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International audience; Consumers generally considered organic products to be healthier and safer but data regarding the contamination of organic products are scarce. This study evaluated the impact of the farming system on the levels of ochratoxin A (OTA) in the tissues of French pigs (muscle and liver) reared following three different types of production (organic, Label Rouge and conventional). Because OTA is present at trace levels in animal products, a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method using stable isotope dilution assay was developed and validated. OTA was detected or quantified (LOQ of 0.10 mu g kg(-1)) in 67% (n = 47) of the 70 pig liver samples analysed, with concentrations ranging from < 0.10 to 3.65 mu g kg(-1). The maximum concentration was found in a sample from organic production but there were no significant differences in the content of OTA between farming systems. OTA was above the LOQ in four out of 25 samples of the pork muscles. A good agreement was found between OTA levels in muscle and liver (liver concentration = 2.9 x OTA muscle concentration, r = 0.981).

TBBPA and HBCDs are the two classes of flame retardants that are still allowed for use by the European Commission. In May 2013, HBCDs were listed as Persistent Organic Pollutants under the Stockholm Convention, and they were banned with an exemption on EPS/XPS for cavity wall insulation. This study describes the development and optimisation of a rapid LC-ESI-MS/MS method using isotopic dilution quantification including a simplified extraction step using a mixture of solvents and sulphuric acid hydrolysis followed by the one-shot analysis of TBBPA and each of the α-, β- and γ-HBCD diastereoisomers. The limits of detection and quantification (LOD and LOQ) were 0.5 and 2.5 ng g−1 (lipid weight, lw) for TBBPA and HBCD diastereoisomers, respectively. The method was applied to analyse 106 samples of individual mature breast milk. TBBPA was quantified in 42% of these samples within a range of

Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. This review, divided into four parts, will focus on the following: (1) the worldwide story of SFP outbreaks, (2) the characteristics and behaviour of S. aureus in food environment, (3) the toxinogenic conditions and characteristics of SEs, and (4) SFP outbreaks including symptomatology, occurrence in the European Union and currently available methods used to characterize staphylococcal outbreaks.

International audience; Mycotoxins in milk are a public health concern and have to be regularly monitored. A survey on the presence of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in raw bulk milk was conducted in 2003 in the northwest of France, the main French milk-producing basin. Randomly selected farms (n = 132) were characterized by a diet based on corn silage and containing a large proportion of on-farm produced cereals, feeding sources that are frequently contaminated by mycotoxins. Farms were surveyed twice in winter and in summer. At each sampling time, a trained surveyor completed a questionnaire recording farm management procedures and production traits. The AFM1 was found in 3 out of 264 samples but at levels (26 ng/L or less) that are below the European legislation limit of 50 ng/L. Traces of AFM1 (less than 8 ng/L) were also found in 6 other samples. The OTA was detected in 3 samples also at low levels, 5 to 8 ng/L. Farms that tested positive to the presence of mycotoxins, 12 in total including 6 farms that had traces of AFM1, differed from negative farms by a more extensive use of total mixed rations, 58 vs. 27%. In addition, the positive farms tended to have lower milk yields. Although the incidence of milk contamination with AFM1 and OTA at the farm level was low during the period studied, production and management data from the surveyed farms suggest a link between feeding management practices and mycotoxin contamination.

From 2010 to 2012, 35 ciguatera fish poisoning (CFP) events involving 87 individuals who consumed locally-caught fish were reported in Guadeloupe (French West Indies). For 12 of these events, the presence of ciguatoxins (CTXs) was indicated in meal remnants and in uncooked fish by the mouse bioassay (MBA). Caribbean ciguatoxins (C-CTXs) were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Using a cell-based assay (CBA), and the only available standard Pacific ciguatoxin-1 (P-CTX-1), the lowest toxins level detected in fish samples causing CFP was 0.022 µg P-CTX-1 equivalent (eq.)·kg(-1) fish. Epidemiological and consumption data were compiled for most of the individuals afflicted, and complete data for establishing the lowest observable adverse effects level (LOAEL) were obtained from 8 CFP events involving 21 individuals. Based on toxin intakes, the LOAEL was estimated at 4.2 ng P-CTX-1 eq./individual corresponding to 48. 4 pg P-CTX-1 eq.kg(-1) body weight (bw). Although based on limited data, these results are consistent with the conclusions of the European Food Safety Authority (EFSA) opinion which indicates that a level of 0.01 µg P-CTX-1 eq.kg(-1) fish, regardless of source, should not exert effects in sensitive individuals when consuming a single meal. The calculated LOAEL is also consistent with the U.S. Food and Drug Administration guidance levels for CTXs (0.1 µg C-CTX-1 eq.kg(-1) and 0.01 µg P-CTX-1 eq.kg(-1) fish).

