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Richard Bos - One of the best experts on this subject based on the ideXlab platform.
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Detection of pyrrolizidine alkaloids using flow analysis with both acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence
Analytica Chimica Acta, 2005Co-Authors: Bree A. Gorman, Neil W. Barnett, Richard BosAbstract:For the first time, analytically useful chemiluminescence was elicited from the reactions of the pyrrolizidine alkaloids. Heliotrine, retronecine, supinine, monocrotaline and echinatine N-oxide yielded chemiluminescence upon reaction with tris(2,2′-bipyridyl)ruthenium(II) whilst Lasiocarpine, its N-oxide and supinine elicited light upon reaction with acidic potassium permanganate. Detection limits for heliotrine were 1.25 × 10−7 M and 9 × 10−9 M for tris(2,2′-bipyridyl)ruthenium(III) perchlorate with flow injection analysis (FIA) and the silica-immobilised reagent (4-[4-(dichloromethylsilanyl)-butyl]-4′-methyl-2,2′-bipyridine)bis(2,2′-bipyridyl)ruthenium(II) with sequential injection analysis (SIA), respectively. Lasiocarpine was detectable at 1.4 × 10−7 M using acidic potassium permanganate with FIA. Additionally, the silica-immobilised reagent was optimised with respect to the oxidant (ammonium ceric nitrate) concentration and the aspiration times which afforded a detection limit for codeine of 5 × 10−10 M using SIA
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Detection of pyrrolizidine alkaloids using flow analysis with both acidic potassium permanganate and tris(2,2’-bipyridyl)ruthenium(II) chemiluminescence
Analytica Chimica Acta, 2004Co-Authors: Bree A. Gorman, Neil W. Barnett, Richard BosAbstract:For the first time, analytically useful chemiluminescence was elicited from the reactions of the pyrrolizidine alkaloids. Heliotrine, retronecine, supinine, monocrotaline and echinatine N-oxide yielded chemiluminescence upon reaction with tris(2,2′-bipyridyl)ruthenium(II) whilst Lasiocarpine, its N-oxide and supinine elicited light upon reaction with acidic potassium permanganate. Detection limits for heliotrine were 1.25 × 10−7 M and 9 × 10−9 M for tris(2,2′-bipyridyl)ruthenium(III) perchlorate with flow injection analysis (FIA) and the silica-immobilised reagent (4-[4-(dichloromethylsilanyl)-butyl]-4′-methyl-2,2′-bipyridine)bis(2,2′-bipyridyl)ruthenium(II) with sequential injection analysis (SIA), respectively. Lasiocarpine was detectable at 1.4 × 10−7 M using acidic potassium permanganate with FIA. Additionally, the silica-immobilised reagent was optimised with respect to the oxidant (ammonium ceric nitrate) concentration and the aspiration times which afforded a detection limit for codeine of 5 × 10−10 M using SIA.
Ivonne M. C. M. Rietjens - One of the best experts on this subject based on the ideXlab platform.
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Monocrotaline-induced liver toxicity in rat predicted by a combined in vitro physiologically based kinetic modeling approach
Archives of Toxicology, 2020Co-Authors: Suparmi Suparmi, Sebastiaan Wesseling, Ivonne M. C. M. RietjensAbstract:The aim of the present study was to use an in vitro–in silico approach to predict the in vivo acute liver toxicity of monocrotaline and to characterize the influence of its metabolism on its relative toxic potency compared to Lasiocarpine and riddelliine. In the absence of data on acute liver toxicity of monocrotaline upon oral exposure, the predicted dose–response curve for acute liver toxicity in rats and the resulting benchmark dose lower and upper confidence limits for 10% effect (BMDL_10 and BMDU_10) were compared to data obtained in studies with intraperitoneal or subcutaneous dosing regimens. This indicated the predicted BMDL_10 value to be in line with the no-observed-adverse-effect levels (NOAELs) derived from availabe in vivo studies. The predicted BMDL_10–BMDU_10 of 1.1–4.9 mg/kg bw/day also matched the oral dose range of 1–3 mg PA/kg bw/day at which adverse effects in human are reported. A comparison to the oral toxicity of the related pyrrolizidine alkaloids (PAs) Lasiocarpine and riddelliine revealed that, although in the rat hepatocytes monocrotaline was less toxic than Lasiocarpine and riddelliine, due to its relatively inefficient clearance, its in vivo acute liver toxicity was predicted to be comparable. It is concluded that the combined in vitro-PBK modeling approach can provide insight in monocrotaline-induced acute liver toxicity in rats, thereby filling existing gaps in the database on PA toxicity. Furthermore, the results reveal that the kinetic and metabolic properties of PAs can vary substantially and should be taken into account when considering differences in relative potency between different PAs.
