Overview of potential adverse health effects of oral exposure to nanocellulose.

Nanocellulose is an emerging material for which several food-related applications are foreseen, for example, novel food, functional food, food additive or in food contact materials. Nanocellulose materials can display a range of possible shapes (fibers, crystals), sizes and surface modifications. For food-related applications in the EU, information on the safety of substances must be assessed. The present review summarizes the current knowledge on (possible) adverse health effects of nanocellulose upon oral exposure, keeping EU regulatory aspects in mind. The overview indicates that toxicity data, especially from in vivo studies, are limited and outcomes are not unambiguous. The hazard assessment is further complicated by: the diversity in morphologies and surface modifications, lack of standard reference materials, limited knowledge about intestinal fate and absorption, analytical difficulties in biological matrices, dispersion issues, the possible presence of impurities and interferences within biological assays. Two subchronic in vivo toxicity studies show no indications of toxicity for two specific nanocellulose materials, even at high doses. However, these studies may have missed certain early or nano-specific toxic effects, such as inflammation potential, for which other, subacute studies provide some indications. Most in vitro studies show no cytotoxicity; however, several indicate that effects on oxidative stress and inflammatory responses depend on differences in size or surface treatments. Further, too few studies assessed genotoxicity of nanocelluloses. Therefore, immunotoxicity, oxidative stress and genotoxicity require further attention, as do absorption and effects on nutrient uptake. Recommendations for future research facilitating the safety assessment and safe-by-design of nanocellulose in food-related applications are provided.

Issues currently complicating the risk assessment of synthetic amorphous silica (SAS) nanoparticles after oral exposure.

Synthetic amorphous silica (SAS) is applied in food products as food additive E 551. It consists of constituent amorphous silicon dioxide (SiO2) nanoparticles that form aggregates and agglomerates. We reviewed recent oral toxicity studies with SAS. Some of those report tissue concentrations of silicon (Si). The results of those studies were compared with recently determined tissue concentrations of Si (and Si-particles) in human postmortem tissues. We noticed inconsistent results of the various toxicity studies regarding toxicity and reported tissue concentrations, which hamper the risk assessment of SAS. A broad range of Si concentrations is reported in control animals in toxicity studies. The Si concentrations found in human postmortem tissues fall within this range. On the other hand, the mean concentration found in human liver is higher than the reported concentrations causing liver effects in some animal toxicity studies after oral exposure to SAS. Also higher liver concentrations are observed in other, negative animal studies. Those inconsistencies could be caused by the presence of other Si-containing chemical substances or particles (which potentially also includes background SAS) and/or different sample preparation and analytical techniques that were used. Other factors which could explain the inconsistencies in outcome between the toxicity studies are the distinct SAS used and different dosing regimes, such as way of administration (dietary, via drinking water, oral gavage), dispersion of SAS and dose. More research is needed to address these issues and to perform a proper risk assessment for SAS in food. The current review will help to progress research on the toxicity of SAS and the associated risk assessment.

Possible effects of titanium dioxide particles on human liver, intestinal tissue, spleen and kidney after oral exposure.

Recent studies reported adverse liver effects and intestinal tumor formation after oral exposure to titanium dioxide (TiO2). Other oral toxicological studies, however, observed no effects on liver and intestine, despite prolonged exposure and/or high doses. In the present assessment, we aimed to better understand whether TiO2 can induce such effects at conditions relevant for humans. Therefore, we focused not only on the clinical and histopathological observations, but also used Adverse Outcome Pathways (AOPs) to consider earlier steps (Key Events). In addition, aiming for a more accurate risk assessment, the available information on organ concentrations of Ti (resulting from exposure to TiO2) from oral animal studies was compared to recently reported concentrations found in human postmortem organs. The overview obtained with the AOP approach indicates that TiO2 can trigger a number of key events in liver and intestine: Reactive Oxygen Species (ROS) generation, induction of oxidative stress and inflammation. TiO2 seems to be able to exert these early effects in animal studies at Ti liver concentrations that are only a factor of 30 and 6 times higher than the median and highest liver concentration found in humans, respectively. This confirms earlier conclusions that adverse effects on the liver in humans as a result of (oral) TiO2 exposure cannot be excluded. Data for comparison with Ti levels in human intestinal tissue, spleen and kidney with effect concentrations were too limited to draw firm conclusions. The Ti levels, though, are similar or higher than those found in liver, suggesting these tissues may be relevant too.

Silicon dioxide and titanium dioxide particles found in human tissues.