The French Food Safety Agency is a public body incorporating 12 laboratories that perform research to support expertise and public decisions taken in the fields of sanitary safety of food, animal health, and veterinary drugs. On the request of the General Management of the Agency a quality-management system in research (QMSR) is being implemented in the Food and Food Process Quality Research Laboratory. The experimental QMSR is based on existing standards and documents, describing the provisions required for scientific and technical competence, quality management, and project management. Furthermore, this QMSR also incorporates specific notions of great importance for research activities such as the positive and negative non-conformities, the non-confirmation of hypotheses, and the principle of evaluation of a research activity by peers for both quality and scientific aspects.

In the 1950s, a series of publications from Bulgaria, Yugoslavia, and Romania locally described a kidney disease called Balkan Endemic Nephropathy (BEN). In Bulgaria, the exposure of populations to ochratoxin A (OTA) was supported by analysis of individual food items demonstrating a higher prevalence and higher levels of OTA in food from the high-incidence areas of BEN. In this work, food consumption from a series of individuals from two villages of the BEN area during 1 month was followed using the duplicate diet method. Meals consumed by volunteers from both villages showed uneven OTA contents, spreading from below the limit of quantification (

Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.

Two different immunoaffinity columns (IACs) were prepared for detection of staphylococcal enterotoxins (SETs) from dairy products. First, a specific IAC for staphylococcal enterotoxin A (SEA), IAC-1, was prepared by coupling monoclonal antibody (mAb) directed against SEA; second, a polyspecific IAC for SEA, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SECs), and staphylococcal enterotoxin D (SED), IAC-2, was prepared by coupling a mixture of mAbs against SEA, SECs, and SED, and rabbit IgG against SEB. These columns were applied for detection of SETs in dairy products, after extraction, immunoaffinity chromatography, and enzyme immunosorbent assay (EIA). Overall recoveries from dairy products spiked with 1 ng SEA/25 g averaged 81.2% (range, 76–85%) on IAC-1. The repeated use of IAC-1 was then determined with good efficiency of 91.5%, in more than 10 runs. On the other hand, a recovery yield of 77%of SETs (SEA, SEB, SEC, and SED) from dairy products spiked with 2.5 ng of each enterotoxin per 25 g, was obtained with IAC-2. IAC-2 was also successfully subjected to the chromatography of naturally contaminated foods implicated in staphylococcal food poisoning outbreaks. This new extraction–concentration–immunoaffinity–chromatography method (ECIC) is very useful for improving staphylococcal enterotoxin detection and eliminating matrix effect in EIA of dairy products.

Diarrhetic Shellfish Poisoning (DSP) is a severe gastro-intestinal disease caused by consumption of seafood contaminated by microalgal toxins, mainly okadaic acid (OA) and structurally related toxins, dinophysistoxins (DTXs). Regulatory monitoring is generally based on rodent bioassays which, however, present some technical and ethical disadvantages. The most promising technique of analysis of these toxins involves an HPLC separation with spectrofluorimetric detection after derivatization of the toxins with a fluorescent reagent. The lack of specificity of the extraction procedure (liquid-liquid partition), and the presence of interfering compounds in the matrix, does not allow the determination and the quantification of low amounts of toxins in seafood. In this paper, the authors report the development and the characterization of immunoaffinity columns (IAC), which were elaborated using anti-okadaic acid monoclonal antibodies, for a specific retention of the OA group of toxins. The coupling yield and the stability of these columns were investigated as well as their capacity to remove interfering compounds. Cross-reactivity was observed between the antibodies and the DTX-1 and the DTX-2, allowing the detection of the different toxins in a single analysis. Different spiked (1 microgram OA/g) or naturally-contaminated (mussel digestive gland: 2 micrograms OA/g; algae: 165 micrograms OA/g) matrices were tested. The recovery for OA varied from 55 to 95% according to the matrices. The IAC purification was then included as a step of a global [IAC/HPLC/spectrofluorimetric detection] method and the performance of the method was evaluated. Estimations of the linearity and the accuracy (percentages of the presumptive response for OA in the range +101% to +114%) were satisfactory in accordance with the method validation criteria.

