Induction of peroxisome proliferator activated receptor γ (PPARγ) mediated gene expression and inhibition of induced nitric oxide production by Maerua subcordata (Gilg) DeWolf
Hiben, Mebrahtom Gebrelibanos ; Haan, Laura de; Spenkelink, Bert ; Wesseling, Sebastiaan ; Vervoort, Jacques ; Rietjens, Ivonne M.C.M. - \ 2020
BMC Complementary Medicine and Therapies 20 (2020)1. - ISSN 2662-7671
The health benefits of botanicals is linked to their phytochemicals that often exert pleiotropic effects via targeting multiple molecular signaling pathways such as the peroxisome proliferator-activated receptors (PPARs) and the nuclear factor kappaB (NFκB). The PPARs are transcription factors that control metabolic homeostasis and inflammation while the NF-κB is a master regulator of inflammatory genes such as the inducible nitric-oxide synthase that result in nitric oxide (NO) overproduction.
Extracts of Maerua subcordata (MS) and selected candidate constituents thereof, identified by liquid chromatography coupled to mass spectroscopy, were tested for their ability to induce PPARγ mediated gene expression in U2OS-PPARγ cells using luciferase reporter gene assay and also for their ability to inhibit lipopolysaccharide (LPS) induced NO production in RAW264.7 macrophages. While measuring the effect of test samples on PPARγ mediated gene expression, a counter assay that used U2OS-Cytotox cells was performed to monitor cytotoxicity or any non-specific changes in luciferase activity.
The results revealed that the fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 g dry weight per litre (gDW/L) and induced PPARγ mediated gene expression but the leaf extract showed some cytotoxicity and exhibited minimal induction. Instead, all extracts showed concentration (1–15 gDW/L) dependent inhibition of LPS induced NO production. The root extract showed weaker inhibition. Among the candidate constituents, agmatine, stachydrine, trigonelline, indole-3-carboxyaldehyde, plus ethyl-, isobutyl-, isopropyl, and methyl-isothiocyanates showed similar inhibition, and most showed increased inhibition with increasing concentration (1–100 μM) although to a lesser potency than the positive control, aminoguanidine.
The present study demonstrated for the first time the induction of PPARγ mediated gene expression by MS fruit, root, and seed extracts and the inhibition of LPS induced NO production by MS fruit, leaf, root, and seed extracts and some candidate constituents thereof.
Evaluation of in vitro models of stem cell-derived cardiomyocytes to screen for potential cardiotoxicity of chemicals
Shi, Miaoying ; Tien, Nguyen T. ; Haan, Laura de; Louisse, Jochem ; Rietjens, Ivonne M.C.M. ; Bouwmeester, Hans - \ 2020
Toxicology in Vitro 67 (2020). - ISSN 0887-2333
Cardiotoxicity - Clinical data - Human induced pluripotent stem cell-derived cardiomyocytes - Mouse embryonic stem cell-derived cardiomyocytes - Multi-electrode array
Cardiotoxicity is an important toxicological endpoint for chemical and drug safety assessment. The present study aims to evaluate two stemcell-based in vitro models for cardiotoxicity screening of chemicals. Eleven model compounds were used to evaluate responses of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) using beating arrest as a readout and the analysis of electrophysiological parameters measured with a multi-electrode array (MEA) platform of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Results revealed that the hiPSC-CM MEA assay responded to all compounds. The mESC-CM beating arrest assay was not responsive to potassium channel blockers and showed a lower sensitivity to sodium channel blockers and Na+/K+ ATPase inhibitors compared to the hiPSC-CM MEA assay. Calcium channel blockers and a β-adrenergic receptor agonist showed comparable potencies in both models. The in vitro response concentrations from hiPSC-CMs were highly concordant with human effective serum concentrations of potassium and sodium channel blockers. It is concluded that both in vitro models enable the cardiotoxicity screening with different applicability domains. The mESC-CM beating arrest assay may be used as a first step in a tiered approach while the hiPSC-CM MEA assay may be the best starting point for quantitative in vitro to in vivo extrapolations.
