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Genotoxic effects of eugenol, isoeugenol and safrole in the wing spot test of Drosophila melanogaster
Abstract
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.
... On the basis of these considerations, the aim of this study was to evaluate peripheral sensitivity of adult females of the spotted-wing D. suzukii toward volatile compounds, which in other invertebrates and populations of D. suzukii have shown a repellent action (Del Fabbro & Nazzi, 2013;Kamsuk et al., 2007;Munerato, Sinigaglia, Reguly, & Rodrigues de Andrade, 2005;Park, Jang, Yoon, & Kim, 2016;Renkema et al., 2017;Renkema, Wright, Buitenhuis, & Hallett, 2016;Sobhy et al., 2017;Zeringota et al., 2013), by means of an electrophysiological and behavioral approach. In this respect, we evaluated the antennal olfactory sensitivity toward increasing concentrations of repellent compounds (both alone and as binary mixtures) and the subsequent ovipositing behavior evoked in adult females of D. suzukii. ...
... These compounds are naturally occurring and showed biological activity in different studies aimed at developing control strategies of different pests. In fact, eugenol exhibited repellent action on larvae and adult ticks (Del Fabbro & Nazzi, 2013;Zeringota et al., 2013), and genotoxic effect on D. melanogaster (Munerato et al., 2005); vanillin increased the repellence times of DEET against mosquitoes (Kamsuk et al., 2007) and evoked the strongest antennal responses in adults of Philaenus spumarius (Germinara et al., 2017); menthol displayed moderate repellent action on D. suzukii populations in Canada (Renkema et al., 2016) and Republic of Korea (Park et al., 2016); finally, plants treated with cis-jasmone were more repellent to aphid Macrosiphum euphorbiae (Sobhy et al., 2017). ...
... Chemoreception plays a key role by regulating essential behaviors such as localization and discrimination of host plants suitable by females for laying eggs and by offspring as food source (Sollai, Biolchini, & Crnjar, 2018b). The goal of this study was to evaluate the olfactory sensitivity of D. suzukii females toward naturally occurring compounds, which showed deterrent or synergistic effects both in other species of insects and in different populations of D. suzukii (Del Fabbro & Nazzi, 2013;Kamsuk et al., 2007;Munerato et al., 2005;Park et al., 2016;Renkema et al., 2016;Renkema et al., 2017;Sobhy et al., 2017;Zeringota et al., 2013). First, we aimed at studying whether a relationship could be found between olfactory sensitivity and behavioral response to tested compounds. ...
Drosophila suzukii Matsumura (Diptera: Drosophilidae) is an invasive, destructive crop pest that originated in South East Asia. D. suzukii recently invaded Western countries and is threatening both European and American fruit industries. It is extremely attracted to otherwise undamaged, ripening fruits, unlike most other Drosophila species that attack only decaying or rotten fruits. Recent studies on different insect species showed that several naturally occurring compounds of easy market availability showing deterrent action may be used to supplement mass catches with food traps. Based on these considerations, the aim of the present work was to test the effects of some natural compounds (alone or in the mixture) on the olfactory system of the D. suzukii and the behavioral responses evoked. We measured by electroantennogram (EAG) recordings, the olfactory sensitivity of antennae to increasing concentrations of eugenol, vanillin, menthol, cis‐jasmone; eugenol + vanillin, +menthol, +cis‐jasmone; vanillin + menthol, +cis‐jasmone. In addition, the behavioral responses to the same compounds and mixtures were evaluated. Our electrophysiological results show a dose‐response relationship between the EAG amplitudes and the increasing concentrations of the olfactory compound. The behavioral results show that the number of laid eggs is significantly different between the standard diet and the standard diet + natural compound. These results underline a specificity in the olfactory sensitivity and in the ovipositing behavior of D. suzukii females; also, they could be valuable for the identification of key chemicals aimed at the future development of strategies in the management and control of this harmful insect for crops.
Highlights
• The olfactory system of Drosophila suzukii is responsive to various naturally occurring volatiles in a dose‐response manner. Some of these compounds affect the choice of the oviposition substrate by D. suzukii females.
... Together with zinc oxide, referred to as zinc oxide-eugenol, it is also greatly used in temporary dental fillings 2 . Despite the beneficial effect of this material, the possibility of genotoxic effects cannot be ignored since genotoxicity is one of the side effects of chemical products 3,4 . ...
... In the last century, dentistry has acquired great technological advances that sought promptness for the professionals of this area and comfort for the patient. Nevertheless, takinga simple restoration as an example, it is necessary to utilize various chemical elements that, depending on the concentration used, can affect human health and are also capable of causing injury to genetic material 3 . ...
... A few well-characterized compounds include (i) 1–2 unsaturated pyrrolizidine alkaloids esters from many Boraginaceae, Asteraceae and Fabaceae (Prakash et al., 1999b; Fang et al., 2011) that exhibit a large variety of genotoxicities, including DNA binding, DNA cross-linking, DNA–protein cross-linking, sister chromatid exchange, and chromosomal aberrations (Roeder, 2000; Fu et al., 2001 Fu et al., , 2002a Fu et al., , 2004); (ii) aristolochic acids (AA), nitro-polyaromatic compounds responsible for terminal nephropathies observed after intoxication by many Aristolochia species (Fang et al., 2011); a series of studies confirmed that they are genotoxic in both bacterial and mammalian cells, yielding highly persistent and non-repaired DNA adducts; (iii) allylalkoxybenzenes (e.g. eugenol, methyleugenol, estragole), safrole (4-allyl-1,2-methylenedioxybenzene) and asarone , potentially genotoxic components from some essential oils (Liu et al., 2004; Munerato et al., 2005; Zhang et al., 2005; Smith et al., 2010). The notion of threshold for genotoxic insult is still a matter of heavy debates; consequently such compounds should be proscribed from herbal medicines or at least severely limited. ...
... In view of minimizing the number of higher organisms used in toxicological research, a somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster ( " wing-spot test " ) has been developed. This test, based on the loss of heterozygosity for two recessive markers (Idaomar et al., 2002; Carmona et al., 2011b), is a tool to evaluate gene mutations, chromosome aberrations and rearrangements related to mitotic recombination (Munerato et al., 2005). Recently, the comet assay has been adapted to be used in vivo in Drosophila melanogaster (Carmona et al., 2011a,b; Sharma et al., 2011). ...
... A few well-characterized compounds include (i) 1–2 unsaturated pyrrolizidine alkaloids esters from many Boraginaceae, Asteraceae and Fabaceae (Prakash et al., 1999b; Fang et al., 2011) that exhibit a large variety of genotoxicities, including DNA binding, DNA cross-linking, DNA–protein cross-linking, sister chromatid exchange, and chromosomal aberrations (Roeder, 2000; Fu et al., 2001 Fu et al., , 2002a Fu et al., , 2004); (ii) aristolochic acids (AA), nitro-polyaromatic compounds responsible for terminal nephropathies observed after intoxication by many Aristolochia species (Fang et al., 2011); a series of studies confirmed that they are genotoxic in both bacterial and mammalian cells, yielding highly persistent and non-repaired DNA adducts; (iii) allylalkoxybenzenes (e.g. eugenol, methyleugenol, estragole), safrole (4-allyl-1,2-methylenedioxybenzene) and asarone , potentially genotoxic components from some essential oils (Liu et al., 2004; Munerato et al., 2005; Zhang et al., 2005; Smith et al., 2010). The notion of threshold for genotoxic insult is still a matter of heavy debates; consequently such compounds should be proscribed from herbal medicines or at least severely limited. ...
... In view of minimizing the number of higher organisms used in toxicological research, a somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster ( " wing-spot test " ) has been developed. This test, based on the loss of heterozygosity for two recessive markers (Idaomar et al., 2002; Carmona et al., 2011b), is a tool to evaluate gene mutations, chromosome aberrations and rearrangements related to mitotic recombination (Munerato et al., 2005). Recently, the comet assay has been adapted to be used in vivo in Drosophila melanogaster (Carmona et al., 2011a,b; Sharma et al., 2011). ...
The increasing use of traditional herbal medicines around the world requires more scientific evidence for their putative harmlessness. To this end, a plethora of methods exist, more or less satisfying. In this post-genome era, recent reviews are however scarce, not only on the use of new "omics" methods (transcriptomics, proteomics, metabonomics) for genotoxicity, teratogenicity, and nephrotoxicity assessment, but also on conventional ones.
The present work aims (i) to review conventional methods used to assess genotoxicity, teratogenicity and nephrotoxicity of medicinal plants and mushrooms; (ii) to report recent progress in the use of "omics" technologies in this field; (iii) to underline advantages and limitations of promising methods; and lastly (iv) to suggest ways whereby the genotoxicity, teratogenicity, and nephrotoxicity assessment of traditional herbal medicines could be more predictive.
Literature and safety reports show that structural alerts, in silico and classical in vitro and in vivo predictive methods are often used. The current trend to develop "omics" technologies to assess genotoxicity, teratogenicity and nephrotoxicity is promising but most often relies on methods that are still not standardized and validated.
Hence, it is critical that toxicologists in industry, regulatory agencies and academic institutions develop a consensus, based on rigorous methods, about the reliability and interpretation of endpoints. It will also be important to regulate the integration of conventional methods for toxicity assessments with new "omics" technologies.
... Another in vitro study revealed no induction of unscheduled DNA synthesis in isolated hepatocytes from male Fischer 344 rats and female B6C3F1 mice [37]. An in vivo mutagenicity and recombination study in Drosophila melanogaster (wing spot somatic and recombination test; SMART) was also negative [38]. Finally, an in vivo study using isoeugenol-treated turkey embryos revealed no DNA strand breaks (COMET assay) or DNA adduct formation in the fetal liver of the turkey embryos [39]. ...
Isoeugenol has recently been evaluated as possibly carcinogenic (Group 2B) by the WHO International Agency for Research on Cancer (IARC). In light of this evaluation, an updated risk assessment of this common food constituent was conducted using the benchmark dose (BMD) approach as recommended by the European Food Safety Authority (EFSA) for point of departure (POD) determination, as an alternative to the no observed adverse effect level (NOAEL). This approach was specifically chosen, as for the relevant neoplastic endpoints only lowest observed adverse effect level (LOAEL) values are available. The toxicological endpoint from the animal studies with the most conservative BMD lower confidence limit (BMDL) value was identified. Using the obtained BMDL value of 8 mg/kg body weight/day as POD, an acceptable daily intake (ADI) of 16 µg/kg body weight/day was obtained, which—despite being more conservative than previous approaches—is still clearly above the estimated daily exposure level to isoeugenol in the USA and in Europe. These results confirm a low risk of the estimated daily exposure levels of isoeugenol.
... Safrole and thiophene are responsible for the unpleasant garlic odour perception. Another possible problem with the irradiation treatment is that animal [42,43] in vitro and in vivo studies [44,45] have confirmed that safrole is carcinogenic. Huo et al. [46] also reported unfavourable alterations in the cheese odour when samples are irradiated at doses of 1.51 kGy and 2 kGy. ...
