Evaluation of Interindividual Human Variation in Bioactivation and DNA Adduct Formation of Estragole in Liver Predicted by Physiologically Based Kinetic/Dynamic and Monte Carlo Modeling.

Estragole is a known hepatocarcinogen in rodents at high doses following metabolic conversion to the DNA-reactive metabolite 1'-sulfooxyestragole. The aim of the present study was to model possible levels of DNA adduct formation in (individual) humans upon exposure to estragole. This was done by extending a previously defined PBK model for estragole in humans to include (i) new data on interindividual variation in the kinetics for the major PBK model parameters influencing the formation of 1'-sulfooxyestragole, (ii) an equation describing the relationship between 1'-sulfooxyestragole and DNA adduct formation, (iii) Monte Carlo modeling to simulate interindividual human variation in DNA adduct formation in the population, and (iv) a comparison of the predictions made to human data on DNA adduct formation for the related alkenylbenzene methyleugenol. Adequate model predictions could be made, with the predicted DNA adduct levels at the estimated daily intake of estragole of 0.01 mg/kg bw ranging between 1.6 and 8.8 adducts in 10(8) nucleotides (nts) (50th and 99th percentiles, respectively). This is somewhat lower than values reported in the literature for the related alkenylbenzene methyleugenol in surgical human liver samples. The predicted levels seem to be below DNA adduct levels that are linked with tumor formation by alkenylbenzenes in rodents, which were estimated to amount to 188-500 adducts per 10(8) nts at the BMD10 values of estragole and methyleugenol. Although this does not seem to point to a significant health concern for human dietary exposure, drawing firm conclusions may have to await further validation of the model's predictions.

Evaluation of the interindividual human variation in bioactivation of methyleugenol using physiologically based kinetic modeling and Monte Carlo simulations.

The present study aims at predicting the level of formation of the ultimate carcinogenic metabolite of methyleugenol, 1'-sulfooxymethyleugenol, in the human population by taking variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Depending on the metabolic route, variation was simulated based on kinetic constants obtained from incubations with a range of individual human liver fractions or by combining kinetic constants obtained for specific isoenzymes with literature reported human variation in the activity of these enzymes. The results of the study indicate that formation of 1'-sulfooxymethyleugenol is predominantly affected by variation in i) P450 1A2-catalyzed bioactivation of methyleugenol to 1'-hydroxymethyleugenol, ii) P450 2B6-catalyzed epoxidation of methyleugenol, iii) the apparent kinetic constants for oxidation of 1'-hydroxymethyleugenol, and iv) the apparent kinetic constants for sulfation of 1'-hydroxymethyleugenol. Based on the Monte Carlo simulations a so-called chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived by dividing different percentiles by the 50th percentile of the predicted population distribution for 1'-sulfooxymethyleugenol formation. The obtained CSAF value at the 90th percentile was 3.2, indicating that the default uncertainty factor of 3.16 for human variability in kinetics may adequately cover the variation within 90% of the population. Covering 99% of the population requires a larger uncertainty factor of 6.4. In conclusion, the results showed that adequate predictions on interindividual human variation can be made with Monte Carlo-based PBK modeling. For methyleugenol this variation was observed to be in line with the default variation generally assumed in risk assessment.

Effects of pre-exercise sucralose ingestion on carbohydrate oxidation during exercise.

Recent studies have demonstrated a direct link between increased exogenous CHO oxidation (CHOexog) and enhanced performance. The limiting factor for CHOexog appears to be at the level of intestinal transporters, with sodium/glucose cotransporter 1 (SGLT1) and glucose transporter Type 5 (GLUT5) responsible for glucose and fructose transport, respectively. Studies in animal models have shown that SGLT1 and intestinal glucose uptake are up-regulated by high carbohydrate diets or noncaloric sweeteners. The aim of this study was to determine the effect of preexercise ingestion of noncaloric sweeteners on CHOexog during exercise in athletes. In a randomized, crossover, double-blind fashion twenty-three healthy male cyclists (age = 29 ± 7 yrs, mass = 73.6 ± 7.4 kg, VO2peak = 68.3 ± 9.3 ml/kg/min) consumed 8 × 50 ml doses of either placebo (CON) or 1mM sucralose (SUCRA) every 15 min starting 120 min before the onset of exercise. This was followed by 2h of cycling at 48.5 ± 8.6% of VO2peak with continual ingestion of a maltodextrin drink (1.2 g/min; 828 ml/ hr). Average CHOexog during the first hour of exercise did not differ between SUCRA and CON conditions (0.226 ± 0.081 g/min vs. 0.212 ± 0.076 g/min, Δ =0.015 g/min, 95% CI -0.008 g/min, 0.038 g/min, p = .178). Blood glucose, plasma insulin and lactate, CHO and fat substrate utilization, heart rate, ratings of perceived exertion, and gastrointestinal symptoms did not differ between conditions. Our data suggest that consumption of noncaloric sweeteners in the immediate period before exercise does not lead to a significant increase in CHOexog during exercise.

Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat.

This study defines a physiologically based kinetic (PBK) model for methyleugenol (ME) in human based on in vitro and in silico derived parameters. With the model obtained, bioactivation and detoxification of methyleugenol (ME) at different doses levels could be investigated. The outcomes of the current model were compared with those of a previously developed PBK model for methyleugenol (ME) in male rat. The results obtained reveal that formation of 1'-hydroxymethyleugenol glucuronide (1'HMEG), a major metabolic pathway in male rat liver, appears to represent a minor metabolic pathway in human liver whereas in human liver a significantly higher formation of 1'-oxomethyleugenol (1'OME) compared with male rat liver is observed. Furthermore, formation of 1'-sulfooxymethyleugenol (1'HMES), which readily undergoes desulfonation to a reactive carbonium ion (CA) that can form DNA or protein adducts (DA), is predicted to be the same in the liver of both human and male rat at oral doses of 0.0034 and 300 mg/kg bw. Altogether despite a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1'-hydroxymethyleugenol (1'HME) between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol (ME) to 1'-sulfooxymethyleugenol (1'HMES) appears to be negligible. Moreover, the PBK model predicted the formation of 1'-sulfooxymethyleugenol (1'HMES) in the liver of human and rat to be linear from doses as high as the benchmark dose (BMD10) down to as low as the virtual safe dose (VSD). This study shows that kinetic data do not provide a reason to argue against linear extrapolation from the rat tumor data to the human situation.

In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling.

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

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.

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.

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes.

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1'-hydroxyestragole. The model was extended by linking the area under the curve for 1'-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1'-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya.

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)-LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC(50) (0.8-3.2 microM) and a comparable induction factor (17-22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC(50) 3.9-6.5 microM, induction factor 17.5-23). After 24h of exposure, on average (+/-SD) 23+/-5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.

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

A physiologically based biokinetic (PBBK) model for estragole bioactivation and detoxification in rat.

The present study defines a physiologically based biokinetic (PBBK) model for the alkenylbenzene estragole in rat based on in vitro metabolic parameters determined using relevant tissue fractions, in silico derived partition coefficients, and physiological parameters derived from the literature. The model consists of eight compartments including liver, lung and kidney as metabolizing compartments, and additional compartments for fat, arterial blood, venous blood, rapidly perfused tissue and slowly perfused tissue. Evaluation of the model was performed by comparing the PBBK predicted dose-dependent formation of the estragole metabolites 4-allylphenol and 1'-hydroxyestragole glucuronide to literature reported levels of these metabolites, which were demonstrated to be in the same order of magnitude. With the model obtained the relative extent of bioactivation and detoxification of estragole at different oral doses was examined. At low doses formation of 4-allylphenol, leading to detoxification, is observed to be the major metabolic pathway, occurring mainly in the lung and kidney due to formation of this metabolite with high affinity in these organs. Saturation of this metabolic pathway in the lung and kidney leads to a relative increase in formation of the proximate carcinogenic metabolite 1'-hydroxyestragole, occurring mainly in the liver. This relative increase in formation of 1'-hydroxyestragole leads to a relative increase in formation of 1'-hydroxyestragole glucuronide and 1'-sulfooxyestragole the latter being the ultimate carcinogenic metabolite of estragole. These results indicate that the relative importance of different metabolic pathways of estragole may vary in a dose-dependent way, leading to a relative increase in bioactiviation of estragole at higher doses.

Glutathione-dependent interaction of heavy metal compounds with multidrug resistance proteins MRP1 and MRP2.

The interactions of three heavy metal-containing compounds, cisplatin (CDDP), arsenic trioxide (As(2)O(3)), and mercury dichloride (HgCl(2)), with the multidrug resistance transporters MRP1 and MRP2 and the involvement of glutathione (GSH)-related processes herein were investigated. In Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, viability, GSH content, calcein efflux and polarized GSH efflux were measured as a function of exposure to CDDP, As(2)O(3) and HgCl(2). In isolated Sf9-MRP1 and Sf9-MRP2 membrane vesicles, the interaction with MRP-associated ATPase activity was measured. In the latter model system adduct formation with GSH is not an issue. The data show that (1) CDDP interacts with both MRP1 and MRP2, and GSH appears to play no major role in this process, (2) As(2)O(3) interacts with MRP1 and MRP2 in which process GSH seems to be essential, and (3) HgCl(2) interacts with MRP1 and MRP2, either alone and/or as a metal-GSH complex.