Okadaic acid and dinophysistoxins are produced by some marine unicellular algae from the plankton and also benthic microalgae and may accumulate in shellfish. These phycotoxins are involved in a gastrointestinal syndrome called diarrhetic shellfish poisoning (DSP), which occurs in humans after consumption of bivalve molluscs. Thousands cases of human poisonings in Europe were caused by consumption of toxic shellfish during the past decade. The rapid detection and the reliable determination of the main phycotoxins implicated in DSP are a major concern for governmental institutions in charge of the sanitary control of seafood safety. Analytical procedures for the detection and determination of DSP toxins can be classified as: bioassays, biochemical methods including immunoassays, or physicochemical methods. Although a large number of methods have been developed, none have been officially validated. A complete panel of tools for DSP toxin analysis should include screening, investigation, and confirmation methods. This paper presents a compilation of recent developments and optimisations of these methods.

Staphylococcal food poisoning is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All SEs have superantigenic activity whereas only a few have been proved to be emetic, representing a potential hazard for consumers. Characterization of staphylococcal food poisoning outbreaks (SFPOs) has considerably progressed compared to 80 years ago, when staphylococci were simply enumerated and only five enterotoxins were known for qualitative detection. Today, SFPOs can be characterized by a number of approaches, such as the identification of S. aureus biovars, PCR and RT-PCR methods to identify the se genes involved, immunodetection of specific SEs, and absolute quantification by mass spectrometry. An integrated gene-to-protein approach for characterizing staphylococcal food poisoning is advocated.

Mycotoxins are small and quite stable molecules which are extremely difficult to remove or eradicate, and which enter the feed chain while keeping their toxic properties. The mycotoxins of major concern as feed contaminants that are potentially removable from feed, are mainly aflatoxins, ochratoxin A, Fusarium toxins (trichothecenes like deoxynivalenol, diacetoxyscirpenol, nivalenol, T2-toxin/HT2-toxin, zearalenone and fumonisins). One of the strategies for reducing the exposure to mycotoxins is to decrease their bioavailability by including various mycotoxin-adsorbing agents in the compound feed, which leads to a reduction of mycotoxin uptake as well as distribution to the blood and target organs. Another strategy is the degradation of mycotoxins into non-toxic metabolites by using biotransforming agents such as bacteria/fungi or enzymes. The specific objectives of the project are to provide a critical and extensive review on mycotoxins detoxifying agents. To this purpose, the consortium performed: The compilation and critical analysis of all published data available in the scientific literature ; The compilation and critical analysis of "grey literature" published worldwide (searches in patents databases); The audition of companies involved in the development and manufacture of mycotoxin-detoxifying agents, as well as stakeholders of feed manufacturer"s; Comparison, validation and synthesis of the reviewed materials. The report made an inventory of agents which may possibly reduce the impact of mycotoxins in feedstuffs by reviewing the literature and by collecting data from feed manufacturer"s or from information published on the Internet. The present report gives also the mechanism of action of the agents listed, by reviewing in vitro studies and identifies the relevant end-points to be studied when testing the efficacy of mycotoxin-detoxifying agents in in vivo trials. Finally this document points out the benefits and risks associated with the potential uses of these agents and gives recommendations for the assessment of such products as feed additives.

Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.

