Prediction of in vivo developmental toxicity of all-trans-retinoic acid based on in vitro toxicity data and in silico physiologycally based kinetic modeling
Louisse, J. ; Bosgra, S. ; Blaauboer, B.J. ; Rietjens, I. ; Verwei, M. - \ 2015
Archives of Toxicology 89 (2015)7. - ISSN 0340-5761 - p. 1135 - 1148.
dose-dependent kinetics - laboratory-animals - response curves - human liver - rat - metabolism - expression - integration - humans - glucuronidation
The use of laboratory animals for toxicity testing in chemical safety assessment meets increasing ethical, economic and legislative constraints. The development, validation and application of reliable alternatives for in vivo toxicity testing are therefore urgently needed. In order to use toxicity data obtained from in vitro assays for risk assessment, in vitro concentration–response data need to be translated into in vivo dose–response data that are needed to obtain points of departure for risk assessment, like a benchmark dose (BMD). In the present study, we translated in vitro concentration–response data of the retinoid all-trans-retinoic acid (ATRA), obtained in the differentiation assay of the embryonic stem cell test, into in vivo dose–response data using a physiologically based kinetic model for rat and human that is mainly based on kinetic model parameter values derived using in vitro techniques. The predicted in vivo dose–response data were used for BMD modeling, and the obtained BMDL10 values [lower limit of the 95 % confidence interval on the BMD at which a benchmark response equivalent to a 10 % effect size (BMR10) is reached (BMD10)] for rat were compared with BMDL10 values derived from in vivo developmental toxicity data in rats reported in the literature. The results show that the BMDL10 values from predicted dose–response data differ about sixfold from the BMDL10 values obtained from in vivo data, pointing at the feasibility of using a combined in vitro–in silico approach for defining a point of departure for toxicological risk assessment.
Toward relevant biomarkers in in vitro developmental toxicity and their extrapolation to the in vivo situation : Reviews
Louisse, J. ; Woutersen, R.A. ; Blaauboer, B.J. ; Verwei, M. ; Rietjens, I. - \ 2012
Expert Opinion on Drug Metabolism and Toxicology 8 (2012)1. - ISSN 1742-5255 - p. 11 - 27.
stem-cell test - whole-embryo culture - gene-expression changes - metabolic-activation system - embryotoxicity tests - alternative methods - end-points - differentiation system - neural differentiation - maternal toxicity
Introduction: Reliable in vitro and in silico assays as alternatives for in vivo developmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivo developmental toxicity in in vitro assays are needed. Areas covered: The present review gives an overview of alternative assays, as described in literature, for in vivo developmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivo developmental toxicity, and how in vitro effect data can be translated to the in vivo situation using physiologically based kinetic (PBK) modeling. Expert opinion: Relevance of readouts in in vitro developmental toxicity assays as predictive biomarkers for in vivo developmental toxicity should be evaluated by comparing the obtained in vitro effect concentrations with in vivo internal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitro effect concentrations to in vivo dose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitro toxicity data in the safety assessment of compounds. Read More: http://informahealthcare.com/doi/abs/10.1517/17425255.2012.639762
Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity
Louisse, J. ; Gonen, S. ; Rietjens, I. ; Verwei, M. - \ 2011
Toxicology Letters 203 (2011)1. - ISSN 0378-4274 - p. 1 - 8.
13-cis-retinoic acid - comparative teratogenicity - embryotoxicity tests - dose-response - mouse limbs - invitro - rat - isotretinoin - validation - etretin
The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicity. The results reveal that the potency ranking obtained in the ES-D3 cell differentiation assay is similar to the reported potency rankings in the two other in vitro assays for developmental toxicity. TTNPB ((E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) was the most potent retinoid, whereas etretinate and retinol had the lowest potency. All-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and acitretin showed an intermediate potency. In vivo potency rankings of the developmental toxicity of retinoids appear to be dependent on the species and/or exposure regimens used. The obtained in vitro potency ranking does not completely correspond with the in vivo potency rankings, although TTNPB is correctly predicted to be the most potent and retinol the least potent congener. The lack of in vivo kinetic processes in the ES-D3 cell differentiation assay might explain the deviating potency predictions of some retinoids. Therefore, knowledge on the species-dependent in vivo kinetics is essential when using in vitro toxicity data for the estimation of in vivo developmental toxicity potencies within series of related compounds
The use of in vitro toxicity data and physiologically based kinetic modeling to predict dose-respomse curves for in vivo developmental toxicity of glycol ethers in rat and man
Louisse, J. ; Jong, E. de; Sandt, J.J.M. van de; Blaauboer, B.J. ; Woutersen, R.A. ; Piersma, A.H. ; Rietjens, I. ; Verwei, M. - \ 2010
Toxicological sciences 118 (2010)2. - ISSN 1096-6080 - p. 470 - 484.
