Bioactivation and Genotoxicity of the Herbal Constituents Safrole, Estragole and Methyleugenol
Total Page：16
File Type：pdf, Size：1020Kb
Bioactivation and genotoxicity of the herbal constituents safrole, estragole and methyleugenol Suzanne Jeurissen PROMOTOREN: Prof. Dr. Ir. I.M.C.M. Rietjens Hoogleraar in de Toxicologie Wageningen Universiteit Prof. Dr. E.J.R. Sudhölter Voormalig Hoogleraar in de Organische Chemie Wageningen Universiteit Hoogleraar in de Nano-Organische Chemie Technische Universiteit Delft COPROMOTOREN: Dr. T.A. van Beek Universitair Hoofddocent, Laboratorium voor Organische Chemie Wageningen Universiteit Dr. G.M. Alink Universitair Hoofddocent, Sectie Toxicologie Wageningen Universiteit SAMENSTELLING PROMOTIECOMMISSIE: Prof. Dr. Ir. M.A.J.S. van Boekel (Wageningen Universiteit) Dr. Ir. N.H.P. Cnubben (TNO Kwaliteit van Leven, Zeist) Prof. Dr. H. Irth (Vrije Universiteit Amsterdam) Prof. Dr. F.J. van Schooten (Universiteit Maastricht) Prof. Dr. R.L. Smith (Imperial College, London, UK) Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG Bioactivation and genotoxicity of the herbal constituents safrole, estragole and methyleugenol Suzanne Maria Francisca Jeurissen Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. M.J. Kropff in het openbaar te verdedigen op vrijdag 15 juni 2007 des namiddags te half twee in de Aula TITEL Bioactivation and genotoxicity of the herbal constituents safrole, estragole and methyleugenol AUTHOR Suzanne Maria Francisca Jeurissen Thesis Wageningen University, Wageningen, The Netherlands (2007) with abstract, with references, with summary in Dutch ISBN 978-90-8504-676-9 ABSTRACT The herbal constituents safrole, estragole, and methyleugenol, belonging to the chemical class of the alkenylbenzenes, are genotoxic and carcinogenic compounds. The genotoxicity of these alkenylbenzenes proceeds via electrophilic metabolites generated by cytochrome P450 enzymes (P450) and sulfotransferases (SULT). Carcinogenicity of these compounds was demonstrated in animal experiments using relatively high doses of single compounds. Human exposure to these compounds is much lower than the doses used in the animal experiments and humans are exposed to alkenylbenzenes in a complex food matrix. The first aim of this thesis was to identify the human P450 enzymes that are responsible for the bioactivation of the alkenylbenzenes into their proximate carcinogenic 1′-hydroxymetabolites. Several in vitro studies using recombinant P450 enzymes and human liver microsomes were undertaken to identify the main enzymes involved in the 1′-hydroxylation of the alkenylbenzenes and to determine their kinetics. These studies showed that at low substrate concentrations, P450 1A2 is the major enzyme in the bioactivation of methyleugenol, P450 1A2 and P450 2A6 are the main enzymes in the bioactivation of estragole, and P450 2A6 is the main enzyme in the bioactivation of safrole. The second objective of this thesis was to study the influence of other herbal constituents on the bioactivation and the genotoxicity of herb-based alkenylbenzenes. An on-line high-performance liquid chromatography detection system was developed for the detection of P450 1A2 inhibitors in herbal extracts. The presence of P450 1A2 inhibitors in basil, a herb that contains methyleugenol and estragole, was demonstrated using this on-line system. In addition to these P450 1A2 inhibitors, also the alkenylbenzenes themselves may act as inhibitors competing for the active site of P450 1A2 (estragole and methyleugenol) or P450 2A6 (estragole and safrole). Furthermore it was demonstrated that basil extract is able to strongly inhibit sulfation and subsequent DNA adduct formation of 1′-hydroxyestragole in incubations with rat and human S9 homogenates and in the human hepatoma HepG2 cell line. These in vitro results suggest that P450- and SULT-catalyzed bioactivation of methyleugenol and/or estragole and subsequent adverse effects may be lower in a matrix of other herbal components than what would be expected on the basis of experiments using single compounds. In vivo experiments have to point out whether the protective effects that are found in these in vitro studies can be extrapolated to the in vivo situation. It may turn out that rodent carcinogenicity data on estragole and methyleugenol considerably overestimate the risks posed when humans are exposed to these compounds in a herbal matrix. TABLE OF CONTENTS PAGE CHAPTER 1 General Introduction 9 CHAPTER 2 Human cytochrome P450 enzyme specificity for bioactivation of 37 safrole to the proximate carcinogen 1′-hydroxysafrole. CHAPTER 3 Human cytochrome P450 enzymes of importance for the bioactivation 51 of methyleugenol to the proximate carcinogen 1′-hydroyxmethyl- eugenol. CHAPTER 4 Human cytochrome P450 enzyme specificity for the bioactivation of 67 estragole and related alkenylbenzenes. CHAPTER 5 Development of an on-line high performance liquid chromatography 89 detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products. CHAPTER 6 Basil extract inhibits the sulfotransferase mediated formation of DNA 109 adducts of the proximate carcinogen 1′-hydroxyestragole by rat and hu- man liver S9 homogenates and in HepG2 Human Hepatoma Cells. CHAPTER 7 Summary and General Discussion. 