DNA damage by Withanone as a potential cause of liver toxicity observed for herbal products of Withania somnifera (Ashwagandha)

Abstract

Withania somnifera, commonly known as Ashwagandha, is a medicinal plant used for thousands of years for various remedies. Extracts of Ashwagandha contain more than 200 metabolites, with withanone (win) being one of the major ones responsible for many of its medicinal properties. Recently, several cases of liver toxicity resulting from commercially available Ashwagandha products have been reported. The first report of Ashwagandha-related liver damage was from Japan, which was quickly resolved after drug-withdrawal. Later, similar cases of liver toxicity due to Ashwagandha consumption were reported from the USA and Iceland. Towards understanding the liver toxicity of Ashwagandha extracts, we studied win, a representative withanolide having toxicophores or structural alerts that are commonly associated with adverse drug reactions. We found that win can form non-labile adducts with the nucleosides dG, dA, and dC. Using various biochemical assays, we showed that win forms adducts in DNA and interfere with its biological property. Win also forms adducts with amines and this process is reversible. Based on the data presented here we concluded that win is detoxified by GSH but under limiting GSH levels it can cause DNA damage. The work presented here provides a potential mechanism for the reported Ashwagandha-mediated liver damage.

Keywords: Ashwagandha; DMEDA, dimethylethylenediamine; DNA damage; EDTA, ethylenediamine tetraacetic acid; GSH, glutathione; Glutathione; HDS, Herbal medicines and dietary supplements; LC–MS, liquid chromatography-mass spectrometry; Toxicity; Win, withanone; Withanone; cys, cysteine.

Graphical abstract

Fig. 1

Detection of win adducts of dG, dA, or dC. A) Win (100 μM) was treated with dG, dA or dC (500 μM) for 16 h at 37 °C in aqueous potassium phosphate buffer (100 mM, pH 7.4) and analyzed by LC-tandem MS. Adducts were detected using SRM. B) MS/MS spectra of the Win-dG, dA and dC adducts.

Scheme 1

Reaction of win with various nucleophiles to form adducts.

Fig. 2

Detection, stability, fragmentation analysis, and reversibility of win-ethylamine (win-NHEt) adducts. Win (100 μM) was treated with ethylamine (1 mM) for 0–6 h in aqueous potassium phosphate buffer (100 mM, pH 7.4) and analyzed by LC-tandem MS. A) LC-MS chromatogram showing the presence of win-NHEt adducts (m/z 516) along with the corresponding CID (MS/MS) of the respective adducts. B) LC-MS chromatogram showing the presence of win-NHEt adducts (m/z 516) at 60 min time interval. C) Fragmentation analysis of the Michael and epoxide adducts. D) LC-MS chromatograms showing the formation win-NHPr adducts following the addition of PrNH₂ to the win-NHEt adducts.

Fig. 3

A) Proposed reaction mechanism of win with EtNH₂ showing the four possible covalent adducts. B) Free energy profiles showing all four possible pathways for the reaction of win with EtNH₂. Relative free energies (ΔG) at 298.15 K were computed at the M06-2X/Def2-TZVP// M06-2X/6-31G* level of theory.

Fig. 4

DNA binding and detection of win-dG adduct from ctDNA treated with win. A) LC–MS chromatogram showing the presence of win in the dialysate. A solution containing 100 µM EDTA, 100 mM potassium phosphate buffer (pH 7.5), 2 µM win, and 1 mM ctDNA (in base pairs) was dialyzed against the same solution without the DNA at room temperature for 16 h. B) LC-MS/MS chromatogram showing the presence of win-dG adduct in ctDNA. 200 μg of sonicated ctDNA and win (200 µM) in 100 mM potassium phosphate buffer (pH 7.5) was incubated for 6 h at 37 °C. The DNA was precipitated, washed, digested, and analyzed by LC-MS/MS following the transition of m/z 738 → 152.

Fig. 5

Formation of win-DNA adducts in the presence of amines and GSH. A) LC-MS extracted ion chromatogram showing the disappearance of the win-NHEt adducts following the addition of DNA over 300 mins. B) Competition between GSH (1 mM) and ctDNA for adduct formation with win. The graph represents the average of 2 independent data sets (N = 2) and error bars represent standard deviation.

Fig. 6

Biological consequences of win exposure A) Ability of win-treated plasmid DNA bearing the ampicillin resistance factor to confer ampicillin resistance phenotype in transformed E. coli cells. B) Ability of win-treated plasmid DNA bearing the green fluorescence protein cDNA to confer green fluorescence phenotype in transfected HEK293T cells. C) Effects of increasing concentrations of win on hepatoma (HepG2), normal mammary epithelium (MCF10A), and mammary carcinoma (MCF-7) cell lines 72 h post-treatment. All graphs represent the average of 2 independent data sets (N = 2) and the error bars represent standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)