The objective of this study was to evaluate the efficacy of gamma irradiation, applied to different cheese sample sizes (250g and 500 g), against Listeria monocytogenes, Escherichia coli, coliforms and aerobic colony counts. The effects on cheese physicochemical and odour properties and all costs involved for the treatment were quantified. The Cobalt-60 γ-irradiator was used at a maximum dose of 5.0 kGy. The values for cheese moisture (28.6%), ash (3.78%), pH (5.1), protein (29.6%), fat (30.7%), salt (1.95%) and water activity (0.92%) were within the acceptable ranges for hard cheese after gamma irradiation treatment. The colour (yellowness, redness, chroma and hue angle) and texture (cohesiveness and springiness) values decreased (p < 0.05) with the treatment. Compounds such as safrole, acetylpyrazine, thiophene, 3,5-octadien-2-one and 1-Octen-3-one were present after the treatment, regardless of sample size. The gamma irradiation treatment resulted in 100%, 87.2%, 85.1% and 77.3% reduction in L. monocytogenes, coliforms, E. coli and aerobic colony counts, respectively. The study highlighted the efficacy of irradiation treatment and its affordability for resource-limited producers.
... Our results were compatible with this opinion, it can be classified as a weak sensitizer according to our results. Eugenol was also found not to be genotoxic (Munerato et al. 2005), which is compatible with our study. The CYP inhibition potency that we found in our study is important from drug-herbal drug or drug-food interactions since it can interact with CYP3A4, CYP2C9, CYP2C19, which have a major role in drug metabolism. ...
Human data on remains sparse and of varying quality and reproducibility. Ex vivo experiments and animal experiments currently is the most preferred way to predict the skin sensitization approved by the regulatory agencies across the world. However, there is a constant need and demand to reduce animal experiments and provide the scope of alternative methods to animal testing. In this study, we have compared the predictive performance of the published computational tools such as ProTox-II, SuperCYPsPred with the data obtained from ex-vivo experiments. From the results of the retrospective analysis, it can be observed that the computational predictions are in agreement with the experimental results. The computational models used here are generative models based on molecular structures and machine learning algorithms and can be applied also for the prediction of skin sensitization. Besides prediction of the toxicity endpoints, the models can also provide deeper insights into the molecular mechanisms and adverse outcome pathways (AOPs) associated with the chemicals used in cosmetic products.
... Observations also showed that eugenol and safrole are able to produce a positive recombinogenic response which is related to a high CYP P450 activation capacity. The genotoxicity of eugenol is related to reactive metabolites and recombinogenic compounds of it [14]. ...
Spontaneous remission of motor neuron disease. A proposed relation to clove oil: a case report.
... Moreover, it was demonstrated that this carcinogenic effect was-at least in parts-mediated via active metabolites, such as 1 -hydroxysafrole or, rather, 1 -sulfoxysafrole [80,98,[101][102][103]. The mutagenic effect of safrole and its metabolites was also verified in vitro and in vivo [7,104]. The toxicological relevance of the genotoxic 1 -sulfoxysafrole is underlined by the finding that co-administration of the SULT-inhibitor PCP drastically reduced the carcinogenic activity of safrole in rodents [80]. ...
Alkenylbenzenes are naturally occurring secondary plant metabolites, primarily present in different herbs and spices, such as basil or fennel seeds. Thus, alkenylbenzenes, such as safrole, methyleugenol, and estragole, can be found in different foods, whenever these herbs and spices (or extracts thereof) are used for food production. In particular, essential oils or other food products derived from the aforementioned herbs and spices, such as basil-containing pesto or plant food supplements, are often characterized by a high content of alkenylbenzenes. While safrole or methyleugenol are known to be genotoxic and carcinogenic, the toxicological relevance of other alkenylbenzenes (e.g., apiol) regarding human health remains widely unclear. In this review, we will briefly summarize and discuss the current knowledge and the uncertainties impeding a conclusive evaluation of adverse effects to human health possibly resulting from consumption of foods containing alkenylbenzenes, especially focusing on the genotoxic compounds, safrole, methyleugenol, and estragole.
... The SMART assay is, therefore, suitable for testing the genotoxic potential of single or complex compounds, food, beverage, herbal extracts, and even polluted water. The SMART assay makes use of three Drosophila strains, which are (i) multiple wing hairs (mwh/mwh); (ii) flare (flr 3 /In(3LR) TM3, ri p p sep l(3)89Aa bx 34e e Bd S ); and (iii) Oregonflare (ORR/ORR;flr 3 /In(3LR) TM3, ri p p sep l(3)89Aa bx 34e e Bd S ) [13,15]. More information on the genetic symbols and descriptions was defined by Lindsley and Zimm [16]. ...
Genomic instability, one of cancer’s hallmarks, is induced by genotoxins from endogenous and exogenous sources, including reactive oxygen species (ROS), diet, and environmental pollutants. A sensitive in vivo genotoxicity test is required for the identification of human hazards to reduce the potential health risk. The somatic mutation and recombination test (SMART) or wing spot test is a genotoxicity assay involving Drosophila melanogaster (fruit fly) as a classical, alternative human model. This review describes the principle of the SMART assay in conjunction with its advantages and disadvantages and discusses applications of the assay covering all segments of health-related industries, including food, dietary supplements, drug industries, pesticides, and herbicides, as well as nanoparticles. Chemopreventive strategies are outlined as a global health trend for the anti-genotoxicity of interesting herbal extract compounds determined by SMART assay. The successful application of Drosophila for high-throughput screening of mutagens is also discussed as a future perspective.
... Eugenol is considered not acutely toxic and has an LD 50 value over 2000 mg/kg bw for rats, and between 1500 and 3000 mg/kg bw for mice, while chronic studies establish a NOAEL value of 300 mg/kg bw/day (ECHA 2021). However, acute in vivo studies found that eugenol causes respiratory distress with hemorrhagic pulmonary edema after injection in rats (Wright et al. 1995), kidney damage, apoptosis, and morphological alteration in renal cells of exposed frogs at esthetic doses (Goulet et al. 2011), and genotoxic effects on Drosophila melanogaster (Munerato et al. 2005). ...
Phenolic compounds carvacrol, thymol, eugenol and vanillin are four of the most thoroughly investigated essential oil components given their relevant biological properties. These compounds are generally considered safe for consumption and have been used in a wide range of food and non-food applications. Significant biological properties, including antimicrobial, antioxidant, analgesic, anti-inflammatory, anti-mutagenic or anti-carcinogenic activity, have been described for these components. They are versatile molecules with wide-ranging potential applications whose use may substantially increase in forthcoming years. However, some in vitro and in vivo studies, and several case reports, have indicated that carvacrol, thymol and eugenol may have potential toxicological effects. Oxidative stress has been described as a main mechanism underlying their cytotoxic behavior, and mutagenic and genotoxic effects have been occasionally observed. In vivo studies show adverse effects after acute and prolonged carvacrol and thymol exposure in mice, rats and rabbits, and eugenol has caused pulmonary and renal damage in exposed frogs. In humans, exposure to these three compounds may cause different adverse reactions, including skin irritation, inflammation, ulcer formation, dermatitis or slow healing. Toxicological vanillin effects have been less reported, although reduced cell viability after exposure to high concentrations has been described. In this context, the possible risks deriving from increased exposure to these components for human health and the environment should be thoroughly revised.
... In addition, eugenol-derived monomers are attractive because they possess antioxidant, antiseptic, and antibacterial properties [16,17], which could be exploited in photopolymers for dentistry or food packaging [18]. In particular, isoeugenol has a higher antibacterial activity than eugenol and is not genotoxic [19]. However, as many other biobased building blocks, eugenol and its derivatives do not possess functional groups that react readily through photoinduced polymerization. ...
... In addition, eugenol-derived monomers are attractive because they possess antioxidant, antiseptic, and antibacterial properties [16,17], which could be exploited in photopolymers for dentistry or food packaging [18]. In particular, isoeugenol has a higher antibacterial activity than eugenol and is not genotoxic [19]. However, as many other biobased building blocks, eugenol and its derivatives do not possess functional groups that react readily through photoinduced polymerization. ...
Biobased monomers have been used to replace their petroleum counterparts in the synthesis of polymers that are aimed at different applications. However, environmentally friendly polymerization processes are also essential to guarantee greener materials. Thus, photoinduced polymerization, which is low-energy consuming and solvent-free, rises as a suitable option. In this work, eugenol-, isoeugenol-, and dihydroeugenol-derived methacrylates are employed in radical photopolymerization to produce biobased polymers. The polymerization is monitored in the absence and presence of a photoinitiator and under air or protected from air, using Real-Time Fourier Transform Infrared Spectroscopy. The polymerization rate of the methacrylate double bonds was affected by the presence and reactivity of the allyl and propenyl groups in the eugenol- and isoeugenol-derived methacrylates, respectively. These groups are involved in radical addition, degradative chain transfer, and termination reactions, yielding crosslinked polymers. The materials, in the form of films, are characterized by differential scanning calorimetry, thermogravimetric, and contact angle analyses.
... Published data has indicated that eugenol may be a potential recombinogen (Munerato et al., 2005) and it seems that even at low doses of 10 μM eugenol is able to induce DSBs in vitro. Eugenol has been shown to produce ROS which can cause oxidative damage and SSBs and induce chromosome breakage (Kligerman et al., 2010). ...
Alkenylbenzenes, such as estragole, myristicin and eugenol, are present is several flavourings, functional foods, plant food supplements (PFS) and in complementary and alternative medicines (CAM) including herbal medicines. The increase in consumption in functional foods observed worldwide requires a strict analysis of the scientific validity of their benefits and risk-benefit ratio associated with their intake. Some instances of acute toxicity have been reported associated with the use of herbal medicines and PFS, in particular because quality control is poor, and this poses a risk especially in internet marketed products. In particular, chronic exposure to low levels of these constituents may pose a hazard. However, given the variability in dietary habits, plant properties, plant misidentification or interaction with pharmaceutical drugs or nutrients, the assessment of risk due to the intake of alkenylbenzenes is difficult. We herein review the regulatory status of the most common alkenylbenzenes and their genotoxic activity and potential carcinogenic activity.
... The genotoxicity of eugenol is related to reactive metabolites and recombinogenic compounds of it. 44 The evaluation of antigenotoxicity effects of eugenol in mice with micronucleus test, suggests that the antigenotoxic effects of eugenol may be dose related. 45 In another experimental study, the effect of eugenol on tobacco-induced genotoxicity was evaluated by the Ames Salmonella/microsome test. ...
Eugenol is a volatile phenolic constituent of clove essential oil obtained from Eugenia caryophyllata buds and leaves. It is a functional ingredient of numerous products which have been used in the pharmaceutical, food and cosmetic industry in restricted concentrations. Its derivatives have been used in medicine as a local antiseptic and anesthetic. The wide range of eugenol activities includes antimicrobial, anti-inflammatory, analgesic and antioxidant. Although eugenol is considered safe as a product, due to the vast range of different applications and extensive use, there has been a great concern about its toxicity in recent years. However, studies about cytotoxicity and genotoxicity of eugenol are very limited and controversial. The pharmacological and toxicological properties of eugenol will be discussed in this review.
