*3.1. Metabolism of Myristicin and Elemicin vs. Safrole and Methyleugenol* 3.1.1. Common Structural Features

Initial steps of the hepatic activation of methylenedioxy- and methoxy-substituted allylic alkenylbenzenes include epoxidation of the exocyclic double bond followed by its cleavage by microsomal or cytosolic epoxide hydrolases or spontaneous hydration to generate 2- ,3- -dihydrodiols [67]. Such metabolites are detected in the urine of animals treated with allylbenzenes [6,68–70]. Another pathway may be the hydroxylation of the 1- -carbon atom adjacent to this 2- ,3- -double bond [71]. Side chain reactions of alkenylbenzenes are catalyzed by various cytochrome P450 monooxygenases (CYPs). Epoxides and dihydrodiols may be derived not only from the allylbenzene compounds but also from some of their metabolites, which still possess an intact allyl group, such as the allylcatechols [72]. However, phenolic and catecholic compounds typically undergo rapid phase II conjugation, which might be a predominant pathway for such metabolites as also shown for the alkenylbenzene eugenol containing a free phenolic group [73]. Thus, in contrast to alkenylbenzenes that bear only methoxy or methylenedioxy substituents, the high first-pass conjugation and rapid elimination may explain why eugenol is deemed to be less toxic as compared to the well-known hepatocarcinogens methyleugenol and safrole.

Following hydroxylation at the 1- -position (Figure 2), the alcoholic metabolite can be sulfonated. Subsequent heterolytic cleavage of the formed sulfate moiety would generate an electrophilic carbenium ion intermediate, which is highly reactive towards nucleophilic sites [74,75], and that may, for example, generate glutathione (GSH) conjugates, as well as adducts with proteins, RNA, or DNA [76]. Since the carbenium ionic charge is delocalized, adducts can be formed at the 1- - or 3- -position, with the 3- -position being the preferred site [77,78].

**Figure 2.** Metabolite excretion of safrole in the rat is reported to be 93% within 72 h, and most of this material (86%; [79]) would consist of metabolites formed via demethylenation of the methylenedioxy moiety to yield carbon monoxide or formate and the dihydroxy-benzene moiety [80]. The other metabolic routes observed were allylic hydroxylation and the epoxide-diol pathway [70,79]. Oxidations of the allylic side chain of safrole may proceed (i) via an epoxide resulting in side chain propane diols during different stages of the metabolic steps [72], or (ii) via 1- -hydroxylation followed by sulfonation that might lead to a reactive carbocation intermediate [5]. Other possible steps of metabolic ways of safrole are (iii) the subsequent oxidation of the 1- -hydroxysafrole to the 1- -oxo-safrole [81], (iv) oxidation at the 3- -position to yield 3- -hydroxy-isosafrole, and (v) the demethylenation of safrole to 4-allylcatechol that may isomerize to its quinone-methide [82–84]. The occurrence of glutathione conjugates at the 1- -position may be indicative of the intermediate formation of *para*-quinone methide tautomers [82], whereas glutathione conjugates at the benzene ring point to reactions with *ortho*-quinone intermediates [82]. CYP: cytochrome P450 monooxygenases; SULT: sulfotransferases; EH: epoxide hydrolases; nuc: nucleophilic structures such as DNA or proteins.

However, the metabolic pathway to the carbenium ions is only one selected pathway, already often discussed with respect to the cyto- and genotoxic activity of alkenylbenzenes. This metabolic pathway presumes the presence of sulfotransferases (SULT) and cofactors such as 3- -phosphoadenosine-5- -phosphosulfate (PAPS) [5].

On the other hand, alkenylbenzenes and their metabolites that bear ortho- and/or para-phenolic groups may form quinone methide intermediates (Figure 2) [82,85] that are also prone to be conjugated by GSH or react directly with other nucleophiles in the cell. The transient formation of a quinone methide of eugenol appears plausible [85] since an eugenol GSH conjugate was detected utilizing rat liver or rat lung microsomes [86]. The cytotoxic effects of eugenol recognized in rat hepatocytes are reasoned to be due to the formation of a reactive quinone methide intermediate [87]. In 1990, Fischer et al. tentatively identified metabolites including thiophenol metabolites (11%) following eugenol ingestion in the urine of humans, presumably formed by GSH conjugation at an aromatic ring position [73]. Thus, methoxylated non-phenolic substances (e.g., methyleugenol and elemicin) may as well undergo CYP enzyme-mediated *O*-demethylation and subsequent quinone methide formation followed by GSH conjugation. Similarly, there can be oxidative demethylation of methoxy groups in elemicin by CYP1B1 [88], creating the possibility to yield also catechols or other phenols and conjugates, as was shown from benzodioxolesubstituted alkenylbenzenes myristicin and safrole in rat and human urine using GC-MS [8].

