Physiologically based kinetic (PBK) modeling as a new approach methodology (NAM) for predicting systemic levels of gut microbial metabolites.

There is a clear need to develop new approach methodologies (NAMs) that combine in vitro and in silico testing to reduce and replace animal use in chemical risk assessment. Physiologically based kinetic (PBK) models are gaining popularity as NAMs in toxico/pharmacokinetics, but their coverage of complex metabolic pathways occurring in the gut are incomplete. Chemical modification of xenobiotics by the gut microbiome plays a critical role in the host response, for example, by prolonging exposure to harmful metabolites, but there is not a comprehensive approach to quantify this impact on human health. There are examples of PBK models that have implemented gut microbial biotransformation of xenobiotics with the gut as a dedicated metabolic compartment. However, the integration of microbial metabolism and parameterization of PBK models is not standardized and has only been applied to a few chemical transformations. A challenge in this area is the measurement of microbial metabolic kinetics, for which different fermentation approaches are used. Without a standardized method to measure gut microbial metabolism ex vivo/in vitro, the kinetic constants obtained will lead to conflicting conclusions drawn from model predictions. Nevertheless, there are specific cases where PBK models accurately predict systemic concentrations of gut microbial metabolites, offering potential solutions to the challenges outlined above. This review focuses on models that integrate gut microbial bioconversions and use ex vivo/in vitro methods to quantify metabolic constants that accurately represent in vivo conditions.

Penetration pathways, influencing factors and predictive models for dermal absorption of exobiotic molecules: A critical review.

This review provides a comprehensive summary of the skin penetration pathways of xenobiotics, including metals, organic pollutants, and nanoparticles (NPs), with a particular focus on the methodologies employed to elucidate these penetration routes. The impacts of the physicochemical properties of exogenous substances and the properties of solvent carriers on the penetration efficiencies were discussed. Furthermore, the review outlines the steady-state and transient models for predicting the skin permeability of xenobiotics, emphasizing the models which enable realistic visualization of pharmaco-kinetic phenomena via detailed geometric representations of the skin microstructure, such as stratum corneum (SC) (bricks and mortar) and skin appendages (hair follicles and sebaceous gland units). Limitations of published research, gaps in current knowledge, and recommendations for future research are highlighted, providing insight for a better understanding of the skin penetration behavior of xenobiotics and associated health risks in practical application contexts.

Biochemical aspects of hemoglobin-xenobiotic interactions and their implications in drug discovery.

Hemoglobin, a homodimeric globular protein, is found predominantly in red blood cells and in a small amount in blood plasma. Along with binding to certain native molecules, it also interacts with various xenobiotics. The present review aims at studying these interactions and the resultant tangible impact on the structure and function of the protein if any. The review also encompasses various analytical and computational approaches which are routinely used to study these interactions. A detailed discussion on types of interaction exhibited by individual xenobiotics has been included herein. Additionally, the effects of xenobiotic binding on the oxygen carrying capacity of hemoglobin have been reviewed. These insights would be of great value in drug design and discovery. Envisaging probable interactions of designed ligands with hemoglobin would help improvise the process of drug development. This would also open up new avenues for studying hemoglobin-mediated drug delivery.

Enzyme Kinetics of Oxidative Metabolism-Cytochromes P450.

The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

The chemical defensome of five model teleost fish.

How an organism copes with chemicals is largely determined by the genes and proteins that collectively function to defend against, detoxify and eliminate chemical stressors. This integrative network includes receptors and transcription factors, biotransformation enzymes, transporters, antioxidants, and metal- and heat-responsive genes, and is collectively known as the chemical defensome. Teleost fish is the largest group of vertebrate species and can provide valuable insights into the evolution and functional diversity of defensome genes. We have previously shown that the xenosensing pregnane x receptor (pxr, nr1i2) is lost in many teleost species, including Atlantic cod (Gadus morhua) and three-spined stickleback (Gasterosteus aculeatus), but it is not known if compensatory mechanisms or signaling pathways have evolved in its absence. In this study, we compared the genes comprising the chemical defensome of five fish species that span the teleosteii evolutionary branch often used as model species in toxicological studies and environmental monitoring programs: zebrafish (Danio rerio), medaka (Oryzias latipes), Atlantic killifish (Fundulus heteroclitus), Atlantic cod, and three-spined stickleback. Genome mining revealed evolved differences in the number and composition of defensome genes that can have implication for how these species sense and respond to environmental pollutants, but we did not observe any candidates of compensatory mechanisms or pathways in cod and stickleback in the absence of pxr. The results indicate that knowledge regarding the diversity and function of the defensome will be important for toxicological testing and risk assessment studies.