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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
Archives of Toxicology, 2019Co-Authors: Lu Chen, Ad Peijnenburg, Laura Haan, Ivonne M. C. M. RietjensAbstract: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.
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Use of an in vitro-in silico testing strategy to predict inter-species and inter-ethnic human differences in liver toxicity of the pyrrolizidine alkaloids Lasiocarpine and riddelliine.
Archives of toxicology, 2019Co-Authors: Jia Ning, Sebastiaan Wesseling, Lu Chen, Jochem Louisse, Marije Strikwold, Ivonne M. C. M. RietjensAbstract:Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) known to cause liver toxicity. The aim of this study was to predict the inter-species and inter-ethnic human differences in acute liver toxicity of Lasiocarpine and riddelliine using physiologically based kinetic (PBK) modelling based reverse dosimetry of in vitro toxicity data. The concentration–response curves of in vitro cytotoxicity of Lasiocarpine and riddelliine defined in pooled human hepatocytes were translated to in vivo dose–response curves by PBK models developed using kinetic data obtained from incubations with pooled tissue fractions from Chinese and Caucasian individuals, providing PBK models for the average Chinese and average Caucasian, respectively. From the predicted in vivo dose–response curves, the benchmark dose lower and upper confidence limits for 5% effect (BMDL5 and BMDU5) were derived and 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. The inter-ethnic human differences varied 2.0-fold for Lasiocarpine and 5.0-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. In conclusion, the present study provides the proof-of-principle to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity data with PBK modelling-based reverse dosimetry.
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use of physiologically based kinetic modelling facilitated reverse dosimetry to convert in vitro cytotoxicity data to predicted in vivo liver toxicity of Lasiocarpine and riddelliine in rat
Food and Chemical Toxicology, 2018Co-Authors: Lu Chen, Sebastiaan Wesseling, Jia Ning, Jochem Louisse, Ivonne M. C. M. RietjensAbstract:Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) present in food and able to cause liver toxicity. The aim of this study was to investigate whether physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry can adequately translate in vitro concentration-response curves for toxicity of Lasiocarpine and riddelliine to in vivo liver toxicity data for the rat. To this purpose, PBK models were developed for Lasiocarpine and riddelliine, and predicted blood concentrations were compared to available literature data to evaluate the models. Concentration-response curves obtained from in vitro cytotoxicity assays in primary rat hepatocytes were converted to in vivo dose-response curves from which points of departure (PODs) were derived and that were compared to available literature data on in vivo liver toxicity. The results showed that the predicted PODs fall well within the range of PODs derived from available in vivo toxicity data. To conclude, this study shows the proof-of-principle for a method to predict in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity assays with in silico PBK modelling-facilitated reverse dosimetry. The approach may facilitate prediction of acute liver toxicity for the large number of PAs for which in vivo toxicity data are lacking.