An earlier intralaboratory validation study based on the EN ISO 16140 Standard conducted by the Community Reference Laboratory for coagulase-positive staphylococci including Staphylocococcus aureus showed that, after an extraction step using dialysis concentration, the Vidas SET2 detection kit could be used to screen staphylococcal enterotoxins in milk and milk products. In order to fully validate Vidas SET2, an interlaboratory study was organized. Six freeze-dried samples and 3 ready-to-use concentrated extracts were analyzed by 21 laboratories according to the method, including a detection with Vidas SET2. Results did not show false-positive or -negative results. Accordance and concordance parameters were equal to 100%, corresponding to a concordance odds ratio of 1. This interlaboratory study confirmed the satisfactory outcome of the preliminary tests and of the intralaboratory study performed previously. The Vidas SET2 detection kit can be used as a method for the detection of staphylococcal enterotoxins in milk and milk products as well as the Transia Plate SET detection kit in the European screening method for official control purposes, after an extraction step followed by dialysis concentration.

Aim: Immunological tools used to detect staphylococcal enterotoxins (SEs) in foods are numerous. The aim of this study was to evaluate, on naturally contaminated milk product samples, the performance of the Vidas SET2, in comparison to the Transia plate SET. Methods and Results: The Vidas SET2 was compared with the Transia plate SET on supernatants of Staphylococcus aureus isolates and on naturally contaminated milk products. It is noteworthy that when using IgG rabbit treatment, both kits can be considered as equivalent to detect enterotoxins in naturally contaminated milk products. Conclusions: This study demonstrated that the Vidas SET2 performance is similar to that of Transia plate SET kit, when a rabbit IgG treatment step is used before detection step. This additional treatment significantly decreased, from 42% to 8%, the rate of positive deviations observed using the Transia plate SET detection kit. Significance and Impact of the Study: The Vidas SET2 was clearly found as more specific, when no preliminary rabbit IgG treatment was used, and which results in a better workflow when a large number of samples have to be analysed within a few days. Considering the results obtained, the Vidas SET2 detection kit can be used to assess the safety of milk products for SEs.

Les mycotoxines sont des molecules de faible masse moleculaire issues du metabolisme secondaire des moisissures. Ces molecules sont elaborees par certaines especes de moisissures colonisant des plantes (cereales, fruits, legumes) ou des aliments. D'apres leur nature chimique, les mycotoxines sont tres stables, en particulier thermostables, et difficiles a eliminer. Compte tenu des effets toxiques qu'elles induisent, notamment toxicite chronique de type cancerogenicite, les mycotoxines introduites dans des chaines alimentaires aboutissant a l'homme peuvent exposer celui-ci a un certain nombre de problemes de sante. Toute une population peut etre concernee par la possible contamination d'un ensemble d'aliments par telle ou telle mycotoxine. Aussi, les pouvoirs publics, considerant des la decouverte des aflatoxines qu'il s'agissait d'un probleme de sante publique, ont organise une surveillance reguliere nationale et des controles officiels sur les lieux de production. Des reglements ou des recommandations ont ete edictes au niveau national puis europeen, et des valeurs guides existent au niveau mondial pour fiabiliser les echanges commerciaux des denrees alimentaires du point de vue de la securite sanitaire. De ce fait, de nombreuses techniques d'analyse ont ete developpees pour detecter et quantifier les mycotoxines, la preference allant vers des methodes chromatographiques ou immunochimiques. Des efforts sont deployes par les analystes pour valider leurs methodes et fiabiliser les resultats analytiques puisque d'importantes decisions de retrait ou d'interdiction d'utilisation de matieres premieres ou de denrees alimentaires peuvent reposer sur ces resultats. Le transfert des mycotoxines dans la chaine alimentaire, la demarche analytique, les methodes d'analyse les plus couramment utilisees et les criteres permettant d'assurer la fiabilite des resultats emis seront egalement decrits dans cet article.

Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.

