Implementation of toxicokinetics in toxicity studies--Toxicokinetics of 4-methylanisole and its metabolites in juvenile and adult rats.

The current risk assessment of compounds is generally based on external exposure and effect relationships. External doses are often not representative for internal exposure concentrations. The aim of this study was to show how the implementation of toxicokinetics in a scheduled toxicity study contributes to improved data interpretation without additional use of animals and to the three goals of the 3R principles for animal testing. Toxicokinetic analyses were implemented in a rat developmental immunotoxicity study with 4-methylanisole without interfering with the outcome of the study and without the use of additional animals. 4-Methylanisole and its metabolites were analysed in plasma of adult rats and in pups at postnatal day 10. 4-Methylanisole has a short half-life in adult animals and the plasma concentrations increased more than proportional with increasing dose. The metabolic profile appeared to be different at low dose as compared to high dose. This information on the dose-proportionality of the internal exposure is crucial for the interpretation of the toxicity data and helps to identify the toxic agent and the appropriate dose metric. The metabolism was similar in adult and juvenile animals. Large inter-individual variability in adult animals, as observed for 4-methylanisole, may hamper dose-response analyses of the results. In addition, 4-metylanisole was excreted via milk, but concentrations in the juvenile animals appeared to be 20- to 100-fold lower than via direct gavage exposure. The toxicokinetic parameters support the data interpretation, among others by providing better insight into internal exposures. Subsequently, it will help to prevent testing of irrelevant exposure scenarios and exposure concentrations. Overall, implementation of kinetics with limited effort provides useful information to support the interpretation of toxicological data and can contribute to reduction and refinement of animal testing.

Tissue distribution and elimination after oral and intravenous administration of different titanium dioxide nanoparticles in rats.

OBJECTIVE: The aim of this study was to obtain kinetic data that can be used in human risk assessment of titanium dioxide nanomaterials. METHODS: Tissue distribution and blood kinetics of various titanium dioxide nanoparticles (NM-100, NM-101, NM-102, NM-103, and NM-104), which differ with respect to primary particle size, crystalline form and hydrophobicity, were investigated in rats up to 90 days post-exposure after oral and intravenous administration of a single or five repeated doses. RESULTS: For the oral study, liver, spleen and mesenteric lymph nodes were selected as target tissues for titanium (Ti) analysis. Ti-levels in liver and spleen were above the detection limit only in some rats. Titanium could be detected at low levels in mesenteric lymph nodes. These results indicate that some minor absorption occurs in the gastrointestinal tract, but to a very limited extent.Both after single and repeated intravenous (IV) exposure, titanium rapidly distributed from the systemic circulation to all tissues evaluated (i.e. liver, spleen, kidney, lung, heart, brain, thymus, reproductive organs). Liver was identified as the main target tissue, followed by spleen and lung. Total recovery (expressed as % of nominal dose) for all four tested nanomaterials measured 24 h after single or repeated exposure ranged from 64-95% or 59-108% for male or female animals, respectively. During the 90 days post-exposure period, some decrease in Ti-levels was observed (mainly for NM-100 and NM-102) with a maximum relative decrease of 26%. This was also confirmed by the results of the kinetic analysis which revealed that for each of the investigated tissues the half-lifes were considerable (range 28-650 days, depending on the TiO(2)-particle and tissue investigated). Minor differences in kinetic profile were observed between the various particles, though these could not be clearly related to differences in primary particle size or hydrophobicity. Some indications were observed for an effect of crystalline form (anatase vs. rutile) on total Ti recovery. CONCLUSION: Overall, the results of the present oral and IV study indicates very low oral bioavailability and slow tissue elimination. Limited uptake in combination with slow elimination might result in the long run in potential tissue accumulation.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Effects of probiotic bacteria on the bioaccessibility of aflatoxin B(1) and ochratoxin A using an in vitro digestion model under fed conditions.

In the present study, we aimed at determining the release of aflatoxin B(1) (AFB(1)) and ochratoxin A (OTA) from different food products in the gastro-intestinal tract in the absence and presence of probiotics, a possible adsorbent. The average bioaccessibility of AFB(1) and OTA without probiotics was about 90%, and 30%, respectively, depending on several factors, such as food product, contamination level, compound and type of contamination (spiked versus naturally contaminated). The six probiotic bacteria showed varying binding capacity to AFB(1) and OTA depending on the bacterial strain, toxin studied, type of food and contamination level. A reduction to a maximum of 37% and 73% as observed for the bioaccessibility of AFB(1) and OTA in the presence of probiotic bacteria, respectively. This is the first report on the effect of probiotic bacteria on reducing the fraction of mycotoxins available for absorption in the gastrointestinal tract from different food products.