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risk assessment for pyrrolizidine alkaloids detected in herbal teas and plant food supplements
Regulatory Toxicology and Pharmacology, 2017Co-Authors: Lu Chen, Jochem Louisse, Ad Peijnenburg, Patrick P J Mulder, Sebas Wesseling, Ivonne M. C. M. RietjensAbstract:Pyrrolizidine alkaloids (PAs) are plant metabolites present in some botanical preparations, with especially 1,2-unsaturated PAs being of concern because they are genotoxic carcinogens. This study presents an overview of tumour data on PAs and points of departure (PODs) derived from them, corroborating that the BMDL10 for Lasiocarpine represents a conservative POD for risk assessment. A risk assessment using this BMDL10 and mean levels of PAs reported in literature for (herbal) teas, indicates that consumption of one cup of tea a day would result in MOE values lower than 10 000 for several types of (herbal) teas, indicating a priority for risk management for these products A refined risk assessment using interim relative potency (REP) factors showed that based on the mean PA levels, 7(54%) of 13 types of (herbal) teas and 1 (14%) of 7 types of plant food supplements (PFS) resulted in MOE values lower than 10 000, indicating a priority for risk management also for these products in particular. This includes both preparations containing PA-producing and non-PA-producing plants. Our study provides insight in the current state-of-the art and limitations in the risk assessment of PA-containing food products, especially (herbal) teas and PFS, indicating that PAs in food presents a field of interest for current and future risk management.
Bree A. Gorman - One of the best experts on this subject based on the ideXlab platform.
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Detection of pyrrolizidine alkaloids using flow analysis with both acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence
Analytica Chimica Acta, 2005Co-Authors: Bree A. Gorman, Neil W. Barnett, Richard BosAbstract:For the first time, analytically useful chemiluminescence was elicited from the reactions of the pyrrolizidine alkaloids. Heliotrine, retronecine, supinine, monocrotaline and echinatine N-oxide yielded chemiluminescence upon reaction with tris(2,2′-bipyridyl)ruthenium(II) whilst Lasiocarpine, its N-oxide and supinine elicited light upon reaction with acidic potassium permanganate. Detection limits for heliotrine were 1.25 × 10−7 M and 9 × 10−9 M for tris(2,2′-bipyridyl)ruthenium(III) perchlorate with flow injection analysis (FIA) and the silica-immobilised reagent (4-[4-(dichloromethylsilanyl)-butyl]-4′-methyl-2,2′-bipyridine)bis(2,2′-bipyridyl)ruthenium(II) with sequential injection analysis (SIA), respectively. Lasiocarpine was detectable at 1.4 × 10−7 M using acidic potassium permanganate with FIA. Additionally, the silica-immobilised reagent was optimised with respect to the oxidant (ammonium ceric nitrate) concentration and the aspiration times which afforded a detection limit for codeine of 5 × 10−10 M using SIA
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Detection of pyrrolizidine alkaloids using flow analysis with both acidic potassium permanganate and tris(2,2’-bipyridyl)ruthenium(II) chemiluminescence
Analytica Chimica Acta, 2004Co-Authors: Bree A. Gorman, Neil W. Barnett, Richard BosAbstract:For the first time, analytically useful chemiluminescence was elicited from the reactions of the pyrrolizidine alkaloids. Heliotrine, retronecine, supinine, monocrotaline and echinatine N-oxide yielded chemiluminescence upon reaction with tris(2,2′-bipyridyl)ruthenium(II) whilst Lasiocarpine, its N-oxide and supinine elicited light upon reaction with acidic potassium permanganate. Detection limits for heliotrine were 1.25 × 10−7 M and 9 × 10−9 M for tris(2,2′-bipyridyl)ruthenium(III) perchlorate with flow injection analysis (FIA) and the silica-immobilised reagent (4-[4-(dichloromethylsilanyl)-butyl]-4′-methyl-2,2′-bipyridine)bis(2,2′-bipyridyl)ruthenium(II) with sequential injection analysis (SIA), respectively. Lasiocarpine was detectable at 1.4 × 10−7 M using acidic potassium permanganate with FIA. Additionally, the silica-immobilised reagent was optimised with respect to the oxidant (ammonium ceric nitrate) concentration and the aspiration times which afforded a detection limit for codeine of 5 × 10−10 M using SIA.
Lu Chen - One of the best experts on this subject based on the ideXlab platform.
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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
Archives of Toxicology, 2019Co-Authors: Lu Chen, Ad Peijnenburg, Laura Haan, Ivonne M. C. M. RietjensAbstract: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.
