Article pp.270-283 du Vol.25 n°4 (2005)

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Focus

Conférence internationale organisée par

la Société scientifique d’hygiène alimentaire (SSHA) à l’occasion de son centenaire

Paris, 17-18 juin 2004

L’alimentation : nouveaux enjeux

après un siècle de progrès

Rédacteur en chef invité

Claude Bourgeois

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SCIENCES DES ALIMENTS, 25(2005) 270-283

Éditorial

J.-L. MULTON

Président de la SSHA

La Société Scientifique d’Hygiène Alimentaire (SSHA), fondée en 1904, a célébré avec faste son centenaire en 2004.

L’idée d’une société savante pluridisciplinaire, orientée sur la nutrition et la qualité des aliments, semble avoir germé en 1903 lors du Congrès International d’Hygiène et de Démographie organisé à Bruxelles, à une époque où les risques liés à l’alimentation, certes moins médiatisés qu’aujourd'hui, n’en étaient pas moins réels et sans doute même beaucoup plus graves.

C’est en effet après ce congrès qu’Émile Roux, sous-directeur de l’Institut Pasteur, et Gabriel Bertrand forment un groupe de réflexion qui se constitue rapidement en un Comité de Fondation comportant quelques un des esprits les plus éminents de l’époque : on citera Arsène d’Arsonval, professeur au Collège de France, Étienne Marey, Armand Gautier, professeur à la Faculté de méde- cine, membre du Conseil d’hygiène et de salubrité de la Seine, Charles Richet, Georges Brouardel, doyen honoraire de la Faculté de médecine, président honoraire du Comité consultatif d’hygiène publique de France, et Auguste Chauveau, auxquels se joindront bientôt Marcelin Berthelot, sénateur et secré- taire perpétuel de l’Académie des sciences, Étienne Marey, professeur au Col- lège de France, et bien d’autres encore.

Le projet est également soutenu par des autorités publiques éminentes : le docteur Henri Ricard, sénateur de Côte d’Or, Georges Clemenceau, Henri Queuille, ministre et médecin, Gaston Meunier, député de Seine-et-Marne, et Émile Levasseur, économiste.

Cette réflexion collective aboutit, fait exceptionnel dans l’histoire des asso- ciations, à une loi votée le 27 juillet 1904 (Journal officiel du 29 juillet 1904) qui institue et reconnaît d’utilité publique la SSHA.

Ainsi parrainée par les plus hautes instances du monde scientifique et de la société civile, l’Association va également bénéficier d’un soutien financier important grâce à une loterie dont le rapport permettra de doter la toute jeune SSHA d’un bâtiment comportant des laboratoires de recherches et de moyens matériels et humains à hauteur des ambitions affichées.

C’est ainsi que la Société va être pendant tout le XIX^e siècle l’un des acteurs les plus dynamiques de l’évolution des sciences physiques, chimiques, micro- biologiques et socio-économiques appliquées à la nutrition et à ce qu’on appelle aujourd’hui la sécurité sanitaire des aliments. Elle y fut souvent

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Éditorial 271

« précurseur et pionnier » (comme l’a écrit Guy Ébrard dans l’Alimentation et la Vie), notamment dans les domaines des vitamines, de la microbiologie alimentaire, de l’analyse sensorielle. On ne saurait évoquer ce prestigieux passé scientifique sans citer le nom de Lucie Randoin (première femme élue à l’Académie de médecine) qui dirigea pendant des années les laboratoires de recherches, sans rappeler les liens contractuels étroits qui lièrent la SSHA et l’INRA (Jean Causeret y débuta sa carrière à l’INRA avant d’aller développer la nutrition dans le Centre de Dijon), sans souligner que le premier laboratoire d’analyse sensorielle y fonctionna à l’initiative de Félix Depledt.