125 List of Abbreviations 137 Samenvatting 139 List of Publications 146 Curriculum Vitae 147 Dankwoord 148 Training and Supervision Plan 150 1General Introduction GENERAL INTRODUCTION Background Herbs and spices have been used for culinary and medical purposes throughout the world for thousands of years. At present, the market for herb-based products such as herbal teas and herbal extracts and the use of herb-based ingredients as food supplements, food flavours, and bioactive ingredients in functional foods is still growing. Also the use of medicinal herbs (phytotherapy) increases. In spite of the common perception among consumers that ‘natural equals safe’, herbs and herbal extracts may contain individual ingredients that are toxic and even genotoxic and carcinogenic. This is even true for some herbs that are used since prehistory. The research in this thesis focuses on one class of herb-based ingredients, the alkenylbenzenes, and in particular on safrole, estragole, and methyleugenol (Fig. 1.1). Combinations of these compounds occur naturally in among others nutmeg, mace, cinnamon, tarragon, basil, star anise, and fennel, and methyleugenol and estragole are also used as food flavoring agents (1-3). Safrole, methyleugenol, and estragole are carcinogenic and genotoxic according to the Scientific Committee on Food of the European Union (EU-SCF) (1-3), and this will lead to restrictions in the use of these compounds in the European Union. Safrole is already banned as a food additive by the US Food and Drug Administration (Federal Register of December 3, 1960, 25 FR 12412) and by the Council of the European Communities (4). On the other hand, an industrial expert panel from the Flavor and Extract Manufacturers’ Association (FEMA) published that present exposure to methyleugenol and estragole, resulting from consumption of food, mainly spices and added as such, does not pose a significant cancer risk (5). For June 2007, a re-evaluation of the safety of estragole is scheduled by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). These different expert judgements mainly result from the absence of adequate scientific data to support unequivocal translation of carcinogenicity data of rodent animal experiments to the human situation. Since carcinogenicity is demonstrated in animal experiments using high doses of single compounds, and since these compounds are naturally present in various herbs and spices, new mechanistic insight in the adverse effects of these ingredients and the possible interaction with other herb-based ingredients is required for a better extrapolation of the animal carcinogenicity data to the human situation. Figure 1.1. Structural formulas of the alkenylbenzenes safrole, estragole, and methyleugenol. 10 CHAPTER 1 Exposure to alkenylbenzenes Occurrence of alkenylbenzenes Safrole occurs naturally in a number of herbs and spices such as nutmeg, mace, cinnamon, anise, black pepper, and sweet basil. Safrole is also present in cola drinks. The most important dietary sources are nutmeg, mace, and their essential oils (3). In addition, in some countries areca (betel nut) quid is chewed, and this can give rise to safrole exposure (420 µM was measured in saliva during chewing (6)) and is associated with the presence of safrole-DNA adducts in human peripheral blood (7). Methyleugenol exposure from food occurs predominantly from a number of herbs and spices such as nutmeg, pimento, lemongrass, tarragon, basil, star anise, and fennel. Methyleugenol is used as a flavoring agent in jellies, baked goods, non-alcoholic beverages, chewing gum, relish, and ice-cream (1) and also occurs at low levels in oranges, bananas, and grapefruit juice (5). Furthermore, it is applied as fragrance in cosmetic products such as creams, lotions, soaps, detergents, and perfumes (8). Methyleugenol is also used as an insect attractant and as an anaesthetic in rodents (1,8). Measurement of blood plasma levels of methyleugenol in a subset of the American population revealed that most people are exposed to methyleugenol. In 98% of the serum samples, methyleugenol was detected at concentrations ranging from < 3.1 (detection limit) up to 390 pg/g wet weight (Table 1.1) (9). When comparing these levels with the peak plasma levels measured in a study in which human volunteers were exposed to