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 1ʹ-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 1ʹ-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 flavonoid

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

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 β-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 cat 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.</p

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.</p

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.</p

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 10⁸ 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 10⁸ 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