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Use of an in vitro-in silico testing strategy to predict inter-species and inter-ethnic human differences in liver toxicity of the pyrrolizidine alkaloids Lasiocarpine and riddelliine.
Archives of toxicology, 2019Co-Authors: Jia Ning, Sebastiaan Wesseling, Lu Chen, Jochem Louisse, Marije Strikwold, Ivonne M. C. M. RietjensAbstract:Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) known to cause liver toxicity. The aim of this study was to predict the inter-species and inter-ethnic human differences in acute liver toxicity of Lasiocarpine and riddelliine using physiologically based kinetic (PBK) modelling based reverse dosimetry of in vitro toxicity data. The concentration–response curves of in vitro cytotoxicity of Lasiocarpine and riddelliine defined in pooled human hepatocytes were translated to in vivo dose–response curves by PBK models developed using kinetic data obtained from incubations with pooled tissue fractions from Chinese and Caucasian individuals, providing PBK models for the average Chinese and average Caucasian, respectively. From the predicted in vivo dose–response curves, the benchmark dose lower and upper confidence limits for 5% effect (BMDL5 and BMDU5) were derived and 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. The inter-ethnic human differences varied 2.0-fold for Lasiocarpine and 5.0-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. In conclusion, the present study provides the proof-of-principle to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity data with PBK modelling-based reverse dosimetry.
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Novel strategies for risk assessment of pyrrolizidine alkaloids
'Wageningen University and Research', 2019Co-Authors: Lu ChenAbstract: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.
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Use of an in vitro–in silico testing strategy to predict inter-species and inter-ethnic human differences in liver toxicity of the pyrrolizidine alkaloids Lasiocarpine and riddelliine
2019Co-Authors: Ning Jia, Lu Chen, Strikwold Marije, Louisse Jochem, Wesseling Sebastiaan, Rietjens, Ivonne M.c.m.Abstract:Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) known to cause liver toxicity. The aim of this study was to predict the inter-species and inter-ethnic human differences in acute liver toxicity of Lasiocarpine and riddelliine using physiologically based kinetic (PBK) modelling based reverse dosimetry of in vitro toxicity data. The concentration–response curves of in vitro cytotoxicity of Lasiocarpine and riddelliine defined in pooled human hepatocytes were translated to in vivo dose–response curves by PBK models developed using kinetic data obtained from incubations with pooled tissue fractions from Chinese and Caucasian individuals, providing PBK models for the average Chinese and average Caucasian, respectively. From the predicted in vivo dose–response curves, the benchmark dose lower and upper confidence limits for 5% effect (BMDL5 and BMDU5) were derived and 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. The inter-ethnic human differences varied 2.0-fold for Lasiocarpine and 5.0-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. In conclusion, the present study provides the proof-of-principle to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity data with PBK modelling-based reverse dosimetry.
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Role of toxicokinetics and alternative testing strategies in pyrrolizidine alkaloid toxicity and risk assessment; state-of-the-art and future perspectives
2019Co-Authors: Ning Jia, Lu Chen, Rietjens, Ivonne M.c.m.Abstract:Toxicokinetics influences the toxicity of chemicals. This also holds for 1,2-unsaturated pyrrolizidine alkaloids (PAs), which need bioactivation to become toxic. Given that only for a limited number of 1,2-unsaturated PAs in vivo toxicity data are available, alternative testing strategies including read-across and quantitative in vitro to in vivo extrapolation (QIVIVE) are important. This paper presents how physiologically-based kinetic (PBK) models for the PAs Lasiocarpine and riddelliine were developed for rat and human, and used for conversion of in vitro data for toxicity in primary hepatocytes to quantitatively predict in vivo acute liver toxicity for both rat and human. Marked differences in toxicokinetics were observed between the two model PAs influencing the predicted in vivo toxicity. In a next step, in vitro toxicokinetic data that predicted relative bioactivation of the PAs, were shown to provide a possible basis for read-across from the BMDL10 for tumor formation by riddelliine of 237 μg/kg bw per day to other PAs for which tumor data are lacking. It is concluded that when comparing toxicity of different PAs, or when extrapolating in vitro toxicity data for PAs to the in vivo situation, differences in toxicokinetics should be taken into account, while future challenges are also discussed.
