Cytotoxicity and genotoxicity of methyleugenol and related congeners-- a mechanism of activation for methyleugenol

Methyleugenol is a substituted alkenylbenzene found in a variety of foods, products, and essential oils. In a 2-year bioassay conducted by the National Toxicology Program, methyleugenol caused neoplastic lesions in the livers of Fischer 344 rats and B6C3F(1) mice. We were interested in the cytotoxicity and genotoxicity caused by methyleugenol and other alkenylbenzene compounds: safrole (a known hepatocarcinogen), eugenol, and isoeugenol. The endpoints were evaluated in cultured primary hepatocytes isolated from male Fischer 344 rats and female B6C3F(1) mice. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, while genotoxicity was determined by using the unscheduled DNA synthesis (UDS) assay. Rat and mouse hepatocytes showed similar patterns of toxicity for each chemical tested. Methyleugenol and safrole were relatively non-cytotoxic, but caused UDS at concentrations between 10 and 500 microM. In contrast, isoeugenol and eugenol produced cytotoxicity in hepatocytes with LC50s of approximately 200-300 microM, but did not cause UDS. Concurrent incubation of 2000 microM cyclohexane oxide (CHO), an epoxide hydrolase competitor, with a non-cytotoxic concentration of methyleugenol (10 microM) resulted in increased cytotoxicity but had no effect on genotoxicity. However, incubation of 15 microM pentacholorophenol, a sulfotransferase inhibitor, with 10 uM methyleugenol resulted in increased cytotoxicity but had a significant reduction of genotoxicity. These results suggest that methyleugenol is similar to safrole in its ability to cause cytotoxicity and genotoxicity in rodents. It appears that the bioactivation of methyleugenol to a DNA reactive electrophile is mediated by a sulfotransferase in rodents, but epoxide formation is not responsible for the observed genotoxicity.     

Screening of safrole, eugenol, their ninhydrin positive metabolites and selected secondary amines for potential mutagenicity.

The mutagenicity of safrole, eugenol, the secondary amines, with which they combine during metabolism, and the ninhydrin positive urinary metabolites of safrole and eugenol was tested. The panel of tests included the direct bacterial assay, a microsomal mutagenesis assay and a host-mediated assay. With the direct bacterial assay employing four mutant strains of Salmonella typhimurium (TA1530, TA1531, TA1532, TA1964), all the compounds gave negative results. In the microsomal mutagenesis assay, employing the same four mutant strains, safrole and safrole metabolite II were mutagenic with strains TA1530 and TA1532. Dimethylamine was also found to be a weak mutagen in the microsomal mutagenesis assay with strain TA1530. Safrole and safrole metabolite II were also mutagenic in the host-mediated assay with strains TA1950 and TA1952. Negative results were observed for safrole metabolites I and III, eugenol, eugenol metabolites I and II, piperidine, pipecolic acid, proline, and pyrrolidine in all three assay systems.     

Genotoxic effects of eugenol, isoeugenol and safrole in the wing spot test of Drosophila melanogaster

In the present study, the phenolic compounds eugenol, isoeugenol and safrole were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The Drosophila wing somatic mutation and recombination test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the SMART in its standard version with normal bioactivation and in its variant with increased cytochrome P450-dependent biotransformation capacity. Eugenol and safrole produced a positive recombinagenic response only in the improved assay, which was related to a high CYP450-dependent activation capacity. This suggests, as previously reported, the involvement of this family of enzymes in the activation of eugenol and safrole rather than in its detoxification. On the contrary, isoeugenol was clearly non-genotoxic at the same millimolar concentrations as used for eugenol in both the crosses. The responsiveness of SMART assays to recombinagenic compounds, as well as the reactive metabolites from eugenol and safrole were considered responsible for the genotoxicity observed.     

Metabolic activation of a natural promutagen, eugenol, by replicative cultures of adult rat liver epithelial cells]

The metabolism of eugenol via the epoxide-diol pathway has been characterized by the incubation of this compound in replicative cultures of epithelial cells from sexed rat liver. It is a naturally occuring compound which has been found as the second metabolite of a known hepatocancerogen: safrole. Eugenol did not appear as a mutagen while epoxyeugenol was mutagenic using the Ames Test.     

Methyleugenol and eugenol variation in Ocimum basilicum cv. Genovese gigante grown in greenhouse and in vitro

The variation of the two main aromatic compounds in Ocimum basilicum cv Genovese Gigante grown in greenhouse and "in vitro" was analyzed. The content of methyleugenol and eugenol was correlated to the plants' height rather than to the plants' age and the growth site. Particularly, methyleugenol was prevalent in plants up to 6.5 cm, as plants grew it was replaced by eugenol that was dominant in taller plants. Analysis of basil 20 cm in height showed that methyleugenol is prevalently localized in the low part while eugenol is prevalent in the upper part of the plant. Moreover, a chronic and acute toxicity of methyleugenol was evidentiated in an assay using chicken embryos.     