Microfluidic chip for culturing intestinal epithelial cell layers: Characterization and comparison of drug transport between dynamic and static models
Kulthong, Kornphimol ; Duivenvoorde, Loes ; Sun, Huiyi ; Confederat, Samuel ; Wu, Jiaqing ; Spenkelink, Bert ; Haan, Laura de; Marin, Victor ; Zande, Meike van der; Bouwmeester, Hans - \ 2020
Toxicology in Vitro 65 (2020). - ISSN 0887-2333
Bioavailability - Dynamic flow - Gut-on-chip - Microfluidics - Transport
Dynamic flow in vitro models are currently widely explored for their applicability in drug development research. The application of gut-on-chip models in toxicology is lagging behind. Here we report the application of a gut-on-chip model for biokinetic studies and compare the observed biokinetics of reference compounds with those obtained using a conventional static in vitro model. Intestinal epithelial Caco-2 cells were cultured on a porous membrane assembled between two glass flow chambers for the dynamic model, or on a porous membrane in a Transwell model. Confocal microscopy, lucifer yellow translocation, and alkaline phosphatase activity evaluation revealed that cells cultured in the gut-on-chip model formed tight, differentiated, polarized monolayers like in the static cultures. In the dynamic gut-on-chip model the transport of the high permeability compounds antipyrine, ketoprofen and digoxin was lower (i.e. 4.2-, 2.7- and 1.9-fold respectively) compared to the transport in the static Transwell model. The transport of the low permeability compound, amoxicillin, was similar in both the dynamic and static in vitro model. The obtained transport values of the compounds are in line with the compound Biopharmaceuticals Classification System. It is concluded that the gut-on-chip provides an adequate model for transport studies of chemicals.
Estrogen receptor alpha (ERα)–mediated coregulator binding and gene expression discriminates the toxic ERα agonist diethylstilbestrol (DES) from the endogenous ERα agonist 17β-estradiol (E2)
Adam, A.H.B. ; Haan, Laura H.J. de; Estruch, Ignacio Miro ; Hooiveld, Guido J.E.J. ; Louisse, Jochem ; Rietjens, Ivonne M.C.M. - \ 2020
Cell biology and toxicology (2020). - ISSN 0742-2091
17β-estradiol - Coregulator binding - Diethylstilbestrol - Estrogen receptor alpha - Transcriptomics
Diethylstilbestrol (DES) is a synthetic estrogen and proven human teratogen and carcinogen reported to act via the estrogen receptor α (ERα). Since the endogenous ERα ligand 17β-estradiol (E2) does not show these adverse effects to a similar extent, we hypothesized that DES’ interaction with the ERα differs from that of E2. The current study aimed to investigate possible differences between DES and E2 using in vitro assays that detect ERα-mediated effects, including ERα-mediated reporter gene expression, ERα-mediated breast cancer cell (T47D) proliferation and ERα-coregulator interactions and gene expression in T47D cells. Results obtained indicate that DES and E2 activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, significant differences between DES- and E2-induced binding of the ERα to 15 coregulator motifs and in transcriptomic signatures obtained in the T47D cells were observed. It is concluded that differences observed in binding of the ERα with several co-repressor motifs, in downregulation of genes involved in histone deacetylation and DNA methylation and in upregulation of CYP26A1 and CYP26B1 contribute to the differential effects reported for DES and E2.
Combining In Vitro Data and Physiologically Based Kinetic Modeling Facilitates Reverse Dosimetry to Define In Vivo Dose–Response Curves for Bixin- and Crocetin-Induced Activation of PPARγ in Humans
Suparmi, Suparmi ; Haan, Laura de; Spenkelink, Albertus ; Louisse, Jochem ; Beekmann, Karsten ; Rietjens, Ivonne M.C.M. - \ 2020
Molecular Nutrition & Food Research 64 (2020)2. - ISSN 1613-4125
bixin - crocetin - peroxisome proliferator-activated receptor γ - physiologically based kinetic modeling - reverse dosimetry
Scope: It is investigated whether at realistic dietary intake bixin and crocetin could induce peroxisome proliferator-activated receptor γ (PPARγ)-mediated gene expression in humans using a combined in vitro–in silico approach. Methods and results: Concentration–response curves obtained from in vitro PPARγ-reporter gene assays are converted to in vivo dose–response curves using physiologically based kinetic modeling-facilitated reverse dosimetry, from which the benchmark dose levels resulting in a 50% effect above background level (BMD50) are predicted and subsequently compared to dietary exposure levels. Bixin and crocetin activated PPARγ-mediated gene transcription in a concentration-dependent manner with similar potencies. Due to differences in kinetics, the predicted BMD50 values for in vivo PPARγ activation are about 30-fold different, amounting to 115 and 3505 mg kg bw−1 for crocetin and bixin, respectively. Human dietary and/or supplemental estimated daily intakes may reach these BMD50 values for crocetin but not for bixin, pointing at better possibilities for in vivo PPARγ activation by crocetin. Conclusion: Based on a combined in vitro–in silico approach, it is estimated whether at realistic dietary intakes plasma concentrations of bixin and crocetin are likely to reach concentrations that activate PPARγ-mediated gene expression, without the need for a human intervention study.
