In vivo genotoxicity of estragole in male F344 rats

Abstract

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio-activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven-week-old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole-induced dose-dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non-target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole-dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical-specific interaction and is also mediated by oxidative species.

<em>In Vivo</em> Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

“…The general protocol is applicable to any tissue from which single cells or nuclei can be isolated with minimal processing-induced DNA damage (i.e., DNA damage induced not by the test agent, but by the handling and processing of the animal tissues). In our research, we have conducted alkaline comet assays on cells from bone marrow 7,9 , stomach 6 , kidney 9 , bladder 9 , lung 9 , heart 10 , mammary gland 5 , uterus 5 , testis 5 , and blood 5 . These assays can provide a quick, simple and inexpensive method for studying xenobiotic-induced DNA damage in multiple organs and tissues of experimental animals.…”

“…Safety Authority (EFSA) 4 regulatory guidelines. In our lab, we have employed the assay for evaluating in vivo DNA damage induced by food ingredients, pharmaceuticals, and nanomaterials [5][6][7][8][9][10] . Rat liver will be used as an example in this protocol, but the comet assay can be performed with other tissues/organs of experimental animals, as long as intact single cells can be isolated from the tissue.…”

“…Thus, the addition of an enzyme-digestion step makes the assay a specific and sensitive method for measuring oxidative DNA damage in vivo 12 . Utilizing these assays, we have demonstrated toxicant-induced oxidative DNA damage in the liver of rats and mice [6][7][8] and in the heart of rats 10 .…”

The safety evaluation of food flavoring substances: the role of genotoxicity studies

“…DNA adducts. DNA adduct studies have been conducted for only a small number of flavoring substances and potential metabolites, including formaldehyde, acetaldehyde, the a,b-unsaturated aldehydes acrolein, crotonaldehyde, and related compounds (Wang et al 2000;Hecht et al 2001;Hecht et al 2011;Yang et al 2019), 2-hexenal and trans, trans-2,4-hexadienal (Frankel et al 1987;Eder et al 1993;Eisenbrand et al 1995;NTP 2003); estragole (Ishii et al 2011;Paini et al 2012;Ding et al 2015), and methyl eugenol (Phillips et al 1984;Herrmann et al 2013;Williams et al 2013;Herrmann et al 2014;Monien et al 2015;Tremmel et al 2017). Most flavoring substances have chemical structures that make them unlikely candidates for DNA adduct formation (e.g.…”

Determination of Estragole in Pharmaceutical Products, Herbal Teas and Herbal Extracts Using GC-FID

“…Estragole is considered a rodent liver carcinogen which requires bio-activation. The mechanism of its carcinogenicity in rat has been reported (Ding et al, 2015). The level of estragole varied according to the origin, harvesting and processing method; estragole was found to constitute 12.36 % of the wild fennel extract.…”