Multixenobiotic resistance mechanism: Organ-specific characteristics in the fish Prochilodus lineatus and its role as a protection against genotoxic agents.

The multixenobiotic resistance mechanism (MXR) can decrease intracellular genotoxic pressure through the efflux of compounds out of the cell. Thus, this work presents a temporal approach to evaluate the MXR activity and the occurrence of genotoxic damage in different organs of the fish Prochilodus lineatus after an intraperitoneal injection of benzo[a]pyrene (B[a]P). Although the liver and brain demonstrated rapid MXR induction (6 h), the occurrence of DNA damage was not prevented. However, these organs presented some return to DNA integrity after MXR activity. The kidney demonstrated the slowest response in the MXR induction (24 h), which may be related to the preferential excretion of B[a]P metabolites by this route. Moreover, the kidney MXR reduction at 96 h may be related to its role in the excretion of metabolites from all other metabolizing organs. The gills did not appear to play an essential role in xenobiotics efflux; however, their participation in biotransformation is exhibited through the occurrence of DNA damage. The integrated response of the organs in the dynamics for the maintenance of the organism integrity could be promoted by the circulation of the xenobiotic through the bloodstream, which corroborates the increase in the DNA damage in the erythrocytes at 6 h. Therefore, the ability to induce MXR was linked to the preservation of DNA integrity in the presence of B[a]P, since MXR acts to avoid the accumulation of xenobiotics inside the cell.

The Pharmacology of Xenobiotics after Intracerebro Spinal Fluid Administration: Implications for the Treatment of Brain Tumors.

The incidence of brain metastasis has been increasing for 10 years, with poor prognosis, unlike the improvement in survival for extracranial tumor localizations. Since recent advances in molecular biology and the development of specific molecular targets, knowledge of the brain distribution of drugs has become a pharmaceutical challenge. Most anticancer drugs fail to cross the blood-brain barrier. In order to get around this problem and penetrate the brain parenchyma, the use of intrathecal administration has been developed, but the mechanisms governing drug distribution from the cerebrospinal fluid to the brain parenchyma are poorly understood. Thus, in this review we discuss the pharmacokinetics of drugs after intrathecal administration, their penetration of the brain parenchyma and the different systems causing their efflux from the brain to the blood.

Biological roles of cytochrome P450 1A1, 1A2, and 1B1 enzymes.

Human cytochrome P450 enzymes (CYPs) play a critical role in various biological processes and human diseases. CYP1 family members, including CYP1A1, CYP1A2, and CYP1B1, are induced by aryl hydrocarbon receptors (AhRs). The binding of ligands such as polycyclic aromatic hydrocarbons activates the AhRs, which are involved in the metabolism (including oxidation) of various endogenous or exogenous substrates. The ligands that induce CYP1 expression are reported to be carcinogenic xenobiotics. Hence, CYP1 enzymes are correlated with the pathogenesis of cancers. Various endogenous substrates are involved in the metabolism of steroid hormones, eicosanoids, and other biological molecules that mediate the pathogenesis of several human diseases. Additionally, CYP1s metabolize and activate/inactivate therapeutic drugs, especially, anti-cancer agents. As the metabolism of drugs determines their therapeutic efficacy, CYP1s can determine the susceptibility of patients to some drugs. Thus, understanding the role of CYP1s in diseases and establishing novel and efficient therapeutic strategies based on CYP1s have piqued the interest of the scientific community.

Intravital Microscopy: A Tool to Investigate the Toxicity in the Immune System, Vessel Rheology, and Xenobiotic Distribution.

Intravital microscopy (IVM) is an essential experimental approach for evaluating, in real time, cell interactions in the blood and rheological parameters in the microcirculation of the living animals. Different tissues are surgically exposed to the visualization of the microvascular network in optical microscopies connected to video cameras and image software. By evaluating in situ microcirculatory network, IVM allows the visualization and quantification of physiological and pathological processes in the blood or in the adjacent tissues considering the whole system. Therefore, IVM has been used to evaluate the effects and mechanisms of actions in the microvascular network caused by pharmacological or toxic chemical agents. In this chapter, different experimental approaches are described to study the toxic effects and mechanisms of xenobiotics in the microcirculatory network.