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry.

The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans.

World Health Organization estimates of the global and regional disease burden of four foodborne chemical toxins, 2010: a data synthesis.

Background Chemical exposures have been associated with a variety of health effects; however, little is known about the global disease burden from foodborne chemicals. Food can be a major pathway for the general population's exposure to chemicals, and for some chemicals, it accounts for almost 100% of exposure.  Methods and Findings Groups of foodborne chemicals, both natural and anthropogenic, were evaluated for their ability to contribute to the burden of disease.  The results of the analyses on four chemicals are presented here - cyanide in cassava, peanut allergen, aflatoxin, and dioxin.  Systematic reviews of the literature were conducted to develop age- and sex-specific disease incidence and mortality estimates due to these chemicals.  From these estimates, the numbers of cases, deaths and disability adjusted life years (DALYs) were calculated.  For these four chemicals combined, the total number of illnesses, deaths, and DALYs in 2010 is estimated to be 339,000 (95% uncertainty interval [UI]: 186,000-1,239,000); 20,000 (95% UI: 8,000-52,000); and 1,012,000 (95% UI: 562,000-2,822,000), respectively.  Both cyanide in cassava and aflatoxin are associated with diseases with high case-fatality ratios.  Virtually all human exposure to these four chemicals is through the food supply.  Conclusion Chemicals in the food supply, as evidenced by the results for only four chemicals, can have a significant impact on the global burden of disease. The case-fatality rates for these four chemicals range from low (e.g., peanut allergen) to extremely high (aflatoxin and liver cancer).  The effects associated with these four chemicals are neurologic (cyanide in cassava), cancer (aflatoxin), allergic response (peanut allergen), endocrine (dioxin), and reproductive (dioxin).

Arsenic: bioaccessibility from seaweed and rice, dietary exposure calculations and risk assessment.

Arsenic is a metalloid that occurs in food and the environment in different chemical forms. Inorganic arsenic is classified as a class I carcinogen. The inorganic arsenic intake from food and drinking water varies depending on the geographic arsenic background. Non-dietary exposure to arsenic is likely to be of minor importance for the general population within the European Union. In Europe, arsenic in drinking water is on average low, but food products (e.g. rice and seaweed) are imported from all over the world including from regions with naturally high arsenic levels. Therefore, specific populations living in Europe could also have a high exposure to inorganic arsenic due to their consumption pattern. Current risk assessment is based on exposure via drinking water. For a good estimation of the risks of arsenic in food, it is important to investigate if the bioavailability of inorganic arsenic from food is different from drinking water. The present study further explores the issue of European dietary exposure to inorganic arsenic via rice and seaweed and its associated health risks. The bioavailability of inorganic arsenic was measured in in vitro digestion experiments. The data indicate that the bioavailability of inorganic arsenic is similar for rice and seaweed compared with drinking water. The calculated dietary intake for specific European Union populations varied between 0.44 and 4.51 µg kg⁻¹ bw day⁻¹. The margins of exposure between the inorganic intake levels and the BMDL0.5 values as derived by JECFA are low. Decreasing the intake of inorganic arsenic via Hijiki seaweed could be achieved by setting legal limits similar to those set for rice by the Codex Alimentarius Commission in July 2014.

Fate in digestion in vitro of several food components, including some toxic compounds coming from omega-3 and omega-6 lipids.

In this study it was proved the formation of oxygenated alpha,beta-unsaturated aldehydes (OαßUAs) of 6, 7, 9 and 10 carbon atoms during the thermal treatment (190°C with aeration) of a commercial vegetable oil rich in omega-3 and omega-6 acyl groups, which also contained small amounts of added proteins and carbohydrates to produce barbecue aroma when heated. The OαßUAs detected by Solid Phase Microextraction (SPME) followed by Gas Chromatography/Mass Spectrometry (GC/MS) were: 4-hydroxy-2-hexenal, 4-oxo-2-hexenal and 4,5-epoxy-2-heptenals, coming from omega-3 acyl groups; and 4-hydroxy-2-nonenal, 4-oxo-2-nonenal and 4,5-epoxy-2-decenals, coming from omega-6 acyl groups. Mixtures of this oil, either thermodegraded or not, with standard food were submitted to an in vitro digestion model. The study of the digestion products obtained revealed that OαßUAs remained unaltered, being bioaccessible in the gastrointestinal tract and so able to reach the systemic circulation. Besides, it was evidenced that during digestion Maillard, esterification and oxidation reactions take place.

