Isolipidic replacement of krabok oil by whole krabok seed reduces in vitro methanogenesis, but negatively affects fermentation
Panyakaew, Paiwan ; Schonewille, J.T. ; Cone, John W. ; Pellikaan, Wilbert F. ; Fievez, Veerle ; Yuangklang, Chalermpon ; Hendriks, Wouter H. - \ 2020
Journal of Animal Physiology and Animal Nutrition 104 (2020)2. - ISSN 0931-2439 - p. 453 - 461.
in vitro - Krabok oil - krabok seed - medium-chain fatty acids - methane
The background of the current in vitro study involves the issue of methane (CH4) production inherent to rumen fermentation. One of the dietary strategies to reduce enteric CH4 production by ruminants involves the supplementation of medium-chain fatty acids in diets. As such, oils containing high amounts of MCFA, such as coconut, palm kernel and krabok oil, are of much interest to formulate energy efficient and environmentally friendly rations for ruminants. Krabok oil (KO) reduces methanogenesis, but the appropriate inclusion level of dietary KO is unclear. We therefore investigated the dose–response relationship between krabok oil and CH4 production. In practice, the use of whole krabok seed (WKS), instead of KO, is easier, but the efficacy of WKS to inhibit methanogenesis was hitherto unknown. Thus, we also investigated whether WKS provides an alternative tool to inhibit CH4 production. The experimental substrates contained either KO, WKS, the residue of WKS after fat extraction residue (FER) or FER + KO. Appropriate amounts of WKS or its derivatives were added to a basal substrate so as to attain either a low, medium or high content of KO, that is, 37–46, 90–94 and 146–153 g/kg dry matter respectively. The experimental substrates were formulated to keep the amounts of incubated fat-free OM, crude protein, neutral detergent fibre and acid detergent fibre constant in order to avoid biased results through potential differences in fermentability between WKS and its derivatives, and the basal substrate. The latter resembled the ingredient composition of a total mixed ration commonly used in Thai dairy cows. Fully automated gas production (GP) equipment was used to measure gas- and CH4 production. Irrespective of the type of substrate (p ≥.115), both the absolute (ml/g fat-free OM) and relative (% of total GP) CH4 production was reduced at the highest inclusion level of WKS or its derivatives (p ≤.019). Total GP (ml/g fat-free OM), however, was reduced after incubation of FER, FER + KO, and WKS, but not KO, at the highest inclusion level of the respective substrates (p =.019). Volatile fatty acids were likewise affected (p ≤.001). Krabok oil can inhibit CH4 production but only when the dietary KO content is at least 9.4% (DM). Supplementation of KO in the form of WKS, however, is considered not opportune because the fat extracted residue of WKS is poorly degraded during fermentation.
Development of an in vitro protocol to screen Clavibacter michiganensis subsp. michiganensis pathogenicity in different Solanum species.
Mohd Nadzir, M.M. ; Vieira Lelis, Flavia ; Thapa, B. ; Ali, Afrida ; Visser, R.G.F. ; Heusden, A.W. van; Wolf, J.M. van der - \ 2019
Plant Pathology 68 (2019)1. - ISSN 0032-0862 - p. 42 - 48.
Clavibacter - Cmm - disease screening - in vitro - PathoScreen - tomato
Clavibacter michiganensis subsp. michiganensis (Cmm) is a quarantine organism in Europe and in many other countries. It is one of the most severe bacterial pathogens affecting tomato. Screening tomato plants for their resistance level
to Cmm requires a large amount of space under quarantine conditions and is therefore costly. This project developed a new inoculation protocol on in vitro tomato plants to facilitate a more economic and higher throughput disease screening. A new method using the PathoScreen system was tested to localize green fluorescent protein-tagged Cmm in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged Cmm in the shoots, but in the roots a high autofluorescence masked detection and thus sensitivity of the assay. The in vitro protocol was tested on several wild relatives of tomato, which were previously screened in a greenhouse assay. The correlation between wilt symptoms in vitro and wilt symptoms in the greenhouse was overall moderate (r = 0.6462). The protocol worked well in differentiating the two parents that were used in the mapping studies. This study shows that the in vitro protocol can be efficiently used for resistance breeding in many tomato genotypes.
In vitro gas and methane production of silages from whole-plant corn harvested at 4 different stages of maturity and a comparison with in vivo methane production
Macome, Felicidade ; Pellikaan, W.F. ; Hendriks, W.H. ; Dijkstra, J. ; Hatew, Bayissa ; Schonewille, J.T. ; Cone, J.W. - \ 2017
Journal of Dairy Science 100 (2017)11. - ISSN 0022-0302 - p. 8895 - 8905.
methane - corn silages - Maturity - in vitro - In vivo
The current study investigated the relationship between in vitro and in vivo CH4 production by cows fed corn silage (CS)-based rations. In vivo CH4 production was measured in climate respiration chambers using 8 rumen-cannulated Holstein-Friesian cows. In vitro CH4 production was measured using rumen fluid from the 8 cows that were fully adapted to their respective experimental rations. The animals were grouped in 2 blocks, and randomly assigned to 1 of the 4 total mixed rations (TMR) that consisted of 75% experimental CS, 20% concentrate, and 5% wheat straw [dry matter (DM) basis]. The experimental CS were prepared from whole-plant corn that was harvested at either a very early (25% DM), early (28% DM), medium (32% DM), or late (40% DM) stage of maturity. The 4 experimental TMR and the corresponding CS served as substrate in 2 separate in vitro runs (each run representing 1 block of 4 animals) using rumen fluid from cows fed the TMR in question. No relationship was found between in vivo CH4 production and in vitro CH4 production measured at various time points between 2 and 48 h. None of the in vitro gas production (GP) and CH4 production parameters was influenced by an interaction between substrate and origin of rumen fluid. In vitro measured 48-h GP was not affected by the maturity of whole-plant corn, irrespective whether CS alone or as part of TMR was incubated in adapted rumen inoculum. Incubation of the experimental TMR did not affect the kinetics parameters associated with gas or CH4 production, but when CS alone was incubated the asymptote of GP of the soluble fraction was slightly decreased with increasing maturity of CS at harvest. In vitro CH4 production expressed as a percent of total gas was not affected by the maturity of whole-plant corn at harvest. Several in vitro parameters were significantly affected (GP) or tended to be affected (CH4) by diet fed to donor cows. It was concluded that the current in vitro technique is not suitable to predict in vivo CH4 production from CS-based rations.
Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
Vissers, Anne M. - \ 2017
Wageningen University. Promotor(en): H. Gruppen; W.H. Hendriks, co-promotor(en): J.P. Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - beta vulgaris - laminaria - eiwitten - catechol oxidase - kernmagnetische resonantiespectroscopie - in vitro - massaspectrometrie - bruinkleuring - fermentatie - diervoedering
Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.
Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
Integrated strategy for the assessment of kidney toxicity : the case of aristolochic acids
Abdullah, Rozaini - \ 2017
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Ans Punt; Sebas Wesseling; Jochem Louisse. - Wageningen : Wageningen University - ISBN 9789463430807 - 207
animal testing alternatives - in vitro - toxicity - models - risk assessment - toxins - carboxylic acids - alternatieven voor dierproeven - in vitro - toxiciteit - modellen - risicoschatting - toxinen - carbonzuren
This PhD thesis aimed to provide additional evidence to demonstrate the potential of an integrated testing strategy using in vitro assays with physiologically based kinetic (PBK) modeling based-reverse dosimetry to predict in vivo toxicity without animal testing. Kidney toxicity was chosen as the toxicity endpoint and aristolochic acids (AAs) were selected as model chemicals. AAs are natural nephrotoxic, genotoxic and carcinogenic chemicals present in Aristolochia species. PBK models for rat, mouse and human were developed for aristolochic acid I (AAI) based on kinetic parameter values derived from in vitro incubations using relevant tissue fractions. Then, in vitro concentration-response curves for cytotoxicity of AAI were obtained in kidney cell lines and translated to in vivo dose-response curves for kidney toxicity using PBK modeling-based reverse dosimetry. The points of departure (PODs) obtained from these predicted in vivo dose-response curves generally fell within the range of PODs derived from in vivo literature data on kidney toxicity of AAI. The same PBK models were subsequently used to translate the in vitro concentration-response curves for AAI-DNA adduct formation to in vivo dose-response curves for kidney AAI-DNA adduct formation. The predicted in vivo AAI-DNA adduct formation in the rat, mouse and human kidney varied within an order of magnitude compared to the in vivo values reported in the literature. The PBK models were also used to predict the dose level that would be required in humans to obtain the level of DNA adducts in rats at the BMD10 (the benchmark dose causing a 10% extra risk above background level) value for AAI-induced tumor formation in the rat kidney. This analysis revealed that the dose level required to induce the level of DNA adduct formation that equals the DNA adduct level at the BMD10 were similar to AA doses estimated to be taken in Belgian patients that developed urinary tract cancer. Given that the exposure to AAI is often accompanied by the presence of AAII, in a next study the relative formation of DNA adducts by these two major AA congeners was investigated. The results revealed that the relative higher formation of AAI-DNA adducts as compared to AAII-DNA adducts observed in vitro was not reflected in vivo where the levels formed upon exposure to equal dose levels were relatively similar. PBK model based translation of the in vitro data to the in vivo situation revealed that PBK model based prediction of in vivo DNA adduct formation is feasible. However, predicted AAI-DNA adduct levels were higher than predicted AAII-DNA adduct levels, indicating that the difference between the in vitro and in vivo AAI-/AAII-DNA adduct ratios could only in part be explained by differences in in vivo kinetics of AAI compared to AAII. The discrepancy between the difference in DNA adduct formation of AAI and AAII in the in vitro and the in vivo situation is an issue that needs further investigation to also adequately predict the relative differences between the two AAs. In a final chapter this thesis aimed to investigate the possible risks associated with exposure to AAs based on AA levels measured in plant food supplements (PFS) and herbal products. This is of interest given the restrictions on the presence of AAs in food, installed in various countries including The Netherlands, after the incidences with induction of Aristolochic Acid Nephropathy upon use of herbal weight loss preparations that accidentally contained AAs. The risk assessment of PFS and herbal products containing AAs purchased via online markets revealed that consumers can still be exposed to AA-containing PFS and herbal products and that the corresponding levels of exposure raise concern especially for people who frequently use the products. Altogether, this thesis presented further support for the use of combined in vitro-PBK modeling based alternative tools for risk assessment and revealed the continued risks posed by AAs present in PFS and herbal products.
In vitro assays for hazard identification of nanoparticles
Kloet, Samantha K. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Jochem Louisse; Nico van den Brink. - Wageningen : Wageningen University - ISBN 9789462579415 - 213
nanotechnology - particles - in vitro - models - hazards - toxicity - toxicokinetics - nanotechnologie - deeltjes - in vitro - modellen - gevaren - toxiciteit - toxicokinetiek
The production of nanoparticles (NPs) has increased in the last decades and the number of products in which NPs are being incorporated is still growing. The rapid increase of nanotechnology has several benefits for society, yet there is an increasing concern that exposure to NPs may result in significant adverse health effects. Since NPs are incorporated in a variety of consumer products, it is likely that the general population will be exposed to NPs. It would be desirable that the safety and risk assessment of NPs could be largely based on studies using in vitro models instead of in vivo models as this would reduce the use of test animals, costs and time required to test the large numbers of NPs. The aim of the present thesis was to investigate the potential of in vitro testing strategies to detect hazards of NPs, focusing on toxicokinetic as well as toxicodynamic endpoints. Toxicokinetic studies focused on translocation of NPs in in vitro models of the placental barrier, while toxicodynamic studies were directed at two endpoints that represent potential hazards of NPs that have not yet been well characterized including: developmental toxicity and immunotoxicity.
In the present thesis different types of NPs were used. Polystyrene nanoparticles (PS-NPs) were selected because of their commercial availability, with high quality and a wide variety of available physicochemical properties like surface charge, and fluorescent labeling enabling easy detection in toxicokinetic (translocation) studies. Several metal (oxide) NPs were selected as well, of which some are possible constituents of food additives like TiO2, Fe2O3, SiO2 and Ag. Other metal oxide NPs that were selected were Mn2O3, CuO, Cr2O3, CoO and NiO to which we may be exposed via products like paints, catalysts, construction materials, coatings and batteries.
Placental translocation of NPs was studied as an important toxicokinetic aspect, since part of the toxicodynamic studies of the present thesis were directed at developmental toxicity testing of NPs. In order to obtain insight in toxicity and translocation of NPs across the placental barrier, cytotoxicity and translocation was studied for one positively and two negatively charged PS-NPs of 50 nm in an in vitro model of the placenta. In this study it appeared that in spite of similar size, surface charge and type of proteins in the protein corona, the differently charged NPs displayed a remarkable difference in cytotoxicity, with only the PS-NPs with an original positive charge inducing cytotoxicity. Translocation of PS-NPs appeared not to be related to PS-NP charge alone. A remarkable difference in translocation was found between the two 50 nm negatively charged PS-NPs that were obtained from different manufacturers. Since none of the characterized parameters, including size, surface charge and protein corona revealed remarkable differences between the two negatively charged NPs, the difference may originate from the chemical groups on the surface of the NPs generating the negative charge. The general conclusion from this study was that the in vitro BeWo b30 model can be used as a fast method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and to set priorities for further in vivo studies on translocation of NPs to the fetus.