Aryl hydrocarbon receptor (AHR), integrating energy metabolism and microbial or obesity-mediated inflammation.

Aryl hydrocarbon receptor (AHR) has been characterized as multifunctional sensor, integrator and ligand-activated transcription factor of the bHLH/PAS family. Regulation of inflammatory diseases and energy metabolism are among the putative functions of AHR. Challenges in AHR research include marked species differences, and cell, tissue and context dependence of AHR functions. The commentary is focused on AHR's role in the integration between energy expenditure and microbial and non-infectious inflammation, the latter exemplified by obesity-mediated nonalcoholic fatty liver disease. One of the mechanisms controlling energy-consuming inflammation is represented by a signalsome that is involved in retinoic acid-triggered neutrophil differentiation and regulation of the NADPH oxidase complex (NOX). Established signalsome components are AHR, CD38, multiple protein kinases and adaptors. To prevent chronic inflammatory diseases, the complex interplay between a range of inflammatory responses and energy expenditure must be precisely regulated. Surviving an infection requires both pathogen clearance and tissue protection from inflammatory damage. Defenses are energy-consuming anabolic programs. Therefore, anti-inflammatory, catabolic tolerance programs by metabolic reprogramming of macrophages have evolved. Therapeutic options of AHR agonists to reduce chronic inflammatory diseases are discussed.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.

Metabolism and pharmacokinetics of pharmaceuticals in cats (Felix sylvestris catus) and implications for the risk assessment of feed additives and contaminants.

In animal health risk assessment, hazard characterisation of feed additives has been often using the default uncertainty factor (UF) of 100 to translate a no-observed-adverse-effect level in test species (rat, mouse, dog, rabbit) to a 'safe' level of chronic exposure in farm and companion animal species. Historically, both 10-fold factors have been further divided to include chemical-specific data in both dimensions when available. For cats (Felis Sylvestris catus), an extra default UF of 5 is applied due to the species' deficiency in particularly glucuronidation and glycine conjugation. This paper aims to assess the scientific basis and validity of the UF for inter-species differences in kinetics (4.0) and the extra UF applied for cats through a comparison of kinetic parameters between rats and cats for 30 substrates of phase I and phase II metabolism. When the parent compound undergoes glucuronidation the default factor of 4.0 is exceeded, with exceptions for zidovudine and S-carprofen. Compounds that were mainly renally excreted did not exceed the 4.0-fold default. Mixed results were obtained for chemicals which are metabolised by CYP3A in rats. When chemicals were administered intravenously the 4.0-fold default was not exceeded with the exception of clomipramine, lidocaine and alfentanil. The differences seen after oral administration might be due to differences in first-pass metabolism and bioavailability. Further work is needed to further characterise phase I, phase II enzymes and transporters in cats to support the development of databases and in silico models to support hazard characterisation of chemicals particularly for feed additives.

Quantification of Proteins Involved in Intestinal Epithelial Handling of Xenobiotics.

The intestinal epithelium represents a natural barrier against harmful xenobiotics, while facilitating the uptake of nutrients and other substances. Understanding the interaction of chemicals with constituents of the intestinal epithelium and their fate in the body requires quantitative measurement of relevant proteins in in vitro systems and intestinal epithelium. Recent studies have highlighted the mismatch between messenger RNA (mRNA) and protein abundance for several drug-metabolizing enzymes and transporters in the highly dynamic environment of the intestinal epithelium; mRNA abundances cannot therefore be used as a proxy for protein abundances in the gut, necessitating direct measurements. The objective was to determine the expression of a wide range proteins pertinent to metabolism and disposition of chemicals and nutrients in the intestinal epithelium. Ileum and jejunum biopsy specimens were obtained from 16 patients undergoing gastrointestinal elective surgery. Mucosal fractions were prepared and analyzed using targeted and global proteomic approaches. A total of 29 enzymes, 32 transporters, 6 tight junction proteins, 2 adhesion proteins, 1 alkaline phosphatase, 1 thioredoxin, 5 markers, and 1 regulatory protein were quantified-60 for the first time. The global proteomic method identified a further 5,222 proteins, which are retained as an open database for interested parties to explore. This study significantly expands our knowledge of a wide array of proteins important for xenobiotic handling in the intestinal epithelium. Quantitative systems biology models will benefit from the novel systems data generated in the present study and the translation path offered for in vitro to in vivo translation.