The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances
Kamelia, Lenny ; Haan, Laura de; Spenkelink, Bert ; Bruyneel, Ben ; Ketelslegers, Hans B. ; Boogaard, Peter J. ; Rietjens, Ivonne M.C.M. - \ 2020
Journal of Applied Toxicology 40 (2020)3. - ISSN 0260-437X - p. 330 - 341.
biotransformation - embryonic stem cell test - petroleum substances - polycyclic aromatic hydrocarbons - prenatal developmental toxicity - UVCBs
In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.
|Human gut-on-a-chip as a predictive model for compound bio availability and toxicity
Zande, M. van der; Kulthong, K. ; Duivenvoorde, L.P.M. ; Rijkers, Deborah ; Grouls, M. ; Haan, L.H.J. de; Bouwmeester, H. - \ 2019
Ethnomedicine and ethnobotany of Maerua subcordata (Gilg) DeWolf
Hiben, Mebrahtom Gebrelibanos ; Louisse, Jochem ; Haan, Laura H.J. de; Rietjens, Ivonne M.C.M. - \ 2019
Journal of Ethnic Foods 6 (2019)1. - ISSN 2352-6181
Ethnobotany - Ethnomedicine - Kunamas - Maerua subcordata - Wild edible plants
Background: Wild edible plants are valuable resources for improving food and nutritional security. Besides, they may provide important health benefits since the health-promoting components of plant-based foods usually exist at higher levels in wild plants. As a result, they are being sought as under-exploited potential sources of a health-promoting diet or a possible strategy to develop novel foods. In such exploration, ethnobotanical and ethnomedicinal data offer a fundamental step. The present study provides ethnomedicinal data on Maerua subcordata (Gilg) DeWolf (Capparidaceae). Methods: The ethnomedicinal data was collected from the Kunama ethnics of northern Ethiopia via focus group discussion and oral interview. Supporting ethnobotanical data from relevant literature was also compiled and systematically reviewed. Results: The results show that M. subcordata tuber is used by the Kunamas to manage malaria, malaria symptoms (fever, pain, gastrointestinal disorders), and seasonal cough while leaves are used for wound healing. In east Africa, its triple potential use as water purifying agent, food item, and herbal medicine was specified. As a herbal medicine, the tuber is used to manage a wide range of disorders including pain, infections, wounds, diabetes, blood pressure, and loss of appetite. Its use as laxative and abortifacient was also indicated. Leaves are used to treat wounds and ophthalmic and respiratory problems. As a food item, fruits are eaten during times of both food scarcity and food abundance while the tuber is used as a famine food. Conclusion: In East Africa, M. subcordata represents a wild food and medicinal plant, which may be developed into a functional food.
In vitro prenatal developmental toxicity induced by some petroleum substances is mediated by their 3- to 7-ring PAH constituent with a potential role for the aryl hydrocarbon receptor (AhR)
Kamelia, Lenny ; Haan, Laura de; Ketelslegers, Hans B. ; Rietjens, Ivonne M.C.M. ; Boogaard, Peter J. - \ 2019
Toxicology Letters 315 (2019). - ISSN 0378-4274 - p. 64 - 76.