Markku Pasanen - One of the best experts on this subject based on the ideXlab platform.
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New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.
Rapid communications in mass spectrometry : RCM, 2018Co-Authors: Fashe Muluneh, Markku Pasanen, Merja R. Häkkinen, Rami El-dairi, Risto O. JuvonenAbstract:RATIONALE The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. METHODS The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, Lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer (pH 7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. RESULTS Retrorsine, senecionine and Lasiocarpine yielded three GSH conjugates producing [M - H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. CONCLUSIONS 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.
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Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine
Chemical research in toxicology, 2015Co-Authors: Muluneh Fashe, Risto O. Juvonen, Aleksanteri Petsalo, Juha Rasanen, Markku PasanenAbstract:There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of Lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1-M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of Lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4-M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4-M7) that could make humans more susceptible to Lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.
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In Silico Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids
Chemical research in toxicology, 2015Co-Authors: Muluneh Fashe, Risto O. Juvonen, Aleksanteri Petsalo, Markku Pasanen, Jouko Vepsäläinen, Minna Rahnasto-rillaAbstract:In humans, the metabolic bioactivation of pyrrolizidine alkaloids (PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation of their necine bases at C3 or C8 of heliotridine- and retronecine-type PAs or at the N atom of the methyl substituent of otonecine-type PAs. However, no attempts have been made to identify which C atom is the most favorable site for hydroxylation in silico. Here, in order to determine the site of hydroxylation that eventually leads to the formation of the toxic metabolites produced from Lasiocarpine, retrorsine, and senkirkin, we utilized the ligand-based electrophilic Fukui function f–(r) and hydrogen-bond dissociation energies (BDEs) as well as structure-based molecular docking. The ligand-based computations revealed that the C3 and C8 atoms of Lasiocarpine and retrorsine and the C26 atom of senkirkin were chemically the most susceptible locations for electrophilic oxidizing reactions. Similarly, according to the predicted binding orientation in the active sit...
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In Silico Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids
2015Co-Authors: Muluneh M. Fashe, Risto O. Juvonen, Aleksanteri Petsalo, Jouko Vepsäläinen, Markku Pasanen, Minna Rahnasto-rillaAbstract:In humans, the metabolic bioactivation of pyrrolizidine alkaloids (PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation of their necine bases at C3 or C8 of heliotridine- and retronecine-type PAs or at the N atom of the methyl substituent of otonecine-type PAs. However, no attempts have been made to identify which C atom is the most favorable site for hydroxylation in silico. Here, in order to determine the site of hydroxylation that eventually leads to the formation of the toxic metabolites produced from Lasiocarpine, retrorsine, and senkirkin, we utilized the ligand-based electrophilic Fukui function f–(r) and hydrogen-bond dissociation energies (BDEs) as well as structure-based molecular docking. The ligand-based computations revealed that the C3 and C8 atoms of Lasiocarpine and retrorsine and the C26 atom of senkirkin were chemically the most susceptible locations for electrophilic oxidizing reactions. Similarly, according to the predicted binding orientation in the active site of the crystal structure of human CYP3A4 (PDB code: 4I4G), the alkaloids were positioned in such a way that the C3 atom of Lasiocarpine and retrorsine and the C26 of senkirkin were closest to the catalytic heme Fe. Thus, it is concluded that the C3 atom of Lasiocarpine and retrorsine and C26 of senkirkin are the most favored sites of hydroxylation that lead to the production of their toxic metabolites
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Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine
2015Co-Authors: Muluneh M. Fashe, Risto O. Juvonen, Aleksanteri Petsalo, Juha Räsänen, Markku PasanenAbstract:There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of Lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1–M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of Lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4–M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4–M7) that could make humans more susceptible to Lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites
