What are the Potential Sites of DNA Attack by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Metabolic bioactivation of small molecules can produce electrophilic metabolites that can covalently modify proteins and DNA. Paracetamol (APAP) is a commonly used over-the-counter analgesic, and its hepatotoxic side effects have been postulated to be due to the formation of the electrophilic metabolite N-acetylbenzoquinone imine (NAPQI). It has been established that NAPQI reacts to form covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. While there have been scattered reports that APAP can form adducts with DNA the nature of these adducts have not yet been fully characterised. Here the four deoxynucleosides, deoxyguanosine (dG), deoxyadenosine (dA), deoxycytidine (dC), and deoxythymidine (dT) were reacted with NAPQI and the formation of adducts was profiled using liquid chromatography–mass spectrometry with positive-ion mode electrospray ionisation and collision-induced dissociation. Covalent adducts were detected for dG, dA, and dC and tandem mass spectrometry (MS/MS) spectra revealed common neutral losses of deoxyribose (116 amu) arising from cleavage of the glyosidic bond with formation of the modified nucleobase. Of the four deoxynucleosides, dC proved to be the most reactive, followed by dG and dA. A pH dependence was found, with greater reactivity being observed at pH 5.5. The results of density functional theory calculations aimed at understanding the relative reactivities of the four deoxynucleosides towards NAPQI are described.

Cancer Hazard Identification Integrating Human Variability: The Case of Coumarin

Coumarin is a naturally occurring sweet-smelling benzopyrone that may be extracted from plants or synthesized for commercial uses. Its uses include as a flavoring agent, fragrance enhancer, and odor-masking additive. We reviewed and evaluated the scientific evidence on the carcinogenicity of coumarin, integrating information from carcinogenicity studies in animals with mechanistic and other relevant data, including data from toxicogenomic, genotoxicity, and metabolism studies, and studies of human variability of a key enzyme, CYP2A6. Increases in tumors were observed in multiple studies in rats and mice in multiple tissues. Our functional pathway analysis identified several common cancer-related biological processes/pathways affected by coumarin in rat liver following in vivo exposure and in human primary hepatocytes exposed in vitro. When coumarin 7-hydroxylation by CYP2A6 is compromised, this can lead to a shift in metabolism to the 3,4-epoxidation pathway and increased generation of electrophilic metabolites. Mechanistic data align with 3 key characteristics of carcinogens, namely formation of electrophilic metabolites, genotoxicity, and induction of oxidative stress. Considerations of metabolism, human variability in CYP2A6 activity, and coumarin hepatotoxicity in susceptible individuals provide additional support for carcinogenicity concern. Our analysis illustrates the importance of integrating information on human variability in the cancer hazard identification process.

Micronuclei in Bone Marrow and Liver in relation to Hepatic Metabolism and Antioxidant Response due to Coexposure to Chloroform, Dichloromethane, and Toluene in the Rat Model

Genotoxicity in cells may occur in different ways, direct interaction, production of electrophilic metabolites, and secondary genotoxicity via oxidative stress. Chloroform, dichloromethane, and toluene are primarily metabolized in liver by CYP2E1, producing reactive electrophilic metabolites, and may also produce oxidative stress via the uncoupled CYP2E1 catalytic cycle. Additionally, GSTT1 also participates in dichloromethane activation. Despite the oxidative metabolism of these compounds and the production of oxidative adducts, their genotoxicity in the bone marrow micronucleus test is unclear. The objective of this work was to analyze whether the oxidative metabolism induced by the coexposure to these compounds would account for increased micronucleus frequency. We used an approach including the analysis of phase I, phase II, and antioxidant enzymes, oxidative stress biomarkers, and micronuclei in bone marrow (MNPCE) and hepatocytes (MNHEP). Rats were administered different doses of an artificial mixture of CLF/DCM/TOL, under two regimes. After one administration MNPCE frequency increased in correlation with induced GSTT1 activity and no oxidative stress occurred. Conversely, after three-day treatments oxidative stress was observed, without genotoxicity. The effects observed indicate that MNPCE by the coexposure to these VOCs could be increased via inducing the activity of metabolism enzymes.