Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells

Schulte-Hubbert, Ruth; K�pper, Jan Heiner; Thomas, Adam D.; Schrenk, Dieter

Abstract

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.

• Journal Article Type: Article
• Acceptance Date: Aug 22, 2020
• Online Publication Date: Aug 25, 2020
• Publication Date: Nov 1, 2020
• Deposit Date: Aug 24, 2020
• Publicly Available Date: Aug 26, 2021
• Journal: Toxicology
• Print ISSN: 0300-483X
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 444
• Article Number: 152566
• DOI: https://doi.org/10.1016/j.tox.2020.152566
• Keywords: Estragole; carcinogenicity; DNA adducts; genotoxicity; hepatoma; liver cells
• Public URL: https://uwe-repository.worktribe.com/output/6631824
• Publisher URL: https://www.sciencedirect.com/journal/toxicology