Methyleugenol Genotoxicity in the Fischer 344 Rat Using the Comet Assay and Pathway-Focused Gene Expression Profiling

Abstract

Methyleugenol (MEG), a constituent of human food, induces malignant tumors in multiple tissues of rats and mice. Although MEG forms DNA adducts and induces unscheduled DNA synthesis in rat liver, it is negative in many in vitro genetic toxicity assays. In the present study, we evaluated MEG-induced DNA damage in the rat using (1) the alkaline Comet assay, (2) the oxidative Comet assay, and (3) expression profiling of genes associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg body weight (bw) MEG and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung 3 h and 24 h later. MEG failed to produce any increase in DNA damage. In addition, rats were given a single oral dose of 2000 mg/kg bw MEG, and Comet assays were performed with liver, bone marrow, and bladder 1, 3, 6, and 8 h later. With one exception (bone marrow at 8 h), no DNA damage was detected. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. Whereas no MEG-induced DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6- and 8-h sampling times. Gene expression analysis on the livers from MEG-exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that MEG induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of MEG in rodents.

In vivo genotoxicity of estragole in male F344 rats

“…Several alkenylbenzenes including safrole [Lord and Ashworth, 2012], estragole [Jones and Go, 2010] and methyleugenol [Williams et al, 2013] form DNA adducts. In addition, safrole and methyleugenol were found to induce oxidative DNA damage in rat liver [Liu et al, 1999;Ding et al, 2011]. Based on these findings, we hypothesize that multiple genotoxic mechanisms, i.e.…”

“…The different genotoxicity results from our studies of structurally similar compounds may be due to the different experimental designs: the methyleugenol study was an acute study (rats were sacrificed 1, 3, 6, 8, and 24 hr after single dose [Ding et al, ] while the present study is a sub‐acute study (rats were dosed at 0, 24, and 45 hr and terminated at 48 hr after the first treatment). A recent study demonstrated a time‐dependence of DNA adduct formation after i.p.…”

Safety assessment of potential food ingredients in canine hepatocytes

“…It has been shown to induce chromosomal aberrations and endoreduplication in vitro (Maralhas et al, 2006). Other research suggests that methyleugenol may cause DNA damage in rat liver (Ding et al, 2011). However, it is unclear if CLO or one of its components could cause hepatotoxicity in dogs.…”

Silicon Dioxide Impedes Antiviral Response and Causes Genotoxic Insult During Calicivirus Replication

“…After dissociating RAW 264.7 cells into single cell suspensions, global cell viabilities were determined using Trypan Blue exclusion tests. For the alkaline comet assay, 26 50 μ l of single-cell suspensions in PBS, derived from each treatment condition were mixed with 450 μ l 0.8% (diluted in PBS) low melting-point (LMP) agarose at 37 °C, and 100 μ l of this suspension was applied to microscope slides (Fisher Scientific, St. Louis, MO,) previously thin-coated with 1% agarose. After solidification of the LMP agarose/embedded cell mixture at 4 °C, the slides were placed in freshly prepared lysis buffer (2.5 M NaCl, 0.1 M ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 10% dimethyl sulfoxide (DMSO) and 1% Triton X-100, pH 10.0) at 4 °C in the dark for 3 h. The slides then were washed once in neutralization buffer (0.4 M Tris, pH 7.5) in the dark for 5 min at 4 °C and then transferred into chilled alkaline unwinding/electrophoresis solution (~300 mM NaOH, 1 mM EDTA, pH > 13) in the dark for 30 min to unwind DNA.…”