... At high dosages (diets containing 12,000 ppm of eugenol), it induced a significant increase in the incidence of liver tumors in female mice, whereas in males, the increase was significant only for those receiving the lower dosage (dietary level of 3,000 ppm of 1983). Cytochrome P450-catalyzed metabolism has been suggested as a possible major bioactivation pathway in vitro (Munerato et al. 2005). Other studies in mice by oral administration, skin application and intraperitoneal injection were inadequate for an evaluation of carcinogenicity, mainly due to the short duration of treatment. ...
In 2012, the World Health Organization (WHO) recorded 14 million new cases of cancer and 8.2 million cancer-related deaths. Remarkably, the WHO estimates that 30 % of cancer mortalities are due to lifestyle choices and environmental factors that can and should be avoided. In line with these recommendations, this chapter discusses the genotoxicity and carcinogenicity of herbal products. Although often perceived as innocuous by the general public, many herbs harbor phytochemicals that are either directly reactive towards DNA or likely to disturb cellular homeostasis, cell cycle, and/or genome maintenance mechanisms; this may translate into genotoxicity, carcinogenicity, or co-carcinogenicity. Genotoxicity refers to the deleterious effect of a chemical compound or a physical event on the genetic material; such genotoxic events are considered hallmarks of cancer risk. Nevertheless, much of the damage to the genetic material can be efficiently bypassed and/or repaired by the numerous genome maintenance mechanisms of the cell and may not lead to cancer. The long-term safety evaluation is probably better investigated through carcinogenicity, which denotes the capacity of a chemical substance or a mixture of chemical substances to induce cancer or increase its incidence. The major mechanisms of carcinogenicity are discussed along with biomarkers and approved regulatory guidelines. The recent development of innovative carcinogenicity testing strategies, especially based on functional genomics, are debated and evaluated for possible application to the precocious evaluation of herbal products' long-term safety. Finally, this chapter provides some examples of proven or suspected carcinogenic herbal products reported in the current literature.
... These genes are expressed differentially in response to various monoterpenes in various tissues and over various time periods. CYPs resulting from a wing somatic mutation in D. melanogaster were induced by eugenol and safrole, but were also involved in the genotoxic activation of terpenes rather than in their detoxification (Munerato et al. 2005). ...
In recent years, botanical insecticides have gradually replaced the traditional synthetic insecticides, with the properties of strong pertinence and environment friendly. Many new botanical insecticides are developed and used, meanwhile insect develop its resistance to these insecticides. Previous studies have proved that Cytochrome P450 monooxygenases (CYPs) play a critical role in insect detoxification. Genomic and proteomic approaches focused on P450 expression and regulation at the transcript and protein levels have made substantial progress. The review covers the recent years’ research of typical botanicals metabolism mediated by CYPs, the relationship between function and structural characters of P450 enzymes, as well as the expression and regulation of related P450 genes in botanicals metabolism.
... Among the most frequently employed in vivo genotoxicity assays developed in Drosophila melanogaster, the somatic mutation recombination test (SMART) can be used to detect a wide spectrum of genetic changes, such as point mutations, deletions, some types of chromosomal aberrations, mitotic recombinations and gene conversions (Idaomar et al. 2002;Munerato et al. 2005;Carmona et al. 2011). The flexibility of the test protocol, as well as the possibility of acute, chronic or combined treatments with several agents, make this test valuable in investigating various biologically active mutagens, promutagens and antimutagens (Heres-Pulido et al. 2004;Cakir & Sarikaya 2005;Costa & Nepomuceno 2006). ...
To gain more information on biological effects of plants, particularly herbs used in human medicine and diet, in vitro and in vivo methods have been developed to predict their genotoxicity and/ or antigenotoxicity in various test systems. The sex-linked recessive lethal (SLRL) and somatic mutations and recombination (SMART) tests are in vivo assays on D. melanogaster that have been used to test both mutagenic and antigenotoxic effects of extracts from numerous plant species used worldwide. The similarity of metabolic pathways between Drosophila and mammals and the ability to activate promutagens make the results of these tests widely applicable. Besides, Drosophila presents significant orthology with human genes that control cancers, which makes the assays on Drosophila reliable and informative for extrapolations onto humans.
... In the present study, the mutagenic and antimutagenic potential of the triterpene betulinic acid was evaluated by the SMART in D. melanogaster. The advantages of this test have been widely demonstrated in several studies (Santos et al., 1999;Vogel et al., 1999;Cunha et al., 2001;Tiburi et al., 2002;De Boeck et al., 2003;Munerato et al., 2005). ...
The mutagenic and antimutagenic activities of triterpene betulinic acid {3b-3-hydroxy-lup-20(29)-en-28-oic} isolated from the roots of Scoparia dulcis (Scrophulariaceae) were analyzed using the somatic mutation and recombination test (SMART) in the wings of Drosophila melanogaster. The mutagenic potential of betulinic acid was evaluated at 3 different concentrations (1.64, 3.28, and 6.57 mM). Antimutagenic activity evaluation was performed by co-treatment trials in which the flies received betulinic acid at 3 different concentrations in addition to 10 mM pro-mutagenic urethane. The results demonstrated that betulinic acid was not capable of causing DNA damage. However, the frequency of small single spots, large spots, and twin spots was significantly reduced. In the high bioactivation cross, betulinic acid was significantly active and exerted enhanced antimutagenic activity, possibly as a desmutagen.
... Según los resultados de la composición química de la fracción volátil del aceite esencial, se puede considerar al estragol (1-Metoxy-4-(2-propenil)-benceno)como el subtipo, el cual fue determinado en una proporción del 9,8%. A este compuesto, y a algunos relacionados estructuralmente, se les ha encontrado propiedades cancerígenas (Telci et al. 2006, Liu et al. 1999, Johnson et al. 2000y Miller et al. 1983) y genotóxicas, Munerato et al. (2005). Estados Unidos y Europa tienen reglamentada la restricción sobre el uso del estragol como aditivo aromatizante en alimentos (Council Directive 88/388/EEC, 1988, y Decreto Legislativo, 1992) debido a su toxicidad. ...
El perfil químico volátil del aceite esencial de hojas de albahaca morada grande, variedad Ocimum, cultivada en la meseta de Ibagué-Colombia, obtenido por hidrodestilación, se determinó mediante cromatografía de gases acoplada a masas (GC-MS) encontrando un contenido en monoterpenos del 47.89% y en sesquiterpenos del 32.39%. Se determinó como quimiotipo al cinamato de metilo con una cantidad relativa del 73.4%, (trans-cinamato de metilo 60.4% y cis-cinamato de metilo 13.0%) y como subtipo el estragol (1-Metoxy-4-(2-propenil)-benceno) determinado en una proporción del 9,8%, el cual se relaciona como una molécula cancerígena.
... With regard to eugenol, studies have demonstrated its capacity for causing mutation and recombination in Drosophila melanogaster [38] and chromosome aberrations in V79 cells [39]. Recently, Martins et al. [40] demonstrated that eugenol was also genotoxic in Chinese hamster cells (DNA repair-proficient-AA8). ...
Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.
... Nesta região, o óleo essencial de Piper hispidinervum é constituído de mais de 90% de safrol, o qual é utilizado como matériaprima para a síntese de substâncias de interesse farmacêutico, como análogos de prostaglandinas, tromboxanas e agentes antiinflamatórios e na indústria de perfumes como fixadores de aroma (Barreiro & Fraga, 1999). Todavia este possui propriedades carcinogênicas e de genotoxicidade que devem ser consideradas (Munerato et al., 2005). ...
Os objetivos desta pesquisa foram a obtenção e caracterização do óleo essencial de folhas de pimenta longa Piper hispidinervum, e avaliação de seu efeito no comportamento e/ou mortalidade da lagarta-do-cartucho do milho Spodoptera frugiperda. O óleo essencial foi obtido pela técnica "arraste a vapor d'água", utilizando-se de um aparelho de Clevenger modificado, e posteriormente submetido, à análise por CG-EM e CG. Foram realizados testes de ingestão e contato tópico em lagartas de 1º e 3º ínstar. Os resultados constataram que o óleo essencial de pimenta-longa possui atividade inseticida sobre S. frugiperda, causando redução alimentar e mortalidade, sendo o safrol (82%) seu constituinte majoritário. Verificou-se mortalidade no teste de ingestão em lagartas de 1º ínstar com CL50 = 16,2 mg/mL e para lagartas de 3º ínstar a CL50 = 9,4 mg/mL com redução alimentar CD50 = 0,72 mg/mL; e de toxicidade aguda no teste de contato tópico com DL50 = 277,91 μg/lagarta, após o intervalo de tempo de 96 horas, sendo também observados sintomas de neurotoxicidade, como o efeito knock-down.
... Some of these NGTX compounds have previously been described in literature to cause DSBs in vitro and/or in vivo (39) For instance, EA, which is generally considered a NGTX compound (38), induced in vitro DSBs at high concentrations on mouse lymphoma cells, which was performed by pulsed-field gel electrophoresis of DNA (67). Also, DZN and EuG have been reported previously as inducers of DNA strand breaks in vitro (68)(69)(70)(71). A plausible explanation has been given by Bradley et al. (72), who have referred to a close correlation of compounds that induce DNA DSBs only at high concentrations and that induce cytotoxicity, which may be underlying their possible misclassification. ...
The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery-Ames test, mouse lymphoma assay (MLA) and CA/MN test-resulted in low accuracy (55-65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62-70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell-based tool for early screening of compounds for in vivo genotoxicity.
... Eugenol and safrole produced a positive recombinagenic response which was related to a high CYP450-dependent activation capacity. It was suggested that the reactive metabolites of eugenol and recombinagenic compounds were responsible for the genotoxicity of eugenol [116]. The genotoxicity of eugenol in V79 cells using chromosomal aberrations (CAs), with and without rat liver biotransformation (S9), was investigated in vitro. ...
Eugenol is a major volatile constituent of clove essential oil obtained through hydrodistillation of mainly Eugenia caryophyllata (=Syzygium aromaticum) buds and leaves. It is a remarkably versatile molecule incorporated as a functional ingredient in numerous products and has found application in the pharmaceutical, agricultural, fragrance, flavour, cosmetic and various other industries. Its vast range of pharmacological activities has been well-researched and includes antimicrobial, anti-inflammatory, analgesic, anti-oxidant and anticancer activities, amongst others. In addition, it is widely used in agricultural applications to protect foods from micro-organisms during storage, which might have an effect on human health, and as a pesticide and fumigant. As a functional ingredient, it is included in many dental preparations and it has also been shown to enhance skin permeation of various drugs. Eugenol is considered safe as a food additive but due to the wide range of different applications, extensive use and availability of clove oil, it is pertinent to discuss the general toxicity with special reference to contact dermatitis. This review summarises the pharmacological, agricultural and other applications of eugenol with specific emphasis on mechanism of action as well as toxicity data.