Centre de recherche et d’innovation, la SSHA a fourni à l’industrie alimentaire émergente au début du XIX^e siècle les moyens de recherche et les moyens analytiques qui lui faisaient défaut pour contrôler et améliorer la qualité des pro- duits et assurer une meilleure sécurité sanitaire et un meilleur équilibre nutrition- nel. Désintéressée et non lucrative au début, cette activité orientée vers l’industrie, la distribution, la restauration collective, est devenue progressive- ment une prestation de service qui a connu un développement spectaculaire lorsque mon prédécesseur, Guy Ébrard, a créé les laboratoires de Champlan et de Massy, bien connus des professionnels de l’agro-alimentaire. Dans le contexte actuel d’une économie libérale mondialisée, cette activité marchande permet de financer en grande partie les activités de recherche et de développe- ment qui sont en retour le garant de la qualité des prestations pour les clients (confirmée par les accréditations COFRAC) et le moyen indispensable de l’innovation dans ces prestations. La qualité et la pertinence des recherches ainsi conduites par la SSHA sont examinées et discutées par le Conseil scientifique dont Bernard Launay (Professeur émérite à l’ENSIA) est actuellement le prési- dent.

Société savante, la SSHA a édité pendant des années le « Bulletin de la Société Scientifique d’Hygiène Alimentaire et d’Alimentation rationnelle de l’Homme », puis la revue « L’alimentation et la vie ». Après plusieurs années d’interruption dues à des contraintes économiques, la publication de ce dernier titre a été reprise sous la forme d’un cahier inséré dans la revue Sciences des aliments, grâce à un partenariat avec les éditions Lavoisier.

Pour célébrer son centenaire, la SSHA a organisé en 2004, sous l’égide de la Commission Européenne et de l’OCDE, un colloque scientifique international, qui comportait en fait deux manifestations complémentaires et coordonnées :

• L’une « L’alimentation, nouveaux enjeux après un siècle de progrès », organisée à Paris, dans les locaux de la SSHA, 16, rue de l’Estrapade, 75015 Paris, les 17 et 18 juin 2004, sous le Haut patronage de Monsieur Jacques Chirac, Président de la République.

• L’autre « Rôle des programmes-cadres européens pour le développe- ment d’une recherche intégrée en sécurité sanitaire au long de la chaîne alimentaire », conférence européenne, co-organisée par la SSHA et l’INRA, à Lille, les 27, 28 et 29 octobre 2004, sous la présidence de Gérard Pascal.

Le colloque « L’alimentation, nouveaux enjeux après un siècle de progrès », ouvert par Marion Guillou, PDG de l’INRA, et clôturé par Kurt Wüthrich, Prix Nobel (ETH, Zürich), se situait clairement dans le contexte de l’espace euro- péen de la recherche.

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272 Sci. Aliments 25(4), 2005 J.-L. Multon

Conçu comme étant un séminaire de réflexion scientifique préparatoire à la conférence européenne qui devait suivre à Lille, il était structuré en quatre ateliers distincts :

– « La chaîne alimentaire : réalités et maîtrise des risques » (The food chain : true risks and control), animé par Bernard Launay (ENSIA) et Laurent Bochereau (Commission Européenne).

– « La Science des aliments : confiance et méfiance des consommateurs » (The food science: trust and mistrust of consumers), animé par Max Fein- berg (INRA / SSHA) et Jean-Pierre Toutant (INRA / OCDE).

– « La diffusion des savoirs alimentaires » (Dissemination of alimentary knowledge), animé par Hervé This (INRA / Collège de France) et Guy Lin- den (CIRAD).

– « Qui influence les choix alimentaires ? » (Who decides of what we eat?), animé par Jean-Blaise Rochette de Lempdes (Consultant) et Jean-Claude Melchior (Hôpital Raymond Poincaré).

