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Molecular mechanisms of toxicity of important food-borne phytotoxins.

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

Alkaloids↗

Species differences of glucuronidation and sulfation in relation to hepatocarcinogenesis.

The role of glucuronidation and sulfation in the control of proximal and ultimate carcinogens is briefly reviewed. In accordance with the adopted practice of tumor risk assessment, data from two rodent species (rat, mouse) and man have been compared. Sulfate esters have been established as ultimate carcinogens in 2-acetylaminofluorene, safrole and estragole induced hepatocarcinogenesis. In interspecies comparisons the tumor incidence paralleled sulfotransferase activity (Miller and Miller 1981). Glucuronides are often stable transport forms of carcinogens and in this way determine their organ specificity, for example in 2-naphthylamine-induced bladder carcinogenesis and in colon carcinogenesis produced by 2',3-dimethyl-4-aminobiphenyl. In contrast to sulfotransferase activity certain UDP-glucuronyltransferase activities are differentially inducible by xenobiotics. A 3-methylcholanthrene-inducible phenol-glucuronyltransferase (GT1), present in rat, mouse and man, appears to be part of an adaptive program to detoxify aromatic hydrocarbons. After initiation of hepatocarcinogenesis permanent alterations of these enzymes occur; GT1 is markedly increased whereas sulfotransferase is decreased. Together with changes of other drug metabolizing enzymes these alterations often lead to toxin-resistance of initiated hepatocytes. This phenomenon may facilitate selective growth of initiated hepatocytes and may enhance the probability of multiple hits in their genome.

Animals↗

Studies on the essential oil from guarana.

The essential oil from guarana [Paullinia cupana H.B.K. var. sorbilis (Mart.) Ducke] was analysed. Nine components were identified, namely (2) methylbenzenes, (1) cyclic monoterpene and (2) cyclic sesquiterpene hydrocarbons, (2) methoxyphenylpropenes and (2) alkylphenol derivatives. The alleged psychoactivity of the essential oil is presumably due to the identified constituents estragole and anethole. Any contribution of aminated metabolites of estragole/anethole to the alleged psychoactivity of the essential oil of guarana can be excluded. Neither the psychoactive 4-methoxyamphetamine nor tert-aminoketones could be traced in human urine after oral application of guarana.

Beverages↗

Genotoxicity of the alkenylbenzenes alpha- and beta-asarone, myristicin and elimicin as determined by the UDS assay in cultured rat hepatocytes.

While the alkenylbenzenes alpha- and beta-asarone are hepatocarcinogenic in rodents, myristicin and elimicin, two other alkenylbenzenes, are not. The present study investigated the mechanism of genotoxicity of the asarones to elucidate the role of cytochrome P-450 and obtain further information about the relationships between the structure, metabolism and genotoxicity of the alkenylbenzenes. The data on the ability of these compounds to induce unscheduled DNA synthesis (UDS) in hepatocytes derived from male Fischer 344 rats are presented in this paper. Cytotoxicity was assessed by lactate dehydrogenase leakage. Elimicin and alpha- and beta-asarone are genotoxic in the UDS assay but myristicin is not. The genotoxicity of the asarones is inhibited by the cytochrome P-450 inhibitor cimetidine but the sulfotransferase inhibitor pentachlorophenol (PCP) is without effect. The major metabolite of the asarones in hepatocytes was identified by liquid chromatography-mass spectrometry as 2,4,5-trimethoxycinnamic acid but this was not genotoxic when tested separately. Simple allylbenzenes such as safrole, estragole and methyleugenol are activated by sequential 1-hydroxylation and sulfation, and this is the likely mechanism of the genotoxicity of elimicin. The propenyl analogues isosafrole, anethole and methylisoeugenol, which cannot undergo 1-hydroxylation, are not genotoxic. The positive results obtained with the asarones suggest the occurrence of a novel activation 'option' for alkenylbenzenes which features a 2-methoxy group in the aromatic ring.

Alkylation↗

32P-postlabeling analysis of adducts formed between DNA and safrole 2',3'-epoxide: absence of adduct formation in vivo.