Aryl hydrocarbon receptor - Embryonic stem cell test - Petroleum substances - Polycyclic aromatic hydrocarbons - Prenatal developmental toxicity - UVCBs
To test the hypothesis that 3–7 ring polycyclic aromatic hydrocarbons (PAHs) are responsible for the prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS), the present study evaluates the PDT potency of DMSO-extracts of 7 heavy fuel oils (HFO), varying in their PAH content, and 1 highly refined base oil (HRBO), containing no aromatics, in the embryonic stem cell test (EST). All DMSO-extracts of HFO inhibit ES-D3 cell differentiation in a concentration-dependent manner and their potency is proportional to the amount of 3–7 ring PAHs they contain. All DMSO-extracts of HFOs also show aryl hydrocarbon receptor (AhR)-mediated activities, as tested in the AhR-CALUX assay. Contrarily, the HRBO-extract tested negative in both assays. Co-exposure of ES-D3 cells with selected DMSO-extracts of PS and the AhR-antagonist trimethoxyflavone, successfully counteracted the PS-induced inhibition of ES-D3 cell differentiation, confirming the role of the AhR in mediating the observed PDT of PS extracts in the EST. A good correlation exists when comparing the in-vitro with the in-vivo PDT potencies of the PS under study. Altogether, our findings corroborate the hypothesis that PS-induced PDT is caused by 3–7 ring PAHs present in these substances and that the observed PDT is partially AhR-mediated.
Prediction of in vivo genotoxicity of lasiocarpine and riddelliine in rat liver using a combined in vitro-physiologically based kinetic modelling-facilitated reverse dosimetry approach
Chen, Lu ; Peijnenburg, Ad ; Haan, Laura de; Rietjens, Ivonne M.C.M. - \ 2019
Archives of Toxicology 93 (2019)8. - ISSN 0340-5761 - p. 2385 - 2395.
Genotoxicity - In vitro–in vivo extrapolation - Lasiocarpine - Physiologically based kinetic (PBK) model - Riddelliine
Pyrrolizidine alkaloids (PAs) are naturally occurring genotoxic compounds, and PA-containing plants can pose a risk to humans through contaminated food sources and herbal products. Upon metabolic activation, PAs can form DNA adducts, DNA and protein cross links, chromosomal aberrations, micronuclei, and DNA double-strand breaks. These genotoxic effects may induce gene mutations and play a role in the carcinogenesis of PAs. This study aims to predict in vivo genotoxicity for two well-studied PAs, lasiocarpine and riddelliine, in rat using in vitro genotoxicity data and physiologically based kinetic (PBK) modelling-based reverse dosimetry. The phosphorylation of histone protein H2AX was used as a quantitative surrogate endpoint for in vitro genotoxicity of lasiocarpine and riddelliine in primary rat hepatocytes and human HepaRG cells. The in vitro concentration–response curves obtained from primary rat hepatocytes were subsequently converted to in vivo dose–response curves from which points of departure (PoDs) were derived that were compared to available in vivo genotoxicity data. The results showed that the predicted PoDs for lasiocarpine and riddelliine were comparable to in vivo genotoxicity data. It is concluded that this quantitative in vitro-in silico approach provides a method to predict in vivo genotoxicity for the large number of PAs for which in vivo genotoxicity data are lacking by integrating in vitro genotoxicity assays with PBK modelling-facilitated reverse dosimetry.
The in vivo developmental toxicity of diethylstilbestrol (DES) in rat evaluated by an alternative testing strategy
Adam, Aziza Hussein Bakheit ; Zhang, Mengying ; Haan, Laura H.J. de; Ravenzwaay, Bennard van; Louisse, Jochem ; Rietjens, Ivonne M.C.M. - \ 2019
Archives of Toxicology 93 (2019)7. - ISSN 0340-5761 - p. 2021 - 2033.