Functional peptides by genome reverse engineering.

Computational biology and chemistry combined with high-throughput analytical technologies contribute to reduce operational costs and foster innovation in every phase of the discovery of bioactive molecules. In order for life science industries to continue to deliver at the required market rate, new concepts need to be implemented in research and development, and new sources of bioactive molecules should be investigated. The genomic revolution provides the necessary information to generate novel bioactive peptides by the computational dissection of genomes.

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from the human NAD(P)H:quinone oxidoreductase (hNQO1) and the mouse glutathione-S-transferase Ya (mGST-Ya) gene, containing one and two tandem EpRE core sequences, respectively. The standard inducer tert-butylhydroquinone (tBHQ), the electrophile benzyl isothiocyanate (BITC), and the antioxidant flavonoid quercetin were found to induce luciferase expression, thereby validating these newly developed reporter cell lines. For tBHQ and BITC, but not for quercetin, higher maximum luciferase induction was found under control of the mGST-Ya EpRE as compared to the hNQO1 EpRE, pointing at different induction mechanisms. Furthermore, we investigated the structure-activity relationship for induction of luciferase expression by flavonoids in EpRE(mGST-Ya)-LUX cells, and also the relation between luciferase induction and flavonoid antioxidant potency. Five different flavonoids with a planar molecular structure were found to induce various levels of luciferase activity, whereas taxifolin, a non-planar flavonoid, did not induce luciferase activity. This suggests that a stereospecific molecular interaction may be important for EpRE-mediated gene activation, possibly with Keap1, a regulator of EpRE-controlled transcription, or with another effector or receptor protein. No consistent relation between luciferase induction level and flavonoid antioxidant potential was observed. Altogether, these results point to differences in induction mechanism between the various chemoprotective compounds tested. The newly developed stably transfected reporter cell lines provide a validated tool for future screening and mechanistic studies of EpRE-mediated gene transcription.

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.

Association between consumption of cruciferous vegetables and condiments and excretion in urine of isothiocyanate mercapturic acids.

A high intake of cruciferous vegetables is associated with a reduced risk of cancer and cardiovascular diseases. This protective effect has been linked to isothiocyanates, enzymatic hydrolysis products of glucosinolates. In this study, the metabolic fate of glucosinolates and isothiocyanates after ingestion of 19 different cruciferous vegetables was studied in three male subjects. After the consumption of 13 cruciferous vegetables (glucosinolate content, 0.01-0.94 mmol/kg) and six condiments (isothiocyanate content, 0.06-49.3 mmol/kg), eight different isothiocyanate mercapturic acids were determined in urine samples. Excretion levels after the consumption of raw vegetables and condiments were higher (bioavailability, 8.2-113%) as compared to cooked vegetables (bioavailability, 1.8-43%), but the excretion rate was similar (t1/2=2.1-3.9 h). Isothiocyanates in urine remain longer at a nonzero level after the consumption of glucosinolates from cooked vegetables, as compared to raw vegetables and condiments, and maximal levels in urine were reached about 4 h later. Isothiocyanate mercapturic acids can be used as a biomarker to reflect the active dose of isothiocyanates absorbed.

Effect of Glucuronidation on the Potential of Kaempferol to Inhibit Serine/Threonine Protein Kinases.

To study the effect of metabolic conjugation of flavonoids on the potential to inhibit protein kinase activity, the inhibitory effects of the dietary flavonol kaempferol and its major plasma conjugate kaempferol-3-O-glucuronide on protein kinases were studied. To this end, the inhibition of the phosphorylation activity of recombinant protein kinase A (PKA) and of cell lysate from the hepatocellular carcinoma cell line HepG2 on 141 putative serine/threonine phosphorylation sites derived from human proteins was assessed. Glucuronidation reduced the inhibitory potency of kaempferol on the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively, but did not appear to affect the target selectivity for kinases present in the lysate. The data demonstrate that, upon glucuronidation, kaempferol retains part of its intrinsic kinase inhibition potential, which implies that K3G does not necessarily need to be deconjugated to the aglycone for a potential inhibitory effect on protein kinases.

Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of alpha,beta-unsaturated carbonyl compounds.

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of alpha,beta-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. Alpha,beta-unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin.

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.