We have used the 32P-postlabeling technique to examine the binding of safrole 2',3'-oxide to DNA. At least 8 covalent adducts are formed when calf thymus DNA is incubated with this oxygenated metabolite of safrole in vitro. However, no corresponding adducts are formed with liver DNA when whole animals are exposed to safrole 2',3'-oxide, or safrole itself. Although safrole 2',3'-oxide is readily formed in vivo, and is sufficiently reactive to covalently bind to DNA, it is probably not a factor in the in vivo genotoxicity of safrole. We also demonstrate that adducts with similar mobility to the major safrole 2',3'-oxide-DNA adduct are formed in vitro between safrole 2',3'-oxide and deoxyguanosine, and also between its chemical analogs allylbenzene 2',3'-oxide or estragole 2',3'-oxide and DNA.

Animals↗

Flavonoids and alkenylbenzenes: mechanisms of mutagenic action and carcinogenic risk.

The present review focuses on the mechanisms of mutagenic action and the carcinogenic risk of two categories of botanical ingredients, namely the flavonoids with quercetin as an important bioactive representative, and the alkenylbenzenes, namely safrole, methyleugenol and estragole. For quercetin a metabolic pathway for activation to DNA-reactive species may include enzymatic and/or chemical oxidation of quercetin to quercetin ortho-quinone, followed by isomerisation of the ortho-quinone to quinone methides. These quinone methides are suggested to be the active alkylating DNA-reactive intermediates. Recent results have demonstrated the formation of quercetin DNA adducts in exposed cells in vitro. The question that remains to be answered is why these genotoxic characteristics of quercetin are not reflected by carcinogenicity. This might in part be related to the transient nature of quercetin quinone methide adducts, and suggests that stability and/or repair of DNA adducts may need increased attention in in vitro genotoxicity studies. Thus, in vitro mutagenicity studies should put more emphasis on the transient nature of the DNA adducts responsible for the mutagenicity in vitro, since this transient nature of the formed DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. For alkenylbenzenes the ultimate electrophilic and carcinogenic metabolites are the carbocations formed upon degradation of their 1'-sulfooxy derivatives, so bioactivation of the alkenylbenzenes to their ultimate carcinogens requires the involvement of cytochromes P450 and sulfotransferases. Identification of the cytochrome P450 isoenzymes involved in bioactivation of the alkenylbenzenes identifies the groups within the population possibly at increased risk, due to life style factors or genetic polymorphisms leading to rapid metaboliser phenotypes. Furthermore, toxicokinetics for conversion of the alkenylbenzenes to their carcinogenic metabolites and kinetics for repair of the DNA adducts formed provide other important aspects that have to be taken into account in the high to low dose risk extrapolation in the risk assessment on alkenylbenzenes. Altogether the present review stresses that species differences and mechanistic data have to be taken into account and that new mechanism- and toxicokinetic-based methods and models are required for cancer risk extrapolation from high dose experimental animal data to low dose carcinogenic risks for man.

Allyl Compounds↗

Interactions of safrole and isosafrole and their metabolites with cytochromes P-450.

The structural features which determine interaction of safrole and related methylenedioxyphenyl compounds with cytochromes P-450 or P-448, and determine the induction of these two classes of the cytochrome, have been studied. All methylenedioxyphenyl compounds studied interact with both cytochromes P-450 and P-448 eliciting type I spectral changes and it has been found that the allyl 4-substituent is important in these interactions. Methylenedioxyphenyl compounds with an oxidised allyl 4-substituent exhibited higher affinity for cytochrome P-448 while those possessing an intact allyl or methylvinyl group generally showed higher affinity for cytochrome P-450. Compounds possessing intact allyl and methylenedioxyphenyl groups (safrole, isosafrole and myristicine) were the most potent inducers of cytochromes P-450 and P-448; compounds containing an intact allyl group only (estragole, allybenzene and eugenol methyl ether) or an oxidized allyl group and an intact methylenedioxyphenyl group (epoxysafrole) were inducers of P-448 only.

Animals↗

Concentrations of nine alkenylbenzenes, coumarin, piperonal and pulegone in Indian bidi cigarette tobacco.

Indian-made bidi cigarettes sold in the United States are available in a variety of exotic (e.g. clove, mango) and candy-like (e.g. chocolate, raspberry) flavors. Because certain tobacco flavorings contain alkenylbenzenes and other toxic or carcinogenic chemicals, we measured the concentration of flavor-related compounds in bidi tobacco using a previously developed method. Twenty-three brands of bidis were sampled using automated headspace solid-phase microextraction and subsequently analyzed for 12 compounds by gas chromatography-mass spectrometry. Two alkenylbenzene compounds, trans-anethole and eugenol, were found in greater than 90% of the brands analyzed. Methyleugenol, pulegone and estragole were each detected in 30% or more of the brands, whereas safrole and elemicin were not detected in any of the brands. The flavor-related compounds with the highest tobacco concentrations were eugenol (12,000 microg/g tobacco) and trans-anethole (2200 microg/g tobacco). The highest eugenol and trans-anethole concentrations found in bidi tobacco were about 70,000 and 7500 times greater, respectively, than the highest levels previously found in US cigarette brands. Measurement of these compounds is crucial to evaluation of potential risks associated with inhaling highly concentrated flavor-related compounds from bidis or other tobacco products.

Allylbenzene Derivatives↗

Chemical composition, toxicity and mosquito repellency of Ocimum selloi oil.

Ocimum spp. (Lamiaceae) and their essential oils have been traditionally used to kill or repel insects, and also to flavor foods and oral products, in fragrances, in folk medicine and as condiments. In Brazil, Ocimum selloi has been used to treat stomachaches and as an anti-inflammatory remedy. This study was performed to provide data on the chemical composition, acute toxicity, mutagenicity, skin irritant potential and mosquito repellency of Ocimum selloi oil. GC/MS analysis of Ocimum selloi oil revealed that its major constituents were methyl-chavicol or estragole (55.3%), trans-anethole (34.2%), cis-anethole (3.9%) and caryophyllene (2.1%). Ocimum selloi oil given by gavage to adult Swiss Webster mice produced no adverse effects at doses as high as 1250 mg/kg body weight. Deaths and symptoms (e.g. hypoactivity, ataxia and lethargy) were observed at doses > or =1500 mg/kg body weight, being females apparently more susceptible than males. Genotoxicity of Ocimum selloi oil was evaluated in the Salmonella/microsome assay without and with S9 mixture. The oil, tested up to the toxicity limit (500-700 microg/plate), was not mutagenic to tester strains TA97a, TA98 and TA100. None of 30 volunteers of either sex exposed to undiluted Ocimum selloi oil (4-h patch test) showed a positive skin irritant reaction. A field test (six volunteers, each individual his/her own control) was carried out to evaluate mosquito (Anopheles braziliensis) repellency of Ocimum selloi oil diluted in ethanol (10% v/v). The median number of mosquito bites on volunteers' skin-recorded for 30 min after application of Ocimum selloi oil (2, range 0-3) was much lower than that noted after application of the solvent alone (19.5, 3-25) (Wilcoxon test, P<0.01). In conclusion, results showed that Ocimum selloi oil is an effective mosquito repellent that presents a low acute toxicity, poses no mutagenic risk and seems not to be irritating to human skin.

Administration, Oral↗

In vitro analysis of the properties of Beiqishen tea.

OBJECTIVE: Chinese Beiqishen tea was studied in an in vitro test system. METHODS: Phytochemical screening, trace element analysis, and the analysis of antioxidant properties were carried out. Characteristic constituents were determined by chromatographic (capillary gas chromatography and GCQ Ion Trap mass spectrometry) and spectrometric (ultraviolet and UV-VIS) methods. Element concentrations were determined by inductively coupled plasma optical emission spectrometry. Antioxidant capacity was studied by spectrophotometric and luminometric techniques using a Berthold Lumat 9501 luminometer. Hydrogen-donating activity, reducing power, and total scavenger capacity were measured. RESULTS: Total polyphenol content was 20.77 +/- 0.52 g/100 g of drug; total flavonoid content was 0.485 +/- 0.036 g/100 g of drug; and tannin content was 9.063 +/- 0.782 g/100 g of drug. Caffeine content was 1.08 mg/100 g of drug. Essential oils were identified by gas chromatography: (+)-limonene (21%), p-cymene (1.7%), estragol (3.2%), beta-ocimene (1.4%), and thymol (2.6%). Metallic ion analysis showed significantly high concentrations of Al, As, Ba, Cr, Cu, Fe, Mn, Ni, and Ti in the drug. Antioxidant and scavenger properties were identified as a function of concentration. CONCLUSIONS: The tea infusion contained some non-desirable trace elements and caffeine in addition to polyphenols and tannins in high concentrations. Therefore, the consumption of this tea may involve risks.

Alkaloids↗

Rapid classification of basil chemotypes by various vibrational spectroscopy methods.

