Evaluation of the interactions of herbal dietary supplements with molecular targets at the intestinal level : a first step toward risk assessment

Since a few years, nutritional habits are changing. We assist to a growing use of herbal dietary supplements, which are sold freely without medical advice, despite their recognized physiological effects. Little is known about the safety of these products. The need for relevant toxicological data, generated at plausible exposure, is therefore encouraging researchers to develop new approaches of risk assessment, adapted to the specificities of herbal dietary supplements, as well as high-throughput in vitro screening tools. Due its high potential of first-pass metabolization, the small intestine, which is exposed to high doses of diverse dietary compounds, plays a key role in food safety. In a first part of the work, a review of the interactions between dietary compounds, including contaminants and phytochemicals, and main first-pass biotransformation enzymes permitted to highlight the major role of cytochrome P-450 (CYP), and especially CYP1A1, in determining bioavailability and potential toxicity at the intestinal level. We therefore dedicated the first experimental part of the work to the study of the modulation of CYP1A1 activity by Ginkgo biloba, a widely used herbal dietary supplement, and by its major active compounds, alone or in mixtures, in the human intestinal Caco-2 cells, at realistic exposure. Ginkgo biloba extracts demonstrated to strongly enhance CYP1A1 activity, suggesting unsuspected side effects for consumers. In a second experimental part, on the basis of our previous experience with Caco-2 cells, we set up an original methodology, involving four distinct in vitro tools packed together in a toolbox, and permitting to easily investigate relevant toxicological effects of dietary compounds at realistic human exposure, with a special interest for the intestinal compartment. This method was validated with four well-known dietary contaminants showing human health concern (ochratoxin A, deoxynivalenol, imazalil and benomyl), in order to be applied in the future to other less studied chemicals, such as active compounds of herbal dietary supplements. Given the necessity to implement our knowledge in the safety of herbal dietary supplements, we have ended up our work by discussing the application of the toolbox as a first step for the risk assessment of these products. Taken as a whole, this thesis opens doors towards the identification of priorities for risk assessment studies and emphasizes the relevance of in vitro tools in food safety.(AGRO 3) -- UCL, 201

Linseed oil presents different patterns of oxidation in real-time and accelerated aging assays

This study aimed at verifying if the hypothesis that one day at 60 °C is equivalent to one month at 20 °C could be confirmed during linseed oil aging for 6 months at 20 °C and 6 days at 60 °C using the “Schaal oven stability test”. Tests were conducted with linseed oil supplemented or not with myricetin or butyl-hydroxytoluene as antioxidants. Oxidation was evaluated with the peroxide and p-anisidine values, as well as the content in conjugated dienes and aldehydes. All four indicators of oxidation showed very different kinetic behaviors at 20 and 60 °C. The hypothesis is thus not verified for linseed oil, supplemented or not with antioxidant. In the control oil, the conjugated dienes and the peroxide value observed were respectively of 41.8 ± 0.8 Absorbance Unit (AU)/g oil and 254.3 ± 5.8 meq. O2/kg oil after 6 months at 20 °C. These values were of 18.2 ± 1.3 AU/g oil and 65.2 ± 20.3 meq. O2/kg after 6 days at 60 °C

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10–400 μg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 μg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 μg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24 h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6 h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements

Modulation of cytochrome P450 1A1 expression and activity in intestinal Caco-2 cells by components of Ginkgo biloba-based dietary supplements

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Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level

The intestinal mucosa is not simply a barrier allowing entry of compounds such as nutrients or chemicals, and restricting that of others. Intestinal cells and activities perform selective absorption, biotransformations and efflux back to the lumen. Furthermore, food substances affect both bioavailability and intestinal function. Some are able to act as transcriptional regulators and enzyme modulators. This review points out plausible interactions between food contaminants and/or natural constituents at molecular and cellular levels and focuses on the effects of classical (pesticides and veterinary drugs), environmental (heavy metals, PCBs, dioxins, etc.) and food processing generated (PAHs, heterocyclic amines, etc.) contaminants on absorption, metabolism and efflux. Special attention is given to secondary metabolites of molds (mycotoxins) and plants (polyphenols). Molecular targets are briefly described as well as regulation mechanisms. Where possible, data referred to deal with human intestinal functions in vivo, and with in vitro studies on human intestinal Caco-2 cells; however, since data related to the intestine are rather scarce, effects on molecular targets in liver are also considered. This review also points out the urgent need for fully validated high throughput in vitro tools to screen combinations of substances, at realistic intestinal concentrations. A higher priority could then be given to combinations of nutrients, xenobiotics and food contaminants, with hazardous or beneficial impacts on human health. (c) 2007 Elsevier Ltd. All rights reserved