... Thus, we can assume that, in eels, the detoxification of Eugenol produces more toxic metabolites, which can impact the mitochondrial muscle metabolism 21 days after recovery. There are two Eugenol metabolization pathways (Munerato et al., 2005): (i) epoxydation, which leads to the formation of 2 0 ,3 0 epoxyeugenol, a mutagen (in vitro) and carcinogen metabolite (in vivo; Delaforge et al., 1977), and (ii) hydroxylation, which generates 1 0 -hydroxyeugenol, rapidly transformed into a methide quinone (Thompson et al., 1990;Tsai et al., 1994) which is known to induce ROS production (Mizutani et al., 1991;Bolton et al., 1994). Furthermore, it was shown that methide quinone could inhibit complex II of the mitochondrial respiratory chain in isolated-mitochondria (Thompson and Perera, 1995). ...
... TEGDMA and UDMA displayed significant increases in total spots, in a non-dependent dose ⁄ effect relationship. The absence of a correlation between genotoxic effect and dose applied is a behaviour also observed for other genotoxins, acting in the SMART assay, especially when the mainly contribution for the total spots significance is related to increases in small single spots [17,18]. ...
The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.
... Maralhas et al. [30] found that eugenol induced chromosomal aberrations in V79 cells, in particular in the presence of rat-liver S9 mix, suggesting bio-transformation of eugenol to reactive metabolites. Munerato et al. [31] studied the genotoxicity of cytochrome P450-bioactivated eugenol by the use of the wingspot test in Drosophila melanogaster and suggested that the positive recombinogenic response of eugenol and safrole was related to a high CYP450-dependent activation capacity. On the other hand, Han et al. [32] studied the chemo-preventive effects of eugenol against 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells and found that eugenol inhibited CYP1A1 and CYP1B1 activities, which catalyze the bio-transformation of DMBA. ...
... Data collected on eugenol has shown that it is genotoxic in various test systems e.g. inducing CAs in eucaryotic cells (5,11) and mutations and recombinations in the Drosophila wing SMART test (12). Concern about the carcinogenicity of eugenol has risen mainly because of structural similarities with the alkenylbenzene safrole, which is classified by IARC as possibly carcinogenic to humans (29), even though carcinogenicity studies by the NTP revealed equivocal results in B6C3F1 male or female mice (5). ...
Eugenol (1-allyl-3-methoxy-4-hydroxybenzene; CAS No. 97-53-0), a compound extracted from clove oil and marjoram, is widely used as a food flavouring substance and is present in spices such as basil, cinnamon and nutmeg. It is also used in dentistry as an antiseptic and analgesic. Structural similarities with the class IIB IARC carcinogen safrole raises questions on its putative carcinogenicity. We evaluated the genotoxicity of eugenol in V79 cells using chromosomal aberrations (CAs), with and without rat liver biotransformation (S9). Eugenol induced CAs, with significant increases (3.5% aberrant cells) at 2500 microM, demonstrating cytotoxicity at higher doses. S9 increased the induction of CAs in a dose-dependent manner to 15% at 2500 microM, with a high frequency of chromatid exchanges. In particular, an increase of endoreduplicated cells was observed, from 0% at control levels to 2.3 and 5% at 2000 microM, without and with S9, respectively. Since endoreduplication has been linked to inhibition of topoisomerase II, the topoisomerase II inhibitor ICRF-193 was used as a control inducer of endoreduplication (0.1-0.5 microM), increasing the number of endoreduplicated cells from 0% (control) to 3.5% (0.5 microM). S9 did not influence endoreduplication by ICRF-193. Both eugenol and ICRF-193 were also assayed for inhibition of topoisomerase II, and both showed a dose-dependent inhibitory effect, with ICRF-193 being a more potent inhibitor. Our results confirm that eugenol is genotoxic and raises the possibility of it having topoisomerase II inhibiting activity.
... The SMART of D. melanogaster has been successfully employed in a number of studies for the detection of genotoxicity of different pesticides (Torres et al., 1992;Kaya et al., 1999Kaya et al., , 2000aKaya et al., , 2000bKaya et al., , 2004, and in comparative studies on genotoxic/antigenotoxic activity of analogous compounds (Cunha et al., 2002a, b;Osaba et al., 2002;Rahden-Staron, 2002;Tiburi et al., 2002;Lehmann et al., 2003;Munerato et al., 2005;Laohavechvanich et al., 2006). The aim of the present study was to evaluate comparatively the genotoxic potential of five analogous IMI herbicides, employing the ST and HB crosses of D. melanogaster. ...
In the present study, five analogous herbicides, namely Imazapyr (IMZR), Imazapic (IMZC), Imazethapyr (IMZT), Imazamox (IMZX) and Imazaquin (IMZQ), were evaluated for genotoxicity (mutagenic and recombinagenic activity) in the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. They are classified as imidazolinone (IMI) herbicides and their mode of action is to inhibit acetohydroxyacid synthase (AHAS), an enzyme involved in the biosynthesis of the amino acids leucine, isoleucine and valine. Two crosses were used: the standard (ST) cross and the high bioactivation (HB) cross. The latter is characterized by high levels of cytochrome P450 conferring increased sensitivity to promutagens and procarcinogens. Three-day-old larvae were exposed by chronic feeding (48 h) to four different concentrations of these herbicides (2.5, 5.0, 10.0 or 20.0 mM). For the evaluation of genotoxic effects, the frequencies of spots per individual in the treated series were compared to the concurrent negative control series (ultrapure water). Imazapyr, Imazapic and Imazethapyr gave negative results with both crosses of the wing spot test. In the ST cross, Imazamox showed positive results only for large single spots (20.0 mM IMZX) and weak positive results for total spots (10.0 and 20.0 mM IMZX), while Imazaquin showed positive results only for large single spots (5.0 and 20.0mM IMZQ) and a weak positive result for total spots (20.0 mM IMZQ). These positive results are mainly due to induced recombination and to a minor extent to mutations. In the HB cross, only Imazamox (5.0 mM IMZX) showed a weak positive result for small single spots. The positive control urethane, a promutagen, caused an increase in the number of all types of spots in both crosses. In conclusion, the results of chronic treatments performed at high doses (toxicity was observed at higher doses) shows the existence of a genotoxic risk for IMZX and IMZQ exposure under these experimental conditions, and indicate the need for further research to delineate the exact mechanisms involved.
To modernize genotoxicity assessment and reduce reliance on experimental animals, new approach methodologies (NAMs) that provide human-relevant dose–response data are needed. Two transcriptomic biomarkers, GENOMARK and TGx-DDI, have shown a high classification accuracy for genotoxicity. As these biomarkers were extracted from different training sets, we investigated whether combining the two biomarkers in a human-derived metabolically competent cell line (i.e., HepaRG) provides complementary information for the classification of genotoxic hazard identification and potency ranking. First, the applicability of GENOMARK to TempO-Seq, a high-throughput transcriptomic technology, was evaluated. HepaRG cells were exposed for 72 h to increasing concentrations of 10 chemicals (i.e., eight known in vivo genotoxicants and two in vivo nongenotoxicants). Gene expression data were generated using the TempO-Seq technology. We found a prediction performance of 100%, confirming the applicability of GENOMARK to TempO-Seq. Classification using TGx-DDI was then compared to GENOMARK. For the chemicals identified as genotoxic, benchmark concentration modeling was conducted to perform potency ranking. The high concordance observed for both hazard classification and potency ranking by GENOMARK and TGx-DDI highlights the value of integrating these NAMs in a weight of evidence evaluation of genotoxicity.
Current environmental concerns and environmental regulations have led to the necessity to synthesize monomers and polymers from renewable resources through environmentally friendly processes. In this work, photoinduced polymerization and aqueous emulsion polymerization were selected as polymerization techniques. Natural phenols have not been widely researched and employed in the synthesis of monomers to be polymerized through the aforementioned polymerization methods. Thus, eugenol, isoeugenol and dihydroeugenol, natural phenols coming from clove oil and lignin depolymerization, were chosen as building blocks. The synthesis of eight novel monomers derived from eugenol bearing polymerizable functional groups such as (meth)acrylate, epoxy and carbonate was achieved. Successful radical polymerization in solution was achieved with the (meth)acrylated eugenol-derivatives. The polymerization rate was affected by secondary reactionsinvolving the allylic and propenyl groups in the eugenol and isoeugenol derivatives (degradative chain transfer and crosspropagation). However, most of the allylic and propenyl groups were preserved for post-polymerization reactions. Photoinduced polymerization was executed with the methacrylate eugenol-derived monomers and monitored in the absence and presence of a photoinitiator and under air or protected from air, using Real-Time Fourier Transform Infrared Spectroscopy. The polymerization rate was again affected by the presence and reactivity of the allyl and propenyl groups in the eugenol- and isoeugenol-derived methacrylates, respectively. These groups are involved in radical addition, degradative chain transfer, and termination reactions, yielding crosslinked polymers. Without photoinitiator and in the presence of air, the formation of peroxides for eugenol and isoeugenol derivatives led to a second polymerization regime. The materials, in the form of films, were characterized by differential scanning calorimetry, thermogravimetric analysis, and contact angle. Eugenol-derived methacrylates were then homopolymerized through aqueous emulsion polymerization using three different initiation systems. Stable latexes of poly(ethoxy dihydroeugenyl methacrylate), poly(ethoxy eugenyl methacrylate) and poly(ethoxy isoeugenyl methacrylate) were successfully obtained. Glass transition temperatures of the resulting polymers ranged between 20 and 72°C. Subsequently, eugenol-derived methacrylates were copolymerized by emulsion polymerization to produce latexes for adhesive applications. Latexes containing ethoxy dihydroeugenyl methacrylate and ethoxy eugenyl methacrylate with high total solids content of 50 wt % were obtained and characterized. Latexes synthesis was carried out using a semibatch process, and latexes with particle diameters in the 159−178 nm range were successfully obtained. Glass transition temperature values of the resulting polymers ranged between −32 and −28 °C. Furthermore, tack and peel measurements confirmed the possibility to use these latexes in adhesive application.
The presence of flavors is one of the commonly cited reasons for use of e-cigarettes by youth; however, the potential harms from inhaling these chemicals and byproducts have not been extensively studied. One mechanism of interest is DNA adduct formation, which may lead to carcinogenesis. We identified two chemical classes of flavors found in tobacco products and byproducts, alkenylbenzenes and aldehydes, documented to form DNA adducts. Using in silico toxicology approaches, we identified structural analogs to these chemicals without DNA adduct information. We conducted a structural similarity analysis and also generated in silico model predictions of these chemicals for genotoxicity, mutagenicity, carcinogenicity, and skin sensitization. The empirical and in silico data were compared, and we identified strengths and limitations of these models. Good concordance (80–100%) was observed between DNA adduct formation and models predicting mammalian mutagenicity (mouse lymphoma sassy L5178Y) and skin sensitization for both chemical classes. On the other hand, different prediction profiles were observed for the two chemical classes for the modeled endpoints, unscheduled DNA synthesis and bacterial mutagenicity. These results are likely due to the different mode of action between the two chemical classes, as aldehydes are direct acting agents, while alkenylbenzenes require bioactivation to form electrophilic intermediates, which form DNA adducts. The results of this study suggest that an in silico prediction for the mouse lymphoma assay L5178Y, may serve as a surrogate endpoint to help predict DNA adduct formation for chemicals found in tobacco products such as flavors and byproducts.