Silicon dioxide (silica, SiO2, SAS) and titanium dioxide (TiO2) are produced in high volumes and applied in many consumer and food products. As a consequence, there is a potential human exposure and subsequent systemic uptake of these particles. In this study we show the characterization and quantification of both total silicon (Si) and titanium (Ti), and particulate SiO2 and TiO2 in postmortem tissue samples from 15 deceased persons. Included tissues are liver, spleen, kidney and the intestinal tissues jejunum and ileum. Low-level analysis was enabled by the use of fully validated sample digestion methods combined with (single particle) inductively coupled plasma high resolution mass spectrometry techniques (spICP-HRMS). The results show a total-Si concentration ranging from <2 to 191 mg Si/kg (median values of 5.8 (liver), 9.5 (spleen), 7.7 (kidney), 6.8 (jejunum), 7.6 (ileum) mg Si/kg) while the particulate SiO2 ranged from <0.2 to 25 mg Si/kg (median values of 0.4 (liver), 1.0 (spleen), 0.4 (kidney), 0.7 (jejunum, 0.6 (ileum) mg Si/kg), explaining about 10% of the total-Si concentration. Particle sizes ranged from 150 to 850 nm with a mode of 270 nm. For total-Ti the results show concentrations ranging from <0.01 to 2.0 mg Ti/kg (median values of 0.02 (liver), 0.04 (spleen), 0.05 (kidney), 0.13 (jejunum), 0.26 (ileum) mg Ti/kg) while particulate TiO2 concentrations ranged from 0.01 to 1.8 mg Ti/kg (median values of 0.02 (liver), 0.02 (spleen), 0.03 (kidney), 0.08 (jejunum), 0.25 (ileum) mg Ti/kg). In general, the particulate TiO2 explained 80% of the total-Ti concentration. This indicates that most Ti in these organ tissues is particulate material. The detected particles comprise primary particles, aggregates and agglomerates, and were in the range of 50-500 nm with a mode in the range of 100-160 nm. About 17% of the detected TiO2 particles had a size <100 nm. The presence of SiO2 and TiO2 particles in liver tissue was confirmed by scanning electron microscopy with energy dispersive X-ray spectrometry.

Considerations for Safe Innovation: The Case of Graphene.

The terms "Safe innovation" and "Safe(r)-by-design" are currently popular in the field of nanotechnology. These terms are used to describe approaches that advocate the consideration of safety aspects already at an early stage of the innovation process of (nano)materials and nanoenabled products. Here, we investigate the possibilities of considering safety aspects during various stages of the innovation process of graphene, outlining what information is already available for assessing potential hazard, exposure, and risks. In addition, we recommend further steps to be taken by various stakeholders to promote the safe production and safe use of graphene.

Nanomedicinal products: a survey on specific toxicity and side effects.

Due to their specific properties and pharmacokinetics, nanomedicinal products (NMPs) may present different toxicity and side effects compared to non-nanoformulated, conventional medicines. To facilitate the safety assessment of NMPs, we aimed to gain insight into toxic effects specific for NMPs by systematically analyzing the available toxicity data on approved NMPs in the European Union. In addition, by comparing five sets of products with the same active pharmaceutical ingredient (API) in a conventional formulation versus a nanoformulation, we aimed to identify any side effects specific for the nano aspect of NMPs. The objective was to investigate whether specific toxicity could be related to certain structural types of NMPs and whether a nanoformulation of an API altered the nature of side effects of the product in humans compared to a conventional formulation. The survey of toxicity data did not reveal nanospecific toxicity that could be related to certain types of structures of NMPs, other than those reported previously in relation to accumulation of iron nanoparticles (NPs). However, given the limited data for some of the product groups or toxicological end points in the analysis, conclusions with regard to (a lack of) potential nanomedicine-specific effects need to be considered carefully. Results from the comparison of side effects of five sets of drugs (mainly liposomes and/or cytostatics) confirmed the induction of pseudo-allergic responses associated with specific NMPs in the literature, in addition to the side effects common to both nanoformulations and regular formulations, eg, with liposomal doxorubicin, and possibly liposomal daunorubicin. Based on the available data, immunotoxicological effects of certain NMPs cannot be excluded, and we conclude that this end point requires further attention.

Oral intake of added titanium dioxide and its nanofraction from food products, food supplements and toothpaste by the Dutch population.

Titanium dioxide (TiO2) is commonly applied to enhance the white colour and brightness of food products. TiO2 is also used as white pigment in other products such as toothpaste. A small fraction of the pigment is known to be present as nanoparticles (NPs). Recent studies with TiO2 NPs indicate that these particles can have toxic effects. In this paper, we aimed to estimate the oral intake of TiO2 and its NPs from food, food supplements and toothpaste in the Dutch population aged 2 to over 70 years by combining data on food consumption and supplement intake with concentrations of Ti and TiO2 NPs in food products and supplements. For children aged 2-6 years, additional intake via ingestion of toothpaste was estimated. The mean long-term intake to TiO2 ranges from 0.06 mg/kg bw/day in elderly (70+), 0.17 mg/kg bw/day for 7-69-year-old people, to 0.67 mg/kg bw/day in children (2-6 year old). The estimated mean intake of TiO2 NPs ranges from 0.19 µg/kg bw/day in elderly, 0.55 µg/kg bw/day for 7-69-year-old people, to 2.16 µg/kg bw/day in young children. Ninety-fifth percentile (P95) values are 0.74, 1.61 and 4.16 µg/kg bw/day, respectively. The products contributing most to the TiO2 intake are toothpaste (in young children only), candy, coffee creamer, fine bakery wares and sauces. In a separate publication, the results are used to evaluate whether the presence of TiO2 NPs in these products can pose a human health risk.

