New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment
Punt, Ans ; Bouwmeester, Hans ; Blaauboer, Bas J. ; Coecke, Sandra ; Hakkert, Betty ; Hendriks, Delilah F.G. ; Jennings, Paul ; Kramer, Nynke I. ; Neuhoff, Sibylle ; Masereeuw, Rosalinde ; Paini, Alicia ; Peijnenburg, Ad A.C.M. ; Rooseboom, Martijn ; Shuler, Michael L. ; Sorrell, Ian ; Spee, Bart ; Strikwold, Marije ; Meer, Andries D. Van der; Zande, Meike Van der; Vinken, Mathieu ; Yang, Huan ; Bos, Peter M.J. ; Heringa, Minne B. - \ 2020
Altex 37 (2020)4. - ISSN 1868-596X - p. 607 - 622.
biokinetics - in silico - in vitro - next-generation risk evaluations - PB(P)K - QIVIVE
For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.
Development of a population of Boswellia elongata Balf. F. in Homhil nature sanctuary, Socotra island (Yemen)
Lvončík, Samuel ; Vahalík, Petr ; Bongers, Frans ; Peijnenburg, Jan ; Hušková, Karolína ; Rensburg, Julian Jansen van; Hamdiah, Salem ; Maděra, Petr - \ 2020
Rendiconti Lincei 31 (2020). - ISSN 2037-4631 - p. 747 - 759.
Age structure - Boswellia elongata - Population - Regeneration - Socotra
We assessed seven decades of change in the largest known population of the endangered endemic Boswellia elongata Balf. F. (Burseraceae) on Socotra Island (Yemen). To quantify the population change we evaluated tree number and locations on digitized images from various sources in the period 1956–2017 and combined this with direct field measurements of the population between 2011 and 2017. Our study reveals that the Homhil Nature Sanctuary B. elongata population shows a continuous decline since 1956. The steady but slow natural decline was strongly accelerated by two catastrophic cyclones in November 2015, when 38% of the trees were directly destroyed by strong winds. During the following 2 years 29% of the remaining trees died additionally. The remaining population has a bell-shaped size distribution; most trees are around 40 cm in diameter (range 18 to 70 cm). Tree ring analysis of 11 dead trees with a diameter of 29 to 44 cm without bark, resulted in estimated tree ages between 80 and 101 years. We estimate that similar-sized trees showing strong signs of senescence have a maximum age of a little over 100 years. The age structure of the Homhil population is, therefore, unbalanced with large sized trees prevailing. Natural regeneration is absent for decades. Viable seeds are available and have been shown to germinate, but the development of seedlings into saplings is a bottleneck. If the decline continues at the current rate, only 30 trees will remain there in 2036. Protection, planting and awareness activities are needed to keep this unique frankincense tree in Homhil Nature Sanctuary.
Perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorononanoic acid (PFNA) increase triglyceride levels and decrease cholesterogenic gene expression in human HepaRG liver cells
Louisse, Jochem ; Rijkers, Deborah ; Stoopen, Geert ; Janssen, Aafke ; Staats, Martijn ; Hoogenboom, Ron ; Kersten, Sander ; Peijnenburg, Ad - \ 2020
Archives of Toxicology 94 (2020). - ISSN 0340-5761 - p. 3137 - 3155.
Cholesterol - HepaRG cells - PFASs - Transcriptomics - Triglycerides
Per- and polyfluoroalkyl substances (PFASs) are omnipresent in the environment, food chain, and humans. Epidemiological studies have shown a positive association between serum levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), and increased serum cholesterol and, in some cases, also triglyceride levels. However, causality has been questioned, as animal studies, as well as a human trial, showed a decrease in serum cholesterol and no effects or a decrease in plasma triglycerides. To obtain more insight into the effects of PFASs on these processes, the present study investigated the effects of PFOA, PFOS, and perfluorononanoic acid (PFNA) on intracellular triglyceride and cholesterol levels in human HepaRG liver cells. DNA microarray analyses were performed to provide insight into underlying mechanisms. All PFASs induced an increase in cellular triglyceride levels, but had no effect on cholesterol levels. Gene set enrichment analysis (GSEA) of the microarray data indicated that gene sets related to cholesterol biosynthesis were repressed by PFOA, PFOS, and PFNA. Other gene sets commonly affected by all PFAS were related to PERK/ATF4 signaling (induced), tRNA amino-acylation (induced), amino acid transport (induced), and glycolysis/gluconeogenesis (repressed). Moreover, numerous target genes of peroxisome proliferator-activated receptor α (PPARα) were found to be upregulated. Altogether, the present study shows that PFOA, PFOS, and PFNA increase triglyceride levels and inhibit cholesterogenic gene expression in HepaRG cells. In addition, the present study indicates that PFASs induce endoplasmic reticulum stress, which may be an important mechanism underlying some of the toxic effects of these chemicals.