The potential of vibrational spectroscopy methods (attenuated total reflectance/Fourier-transform-infrared (ATR/FT-IR), FT-Raman and near infrared (NIR) spectroscopy) for the identification and quantification of valuable as well as carcinogenic substances in different basil chemotypes is described. It is shown that all main volatile components occurring in different basil accessions can be reliably determined in the isolated essential oils or solvent extracts but also in the air-dried herbs. While NIR data can be interpreted only by chemometric methods, IR and Raman spectra present characteristic key bands of the individual volatiles; therefore, in the latter case, a discrimination of basil chemotypes is frequently possible without applying chemometric algorithms. NIR calibrations are successfully established for various terpenoids and phenylpropanoids; on the basis of these data, the content of the two carcinogenic compounds methyleugenol (range: 2-235 microg/100 g) and estragole (range: 34-138 microg/100 g) can be reliably predicted in air-dried basil leaves (R (2) (coefficient of determination) = 0.951; SECV (standard error of cross validation) = 19.1 microg/100 g and R (2) = 0.890; SECV = 12.8 microg/100 g, respectively). The described methods were found to be very useful tools for the efficient selection of special basil single plants, adapted to the new demands set by the legislator and the consumer. Furthermore, they can be applied in industry to very easily control the purifying, blending, and redistilling processes of basil oil.

Calibration↗

Quantification of flavor-related compounds in the unburned contents of bidi and clove cigarettes.

Bidi cigarettes, small hand-rolled cigarettes produced primarily in India, are sold in the United States in a wide variety of candy-like flavors (e.g. dewberry, chocolate, clove) and are popular with adolescents. Many flavored bidis contain high concentrations of compounds such as eugenol, anethole, methyleugenol, pulegone, and estragole; several of these compounds have known toxic or carcinogenic properties. Clove cigarettes, or kreteks, are another highly flavored tobacco product with high levels of eugenol due to clove buds present in the tobacco filler. In this study, compounds in the burnable portion-the filler and wrapper material actually consumed during the smoking of bidis, kreteks, and U.S. cigarettes-were analyzed. Flavor-related compounds were solvent extracted from the burnable portion of each cigarette with methanol. An aliquot of the methanol extract was heated, and the sample headspace was sampled with a solid-phase microextraction fiber and introduced into a gas chromatograph-mass spectrometer for analysis in selected-ion monitoring mode. High levels of eugenol were detected in five clove-flavored bidi brands ranging from 78.6 to 7130 microg/cigarette (microg/cig), whereas diphenyl ether (128-3550 microg/cig) and methyl anthranilate (154-2360 microg/cig) were found in one grape-flavored bidi brand. A nontobacco herbal bidi brand contained the greatest variety of compounds, including anethole (489-665 microg/cig), eugenol (1670-2470 microg/cig), methyleugenol (27.7-36.6 microg/cig), safrole (32.4-34.4 microg/cig), myristicin (170-247 microg/cig), and elemicin (101-109 microg/cig). Filler from kreteks was found to contain high levels of eugenol, anethole, and coumarin. Flavored bidis and clove cigarettes contain a number of compounds that are present at levels far exceeding those reported in U.S. cigarette tobacco. Research is underway to determine the levels of these compounds delivered in smoke. It is not known what effect inhalation of these compounds has on smokers.

Filtration↗

Floral Origin Markers of Chestnut and Lime Tree Honeys.

The apiculture industry is more and more interested in finding typical markers to authenticate floral origin of honeys. With this aim, some reliable volatile compounds were proposed to identify origin of lime tree and chestnut samples. A dichloromethane extraction followed by a Likens-Nickerson simultaneous steam distillation/solvent extraction led to representative honey extracts. About 400 volatile compounds were separated by gas chromatography, but only a few authenticated the floral origin of honeys. Chestnut honeys are distinguishable from other origins by high concentrations of acetophenone, 1-phenylethanol (>88 ppb), and 2-aminoacetophenone (>154 ppb). Lime tree honeys are characterized by enhanced amounts of shikimate pathway derivatives (ethylmethylphenol isomer (>31 ppb), 4-tert-butylphenol, estragole (>51 ppb), and p-methylacetophenone but also by high concentrations of monoterpene-derived compounds (menthol, thymol, 8-p-menthene-1,2-diol, and carvacrol (>76 ppb)) and methyl(1-methylethenyl)benzene.

Journal Article↗

Flavor volatiles and physical properties of vacuum-microwave- and air-dried sweet basil (Ocimum basilicum L.).