HuskinDB, a database for skin permeation of xenobiotics.

Skin permeation is an essential biological property of small organic compounds our body is exposed to, such as drugs in topic formulations, cosmetics, and environmental toxins. Despite the limited availability of experimental data, there is a lack of systematic analysis and structure. We present a novel resource on skin permeation data that collects all measurements available in the literature and systematically structures experimental conditions. Besides the skin permeation value kp, it includes experimental protocols such as skin source site, skin layer used, preparation technique, storage conditions, as well as test conditions such as temperature, pH as well as the type of donor and acceptor solution. It is important to include these parameters in the assessment of the skin permeation data. In addition, we provide an analysis of physicochemical properties and chemical space coverage, laying the basis for applicability domain determination of insights drawn from the collected data points. The database is freely accessible under https://huskindb.drug-design.de or https://doi.org/10.7303/syn21998881 .

Assessing the impacts on fetal dosimetry of the modelling of the placental transfers of xenobiotics in a pregnancy physiologically based pharmacokinetic model.

The developmental origin of health and diseases theory supports the critical role of the fetal exposure to children's health. We developed a physiologically based pharmacokinetic model for human pregnancy (pPBPK) to simulate the maternal and fetal dosimetry throughout pregnancy. Four models of the placental exchanges of chemicals were assessed on ten chemicals for which maternal and fetal data were available. These models were calibrated using non-animal methods: in vitro (InV) or ex vivo (ExV) data, a semi-empirical relationship (SE), or the limitation by the placental perfusion (PL). They did not impact the maternal pharmacokinetics but provided different profiles in the fetus. The PL and InV models performed well even if the PL model overpredicted the fetal exposure for some substances. The SE and ExV models showed the lowest global performance and the SE model a tendency to underprediction. The comparison of the profiles showed that the PL model predicted an increase in the fetal exposure with the pregnancy age, whereas the ExV model predicted a decrease. For the SE and InV models, a small decrease was predicted during the second trimester. All models but the ExV one, presented the highest fetal exposure at the end of the third trimester. Global sensitivity analyses highlighted the predominant influence of the placental transfers on the fetal exposure, as well as the metabolic clearance and the fraction unbound. Finally, the four transfer models could be considered depending on the framework of the use of the pPBPK model and the availability of data or resources to inform their parametrization.

In Silico Simulation of Simultaneous Percutaneous Absorption and Xenobiotic Metabolism: Model Development and a Case Study on Aromatic Amines.

PURPOSE: To advance physiologically-based pharmacokinetic modelling of xenobiotic metabolism by integrating metabolic kinetics with percutaneous absorption. METHOD: Kinetic rate equations were proposed to describe the metabolism of a network of reaction pathways following topical exposure and incorporated into the diffusion-partition equations of both xenobiotics and metabolites. The published ex vivo case study of aromatic amines was simulated. Diffusion and partition properties of xenobiotics and subsequent metabolites were determined using physiologically-based quantitative structure property relationships. Kinetic parameters of metabolic reactions were best fitted from published experimental data. RESULTS: For aromatic amines, the integrated transdermal permeation and metabolism model produced data closely matched by experimental results following limited parameter fitting of metabolism rate constants and vehicle:water partition coefficients. The simulation was able to produce dynamic concentration data for all the dermal layers, as well as the vehicle and receptor fluid. CONCLUSION: This mechanistic model advances the dermal in silico functionality. It provides improved quantitative spatial and temporal insight into exposure of xenobiotics, enabling the isolation of governing features of skin. It contributes to accurate modelling of concentrations of xenobiotics reaching systemic circulation and additional metabolite concentrations. This is vital for development of both pharmaceuticals and cosmetics.

In Vitro Human Cell-Based Experimental Models for the Evaluation of Enteric Metabolism and Drug Interaction Potential of Drugs and Natural Products.