P-gp efflux pump inhibition potential of common environmental contaminants determined in vitro.

Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp-overexpressing Madin-Darby canine kidney cells (MDCKII-MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM-based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp-mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices.

Sample preparation for combined chemical analysis and in vitro bioassay application in water quality assessment.

The combination of in vitro bioassays and chemical screening can provide a powerful toolbox to determine biologically relevant compounds in water extracts. In this study, a sample preparation method is evaluated for the suitability for both chemical analysis and in vitro bioassays. A set of 39 chemicals were spiked to surface water, which were extracted using Oasis MCX cartridges. The extracts were chemically analyzed by liquid chromatography linear ion trap Orbitrap analysis and recoveries appeared to be on average 61% Compounds with logK(ow) values in the range between 0 and 4 are recovered well using this method. In a next step, the same extracts were tested for genotoxic activity using the Comet assay and Ames fluctuation test and for specific endocrine receptor activation using a panel of CALUX assays, for estrogenic (ER), androgenic (AR), glucocorticoid (GR), progestagenic (PR), and thyroidogenic (TR) agonistic activities. The results of the genotoxicity assays indicated that spiked genotoxic compounds were preserved during sample preparation. The measured responses of the GR CALUX and ER CALUX assays were similar to the predicted responses. The measured responses in the AR CALUX and PR CALUX assays were much lower than expected from the analytical concentration, probably due to antagonistic effects of some spiked compounds. Overall, the presented sample preparation method seems to be suitable for both chemical analysis and specific in vitro bioassay applications.

Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays.

To screen for hormonal activity in water samples, highly sensitive in vitro CALUX bioassays are available which allow detection of estrogenic (ERα), androgenic (AR), progestagenic (PR), and glucocorticoid (GR) activities. This paper presents trigger values for the ERα, AR, PR, and GR CALUX bioassays for agonistic hormonal activities in (drinking) water, which define a level above which human health risk cannot be waived a priori and additional examination of specific endocrine activity may be warranted. The trigger values are based on 1) acceptable or tolerable daily intake (ADI/TDI) values of specific compounds, 2) pharmacokinetic factors defining their bioavailability, 3) estimations of the bioavailability of unknown compounds with equivalent hormonal activity, 4) relative endocrine potencies, and 5) physiological, and drinking water allocation factors. As a result, trigger values of 3.8ng 17ß-estradiol (E2)-equivalents (eq)/L, 11ng dihydrotestosterone (DHT)-eq/L, 21ng dexamethasone (DEX)-eq/L, and 333ng Org2058-eq/L were derived. Benchmark Quotient (BQ) values were derived by dividing hormonal activity in water samples by the derived trigger using the highest concentrations detected in a recent, limited screening of Dutch water samples, and were in the order of (value) AR (0.41)>ERα (0.13)>GR (0.06)>PR (0.04). The application of trigger values derived in the present study can help to judge measured agonistic hormonal activities in water samples using the CALUX bioassays and help to decide whether further examination of specific endocrine activity followed by a subsequent safety evaluation may be warranted, or whether concentrations of such activity are of low priority with respect to health concerns in the human population. For instance, at one specific drinking water production site ERα and AR (but no GR and PR) activities were detected in drinking water, however, these levels are at least a factor 83 smaller than the respective trigger values, and therefore no human health risks are to be expected from hormonal activity in Dutch drinking water from this site.

Effect of combining in vitro estrogenicity data with kinetic characteristics of estrogenic compounds on the in vivo predictive value.

With the ultimate aim of increasing the utility of in vitro assays for toxicological risk assessment, a method was developed to calculate in vivo estrogenic potencies from in vitro estrogenic potencies of compounds by taking into account systemic availability. In vitro estrogenic potencies of three model compounds (bisphenol A, genistein, and 4-nonylphenol) relative to ethinylestradiol (EE2), determined with the estrogen receptor alpha (ERα) transcriptional activation assay using hER-HeLa-9903 cells, were taken from literature and used to calculate the EE2 equivalent (EE2EQ) effect doses in the predominantly ERα-dependent rat uterotrophic assay. Compound-specific differences in hepatic clearance relative to the reference compound EE2 were determined in vitro to examine whether in vivo estrogenic potencies reported in literature could be more accurately estimated. The EE2EQ doses allowed to predict in vivo uterotrophic responses within a factor of 6-25 and the inclusion of the hepatic clearance further improved the prediction with a factor 1.6-2.1 for especially genistein and bisphenol A. Yet, the model compounds still were less potent in vivo than predicted based on their EE2 equivalent estrogenic potency and hepatic clearance. For further improvement of the in vitro to in vivo predictive value of in vitro assays, the relevance of other kinetic characteristics should be studied, including binding to carrier proteins, oral bioavailability and the formation of estrogenic metabolites.

Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells.

The citrus flavonoid hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of hesperetin, was studied using Sf9 membranes from cells overexpressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transporters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC(50) of 0.45 µM, suggesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRP ATPase activity at high H3'G concentrations was observed. This study provides information on the potential of hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of hesperetin.

Stereoselective conjugation, transport and bioactivity of s- and R-hesperetin enantiomers in vitro.

The flavanone hesperetin ((+/-)-4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, which is the major flavonoid present in sweet oranges. Hesperetin contains a chiral C-atom and so can exist as an S- and R-enantiomer, however, in nature 2S-hesperidin and its S-hesperetin aglycone are predominant. The present study reports a chiral HPLC method to separate S- and R-hesperetin on an analytical and semipreparative scale. This allowed characterization of the stereoselective differences in metabolism and transport in the intestine and activity in a selected bioassay of the separated hesperetin enantiomers in in vitro model systems: (1) with human small intestinal fractions containing UDP-glucuronosyl transferases (UGTs) or sulfotransferases (SULTs); (2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; (3) with mouse Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-mediated gene expression. The results obtained indicate some significant differences in the metabolism and transport characteristics and bioactivity between S- and R-hesperetin, however, these differences are relatively small. This indicates that for these end points, including intestinal metabolism and transport and EpRE-mediated gene induction, experiments performed with racemic hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer. This is an important finding since at present hesperetin is only commercially available as a racemic mixture, while it exists in nature mainly as an S-enantiomer.

The effect of co-administered flavonoids on the metabolism of hesperetin and the disposition of its metabolites in Caco-2 cell monolayers.

Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers in a two-compartment transwell system. Flavonoids may interfere with hesperetin metabolism and can also inhibit the apically located ABC transporter breast cancer resistance protein (ABCG2) which was previously shown to be responsible for the apical transport of hesperetin metabolites. Co-exposure of Caco-2 cell monolayers to hesperetin with specific flavonoids reduced the ratio of apical efflux to basolateral transport of hesperetin metabolites, and in some cases, also reduced the amount of hesperetin metabolites detected extracellularly. As intracellular accumulation of hesperetin metabolites did not account for this decrease, inhibition of metabolism of hesperetin is likely the underlying mechanism for the reduced metabolite formation and excretion. In spite of the reduction in metabolism the amount of hesperetin metabolites transported to the basolateral side significantly increased upon co-exposure with specific flavonoids and therefore co-administration of specific flavonoids could be a strategy to improve the bioavailability of hesperetin.

Phase II metabolism of hesperetin by individual UDP-glucuronosyltransferases and sulfotransferases and rat and human tissue samples.

Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.

Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers.

Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied hesperetin (10 microM) was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm(2)), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 micro M), the apical efflux of hesperetin conjugates was 1.9-fold reduced (p

Flavonoid-mediated inhibition of intestinal ABC transporters may affect the oral bioavailability of drugs, food-borne toxic compounds and bioactive ingredients.

The transcellular transport of ingested food ingredients across the intestinal epithelial barrier is an important factor determining bioavailability upon oral intake. This transcellular transport of many chemicals, food ingredients, drugs or toxic compounds over the intestinal epithelium can be highly dependent on the activity of membrane bound ATP binding cassette (ABC) transport proteins, able to export the compounds from the intestinal cells. The present review describes the ABC transporters involved in the efflux of bioactive compounds from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, reducing bioavailability. The role of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The present paper focuses on the role of flavonoids as important modulators or substrates of intestinal ABC transport proteins. Several examples of such an effect of flavonoids are presented. It can be concluded that flavonoid-mediated inhibition of ABC transporters may affect the bioavailability of drugs, bioactive food ingredients and/or food-borne toxic compounds upon oral uptake. All together it appears that the flavonoid-mediated interactions at the level of the intestinal ABC transport proteins may be an important mechanism for unexpected food-drug, food-toxin or food-food interactions. The overview also indicates that future studies should focus on i) in vivo validation of the flavonoid-mediated effects on bioavailability of drugs, toxins and beneficial bioactive food ingredients detected in in vitro models, and on ii) the role of flavonoid phase II metabolism in modulating the activity of the flavonoids to act as ABC transporter inhibitors and/or substrates.

Human cytochrome p450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1'-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1'-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1'-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.