The same PS-NPs as tested for placental translocation were investigated whether they are able to cause in vitro developmental toxicity in the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) focusing also on the effect that charge may have. The study showed that the two negatively charged PS-NPs did not show any effect in the ES-D3 cell differentiation assay up to the highest concentration tested while the positively charged PS-NP showed a concentration-dependent inhibition of ES-D3 cell differentiation. However, effect concentrations in the ES-D3 cell differentiation assay were close to cytotoxic concentrations, which indicated that the inhibition of the ES-D3 cell differentiation may be due to cytotoxic effects of the positively charged PS-NPs. This indicated that the inhibition of the ES-D3 cell differentiation by the positively charged PS-NPs may be caused by non-specific effects. Although the experiments on placental translocation of the present thesis showed that positively charged PS-NPs are more toxic than negatively charged PS-NPs, it appeared that this may not be generalizable to other NPs. This follows from the fact that in SiO2, Ag and TiO2 NPs that were reported in other studies to inhibit ES-D3 cell differentiation were negatively charged, while the negatively charged PS-NPs of the present study did not affect ES-D3 cell differentiation. Although the limited data available indicate that charge, size and coating of NPs may be important characteristics that determine the developmental toxicity potential of NPs, more (systematic) studies are needed to assess how physicochemical characteristics of NPs relate to their developmental toxicity. This information may help to prioritize NPs for in vitro and in vivo developmental toxicity testing.
In addition, toxic effects of a series of metal (oxide) NPs were tested in macrophage RAW264.7 cells in order to obtain insight in effect of these NPs on cells of the innate immune response. In these macrophage RAW264.7 cells the effects of the metal (oxide) NPs were characterized on cell viability, TNF-α production and mitochondria-related parameters like production of reactive oxygen species (ROS), mitochondrial permeability transition pore (MPTP) opening, and intracellular ATP levels. Altogether, results obtained showed no or limited effects of the NP formulations of metal (oxide) food additives on cell viability, ROS production, MPTP opening, ATP levels and TNF-α production in RAW264.7 macrophages. Effects were only observed at high concentrations that may not be physiologically relevant, indicating that related adverse effects upon exposure to the respective NPs in vivo may be limited.
Taken together, the present thesis provided further evidence of the influence of physicochemical properties of NPs in driving toxicity in in vitro models. However, the determination of the fate and toxicity of NPs using in vitro or in vivo models is a challenge that needs further evaluation. A combination of several factors likely play a role in determining the outcome of exposure including factors like NP core material and presence and type of coating agents resulting in various physicochemical properties (size, charge, etc.). This appears to hamper conclusive evaluation of the role of physicochemical characteristics of NPs in their potential hazards and risks so far. The results obtained do show however that in vitro assays can detect differences in potential hazards posed by NPs. Therefore it is concluded that the results of the work presented in this thesis will contribute to the further development and use of non-animal based testing strategies for safety testing of NPs providing insight into selected potential hazards of the tested NPs.
Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action
Zande, Meike van der; Undas, Anna K. ; Kramer, Evelien ; Monopoli, Marco P. ; Peters, Ruud J. ; Garry, David ; Antunes Fernandes, Elsa C. ; Hendriksen, Peter J. ; Marvin, Hans J.P. ; Peijnenburg, Ad A. ; Bouwmeester, Hans - \ 2016
Nanotoxicology 10 (2016)10. - ISSN 1743-5390 - p. 1431 - 1441.
Caco-2 - in vitro - MCF-7 - silver nanoparticles - toxicogenomics
The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study, we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3 were exposed for 6 h and 24 h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism.
Aspects of bulblet growth of lily in vitro
Askari Rabori, N. - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk. - Wageningen : Wageningen University - ISBN 9789462577602 - 130
lilium - in vitro - growth - in vitro regeneration - abiotic conditions - stress - bulbs - lilium - in vitro - groei - in vitro regeneratie - abiotiek - stress - bollen
Many geophytes have a high ornamental value. They are preferably propagated by micropropagation because in this way large quantities of uniform, disease-free starting material are produced in a short period of time. In comparison with shoots, bulblets have several clear advantages as starting material. Therefore, in vitro bulblet formation is an important target for improvement of tissue culture of geophytes. The research described in this thesis was carried out with the lily cultivars ‘Santander’ and ‘Stargazer’.
Commercially, lily is the second geophyte in the global flower industry. The size of lily bulblets regenerated in vitro has a direct effect on the performance in the field. After planting, large bulblets sprout with a stem and gain twice as much weight compared with small bulblets that sprout with a rosette. In the present study we studied basic and applied aspects of the following topics: (1) new methods for sterilization during initiation, (2) the effect of scale-related factors on bulblet growth, (3) growth enhancement by moderate abiotic stresses, and (4) the effect of CO2 removal from headspace of tissue culture containers on lily bulblet growth and as a control Arabidopsis thaliana seedling growth.
Contamination is an everlasting problem in tissue culture laboratories. Lily bulbs are underground organs and contain therefore more contaminants as compared with aerial organs. During initiation, operators cause additional contamination in two ways that have as yet not been recognized adequately. (1) Rinsing explants with sterile water after surface-sterilization is the generally advised method to remove the residues of decontaminants. However, when scales are heavily contaminated, the surface-sterilization does not kill microorganisms in all scales. Surface-sterilization is usually done with batches of 10 to 30 scales and the contaminated scales may cross-contaminate uninfected scales during rinsing in water. We have tested the rinsing water and found heavy bacterial contamination in the 2th and especially the 3rd rinse. The contaminated rinsing water resulted in a high incidence of cross-contamination. Cross contamination was reduced almost fully by rinsing in diluted NaClO solution (0.03%) instead of sterile water. There was no negative effect of diluted NaClO on growth. (2) A second way of introducing contamination by the operator is the entering of microorganisms during detachment from mother bulbs via the vascular bundles caused by negative hydrostatic pressure within the bulb tissue. By detaching scales from bulbs submerged in 0.03% NaClO, hydrostatic-pressure related contamination was strongly reduced. The growth of bulblets increased by 22% and 17% when cross contamination and negative-hydrostatic pressure related contamination were prevented.
Storage organ formation is controlled by interacting environmental, biochemical and genetic factors. We studied various aspects of the effect of the scale explant. We found that large explants produced larger bulblets than small explants. When bulblets were excised and cultured in vitro, growth was improved by 33% when a small piece of the original scale explant was left attached to the bulblet. The position in the scale from where the explant was excised affected the growth of the regenerating bulblets. Basal-scale explants improved bulblet growth by 40-50 % compared with apical-scale explants. This might be related to the physiological state of the tissues: there was more starch and there were more vascular bundles present in basal scale explants. Furthermore, excision of an explant from the middle of the scale improved bulblet growth by 40-50 % compared with explants excised from the edge of the scale. In general, the middle scale explants were heavier and contained wider vascular bundles.
Plants in stressful conditions tend to allocate a higher proportion of biomass to below-ground biomass (roots and storage organs) as compared to above ground biomass. We investigated the effect of moderate abiotic stresses on lily bulblets grown in vitro. In general, lily bulblets showed an increased growth after moderate stresses. Hot air increased growth by 30%, hot water by 40%. We also examined the effect of drought and anaerobiosis. Drought stress increased growth of bulblets by 40% in the cultivar ‘Stargazer’, but significantly decreased bulblet growth in cv ‘Santander’. Anaerobiosis increased growth in ‘Stargazer’ and ‘Santander’ by 32% and 65%, respectively. We also showed that a moderate stresses treatment protects lily bulblets against future severe abiotic stresses.