Elements of key enteric drug metabolism and disposition pathways are reviewed to aid the assessment of the applicability of current cell-based enteric experimental systems for the evaluation of enteric metabolism and drug interaction potential. Enteric nuclear receptors include vitamin D receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, liver X receptor, aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor. Enteric drug metabolizing enzyme pathways include both cytochrome P450 (P450) and non-P450 drug metabolizing enzymes based on gene expression, proteomics, and activity. Both uptake and efflux transporters are present in the small intestine, with P-glycoprotein found to be responsible for most drug-drug and food-drug interactions. The cell-based in vitro enteric systems reviewed are 1) immortalized cell line model: the human colon adenocarcinoma (Caco-2) cells; 2) human stem cell-derived enterocyte models: stem cell enteric systems, either from intestinal crypt cells or induced pluripotent stem cells; and 3) primary cell models: human intestinal slices, cryopreserved human enterocytes, permeabilized cofactor-supplemented (MetMax) cryopreserved human enterocytes, and cryopreserved human intestinal mucosa. The major deficiency with both immortalized cell lines and stem cell-derived enterocytes is that drug metabolizing enzyme activities, although they are detectable, are substantially lower than those for the intestinal mucosa in vivo. Human intestine slices, cryopreserved human enterocytes, MetMax cryopreserved human enterocytes, and cryopreserved human intestinal mucosa retain robust enteric drug metabolizing enzyme activity and represent appropriate models for the evaluation of metabolism and metabolism-dependent drug interaction potential of orally administered xenobiotics including drugs, botanical products, and dietary supplements. SIGNIFICANCE STATEMENT: Enteric drug metabolism plays an important role in the bioavailability and metabolic fate of orally administered drugs as well as in enteric drug-drug and food-drug interactions. The current status of key enteric drug metabolism and disposition pathways and in vitro human cell-based enteric experimental systems for the evaluation of the metabolism and drug interaction potential of orally administered substances is reviewed.

Human variability in isoform-specific UDP-glucuronosyltransferases: markers of acute and chronic exposure, polymorphisms and uncertainty factors.

UDP-glucuronosyltransferases (UGTs) are involved in phase II conjugation reactions of xenobiotics and differences in their isoform activities result in interindividual kinetic differences of UGT probe substrates. Here, extensive literature searches were performed to identify probe substrates (14) for various UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) and frequencies of human polymorphisms. Chemical-specific pharmacokinetic data were collected in a database to quantify interindividual differences in markers of acute (Cmax) and chronic (area under the curve, clearance) exposure. Using this database, UGT-related uncertainty factors were derived and compared to the default factor (i.e. 3.16) allowing for interindividual differences in kinetics. Overall, results show that pharmacokinetic data are predominantly available for Caucasian populations and scarce for other populations of different geographical ancestry. Furthermore, the relationships between UGT polymorphisms and pharmacokinetic parameters are rarely addressed in the included studies. The data show that UGT-related uncertainty factors were mostly below the default toxicokinetic uncertainty factor of 3.16, with the exception of five probe substrates (1-OH-midazolam, ezetimibe, raltegravir, SN38 and trifluoperazine), with three of these substrates being metabolised by the polymorphic isoform 1A1. Data gaps and future work to integrate UGT-related variability distributions with in vitro data to develop quantitative in vitro-in vivo extrapolations in chemical risk assessment are discussed.

The role of xenobiotic-metabolizing enzymes in the placenta: a growing research field.

Introduction: The placenta is a temporary and unique organ that allows for the physical connection between a mother and fetus; this organ regulates the transport of gases and nutrients mediating the elimination of waste products contained in the fetal circulation. The placenta performs metabolic and excretion functions, on the basis of multiple enzymatic systems responsible for the oxidation, reduction, hydrolysis, and conjugation of xenobiotics. These mechanisms give the placenta a protective role that limits the fetal exposure to harmful compounds. During pregnancy, some diseases require uninterrupted treatment even if it is detrimental to the fetus. Drugs and other xenobiotics alter gene expression in the placenta with repercussions for the fetus and mother's well-being.Areas covered: This review provides a brief description of the human placental structure and function, the main drug and xenobiotic transporters and metabolizing enzymes, placenta-metabolized substrates, and alterations in gene expression that the exposure to xenobiotics may cause.Expert opinion: Research should be focused on the identification and validation of biological markers for the assessment of the harmful effects of some drugs in pregnancy, including the evaluation of polymorphisms and methylation patterns in chorionic villous samples and/or amniotic fluid.

Genetic studies of urinary metabolites illuminate mechanisms of detoxification and excretion in humans.

The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite-locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research.