Developmental toxicity - Diethylstilbestrol - Estrogen receptor alpha (ERα) - Physiologically based kinetic modelling - Reverse dosimetry
In the present study, we evaluated an alternative testing strategy to quantitatively predict the in vivo developmental toxicity of the synthetic hormone diethylstilbestrol (DES). To this end, a physiologically based kinetic (PBK) model was defined that was subsequently used to translate concentration–response data for the in vitro developmental toxicity of DES, obtained in the ES-D3 cell differentiation assay, into predicted in vivo dose–response data for developmental toxicity. The previous studies showed that the PBK model-facilitated reverse dosimetry approach is a useful approach to quantitatively predict the developmental toxicity of several developmental toxins. The results obtained in the present study show that the PBK model adequately predicted DES blood concentrations in rats. Further studies revealed that DES tested positive in the ES-D3 differentiation assay and that DES-induced inhibition of the ES-D3 cell differentiation could be counteracted by the estrogen receptor alpha (ERα) antagonist fulvestrant, indicating that the in vitro ES-D3 cell differentiation assay was able to mimic the role of ERα reported in the mode of action underlying the developmental toxicity of DES in vivo. In spite of this, combining these in vitro data with the PBK model did not adequately predict the in vivo developmental toxicity of DES in a quantitative way. It is concluded that although the EST qualifies DES as a developmental toxin and detects the role of ERα in this process, the ES-D3 cell differentiation assay of the EST apparently does not adequately capture the processes underlying DES-induced developmental toxicity in vivo.
Hazard assessment of Maerua subcordata (Gilg) DeWolf. for selected endpoints using a battery of in vitro tests
Gebrelibanos Hiben, Mebrahtom ; Kamelia, Lenny ; Haan, Laura de; Spenkelink, Bert ; Wesseling, Sebastiaan ; Vervoort, Jacques ; Rietjens, Ivonne M.C.M. - \ 2019
Journal of Ethnopharmacology 241 (2019). - ISSN 0378-8741
CALUX assays - Embryonic stem cell test - Hazard - In vitro - Maerua subcordata - Zebrafish embryotoxicity test
Ethnopharmacological relevance: Maerua subcordata (Gilg) DeWolf is a medicinal and wild food plant growing mainly in east Africa. Especially its root tuber is widely used in traditional medicine to treat several infectious and chronic diseases but also in some toxicity implications like use as abortifacient. Aim of the study: the present study applied in silico and in vitro tests to identify possible hazards of M. subcordata (fruit, leaf, root, seed) methanol extracts focussing on developmental toxicity. Materials and methods: Ames test, estrogen receptor alpha (ERα) assay, aryl hydrocarbon receptor (AhR) assay, embryonic stem cell test (EST), and zebrafish embryotoxicity test (ZET) were employed. Besides, a Derek Nexus toxicity prediction was performed on candidate structures obtained from metabolomics profiling of the extracts using liquid chromatography coupled to multistage mass spectroscopy (LC/MSn) and a MAGMa software based structural annotation. Results: Glucosinolates, which degrade to isothiocyanates, and biogenic amines were among the candidate molecules identified in the extracts by LC/MSn - MAGMa software structural annotation. Isothiocyanates and some other candidate molecules suggested a positive mutagenicity alert in Derek toxicity predictions. All the extracts showed negative mutagenicity in the Ames test. However, the Derek predictions also identified endocrine and developmental toxicity as possible endpoints of concern. This was further assessed using in vitro tests. Results obtained reveal that leaf extract shows AhR and ERα agonist activities, inhibited differentiation of ES-D3 stem cells into contracting cardiomyocytes in the EST (p < 0.001) as well as inhibited hatching (p < 0.01) and showed acute toxicity (p < 0.01) in the ZET. Also, the fruit extract showed toxicity (p < 0.05) towards zebrafish embryos and both fruit and seed extracts showed AhR agonist activities while root extract was devoid of activity in all in vitro assays. Conclusion: The leaf extract tests positive in in vitro tests that may point towards a developmental toxicity hazard. The current evaluations did not raise concerns of genotoxicity or developmental toxicity for the fruit, seed and root extracts. This is important given the use of especially these parts of M. subcordata, in traditional medicine and/or as (famine) food.