In general, the in vitro situation is not favorable for plants. Composition of the headspace (high humidity, strongly fluctuating CO2- and O2-levels and accumulation of gases like ethylene), low light, and wounding are unfavorable conditions that plants have to deal with. We examined the effect of CO2 starvation on growth in vitro with and without addition of 3% sucrose to the medium. A CO2-poor headspace reduced the growth of bulblets, leaves, roots and scale explants strongly, also in the presence of 3% sucrose. CO2 removal from the headspace decreased growth of Arabidopsis seedlings by 50 % on medium with 3% sucrose. It seems unlikely that the growth reduction on medium with 3% sucrose is caused solely by the lack of sucrose production in photosynthesis when CO2 is removed. Indeed, we found evidence that the low CO2 resulted in heavy stress that in turn may reduce growth. Fv/Fm in lily and Arabidopsis dropped when CO2 was removed. Occurrence of reactive oxygen radicals (ROS) was examined in Arabidopsis seedlings by staining with nitroblue tetrazolium (NBT). ROS was virtually absent in ex vitro growing seedlings and very abundant in seedlings grown under CO2 starvation. Seedlings grown under normal tissue culture conditions showed an intermediate presence of ROS. We hypothesize that low levels of CO2 may results in ROS in in vitro seedlings which reduces growth.
The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - in vitro - biosynthese - peroxisomen - microarrays - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - kaempferol - serine proteïnasen - threonine
Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.
A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.
As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.
To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.
Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.
Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.
The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.
Dermal absorption and toxicological risk assessment : pitfalls and promises
Buist, H. - \ 2016
Wageningen University. Promotor(en): Ruud Woutersen; Ivonne Rietjens, co-promotor(en): J.J.M. van de Sandt. - Wageningen : Wageningen University - ISBN 9789462577275 - 200
skin - absorption - permeability - in vitro - experiments - exposure assessment - risk assessment - toxicology - biocides - rodenticides - preservatives - disinfection - huid - absorptie - permeabiliteit - in vitro - experimenten - blootstellingsbepaling - risicoschatting - toxicologie - biociden - rodenticiden - conserveermiddelen - desinfectie
Absorption of toxic substances via the skin is an important phenomenon in the assessment of the risk of exposure to these substances. People are exposed to a variety of substances and products via the skin, either directly or indirectly, while at work, at home or in public space. Pesticides, organic solvents and metalworking fluids are seen to be important contributors to adverse health effects due to occupational exposure via the skin. In daily life, cosmetics, clothing and household products are the most relevant commodities with respect to exposure via the skin.
Given the importance of skin exposure in the assessment of the risk of toxic substances, the objective of this thesis was to further develop, evaluate and improve methods for including skin absorption data this assessment.
In this thesis, four factors influencing dermal absorption, namely dermal loading (chapters 3 and 6), irritative/corrosive potential (chapters 3 and 4), frequency of exposure (chapters 3, 4 and 5) and the vehicle used (chapter 5), were investigated in more detail. Furthermore, a model to extrapolate infinite dose absorption data to finite dose conditions, baptized Dermal Absorption Model for Extrapolation (DAME), was developed and tested.
n chapter 2 of this thesis, the relationship between relative dermal absorption and dermal loading was investigated. Hundred-and-thirty-eight dermal publicly available absorption experiments with 98 substances were evaluated. The results obtained revealed that dermal loading ranged mostly between 0.001 and 10 mg/cm2. In 87 experiments (63%), an inverse relationship was observed between relative dermal absorption and dermal loading. On average, relative absorption at high dermal loading was 33 times lower than at low dermal loading. Known skin irritating and volatile substances less frequently showed an inverse relationship between dermal loading and relative absorption. It was concluded that when using relative dermal absorption in regulatory risk assessment, its value should be determined at or extrapolated to dermal loadings relevant for the exposure conditions being evaluated.
n chapter 3 of this thesis, a literature search was presented with the aim to investigate whether neglecting the effects of repeated exposure may lead to an incorrect estimate of dermal absorption. The results demonstrated that the effect of repeated versus single exposure does not demonstrate a unique trend. Nevertheless, an increase in daily absorption was frequently observed upon repeated daily exposure. The little information available mostly concerned pharmaceuticals. However, consumers and workers may be repeatedly exposed to other types of chemicals, like disinfectants and cleaning products, which often contain biocidal active substances that may decrease the barrier function of the skin, especially after repeated exposure. These biocidal products, therefore, may present a safety risk that is not covered by the current risk assessment practice since absorption data are usually obtained by single exposure experiments. Consequently, it was decided to investigate the importance of this issue for biocide safety evaluation. As the literature search revealed that hardly any data on absorption upon repeated dermal exposure to biocides are available, it was concluded that data need to be generated by testing.
To cover the entire range of biocidal products in such testing, a representative series of biocidal substances should be tested, making in vitro testing of dermal absorption the preferred choice over in vivo testing. Based on an inventory made, it appeared that the 16 product types represented among the biocidal products authorised in the Netherlands could be clustered into 6 more or less homogeneous categories based on similarity in active substances. This result could facilitate experimental testing by providing a basis for selection of a limited number of representative compounds to be evaluated.
n chapter 4 of this thesis, the importance of the effect of repeated dermal exposure on skin permeability for biocide safety evaluation was investigated, using a selection of nine representative biocides from the inventory made in chapter 3. The in vitro dermal penetration of tritiated water and [14C]propoxur was chosen as a measure of the permeability and integrity of human abdominal skin after single and repeated exposure. The results indicated that single and repeated exposure to specific biocidal products (e.g. the quaternary ammonium chlorides DDAC and ADBAC) may significantly increase skin permeability, especially when the compounds are applied at high concentrations, while a substance like formaldehyde may reduce skin permeability under specific conditions.
n chapter 5 of this thesis, the in vitro dermal absorption kinetics of the quaternary ammonium compound didecyldimethylammonium chloride (DDAC) during single and repeated exposure was studied in more detail. In addition, the influence of biocidal formulations on the absorption of DDAC was investigated, because it was expected that formulation characteristics may be another factor influencing its dermal absorption. The analysis of biocidal products on the Dutch market, reported in chapter 3, indicated that DDAC is often used in combination with other active ingredients. DDAC was most frequently combined with formaldehyde, glutaraldehyde and/or alkyldimethylbenzylammonium chloride (ADBAC). Consequently, commercial formulations containing one or more of these additional active ingredients were selected, in addition to one formulation containing only DDAC as an active ingredient. The selected commercial formulations tended to reduce skin penetration of DDAC. This was most pronounced with the formulation containing the highest concentration of formaldehyde (196 mg/mL) and glutaraldehyde (106 mg/mL), which reduced the flux of DDAC across the skin by 95%. The reduction caused by the only tested formulation containing no other active ingredients than DDAC, and thus incorporating no aldehydes, was smallest, and did not reach statistical significance.
n chapter 6 of this thesis, a simple in silico model to predict finite dose dermal absorption from infinite dose data (kp and lag time) and the stratum corneum/water partition coefficient (KSC,W) was developed. This model was tentatively called Dermal Absorption Model for Extrapolation (DAME). As dermal exposure may occur under a large variety of conditions leading to quite different rates of absorption, such a predictive model using simple experimental or physicochemical inputs provides a cost-effective means to estimate dermal absorption under different conditions.