Safrole, a phenylpropene with a ‘candy shop’ aroma, is abundant in nature among
diverse plant genera such as Sassafras, Ocotea, Cinnamomum, Myristica, and Piper.
Sassafras oil has been used extensively for a long time, first by Native Americans and
later by European settlers in traditional medicine and as a flavouring agent. Until 1960
the consumption of safrole by the western population, as a flavouring agent in beer,
meat, and soft drinks, was unregulated. Later, the recognition of this phytochemical
as a weak hepatocarcinogen with demonstrated genotoxicity in rodents led to strict
restrictions on its use in food by various regulatory bodies globally. Moreover, in
Asian countries oral carcinogenesis has been linked to safrole through the habit of
chewing betel quid. As a separate issue, safrole is an inexpensive synthetic precursor
to the illicit recreational drug Ecstasy. Accelerating demand for this party drug during
the last few decades has encouraged the unscientific harvesting, illegal production,
and trading of safrole‐rich oils, leading to massive deforestation. Recently, many government
authorities have enforced laws to restrict the production and harvesting
of safrole‐bearing plants. Thus, the identity of safrole has altered with time from a
pleasant flavouring agent to a hepatocarcinogen, and more recently as a driver of the
destruction of biodiversity. Law enforcement has not only hampered its availability
but has also extensively affected industrial use. Our review describes the research
progress (1960–2018) on its natural distribution, carcinogenicity, usage as a natural
synthon, and the regulations imposed on safrole and safrole‐rich oils worldwide.
Finally, we draw our readers’ attention to the sustainable use of this phytochemical
for a better future.
Safrole, a phenylpropene with a ‘candy shop’ aroma, is abundant in nature among diverse plant genera such as Sassafras, Ocotea, Cinnamomum, Myristica, and Piper. Sassafras oil has been used extensively for a long time, first by Native Americans and later by European settlers in traditional medicine and as a flavouring agent. Until 1960 the consumption of safrole by the western population, as a flavouring agent in beer, meat, and soft drinks, was unregulated. Later, the recognition of this phytochemical as a weak hepatocarcinogen with demonstrated genotoxicity in rodents led to strict restrictions on its use in food by various regulatory bodies globally. Moreover, in Asian countries oral carcinogenesis has been linked to safrole through the habit of chewing betel quid. As a separate issue, safrole is an inexpensive synthetic precursor to the illicit recreational drug Ecstasy. Accelerating demand for this party drug during the last few decades has encouraged the unscientific harvesting, illegal production, and trading of safrole‐rich oils, leading to massive deforestation. Recently, many government authorities have enforced laws to restrict the production and harvesting of safrole‐bearing plants. Thus, the identity of safrole has altered with time from a pleasant flavouring agent to a hepatocarcinogen, and more recently as a driver of the destruction of biodiversity. Law enforcement has not only hampered its availability but has also extensively affected industrial use. Our review describes the research progress (1960–2018) on its natural distribution, carcinogenicity, usage as a natural synthon, and the regulations imposed on safrole and safrole‐rich oils worldwide. Finally, we draw our readers’ attention to the sustainable use of this phytochemical for a better future. Review on safrole: a retrospection.
Es wurde eine neue Route zur Synthese funktioneller wässriger Nanogele mit kontrollierter Isoeugenol-Oberflächenbeladung entwickelt. Die so erhaltenen Nanogele haben zwei Schlüsselfunktionen: a) antibakterielle Aktivität gegen diverse orale Krankheitserreger sowie b) zelladhäsive und zellwachstumsfördernde Eigenschaften. Diese funktionellen Nanogele sind potenzielle Bausteine für bioaktive Beschichtungen auf verschiedenen Implantaten, die zur Verhinderung von Infektionen und zur Förderung von Zellwachstum genutzt werden können.
A new route for the synthesis of functional aqueous nanogels decorated with a controlled amount of surface-drafted isoeugenol molecules has been developed. Obtained nanogels exhibit two key functions: a) antibacterial activity against different oral pathogens and b) cell-adhesive and -growth-promoting properties. Functional nanogels can be potentially used as building blocks in the design of bioactive coatings on various implants preventing infections and accelerating tissue regeneration.
Anaesthetic properties and potential genotoxicity of six essential oils extracted from the medicinal plants Origanum vulgare, Eugenia aromatica, Aloysia triphylla, Melaleuca alternifolia, Juniperus communis, Cinnamomum zeylanicum, are presently assessed in gilthead seabream. Essential oils are an alternative option for fish anaesthesia, which is necessary in order to ensure welfare under intensively reared conditions. Their anaesthetic efficacy was assessed in comparison with chemical agents, by monitoring fish behavior throughout the anaesthesia and recovery stages, while the post-treatment mortality rate was also recorded. Genotoxicity was evaluated using the single cell gel comet assay in vitro and in vivo. DNA migration (tail moment) and the percentage of cells with increased level of DNA damage in hepatocytes were assessed. All essential oils proved to qualify as anaesthetic agents achieving deep narcosis and behavioural recovery. Essential oils indicated higher efficiency compared to chemicals, mitigating the side-effects that are often associated with synthetic substances. E. aromatic, O. vulgare and A. triphylla induced genotoxic effects at applied doses. However, as genotoxicity is dose dependent and most essential oils usually devoid of long-term genotoxic risk, lower anaesthetic doses have to be tested in order to be proposed for commercial use in aquaculture species. M. alternifolia and J. communis did not induced genotoxicity, maintaining DNA damage at control group level. Finally, C. zeylanicum was evaluated as equal competent anaesthetic agent satisfying the criteria of the ideal anaesthetic, reducing DNA strand breakage and indicating anti-stress, anti-genotoxic and geno-protective effect.
Antioxidant capacity of nutmeg (Myristica fragrans Houttuyn) oil was investigated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical scavenging assay and the β-carotene-linoleic acid assay. The antioxidant EC50 values of the crude nutmeg oil dissolved in methanol were 2.4 μL/mL and 0.4 μL/mL, respectively. The former value was approximately equivalent to the free radical scavenging capacities of 462 μM BHT and 656 μM α-tocopherol, and the latter one was comparable to the inhibitive capacities of 43 μM BHT and 9 μM α-tocopherol against the oxidation of β-carotene and linoleic acid. Further investigations discovered three major antioxidant constituents of the nutmeg oil (i.e., eugenol, isoeugenol, and methoxyeugenol), which were sequentially separated and identified by silica open column chromatography, HPLC and GC-MS. Their antioxidant activities in the DPPH• assay decreased in the following order: eugenol > methoxyeugenol > BHT > isoeugenol > α–tocopherol, while in the β-carotene-linoleic acid assay, the antioxidant activities of the chemicals were in the following order: α–tocopherol > BHT > isoeugenol > methoxyeugenol > eugenol.
The aim of this study was to analyze the eugenol content of some eugenol based commercially available dental cements. Eugenol analysis was made by gas chromatography equipped with flame ionization detector (GC-FID) and also results confirmed by gas chromatography-mass spectrometry (GC-MS). In all products, eugenol was found in different concentrations. It is noteworthy that there were variations in the concentration of eugenol even in products manufactured by the same company. Due to the fact that anaesthetic, sedative and analgesic effects are related to the eugenol concentration. It is critical to have eugenol content standardized.
Isomerization of allylbenzenes have been used as starting materials in many multistep syntheses. Many naturally occurring 1-propenylbenzenes have therefore been directly isolated from essential oils in which they occur and subsequently synthetically transformed to afford a wide range of diversified products, most of which display interesting bioactivities. It should be noted that 1-propenylbenzenes have also been very popular substrates for the development of new synthetic methods, requiring, in particular, the synthesis of purecis and trans isomers of these compounds.
The second edition of this book is virtually a new book. It is the only comprehensive text on the safety of essential oils, the first review of essential oil/drug interactions, and it provides detailed essential oil constituent data not found in any other text. Much of the existing text has been re-written, and 80% of the text is completely new. There are 400 comprehensive essential oil profiles and almost 4000 references. There are new chapters on the respiratory system, the cardiovascular system, the urinary system, the digestive system and the nervous system. For each essential oil there is a full breakdown of constituents, and a clear categorization of hazards and risks, with recommended maximum doses and concentrations. There are also 206 Constituent Profiles. There is considerable discussion of carcinogens, the human relevance of some of the animal data, the validity of treating an essential oil as if it was a single chemical, and the arbitrary nature of uncertainty factors. There is a critique of current regulations.
Several medicinal plants are known to act as mutagenic, cocarcinogenic, and/or carcinogenic agents. Prevention of cancer and other related diseases can be pursued by avoiding exposure to these agents. This chapter reports scientific progress in detecting mutagenic and carcinogenic plants used in African traditional medicine. Over 400 African medicinal plants were screened mainly in South Africa, Nigeria, and Tunisia, for their mutagenicity and carcinogenicity for the period of 2003–2013 by in vitro and in vivo methods. The results showed that at least 64 plant species used in African traditional medicine are potentially mutagenic/carcinogenic based on their ability to induce genetic changes. Although plants used medicinally are widely assumed to be safe, the issue of quality control may, in the interim, be addressed. This chapter aims: (i) to report African medicinal plants with mutagenic/carcinogenic effects and (ii) to highlight the advanced in the methods used to detect these mutagenic/carcinogenic plants. Most of the extracts reported in this chapter were prepared from plant species with recognized toxicity, but widely used in African traditional medicine. Such plant extracts should consequently be examined carefully for their safety perspective and caution to their use should be taken.
Keywords: African medicinal plants; genetic bioassays; mutagenicity; carcinogenicity
Toxic medicinal plants can be defined as any plant that in one or more of its organs contains toxin that can induce adverse side effects in animal/humans upon consumption or administration for therapeutic purposes. The use of plants in African traditional medicine (ATM) is currently not regulated in most countries of the continent, resulting in a serious danger of misadministration of toxic plants. The potential toxic effects following prolonged use of some of the popular medicinal plants are to be scientifically investigated. Of the 120 African medicinal plants screened for their toxic effects and examined in this chapter, 49 of them were potentially toxic. Toxic symptoms mainly included neurological, hepatic, renal, gastrointestinal, and cardiovascular signs. The issues related to the use of toxic plants in ATM should now be addressed and taken as a priority in all African countries. This chapter aims: (i) to report toxic or potentially toxic plants used in ATM based on the LD50 values, physical and biochemical changes; (ii) to highlight the advanced in the methods used to detect these toxic plants; (iii) to provide baseline information in order to warn healer and patient on the side effects of some commonly used herbal drugs. Most of the plants mentioned in this chapter are used in ATM for treatment of several diseases in humans and it is essential to be aware of their toxic effects.