An inter-laboratory trial of the unified BARGE bioaccessibility method for arsenic, cadmium and lead in soil.

The Bioaccessibility Research Group of Europe (BARGE) has carried out an inter-laboratory trial of a proposed harmonised in vitro physiologically based ingestion bioaccessibility procedure for soils, called the Unified BARGE Method (UBM). The UBM includes an initial saliva phase and simulated stomach and intestine compartments. The trial involved the participation of seven laboratories (five European and two North American) providing bioaccessibility data for As (11 samples), Cd (9 samples) and Pb (13 samples) using soils with in vivo relative bioavailability data measured using a swine model. The results of the study were compared with benchmark criteria for assessing the suitability of the UBM to provide data for human health risk assessments. Mine waste and slag soils containing high concentrations of As caused problems of poor repeatability and reproducibility which were alleviated when the samples were run at lower soil to solution ratios. The study showed that the UBM met the benchmark criteria for both the stomach and stomach & intestine phase for As. For Cd, three out of four criteria were met for the stomach phase but only one for the stomach & intestine phase. For Pb two, out of four criteria were met for the stomach phase and none for the stomach & intestine phase. However, the study recommends tighter control of pH in the stomach phase extraction to improve between-laboratory variability, more reproducible in vivo validation data and that a follow up inter-laboratory trial should be carried out.

Effect of administration route on the biodistribution and shedding of replication-deficient HAdV-5: a qualitative modelling approach.

Gene therapy is a rapidly developing field in which recombinant nucleic acid sequences are introduced to individuals. Its therapeutic, prophylactic or diagnostic effect relates directly to the sequence it contains or to the product of genetic expression of this sequence. Recombinant adenoviral vectors (in particular HAdV-5 vectors) are frequently used in gene therapy. Knowledge on biodistribution and shedding is crucial in the risk assessment for the patient and the patient's environment. This review presents a critical overview on biodistribution and shedding data of non-replicating viral vector HAdV-5, related to the used administration route. Based on these data, a qualitative model for the biodistribution and shedding of HAdV-5 based viral vectors is presented. Biodistribution and shedding depend on the route of administration. Some routes lead to local biodistribution and no shedding or one shedding route only. Other routes lead to systemic biodistribution and to shedding via several excreta. Shedding via semen and transport across the blood-brain barrier is not expected for HAdV-5. The presented qualitative model can help researchers and risk assessors to determine the possible distribution in the body and the risk of shedding via the different excretion routes. Furthermore, it can help regulators to predict the different shedding routes after a certain administration route and thus in deciding which studies are warranted or which safety precautions are needed after administration to patients.

Effect of administration route on the biodistribution and shedding of replication-deficient AAV2: a qualitative modelling approach.

Gene therapy is a rapidly developing field in which recombinant nucleic acid sequences are introduced to individuals to regulate, repair, replace, add or delete a genetic sequence. Recombinant adeno-associated viral (AAV) vectors, especially AAV2, are frequently used in gene therapy. Knowledge on the biodistribution and potential shedding of AAV2 is crucial to evaluate the risks of infection with the viral vector for the patient and the environment. Literature was analysed for biodistribution and shedding data for AAV2. Preclinical and clinical studies were included with a focus on the influence of the administration route on spreading. Based on biodistribution and shedding data, a qualitative model for the biodistribution and shedding of AAV2 related to the administration route is presented. It is concluded that biodistribution and shedding of AAV2 depends on the route of administration. Some routes lead to local biodistribution and thus to no shedding or shedding via one route only. Other routes lead to systemic biodistribution and to shedding via several excretion routes. The qualitative model presented can help to determine the possible biodistribution in the body and the risk of shedding via the different excretion routes. In addition, it can help to predict the different shedding routes after a certain administration route of AAV2 and thus in deciding which studies are warranted or which safety precautions are needed after administration to patients.

Use of an in vitro digestion model to study the bioaccessibility of 4-hydroxy-2-nonenal and related aldehydes present in oxidized oils rich in omega-6 acyl groups.