Après les séances de restitutions tenues par les animateurs des ateliers, les conclusions scientifiques ont été synthétisées par Bernard Chevassus-Au- Louis, ancien Directeur de l’INRA et actuel Président du Muséum National d’Histoire Naturelle de Paris et présentées à la conférence de Lille par Gérard Pascal, Directeur de recherches et pendant des années le responsable de la direction scientifique « Nutrition Humaine et Sécurité Sanitaire des Aliments » de l’INRA.

Les lecteurs de Sciences des Aliments trouveront une sélection des commu- nications présentées au colloque de Paris. Toute sélection a sa part d’arbitraire et celle-ci n’échappe pas à la règle. Notre choix a été guidé uniquement par le souci de la meilleure adéquation possible entre les thèmes de ces communica- tions et les objectifs de la revue et par l’accord des orateurs pour préparer une version publiable de leurs interventions orales.

J’espère que les lecteurs y trouveront une illustration de l’état de l’art dans quelques domaines importants de ces sciences qui sont la raison d’être de la SSHA depuis un siècle.

Dans chaque numéro, la rubrique « L’alimentation et la vie » met en avant un article traitant d’un des aspects de la nutrition, du rôle des technologies agroalimentaires sur la qualité des aliments jusqu’à la « cuisine », en passant par les problèmes nutritionnels, la toxicologie alimentaire, et plus généralement les conséquences sur la santé des pratiques alimentaires. Les articles retenus sont soit des travaux de synthèse de haut niveau faisant le point sur une question, soit des publications originales rendant compte de travaux de recherche appliquée récents apportant un regard nouveau.

La Société scientifique d’hygiène alimentaire (SSHA), société savante créée en 1904 pour contri- buer à la diffusion des connaissances en nutrition et sécurité sanitaire, est aujourd’hui formée de deux départements : l’Institut supérieur de l’alimentation (ISA) développe des actions de formation, d’information et de conseil ; l’Institut supérieur d’hygiène alimentaire (ISHA) propose un catalogue complet d’analyses (composants nutritionnels, contaminants, analyse sensorielle, microbiologie…).

Les propositions d’articles, remarques et suggestions peuvent être envoyées à : Claude Bourgeois

SSHA

Rue du Chemin-Blanc, BP 138, Champlan F-91163 Longjumeau cedex Tél. : + 33 (0)1 69 79 31 50

Fax : + 33 (0)1 64 48 82 49 http ://www.ssha.asso.fr [email protected]

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SCIENCES DES ALIMENTS, 25(2005) 273-283

FOCUS : L’alimentation : nouveaux enjeux

Detection of xenobiotics and chemical contaminants in the food chain:

focus on mass spectrometry based methods and their use for toxicological risk assessment

in the field of food safety

L. Debrauwer, S. Chevolleau, D. Zalko, A. Paris, J. Tulliez

This article is dedicated to Dr G.F. Bories at the occasion of his retirement.

RÉSUMÉ

Détection de xénobiotiques et de contaminants chimiques dans la chaîne alimentaire par spectrométrie de masse et son rôle dans l’éva- luation des risques toxicologiques en sécurité des aliments

La contamination chimique des aliments est devenue une préoccupation majeure chez les consommateurs des pays développés. Sur la base de plusieurs exemples d’application, le rôle central des méthodes reposant sur la spectrométrie de masse pour la détermination des contaminants chimiques dans les aliments est montré. Cette technique d’analyse est maintenant utili- sée en routine pour la détermination des résidus de contaminants dans les aliments et est également incontournable dans les études de métabolisme liées à ces composés. Dans ce contexte, l’accent est également mis sur le besoin d’approches plus intégratives incluant non seulement les résultats quantitatifs des analyses mais aussi la mesure de l’impact biologique et physiologique (direct ou indirect) des contaminants pour une meilleure évaluation du risque.

Mots clés

spectrométrie de masse, sécurité des aliments, évaluation du risque, conta- minants chimiques.

