Physiologically based kinetic (PBK) models to give insight into dose-, species-, matrix- and interindividual human variation-dependent effects on bioactivation and detoxification of methyleugenol

Methyleugenol, which occurs naturally in various herbs such as tarragon, basil, nutmeg and allspice, is added to food either directly as a flavoring substance or as a constituent of added essential oils (Smith et al., 2002). The interest in the risk of methyleugenol as a food constituent came from its widespread use in a variety of foods and beverages as well as its structural resemblance to the known carcinogen safrole (Johnson et al., 2000). In addition, methyleugenol has been reported to be DNA reactive and carcinogenic, inducing malignant tumors in multiple tissues of rats and mice as well as inducing unscheduled DNA synthesis in rat liver (Ding et al., 2011; NTP, 2000; Smith et al., 2002). The safety of human exposure to methyleugenol at low dietary intake levels has been assessed several times (Hall and Oser, 1965; NTP, 2000; SCF, 2001; Smith et al., 2002) without reaching a scientific agreement on how to translate the carcinogenicity data of rodent animal experiments obtained at high levels of exposure to the relevant human situation. A recent evaluation, performed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, has indicated that although evidence of carcinogenicity to rodents given high doses of methyleugenol exists, further research is needed to assess the potential risk to human health at relevant dietary exposure resulting from the presence of methyleugenol in foods and essential oils and its use as flavoring agent (JECFA, 2008). Predicting the cancer risk in humans at relevant dietary intake levels requires extrapolation of the animal carcinogenicity data taking in consideration dose, species, and interindividual variation. Furthermore, it implies extrapolation from rat or mouse studies with high dose levels of the pure compound to the human situation in which exposure at low dose levels occurs within the context of a complex food matrix. The aim of the present PhD project was to obtain quantitative insight into the consequences of dose- and species-dependent effects and of interindividual differences and matrix effects for the bioactivation and detoxification of methyleugenol by using physiologically based kinetic (PBK) modeling. The first chapter of this thesis presents background information to the topic. In chapter 2, a physiologically based kinetic (PBK) model for the alkenylbenzene methyleugenol in rat was defined based on in vitro metabolic parameters determined using relevant tissue fractions, in silico derived partition coefficients (Payne and Kenny, 2002 and reference therin), and physiological parameters (Brown et al., 1997) derived from the literature. The model was based on the model previously developed for the related alkenylbenzene estragole and consists of eight compartments including liver, lung, and kidney as metabolizing compartments, and separate compartments for fat, arterial blood, venous blood, richly perfused and slowly perfused tissues (Punt et al., 2008). Evaluation of the model was performed by comparing the PBK predicted concentration of methyleugenol in the venous compartment to methyleugenol plasma levels reported in the literature, by comparing the PBK predicted dose-dependent % of formation of 2-hydroxy-4,5-dimethoxyallylbenzene, 3-hydroxy-4-methoxyallylbenzene, and 1′- hydroxymethyleugenol glucuronide to the corresponding % of metabolites excreted in urine reported in the literature, which were demonstrated to be in the same order of magnitude (Solheim and Scheline, 1976). With the model obtained the relative extent of bioactivation and detoxification of methyleugenol at different oral doses was examined. At low doses, formation of3-(3,4-dimethoxyphenyl)-2-propen-1-olandmethyleugenol-2′,3′-oxideleadingto detoxification appear to be the major metabolic pathways, occurring in the liver. At high doses, the model reveals a relative increase in the formation of the proximate carcinogenic metabolite 1′- hydroxymethyleugenol, occurring in the liver. This relative increase in formation of 1′- hydroxymethyleugenol leads to a relative increase in formation of 1′-hydroxymethyleugenol glucuronide, 1′-oxomethyleugenol, and 1′-sulfooxymethyleugenol the latter being the ultimate carcinogenic metabolite of methyleugenol. These results indicate that the relative importance of different metabolic pathways of methyleugenol may vary in a dose-dependent way, leading to a relative increase in bioactiviation of methyleugenol at higher doses. In subsequent studies described in chapter 3 a physiologically based kinetic (PBK) model for methyleugenol in human based on in vitro and in silico derived parameters was identified based on the model previously developed for the related alkenylbenzene estragole. The model consists of six compartments including liver as metabolizing compartment, and