experimental human exposure - human health-risk - stem-cell test - monomethyl ether - monoethyl ether - pharmacokinetic model - inhalation exposure - methoxyacetic acid - ethoxyacetic acid - embryotoxicity tests
At present, regulatory assessment of systemic toxicity is almost solely performed using animal models. The EU REACH legislation stimulates the use of animal-free approaches to obtain information on the toxicity of chemicals. In vitro toxicity tests provide in vitro concentration-response curves for specific target cells, whereas in vivo dose-response curves are regularly used for human risk assessment. The present study shows an approach to predict in vivo dose-response curves for developmental toxicity by combining in vitro toxicity data and in silico kinetic modeling. A physiologically based kinetic (PBK) model was developed, describing the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites in rat and man. In vitro toxicity data of these metabolites derived in the embryonic stem cell test were used as input in the PBK model to extrapolate in vitro concentration-response curves to predicted in vivo dose-response curves for developmental toxicity of the parent glycol ethers in rat and man. The predicted dose-response curves for rat were found to be in concordance with the embryotoxic dose levels measured in reported in vivo rat studies. Therefore, predicted dose-response curves for rat could be used to set a point of departure for deriving safe exposure limits in human risk assessment. Combining the in vitro toxicity data with a human PBK model allows the prediction of dose-response curves for human developmental toxicity. This approach could therefore provide a means to reduce the need for animal testing in human risk assessment practices.
Decrease of intercellular pH as possible mechanism of action of embryotoxicity of glycol ether alkoxyacetic acid metabolites
Louisse, J. ; Yanquin Bai, ; Verwei, M. ; Sandt, J.J.M. van de; Blaauboer, B.J. ; Rietjens, I. - \ 2010
Toxicology and Applied Pharmacology 245 (2010)2. - ISSN 0041-008X - p. 236 - 243.
stem-cell test - induced limb malformations - carbonic-anhydrase-ii - developmental toxicity - monomethyl ether - in-vitro - methoxyacetic acid - monoethyl ether - rats - mice
Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH,) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH(i) of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na+/H+-antiporter, corroborating an important role of the pHi in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH; may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers. (C) 2010 Elsevier Inc. All rights reserved.
Relative developmental toxicity of glycol ether alkoxy acid metabolites in the embryonic stem cell test as compared to the in vivo potency of their parent compounds
Jong, E. de; Louisse, J. ; Verwei, M. ; Blaauboer, B.J. ; Sandt, J.J.M. van de; Woutersen, R.A. ; Rietjens, I. ; Piersma, A.H. - \ 2009
Toxicological sciences 110 (2009)1. - ISSN 1096-6080 - p. 117 - 124.
vitro embryotoxicity tests - methoxyacetic acid - ethoxyacetic acid - monomethyl ether - monoethyl ether - teratogenicity - elimination - exposure - rats - mice
The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds
Development of a QSAR for worst case estimates of acute toxicity of chemically reactive compounds
Freidig, A.P. ; Dekkers, S. ; Verwei, M. ; Zvinavashe, E. ; Bessems, J.G.M. ; Sandt, J.J.M. van de - \ 2007
Toxicology Letters 170 (2007)3. - ISSN 0378-4274 - p. 214 - 222.
in-vitro - skin irritation - electrophilic chemicals - systemic toxicity - structural alerts - prediction - classification - corrosion - applicability - mechanisms
Future EU legislations enforce a fast hazard and risk assessment of thousands of existing chemicals. If conducted by means of present data requirements, this assessment will use a huge number of test animals and will be neither cost nor time effective. The purpose of the current research was to develop methods to increase the acceptability of in vitro data for classification and labelling regarding acute toxicity. For this purpose, a large existing database containing in vitro and in vivo data was analysed. For more than 300 compounds in the database, relations between in vitro cytotoxicity and rat or mouse intravenous and oral in vivo LD50 values were re-evaluated and the possibilities for definition of mechanism based chemical subclasses were investigated. A high in vitro¿in vivo correlation was found for chemicals classified as irritants. This can be explained by a shared unspecific cytotoxicity of these compounds which will act as the predominant mode of action for both endpoints, irritation and acute toxicity. For this subclass, which covered almost 40% of all compounds in the database, the LD50 values after intravenous dosing could be predicted with high accuracy. A somewhat lower accuracy was found for the prediction of oral LD50 values based on in vitro cytotoxicity data. Based on this successful correlation, a classification and labelling scheme was developed, that includes a hazard based definition of the applicability domain (irritants) and a prediction of the labelling of compounds for their acute iv and oral toxicity. The scheme was tested by an external validation.