SUMMARY

Chemical contamination of the food chain has become a central concern for developed countries consumers. On the basis of several application examples, the essential role of mass spectrometry based methods for the determination of chemical contaminants in food is shown. This analytical

INRA, UMR 1089 Xénobiotiques – 180, chemin de Tournefeuille – BP 3 – 31931 Toulouse Cedex 9 – France.

Correspondence: [email protected]

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274 Sci. Aliments 25(4), 2005 L. Debrauwer et al.

technique is now routinely used for the determination of residues of contaminants in food and is also of great interest in the field of metabolism studies undertaken on such compounds. In this context, the need for more integrative approaches including not only quantitative analytical results but also biological and global physiological effects measurements for a better risk assessment is emphasized.

Keywords

mass spectrometry, food safety, risk assessment, chemical contaminants.

1 – INTRODUCTION

In developed countries, modern way of life features have turned foodstuff consumption customs towards the use of more and more ready-cooked dishes elaborated using world-wide raw materials supplying. This feature, together with an increasing social pressure of consumers, has implied the promulgation of very strict sanitary regulations concerning food products for a better control of food production and distribution in developed countries. Besides this important social demand, one of the main factors having favoured the establishment of always lower tolerance thresholds is the expanded capabilities of analytical methodologies, allowing the detection of lower and lower amounts of residues.

Food safety management imposes to supervise various contamination types, which can schematically be divided into three main groups: (i) contamination of microbial origin (bacteria, micro-organisms…), (ii) contamination of inorganic origin (heavy metals) and finally (iii) contamination of organic origin (pesticides, industrial contaminants…). Difficulties in the risk assessment related to these three categories mainly lies in the fact that they involve different approaches, and in addition, that the nature of a food contamination can change along the food chain. For example, this is the case for aquatic micro-organisms that can turn mercury (inorganic) into methyl-mercury (organic), an extremely toxic agent for man eating contaminated fishes, or for the fungal (microbial) contamination of cereals responsible for the occurrence of mycotoxins (organic) in food.

In this article, we will focus on the assessment of food contamination by organic chemical compounds, which can also be of various origins. Indeed, they can be natural compounds occurring in raw materials and can be responsible for biological effects (isoflavones, mycotoxins…), or compounds formed during transformation and processing steps in agro-food industrial plants (e.g.

acrylamide), as well as contaminants introduced intentionally (pesticides, growth promoting agents…) or not (pollutants) all along the food production and supply chain. Thus, xenobiotic compounds, non nutritional and potentially toxic for man, can occur in many types of foodstuffs and need to be detected as early as possible. Depending on the physico-chemical characteristics and the toxicity of the compound of interest (molecular weight, polarity…), the analytical challenge may be very different. In addition, it is also of great importance to have information on the metabolic fate of these xenobiotics since their biological effects may be mainly due to one of their metabolites. Furthermore, several

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Mass spectrometric chemical analysis and food safety 275

xenobiotic compounds belonging to different chemical classes and originating in various sources may induce similar biological effects. For example, estrogenic compounds can be of various origin and may penetrate the food chain at different levels (figure 1). Their metabolic fate may also be different, and although these biotransformations often lead to the elimination of the formed (phase I and phase II) metabolites, they can also generate reactive species, which may disrupt the cellular functioning (see figure 1). It is thus important to assess not only the exposition of living organisms to contaminants but also the metabolic fate of these compounds as well as the biological activities of their biotransformation products. In this article, we will illustrate from some examples of our recent work, how modern analytical chemistry and especially mass spectrometry, can provide interesting solutions to the very various problems posed by chemical contamination in the field of food safety.