Okadaic acid (OA) is a major toxin involved in diarrhetic shellfish poisoning in humans and has been shown to be both a potent tumor promoter in rodent skin and stomach and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of OA amounts to only a few studies, which give conflicting results. In order to evaluate the ability of OA to induce DNA damage, the cytokinesis-block micronucleus assay was performed in the CHO-K1 cell line. A statistically significant induction of micronuclei without strong cytotoxicity was obtained after a 24 h treatment with 20 (approximately 5-fold) and 30 nM (approximately 10-fold) OA. Then, in order to discriminate between a clastogenic or aneugenic effect of OA, the micronucleus assay was carried out in combination with fluorescence in situ hybridization (FISH) using a (TTAGGG)(n) DNA probe for centromere detection. FISH analysis showed that OA mainly induced centromere-positive micronuclei (68.9% induction with 20 nM OA and 77.0% with 30 nM). Therefore, OA can be considered aneugenic. Using the same assay, biotransformation of OA was studied after a 4 h treatment with and without metabolic activation. The results show that reactive metabolites of OA were generated with a significant increase in genotoxic potential. The relationship between the different components involved in the mitotic process and OA inhibition of protein phosphatase is also discussed.

Among several batches of clams harvested in Tunisia and imported to France, a small number of them were found to be neurotoxic to mice (intraperitoneal injection) as determined by the diarrhetic shellfish poisoning (DSP) bioassay developed by Yasumoto et al. (1978). The present study was conducted to confirm the nature of the toxic agent, suspected to be gymnodimine. Liquid chromatography tandem mass spectrometry analyses unequivocally revealed the presence of gymnodimine in the shellfish, making Tunisia the second country, after New Zealand, where shellfish contamination with this phycotoxin is reported. Gymnodimine B and C analogues were not detected in the clam samples. Gymnodimine preferentially accumulates in the digestive gland of the Tunisian clams, although substantial amounts are also found in the meat.

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic method for determination of aflatoxin M1 in milk at proposed European regulatory limits. The test portion of liquid milk was centrifuged, filtered, and applied to an immunoaffinity column. The column was washed with water, and aflatoxin was eluted with pure acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography (LC) with fluorescence detection. Frozen liquid milk samples both naturally contaminated with aflatoxin M1 and blank samples for spiking, were sent to 12 collaborators in 12 different European countries. Test portions of samples were spiked at 0.05 ng aflatoxin M1 per mL. After removal of 2 noncompliant sets of results, the mean recovery of aflatoxin M1 was 74%. Based on results for spiked samples (blind pairs at 1 level) and naturally contaminated samples (blind pairs at 3 levels) the relative standard deviation for repeatability (RSDr) ranged from 8 to 18%. The relative standard deviation for reproducibility (RSDR) ranged from 21 to 31%. The method showed acceptable within- and between-laboratory precision data for liquid milk, as evidenced by HORRAT values at the low level of aflatoxin M1 contamination.

Glucuronidation of 4-nitrophenol, nopol (a monoterpenoid alcohol) and bilirubin, which in the rat, are catalyzed by three different enzymes, has been examined in liver biopsies from patients with various liver diseases, in particular cholestasis. These different activities were not correlated, which strongly suggests that at least three independently regulated forms of UDP-glucuronosyltransferases were present in the microsomes. Non ionic detergents (Triton X100, Emulgen 911) and deoxycholate produced similar activation (more than 2-fold) of the glucuronidation of 4-nitrophenol. Amphipathic substances, such as CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane sulfonate), and lysophosphatidylcholines maximally increased this UDP-glucuronosyltransferase activity, the most potent being oleoyl lysophosphatidylcholine (4-fold increase). Discriminant analysis of the data revealed no correlation between the three different UDP-glucuronosyltransferase activities and the age or sex of the patients. A good correlation was found on multidimensional analysis between form 1 of the enzyme (4-nitrophenol glucuronidation) and, in decreasing order of magnitude, epoxide hydrolase (measured with benzo(a)pyrene-4,5-oxide as substrate), cytochrome P-450, 7-ethoxycoumarin deethylase, aspartate aminotransferase and gamma-glutamyltransferase (r = 0.89); and between Form 3 of the enzyme (bilirubin glucuronidation) and NADPH cytochrome c reductase, alkaline phosphatase, (r = 0.81). These relationships may reflect the differential variation in enzymatic activities in various hepato-biliary diseases.

