Diethylnitrosamine dena

Luteolin, soy lecithin (SL), cholesterol, and 12,000 molecular cuts off cellophane membrane were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Propylene glycol (PG), EtOH, potassium di-hydrogen orthophosphate anhydrous, and di-potassium hydrogen orthophosphate anhydrous, were purchased from Elnasr pharmaceutical chemical Co. (Cairo, Egypt). Diethylnitrosamine (DENA) and carbon tetrachloride (CCL₄) were obtained from Sigma Aldrich (USA). Hydrophobic filter, membrane filter, diameter pore 0.2 µm was obtained from Versapor, German Sciences (Primo Heraeus, Hanau, Germany). All other reagents used were of analytical grade.

Diethylnitrosamine (DENA) was purchased from Sigma Chemical Co. (St. Louis, MO). Phenobarbitone was kindly supplied by the Egyptian International Pharmaceutical Industry Co. (EIPICO). Cisplatin was purchased from El Azaby pharmacy under the commercial name of cisplatin 10 mg.

Diethylnitrosamine (DENA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fresh pomegranate fruits, free of obvious defects, were purchased from Dina farms (Cairo-Alexandria Desert Road, Egypt) and kindly were authenticated by our colleagues in the Botany Department.; fruits were manually washed and peeled, and separated arils were processed using a commercial blender to obtain PJ, and the juice was filtered and was provided orally to the animals within 1 week after squeezing. PCG (purity ≥ 80% HPLC) was purchased from Xi’an Rongsheng Biotechnology Co., Ltd. (82 Keji Road, Xi’an Hi-tech Zone, China).

Guidelines for Care and Use of Laboratory Animals were followed during the investigation. All animal procedures were approved by the Ethical Commission of the University of Cagliari and the Italian Ministry of Health. Male Fischer F-344 rats (100-125 g) purchased from Charles River (Milan, Italy) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (γNull) were originally by Jackson Laboratories (Bar Harbor, Maine, USA). Animals were kept on a laboratory diet (Ditta Mucedola, Milan, Italy) and given food and water ad libitum with a 12-hour light/dark daily cycle.
HCC was induced according to the Resistant Hepatocytes (R-H) model [7 (link)]. Rats were injected intraperitoneally with the chemical carcinogen diethylnitrosamine (DENA, Sigma, MO) at a dose of 150 mg/kg body weight. After a 2-week recovery, rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF, Sigma, MO) for 1 week followed by a two-thirds partial hepatectomy (2/3 PH), and an additional week of 2-acetylaminofluorene diet. The animals were then returned to the basal diet and euthanized at 14 months (Supplementary Figure 1). Rats exposed to 2-AAF and 2/3 PH, but in the absence of carcinogen, were used as controls.

Diethylnitrosamine (DENA) and carbon tetrachloride (CCl₄) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). DENA at a dose 15 mg/kg b.wt (single i.p. injection in normal saline) was injected to initiate hepatic carcinogenesis, while CCl₄ (1.6 g/kg b.wt.) in 1 : 1 dilution with corn oil was given orally to animals by gavage to stimulate liver cell proliferation and regeneration.