Stability and repair of DNA adducts formed by food-borne alkenylbenzene liver carcinogens; consequences for hazards and risks

Summary

DNA adduct formation upon exposure to genotoxic carcinogens is often referred to as a biomarker of exposure rather than as a biomarker of effect. In spite of this, increased levels of DNA adducts of a specific genotoxic carcinogen are generally assumed to increase the tumor incidences. However, the relation between the levels of DNA adducts formed and the levels of mutations or tumor formation is by no means well defined and may vary from one compound to another. This may in part be related to the fact that cells have quite efficient DNA repair systems, which may prevent the conversion of DNA lesions into mutations. At the present state-of-the-art DNA adduct persistence was found to be substantial for different genotoxic carcinogens including for example, DNA adducts derived from a pyrrolizidine alkaloid, aflatoxin B1 and some polycyclic aromatic hydrocarbons (Croy and Wogan 1981; Geacintov and Broyde 2017; Zhu et al. 2017), providing opportunities for adduct accumulation upon chronic exposure. The potential adduct accumulation may increase the chances of mutations and subsequent induction of tumors.

The aim of the present thesis was to obtain better insight in the relative hazards and risks of DNA adducts formed by alkenylbenzenes, a group of compounds naturally occurring in many spices and herbs, by studying their DNA adduct formation, stability, and repair.

Chapter 1 provided general information on estragole and safrole, their bioactivation resulting in DNA adduct formation, DNA repair, molecular dynamics (MD) simulations to study structural perturbations upon DNA adduct formation, and the current state-of-the-art on the risk assessment related to these genotoxic carcinogens. In Chapter 2 and 3, formation and repair of the major DNA adducts of estragole and safrole was quantified in different cell models. Results showed a limited DNA repair efficiency for both DNA adducts. Molecular dynamics simulations were used to investigate the potential conformation dependent (in)efficiency of repair of the major estragole and safrole DNA adducts. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA upon formation of these adducts were small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure to activate recognition and subsequent repair. In Chapter 4, accumulation of the major DNA adducts derived from the alkenylbenzene estragole upon repeated exposure was investigated in HepaRG cells treated in repeated cycles of 2 h exposure and 22 hours repair. The results obtained showed accumulation of adducts at a rate of 17.53 adducts / 108 nts / cycle. This rate was converted to a rate expected at average human daily intake of estragole. Based on these data it was estimated that it would take 6-57 years intake at the estimated daily intake to reach levels of 10-100 adducts /108 nts, a level of DNA adducts reported at the BMD10 of the related alkenylbenzene methyleugenol. These findings revealed that the persistent nature of the major estragole DNA adducts may contribute to accumulation of substantial levels of DNA adducts upon prolonged dietary exposure. In Chapter 5, potential consequences of combined exposure to the proximate carcinogenic metabolites of the selected foodborne alkenylbenzenes safrole and estragole were evaluated in vitro and in silico. Results indicate that concentration addition adequately described the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts upon combined as compared to single exposures. The absence of any interaction on DNA adduct formation was also predicted for combined exposure to estragole and safrole at normal dietary intake. The prediction revealed that the simultaneous presence of the two proximate carcinogens does not affect their DNA adduct formation. Chapter 6 presented a summary of the results obtained in this thesis and an overall discussion regarding to 1) considerations on the use of MD simulations to investigate the potential underlying mechanisms for the NER resistance of alkenylbenzene DNA adducts, 2) impact of the base sequence within which DNA adduct binding occurs, and the consequences for DNA structure distortion and the efficiency of NER mediated repair, 3) the consequences of DNA damage caused by estragole and safrole DNA adduct formation when not repaired, 4) accumulation of the persistent estragole DNA adducts, 5) potential combination effects affecting the DNA adduct formation, 6) implications for risk assessment.

Altogether, the present thesis provides further insight in the relative hazards and risks of DNA adduct formation by elucidating the mode of action of estragole and safrole DNA adduct formation and repair in more details via combined use of in vitro and in silico novel testing strategies. The in vitro and in silico methods of the present thesis provide a way forward to study the accumulation of the DNA adducts at low repeated dose exposure levels to a further extent.