Bioavailability of folic acid from fortified pasteurised and UHT-treated milk in humans
Jong, R.J. ; Verwei, M. ; West, C.E. ; Vliet, T. van; Siebelink, E. ; Berg, H. van den; Castenmiller, J.J.M. - \ 2005
European Journal of Clinical Nutrition 59 (2005)8. - ISSN 0954-3007 - p. 906 - 913.
folate-binding-protein - plasma homocysteine concentrations - neural-tube defects - methylenetetrahydrofolate reductase - food fortification - vascular-disease - common mutation - dietary-folate - risk factor - cows milk
Objective The aim of this study was to investigate whether milk fortified with folic acid enhances the folate status of humans and whether the presence of folate-binding proteins (FBP) in pasteurised milk affects the bioavailability of folic acid from fortified milk. In untreated and pasteurised milk, folate occurs bound to FBP, while FBP is (partly) denatured in ultra-high-temperature (UHT)-treated milk. The effect of FBP on folate bioavailability is still unclear. Design, subjects and setting Healthy, free-living subjects (n=69) aged 18-49 y participated in a 4-week double-blind, placebo-controlled dietary intervention study. Intervention In addition to a fully controlled diet, the subjects consumed each day 500 ml of pasteurised or UHT milk, either fortified or not with 200 g folic acid. Results Consumption of fortified milk increased folate concentrations in serum and in red blood cells (RBC) by 6.6-7.0 nmol/l (P
Bioavailability of folate from fortified milk products
Verwei, M. - \ 2004
Wageningen University. Promotor(en): G. Schaafsma; C.E. West, co-promotor(en): John Groten. - [S.I.] : S.n. - ISBN 9789085040804
foliumzuur - biologische beschikbaarheid - melk - melksamenstelling - melkconsumptie - spijsvertering - folic acid - bioavailability - milk - milk composition - milk consumption - digestion
The gap between actual intake and recommended intake of folate could be bridged by the consumption of fortified food products. Milk is considered as a potential food matrix for folate fortification in countries (such as the
The Binding of Folic acid and 5-methyltetrahydrofolate to Folate-Binding Proteins during Gastric Passage Differs in a dynamic in vitro gastrointestinal model
Verwei, M. ; Arkbåge, K. ; Mocking, H. ; Havenaar, R. ; Groten, J. - \ 2004
The Journal of Nutrition 134 (2004)1. - ISSN 0022-3166 - p. 31 - 37.
neural-tube defects - red-cell folate - bovine-milk - cows milk - plasma homocysteine - vascular-disease - dairy-products - dietary-folate - bioavailability - prevention
Despite its low natural folate concentration, milk is responsible for 10-15% of the daily folate intake in countries with a high dairy consumption. Milk products can be considered as a potential matrix for folate fortification, e.g., with synthetic folic acid, to enhance the daily intake of folate. In untreated milk, the natural folate, 5-methyltetrahydrofolate (5-CH3-H(4)folate), is bound to folate-binding proteins (FBP). In this study, the extent of binding to FBP for folic acid and 5-CH3-H(4)folate was investigated in a dynamic in vitro model simulating human gastric passage. Protein binding of folic acid and 5-CH3-H(4)folate was characterized using gel-exclusion chromatography. Before gastric passage, folic acid and 5-CH3-H(4)folate were bound mainly to FBP (76-79%), whereas 7% was free. Folic acid remained bound to FBP to a similar extent after gastric passage. For 5-CH3-H(4)folate, the FBP-bound fraction gradually decreased from 79 to 5% and the free fraction increased from 7 to 93%. Although folic acid enters the proximal part of the intestine bound to FBP, 5-CH3-H(4)folate appears to be present mainly as free folate in the duodenal lumen. The stability of FBP was similar in both folate/FBP mixtures, i.e., 70% of the initial FBP content was retained after gastric passage. This study indicated that FBP are partly stable during gastric passage but have different binding characteristics for folic acid and 5-CH3-H(4)folate in the duodenal lumen. This could result in different bioavailability from folic acid- and 5-CH3-H(4)folate-fortified milk products.