Development of a QSAR model to predict hepatic steatosis using freely available machine learning tools
Cotterill, J. ; Price, N. ; Rorije, E. ; Peijnenburg, A. - \ 2020
Food and Chemical Toxicology 142 (2020). - ISSN 0278-6915
Non-alcoholic fatty liver disease - QSAR model - Steatosis
There are various types of hepatic steatosis of which non-alcoholic fatty liver disease, which may be caused by exposure to chemicals and environmental pollutants is the most prevalent, representing a potential major health risk. QSAR modelling has the potential to provide a rapid and cost-effective method to identify compounds which may trigger steatosis. Although models exist to predict key molecular initiating events of steatosis such as nuclear receptor binding, we are aware of no models to predict the apical effect steatosis. In this study, we describe the development of a QSAR model to predict steatosis using freely available machine learning tools. It was built using a dataset of 207 pharmaceuticals and pesticides which were identified as steatotic or non-steatotic from existing data from in vivo human and animal studies. The best performing model developed using the linear discriminant analysis module in TANAGRA, based on four chemical descriptors, had an accuracy of 70%, a sensitivity of 66% and a specificity of 74%. The expansion of the steatosis dataset to other chemical types, to enable the development of further models, would be of benefit in the identification of compounds with a range of mechanisms of action contributing to steatosis.
First come, first served : Possible role for priority effects in marine populations under different degrees of dispersal potential
Leeuw, Christiaan A. de; Peijnenburg, Katja T.C.A. ; Gillespie, Rosemary G. ; Maas, Diede L. ; Hanzawa, Naoto ; Tuti, Yosephine ; Toha, Abdul Hamid A. ; Aji, Ludi Parwadani ; Becking, Leontine E. - \ 2020
Journal of Biogeography 47 (2020)8. - ISSN 0305-0270 - p. 1649 - 1662.
anchialine ecosystems - Brachidontes mussels - coral triangle - isolation-by-distance - marine biodiversity - phylogeography
Aim: Studying clearly delineated populations in marine lakes, islands of sea, we investigated the interplay of habitat size, dispersal potential, and priority effects in shaping marine population genetic structure. Location: Marine lakes and coastal locations in Indonesia, Palau, Papua New Guinea and Australia. Taxon: Mussels (Mytilidae, Brachidontes spp.). Methods: Populations were sampled from four coastal locations and 22 marine lakes of similar age (~8,000 years), yet differing in size (0.04–4.7 km2) and degree of connection to the adjacent sea. While some lakes are highly connected, allowing potential influx of larvae from the sea, others have very limited water exchange. We assessed the phylogeographical structure and demographic history using mitochondrial and nuclear DNA sequence data, and combined this with geometric morphometrics. The effects of lake characteristics on population genetic diversity and structure were tested using linear regression and Mantel tests. Results: Each lake contained one of six distinct genetic lineages, which were characterized by deep phylogenetic splits and significant morphometric differences. These lineages likely represent separate species. The lineages showed similar demographic patterns, with lakes containing founder populations that rapidly expanded and diverged. Genetic diversity within lake populations was significantly correlated with lake area, but not with physical connection to the adjacent sea. Within lineages that occurred in multiple lakes there was strong population structure (average ΦST 0.65), which did not conform to an isolation-by-distance pattern or to the degree of dispersal potential. Main Conclusions: Marine lakes across a gradient of physical isolation show strong population structure and evidence for in situ divergence. We hypothesize that the observed genetic structure is the result of priority effects. In addition, reduction of habitat size appears to reduce genetic diversity, even at very small spatial scales. Our findings are relevant in the context of ongoing alterations to coastal hydrodynamics, which lead to habitat reduction and influence migration among populations at fine spatial scales.