Effects of Maerua subcordata (Gilg) DeWolf on electrophile-responsive element (EpRE)mediated gene expression in vitro
Hiben, Mebrahtom Gebrelibanos ; Haan, Laura De; Spenkelink, Bert ; Wesseling, Sebas ; Louisse, Jochem ; Vervoort, Jacques ; Rietjens, Ivonne M.C.M. - \ 2019
PLoS ONE 14 (2019)4. - ISSN 1932-6203
Plant extracts and phytochemicals may prevent chronic diseases via activation of adaptive cellular stress response pathways including induction of antioxidant and phase II detoxifying enzymes. The regulatory regions of these inducible genes encode the electrophile-response element (EpRE). This study tested the EpRE induction ability of Maerua subcordata (fruit, leaf, root, seed) methanol extracts and selected candidate constituents thereof, identified by liquid chromatography coupled with multistage mass spectroscopy, employing an EpRE luciferase reporter gene assay using hepa-1c1c7 mouse hepatoma cells. A parallel Cytotox CALUX assay using human osteosarcoma U2OS cells was used to monitor any non-specific changes in luciferase activity or cytotoxicity. Results showed that fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 gram dry weight per litre but the leaf extract exhibited some cytotoxicity and that the leaf (despite some cytotoxicity), fruit, and seed extracts showed strong induction of EpRE mediated gene expression while induction by the root extract was minimal. Selected candidates included glucosinolates, isothiocyanates, and some biogenic amines. Subsequent studies showed that methyl-, ethyl-, isopro-pyl-, isobutyl- isothiocyanates, and sec-butyl thiocyanate as well as glucobrassicin induced concentration (1–100 μM) dependent EpRE-mediated gene expression while the biogenic amines stachydrine and trigonelline acted as inhibitors of EpRE-mediated gene expression at 100 μM. The identification of glucolepidiin, glucobrassicin, glucocapparin, stachydrine, and trigonelline in all extracts was confirmed using standards and based on multiple reaction monitoring; yet, glucobrassicin level in the root extract was negligible. In conclusion, this study provided a first report on EpRE mediated gene expression effects of M. subcordata; and despite detection of different glucosinolates in all extracts, those containing glucobrassicin particularly displayed high EpRE induction. Because EpRE inducers are cytoprotective and potential chemopreventive agents while inhibitors are suggested adjuvants of chemotherapy, results of this study imply that process manipulation of this plant may result in herbal preparations that may be used as chemopreventive agents or adjuvants of chemotherapies.
Prenatal developmental toxicity testing of petroleum substances using the zebrafish embryotoxicity test
Kamelia, Lenny ; Brugman, S. ; Haan, L.H.J. de; Ketelslegers, H.B. ; Rietjens, I.M.C.M. ; Boogaard, Peter - \ 2018
Altex 36 (2018)2. - ISSN 0946-7785 - p. 245 - 260.
The present study evaluates the applicability of the zebrafish embryotoxicity test (ZET) to assess prenatal developmental toxicity (PDT) potency of the DMSO-extracts of 9 petroleum substances (PS), with variable polycyclic aromatic hydrocarbon (PAH) content, and 2 gas-to-liquid (GTL) products, without any PAHs but otherwise similar properties to PS. The results showed that all PS extracts induced concentration-dependent in vitro PDT, as quantified in the ZET and that this potency is associated with their 3-5 ring PAH content. In contrast and as expected, GTL products did not induce any effect at all. The potencies obtained in the ZET correlated with those previously reported for the embryonic stem cell test (EST) (R2=0.61), while the correlation with potencies reported in in vivo studies were higher for the EST (R2=0.85) than the ZET (R2=0.69). Combining the results of the ZET with those previously reported for the EST (Kamelia et al., 2017), the aryl hydrocarbon (AhR) CALUX assay (Kamelia et al., 2018), and the PAH content, ranked and clustered the test compounds in line with their in vivo potencies and chemical characteristics. To conclude, our findings indicate that the ZET does not outperform the EST as a stand-alone assay for testing PDT of PS, but confirms the hypothesis that PAHs are the major inducers of PDT by some PS, while they also indicate that the ZET is a useful addition to a battery of in vitro tests able to predict the in vivo PDT of PS.
The Role of Endocrine and Dioxin-Like Activity of Extracts of Petroleum Substances in Developmental Toxicity as Detected in a Panel of CALUX Reporter Gene Assays
Kamelia, Lenny ; Louisse, Jochem ; Haan, Laura de; Maslowska-Gornicz, Anna ; Ketelslegers, Hans B. ; Brouwer, Abraham ; Rietjens, Ivonne M.C.M. ; Boogaard, Peter J. - \ 2018
Toxicological sciences 164 (2018)2. - ISSN 1096-6080 - p. 576 - 591.
dioxin-like activity - endocrine activity - petroleum substances - polycyclic aromatic hydrocarbons - prenatal developmental toxicity - reporter gene assays
Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the dioxin (aryl hydrocarbon receptor [AhR]) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, onlyminor effects of specific extracts, particularly those with a substantial amount of 4-5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stemcell test, suggesting an important role of the AhR inmediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.