To evaluate the DAME, a series of in vitro dermal absorption experiments was performed under both infinite and finite dose conditions using a variety of different substances. The kp’s and lag times determined in the infinite dose experiments were entered into DAME to predict relative dermal absorption value under finite dose conditions. For six substances, the predicted relative dermal absorption under finite dose conditions was not statistically different from the measured value. For all other substances, measured absorption was overpredicted by DAME, but most of the overpredicted values were still lower than 100%, the European default absorption value for the tested compounds.
In conclusion, our finite dose prediction model (DAME) provides a useful and cost-effective estimate of in vitro dermal absorption, to be used in risk assessment for non-volatile substances dissolved in water at non-irritating concentrations.
n chapter 7 of this thesis, the results of the research reported in chapters 2 to 6 were put into perspective, the pitfalls and promises emanating from them discussed and general conclusions drawn. The possible influence of vehicles on absorption and the possible impact of irritative or corrosive vehicles or chemicals on the skin barrier have been demonstrated in this thesis. An in silico predictive model tentatively called DAME was developed, which enables the user to evaluate a variety of dermal exposure scenarios with limited experimental data (kp and lag time) and easy to obtain physicochemical properties (MW and log KOW). The predictions of our experiments reported in chapter 6 were compared to those of the Finite Dose Skin Permeation (FDSP) model published on the internet by the US Centers for Disease Control and Prevention (CDC). DAME outperformed FDSP (R2 of the correlation predicted/measured potential absorption 0.64 and 0.12, respectively). At present, the applicability domain of DAME is limited to non-volatile substances dissolved in aqueous solvents. However, in future the model will be adapted to include volatile substances as well.
Altogether, it is concluded that dermal exposure can be an important factor in risks posed by chemicals and should be taken into account in risk assessment. The methods to actually do this are still open for further improvement to better account for the various factors influencing skin penetration and to develop adequate combinations of in vitro and in silico models that can accurately predict human dermal absorption.
Replacing animal experiments in developmental toxicity testing of phenols by combining in vitro assays with physiologically based kinetic (PBK) modelling
Strikwold, Marije - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Ruud Woutersen, co-promotor(en): Ans Punt. - Wageningen : Wageningen University - ISBN 9789462576926 - 169
animal experiments - animal testing alternatives - toxicity - testing - phenols - in vitro - embryonic stem cells - tissues - cells - dosage - toxicology - animal health - dierproeven - alternatieven voor dierproeven - toxiciteit - testen - fenolen - in vitro - embryonale stamcellen - weefsels - cellen - dosering - toxicologie - diergezondheid
Intestinal nutrient sensing : a gut feeling for food
Wielen, N. van der - \ 2016
Wageningen University. Promotor(en): Renger Witkamp, co-promotor(en): Jocelijn Meijerink; Henk F.J. Hendriks. - Wageningen : Wageningen University - ISBN 9789462576995 - 200
obesity - hormones - intestines - gastrointestinal hormones - pancreozymin - vasoactive intestinal peptide - sensing - in vivo experimentation - animal models - in vitro - gastric bypass - food - weight reduction - stevia rebaudiana - release - obesitas - hormonen - darmen - maagdarmhormonen - pancreozymine - vasoactief intestinaal peptide - aftasten - in vivo experimenten - diermodellen - in vitro - buik bypass - voedsel - gewichtsvermindering - stevia rebaudiana - vrijgeven
The alarming increase in obesity rates creates an urgent need for effective prevention and treatment strategies. The most effective treatment for obesity today is bariatric surgery. Bariatric surgery comprises a number of different procedures having in common that they induce weight loss and alter gut hormone release. Gut hormones are well known for their effects on food intake behavior and their role in weight loss after bariatric surgery is undeniable. In addition, the therapeutic use of GLP-1 (Glucagon-Like Peptide-1) analogues including liraglutide in type II diabetes and obesity is on the rise. This underlines why gut hormones are considered promising targets for the development of new treatment strategies against obesity and its comorbidities.
The secretion of gut hormones, among which GLP-1, is influenced by nutrient ingestion. The interactions of dietary components or their breakdown products with receptors and transporters located on the enteroendocrine cells of the intestinal tract can induce their release, a process called intestinal nutrient sensing. In this thesis, we aimed to further elucidate intestinal nutrient sensing mechanisms on a cellular level. First, the regional expression of several gut nutrient sensing related genes along the intestinal tract was assessed in three commonly studied species, namely mouse, pig and man. Gene expression of receptors, transporters and peptides involved in nutrient sensing shows a distinctive distribution pattern along the small intestine, which is in the distal small intestine highly similar between the species. Subsequently, we sought to investigate if this expression was changed after a weight loss inducing bariatric procedure. By whole transcriptome analysis, we showed that upper gastrointestinal tissue expression of genes associated with nutrient sensing was hardly changed. In contrast, a considerable reduction in inflammatory pathways was observed.
Next, we sought to investigate the effects of the non-caloric sweetener rebaudioside A. This Stevia rebaudiana-derived compound was approved on the European market in 2011. As there is still some controversy about the effects of sweeteners in general on GLP-1 release, we investigated the effects of this specific sweetener. Because of the short half-life of GLP-1, the effect of nutrient stimulation was mainly studied in ex vivo and in vitro models in which local intestinal hormone release could be determined. A two dimensional gut model using intestinal organoids derived from murine intestinal crypts was developed to study location-specific hormone secretion. Rebaudioside A was found to induce GLP-1 and PYY release ex vivo from porcine intestinal tissue and in two dimensional organoids. This induction of the release was specific for the intestinal location, with the ileum being most potently stimulated by rebaudioside A. Moreover, prolonged exposure to rebaudioside A increased enteroendocrine cell numbers in two dimensional organoids. When studying the underlying mechanism in enteroendocrine STC-1 cells, we concluded that rebaudioside A-induced GLP-1 release was independent of the sweet taste receptor.
The studies presented in this thesis add to our understanding the role of receptors and other molecular structures that are likely to be involved in nutrient sensing and the modulation of gut hormone release. What we know now is that several factors play a role in gut hormone release. This includes not only the nature and dose of the active compound(s), but also the location and timing of its (their) interactions with receptors and other targets along the gastrointestinal tract. We have shown that rebaudioside A may be a potential compound to induce gut hormone release in vivo, especially when applied to the distal small intestine. Therefore, rebaudioside A may be a promising compound to influence food intake, possibly most potent when delivered in the ileum.
Alternative testing strategies for predicting developmental toxicity of antifungal compound
Li, H. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens; Bennard van Ravenzwaay, co-promotor(en): Jochem Louisse. - Wageningen : Wageningen University - ISBN 9789462576780 - 197
toxicity - fetal development - transfer - infant development - adolescent development - child development - pregnancy - in vivo experimentation - modeling - placenta - in vitro - risk assessment - tebuconazole - conazole fungicides - antifungal agents - alternative methods - toxiciteit - foetale ontwikkeling - overdracht - zuigelingenontwikkeling - adolescentenontwikkeling - kinderontwikkeling - zwangerschap - in vivo experimenten - modelleren - placenta - in vitro - risicoschatting - tebuconazool - conazoolfungiciden - antimycotica - alternatieve methoden
Determination of safe human exposure levels of chemicals in toxicological risk assessments largely relies on animal toxicity data. In these toxicity studies, the highest number of animals are used for reproductive and developmental toxicity testing. Because of economic and ethical reasons, there is large interest in the development of in vitro and/or in silico test systems as alternatives for the animal studies. The aim of the present thesis was to evaluate the applicability of combined in vitro approaches taking toxicokinetic and toxicodynamic aspects into account, as well as of an integrated in vitro and in silico approach for prediction of developmental toxicity using a series of antifungal compounds as the model compounds.