Hepatotoxicity of the pesticides imazalil, thiacloprid and clothianidin – Individual and mixture effects in a 28-day study in female Wistar rats
Alarcan, Jimmy ; Waizenegger, Julia ; Lourdes Marzo Solano, Marize de; Lichtenstein, Dajana ; Luckert, Claudia ; Peijnenburg, Ad ; Stoopen, Geert ; Sharma, Raju Prasad ; Kumar, Vikas ; Marx-Stoelting, Philip ; Lampen, Alfonso ; Braeuning, Albert - \ 2020
Food and Chemical Toxicology 140 (2020). - ISSN 0278-6915
EuroMix - Liver hypertrophy - Mixture effects - Pesticides
Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.
An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro
Lichtenstein, Dajana ; Luckert, Claudia ; Alarcan, Jimmy ; Sousa, Georges de; Gioutlakis, Michail ; Katsanou, Efrosini S. ; Konstantinidou, Parthena ; Machera, Kyriaki ; Milani, Emanuela S. ; Peijnenburg, Ad ; Rahmani, Roger ; Rijkers, Deborah ; Spyropoulou, Anastasia ; Stamou, Marianna ; Stoopen, Geert ; Sturla, Shana J. ; Wollscheid, Bernd ; Zucchini-Pascal, Nathalie ; Braeuning, Albert ; Lampen, Alfonso - \ 2020
Food and Chemical Toxicology 139 (2020). - ISSN 0278-6915
AOP-Wise testing - Liver - Pesticide mixtures - Relative potency factors - Triglyceride accumulation
Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.
In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX)
Arantes, Mariana Reis ; Peijnenburg, Ad ; Hendriksen, Peter J.M. ; Stoopen, Geert ; Almeida, Thiago Silva ; Souza, Terezinha Maria ; Farias, Davi Felipe ; Carvalho, Ana Fontenele Urano ; Rocha, Talita Magalhães ; Leal, Luzia Kalyne Almeida Moreira ; Vasconcelos, Ilka Maria ; Oliveira, Jose Tadeu Abreu - \ 2020
Toxicology in Vitro 65 (2020). - ISSN 0887-2333
Antifungal agent - Cytotoxicity - SBTX - Toxicogenomics
Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 μg/mL and 200 μg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 μg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 μg/mL or 200 μg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.
Are Technological Developments Improving the Environmental Sustainability of Photovoltaic Electricity?
Blanco, Carlos Felipe ; Cucurachi, Stefano ; Peijnenburg, Willie J.G.M. ; Beames, Alistair ; Vijver, Martina G. - \ 2020
Energy Technology (2020). - ISSN 2194-4288
environmental impacts - life-cycle assessments - photovoltaics - solar - sustainability
Innovation in photovoltaics (PV) is mostly driven by the cost per kilowatt ratio, making it easy to overlook environmental impacts of technological enhancements during early research and development stages. As PV technology developers introduce novel materials and manufacturing methods, the well-studied environmental profile of conventional silicon-based PV may change considerably. Herein, existing trends and hotspots across different types of emerging PV technologies are investigated through a systematic review and meta-analysis of life-cycle assessments (LCAs). To incorporate as many data points as possible, a comprehensive harmonization procedure is applied, producing over 600 impact data points for organic, perovskite (PK), dye-sensitized, tandem, silicon, and other thin-film cells. How the panel and balance of system components affect environmental footprints in comparable installations is also investigated and discussed. Despite the large uncertainties and variabilities in the underlying LCA data and models, the harmonized results show clear positive trends across the sector. Seven potential hotspots are identified for specific PV technologies and impact categories. The analysis offers a high-level guidance for technology developers to avoid introducing undesired environmental trade-offs as they advance to make PV more competitive in the energy markets.