Keywords: African traditional medicine; medicinal plants; toxic effects; physical symptoms; LD50 values; biochemical and hematological changes
Dental materials can induce local and systemic effects. The Allium cepa assay was used to evaluate the genotoxicity and/or cytotoxicity of zinc oxide and eugenol (ZOE) at different proportions. The ZOE solution was tested at the concentration of 1 drop of eugenol (in each drop of liquid, the approximate concentration of eugenol is 85%) and 1 portion of zinc oxide cement (treatment I), and twice the concentration of eugenol (treatment II). Treated roots appeared to be yellowish-brown, fewer in number, thicker and less turgid compared with the control, suggesting a cytotoxic activity of ZOE. A significant difference was found in the root size between the control and treatment II. This treatment reduced by 79% the size of the root compared with the control, and the mitotic index was 66%, indicating a 22.4% reduction relative to the control, which in turn evidenced the cytotoxicity of ZOE. The significant increase in anaphase bridges suggests a genotoxic effect.
Introduction: Since some dental materials may be aggressive to a person’s body, studies involving such materials seem to be necessary. Objective: This study was conducted to evaluate the genotoxicity of dental materials through micronucleus (MN) test. Material and methods: Exfoliated buccal cells of 4-to-12
year-old children, who were on some type of dental treatment, were collected either before or after the treatment ending. Each sample was composed of 1,000 cells per patient. Student’s t test was used for comparison. Results: The dental materials were divided into 3 groups, as follows: cement, monomers, and
their combination. Treatments using monomer + cement-based materials were found to increase significantly the number of binucleated (BN) cells, (p < 0.05) which indicate several degenerative nuclear changes. Conclusion: The combination of cement-based dental material with monomers increases the
cytotoxic action of dental materials.
In the last decades, cases of poisoning due to herbal medicines have occurred in many countries; Chinese herbal medicines (CHMs) are occasionally involved. The experience gained from traditional use is efficient to detect immediate or near-immediate relationship between administration and toxic effects but is quite unlikely to detect medium- to long-term toxicities; thorough investigations of herbal medicines (toxicity assessments, active pharmacovigilance) appear then essential for their safe use. Genotoxicity is an especially insidious toxicity that may result in carcinoma development years after exposure; it can arise from multiple compounds, with or without metabolic activation. The present work reviews traditional CHMs and phytochemicals that have been shown to present a genotoxic hazard. Copyright © 2013 John Wiley & Sons, Ltd.
A pressurized liquid extraction and GC-MS method was developed for simultaneous quantitative determination of the seven components, including cinnamaldehyde, copaene, cinnamic acid, coumarin, 2-methoxycinnamaldehyde, 2-methoxycinnamic acid and safrole in Cinnamomum cassia. The results showed that methanol and ethanol was not available for extraction of cinnamaldehyde and 2-methoxycinnamaldehyde due to aldol reaction. The developed method was validated to be sensitive, accurate and simple, and was successfully employed for the analysis of 15 samples of C. cassia. The contents of the investigated components were significantly variant and cinnamaldehyde is the most abundant compound, but safrole was not detected in all samples.
Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr ∼52,000) was expressed in each of 40 individual specimens examined. In about 10–20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr ∼52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a λgt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with λmax at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6β-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15β-hydroxy testosterone), comprising up to ∼20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (Ml and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substratespecificity compared to hPCN1 for metabolism of steroid and drug substrates.
Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.
We have investigated the activation of eugenol to form DNA adducts and oxidative base damage. Treatment of myeloperoxidase containing HL-60 cells with eugenol, produced a dose-dependent formation of three DNA adducts as detected with P1-enhanced 32P-post-labeling. Incubation of HL-60 cells with the combination of 100 microM eugenol and 100 microM H2O2 potentiated the levels of DNA adduct in HL-60 cells by 14-fold, which suggests peroxidase activation in adduct formation. In vitro activation of eugenol with either horseradish peroxidase or myeloperoxidase and H2O2 produced three DNA adducts that were inhibited by the addition of either ascorbic acid or glutathione, by 66 and 90%, respectively. The DNA adducts formed in HL-60 cells treated with eugenol were the same as those formed by in vitro peroxidase activation. In addition to adduct formation, peroxidase activation of eugenol produced a 2- to 3-fold increase in the level of oxidative base damage. Eugenol quinone methide was prepared by Ag(I)oxide oxidation of eugenol. Peroxidase activation of eugenol gave a product that had the same UV spectrum as eugenol quinone methide, which suggests that it was one of the products. Reaction of eugenol quinone methide with either DNA or deoxyguanosine-3'-phosphate produced two principal adducts (2 and 4). When DNA adduct 2 formed by incubation of eugenol quinone methide with deoxyguanosine-3'-phosphate was compared with DNA 2 adduct formed in HL-60 cells treated with eugenol results demonstrated that they were the same. This suggests that eugenol quinone methide is one of the reactive intermediates leading to DNA adduct formation in cells. Activation of eugenol with 10 microM copper sulfate resulted in the production of one principal (2) and several minor adducts. DNA adduct 2 formed by activation of eugenol with copper sulfate was the same as DNA adduct 2 formed by either peroxidase activation of eugenol or by reactions with eugenol quinone methide, which indicates that the reactive intermediates generated by these activation systems were similar. Copper sulfate produced a 95-fold increase in the level of oxidative base damage, which was significantly inhibited by the addition of either bathocuproinedisulphonic acid or catalase. The formation of oxidative base damage was consistent with a Fenton reaction mechanism. Our results demonstrate that eugenol can be activated to form both DNA adducts and oxidative base damage. We propose that the formation of this DNA damage may contribute to the observed toxic properties of eugenol.
We have investigated the activation of eugenol to form DNA adducts and oxidative base damage. Treatment of myeloperoxidase containing HL-60 cells with eugenol, produced a dose-dependent formation of three DNA adducts as detected with P1-enhanced 32 P-post-labeling. Incubation of HL-60 cells with the combination of 100 μM eugenol and 100 μM H 2 O 2 potentiated the levels of DNA adduct in HL-60 cells by 14-fold, which suggests peroxidase activation in adduct formation. In vitro activation of eugenol with either horseradish peroxidase or myeloperoxidase and H 2 O 2 produced three DNA adducts that were inhibited by the addition of either ascorbic acid or glutathione, by 66 and 90%, respectively. The DNA adducts formed in HL-60 cells treated with eugenol were the same as those formed by in vitro peroxidase activation. In addition to adduct formation, peroxidase activation of eugenol produced a 2-to 3-fold increase in the level of oxidative base damage. Eugenol quinone methide was prepared by Ag(I)oxide oxidation of eugenol. Peroxidase activation of eugenol gave a product that had the same UV spectrum as eugenol quinone methide, which suggests that it was one of the products. Reaction of eugenol quinone methide with either DNA or deoxyguanosine-3'-phosphate produced two principal adducts (2 and 4). When DNA adduct 2 formed by incubation of eugenol quinone methide with deoxyguanosine-3'-phosphate was compared with DNA 2 adduct formed in HL-60 cells treated with eugenol results demonstrated that they were the same. This suggests that eugenol quinone methide is one of the reactive intermediates leading to DNA adduct formation in cells. Activation of eugenol with 10 μM copper sulfate resulted in the production of one principal (2) and several minor adducts. DNA adduct 2 formed by activation of eugenol with copper sulfate was the same as DNA adduct 2 formed by either peroxidase activation of eugenol or by reactions with eugenol quinone methide, which indicates that the reactive intermediates generated by these activation systems were similar. Copper sulfate produced a 95-fold increase in the level of oxidative base damage, which was significantly inhibited by the addition of either bathocuproinedisulphonic acid or catalase. The formation of oxidative base damage was consistent with a Fenton reaction mechanism. Our results demonstrate that eugenol can be activated to form both DNA adducts and oxidative base damage. We propose that the formation of this DNA damage may contribute to the observed toxic properties of eugenol.
To increase the number of chemicals tested using the zeste-white (UZ) somatic mutation assay, ten selected carcinogens (acetamide, acrylamide, benzo(α)pyrene, cyclophospha-mide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) have been evaluated in this assay. Our results show that all the compounds tested produce significant increases in the eye spot frequency at, at least, one of the concentrations assayed, indicating that the zeste-white assay appears to be highly sensitive to these carcinogenic compounds. That is in agreement with data previously reported by other authors.
To provide further background data on the wing spot somatic mutation and recombination assay, 10 selected carcinogens (acetamide, acrylamide, benzo(a)pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) were tested in this assay. 72-h-old third-instar larvae, trans-heterozygous for 2 recessive wing cell markers:multiple wing hairs (mwh) andflare
3 (flr
3) were fed with 3 concentrations of each carcinogen during the rest of their development until pupation, and the genotoxic effects were measured as significant increases in the appearance of visible mutant hair clones on the adult wing blade. Our results show that 6 of the carcinogens tested produce significant increases in wing spot frequency, at least at one of the concentrations assayed. Benzo(a)pyrene, diethylstilbestrol, safrole and thiourea were the compounds that did not increase the incidence of mutant clones.
Safrole, estragole, anethole, and eugenol and some of their known or possible metabolites were tested for mutagenic activity for S. typhimurium TA1535, TA100, and TA98. Highly purified 1'-hydroxyestragole and 1'-hydroxysafrole were mutagenic (approximately 15 and 10 revertants/micromole, respectively) for strain TA100 in the absence of fortified liver microsomes; trans-anethole and estragole appeared to have very weak activity. 3'-Hydroxyanethole was too toxic for an adequate test. Supplementation with NADPH-fortified rat-liver microsomes and cytosol converted 3'-hydroxyanethole to a mutagen(s) and increased the mutagenic activities for strain TA100 of 1'-hydroxyestragole, 1'-hydroxysafrole, estragole, and anethole. No mutagenicity was detected for safrole or eugenol with or without added NADPH-fortified liver preparations. The electrophilic 2',3'-oxides of safrole, 1'-hydroxysafrole, 1'-acetoxysafrole, 1'-oxosafrole, estragole, 1'-hydroxyestragole, and eugenol showed dose-dependent mutagenic activities for strain TA1535 in the absence of fortified liver microsomes. These mutagenic activities ranged from about 330 revertants/micromole for 1'-oxosafrole-2',3'-oxide to about 7000 revertants/micromole for safrole-2',3'-oxide. The arylalkenes, their hydroxylated derivatives, or their epoxides did not show mutagenic activity for strain TA98, except for 1'-oxosafrole-2',3'-oxide, which had weak activity. Since the arylalkenes are hydroxylated and/or epoxidized by hepatic microsomes, hydroxy and epoxide derivatives appear to be proximate and ultimate mutagenic metabolites, respectively, of the arylalkenes.