Transplacental transfer of compounds is highly likely to play an important role in developmental toxicity, so we developed and validated an in vitro placental barrier model using BeWo b30 cells to predict placental transfer. Then we investigated the applicability of the ES-D3 cell differentiation assay combined with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies of two sets of selected antifungal compounds. The data obtained show that the combined in vitro approach provided a correct prediction for the relative in vivo developmental toxicity, whereas the ES-D3 cell differentiation assay as stand-alone did not. In order to detect specific structural alterations induced by chemicals, we investigated the applicability of the ex ovo assay of chicken embryos to predict the specific alterations induced by the antifungal compounds. Data revealed that the ex ovo assay of chicken embryos is able to assess the teratogenic potential of antifungal compounds, and, when combined with the in vitro BeWo transport model, is able to better predict relative in vivo prenatal developmental toxicity potencies.
Subsequently, we translated in vitro concentration–response data of the antifungal compound tebuconazole, obtained in the ES-D3 cell differentiation assay and the ex ovo assay of chicken embryos, into predicted in vivo dose–response data using physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry. The results show that the BMD10 values from predicted dose–response data from both assays are in concordance with BMD10 values derived from in vivo data (within 5-fold difference). This revealed that PBK modeling is a promising tool to predict in vivo dose-response curves based on the results of in vitro toxicity assays, and may therefore be used to set a point of departure for deriving safe exposure limits in risk assessment.
It is concluded the combined in vitro approaches and the integrated in vitro-in silico approaches appear to be promising for the screening and prioritization of chemicals and to provide reference values, such as BMD10 values, without using animals, therefore contributing to the 3R principle of animal testing.
Estrogenicity and metabolism of prenylated flavonoids and isoflavonoids
Schans, M.G.M. van de - \ 2015
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken; Toine Bovee. - Wageningen : Wageningen University - ISBN 9789462574748 - 180
flavonoïden - isoflavonoïden - glycine max - sojabonen - oestrogeenreceptoren - zoethout - glycyrrhiza glabra - in vitro - flavonoids - isoflavonoids - glycine max - soyabeans - oestrogen receptors - liquorice - glycyrrhiza glabra - in vitro
Binding of (prenylated) flavonoids and isoflavonoids to the human estrogen receptors (hERs) might result in beneficial health effects in vivo. To understand structure-activity relationships of prenylated (iso)flavonoids towards the hERs, prenylated (iso)flavonoids were purified from extracts of licorice roots and elicited soybean seedlings. It was observed that prenylation can modulate estrogenicity. Unprenylated, chain and δ-position pyran prenylated (iso)flavonoids show an agonistic mode of action, whereas α/β-position pyran, α/β-position furan and double chain prenylated (iso)flavonoids show an antagonistic mode of action towards hERα in the yeast bioassay. The mode of estrogenic action of prenylated (iso)flavonoids could be related to structural features of the hER. In particular, the increase in length of α/β-position pyran prenylated compounds was related to indirect antagonism. It was also shown that heat and acid affected the stability of 6a-hydroxy-pterocarpans, converting them into their respective 6a,11a-pterocarpenes and consequently modulate their estrogenicity. Six prenylated isoflavonoids acted as SERMs and eight prenylated isoflavonoids showed ER subtype-selective behavior. The kind of prenylation (chain, furan or pyran) was most important for determining SERM activity, whereas additionally the backbone structure, i.e. the presence of an additional D-ring, was of importance for determining ER subtype-selectivity. To determine structure-metabolism relationships, in vitro conversion of purified prenylated (iso)flavonoids by liver enzymes was studied. These compounds can be extensively metabolized by phase I and II enzymes. A glucuronidation yield between 70-80% was observed. It was also shown that pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, compared to unprenylated compounds (only 1 hydroxyl isomer).
Biological processes induced by ZnO, Amoxicillin, Rye and Fructooligosaccharides in cultured Intestinal Porcine Epithelial Cells : VDI-4; In-vitro tests 2013-2014
Hulst, M.M. ; Hoekman, A.J.W. ; Wijers, I. ; Schokker, D. ; Smits, M.A. - \ 2015
Wageningen : Wageningen UR Livestock Research (Livestock Research report 882) - 42
in vitro - bioassays - epithelium - livestock - feed additives - genes - immunology - in vitro - biotesten - epitheel - vee - voedertoevoegingen - genen - immunologie
The objective of this study was to develop an in-vitro bioassay using cultured Intestinal Porcine Epithelial Cells (IPEC-J2) and evaluate the capability of this assay to predict enterocyte-specific physiological and immunological processes induced by nutrients/additives in the intestines of farm animals. Responses to five nutrients/feed-additives, similar to those studied in animal trials, performed in the Feed4Foodure framework, were measured by gene expression analysis of IPEC-J2 cells either under stressed (Salmonella) or non-stressed conditions. Response genes were analysed using bioinformatics web-tools in order to identify dominant biological processes induced by these nutrients/feed-additives and to predict key-genes/proteins important for regulation of these biological proc
Thyroid in a jar: towards an integrated in vitro testing strategy for thyroid-active compounds
Jomaa, B. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Jac Aarts; Ad Peijnenburg. - Wageningen : Wageningen University - ISBN 9789090290089 - 187
schildklierziekten - schildklierhormonen - hormoonverstoorders - in vitro - assays - celgroei - thyroid diseases - thyroid hormones - endocrine disruptors - in vitro - assays - cell growth
Jomaa, B. (2015). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. PhD thesis, Wageningen University, the Netherlands
The aim of this thesis was to find in vitro and toxicogenomics-based alternatives to in vivo thyroid hormone disruption tests. In vitro alternatives can help reduce the amount of animal testing required under the European Union regulation for the registration, evaluation, authorization and restriction of chemicals (REACH). Moreover, with the use of human cell lines and human-identical synthetic proteins, interspecies differences can be reduced and in some cases eliminated. This thesis has shed light on the relevance of current in vitro assays for thyroid and pituitary cell proliferation, has led to the development of the TSH screen, a luminol-based thyroid peroxidase inhibition assay and the zebrafish-based general development score (GDS) for the detection of developmental toxicants, including those that act through the thyroid hormone system. Moreover, the microarray assay for real-time coregulator-nuclear receptor interaction (MARCoNI) assay was used to reveal the modulating effects of thyroid-active compounds on TRα and TRβ interactions with a peptide array representing 66 different coregulators. These developments along with an in-depth analysis of the thyroid hormone system and the presentation of the state of the art in thyroid disruption testing have highlighted the progress made and at the same time have underlined the challenges that lay ahead.