Pyrrolizidine alkaloids in food and phytomedicine : Occurrence, exposure, toxicity, mechanisms, and risk assessment - A review
Schrenk, Dieter ; Gao, Lan ; Lin, Ge ; Mahony, Catherine ; Mulder, Patrick P.J. ; Peijnenburg, Ad ; Pfuhler, Stefan ; Rietjens, Ivonne M.C.M. ; Rutz, Lukas ; Steinhoff, Barbara ; These, Anja - \ 2020
Food and Chemical Toxicology 136 (2020). - ISSN 0278-6915
Food safety - Hepatocarcinogenicity - Liver toxicity - Natural toxins - Pyrrolizidine alkaloids
Among naturally occurring plant constituents, the 1,2-unsaturated pyrrolizidine alkaloids (in the following termed ‘PAs’) play a distinct role because of the large number of congeners occurring in nature and the pronounced toxicity of some congeners. Several PAs are hepatotoxic in humans, experimental and farm animals and were shown to be potent hepatocarcinogens in laboratory rodents. Although the general mode of action leading to toxicity has been elucidated, i.e., being mediated by metabolic conversion of the parent molecule into a highly reactive electrophile capable of attacking cellular target molecules, major questions related to the risk assessment of PAs remain unresolved. It was the aim of a workshop held in September 2018 to shed more light on the occurrence, exposure, mode of action, toxicokinetics and –dynamics of PAs to improve the scientific basis for an advanced toxicological risk assessment. The contributions in nine chapters describe the scientific progress using advanced analytical methods, studies in subcellular fractions, cell culture, experimental animals and humans and the use of PBPK modeling and structure-activity relationship considerations aiming at a better understanding of PA toxicity and genotoxicity. Since PAs differ considerably in their toxic potencies and substantial species differences in sensitivity towards PA exposure exist, a special emphasis was placed on these issues.
Towards harmonization of test methods for in vitro hepatic clearance studies
Louisse, Jochem ; Alewijn, Martin ; Peijnenburg, Ad A.C.M. ; Cnubben, Nicole H.P. ; Heringa, Minne B. ; Coecke, Sandra ; Punt, Ans - \ 2020
Toxicology in Vitro 63 (2020). - ISSN 0887-2333 - 1 p.
Non-animal methods for toxicokinetics, such as in vitro hepatic metabolic clearance studies, play an important role in chemical risk evaluations. To gain regulatory acceptance of such clearance data, the development of a test guideline for performing in vitro hepatic clearance studies is crucial. The aim of the present study was to obtain insight in the experimental conditions of clearance studies that influence obtained intrinsic clearance (CLint) values. To that end, in vitro hepatic CLint data obtained with rat or human hepatocytes and methodological aspects of the experiments, were collected from 42 different suitable studies published between 1995 and 2018. The CLint values for the majority of chemicals differed by more than one order of magnitude. We estimated the systematic effect of different experimental setups on the CLint values using a random forest regression analysis, revealing that 'hepatocyte concentration', 'species' (rat or human hepatocytes) and 'culture medium' have the largest impact. Calculating unbound CLint (CLint,u) values slightly reduced the variation for most chemicals. Given that in vivo clearance is in general underpredicted based on in vitro clearance data, a harmonized protocol is preferably based on a protocol that provides relatively high in vitro CLint values.
Current insights in monitoring, bioaccumulation, and potential health effects of microplastics present in the food chain
Raamsdonk, L.D.W. van; Zande, M. van der; Koelmans, A.A. ; Hoogenboom, R.L.A.P. ; Peters, R.J.B. ; Groot, M.J. ; Peijnenburg, A.C.M. ; Weesepoel, Y.J.A. - \ 2020
Foods 9 (2020)1. - ISSN 2304-8158
Microplastics (MPs) are considered an emerging issue as environmental pollutants and a potential health threat. This review will focus on recently published data on concentrations in food, possible effects, and monitoring methods. Some data are available on concentrations in seafood (fish, bivalves, and shrimps), water, sugar, salt, and honey, but are lacking for other foods. Bottled water is a considerable source with numbers varying between 2600 and 6300 MPs per liter. Particle size distributions have revealed an abundance of particles smaller than 25 µm, which are considered to have the highest probability to pass the intestinal border and to enter the systemic circulation of mammals. Some studies with mice and zebrafish with short- or medium-term exposure (up to 42 days) have revealed diverse results with respect to both the type and extent of effects. Most notable modifications have been observed in gut microbiota, lipid metabolism, and oxidative stress. The principal elements of MP monitoring in food are sample preparation, detection, and identification. Identified data gaps include a lack of occurrence data in plant- and animal-derived food, a need for more data on possible effects of different types of microplastics, a lack of in silico models, a lack of harmonized monitoring methods, and a further development of quality assurance.