separate compartments for fat, arterial blood, venous blood, richly perfused and slowly perfused tissues (Punt et al., 2009). With the model obtained, bioactivation and detoxification of methyleugenol at different dose levels could be investigated. The outcomes of this human model were compared with those of the PBK model for methyleugenol in male rat. The results obtained reveal that formation of 1′-hydroxymethyleugenol glucuronide, 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 compared with male rat liver is observed. Furthermore, formation of 1′-sulfooxymethyleugenol, which readily undergoes desulfonation to a reactive carbo-cation that can form DNA or protein adducts, is predicted to be the same in the liver of both human and male rat at oral doses of 0.0034 up to 300 mg/(kg bw). Altogether it was concluded that despite a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1′-hydroxymethyleugenol between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol to 1′-sulfooxymethyleugenol appears to be negligible. Moreover, the PBK model predicted the formation of 1′-sulfooxymethyleugenol 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 shows that kinetic data do not provide a reason to argue against linear extrapolation from the rat tumor data to the human situation. Another aim of the present PhD study was to study the effect of the basil constituent nevadensin on the bioactivation and genotoxicity of herb based methyleugenol. The results presented in chapter 4 show that nevadensin is able to inhibit DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1′-hydroxymethyleugenol in the presence of this flavonoid. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxymethyleugenol. In order to investigate possible in vivo implications the SULT inhibition by nevadensin was integrated into the male rat and human PBK models for bioactivation and detoxification of methyleugenol. The results thus obtained reveal that coadministration of methyleugenol with nevadensin may reduce the levels of bioactivation of 1′- hydroxymethyleugenol to the DNA reactive metabolite, without reducing its detoxification via glucuronidation or oxidation. This effect may be significant even at realistic low dose human exposure levels. The results obtained point at a potential reduction of the cancer risk when methyleugenol exposure occurs by oral intake within a relevant food matrix containing SULT inhibitors compared to what is observed in rodent bioassays upon exposure to pure methyleugenol dosed by gavage. Besides dose-dependent effects, species differences effects, and matrix effects on the bioactivation of methyleugenol the effect of interindividual variation on methyleugenol detoxification and bioactivation was investigated in chapter 5. To this end we predicted the level of formation of the ultimate carcinogenic metabolite 1′-sulfooxymethyleugenol in the human population by taking the variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Insight in the variation in relevant metabolic routes was obtained by determining kinetic constants for the metabolic reactions by specific isoenzymes or by measuring the kinetic constants in incubations with a range of individual human liver fractions. The results of the study indicate that formation of 1′-sulfooxymethyleugenol is predominantly affected by i) P450 1A2 catalyzed bioactivation of methyleugenol to 1′- hydroxymethyleugenol ii) P450 2B6 catalyzed epoxidation of methyleugenol and iii) the apparent kinetic constants for detoxification of 1′-hydroxymethyleugenol via oxidation and iv) the apparent kinetic constants for bioactivation of 1′-hydroxymethyleugenol to 1′- sulfooxymethyleugenol. Based on the Monte Carlo simulation a chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived which is defined as the 95th or 99th percentile divided by the 50th percentile of the predicted distribution of the formation of 1′- sulfooxymethyleugenol in the liver. The obtained CSAF value at the 95th percentile was 3.7 indicating that the default uncertainty factor of 3.16 for human variability in kinetics (WHO, 1999) may adequately protect 95% of the population. While protecting 99% of the population requires a larger uncertainty factor of 5.8. Altogether, the results shown in this thesis reveal that integrating in vitro metabolic parameters within a framework of a PBK model provides a good method to evaluate the occurrence of dose-dependent effects, species differences, and human variability in detoxification and bioactivation of a genotoxic carcinogen. Moreover, the results presented in this thesis show the possible protective effect of the basil constituent nevadensin on SULT catalysed bioactivation and DNA adduct formation of methyleugenol in vitro. Upon validation of these effects in vivo, it may turn out that rodent carcinogenicity data on methyleugenol substantially overestimate the risks posed when humans are exposed to methyleugenol within a nevadensin containing food matrix.</p

