal tar pitch, and various cutting oils [4] may cause skin cancer in a multistage fashion [5,6]. The first stage of skin cancer is initiation in which some basal epidermal cells are converted into latent neoplastic cells [7]. These latent cells may persist for lifetime without causing any toxic man- ifestation unless a tumor promoter acts on these cells to transform into malignant squamous cell carci- noma (SCC) [8]. Oxidative stress and inflammation have been reported to be closely associated with tumor promo- tion stage of carcinogenesis [8]. Many known inflammatory agents such as phorbol esters, for example, 12-O-tetradecanoyl-13-phorbol acetate MOLECULAR CARCINOGENESIS 45:741–751 (2006) ß 2006 WILEY-LISS, INC. Abbreviations: RFFE, repeated fish fried oil extract; PAHs, polycyclic aromatic hydrocarbons; RFFO, repeated fish fried oil; MCA, methylcholanthrene; DMBA, dimethylbenzanthracene; TPA, tetradecanoyl phorbol myristate acetate; AHH, aryl hydrocarbon hydroxylase; SCC, squamous cell carcinoma; ODC, orinithine decarboxylase; EROD, ethoxyresorufin-O-deethylase; SOD, super- oxide dismutase; GR, glutathione reductase; LPO, lipid peroxidation. *Correspondence to: Food Toxicology Division, Industrial Toxicol- ogy Research Centre, Mahatma Gandhi Marg, P.O. Box # 80, Lucknow 226001, India. Received 10 November 2005; Revised 16 February 2006; Accepted 17 March 2006 DOI 10.1002/mc.20238