Imazalil modulates CYPs 1A1 and 3A4 activities in the human Caco-2 cells as an intestinal model to assess food safety

There are increasing evidences that ingestion of contaminated food provokes interactions at the intestinal level of major concern for food safety and appearance of drug interactions. We currently use human intestinal Caco-2 cells to evaluate interactions between chemicals and intestinal xenobiotic-metabolizing enzymes, mainly CYPs 1A1 and 3A4 isoforms, as well as apical efflux transporters. Imazalil (IMA) is a widely used post-harvest fungicide and, as such, a relevant food contaminant. IMA strongly induced CYP1A1 activity in a dose- and time-dependent manner. At low, but realistic intestinal concentrations (25 ppb), CYP1A1 activity increased rapidly, but only transiently, suggesting IMA biotransformation and/or (intralysosomal) accumulation by Caco-2 cells. In these cells, IMA was not an aryl hydrocarbon receptor (AhR) agonist, as revealed by reporter gene assay. In TCDD-induced cells, IMA showed no antagonistic effect towards CYP1A1 activity. Finally, some polyphenols reduced the IMA-induced CYP1A1 activity, whereas an additive effect was observed upon coincubation of IMA with benzo(a)pyrene. IMA also appeared as a CYP3A4 inhibitor as potent as ketoconazole, a classical pharmacological inhibitor. In addition, IMA seems to be without effect on either PgP or MRP-2 efflux pumps. HPLC analyses were carried out in order to evaluate IMA absorption by Caco-2 cells. These data reveal that IMA is a potent inducer of CYP1A1 activity in human intestinal Caco-2 cells, occurring by an AhR-independent mechanism, as well as a potent inhibitor of CYP3A4. These CYPs interactions may potentially cause adverse effects on human health by activating procarcinogenic food contaminants or by modulating drug bioavailability

Validation of the Analytical Procedure for the Determination of Malondialdehyde and Three Other Aldehydes in Vegetable Oil Using Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS) and Application to Linseed Oil

Secondary oxidation products of fatty acids, mainly aldehydes, are susceptible to cause significant deterioration in chemical, sensory and nutritional food properties, as well as adverse health effects. An analytical method involving separation by liquid chromatography coupled to the detection by tandem mass spectrometry (LC-MS/MS) has been developed to evaluate the concentration of four aldehydes in oil samples: malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), 4-hydroxy-2-hexenal (4-HHE) and 2,4-decadienal (2,4-DECA). The optimisation of the extraction, derivation, detection and quantification has been finalised for coconut oil, used as a model of vegetable oils. The method has been validated according to the criteria and procedure described in international standards. The evaluated parameters include specificity/selectivity, recovery, precision, accuracy, uncertainty, limits of detection and quantification, using the concept of accuracy profiles. These parameters have been evaluated during experiments planned on different non-consecutive days with coconut oil spiked at different levels of concentration. The validation of the developed analytical method showed that it is possible to analyse MDA, 4-HHE, 4-HNE and 2,4-decadienal in oil samples, in the same run, with a very good accuracy for MDA, and a defined accuracy at specified concentrations for the three other aldehydes. The accuracy profile of MDA showed a recovery rate of 100 % (±1) and a maximum coefficient of variation for the intermediate precision of 14 % at 0.15 mg kg−1. For the three other aldehydes, recovery rates ranged between 79 and 101 % and coefficient of variation for the intermediate precision between 13 and 23 %. In first pressure linseed oil, stored for several days at 60 °C according to the Schaal oven test, it was shown that 4-HHE was the most produced aldehyde, reaching levels of 85 and 382 μmol kg−1 after 12 and 24 days, respectively, versus levels of 18 and 28 μmol MDA kg−1 of oil, respectively, and 17 and 51 μmol 4-HNE kg−1 of oil. © 2014, Springer Science+Business Media New York