2 – SOME EXAMPLES OF THE USE OF MASS SPECTROMETRY FOR THE ASSESSMENT OF CHEMICAL CONTAMINATION IN THE FOOD CHAIN

2.1 Toxic compounds formed during food processing:

the example of acrylamide

The first example we will discuss in this article is the case of acrylamide, a small molecular weight and very polar molecule, which is formed during thermal treatments of some categories of food (deep-fat frying, oven-baking…). This compound is known to be neurotoxic and has been classified as probable human carcinogen by the International Agency for Research on Cancer in 1994 (IARC, 1994). The possible exposure of humans to acrylamide via the diet was first reported by Swedish researchers (TAREKE et al., 2000) and since that time, a flurry of work has been carried out for understanding the conditions of its for-

Environment (Xenoestrogens)

Food (Phytoestrogens)

Treatments (Estrogens)

Living organism (Food chain)

Interaction with cellular receptors

Physiological action Elimination Toxic effect Reaction with endogeneous macromolecules Biotransformation

(detoxification, bioactivation…)

Figure 1

General description of xenobiotic fate in living organisms (case of estrogenic compounds).

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mation, the extent of its occurrence as well as the likely dietary exposure of human consumers. In this context, powerful analytical methods had to be developed. Mass spectrometry based methods allow the quantification of contaminants at the ppb level (one nanogram per gram) or less, from various food matrices (biscuits, coffee…) and using a small sample weight (few grams). Thus, this technique, coupled to either gas (GC-MS) or liquid chromatography (LC- MS) proved to be the most efficient analytical tool for the unambiguous characterisation and quantification of acrylamide, and accounted for more 90% of the analyses carried out within inter-laboratory ring tests, as mentioned by WENZL

et al. in a recent review article (WENZL et al., 2003). Owing to the wide variety of ionisation methods and coupling capabilities of MS, different approaches have been proposed for the analysis of acrylamide in food matrices. Almost all of them use tandem mass spectrometry (MS/MS) allowing the detection of the compound of interest not only with a great sensitivity but also with a very high degree of confidence by both providing structural confirmation of the analyte and eliminating most of the interferences.

GC-MS based methods offer a great detection sensitivity but generally require more complex sample treatment procedures. The high polarity of acrylamide requires the use of adapted capillary columns (HOENICKE et al., 2004) unless a derivatisation step of the molecule (by means of bromination of the ethylenic bond) is used (CASTLE et al., 1991). For these reasons, most of methods for the determination of acrylamide in food have been developed using LC-MS. Electrospray has been widely used as the ionisation technique (TAREKE

et al., 2002; BECALSKI et al., 2003; AHN et al., 2002, ONO et al., 2003) but atmos- pheric pressure chemical ionisation (APCI), which is less sensitive towards ion suppression or matrix effects, can also be used. In this case, MH⁺ (m/z 72) ions are produced, which give characteristic fragment ions at m/z 55 [MH-NH₃]⁺ and m/z 44 [MH-CO]⁺ when using MS/MS. These fragment ions are used for the specific detection of the compound in complex matrices. As an example, LC-APCI-MS/MS chromatograms resulting from the analysis of roasted almonds are presented in figure 2, for acrylamide and its deuterated analogue used as the internal standard for quantification (CHEVOLLEAU et al., 2003).

These powerful methodologies allow the precise determination of acrylamide contents in various food matrices and several reports have been published on this topic. However, another feature which has to be considered is the metabolic fate of this compound. Acrylamide is known to be metabolised mainly into glycidamide, an epoxyde reactive intermediate which covalently binds to DNA. Acrylamide also leads to the formation of adducts with haemoglobin which measurement can be considered as a good indicator of exposure to acrylamide. For this purpose, mass spectrometry based methods are of great usefulness. Acrylamide-haemoglobin adducts have mostly been determined using LC-ESI-MS/MS (FENNELL et al., 2003). For that, the protein is hydrolysed into single amino acids among which the modified valine residue is specifically quantified after derivatisation with phenylisothiocyanate (CHEVOLLEAU et al., 2004). This methodology constitutes a valuable tool for the assessment of human exposure to acrylamide, and especially for measuring the impact of daily dietary low levels of this compound in people who are not exposed to other acrylamide sources like smoking or special working activities. Complementary to the worldwide surveillance of this substance in various food products, the determination of acrylamide- haemoglobin adducts in relation to diet customs

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may give valuable information on the real impact of the consumption of acrylamide contaminated food on human health.

