Species Differences in in vitro and Estimated in vivo Kinetics for Intestinal Microbiota Mediated Metabolism of Acetyl-deoxynivalenols.

SCOPE: Deoxynivalenol (DON) and its acetylated derivatives 3-acetyl-DON (3-Ac-DON) and 15-acetyl-DON (15-Ac-DON) are important mycotoxins of concern in the modern food chain. METHODS AND RESULTS: The present study reveals that the rate of de-acetylation in in vitro anaerobic fecal incubations decreased in the order rat > mouse > human > pig for 3-Ac-DON, and mouse > human > rat > pig for 15-Ac-DON. The ratio between the de-acetylation rate of 3-Ac-DON and 15-Ac-DON varies with the species. Scaling of the kinetic parameters to the in vivo situation results in catalytic efficiencies decreasing in the order human > rat > pig > mouse for 3-Ac-DON and human > pig > rat > mouse for 15-Ac-DON. The results obtained indicate that in mice, 3-Ac-DON can be fully deconjugated while 15-Ac-DON cannot. In rats, pigs, and humans, both 3-Ac-DON and 15-Ac-DON can be totally transformed by gut fecal microbiota during the estimated intestinal residence time. A correlation analysis between the deacetylation rate and the relative abundance of the microbiome suggests Lachnospiraceae may be involved in the deacetylation process. CONCLUSION: It is concluded that interspecies differences in deacetylation of acetylated DONs exist but that in risk assessment assumption of complete intestinal deconjugation provides an adequate approach.

An in vitromodel to quantify interspecies differences in kinetics for intestinal microbial bioactivation and detoxification of zearalenone.

Zearalenone (ZEN) is a mycotoxin known for its estrogenic activities. The metabolism of ZEN plays a role in the interspecies differences in sensitivity to ZEN, and is known to occur in the liver and via the intestinal microbiota, although the relative contribution of these two pathways remains to be characterized. In the present study a fecal in vitro model was optimized and used to quantify the interspecies differences in kinetics of the intestinal microbial metabolism of ZEN in rat, pig and human. Vmax, Km, and catalytic efficiencies (kcat) were determined, and results obtained reveal that the kcat values for formation of α-ZEL and ß-ZEL amounted to 0.73 and 0.12 mL/h/kg bw for human microbiota, 2.6 and 1.3 mL/h/kg bw for rat microbiota and 9.4 and 6.3 mL/h/kg bw for pig microbiota showing that overall ZEN metabolism increased in the order human < rat < pig microbiota. Expressed per kg bw the kcat for ZEN metabolism by the liver surpassed that of the intestinal microbiota in all three species. In conclusion, it is estimated that the activity of the intestinal colon microbiome may be up to 36 % of the activity of the liver, and that it can additionally contribute to the species differences in bioactivation and detoxification and thus the toxicity of ZEN in pigs and rats but not in humans. The results highlight the importance of the development of human specific models for the assessment of the metabolism of ZEN.

Combining In Vitro Data and Physiologically Based Kinetic Modeling Facilitates Reverse Dosimetry to Define In Vivo Dose-Response Curves for Bixin- and Crocetin-Induced Activation of PPARγ in Humans.

SCOPE: It is investigated whether at realistic dietary intake bixin and crocetin could induce peroxisome proliferator-activated receptor γ (PPARγ)-mediated gene expression in humans using a combined in vitro-in silico approach. METHODS AND RESULTS: Concentration-response curves obtained from in vitro PPARγ-reporter gene assays are converted to in vivo dose-response curves using physiologically based kinetic modeling-facilitated reverse dosimetry, from which the benchmark dose levels resulting in a 50% effect above background level (BMD50 ) are predicted and subsequently compared to dietary exposure levels. Bixin and crocetin activated PPARγ-mediated gene transcription in a concentration-dependent manner with similar potencies. Due to differences in kinetics, the predicted BMD50 values for in vivo PPARγ activation are about 30-fold different, amounting to 115 and 3505 mg kg bw-1 for crocetin and bixin, respectively. Human dietary and/or supplemental estimated daily intakes may reach these BMD50 values for crocetin but not for bixin, pointing at better possibilities for in vivo PPARγ activation by crocetin. CONCLUSION: Based on a combined in vitro-in silico approach, it is estimated whether at realistic dietary intakes plasma concentrations of bixin and crocetin are likely to reach concentrations that activate PPARγ-mediated gene expression, without the need for a human intervention study.