Mixtures of either sunflower oil or thermodegraded sunflower oil and a standard meal were submitted to an in vitro digestion model. The same experiment was carried out with fluid deep-frying fat and thermodegraded fluid deep-frying fat. The thermodegradation of the oil and fat was provoked by submitting them to 190 degrees C with aeration in a convection oven, and the presence in the headspace of the thermodegraded oil and fat of oxygenated alpha,beta-unsaturated aldehydes (OalphabetaUAs), such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and 4,5-epoxy-2-decenal (EDE), was monitored by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS). The digestion products were separated by centrifugation in a lipidic phase, an aqueous phase, and a pellet phase. The headspace of these three phases was also studied by SPME/GC-MS to check if the toxic and very reactive OalphabetaUAs above-mentioned remained unaltered after the in vitro digestion process or if they had reacted with the various compounds present in the digestion products, so disappearing from the samples. With the same aim the extract in ethyl acetate of the aqueous and pellet phases, and of the lipidic phase after dilution, were studied by GC-MS. All results obtained showed that a certain proportion of the toxic OalphabetaUAs remains unaltered after digestion, dispersed in the three phases above-mentioned, and thus are bioaccessible in the gastrointestinal tract and so could reach the systemic circulation. Compounds that may originate in Maillard type reactions (2-pentylpyridine) are found among digestion products, proving that these reactions are possible in this process if adequate substrates are present. In addition, it has been shown that toxic metabolites from the synthetic antioxidant BHT, present in fat before digestion, remain unaltered after this process and could reach the systemic circulation.

In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug.

Aplidine is a potent marine anti-cancer drug and is currently being investigated in phase II clinical trials. However, the enzymes involved in the biotransformation of aplidine and thus its pharmacokinetics are not known yet. To assess the biotransformation pathways of aplidine and their potential implications for human pharmacology and toxicology, the in vitro metabolism of aplidine was characterized using incubations with human plasma, liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes in combination with HPLC analysis and cytotoxicity assays with cell lines. Aplidine was metabolised by carboxyl esterases in human plasma. Using CYP supersomes and liver microsomes, it was shown that aplidine was metabolised mainly by CYP3A4 and also by CYP2A6, 2E1 and 4A11. Four metabolites were observed after incubation with human liver microsomes, one formed by CYP2A6 (C-demethylation) and three by CYP3A4 (hydroxylation and/or C-dealkylation). No conjugation was observed in human liver S9 fraction. However, the aplidine metabolites formed by CYP were further conjugated by the phase II enzymes UGT, GST and SULT. In accordance with the findings in microsomes and CYP supersomes, a significant effect of specific CYP2A6, 2E1, 3A4 and 4A11 inhibitors on the cytotoxicity of aplidine in Hep G2 and IGROV-1 cells could be observed. These results provide evidence that CYP3A4 has a major role in metabolising aplidine in vitro with additional involvement of CYP2A6, 2E1, and 4A11. Further, the metabolites formed by CYPs can be conjugated by UGT, SULT and GST. These findings could help interpret the in vivo pharmacokinetics of aplidine.

In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis, Trabectidin), a novel marine anti-cancer drug.

ET-743 is a potent marine anti-cancer drug and is currently being investigated in phase I and II clinical trials, e.g. in combination with other anti-cancer agents. To assess the biotransformation and CYP reaction phenotype and their potential implications for human pharmacology and toxicology, the in vitro metabolism of ET-743 was characterized using incubations with human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes.CYP supersomes and liver microsomes showed that ET-743 was metabolized mainly by CYP3A4, but also by CYP2C9, 2C19, 2D6, and 2E1. ET-743 showed the highest affinity for CYP3A4 and the highest maximal metabolic rate for CYP2D6 among the CYPs shown to metabolize ET-743. In addition, the Km value of ET-743 in female microsomes was significantly lower compared to male microsomes, while the Vmax values did not differ. ET-743 glucuronidation, catalyzed by UGT2B15, was observed in microsomes and S9 fraction. In addition, conjugation by glutathione-S-transferase and no sulphation was observed for ET-743 in cytosol and S9 fraction. ET-743 was more extensively metabolized when CYP activity was combined with phase II enzymes UGT and glutathione-S-transferase (GST), indicating that CYP, UGT, and GST simultaneously metabolize ET-743 in the S9 fraction. These results provide evidence that CYP3A4 has a major role in the metabolism of ET-743 in vitro with additional involvement of CYP2C9, 2C19, 2D6, and 2E1. Furthermore, ET-743 is conjugated by UGT and GST. This information could be important for interpretation of the pharmacokinetic data of clinical trials and prediction of drug-drug interactions.

Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment.

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.

Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines.

