1 Zagazig Veterinary Journal, ©Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt. Volume 49, Number 4, p. 462-478, December 2021 RESEARCH ARTICLE Ethoxyquin and Butylated Hydroxyl Toluene Induced Hepatotoxic Effect Via Apoptosis, Oxidative Stress in Rats: Tissue Injury-Related CYP1A1 Gene Expression Amany T. Mohammed, Mohamed M.M. Metwally, Dina Y. Hegab*, Mervat H. Ghoneim, and Ali H. Abou-Hadeed Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt. Pathology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt. *Corresponding author e-mail: dinahegab23@gmail.com Abstract Animal feed may contain different phenolic antioxidants, such as butylated hydroxyanisole, butylated hydroxy toluene (BHT), and ethoxyquin (EQ). EQ and BHT at high concentrations can have a pro-oxidant effect and can cause adverse health effects in animals. This study was designed to evaluate the hepatotoxic effect of EQ and / or BHT in rats. Fifty male Sprague– Dawley rats were assigned to five groups of 10 rats each as following: the first group served as control and did not receive any treatments; the second group served as vehicle control and was orally given corn oil. The third group was orally administered EQ day after day in a dose of 1/5 of LD50, the fourth group was orally received BHT day after day in a dose of 1/5 LD50. The fifth group was orally administered both EQ and BHT at the same doses and durations described above. The duration of this study was 45 and 90 days. The results revealed that EQ, BHT and their co-exposure caused a significant decrease in levels of antioxidant enzymes (superoxide dismutase (SOD), catalase enzyme (CAT) and reduced glutathione (GSH)) in rats. Also a significant increase in malondialdehyde (MDA) and 8-hydroxyguanosine (8-OH-dG) levels was recorded in serum of rats. Co-exposure group had a significant increase in DNA damage variables (% tail DNA, tail length, and Olive Tail Moment (OTM)), strong immuno positive reactivity of caspase3 and up regulation of CYP1A1 gene expression in the liver cells of exposed rats. In conclusion, we can state that EQ and BHT are potentially hepatotoxic compounds where the oxidative stress could be accepted as a possible mechanism of their toxicity. Therefore, the utilization of such compounds as feed additives should be more controlled and limited. Keywords: Ethoxyquin, Butylated hydroxyl toluene, DNA damage, CYP1A1. Introduction Antioxidants are the most suitable additives used to prolong the shelf-life of feeds by preventing oxidation deteriorations which might result in fat rancidity and color changes [1]. Antioxidants that are used in animal feeds can be generally divided into natural and synthetic ones. The most common natural antioxidants used for protecting feed against peroxidation are vitamin E and vitamin C [2]. While the commonest synthetic ones are butylated hydroxy anisole (BHA), tert-butyl hydroquinone (TBHQ), butylated hydroxy toluene (BHT), propyl gallate, and ethoxyquin (EQ) [3]. Ethoxyquin (6-ethoxy-2,2,4-trimethyl- 1,2-dihydroquinoline, EQ) is a lipid-soluble, quinolone-based synthetic antioxidant that accumulates in human and animal tissues. In addition, it is used as a colour preservative in spices and as an anti-degradation agent in rubber [4]. It is widely used as a technological additive in food for pets and livestock animals and farmed fish feed to protect it from lipid peroxidation and preserve the stability of fat-soluble vitamins [5]. It affect the animal’s well-being and