2.2 Food contamination by environmental pollutants:

the case of endocrine disruptors

During the past few years, the scientific community has drawn attention to numerous chemical compounds which penetrate the food chain at various levels, and which are suspected to interfer with natural hormone receptors and disrupt the endocrine system, thus inducing perturbation of the sexual function and the reproduction in animal and human (LEVI, 1999). These compounds are called endocrine disruptors and can be pesticides, plastics or other pollutants released into various environmental compartments (water, earth, air particles).

Although inducing similar biological effects, they may belong to very different chemical classes as indicated in table 1, and here again, their analysis is often very complex (PETROVIC et al., 2002). For that reason, the development of powerful methods is required for the simultaneous determination of a high number of compounds within a single analysis. In the field of pesticide residue analysis, GC-MS et LCMS couplings have proved to be the techniques of reference by providing non ambiguous data that fit the MRLs imposed by official regulations (NIESSEN, 1999). Most of MS manufacturers now propose methodologies using triple quadrupole mass spectrometers and allowing the simultaneous determination of almost a hundred different pesticides at the ppb level in various food matrices (tomato, strawberry…) within analysis times of no more than 20 minutes (OZEKI et al., 2004; PATEL et al., 2003). In the case of other endocrine disrupting molecules from e.g. the chemical and polymer industry (alkylphenols,

0 1 2 3 4 5 6 7 8 9 10

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

7,19

2,93

3,33

7,03 MS/MS

m/z 72 –> m/z 55

MS/MS

m/z 75 –> m/z 58

a

b

Time (min) Figure 2

Mass fragmentograms obtained from a roasted almond sample after LCMS/MS analysis. A: analysed acrylamide and B: internal standard d3-acrylamide

(concentration of acrylamide was 167 ng/g).

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bisphenol A, phtalates…), mass spectrometry provides information on the nature and level of exposition for the organism of interest, but is also used as a powerful tool for the exploration on the metabolic fate of these compounds by means of structural identification of their various metabolites (DEBRAUWER, 2000;

THIBAUT et al., 1998, 2000; ZALKO et al., 2003). This aspect is very important, especially in the case of compounds for which the biological effects are not well known. Providing information on the fate of the molecule allows a better evaluation of the toxic potential of the compound, thus providing some useful information to experts and lawyers which regulate maximum allowable daily doses or assess contamination levels. For example, human exposure to bisphenol A is due to its migration from packaging materials (lacquered coated cans, polycar- bonate bottles…) into food and beverages (GOODSON et al., 2002). The estrogenic activity of this compound is several orders of magnitude lower than 17β-estradiol, and the exposure is expected to be low but of daily occurrence.

However, several studies have demonstrated biological effects in mouse pro- geny after exposure of pregnant mice to low doses of bisphenol A (WELSHONS et al., 1999). Thus, knowledge of the metabolic fate of this compound is essential for a better understanding of the biologic consequences of exposure. Indeed, when studied in pregnant mice, the analysis of excreta and tissues, including fetuses, showed that bisphenol A easily crossed the placental barrier and that its metabolic pathways were more complex than expected (ZALKO et al., 2003).

Here again, mass spectrometry is of essential utility for the structural identification of metabolites occurring in the low nanogram range in the studied biofluids or tissues. Proposed metabolic pathways in pregnant mouse are reported in figure 3 for bisphenol A (ZALKO, 2003). The identification of several metabolites suggests that this xenoestrogen could be metabolised into reactive intermedia- tes such as catechol structures. This highlights new aspects on potential biological effects of bisphenol by raising the question of cellular macromolecule (proteins, DNA) adulteration. This illustrates the need of metabolic studies on a number of contaminants, for which the daily exposure of consumers is not well known. Long term low dose effects should also be investigated in order to better reflect real life situations which certainly does not fit the case of acute poi- soning.