Basil (Ocimum basilicum L.) was dried using conventional hot air or the recently developed vacuum-microwave dryers. The effect of the drying method on the relative abundance of major flavor volatiles, rehydration rate, color, and structural integrity of the plant was evaluated. Dynamic headspace analysis of volatiles present in fresh or dried basil revealed that linalool and methylchavicol (estragole) were the two major headspace volatile compounds of the plant sample. Vacuum-microwave dehydrated basil yielded approximately 2.5 times the linalool and 1.5 times the methylchavicol of the air-dried samples. Furthermore, the vacuum-microwave-treated samples yielded more volatiles than fresh basil, due to chemical reactions during drying. Air-dried samples of basil had darker and fewer green hues than those prepared by vacuum microwave. Vacuum-microwave-dried samples had a higher rehydration rate, whereas the potential of the plant material to rehydrate was hindered in air-dried samples. This is likely attributed to the dramatic and pronounced structural collapse of the air-dried cells as revealed by the scanning electron microscope.

Food Handling↗

Structure-genotoxicity relationships of allylbenzenes and propenylbenzenes: a quantum chemical study.

Quantum mechanical calculations at the semiempirical level (AM1 method) were conducted for estragole (1), methyleugenol (2), safrole (3), alpha-asarone (4), beta-asarone (5), elemicin (6), allylbenzene (7), eugenol (8), trans-anethole (9), isosafrole (10), and myristicin (11), and the results compared with the known genotoxicity of 1-6 and the absence of genotoxicity of 7-11 (unscheduled DNA synthesis assay). The various compounds showed no significant differences in the relative stability of the radical species formed as intermediates in C-sp3 hydroxylation (delta HR(radical)) and in the corresponding enthalpy of activation (delta H++). In contrast, the carbonium ions of the genotoxic congeners 1-6 were shown to be comparatively more stable than those of the inactive compounds 7-11, with the exception of eugenol (8). The inactivity of this compound could be due to a very rapid stabilization of the carbonium ion by deprotonation to form a quinone methide, as suggested by quantum chemical calculations. The relative stability of the carbonium ion thus appears to be one of the key factors in the genotoxicity of allylbenzenes and propenylbenzenes.

Allylbenzene Derivatives↗

Expeditious synthesis of bioactive allylphenol constituents of the genus Piper through a metal-free photoallylation procedure.

Nine bioactive allylphenol (anisole) derivatives (e.g. eugenol, safrole and asaricin) present in several plants of the genus Piper have been synthesized in medium to high yield via aryl cation intermediates. This expeditious metal-free procedure involves the irradiation of the corresponding chlorophenols or chloroanisoles in a polar solvent (MeCN or, better, TFE or aqueous acetonitrile) in the presence of allyltrimethylsilane. Estragole has also been synthesized starting from the corresponding fluoroderivative and diazonium salt, though in a lower yield.

Metals↗

Studies on the genotoxic properties of essential oils with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay.

Genotoxic properties of essential oils from Anthemis nobilis L., Artemisia dracunculus L., Salvia officinalis L., Salvia sclarea L., Satureja hortensis L., Satureja montana L., Thymus capitatus L., Thymus citriodorus Schreb., Thymus vulgaris L., Citrus bergamia Risso, were studied with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay. The essential oil of Artemisia dracunculus L. "Piemontese" turned out to be active in the rec-assay but not in the Salmonella test. DNA-damaging activity was demonstrated to be due to the estragol component of the oil. Advantages of the combined use of these two short-term microbial assays in genotoxic studies are discussed.

Animals↗

Comparative essential oil composition and antifungal effect of bitter fennel (Foeniculum vulgare ssp. piperitum) fruit oils obtained during different vegetation.

The chemical composition of the flower and unripe and ripe fruits from fennel (bitter) (Foeniculum vulgare ssp. piperitum) has been examined by gas chromatography and gas chromatography-mass spectrometry. The main identified components of the flower and unripe and ripe fruit oils were estragole (53.08%, 56.11%, and 61.08%), fenchone (13.53%, 19.18%, and 23.46%), and alpha-phellandrene (5.77%, 3.30%, and 0.72%), respectively. Minor qualitative and major quantitative variations for some compounds of essential oils were determined with respect to the different parts of F. vulgare. The oils exerted varying levels of antifungal effects on the experimental mycelial growth of Alternaria alternata, Fusarium oxysporum, and Rhizoctonia solani. The 40 ppm concentrations of fennel oils showed inhibitory effect against mycelial growth of A. alternaria, whereas 10 ppm levels were ineffective. The analyses show that fennel oils exhibited different degrees of fungistatic activity depending on the doses.

Allylbenzene Derivatives↗