In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (.OH) scavenging ability. A Fenton system was used to produce .OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for .OH were studied. The relative yield of the spin adduct of .OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and .OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10(9) M-1 s-1 (diffusion-controlled). Also, using the TBA tests, the reactions between .OH and several compounds reactive with .OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for .OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by .OH generated by UV-light.
The anti-peroxidative activity of eugenol on Fe(2+)-ascorbate- and Fe(2+)-H2O2-induced lipid peroxidation was studied using rat liver mitochondria. Eugenol inhibited thiobarbituric acid reactive substance (TBARS) formation induced by both the systems in addition to oxygen uptake and mitochondrial swelling induced by Fe(2+)-ascorbate. Time course studies on TBARS formation indicated the ability of eugenol to inhibit initiation and propagation reactions. There was no measurable chemical modification of eugenol during the course of mitochondrial peroxidation by both the systems. Mitochondrial peroxidation by Fe(2+)-H2O2 was inhibited by hydroxyl radical (OH) scavengers like mannitol, benzoate, formate and dimethyl sulfoxide apart from eugenol. The OH scavenging ability of eugenol was evident from its inhibitory effect on OH-mediated deoxyribose degradation. The second-order rate constant for the reaction of OH with eugenol was about 4.8 x 10(10) M-1 sec-1. Eugenol reduced Fe3+ ions and Fe3+ chelated to citrate or ADP but it did not exhibit pro-oxidant activity in OH-mediated deoxyribose degradation. Incubation of mitochondria with eugenol resulted in the uptake of small but significant quantities of eugenol which inhibited subsequent lipid peroxidation by acting as a chain breaking antioxidant.
2-Chlorethanol, 8-hydroxyquinoline, 2,6-toluenediamine, and eugenol, previously found to behave as genotoxins in in vitro systems and as noncarcinogens in rodents, were evaluated for their ability to induce genotoxic effects in vivo. Rats were given by gavage a single or two successive doses equal to one-half the corresponding LD50, killed at different times after treatment, and examined for the following end points: the frequency of both micronucleated polychromatic erythrocytes in the bone marrow and micronucleated hepatocytes (after partial hepatectomy); the in vivo-in vitro induction of DNA fragmentation, as measured by the alkaline elution technique, and of unscheduled DNA synthesis, as measured by autoradiography, in hepatocyte primary cultures. The two latter end points were also evaluated after in vitro exposure of hepatocytes to log-spaced subtoxic concentrations. 2-Chloroethanol, 8-hydroxyquinoline, and eugenol never produced effects indicative of genotoxic activity. The same happened with 2,6-toluenediamine, with the exception of a significant increase over controls in the amounts of DNA damage and repair displayed by hepatocyte cultures obtained from rats given two 1/2 LD50 separated by a 24 h interval. Our results, which, apart the above mentioned exception, are in concordance with the rodent carcinogenicity results, contribute to underline the role of in vivo short-term tests for the detection of potential genotoxic carcinogens.
Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.
The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Frölich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibits a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross (ORR; flr3 females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.
The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.
The allylbenzenes estragole, methyleugenol and safrole are hepatocarcinogens in rodents at very high doses, but allylbenzene itself is neither hepatotoxic nor hepatocarcinogenic. To elucidate further the significance of metabolic 1'-hydroxylation in the carcinogenicity of the allylbenzenes and to give further insights into the structure-metabolism-genotoxicity relationships of these compounds, comparative data were established on the ability of estragole, methyleugenol, safrole, allylbenzene and their 1'-hydroxy metabolites to induce unscheduled DNA synthesis (UDS) in hepatocytes derived from male Fischer 344 rats. Cytotoxicity was assessed by lactate dehydrogenase leakage. The first three compounds increased UDS in a dose-related fashion but allylbenzene was non-genotoxic. 1'-Hydroxyestragole, -methyleugenol and -safrole were more potent genotoxins than their parent compounds. This difference in genotoxicity indicates the importance of the attachment of the electron-withdrawing methoxy or methylenedioxy substituents to the benzene ring. The non-linear dose-response curves for genotoxicity obtained with the allylbenzenes and their 1'-hydroxy metabolites indicate that it is important to consider dose-dependence in metabolism when interpreting the significance to humans of animal data obtained with very high doses of the compounds studied. It is likely that the use of these high doses markedly overestimates the potential hazard to humans of low doses of allylbenzenes, which generate only very small quantities of genotoxic metabolites.
Eugenol produces hepatic injury in mice depleted of glutathione (GSH) by pretreatment with buthionine sulfoximine (BSO). Several eugenol analogs were examined for their ability to cause hepatic injury after administration to mice in combination with BSO. Hepatotoxicity was assessed by measuring relative liver weight, liver blood volume, and serum GPT activity in mice. Comparison of the tested compounds showed that the structural requirements for toxic potency was a phenolic ring having an allyl substituent at the 4-position. These structural requirements can be explained by assuming that a vinylogous quinone methide formed by metabolic oxidation of eugenol plays a role in inducing hepatotoxicity in GSH-depleted mice.
To increase the number of chemicals tested using the zeste-white (UZ) somatic mutation assay, ten selected carcinogens (acetamide, acrylamide, benzo(alpha)pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) have been evaluated in this assay. Our results show that all the compounds tested produce significant increases in the eye spot frequency at, at least, one of the concentrations assayed, indicating that the zeste-white assay appears to be highly sensitive to these carcinogenic compounds. That is in agreement with data previously reported by other authors.
Rat hepatic and pulmonary microsomes catalyzed the formation of at least three distinct glutathione conjugates with eugenol (4-allyl-2-methoxyphenol). These three conjugates were identical with the products obtained from the chemical reaction of synthetic eugenol quinone methide and glutathione. The microsomal reaction was dependent on NADPH and oxygen and was inhibited by cytochrome P450 inhibitors such as metyrapone, 2-diethylaminoethyl-2,2'-diphenylvalerate (SKF 525-A), alpha-naphthoflavone and piperonyl butoxide. The enzyme responsible for eugenol oxidation was inducible with 3-methylcholanthrene but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by the presence of glutathione-depleted cytosol which contained active glutathione transferase, even at low glutathione concentrations, suggesting that conjugation occurs nonenzymatically with an electrophilic metabolite of eugenol. Covalent binding to microsomal protein was observed using [3H]eugenol. Cumene hydroperoxide catalyzed the formation of these same glutathione conjugates via the formation of a quinone methide-like intermediate which was detected by spectroscopic means. Our results suggest that eugenol is oxidized by cytochrome P450 to a reactive quinone methide intermediate which can then covalently modify protein or conjugate with glutathione.
Deletion of an integrated plasmid, a specific type of intrachromosomal recombination, was evaluated for inducibility with the phenylpropenes safrole, eugenol and methyleugenol in the yeast Saccharomyces cerevisiae. These phenylpropenes are found in food products, spices, pharmaceuticals and clove cigarettes. Safrole and eugenol are known carcinogens in animals and methyleugenol is a suspected carcinogen. These phenylpropenes are not detectable by the Ames assay and most other short-term tests used currently in predictive carcinogenesis. Like safrole, which has been shown to be nonmutagenic with the Ames assay, eugenol and methyleugenol were found to be nonmutagenic with the Ames assay. In contrast, with the yeast assays which screen for intra- and inter-chromosomal recombination in logarithmic phase cultures, all 3 compounds gave a positive dose-related response. These results demonstrate further that the yeast system can be modified easily to detect various genetic endpoints and that it deserves serious consideration as a test system for predictive carcinogenesis.
The DNA-damaging, mutagenic and chromosomal effects of eugenol were assayed by the DNA alkaline elution technique, the granuloma pouch assay and the bone marrow micronucleus test in rats. With all the techniques used, eugenol did not show any genotoxic activity.
Male Fisher rats were fed a diet ad lib. containing eugenol (4-allyl-2-methoxyphenol) to observe its effects on liver drug-detoxifying enzymes such as UDP-glucuronyltransferase (GT), UDP-glucose dehydrogenase (DH) and glutathione S-transferase (GST). Liver weights were not affected significantly by a diet containing 3% eugenol (w/w) for 13 weeks. The activities of GT of liver microsomes toward various xenobiotic substances such as 4-nitrophenol, 1-naphthol, 4-hydroxybiphenyl and 4-methylumbelliferone were enhanced by dietary administration of eugenol, but the activity of GT toward its endogenous substrate, bilirubin, was not changed. Dose-response relationships between the enhancement of GT activities toward these xenobiotics and the dose of eugenol were observed. The induced higher activities of GT toward these xenobiotics were maintained during 13 weeks of eugenol treatment. Similar results on DH and GST activities in the liver cytosol were obtained by dietary administration of eugenol, while no effect on cytochrome P-450 content in the liver microsomes from the rats fed the eugenol diet was observed during 13 weeks. These results suggest that the intracellular content of the active intermediates of various drugs or carcinogens would be reduced by this specific enhancement of drug-detoxifying enzymes in the liver of rats given a diet containing eugenol, as previously described for a diet containing 2(3)-tert-butyl-4-hydroxyanisole (BHA) [Y-N. Cha and H. S. Heine, Cancer Res. 42, 2609 (1982)].
Two alternative hypotheses are used to distinguish among the possibilities of a positive, inconclusive, or negative result in Drosophila mutagenicity tests. In the null hypothesis one assumes that there is no difference in the mutation frequency between control and treated series. The alternative hypothesis postulates a priori that the treatment results in an increased mutation frequency that is m times the spontaneous frequency. To test against the hypotheses, the conditional binomial test according to Kastenbaum and Bowman or the chi 2 test for proportions may be applied. These 2 methods are in principle equivalent. An alternative method which is based on determining confidence limits of observed mutation frequencies also leads to the same conclusions. The practical calculations are formulated and an application is shown with a test example demonstrating the genotoxicity of the pyrrolizidine alkaloid 7-acetylintermedine in the somatic wing mosaic test. In the Appendix, the calculus for the 3 testing methods is explained with a numerical example.
Since our earlier studies of 23 individual weakly acidic constituents of cigarette smoke indicated that benzenes having vicinal oxygenation or a conjugated double bond induce sister-chromatid exchanges (SCE), we have now selected and examined a complementary set of 27 smoke constituents for their SCE-inducing properties. Of the 50 compounds tested in all, 23 were found to induce SCE, and these include all benzaldehydes but one and the majority of the compounds having a conjugated carbon-carbon double bond as well as several of the guaiacols. These groups of active compounds comprise important flavourants such as vanillin, ethylvanillin, isoeugenol and guaiacol. The structure-activity relationships encountered here may be useful in predicting the SCE-inducing activity of related compounds.