Hepatic microsomal glucuronoconjugation of the hypolipidemic drug clofibric acid was characterized in human liver and compared to the acylglucuronide formation of an endogenous substrate, bilirubin. The affinity of UDP-glucuronosyltransferase for bilirubin was 15-fold higher than for clofibric acid; the V max , for the transformation of the two substrates were similar. The analysis of the specific activity in 32 liver biopsies showed that glucuronidation of clofibric acid or bilirubin were comparable in man and in rat. However, UDP-glucuronosyltransferase activity towards clofibric acid exhibited a large interindividual variation in man. Sex or age did not influence the glucuronidation of bilirubin and clofibric acid. Among the drugs given to the patients only clofibrate was able to increase the bilirubin conjugation. No effect of alcohol or smoking on the conjugation of the two substrates was observed. The absence of correlation between UDP-glucuronosyltransferase activities towards clofibric acid and bilirubin together with the specific induction of bilirubin glucuronidation by clofibrate suggested that these arylcarboxylic substrates were conjugated by separate forms of UDP-glucuronosyltransferase in human.

After induction by phenobarbital and 3-methylcholanthrene, UDP-glucuronosyltransferase involved mainly in the conjugation of planar substrates was purified. Compared to the microsomal enzyme, the purified protein exhibited less affinity towards the substrates, but the corresponding Vmaxs were increased. These results were attributed to a change in the lipid environment of the purified enzyme. The conjugation rate for 4-hydroxycoumarine was 15-45 times less than that measured for the 7-hydroxyisomer with the microsomal or the purified enzymes. Immunoprecipitation studies of the enzyme revealed that the two compounds were transformed by the same enzyme, or metabolized by two separate enzymes presenting the same antigenic site. The orientation of the hydroxyl group of planar aglycones in the active site is the determinant for the efficiency of catalysis.

Staphylococcal food poisoning is one of the most common food-borne diseases resulting from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of coagulase-positive staphylococci, mainly Staphylococcus aureus. To date, more than twenty SEs have been described: SEA to SElV. Each of them share superantigenic activity whereas only few of them have been proved to be emetic and thus represent a potentiel hazard for consumers. Characterisation of staphylococcal food poisoning outbreaks are dependent of various parameters such as isolation of coagulase positive staphylococci and detection of staphylococcal enterotoxins. However, various drawbacks such as heat treatment sensitivity of staphylococci or availability of antibodies against all staphylococcal enterotoxins lead to false diagnosis or to a lack of characterisation of food poisoning outbreaks. Thus, in order to characterise and elucidate staphylococcal food poisoning, we developed an integrated approach from gene to protein using the combination of PCR-based tools, ELISA tests and quantitative MS., Les toxi-infections alimentaires collectives staphylococciques représentent ces dernières années en France la seconde cause de toxi-infections alimentaires bactériennes. Ces dernières sont dues à l'ingestion de toxines préformées dans les aliments : les entérotoxines. Actuellement, une vingtaine d'entérotoxines (SEA àSElV) ont été décrites dans la littérature ; parmi celles-ci, toutes possédent une activité superantigéniques mais seules quelques unes possèdent une activité émétique prouvée et présentent donc un risque sanitaire pour le consommateur. Deux problèmes majeurs se posent pour confirmer le diagnostic des épisodes toxiques à staphylocoques. D'une part, les staphylocoques sont sensibles aux traitements thermiques appliqués aux aliments alors que les entérotoxines ne le sont pas, et d'autre part les méthodes de détection des entérotoxines staphylococciques actuellement disponibles ne couvrent pas l'ensemble des entérotoxines décrites à ce jour. Ainsi, et afin de proposer une alternative innovante aux tests immuno-enzymatiques utilisés pour la recherche des entérotoxines staphylococciques dans les matrices alimentaires, nous avons mis en place une démarche intégrée « du gène à la protéine » utilisant des outils de biologie moléculaire, d'immunochimie et de physisco-chimie des protéines pour caractériser, comprendre et élucider les toxi-infections alimentaires collectives à staphylocoques.