Table 1

Some chemical compounds known as or suspected to be endocrine disruptors (LEVI, 1999).

Pesticides 2,4,5-T; 2,4-D; Alachlor, Aldicarb, Amitrole, Atrazine, Benomyl, Carbaryl, Chlordane, Cypermethrine, DDT, Dicofol, Dieldrine, Endosulfan, Ethylparathion, Lindane, Heptachlore, Kelthane, Kepone, Malathion, Mancozebe, Maneb, Methomyl, Mirex, Parathion, Permethrine, Toxaphene, Zinab, Ziram…

Phtalate esters DEHP, BBP, DBP, DPP, DHP, DPrP, DCHP, DEP…

Heavy metals Cd, Pb, Hg

Others Styrene, Benzo(a)pyrene, Alkylphenols (penta to nonyl), Bisphenol A, 2,4-dichlorophenol, Diethylhexyladipate, 2,3,7,8-TCDD,

2,3,7,8-tetrachlorodibenzofurane, PCBs, Octachlorostyrene, Hexachlorobenzene, Pentachlorophenol…

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More generally, for this class of chemical compounds, an efficient risk assessment involves not only sophisticated separation or structural analysis techniques for the evaluation of the exposition of consumers, but also biological tools aimed at the evaluation of their toxicity and endocrine disrupting effects.

2.3 More global tools for the assessment of metabolic network disruptions by xenobiotics: the metabonomic approach

The structural characterisation of xenobiotic metabolites provides valuable information on detoxification mechanisms as well as on problematics related to the metabolic activation of some of these compounds. The use of complementary ionisation techniques and mass analysers offered by modern mass spectrometry allows to investigate both the identification of small organic molecules and the investigation of cellular macromolecule modifications by reactive metabolites. The extreme sensitivity of mass spectrometry now allows to evidence very low contamination levels by detecting residues of the xenobiotic compound as such or as its metabolised form. In the case of breeding animals, the use of some compounds may be very difficult to evidence, especially after long withdrawal periods. This is of particular importance since the use of growth promoting agents in farm animals represents an important source of biologically active compounds in food. Regulations of the producing countries are often different and powerful detection and/or quantification techniques have to be used for an efficient control of these molecules, which use is sometimes forbidden and always regulated. Thus, mass spectrometry is widely used as a reference technique for the control of growth promoting agents (steroids,

OH HO

O-GlcADH OH HO3S-O

OH

O-Glc HO3S-O

H3C-O O-GlcA HO

O-GlcADH HO

O-GlcA HO

OH O-GlcA Glu-O

O-GlcADH HO3S-O

O-CH3

O-Glc HO3S-O

H3C-O OH

O-GlcA HO

GlcNAc.

OH

HO HO OSO3H

Bisphenol A OH

O-GlcA HO

Figure 3

Metabolic pathways proposed for bisphenol A in pregnant mouse.

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anabolisers, β-agonists, growth hormone…) in breeding animals, with detection selectivities and sensitivities compatible with searched contamination levels.