The Effect of {alpha}-Naphthalene Acetic Acid (NAA) and Benzylaminopurine (BAP) on the Accumulation of Volatile Oil Components in Cell Cultures of Tarragon (Artemisia dracunculus)

Disorganized cells of A. dracunculus have been demonstratedto be competent to synthesize the four phenylpropene compoundscharacteristic of tarragon essential oil. Allylanisole and methyleugenol, the major components, are accumulated at particularlylow levels and are inhibited largely by the presence of exogenousNAA. Volatilization, active metabolism, and autotoxicity ofthese compounds are involved in limiting their accumulation.The data reported here suggest that both limited expressionof specific synthetic pathways, and the absence of suitable(inert) accumulation sites can restrict accumulation of phenylpropenesin disorganized cultures of A. dracunculus. Key words: Artemisia dracunculus, tarragon, tissue culture, essential oil, phenylpropenes, allylanisole, methyl eugenol     

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.     

The O‐methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit

Phenylpropenes, such as eugenol and trans‐anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non‐producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co‐located with seven candidate genes for phenylpropene O‐methyltransferases (MdoOMT1–7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over‐expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.     

Bioactivity of natural O‐prenylated phenylpropenes from Illicium anisatum leaves and their derivatives against spider mites and fungal pathogens

A variety of volatile phenylpropenes, C₆‐C₃ compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O‐prenylated phenylpropenes, 4‐allyl‐2‐methoxy‐1‐[(3‐methylbut‐2‐en‐1‐yl)oxy]benzene [O‐dimethylallyleugenol ( 9 )] and 5‐allyl‐1,3‐dimethoxy‐2‐(3‐methylbut‐2‐en‐1‐yl)oxy]benzene [O‐dimethylallyl‐6‐methoxyeugenol ( 11 )] as major constituents. The structure–activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol ( 1 ), isoeugenol ( 2 ) and 6‐methoxyeugenol ( 6 ), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O‐dimethylallyleugenol ( 9 ) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure‐based design of new functional properties of phenylpropenes.     

Chemical Constituents and Activities of the Essential Oil from Myristica fragrans against Cigarette Beetle Lasioderma serricorne

Essential oil extracted from nutmeg seeds (Myristica fragrans Houtt .) by hydrodistillation was subjected to GC/MS and GC analysis. A total of 27 constituents were identified, of which eugenol (19.9%), methylisoeugenol (16.8%), methyleugenol (16.7%), sabinene (11.8%), and terpinen‐4‐ol (8.5%) were the major components. The essential oil was tested against Lasioderma serricorne for insecticidal and repellent activity, the LD₅₀ value at the end of 24 h exposure period was 19.3 μg/adult. Six active compounds were isolated by bioassay‐guided fractionation. They were identified as eugenol ( 1 ), methyleugenol ( 2 ), methylisoeugenol ( 3 ), elemicin ( 4 ), myristicin ( 5 ), and safrole ( 6 ). Among these isolates, 4 showed the strongest contact toxicity against L. serricorne adults with an LD₅₀ value of 9.8 μg/adult. Repellency of crude oil and active compounds were also determined. Compounds 1, 2, 4 , and 5 were strongly repellent against the cigarette beetle and exhibited the same level of repellency compared with the positive control, DEET. The results indicate that the essential oil of M. fragrans and its active constituents have potential for development as natural insecticides and repellents to control L. serricorne.     

Anticonvulsant activity of the leaf essential oil of Laurus nobilis against pentylenetetrazole- and maximal electroshock-induced seizures.

The leaf essential oil of Laurus nobilis Linn., Lauraceae, which has been used as an antiepileptic remedy in Iranian traditional medicine, was evaluated for anticonvulsant activity against experimental seizures. The essential oil protected mice against tonic convulsions induced by maximal electroshock and especially by pentylenetetrazole. Components responsible for this effect may be methyleugenol, eugenol and pinene present in the essential oil. At anticonvulsant doses, the essential oil produced sedation and motor impairment. This effect seems to be related in part to cineol, eugenol and methyleugenol. Although the essential oil had an acceptable acute toxicity, further studies are required before any absolute conclusions can be drawn.     

Floral scent production in Clarkia breweri (Onagraceae). II. Localization and developmental modulation of the enzyme S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase and phenylpropanoid emission.

We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55-67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4'-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.     

Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells

Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four active compounds (-)-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), kakuol (3), and '3,4,5-trimethoxytoluene' (=1,2,3-trimethoxy-5-methylbenzene; 4), in terms of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compounds 1-4 showed potent inhibition of NO production, with IC(50) values in the low nanomolar-to-micromolar range. Also isolated were the known compounds methylkakuol (5), '3,5-dimethoxytoluene', safrole, asaricin, methyleugenol, and (-)-asarinin, which were found to be inactive in the above assay. Among the ten known isolates, compounds 1, 2, and 5 were found for the first time in this plant.     

Effect of Japanese seaweed (Laminaria angustata) extracts on the mutagenicity of 7,12-dimethylbenz[a]anthracene, a breast carcinogen, and of 3,2'-dimethyl-4-aminobiphenyl, a colon and breast carcinogen

Animal model studies suggest that diets containing Laminaria angustata, a brown seaweed commonly eaten in Japan, inhibit breast carcinogenesis. In order to identify the compound(s) in the seaweed responsible for tumor-inhibiting activity, we used Ames/mammalian microsome assay system to determine the antimutagenic (or anticarcinogenic) effect of various solvents and water extracts of Laminaria angustata. The antimutagenic effects of acetone, ether, chloroform, chloroform + methanol, hot water and cold water extracts on the mutagenicity induced by 7,12-dimethylbenz[a]anthracene (DMBA), a breast carcinogen, and 3,2'-dimethyl-4-aminobiphenyl (DMAB), a colon and breast carcinogen, was studied using the Salmonella typhimurium strains TA98 and TA100. All extracts were nonmutagenic in both bacterial tester strains. The addition of 10-100 mg solvent extracts of seaweed/plate greatly inhibited DMAB-induced mutagenicity in both tester strains (80-96% inhibition) and DMBA-induced mutagenicity in TA100 (about 82%), whereas hot and cold water extracts produced a moderate inhibition in a dose-related manner in both strains.     

Evaluation of alternariol and alternariol methyl ether for mutagenic activity in Salmonella typhimurium.

Alternariol and alternariol methyl ether were tested in the Ames Salmonella typhimurium assay, and both were shown, with and without metabolic activation, to be nonmutagenic to strains TA98 and TA100. The finding of other investigators that alternariol methyl ether is weakly mutagenic to TA98 without metabolic activation could have resulted from the presence of a small amount of one of the highly mutagenic altertoxins in the alternariol methyl ether originally tested.     

Mutagenicity of the metabolites of the epoxide-diol pathway of safrole and its analogs. Study on Salmonella typhimurium]

Mutagenicity of the metabolites of the expoxide-diol pathway of safrole and analogues was studied on Ames' strains with Ames' method. Safrole, eugenol, eugenolmethylether, estragol, allylbenzene and 1'-hydroxysafrole, are not mutagen on TA 1535, TA 100 (point mutation) and TA 1537, TA 1538, TA 98 (frameshift mutations) without activation system. The corresponding epoxides that we have synthetized, are mutagens and inducers of point mutation in TA 1535 and TA 100. Dose-effect curves show differences between the mutagen efficiencies of these epoxides probably in relation with their electrophilic properties. On the other hand the 2', 3'-dihydro-2',3'-dihydroxisafrole was not mutagen in Ames' test. These results confirm the promutagen character of safrole and analogues and the role of the epoxides as proximate carcinogens.     

An investigation of the storage and biosynthesis of phenylpropenes in sweet basil

Plants that contain high concentrations of the defense compounds of the phenylpropene class (eugenol, chavicol, and their derivatives) have been recognized since antiquity as important spices for human consumption (e.g. cloves) and have high economic value. Our understanding of the biosynthetic pathway that produces these compounds in the plant, however, has remained incomplete. Several lines of basil (Ocimum basilicum) produce volatile oils that contain essentially only one or two specific phenylpropene compounds. Like other members of the Lamiaceae, basil leaves possess on their surface two types of glandular trichomes, termed peltate and capitate glands. We demonstrate here that the volatile oil constituents eugenol and methylchavicol accumulate, respectively, in the peltate glands of basil lines SW (which produces essentially only eugenol) and EMX-1 (which produces essentially only methylchavicol). Assays for putative enzymes in the biosynthetic pathway leading to these phenylpropenes localized many of the corresponding enzyme activities almost exclusively to the peltate glands in leaves actively producing volatile oil. An analysis of an expressed sequence tag database from leaf peltate glands revealed that known genes for the phenylpropanoid pathway are expressed at very high levels in these structures, accounting for 13% of the total expressed sequence tags. An additional 14% of cDNAs encoded enzymes for the biosynthesis of S-adenosyl-methionine, an important substrate in the synthesis of many phenylpropenes. Thus, the peltate glands of basil appear to be highly specialized structures for the synthesis and storage of phenylpropenes, and serve as an excellent model system to study phenylpropene biosynthesis.