Relationship Between Glutathione Concentration and Metabolism of the Pyrrolizidine Alkaloid, Monocrotaline, in the Isolated, Perfused Liver

doi:10.1006/taap.1995.1017

Toxicology and Applied Pharmacology

Volume 130, Issue 1, January 1995, Pages 132-139

https://doi.org/10.1006/taap.1995.1017 Get rights and content

Abstract

The influence of GSH concentration on metabolism of monocrotaline was examined in the isolated, perfused rat liver. Chloroethanol (0.37 mmol/kg), diethyl maleate (5.6 mmol/kg), and buthionine sulfoximine (72.9 mmol/kg) given in vivo reduced hepatic GSH from 3.7 μmol/g wet weight to 1.5, 0.6 and 0.9 μmol/g, respectively. Livers were then perfused in vitro for 1 hr with monocrotaline (0.5 mM). GSH depletion had no effect on the total release of pyrrolic metabolites of monocrotaline. Depletion, however, markedly affected the pattern of pyrrole release. Biliary release of 7-glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSDHP) was reduced by up to 72%. Pretreatment with diethyl maleate or buthionine sulfoximine increased the level of protein-bound pyrroles in the liver by 107 and 84%, respectively. Such pyrroles are probably responsible for liver toxicity. GSH depletion also led to a doubling of dehydromonocrotaline release into the perfusate. This metabolite is probably responsible for the extrahepatic toxicity of monocrotaline. Release into perfusate of the relatively nontoxic metabolite, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) was correspondingly decreased. Hepatic GSH content was increased to 4.4 μmol/g by pretreatment with oxo-4-thiazolidine carboxylate (4.76 mmol/kg). This agent increased total pyrrolic metabolites by 54%. Biliary release of GSDHP and perfusate release of dehydromonocrotaline and DHP were all increased. Thus, hepatic GSH levels regulate the metabolism of monocrotaline and dehydromonocrotaline and, consequently, the hepatic and extrahepatic toxicity of monocrotaline. GSH depletion leads to a switch from the biliary release of the mildly toxic GSDHP to the perfusate release of the highly toxic dehydromonocrotaline. GSH depletion also permits more dehydromonocrotaline in the liver to become available for macromolecular alkylation. These findings suggest that nutritional intake of sulfur-containing amino acids can influence the severity of pyrrolizidine poisoning.

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In addition, bile acids are also important biomarkers for the evaluation of hepatotoxicity that is caused by exposure to PAs-containing herbs.122,123 Decreased release of bile acids in PAs-perfused livers and an increased serum level of bile acids were observed in PAs-treated animals.124–127 Previous studies have suggested that increased serum bile acids could be a sensitive index of hepatic function caused by PAs.125,126
Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural compounds that are widely distributed throughout the world. Most PAs are metabolically activated to trigger toxicity. Exposure to herbal medicine containing PAs and food supplements contaminated by PAs is considered to be one of the two main causes of hepatic sinusoidal obstruction syndrome (HSOS), which is a rare hepatic vascular disease with a high mortality rate. PAs-induced HSOS cases have been reported worldwide. However, there is no clinically effective therapy for PAs-induced HSOS, which is partially because the toxic mechanism is not fully understood. This review focuses on updating the information on the metabolism and the molecular mechanisms of PAs hepatotoxicity, including oxidative stress, apoptosis, and dysfunction of bile acid metabolism, and their interactions.
Role of toxicokinetics and alternative testing strategies in pyrrolizidine alkaloid toxicity and risk assessment; state-of-the-art and future perspectives
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Given the unstable nature of these metabolites such methods should be based on scavenging these metabolites by for example thiol reagents like GSH or cysteine. Cysteine-DHP adducts in hepatic proteins have been isolated from PA-exposed perfused livers (Yan and Huxtable, 1995) and experimental animals (Mattocks and White, 1970). In addition, DHP adducts of blood proteins have been observed in patients who were accidently exposed to PAs (Lin et al., 2011; Ruan et al., 2015).
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.
Development of a two-layer transwell co-culture model for the in vitro investigation of pyrrolizidine alkaloid-induced hepatic sinusoidal damage
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Dehydro-PAs also react with glutathione (GSH) to form readily excreted pyrrole-GSH conjugates (Fig. 1). Thus, glutathione conjugation is generally considered as a detoxification pathway (Fu et al., 2004; Li et al., 2011; Yan and Huxtable, 1995). On the other hand, excessive reaction with GSH may severely deplete cellular GSH.
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Pyrrole-protein adducts – A biomarker of pyrrolizidine alkaloid-induced hepatotoxicity
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Citation Excerpt :
Similar to the metabolic activation of many other chemical toxins, the metabolic activation of PAs significantly diminishes cellular GSH levels in different cell lines and animals [33,49,50], which causes the disturbance of cellular redox homeostasis and leads to PA-induced cytotoxicity [51]. Restoration of cellular GSH to detoxify the reactive pyrrolic metabolites has proved to be an effective approach for PA detoxification [50,52], although it is only effective with the pretreatment of GSH or its precursors, such as N-acetylcysteine and γ-glutamylcysteinylglycyl ethyl ester, or with the treatment at very early stage after PA ingestion. In addition to the study of these mechanisms leading to cytotoxicity, only a limited number of studies have aimed at identifying the cellular proteins which form the pyrrole-protein adducts associated with PA-induced cytotoxicity [53–55].
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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.
Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: A whole genome transcriptome analysis
2015, Toxicology in Vitro
1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 μM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited.
In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.

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Regular ArticleRelationship Between Glutathione Concentration and Metabolism of the Pyrrolizidine Alkaloid, Monocrotaline, in the Isolated, Perfused Liver

Abstract

Regular Article
Relationship Between Glutathione Concentration and Metabolism of the Pyrrolizidine Alkaloid, Monocrotaline, in the Isolated, Perfused Liver