Development of an integrated in vitro model for the prediction of oral bioavailability of nanoparticles
Walczak, A.P. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Hans Bouwmeester; Peter Hendriksen. - Wageningen : Wageningen University - ISBN 9789462572201 - 153
nanotechnologie - deeltjes - in vitro - inname - biologische beschikbaarheid - voedingsonderzoek bij de mens - risicoschatting - nanotechnology - particles - in vitro - ingestion - bioavailability - human nutrition research - risk assessment
Title of the PhD thesis: Development of an integrated in vitro model for the prediction of oral bioavailability of nanoparticles
The number of food-related products containing nanoparticles (NPs) increases. To understand the safety of such products, the potential uptake of these NPs following consumption needs to be assessed. In normal safety assessment studies this is investigated using animal models. For scientific, ethical and economical reasons, there is a demand to refine, reduce and replace animal testing by developing in vitro alternatives for hazard characterization. In this thesis an in vitro model for the prediction of the uptake of NPs in the human body after consumption was developed. The model consists of two parts. The first part is a laboratory incubation model mimicking human digestion in mouth, stomach and intestine. For the second part, human intestinal wall cells are used to assess the uptake of nanoparticles. The two models were combined into the integrated in vitro model to take into consideration the potential effect of digestion on nanoparticle uptake in the gut. The main outcome of the work is that the cell-based integrated in vitro model can be used to evaluate which NPs are likely taken up by the body at the highest rate. The size of NPs and the type of chemical groups on their surface greatly influenced the uptake of NPs. The developed model can be used to prioritize the NPs for additional investigations. Using this model in the safety assessment of NPs would reduce the number of animals used in safety assessment.
Innovative approaches to reduce animal testing : replace whenever possible, reduce through refinement and mechanistic understanding
Ravenzwaay, B. van - \ 2013
Wageningen : Wageningen University - ISBN 9789461736161 - 24
alternatieven voor dierproeven - dierproeven - in vitro - toxische stoffen - toxicologie - kinetica - modellen - dierenwelzijn - animal testing alternatives - animal experiments - in vitro - toxic substances - toxicology - kinetics - models - animal welfare
'Many of the in vitro toxicological studies have not been sufficiently validated to determine their applicability domain, even less have gained regulatory acceptance. Major advantage of in vitro testing today is the early identification of significant hazards in compound development and reduced and targeted animal testing. Replacing complex animal tests may be achieved by a battery of in vitro test addressing the adverse outcome pathway in question. Kinetics models are needed to translate in vitro results into in vivo values.'
Towards a realistic risk characterization of complex mixtures using in vitro bioassays
Montano Garces, M. - \ 2013
Wageningen University. Promotor(en): Tinka Murk, co-promotor(en): A.C. Gutleb. - [S.l.] : s.n. - ISBN 9789461736598
risicoschatting - persistente organische verontreinigende stoffen - verontreinigde sedimenten - mengsels - biotesten - in vitro - toxiciteit - schildklierhormonen - risk assessment - persistent organic pollutants - contaminated sediments - mixtures - bioassays - in vitro - toxicity - thyroid hormones
This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments.
In Chapter 1 the importance of complex mixture characterization in modern society is introduced. The methods available, their current advantages and their disadvantages for complex mixture testing are described. With the shift from policy oriented chemical testing towards the inclusion of in vitro bioanalysis, important challenges have to be overcome to ensure a relevant and reliable quantification of the toxic potency of complex mixtures. These challenges are explained in the introduction, including the status of development and validation of those aspects for reliable testing. One of the main advantages that in vitro bioanalysis has to offer is the possibility to quantify the toxic potency of compounds for which chemical analytical methods have not or hardly been developed, for example because standards do not yet exist. Hydroxylated metabolites of POPs are an example of a toxicologically relevant group of compounds that can exert endocrine disrupting effects, but they cannot yet be routinely analysed. A selection of yet unsolved issues are further studied and discussed in this thesis, as outlined in the “approach and structure of the thesis”.
In Chapter 2 a meta-analysis is performed to study the occurrence and relevance of hydroxylated (OH) compounds in humans and wildlife. Reported body burdens of halogenated phenolic contaminants (HPCs), including OH-POP in different tissues from humans and wildlife species, are reviewed in relation to the concentration of their putative parent compounds to be able to reveal relevant exposure routes and sub-populations at risk. Highest OH-POP levels were found in blood plasma, and highly perfused and fetal tissues. Plasma concentrations of analysed known HPCs ranged from 0.1-100 nM in humans and up to 240, 454, 800 and 7650 nM for birds, fish, cetaceans and other mammals, respectively. Reported metabolite blood plasma levels also are compared with relevant toxicological threshold concentrations from toxicological studies, and appeared to fully fall within the in vitro (0.05–10000 nM) and in vivo (3-940 nM) effect concentrations reported for OH-POPs. Given the sensitivity of early developmental stages, and information lacking about the general population, it is advisable to determine HPC background blood levels in children and fetal tissue .
Given the toxicological relevance of the OH-POPs, Chapter 3 aims at providing solutions to the long standing problem of the in vitro production and analysis of OH-POP metabolite thyroid hormone disrupting (THD) potency via binding to plasma thyroid hormone binding proteins (THBPs). In sediments and for example seafood, the POPs occur as parent compounds that would only become THD after metabolisation (hydroxylation). Several methods have shown the competitive thyroxine (T4) T4 displacement potency of pure metabolites. However, in vitro metabolization of, among others, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers(PBDEs) followed by in vitro quantification of their potency has encountered drawbacks related to the co-extraction of compounds disturbing the T4-TTR competitive binding assay. The present study identifies and quantifies the major co-extractants, cholesterol and saturated and non-saturated fatty acids (SFA and NSFA), at levels above 20 μM (20 nmol per mg protein in the incubation mixture) following various extraction methods. A new method is presented to in vitro metabolise parent compounds into OH-metabolites followed by selective extraction of metabolites while four-fold reducing co-extraction of the disturbing compounds. In addition a microplate-format non-radioactive fluorescence displacement assay was developed to quantify the TTR binding potency of the metabolites formed. The effectiveness of the in vitro metabolism and extraction of the OH-metabolites of the model compounds CB 77 and BDE 47 was chemically quantified with a newly developed chromatographic method analyzing silylated derivatives of the OH-metabolites and co-extractants. Due to the mentioned improvements, it is now possible to make a dose-response curve up to 50% inhibition with OH-metabolites extracted from bioactivated CB 77 and BDE 47. Without taking the toxic potencies of bio-activated POPs into account with bioanalysis, the hazard and risk posed by POPs will be seriously underestimated.
The chapters 4 and 5 are committed to tackle the issues of supramaximal (SPMX) responses and sample extract concentration which are crucial to reliably quantify of the toxic potencies of complex mixtures with in vitro bioassays.