Assessment of the predictive capacity of a physiologically based kinetic model using a read-across approach

With current progress in science, there is growing interest in developing and applying Physiologically Based Kinetic (PBK) models in chemical risk assessment, as knowledge of internal exposure to chemicals is critical to understanding potential effects in vivo. In particular, a new generation of PBK models is being developed in which the model parameters are derived from in silico and in vitro methods. To increase the acceptance and use of these “Next Generation PBK models”, there is a need to demonstrate their validity. However, this is challenging in the case of data-poor chemicals that are lacking in kinetic data and for which predictive capacity cannot, therefore, be assessed. The aim of this work is to lay down the fundamental steps in using a read across framework to inform modellers and risk assessors on how to develop, or evaluate, PBK models for chemicals without in vivo kinetic data. The application of a PBK model that takes into account the absorption, distribution, metabolism and excretion characteristics of the chemical reduces the uncertainties in the biokinetics and biotransformation of the chemical of interest. A strategic flow-charting application, proposed herein, allows users to identify the minimum information to perform a read-across from a data-rich chemical to its data-poor analogue(s). The workflow analysis is illustrated by means of a real case study using the alkenylbenzene class of chemicals, showing the reliability and potential of this approach. It was demonstrated that a consistent quantitative relationship between model simulations could be achieved using models for estragole and safrole (source chemicals) when applied to methyleugenol (target chemical). When the PBK model code for the source chemicals was adapted to utilise input values relevant to the target chemical, simulation was consistent between the models. The resulting PBK model for methyleugenol was further evaluated by comparing the results to an existing, published model for methyleugenol, providing further evidence that the approach was successful. This can be considered as a “read-across” approach, enabling a valid PBK model to be derived to aid the assessment of a data poor chemical

The Extent of the Acquisition of Geography Teachers of the Concepts and Skills Found in the First-Secondary Economic Geography Textbook in Jordan

This study aimed at answering the following questions: 1- To what extent did geography teachers acquire the concepts and skills found in the economic geography textbook? The following two sub-questions were also discussed: a- Is there a statistically significant difference related to sex between the acquisition of geography teachers of the concepts and skills of economic geography and the educationally - accepted standard? b- Is there a statistically significant difference related to teaching experience between the acquisition of geography teachers of the concepts and skills of economic geography and the educationally - accepted standard? 2- Are there any statistically significant differences related to sex and teaching experience and their interaction in the teachers' acquisition of the concepts and skills of economic geography? 3- The population of the study was all the female and male teachers in Al-Koora, Bani Kenana and Irbid First districts , N=118, Selected sample of 30 males and females were randomly selected. The study used a sixty-item achievement test to measure the extent of acquisition of these concept and skills, and the results of the statistical analysis were as follows: 1- The acquisition of geography teachers of the concept and skills of economic geography is below the educationally-accepted standard , 75%, for males, females and teachers with a long or short teaching experience. 2- There are no statistically significant differences due to sex , teaching experience and their interaction in the acquisition of these concepts and skills

The Extent of the Acquisition of Geography Teachers of the Concepts and Skills Found in the First-Secondary Economic Geography Textbook in Jordan

This study aimed at answering the following questions: 1- To what extent did geography teachers acquire the concepts and skills found in the economic geography textbook? The following two sub-questions were also discussed: a- Is there a statistically significant difference related to sex between the acquisition of geography teachers of the concepts and skills of economic geography and the educationally - accepted standard? b- Is there a statistically significant difference related to teaching experience between the acquisition of geography teachers of the concepts and skills of economic geography and the educationally - accepted standard? 2- Are there any statistically significant differences related to sex and teaching experience and their interaction in the teachers' acquisition of the concepts and skills of economic geography? 3- The population of the study was all the female and male teachers in Al-Koora, Bani Kenana and Irbid First districts , N=118, Selected sample of 30 males and females were randomly selected. The study used a sixty-item achievement test to measure the extent of acquisition of these concept and skills, and the results of the statistical analysis were as follows: 1- The acquisition of geography teachers of the concept and skills of economic geography is below the educationally-accepted standard , 75%, for males, females and teachers with a long or short teaching experience. 2- There are no statistically significant differences due to sex , teaching experience and their interaction in the acquisition of these concepts and skills