Speurtocht naar gifmengsels
Peijnenburg, Ad - \ 2019
Novel strategies for risk assessment of pyrrolizidine alkaloids
Chen, Lu - \ 2019
Wageningen University. Promotor(en): I.M.C.M. Rietjens, co-promotor(en): A.A.C.M. Peijnenburg. - Wageningen : Wageningen University - ISBN 9789463950374 - 245
Scope: Botanicals and botanical preparations may contain natural constituents that are of concern for human health. One group of such natural toxic compounds that may raise a concern is the group of pyrrolizidine alkaloids (PAs). Especially 1,2-unsaturated PAs are hepatotoxic and may act as genotoxic carcinogens in humans. At the current state-of-the-art, risk assessment of botanicals and botanical preparations is generally not required before they can enter the market, thus may pose a potential risk to human health.
Objective: The present thesis aimed to perform the risk assessment for PAs derived from botanical products following daily life-time exposure and also more realistic exposure scenarios. Another aim of the present thesis was to investigate whether animal-free testing strategies could be of use in tackling data and knowledge gaps for PAs by predicting in vivo toxicity of different PAs and whether proofs-of-principle for applying such alternative testing approaches could be provided for two selected PA model compounds, riddelliine and lasiocarpine.
Material and methods: Herbal teas, herbal medicines and plant food supplements (PFS) were bought from different counties. LC-MS/MS was used to detect the PA levels in these botanical and botanical preparations. Because PAs are genotoxic carcinogens, Margin of exposure (MOE) approach was applied for risk assessment. For development of the physiologically based kinetic (PBK) modelling to predict in vivo liver toxicity for lasiocarpine and riddelliine in rat and human, the kinetic parameters were obtained from in vitro incubations assays. The microsomal incubation assays were performed with rat and human tissue fractions to determine Vmax and Km values for lasiocarpine and riddelliine clearance, using a substrate depletion approach. The MTT assay was used to detect in vitro liver toxicity for lasiocasrpine and riddelliine. In a subsequent step, the same approach was used for prediction of genotoxicity in rat, another endpoint relevant for PA toxicity. To this end, the in vitro concentration-responses curves obtained from in vitro genotoxicity studies using the ƴH2AX assay were translated into in vivo dose-response curves using PBK modelling-facilitated reverse dosimetry.
Main results: When consumption of one cup of tea a day during a whole lifetime would result in MOE values lower than 10000 for several types of herbal teas, indicating a priority for risk management for these products, these products not only derived from PA-producing plants but also derived from non-PA-producing plants. Using MOE approach, combined with Haber’s rule was employed to analyse the risks of shorter-than-lifetime exposure for total of 39 herbal teas, 8 herbal medicines, and 19 PFS. This analysis revealed that shorter-than-lifetime use would result in MOE values lower than 10000 upon use for 40 up to 3450 weeks during a lifetime (depending on the preparation). Only for a limited number of herbal teas and medicines, use of two weeks a year (150 weeks during a 75 year lifetime) would still raise a concern. The PBK models were used for translation of in vitro concentration-response curves to in vivo dose-response curves in rat. From these in vivo dose-response curves, the predicted BMDL5-BMDU5 (lower/upper limit of the 90% confidence interval of the benchmark dose that gives a 5% response) of lasiocarpine and riddelline were obtained which were 23.0-34.4 and 4.9-8.4 mg/kg bw/day, respectively. The predicted BMDL5-BMDU5 of lasiocarpine falls well within the range of the point of departures (PoDs) derived from available in vivo toxicity data. The same Quantitative in vitro to in vivo extrapolation (QIVIVE) method was subsequently used to predict the inter-species and inter-ethnic human differences in liver toxicity of lasiocarpine and riddelliine using a human PBK model. The BMDL5-BMDU5 of lasiocarpine were found to amount to 14.7-41.2 mg/kg bw/day for Chinese and 7.4-23.7 mg/kg bw/day for Caucasian, indicating the Chinese to be less sensitive. The predicted BMDL5-BMDU5 of riddelliine were 1.0-5.9 mg/kg bw/day for Chinese and 0.2-1.2 mg/kg bw/day for Caucasian. These values were subsequently compared to those previously obtained in rat to evaluate inter-species differences. The inter-species differences amounted to 2.0-fold for lasiocarpine and 8.2-fold for riddelliine with humans being more sensitive than rats. When extending the developed PBK modelling to predicted in vivo genotoxicity for lasiocarpine and riddelliine in rat, the predicted BMD10 for lasiocarpine and riddelliine amounted to 8.82 and 3.41 mg/kg bw/day, respectively, and were in line with the experimental data on in vivo genotoxicity available in the literature for these two PAs.