Human cell lines are often used for in vitro biotransformation and transport studies of drugs. In vivo, genetic polymorphisms have been identified in drug-metabolizing enzymes and ABC-drug transporters leading to altered enzyme activity, or a change in the inducibility of these enzymes. These genetic polymorphisms could also influence the outcome of studies using human cell lines. Therefore, the aim of our study was to pharmacogenotype four cell lines frequently used in drug metabolism and transport studies, HepG2, IGROV-1, CaCo-2 and LS180, for genetic polymorphisms in biotransformation enzymes and drug transporters. The results indicate that, despite the presence of some genetic polymorphisms, no real effects influencing the activity of metabolizing enzymes or drug transporters in the investigated cell lines are expected. However, this characterization will be an aid in the interpretation of the results of biotransformation and transport studies using these in vitro cell models.

In-vitro cytotoxicity of ET-743 (Trabectedin, Yondelis), a marine anti-cancer drug, in the Hep G2 cell line: influence of cytochrome P450 and phase II inhibition, and cytochrome P450 induction.

ET-743 is a marine anti-cancer drug and is currently in phase I trials in which the effect of combination therapies will be investigated. Its dose-limiting toxicity in patients is hepatotoxicity. In-vitro studies have shown that ET-743 is mainly metabolized by cytochrome P450 (CYP) 3A4, but also by 2C9, 2C19, 2D6 and 2E1, and the phase II enzymes uridine diphosphoglucuronosyl transferase and glutathione-S-transferase. Based on this metabolic profile, there is a risk of drug-drug interactions possibly influencing the hepatotoxicity of ET-743. Therefore, the effect of CYP and phase II activity on the cytotoxicity of ET-743 was investigated in vitro in a human cell line model system. The effect of different CYP and phase II inhibitors and CYP inducers on ET-743 cytotoxicity was studied after 48 and 120 h of treatment in Hep G2 cells using different assays. Furthermore, the toxicity of ET-743 metabolites was investigated. Potent cytotoxic activity of ET-743 after 120 h treatment was observed, which could be increased in combination with the CYP inhibitors metyrapone (3A4), phenanthrene (substrate for 2E1, 3A4), piperonyl butoxide (3A), proadifen (2C9, 2E1, 3A4), ritonavir (3A4), and warfarin (2C9, 2C19). No effect on the cytotoxicity of ET-743 was observed in combination with phase II enzyme inhibition and CYP induction. CYP metabolites of ET-743 were less toxic compared with ET-743. These findings indicate that combination therapy of ET-743 with CYP inhibitors, e.g. other anti-cancer drugs, could lead to changes in the hepatotoxicity of ET-743 and are therefore of clinical importance.

In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug.

Thiocoraline is a potent new marine anti-cancer drug in vitro, which will be tested in phase I clinical studies shortly. To assess the biotransformation and the potential implications for human pharmacology and toxicology, the in vitro metabolism of thiocoraline was characterized using human plasma, human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes and human cell lines. Thiocoraline is significantly metabolized by enzymes present in human plasma; t (1/2) shifted from 25.2 h in phosphate buffered saline to 4.3 h in human plasma. Using CYP supersomes it was shown that thiocoraline is mainly metabolized by CYP3A4, with CYP1A1, CYP2C8 and CYP2C9 playing a minor role in the biotransformation (<3%). Only minor glucuronidation was observed for thiocoraline by UGT1A1 and UGT1A9 and no glucuronidation was observed in human liver S9 fraction. In addition, no glucosidation and sulfation were observed for thiocoraline in human liver cytosol and S9 fraction. However, the metabolites formed by cytochrome P450 were further conjugated by UGT, glutathione-S-transferase (GST) and sulfotransferase (ST). In contrast to the CYP metabolism observed in supersomes, no effect could be observed from the CYP3A4 inhibitors on the cytotoxicity of thiocoraline in Hep G2 cells. However, this could be due to low CYP expression levels in the Hep G2 and IGROV-1 cell line. These results provide evidence that human CYP3A4 plays a major role in the metabolism of thiocoraline in vitro and that the metabolites formed by CYP are conjugated by the phase II enzymes UGT, ST and GST.

An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons.

The liver is the predominant organ in which biotransformation of foreign compounds takes place, although other organs may also be involved in drug biotransformation. Ideally, an in vitro model for drug biotransformation should accurately resemble biotransformation in vivo in the liver. Several in vitro human liver models have been developed in the past few decades, including supersomes, microsomes, cytosol, S9 fraction, cell lines, transgenic cell lines, primary hepatocytes, liver slices, and perfused liver. A general advantage of these models is a reduced complexity of the study system. On the other hand, there are several more or less serious specific drawbacks for each model, which prevents their widespread use and acceptance by the regulatory authorities as an alternative for in vivo screening. This review describes the practical aspects of selected in vitro human liver models with comparisons between the methods.