Ex vivo/in vitro evaluation of the effects of Nigella sativa aqueous extract utilizing rat hepatocytes

Aqueous extracts of Nigella sativa (Ranunculaceae), has been traditionally used for the treatment of several ailments such as inflammations, liver disorders, and arthritis. Several studies on N. sativa revealed antioxidant, anti-inflammatory, hepatoprotective and antimutagenic activities. In this study the protective effects of aqueous extracts of Nigella sativa were evaluated against four toxicants by ex vivo/in vitro treatment of rat hepatocytes. Rats were supplied with N. sativa aqueous extracts instead of drinking water for two weeks prior to hepatocyte isolation by collagenase in situ two step liver perfusion. Primary cultures of hepatocytes from fed and unfed rats were established, and treated with benzo[a]pyrene (B[a]P), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), and tert-Butyl hydroperoxide (TBHP). Controls from both fed and unfed rats were either untreated or treated with the solvent dimethylsulfoxide (DMSO). Thereafter, the treated and control cultures were stimulated for proliferation, fixed and analyzed for cyto- and genotoxic effects. Mitotic indices and the percentages of necrotic and apoptotic cells were evaluated for cytotoxicity testing, while the percentages of micronucleated cells (MN) and the frequency of chromosomal aberrations (CA) were analyzed as endpoints of genotoxicity. Control cultures from extract fed rats showed a significant increase in the frequency of CA, and the percentages of MN and necrotic cells as well as the mitotic indices, when compared to controls from unfed rats, indicating potential cyto- and genotoxic as well as mitogenic effects of N. sativa extracts by themselves. Furthermore, DMNQ treatment of cultures from fed rats demonstrated a significant increase in CA and MN, and a reduction of the mitotic indices compared to DMNQ treatment of cultures from unfed rats, which indicates an increased susceptibility of hepatocytes to DMNQ. Additionally, in TBHP-treated cultures from fed rats significantly increased percentages of MN were observed indicating an enhancement of its toxicity. In contrast, MNNG-treated cultures from fed rats showed significantly reduced CA compared to those from unfed rats, indicating a protective effect against this directly acting mutagen. Finally treatment with B[a]P of cultures from fed rats did not influence any of the parameters compared to unfed rats. In conclusion, N. sativa extract constituents or their metabolites seem to render hepatocytes sensitive to redox cycling agents, but induce potential protection from the clastogenic effects of the directly acting mutagen MNNG.