However, this approach is limited to the detection of several known compounds and the complexity of multiresidue determinations increases with the number of compounds to be analysed simultaneously. Unfortunately, this approach is not adapted for the detection of unknown compounds and (even if the food safety problem can be considered as over) for evidencing the use of forbidden molecules after long withdrawal periods. In such situations, more integrative approaches may give a more global vision of a biological situation. Recently, the emergence of metabonomics took in consideration the whole low molecular weight molecules and metabolites (called metabolome) of a cell, a tissue or an organism in a given physiological state, allowing to investigate biological con- trasts (NICHOLSON et al., 1999) by analysing spectroscopic data. In the domain of toxicology, this approach can be used for evidencing indirect physiological effects due to pathological states or to the action of xenobiotics (occurring either as contaminants or as medicines). This requires the tracking of weak disruptions of the metabolic network and necessitates the generation of data sets containing as much information as possible. This also requires the use of robust data generation tools in order to give reproducible spectra which can be considered, in this case, as a photograph of a given biological situation. For example, indirect long term effects related to bovine treatment by anabolisers can be detected using such a methodology (DUMAS et al., 2002). In this case, the withdrawal period was three months and no more trace of the administered molecule was detectable using classical analytical processes. The experimental device used in this work was a prototype time of flight mass spectrometer coupled to a pyrolysis system and to metastable atom bombardment ionisation (FAUBERT et al., 1993). This special ionisation technique is known to provide very repeatable mass spectra due to the discrete and tunable energy transferred to the analytes during the ionisation process. Mass spectra generated from crude bovine urine samples (control and treated steers) are reported in figure 4a and 4b. Sets of crude bovine urine samples were analysed in order to generate mass spectra which can be considered as fingerprints, reflecting a physiological situation at a given time. Data sets were then treated using multidimensional statistics (pattern recognition and linear discriminant analysis). Figure 4c shows a linear discriminant analysis 2D-factorial plot obtained from the analysis of different animal groups. The various animal groups can be discriminated accor- ding to the nature of the treatment they have received. This shows that the statistical interpretation of the metabolic variations induced by steroids could give a clear description of the physiological disruption which occurred for treated animals. This approach seems very encouraging for the generation of metabonomic databases aimed at the screening of xenobiotic use, or at the detection of pathologies or other physiological disruptions.

In other application fields, the same methodology has also been success- fully used for the differentiation of various animal fats (BEAUDET et al., 2003) or for the rapid identification of vegetable oils (SANCHEZ et al., 2002) by generating lipid profiles which were characteristic of the animal or vegetal species. This opens new future possibilities for rapid food authentication or certification. The constitution of databases generated with such analytical devices may also be considered and should provide an interesting alternative to complex analytical methodologies for screening purposes.

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50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

m/z 0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative AbundanceRelative Abundance LD3 (14.8%) 108. 00

262 .00 121. 00

251. 00 59.00

263. 00 133. 00

277. 00 67. 00

287. 00 159. 00

312. 00 313. 00

332. 00 354.00 173. 00

379. 00416. 00 483. 00 547. 00 657. 00676. 00

235. 00 731. 00

186. 00 583. 00 799. 00

x50

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

m/z 0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 59.00

108. 00

117.00 121. 00 67. 00

75. 00

78. 00 255 .00275. 00

362. 00

312. 00 585. 00

122.00 625. 00

467.00 569. 00

432.00 611. 00 779.00

407. 00 495. 00 655. 00 709. 00

159. 00173. 00221. 00 521. 00 774. 00

x50

LD1 (57.8%)

– 5 0 5 10

– 6– 4– 202468 C

1 2 4

Dose effect

Control 90 days after

treatment

a

b

c

Figure 4

Metabonomic analyses of cattle urine samples: A: MAB mass spectrum obtained from the pyrolysis of a urine sample from a control steer, B: same MAB mass spectrum

from a treated steer and C: LDA 2-D factorial plots from data generated with MAB-MS of various cattle urine samples.

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3 – CONCLUSION

In conclusion, growing capabilities of analytical chemistry in general and of mass spectrometry in particular had allowed for the detection and quantification of more and more compounds, occurring at lower and lower levels, and determined in more and more complex matrices. All these performances contribute to a better efficiency of the analysis and control processes related to the risk assessment in food safety. In addition, investigation of the metabolic fate of chemical contaminants provide useful data for the toxicological evaluation of these compounds. This contributes to provide some objective scientific data to experts and lawyers, which can be very helpful in their missions of chemical risk evaluation in the field of sanitary quality of food.

However, despite the powerfulness of such methodologies using very sophisticated technologies, the need for more general approaches including biological activity evaluation or global metabolic network disruption studies for a better chemical risk assessment has to be considered.

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