Conclusions and implications: The risk assessment of herbal products revealed that daily life-time consumption of some of these products would be a priority for risk management. When considering realistic exposure scenarios, exposure to most of these herbal products would have MOE values higher than 10000 indicating a low priority for risk management. Moreover, this thesis demonstrated that the combined in vitro PBK modelling-based reverse dosimetry approach could adequately predict in vivo liver toxicity and genotoxicity for two model PAs, lasiocarpine and riddelliine. Such QIVIVE methods may prove to be of use in defining more realistic relative potency values for the different food/feed-related PAs. The results obtained reveal the feasibility of this combined quantitative in vitro-in silico approach to determine a PoD for a chemical without the use of experimental animals and to address the issue of how to use in vitro data for risk assessment.
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.
Predicting estrogen receptor binding of chemicals using a suite of in silico methods – Complementary approaches of (Q)SAR, molecular docking and molecular dynamics
Cotterill, J.V. ; Palazzolo, L. ; Ridgway, C. ; Price, N. ; Rorije, E. ; Moretto, A. ; Peijnenburg, A. ; Eberini, I. - \ 2019
Toxicology and Applied Pharmacology 378 (2019). - ISSN 0041-008X
Estrogen receptor - In Silico - Low-mode molecular dynamics simulation - Molecular docking - QSAR
With the aim of obtaining reliable estimates of Estrogen Receptor (ER) binding for diverse classes of compounds, a weight of evidence approach using estimates from a suite of in silico models was assessed. The predictivity of a simple Majority Consensus of (Q)SAR models was assessed using a test set of compounds with experimental Relative Binding Affinity (RBA) data. Molecular docking was also carried out and the binding energies of these compounds to the ERα receptor were determined. For a few selected compounds, including a known full agonist and antagonist, the intrinsic activity was determined using low-mode molecular dynamics methods. Individual (Q)SAR model predictivity varied, as expected, with some models showing high sensitivity, others higher specificity. However, the Majority Consensus (Q)SAR prediction showed a high accuracy and reasonably balanced sensitivity and specificity. Molecular docking provided quantitative information on strength of binding to the ERα receptor. For the 50 highest binding affinity compounds with positive RBA experimental values, just 5 of them were predicted to be non-binders by the Majority QSAR Consensus. Furthermore, agonist-specific assay experimental values for these 5 compounds were negative, which indicates that they may be ER antagonists. We also showed different scenarios of combining (Q)SAR results with Molecular docking classification of ER binding based on cut-off values of binding energies, providing a rational combined strategy to maximize terms of toxicological interest.
Determination of genotoxic potencies of pyrrolizidine alkaloids in HepaRG cells using the γH2AX assay
Louisse, Jochem ; Rijkers, Deborah ; Stoopen, Geert ; Holleboom, Wendy Jansen ; Delagrange, Mona ; Molthof, Elise ; Mulder, Patrick P.J. ; Hoogenboom, Ron L.A.P. ; Audebert, Marc ; Peijnenburg, Ad A.C.M. - \ 2019
Food and Chemical Toxicology 131 (2019). - ISSN 0278-6915
Genotoxicity - HepaRG - Pyrrolizidine alkaloids (PAs) - Relative potency factor (RPF) - γH2AX assay
Pyrrolizidine alkaloids (PAs) are secondary metabolites from plants that have been found in substantial amounts in herbal supplements, infusions and teas. Several PAs cause cancer in animal bioassays, mediated via a genotoxic mode of action, but for the majority of the PAs, carcinogenicity data are lacking. It is assumed in the risk assessment that all PAs have the same potency as riddelliine, which is considered to be one of the most potent carcinogenic PAs in rats. This may overestimate the risks, since many PAs are expected to have lower potencies. In this study we determined the concentration-dependent genotoxicity of 37 PAs representing different chemical classes using the γH2AX in cell western assay in HepaRG human liver cells. Based on these in vitro data, PAs were grouped into different potency classes. The group with the highest potency consists particularly of open diester PAs and cyclic diester PAs (including riddelliine). The group of the least potent or non-active PAs includes the monoester PAs, non-esterified necine bases, PA N-oxides, and the unsaturated PA trachelanthamine. This study reveals differences in in vitro genotoxic potencies of PAs, supporting that the assumption that all PAs have a similar potency as riddelliine is rather conservative.