In vitro-in silico-based analysis of the dose-dependent in vivo oestrogenicity of the soy phytoestrogen genistein in humans.

BACKGROUND AND PURPOSE: The in vivo oestrogenicity of genistein and its glycoside genistin is still under debate. The present study aimed to develop a physiologically based kinetic (PBK) model that provides insight in dose-dependent plasma concentrations of genistein aglycone and its metabolites and enables prediction of in vivo oestrogenic effective dose levels of genistein and genistin in humans. EXPERIMENTAL APPROACH: A PBK model for genistein and genistin in humans was developed based on in vitro metabolic parameters. The model obtained was used to translate in vitro oestrogenic concentration-response curves of genistein to in vivo oestrogenic dose-response curves for intake of genistein and genistin. KEY RESULTS: The model predicted that genistein-7-O-glucuronide was the major circulating metabolite and that levels of the free aglycone were generally low [0.5-17% of total plasma genistein at oral doses from 0.01 to 50 mg (kg·bw)-1 ]. The predicted in vivo benchmark dose for 5% response values for oestrogenicity varied between 0.06 and 4.39 mg kg-1 genistein. For genistin, these values were 1.3-fold higher. These values are in line with reported human data and show that oestrogenic responses can be expected at an Asian dietary and a supplementary intake, while intake resulting from a Western diet may not be effective. CONCLUSIONS AND IMPLICATIONS: The present study shows how plasma concentrations of genistein and its metabolites and oestrogenic dose levels of genistein in humans can be predicted by combining in vitro oestrogenicity with PBK model-based reverse dosimetry, eliminating the need for human intervention studies.

Physiologically based kinetic modeling of hesperidin metabolism and its use to predict in vivo effective doses in humans.

SCOPE: To develop a physiologically based kinetic (PBK) model that describes the absorption, distribution, metabolism, and excretion of hesperidin in humans, enabling the translation of in vitro concentration-response curves to in vivo dose-response curves. METHODS AND RESULTS: The PBK model for hesperidin in humans was developed based on in vitro metabolic parameters. Hesperidin was predicted to mainly occur in the systemic circulation as different monoglucuronides. The plasma concentrations of hesperidin aglycone (hesperetin) was predicted to be <0.02 mg/L at an oral dose of 50 mg/kg bw. The developed PBK model allowed conversion of in vitro concentration-response curves for different effects to in vivo dose-response curves. The BMD05 (benchmark dose for 5% response) values for protein kinase A inhibition ranged between 135 and 529 mg/kg bw hesperidin, and for inhibition of endothelial cell migration and prostaglandin E2 and nitric oxide production ranged between 2.19 and 44 mg/kg bw hesperidin. These values are in line with reported human data showing in vivo effects by hesperidin and show that these effects may occur at Western dietary and supplementary intake of hesperidin. CONCLUSIONS: The developed PBK model adequately predicts absorption, distribution, metabolism, and excretion of hesperidin in humans and allows to evaluate the human in vivo situation without the need for human intervention studies.

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.

Use of physiologically based kinetic (PBK) modeling to study interindividual human variation and species differences in plasma concentrations of quercetin and its metabolites.

Biological activities of flavonoids in vivo ultimately depend on the systemic bioavailability of the aglycones and their metabolites. We aimed to develop physiologically based kinetic (PBK) models to predict plasma concentrations of the flavonoid quercetin and its metabolites in individual human subjects and to define species differences compared with male rat. The human models were developed based on in vitro metabolic parameters derived from incubations with pooled and 20 individual human tissue fractions and by fitting kinetic parameters to available in vivo data. The outcomes obtained were compared to a previously developed model for quercetin and its metabolites formation in male rat. Quercetin-3'-O-glucuronide was predicted to be the major circulating metabolite in 19 out of 20 individuals, while in male rat di- and tri-conjugates of quercetin containing a glucuronic acid, sulfate and/or methyl moieties are the major metabolites. Significant species differences occur in major circulating metabolites of quercetin suggesting that rat is not an adequate model to study effects of quercetin in man. The defined PBK models can be used to guide the experimental design of in vitro experiments with flavonoids, especially to better take into account the relevance of metabolism and the contribution of metabolites to the biological activity in humans.

A physiologically based kinetic (PBK) model describing plasma concentrations of quercetin and its metabolites in rats.