A number of CYP isoenzymes are capable of catalyzing the 1- -hydroxylation of akenylbenzenes [88–93]. An overview of CYPs that are demonstrated to be involved in the oxidation of methyleugenol, elemicin, safrole, and myristicin is shown in Table 2.


**Table 2.** Human cytochrome P450 isoenzymes mediating the 1- -hydroxylation of alkenylbenzenes.

Main human CYPs and, in brackets, contributing human CYPs involved in the metabolism of methyleugenol, elemicin, safrole, and myristicin.

Apart from the epoxide, the carbenium ion, and the quinone methide metabolic pathways of the alkenylbenzenes already discussed, another metabolic pathway that may occur after rearrangement of the double bond from 2- ,3- -position to 1- ,2- -position is the oxidation of 3- -hydroxy metabolites of alkenylbenzenes leading to cinnamic acids and propionic acids [6,68,69]. In principle, 3- -hydroxy-1- ,2- -propenylbenzenes may be equivalent to 1- -hydroxy-allylbenzenes as substrates for hepatic SULTs. On the other hand, due to steric reasons further side chain oxidation of the 3- -hydroxy-propenylbenzenes yielding cinnamaldehydes and cinnamic acids, which can be conjugated with GSH or glycine, appear to dominate. Further oxidation, probably via the fatty acid *β*-oxidation cycle, would lead to side chain cleavage and the formation of benzoic acids and its glycine conjugates [5].

Seemingly small, but relevant structural molecular differences in benzene ring substituents of the parent alkenylbenzenes call for a closer look at the potential metabolic pathways of elemicin, myristicin, methyleugenol, and safrole. In an attempt to identify similarities and possible differences of elemicin and myristicin, we compared their metabolic features to the closely related derivatives methyleugenol and safrole. Those two compounds bearing only methoxy groups at the benzene ring without methylene bridge are methyleugenol and elemicin. The two compounds with a methylenedioxy moiety are safrole and myristicin, which are categorized as benzodioxoles.

#### 3.1.2. Metabolism of Methyleugenol

Results of ADME experiments performed in 2000 within a study of the National Toxicology Program (NTP) led to the conclusion that absorption of orally ingested methyleugenol in rats and mice is rapid and complete, and that the distribution of methyleugenol to tissues is fast. In rodents, methyleugenol is extensively metabolized in the liver and more than 70% of the dose administered is found in the urine of rats and mice as hydroxylated, sulfated, or glucuronidated metabolites [92].

With view on the toxicity of methyleugenol, it is generally assumed that bioactivation is mainly mediated via 1- -hydroxylation at the allylic side chain followed by sulfo conjugation, yielding a highly reactive sulfate ester [10,94].

In the NTP study, it was shown that repeated ingestion of methyleugenol may saturate metabolic enzymes [92], leading to greater tissue accumulation and thus higher probability for genotoxicity, mutations, and malignant cell transformations. Saturability of metabolism is of special concern in cases when 1- -hydoxylation of the allylic side chain becomes more prominent over other pathways. This may enhance hepatocarcinogenesis in rodents at higher dose levels [95].

In rat bile, methyleugenol could be found in the form of GSH conjugates. These conjugates detected by Yao and colleagues potentially resulted from reactions with methyleugenolderived epoxide metabolites, α,β-unsaturated aldehydes, carbenium ions, and quinone methides [96]. These conjugates were further metabolized, yielding the cysteine conjugates found in rat urine. In GSH-fortified microsomal preparations that lack SULT and PAPS, it was generally not expected that carbenium intermediates would be formed. However, Yao et al. found 1- -bound GSH and related cysteine conjugates in such incubations [96]. Thus, it is hypothesized that 1- -hydroxy metabolites or other metabolites than the sulfate esters may directly react with GSH under certain conditions.

Beside 1- -hydroxylation, the metabolites observed in rats and mice suggest that methyleugenol can also undergo demethylation, ring, and/or further side chain oxidations [92].

The NTP authors further concluded that the risk to humans ingesting methyleugenol is expected to be subject to marked inter-individual metabolic variability. Indeed, hydroxylation of methyleugenol investigated in human liver microsomes varied considerably (37-fold), with the highest hydroxylation rate being similar to that observed with liver microsomes from rats [97]. Moreover, one study by Tremmel et al. demonstrated that methyleugenol-induced DNA adduct levels in human liver samples were dependent on the SULT1A1 copy number [94].