Bioaccessibility of Folic Acid and (6S)-5-Methyltetrahydrofolate Decreases after the Addiction of Folate-Binding Protein to Yogurt as Studied in a Dynamic In Vitro Gastrointestinal Model
Arkbåge, K. ; Verwei, M. ; Havenaar, R. ; Witthöft, C. - \ 2003
The Journal of Nutrition 133 (2003)11. - ISSN 0022-3166 - p. 3678 - 3683.
neural-tube defects - small-intestine - cows milk - bovine-milk - 5-methyltetrahydrofolate - bioavailability - absorption - homocysteine - transport - diets
Milk products are only moderate sources of folate. Nevertheless, they are of interest due to their content of folate-binding proteins (FBP), which in some studies have been reported to increase folate bioavailability. The effect of FBP on folate bioavailability has been widely discussed. The aim of this study was to investigate the bioaccessibility of folic acid and (6S)-5- methyltetrahydrofolate (5-CH3-H(4)folate) from fortified yogurt using a dynamic in vitro gastrointestinal model (TIM). In addition, the effect of FBP on folate bioaccessibility and the stability of FBP added to yogurt during gastrointestinal passage were investigated. Folate bioaccessibility was 82% from yogurt fortified with folic acid and 5-CH3-H(4)folate. The addition of FBP to yogurt decreased (P <0.05) folate bioaccessibility. The lowering effect of FBP was more pronounced in yogurt fortified with folic acid (34% folate bioaccessibility) than from yogurt fortified with 5-CH3-H(4)folate (57% folate bioaccessibility). After gastrointestinal passage, 17% of the FBP in yogurt fortified with 5-CH3-H(4)folate and 34% of the FBP in yogurt fortified with folic acid were recovered. No difference in folate bioaccessibility was found between folate-fortified yogurt and folate-fortified pasteurized milk (P = 0.10), whereas the lowering effect of FBP was (P <0.05) greater in yogurt compared with pasteurized milk. In conclusion, based on the high bioaccessibility of folic acid and 5-CH3-H(4)folate, yogurt without active FBP can be considered to be an appropriate food matrix for folate fortification.
Folic acid and 5-methyltetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model
Verwei, M. ; Arkbåge, K. ; Havenaar, R. ; Berg, H. van den; Witthöft, C. ; Schaafsma, G. - \ 2003
The Journal of Nutrition 133 (2003)7. - ISSN 0022-3166 - p. 2377 - 2383.
folate-binding-protein - neural-tube defects - red-cell folate - cows milk - plasma homocysteine - small-intestine - dietary-folate - bovine-milk - bioavailability - prevention
Dairy products are a potential matrix for folate fortification to enhance folate consumption in the Western world. Milk folate-binding proteins (FBP) are especially interesting because they seem to be involved in folate bioavailability. In this study, folate bioaccessibility was investigated using a dynamic computer-controlled gastrointestinal model [TNO gastrointestinal model (TIM)]. We used both ultrahigh temperature (UHT)-processed milk and pasteurized milk, differing in endogenous FBP concentrations and fortified with folic acid or 5-methyltetrahydrofolate (5-CH3-H(4)folate). To study FBP stability during gastrointestinal passage and the effect of additional FBP on folate bioaccessibility, FBP-fortified UHT and pasteurized milk products were also tested. Folate bioaccessibility and FBP stability were measured by taking samples along the compartments of the gastrointestinal model and measuring their folate and FBP concentrations. Folate bioaccessibility from folic acid-fortified milk products without additional FBP was 58-61%. This was lower (P <0.05) than that of the 5-CH3-H(4)folate-fortified milk products (71%). Addition of FBP reduced (P <0.05) folate bioaccessibility from folic acid-fortified milk (44-51%) but not from 5-CH3-H(4)folate-fortified milk products (72%). The residual FBP levels in the folic acid- and 5-CH3-H(4)folate-fortified milk products after gastrointestinal passage were 13-16% and 0-1%, respectively, of the starting amounts subjected to TIM. In conclusion, milk seems to be a suitable carrier for folate, because both folic acid and 5-CH3-H(4)folate are easily released from the matrix and available for absorption. However, our results suggest that folic acid remains partly bound to FBP during passage through the small intestine, which reduces the bioaccessibility of folic acid from milk in this model.