The mutagenicities of malondialdehyde and formaldehyde were tested by screening each for genetic mosaics of Drosophila melanogaster and by the Muller-5 test for sex-linked recessive lethal mutations. For comparison, the effects of X-rays were also assayed by the above technique. Malondialdehyde, a degradation product of polyunsaturated fatty acids, was found to be a weak mutagen by the above criteria; it induced point mutations and chromosome exchanges at low frequency, as proved by the mosaic test, but failed to induce detectable sex-linked lethality. Formaldehyde was more mutagenic than malondialdehyde; beside induction of mosaic spots it induced sex-linked recessive lethal mutations, but only in the larval testes of Drosophila. Formaldehyde also induced disintegration of the clones. Formaldehyde treatment (feeding larvae with formaldehyde-containing food for about 4 days) was 5 times more mutagenic than malondialdehyde treatment and 5 times less effective than irradiation by 1000 R of X-rays. Wing mosaicism offers a more sensitive way to detect mutagenesis as compared with eye mosaicism. It is suggested that aldehyde-induced mosaic spots derive from mitotic recombination and point mutations.
A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans-heterozygous for the mutations multiple wing hairs (mwh) and flare (flr) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well-known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B1), DNA breaking agents (bleomycin), intercalating agents (5-aminoacridine, ICR-170), spindle poisons (vinblastine), and antimetabolites (methotrexate). In addition, the test detects mutagens unstable in aqueous solution (beta-propiolactone), gaseous mutagens (1,2-dibromoethane), as well as promutagens needing various pathways of metabolic activation (aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short-term test.
The genotoxicity of safrole, 9 compounds that are structurally similar to safrole (anethole, cinnamaldehyde, cinnamyl alcohol, estragole, methyl eugenol, eugenol, isoeugenol, isosafrole, piperonal), 5 essential oils, cassia oil, cinnamon bark oil, clove oil, fennel oil) which contain the chemicals tested, and 1 oleoresin was studies in 3 microbial test systems. Only anethole showed mutagenicity in the Ames Salmonella reversion assay. All chemicals except anethole, estragole and isosafrole were positive in the Bacillus subtilis DNA-repair test (Rec assay) without S9. All samples tested were negative in the Escherichia coli WP2 uvrA reversion test. The essential oils and pimenta oleoresin were positive in the DNA-repair test. The results obtained are discussed in relation to the nature of the problems encountered with each test method.
Eugenol, a widely used chemical in clinical dentistry, was evaluated for genotoxicity using the bone marrow micronucleus (Mn) assay in mice. Three doses (100, 400 or 600 mg/kg) were administered intraperitoneally (i.p.). The animals were killed 30 h post-treatment and the frequency of Mn in 1000 polychromatic erythrocytes (PCE) was determined. Corn oil and methyl methanesulfonate (MMS) were used as negative and positive controls, respectively. Only 400 and 600 mg/kg doses showed significant induction of Mn. We also studied the effect of eugenol at different recovery times (24, 30 or 48 h) with a dose of 400 mg/kg. A positive Mn response was seen at all three post-treatment times, but the differences between them were not significant. For treatment and control groups, the cytotoxicity was in the normal range, measured as the ratio of PCE/NCE (normochromatic erythrocytes).
The Saccharomyces cerevisiae DEL assay detects a wide variety of nonmutagenic carcinogens (Schiestl et al. (1989) Carcinogenesis, 10, 1445-1455). This study shows the effect on DEL recombination of 8 carcinogenic compounds (o-toluidine, hexamethylphosphoramide, safrole, acrylonitrile, benzene, diethylhexylphthalate, phenobarbital and diethylstilbestrol) and 2 noncarcinogenic compounds (caprolactam and benzoin). These chemicals have been selected by the Program on Chemical Safety for the evaluation of short-term tests for carcinogens, because sufficient carcinogenicity data for these compounds exist, and because they are difficult to detect with the Salmonella assay. 5 of 8 carcinogens reproducibly gave a strong positive response and the noncarcinogen benzoin was negative. Thus, 60% of the chemicals tested in this study have been correctly identified with the DEL assay compared to only 20% with the Salmonella assay.
The hapatotoxicity of safrole and related compounds has been attributed to the electrophilic compounds generated from the oxidation of these chemicals. In this paper, for the first time using quasi pre-steady state conditions, we present direct evidence for the rapid production of electrophilic allyl o-quinone and its tautomerization to more stable and reactive vinyl p-quinone methide during both the enzyme catalyzed and chemical mediated oxidation of 4-allylcatechol, an in vivo product of safrole metabolism.
Harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In CHO cells we have found that for a variety of chemicals one harvest time near 20 h is optimal following a 3-h treatment (Bean et al., 1992). However, some guidelines for testing for regulatory purposes recommend an additional late harvest time 24 h after the first. We tested 10 diverse chemicals in CHO-WBL cells harvested 20-21 h and 42-44 h from the beginning of a 3-hr treatment. We added BrdUrd after treatment and recorded the total% of aberrant cells, and the proportions of aberrations (abs) in first (M1), second (M2) or later metaphases. The chemicals fell into 3 categories: ab yield greatly decreased at 44 h: benzo[a]pyrene, cadmium sulfate, chlorambucil, 2,6-diaminotoluene, 4-nitroquinoline N-oxide and mitomycin C (e.g., 37.0% cells with abs at 20 h and 1.0% at 44 h); ab yields similar at 20 and 44 h: 2-aminobiphenyl, eugenol and 8-hydroxyquinoline (e.g., 8.5% at 20 h and 7.0% at 44 h); and one, dimethylnitrosamine (DMN), which was detected at both times but gave a stronger response at 44 h than at 20 h (e.g., at 10 mM: 6.2% at 20 h and 25.0% at 44 h). This DMN effect was not seen in normal diploid human cells. For DMN the higher ab levels at 44 h than at 20 h were contributed by abs in M3 cells. Thus, while for some chemicals ab yields decrease with successive division, further increases can be seen in CHO in later metaphases, notably for DMN. Overall, however, after a 3-h pulse treatment of CHO cells a positive ab result could be obtained at the early harvest time (20 h) for all 10 chemicals.
A complementary DNA clone coding for a novel form of cytochrome P450, Cyp3a-16, in mouse fetal livers was isolated and completely sequenced. This clone encoded a polypeptide of 504 deduced amino acids and showed 87.3% and 66.6% amino acid identities with mouse Cyp3a-11 and Cyp3a-13, respectively. Cyp3a-16 transcript was detectable before birth and remarkably diminished five weeks after birth in mice. We conclude that Cyp3a-16 is a fetal- and puberty-specific cytochrome P450 in mice.
To provide further background data on the wing spot somatic mutation and recombination assay, 10 selected carcinogens (acetamide, acrylamide, benzo(a)pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) were tested in this assay. 72-h-old third-instar larvae, trans-heterozygous for 2 recessive wing cell markers: multiple wing hairs (mwh) and flare3 (flr3) were fed with 3 concentrations of each carcinogen during the rest of their development until pupation, and the genotoxic effects were measured as significant increases in the appearance of visible mutant hair clones on the adult wing blade. Our results show that 6 of the carcinogens tested produce significant increases in wing spot frequency, at least at one of the concentrations assayed. Benzo(a)pyrene, diethylstilbestrol, safrole and thiourea were the compounds that did not increase the incidence of mutant clones.
The antimutagenic effect of eugenol on the mutagenicity of cyclophosphamide (CP), mitomycin C (MMC), ethyl methanesulfonate (EMS) and benzo[a]pyrene (B[a]P) was assessed in the rodent bone marrow micronucleus test using male Swiss mice. Oral administration of eugenol (0.4% in the diet) for 15 days was found to decrease significantly the frequency of micronucleated polychromatic erythrocytes (MPEs) elevated by CP. No effect was found on the frequency of MPEs elevated by MMC, EMS and B[a]P. The results provide some support for antimutagenic potency of eugenol in vivo.
Pathobiological effects of eugenol (4-allyl-2-methoxyphenol), a major constituent of betel quid (BQ), were studied on oral mucosal fibroblasts. At a concentration higher than 3 mmol/L, eugenol was cytotoxic to oral mucosal fibroblasts in a concentration- and time-dependent manner. Cell death was associated with intracellular depletion of glutathione (GSH). Most of the GSH was depleted prior to the onset of cell death. At concentrations of 3 mmol/L and 4 mmol/L, eugenol depleted about 45% and 77% of GSH after one-hour incubation. In addition, eugenol decreased cellular ATP level in a concentration- and time-dependent manner. Eugenol also inhibited lipid peroxidation. Inhibition of lipid peroxidation was partially explained by its dose-dependent inhibition of xanthine oxidase activity. The IC50 of eugenol on xanthine oxidase activity was about 0.3 mmol/L. No DNA strand break activity for eugenol was found at concentrations between 0.5 and 3 mmol/L. Taken together, frequent exposure of oral mucosa to a high concentration of eugenol during the chewing of BQ might be involved in the pathogenesis of oral submucous fibrosis and oral cancer via its cytotoxicity. In contrast, eugenol at a concentration less than 1 mmol/L might protect cells from the genetic attack of reactive oxygen species via inhibition of xanthine oxidase activity and lipid peroxidation.
Several naturally occurring aromatic ethers, of which safrole [1-allyl-3,4-(methylenedioxy)-benzene] is one example, are hepatocarcinogens. One bioactivation pathway previously proposed for safrole involves hydroxylation of the benzyl carbon, conjugation with sulfate, and then alkylation of DNA with displacement of the sulfate group [Miller, J.A., and Miller, E.C. (1983) Br. J. Cancer 48, 1-15]. The fact that safrole is O-dealkylated to the corresponding catechol (hydroxychavicol, 1-allyl-3,4-dihydroxybenzene) indicates that quinoid formation is also possible and may contribute to the genotoxic and/or cytotoxic activity of this compound. In the present investigation we selectively oxidized hydroxychavicol to the corresponding o-quinone (HC-quinone, 4-allyl-3,5-cyclohexadiene-1,2-dione) or p-quinone methide (HC-QM, 2-hydroxy-4-allylidene-2,5-cyclohexadien-1-one) and trapped these reactive electrophiles with glutathione (GSH). The GSH adducts were fully characterized by UV, NMR, and mass spectrometry. Microsomal incubations with safrole or hydroxychavicol in the presence of glutathione produced only o-quinone glutathione conjugates. However, if the trapping agent (GSH) was added after an initial incubation of 10 min, both o-quinone and p-quinone methide GSH conjugates were observed. The first-order rate constant of isomerization was estimated from the decrease in HC-quinone GSH adducts to be 1.9 x 10(-3) s-1 (t1/2 = 9 min). Kinetic studies showed that while HC-QM reacts rapidly with water, the model o-quinone (4-tert-butyl-3,5-cyclohexadiene-1,2-dione), which cannot isomerize to a quinone methide, was remarkably resistant to hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Mouse liver DNA adducted with metabolites of the spice constituent safrole (1-allyl-3,4-methylenedioxybenzene), when analyzed via the bisphosphate version of the 32P-postlabeling assay, exhibits two major adducts, which had been previously identified as N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 1) and N2-(safrol-1'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 2). However, analysis of the same DNA preparation by the dinucleotide/monophosphate version of the assay gave two additional spots on PEI-cellulose TLC whose nature was clarified in the present study. Several enzymes (T4 polynucleotide kinase, nuclease P1, venom phosphodiesterase and spleen phosphodiesterase) were utilized to hydrolyze these compounds, and t