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

Methyleugenol (ME), found in numerous plants and spices, is a rodent carcinogen and is classified as “possibly carcinogenic to humans”. The hypothesis of a carcinogenic risk for humans is supported by the observation of ME-derived DNA adducts in almost all human liver and lung samples examined. Therefore, a risk assessment of ME is needed. Unfortunately, biomarkers of exposure for epidemiological studies are not yet available. We hereby present the first detection of N-acetyl-l-cysteine conjugates (mercapturic acids) of ME in human urine samples after consumption of a popular ME-containing meal, pasta with basil pesto. We synthesized mercapturic acid conjugates of ME, identified the major product as N-acetyl-S-[3′-(3,4-dimethoxyphenyl)allyl]-l-cysteine (E-3′-MEMA), and developed methods for its extraction and LC–MS/MS quantification in human urine. For conducting an exposure study in humans, a basil cultivar with a suitable ME content was grown for the preparation of basil pesto. A defined meal containing 100 g of basil pesto, corresponding to 1.7 mg ME, was served to 12 participants, who collected the complete urine at defined time intervals for 48 h. Using d6-E-3′-MEMA as an internal standard for LC–MS/MS quantification, we were able to detect E-3′-MEMA in urine samples of all participants collected after the ME-containing meal. Excretion was maximal between 2 and 6 h after the meal and was completed within about 12 h (concentrations below the limit of detection). Excreted amounts were only between 1 and 85 ppm of the ME intake, indicating that the ultimate genotoxicant, 1′-sulfooxy-ME, is formed to a subordinate extent or is not efficiently detoxified by glutathione conjugation and subsequent conversion to mercapturic acids. Both explanations may apply cumulatively, with the ubiquitous detection of ME DNA adducts in human lung and liver specimens arguing against an extremely low formation of 1′-sulfooxy-ME. Taken together, we hereby present the first noninvasive human biomarker reflecting an internal exposure toward reactive ME species.


Table of Contents
Table S1.Methyleugenol and eugenol contents in basil cultivars S3 Table S2.Recipe of the basil pesto used for the controlled exposure study S4   Table S2.Recipe of the basil pesto used for the controlled exposure study in humans.
The listed ingredients resulted in a total quantity of ~332 g pesto.For the human intervention study, a total quantity of 3 kg basil pesto was produced by scaling up.

Ingredient Amount
Basil

Figure S1 :Figure S3 :Figure S4 :Figure S5 :Figure S6 :
Figure S1: Product ion mass spectra of peaks 1-3 in the MEMA isomer mixture S5 Figure S2: 1 H NMR, 13 C NMR and MS spectrum of synthesized E-3'-MEMA S6 Figure S3: LC-MS characterization of synthesized d 6 -MEMA S7 Figure S4: GC-MS/MS characterization of ME and d 3 -ME S8 Figure S5: GC-MS/MS analysis of ME in basil leaves and pesto S9 Figure S6: Serial dilution of d 6 -E-3'-MEMA in water or urine matrix S10 Methods: Details of initial GC-MS analysis for ME determination S11

Figure S3 :
Figure S3: LC-MS characterization of synthesized d 6 -MEMA using instrumental setup "system 1".(A) High-resolution mass spectrometry (HRMS) chromatogram of d 6 -MEMA in ESI-single ion monitoring (SIM) mode.Three prominent signals (Peaks 1, 2, and 3) were detected at m/z 344.1444.(B) Isotopic pattern of isomeric d 6 -MEMA Peaks 1-3 and corresponding mass error (Δm/z).(C) Product ion mass spectrum of Peak 3 obtained at a collision energy of 20 eV.The associated precursor ion was set at m/z 344.2.(D) Chemical structure of d 6 -E-3'-MEMA and suspected fragmentation pattern.(E) Multiple reaction monitoring (MRM) chromatogram of d 6 -MEMA.An overlay of multiple transitions is given.

Figure S4 :
Figure S4: GC-MS/MS characterization of ME (upper panel) and d 3 -ME (lower panel).(A) Product ion mass spectrum of ME and d 3 -ME obtained at a collision energy of 10 eV.The associated precursor ion was set at m/z 178 and 181, respectively.(B) Chemical structure of ME and d 3 -ME including the presumed fragmentation pattern for the three most prominent product ions.(C) Multiple reaction monitoring (MRM) chromatogram for ME and d 3 -ME.An overlay of the different transitions used for analysis is shown.

Figure S5 :
Figure S5: GC-MS/MS analysis of ME in basil leaves and in pesto made from them.Shown are the quantifier mass transitions of ME (upper panel; m/z 178.1 → 107.0) and d 3 -ME (lower panel; m/z 181.1 → 107.0).Corresponding signals of ME and its internal standard are shaded black.

Figure S6 :
Figure S6: Serial dilution of d 6 -E-3'-MEMA in water (open circles) or urine matrix (closed circles) measured with the optimized LC-MS/MS method ("system 2"), which was applied to urine samples of the intervention study including twelve participants.Equations of linear regression together with coefficients of determination are given as insets.

Table S1 .
Methyleugenol and eugenol content [µg/g fresh weight, mean ± SEM] in cultivars used for the screening experiment.