p53 triggers mitochondrial apoptosis following DNA damage-dependent replication stress by the hepatotoxin methyleugenol

Abstract

Liver cancer is one of the most frequent tumor entities worldwide, which is causally linked to viral infection, fatty liver disease, life-style factors and food-borne carcinogens, particularly aflatoxins. Moreover, genotoxic plant toxins including phenylpropenes are suspected human liver carcinogens. The phenylpropene methyleugenol (ME) is a constituent of essential oils in many plants and occurs in herbal medicines, food, and cosmetics. Following its uptake, ME undergoes Cytochrome P450 (CYP) and sulfotransferase 1A1 (SULT1A1)-dependent metabolic activation, giving rise to DNA damage. However, little is known about the cellular response to the induced DNA adducts. Here, we made use of different SULT1A1-proficient cell models including primary hepatocytes that were treated with 1′-hydroxymethyleugenol (OH-ME) as main phase I metabolite. Firstly, mass spectrometry showed a concentration-dependent formation of N2-MIE-dG as major DNA adduct, strongly correlating with SULT1A1 expression as attested in cells with and without human SULT1A1. ME-derived DNA damage activated mainly the ATR-mediated DNA damage response as shown by phosphorylation of CHK1 and histone 2AX, followed by p53 accumulation and CHK2 phosphorylation. Consistent with these findings, the DNA adducts decreased replication speed and caused replication fork stalling. OH-ME treatment reduced viability particularly in cell lines with wild-type p53 and triggered apoptotic cell death, which was rescued by pan-caspase-inhibition. Further experiments demonstrated mitochondrial apoptosis as major cell death pathway. ME-derived DNA damage caused upregulation of the p53-responsive genes NOXA and PUMA, Bax activation, and cytochrome c release followed by caspase-9 and caspase-3 cleavage. We finally demonstrated the crucial role of p53 for OH-ME triggered cell death as evidenced by reduced pro-apoptotic gene expression, strongly attenuated Bax activation and cell death inhibition upon genetic knockdown or pharmacological inhibition of p53. Taken together, our study demonstrates for the first time that ME-derived DNA damage causes replication stress and triggers mitochondrial apoptosis via the p53-Bax pathway.

Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery

“…Saponine (0.1%) served as positive control and solvent as negative control. Cell viability was determined as described previously (Carlsson et al 2022 ). After 24 h and 72 h, cells were washed with PBS and incubated for 1 h with DMEM low glucose (-FCS; -P/S) or William’s E Medium (1X) supplemented with 10% 440 µM Resazurin-NaCl-Pi-solution (0,1% dimethylformamide; 1.1 mM KH 2 PO 4 , 154 mM NaCl, 3.7 mM Na 2 HPO 4 ) at 37 °C in humidified atmosphere of 5% CO 2 .…”

A Novel Inhibitor of Poly(ADP-Ribose) Polymerase-1 Inhibits Proliferation of a BRCA-Deficient Breast Cancer Cell Line via the DNA Damage–Activated cGAS–STING Pathway

“…DSBs are the main causative factor for genome instability . Upon encountering DSBs in the DNA, the cells maintain the stability of the genome through a series of responses, including cell cycle arrest, DNA repair, and apoptosis. , Previous studies have shown that PARP1 inhibitors cause DSBs in BRCA-deficient tumor cells, thereby inducing apoptosis and cell cycle arrest.…”

Urinary Excretion of Mercapturic Acids of the Rodent Carcinogen Methyleugenol after a Single Meal of Basil Pesto: A Controlled Exposure Study in Humans

“…8 In a recent study, it was shown that ME caused DNA damage-dependent replication stress resulting in mitochondrial apoptosis via the p53-Bax pathway. 9 ME-derived DNA adducts, found in cell culture experiments, 10,11 were also detected in mice, 12−15 and, most importantly, in humans. 16−18 To be precise, the presence of DNA adducts, primarily N 2 -(methylisoeugenol-3′-yl)-2′-deoxyguanosine, was demonstrated in 150 out of 151 surgical human liver samples 16,17 and 10 out of 10 lung samples 18 investigated.…”

“…Furthermore, ME increased the mutational burden in hepatocellular carcinomas of mice in a dose-dependent manner, leading to mutational signatures similar to those in human hepatocellular carcinomas with known exposure to the carcinogens aflatoxin or benzo­[ a ]­pyrene . In a recent study, it was shown that ME caused DNA damage-dependent replication stress resulting in mitochondrial apoptosis via the p53-Bax pathway …”