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

Mode of action based risk assessment of the botanical food-borne alkenylbenzene apiol from parsley using physiologically based kinetic (PBK) modelling and read-across from safrole

The present study developed physiologically-based kinetic (PBK) models for the alkenylbenzene apiol in order to facilitate risk assessment based on read-across from the related alkenylbenzene safrole. Model predictions indicate that in rat liver the formation of the 1'-sulfoxy metabolite is about 3 times lower for apiol than for safrole. These data support that the lower confidence limit of the benchmark dose resulting in a 10% extra cancer incidence (BMDL10) that would be obtained in a rodent carcinogenicity study with apiol may be 3-fold higher for apiol than for safrole. These results enable a preliminary risk assessment for apiol, for which tumor data are not available, using a BMDL10 value of 3 times the BMDL10 for safrole. Based on an estimated BMDL10 for apiol of 5.7-15.3 mg/kg body wt per day and an estimated daily intake of 4 × 10-5 mg/kg body wt per day, the margin of exposure (MOE) would amount to 140,000-385,000. This indicates a low priority for risk management. The present study shows how PBK modelling can contribute to the development of alternatives for animal testing, facilitating read-across from compounds for which in vivo toxicity studies on tumor formation are available to compounds for which these data are unavailable.</p

Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modeling.

Methyleugenol (ME) occurs naturally in a variety of spices, herbs, including basil, and their essential oils. ME induces hepatomas in rodent bioassays following its conversion to a DNA reactive metabolite. In the present study, the basil constituent nevadensin was shown to be able to inhibit SULT-mediated DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1'-hydroxymethyleugenol in the presence of nevadensin. To investigate possible in vivo implications of SULT inhibition by nevadensin on ME bioactivation, the rat physiologically based kinetic (PBK) model developed in our previous work to describe the dose-dependent bioactivation and detoxification of ME in male rat was combined with the recently developed PBK model describing the dose-dependent kinetics of nevadensin in male rat. The resulting binary ME–nevadensin PBK model was used to predict the possible nevadensin mediated reduction in ME DNA adduct formation and resulting carcinogenicity at the doses of ME used by the NTP carcinogenicity study. Using these data an updated risk assessment using the Margin of Exposure (MOE) approach was performed. The results obtained point at a potential reduction of the cancer risk when rodents are orally exposed to ME within a relevant food matrix containing SULT inhibitors compared to exposure to pure ME

Physiologically based kinetic modeling of the bioactivation of myristicin

The present study describes physiologically based kinetic (PBK) models for the alkenylbenzene myristicin that were developed by extension of the PBK models for the structurally related alkenylbenzene safrole in rat and human. The newly developed myristicin models revealed that the formation of the proximate carcinogenic metabolite 1′-hydroxymyristicin in liver is at most 1.8 fold higher in rat than in human and limited for the ultimate carcinogenic metabolite 1′-sulfoxymyristicin to (2.8–4.0)-fold higher in human. In addition, a comparison was made between the relative importance of bioactivation for myristicin and safrole. Model predictions indicate that for these related compounds, the formation of the 1′-sulfoxy metabolites in rat and human liver is comparable with a difference of 10 derived for safrole of 1.9–5.1 mg/kg bw per day. Using an estimated daily intake of myristicin of 0.0019 mg/kg bw per day resulting from the use of herbs and spices, this results in MOE values for myristicin that amount to 1000–2700, indicating a priority for risk management. The results obtained illustrate that PBK modeling provides insight into possible species differences in the metabolic activation of myristicin. Moreover, they provide an example of how PBK modeling can facilitate a read-across in risk assessment from a compound for which in vivo toxicity studies are available to a related compound for which tumor data are not reported, thus contributing to alternatives in animal testing.</p