Biological activities of flavonoids in vivo are ultimately dependent on the systemic bioavailability of the aglycones as well as their metabolites. In the present study, a physiologically based kinetic (PBK) model was developed to predict plasma concentrations of the flavonoid quercetin and its metabolites and to tentatively identify the regiospecificity of the major circulating metabolites. The model was developed based on in vitro metabolic parameters and by fitting kinetic parameters to literature available in vivo data. Both exposure to quercetin aglycone and to quercetin-4'-O-glucoside, for which in vivo data were available, were simulated. The predicted plasma concentrations of different metabolites adequately matched literature reported plasma concentrations of these metabolites in rats exposed to 4'-O-glucoside. The bioavailability of aglycone was predicted to be very low ranging from 0.004%-0.1% at different oral doses of quercetin or quercetin-4'-O-glucoside. Glucuronidation was a crucial pathway that limited the bioavailability of the aglycone, with 95-99% of the dose being converted to monoglucuronides within 1.5-2.5h at different dose levels ranging from 0.1 to 50mg/kg bw quercetin or quercetin-4'-O-glucoside. The fast metabolic conversion to monoglucuronides allowed these metabolites to further conjugate to di- and tri-conjugates. The regiospecificity of major circulating metabolites was observed to be dose-dependent. As we still lack in vivo kinetic data for many flavonoids, the developed model has a great potential to be used as a platform to build PBK models for other flavonoids as well as to predict the kinetics of flavonoids in humans.

Malabaricone C-containing mace extract inhibits safrole bioactivation and DNA adduct formation both in vitro and in vivo.

Safrole, present in mace and its essential oils, causes liver tumors in rodents at high dose levels due to formation of a DNA reactive 1'-sulfooxysafrole. The present study identifies malabaricone C as a mace constituent able to inhibit safrole DNA adduct formation at the level of sulfotransferase mediated bioactivation. This inhibition was incorporated into physiologically based biokinetic rat and human models. Dosing safrole at 50mg/kg body weight and malabaricone C-containing mace extract at a ratio reflecting the relative presence in mace, and assuming 100% or 1% uptake of malabaricone C-containing mace extract, the model predicted inhibition of 1'-sulfooxysafrole formation for rats and humans by 90% and 100% or 61% and 91%, respectively. To validate the model, mace extract and safrole were co-administered orally to Sprague-Dawley rats. LC-ECI-MS/MS based quantification of DNA adduct levels revealed a significant (p<0.01) 55% reduction of safrole DNA adduct formation by malabaricone C-containing mace extract in the liver of rats exposed to safrole. The data obtained were used to perform a refined risk assessment of safrole. Overall, the results suggest a lower tumor incidence when safrole would be tested within a relevant food matrix containing sulfotransferase inhibitors compared to dosing pure safrole.

Matrix modulation of the bioactivation of estragole by constituents of different alkenylbenzene-containing herbs and spices and physiologically based biokinetic modeling of possible in vivo effects.

The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1'-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1'-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoids.

Physiologically based biokinetic (PBBK) modeling of safrole bioactivation and detoxification in humans as compared with rats.

A physiologically based biokinetic (PBBK) model for the alkenylbenzene safrole in humans was developed based on in vitro- and in silico-derived kinetic parameters. With the model obtained, the time- and dose-dependent formation of the proximate and ultimate carcinogenic metabolites, 1-hydroxysafrole and 1-sulfooxysafrole in human liver were estimated and compared with previously predicted levels of these metabolites in rat liver. In addition, Monte Carlo simulations were performed to predict interindividual variation in the formation of these metabolites in the overall population. For the evaluation of the model performance, a comparison was made between the predicted total amount of urinary metabolites of safrole and the reported total levels of metabolites in the urine of humans exposed to safrole, which adequately matched. The model results revealed no dose-dependent shifts in safrole metabolism and no relative increase in bioactivation at dose levels up to 100mg/kg body weight/day. Species differences were mainly observed in the detoxification pathways of 1-hydroxysafrole, with the formation of 1-oxosafrole being a main detoxification pathway of 1-hydroxysafrole in humans but a minor pathway in rats, and glucuronidation of 1-hydroxysafrole being less important in humans than in rats. The formation of 1-sulfooxysafrole was predicted to vary 4- to 17-fold in the population (fold difference between the 95th and median, and 95th and 5th percentile, respectively), with the median being three to five times higher in human than in rat liver. Comparison of the PBBK results for safrole with those previously obtained for the related alkenylbenzenes estragole and methyleugenol revealed that differences in 1-sulfooxy metabolite formation are limited, being only twofold to fivefold.

Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays.

A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay >> YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.