A SPMX effect is the phenomenon that compounds induce a maximum response in an assay that is significantly higher than that of the positive control. As the positive control is used to quantify the toxic potency of a sample, this could result in over-estimation of its toxic potency. As this has been most elaborately reported for in vitro estrogenicity assays, a meta-analysis was performed of such assays, compounds and conditions in which the effect is observed (Chapter 4a).For the 21 natural and industrial chemicals that could be identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Relevant information on assay characteristics, however, sometimes lacked. Diethylstilbestrol (DES), genistein (GEN) and bisphenol A (BPA) were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects, could be related to a number of specific assay characteristics: 1) the type and concentration of the serum used to supplement the exposure medium; 2) the endpoint used to quantify the estrogenic potency (endogenous or transfected reporter gene), 3) the number of EREs (estrogen responsive elements) used before the reporter gene, and 4) the nature of the promoter’s. There were no indications that solvent concentration in culture, exposure period or cell model influenced the occurrence of SPMX. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts.
Several SPMX inducers also have been reported to block cellular efflux pumps in vivo and in vitro (Anselmo et al. 2012; Georgantzopoulou et al. 2013). Therefore it was hypothesized that efflux pump blockers present in environmental matrices could increase the internal concentration of bioassay agonists and thus cause the SPMX. In Chapter 4b this hypothesis was tested by adapting a 96-well plate cellular efflux pump inhibition assay (CEPIA) to the H4IIE rat hepatoma cell line used for the DR.Luc reporter gene assay for dioxin-like compounds. The influence of various environmentally relevant efflux pump inhibitors on the 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) response was tested. Under the DR.Luc assay conditions there was no evidence that P-gp efflux pump inhibitors modified or potentiated the activity of TCDD. Neither genistein nor quercetin, two potent SPMX inducers on ER-mediated assays, induced any signal on the DR.Luc assay, nor influenced the luciferase induction by TCDD. Future work should be focused on testing the consequences of efflux pump inhibition with an AhR-agonist which is a P-gp substrate, as this could result in intracellular accumulation of this AhR-agonist.
It is standard practice to use a high single stock concentration of extracts to further dilute test concentrations from and perform the analysis. However, a high contaminant load in an extract may oversaturate the solubility of the extracted compounds in carrier solvents and overload the clean-up columns which may reduce the efficiency of polyaromatic hydrocarbons (PAHs) elimination from the extract. These problems may cause respectively under- or over-estimation of the quantified dioxin-like toxic potency. Therefore Chapter 5 focuses on the effects of initial stock concentrations, including sonication assisted dissolution and exposure time, on the quantified dioxin-like potency of cleaned nonpolar sediment extracts. Indeed, more than 20 g sediment equivalents (SEQ)/mL DMSO) as initial stock concentrations resulted in underestimation of bio-TEQ levels in the sediments as observed for cleaned nonpolar sediment extracts from various locations in Luxembourg. An overload of extract on clean-up columns caused an over-estimation of the dioxin-like potency at 24 hours of exposure, probably due to limited removal of PAHs that can induce false positive responses in the in vitro assays. Sonication assisted dissolution of the stock before serial dilution strongly reduced the standard variation of the outcomes. Taking into account the aspects revealed in this study, in addition to already described important issues for quality control, the in vitro bioassays based bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment. For the generally applied sediment quality criteria, advices are given about maximum initial stock concentrations to achieve reliable bioassay outcomes.
The methods and concepts developed for metabolic activation of compounds in non-polar sediment extracts and in in vitro analysis of the TTR-competitive binding are applied in Chapter 6 to extracts from highly or less contaminated sediments collected in Luxembourg. Nonpolar fractions of sediment extracts were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the non-radioactive 96-well plate transthyretin (TTR) competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). Without bioactivation, medium polar and polar fractions also contained potent TTR-binding compounds with potencies from 1.6 to 17 nmol T4-Eq/g SEQ. This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing ofboth polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions. Without bioactivation THD potency is not observed in nonpolar sediment extracts, although in in vivo experiments PCBs and PBDEs, and not with dioxins or PAHs, have shown to be thyroid hormone disrupting (THD), demonstrating this bio-activation is toxicologically relevant and therefore required for sediment hazard characterisation.
Chapter 7 discusses the implications of our results to improve the relevance and reliability of in vitro bioassay applied for risk characterisation of complex mixtures from sediments and other matrices. The evidence obtained to support the relevance of POP bio-activation is considered both from the exposure perspective as well as the toxicity perspective. Various features of the newly developed methods and knowledge acquired within this PhD project are discussed in relation to in vitro bioassay risk characterization of sediments towards a realistic in vitro bioassay-based risk characterization of complex mixtures. Some important aspects for the inclusion of metabolizing systems within in vitro bioassay are discussed. In addition, alternatives to deal with the SPMX effect and the definition of suitable sample amounts to improve in vitro bioassay reliability are offered. The suitability of the developed approach application is considered for the risk characterization of sediments. Furthermore, an analysis is made to decide whether this thesis have made in vitro bioassays more reliable and relevant for risk characterization of complex mixtures. Finally, it provides some concluding remarks and aspects for further applications and research.
Toxicogenomics-based in vitro alternatives for estrogenicity testing
Wang, S. - \ 2013
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Toine Bovee; Jac Aarts. - S.l. : s.n. - ISBN 9789461735812 - 193
oestrogene eigenschappen - oestrogenen - in vitro - toxicogenomica - testprocedure - alternatieve methoden - methodologie - oestrogenic properties - oestrogens - in vitro - toxicogenomics - test procedure - alternative methods - methodology
Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor signaling, is an important aspect to assess the safety of currently used and newly developed chemicals. The standard test for disruption of normal estrogen function is the in vivo uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. Due to the high costs, ethical objections and labour intensiveness of the in vivo uterotrophic assay, the development of an in vitro test battery for in vivo estrogenicity has high priority. The aim of the present thesis was to develop an integrated testing strategy (ITS), based on existing and newly developed in vitro assays for estrogenicity testing, allowing easy high-throughput screening and prioritization of chemicals. An ITS preferentially based on in vitro assays would be a crucial step towards refinement, reduction, and ultimately replacement of current animal testing for estrogenic and other endocrine disrupting effects.
To reach this aim, several presently available and newly developed in vitro bioassays were selected and evaluated for optimal representation of the estrogenic effects occurring in the uterus/endometrium in vivo. Results show that the yeast estrogen bioassay revealed the best correlation with the in vivo uterotrophic assay(R2=0.87). The estrogenic potencies predicted by the peptide microarray also correlated very well with the uterotrophic assay and 30 coactivators on the peptide microarray resulted in correlation coefficient values higher than 0.85. The present thesis thus provides proof-of-principle that combining in vitro assays measuring different steps in the estrogen receptor signaling pathway enables accurate prediction of the estrogenic effects in vivo. By including the androgen reporter gene assays as well as the H295R steroidogenesis assay, the extended testing panel even goes beyond estrogenicity testing, as it can detect possible (anti)androgenic effects and effects on steroidogenesis that are not covered by the in vivo uterotrophic assay. The integrated in vitro testing strategy presented in this thesis may therefore allow easy high-throughput screening and prioritization of chemicals, thereby contributing to refinement, reduction and to some extent even replacement of current animal testing for estrogenic effects.