The effects of all-trans retinoic acid on estrogen receptor signaling in the estrogen-sensitive MCF/BUS subline
Miro Estruch, Ignacio ; Haan, Laura H.J. de; Melchers, Diana ; Houtman, René ; Louisse, Jochem ; Groten, John P. ; Rietjens, Ivonne M.C.M. - \ 2018
Journal of Receptors and Signal Transduction 38 (2018)2. - ISSN 1079-9893 - p. 112 - 121.
breast cancer - coregulator - crosstalk - ERα - proliferation - RAR
Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17β-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.
Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells
Bodero Baeza, Marcia ; Hoogenboom, Ron L.A.P. ; Bovee, Toine F.H. ; Portier-Onstenk, Liza ; Haan, Laura de; Peijnenburg, Ad ; Hendriksen, Peter J.M. - \ 2018
Toxicology in Vitro 46 (2018). - ISSN 0887-2333 - p. 102 - 112.
Azaspiracid-1 - Caco-2 cells - Dinophysis toxin-1 - Mode of action - Okadaic acid - Transcriptomics
A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.
The Regulatory Framework Across International Jurisdictions for Risks Associated with Consumption of Botanical Food Supplements
Low, Teng Yong ; Wong, Kwok Onn ; Yap, Adelene L.L. ; Haan, Laura H.J. De; Rietjens, Ivonne M.C.M. - \ 2017
Comprehensive Reviews in Food Science and Food Safety 16 (2017)5. - ISSN 1541-4337 - p. 821 - 834.
botanical food supplements - botanicals - regulatory challenges - regulatory framework - risk assessment
Dietary supplements, including those containing botanical ingredients and botanical-derived compounds, have been marketed to consumers globally for many decades. However, the legislative framework for such products remains inconsistent across jurisdictions internationally. This study aims to compare the regulatory framework of botanical food supplements in the EU, USA, Canada, Australia, New Zealand, India, Japan, and China. The study also aims to investigate and describe safety assessment criteria for botanical food supplements where they are present in the above said jurisdictions, and attempts to analyze whether these criteria are suitable for addressing the toxicological risks associated with the use of botanical food supplement products, based on the evaluation of reported adverse effects related to botanical food supplement use as examples. Finally, this study discusses some future issues that need further attention, such as the consideration of less than lifetime exposures, potential for misidentification, and adulteration of botanical supplements by pharmacologically active substances. It is concluded that the regulatory approaches towards botanical food supplements differ significantly across jurisdictions. In addition, national authorities are increasingly considering having more regulatory oversight for such products. Further consideration of the actual impact of adverse events arising from botanical food supplement usage will be helpful in guiding such decisions.
Prenatal developmental toxicity testing of petroleum substances : Application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo
Kamelia, Lenny ; Louisse, Jochem ; Haan, Laura de; Rietjens, Ivonne M.C.M. ; Boogaard, Peter J. - \ 2017
Toxicology in Vitro 44 (2017). - ISSN 0887-2333 - p. 303 - 312.
Embryonic stem cell test - Gas-to-liquid products - Petroleum substances - Polycyclic aromatic hydrocarbons - Prenatal developmental toxicity - UVCBs
Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3–7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R2 = 0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS.
Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action
Miro Estruch, Ignacio ; Melchers, Diana ; Houtman, René ; Haan, Laura H.J. de; Groten, John P. ; Louisse, Jochem ; Rietjens, Ivonne M.C.M. - \ 2017
Biochimica et Biophysica Acta. Proteins & Proteomics 1865 (2017)9. - ISSN 1570-9639 - p. 1195 - 1206.
Agonist - Antagonist - Coregulator - Ligand binding domain (LBD) - RAR - Subtype
Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARβ/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to > 60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARβ compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.