Quantitative in vitro-to-in vivo extrapolation (QIVIVE) of estrogenic and anti-androgenic potencies of BPA and BADGE analogues
Punt, Ans ; Aartse, Aafke ; Bovee, Toine F.H. ; Gerssen, Arjen ; Leeuwen, Stefan P.J. van; Hoogenboom, Ron L.A.P. ; Peijnenburg, Ad A.C.M. - \ 2019
Archives of Toxicology 93 (2019)7. - ISSN 0340-5761 - p. 1941 - 1953.
Androgenic - Bisphenol A - BP analogues - Estrogenic - QIVIVE - Relative potencies
The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds.
Risk assessment of intake of pyrrolizidine alkaloids from herbal teas and medicines following realistic exposure scenarios
Chen, Lu ; Mulder, Patrick P.J. ; Peijnenburg, Ad ; Rietjens, Ivonne M.C.M. - \ 2019
Food and Chemical Toxicology 130 (2019). - ISSN 0278-6915 - p. 142 - 153.
Extraction efficiency - Herbal medicines - Herbal teas - Margin of exposure - Pyrrolizidine alkaloids - Shorter-than-lifetime exposure
In this study five types of herbal teas were used to quantify the effect of comminution of the leaves on resulting PA exposure. Results show that PA levels extracted from intact leaves were consistently lower than from comminuted tea leaves. The Margin of Exposure (MOE) approach was applied to evaluate the consequences of this difference for the associated risks in the scenario of lifetime exposure. Furthermore, we considered medicinal use of these teas for shorter-than-lifetime exposure scenarios, and also analysed the risks of shorter-than-lifetime use of eight herbal medicines and 19 previously analysed plant food supplements. This analysis revealed that shorter-than-lifetime use resulted in MOE values < 10,000 upon use for 40–3450 weeks during a lifetime, with for only a limited number of herbal teas and medicines use of two weeks a year (150 weeks during a 75 year lifetime) would still raise a concern. It is concluded that taking more realistic conditions into account markedly reduces the concerns raised for these herbal preparations. These results also illustrate the need for development of a generally accepted method for taking short term exposure into account in risk assessment of compounds that are genotoxic and carcinogenic.
Method for Extraction and Quantification of Metal-Based Nanoparticles in Biological Media : Number-Based Biodistribution and Bioconcentration
Abdolahpur Monikh, Fazel ; Chupani, Latifeh ; Zusková, Eliska ; Peters, Ruud ; Vancová, Marie ; Vijver, Martina G. ; Porcal, Petr ; Peijnenburg, Willie J.G.M. - \ 2019
Environmental Science and Technology 53 (2019)2. - ISSN 0013-936X - p. 946 - 953.
A multistep sample preparation method was developed to separate metal-based engineered nanoparticles (ENPs) from biological samples. The method was developed using spiked zebrafish tissues and standard titanium dioxide (TiO2) and cerium dioxide (CeO2) ENPs. Single-particle inductively coupled plasma mass spectrometry was used to quantify the separated particles in terms of number concentration. This method demonstrated mass recoveries of more than 90% and did not strikingly alter the median particles size. High number recoveries were calculated for CeO2 ENPs (>84%). Particle number recoveries were poor for TiO2 ENPs (<25%), which could be due to the interference of 48Ca with the measured isotope 48Ti. The method was verified using zebrafish exposed to CeO2 ENPs to test its applicability for nanotoxicokinetic investigations. Total mass of Ce and particle number concentration of CeO2 ENPs were measured in different tissues. Notably, the mass-based biodistribution of Ce in the tissues did not follow the number-based biodistribution of CeO2. Moreover, the calculated mass-based bioconcentration factors showed a different pattern in comparison to the number-based bioconcentration factors. Our findings suggest that considering mass as the sole dose-metric may not provide sufficient information to investigate toxicity and toxicokinetics of ENPs.