RESUMO
In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.
Assuntos
Plantas Medicinais , Alcaloides de Pirrolizidina , Alcaloides de Pirrolizidina/toxicidadeRESUMO
In October 2022, the World Health Organization (WHO) convened an expert panel in Lisbon, Portugal in which the 2005 WHO TEFs for chlorinated dioxin-like compounds were reevaluated. In contrast to earlier panels that employed expert judgement and consensus-based assignment of TEF values, the present effort employed an update to the 2006 REP database, a consensus-based weighting scheme, a Bayesian dose response modeling and meta-analysis to derive "Best-Estimate" TEFs. The updated database contains almost double the number of datasets from the earlier version and includes metadata that informs the weighting scheme. The Bayesian analysis of this dataset results in an unbiased quantitative assessment of the congener-specific potencies with uncertainty estimates. The "Best-Estimate" TEF derived from the model was used to assign 2022 WHO-TEFs for almost all congeners and these values were not rounded to half-logs as was done previously. The exception was for the mono-ortho PCBs, for which the panel agreed to retain their 2005 WHO-TEFs due to limited and heterogenous data available for these compounds. Applying these new TEFs to a limited set of dioxin-like chemical concentrations measured in human milk and seafood indicates that the total toxic equivalents will tend to be lower than when using the 2005 TEFs.
Assuntos
Dioxinas , Bifenilos Policlorados , Dibenzodioxinas Policloradas , Animais , Humanos , Teorema de Bayes , Dibenzofuranos/toxicidade , Dibenzofuranos Policlorados/toxicidade , Dioxinas/toxicidade , Mamíferos , Bifenilos Policlorados/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Organização Mundial da SaúdeRESUMO
Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.
Assuntos
Neoplasias Hepáticas , Alcaloides de Pirrolizidina , Humanos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Alcaloides de Pirrolizidina/metabolismo , Hepatócitos , Testes de Mutagenicidade , Neoplasias Hepáticas/metabolismoRESUMO
Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.
Assuntos
Antineoplásicos , Alcaloides de Pirrolizidina , Humanos , Citocromo P-450 CYP3A/metabolismo , Fígado , Hepatócitos , Alcaloides de Pirrolizidina/metabolismo , Dano ao DNA , Antineoplásicos/farmacologiaRESUMO
This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.
Assuntos
Alcaloides de Pirrolizidina , Chás de Ervas , Animais , Contaminação de Alimentos/análise , Alcaloides de Pirrolizidina/toxicidade , Medição de Risco , ToxicocinéticaRESUMO
Contamination with 1,2-unsaturated pyrrolizidine alkaloids (PAs) is a serious problem for certain phytomedicines, foods, and animal feeds. Several of these PAs are genotoxic and carcinogenic, primarily in the liver, upon cytochrome P450 (CYP)-catalyzed activation into reactive (pyrrolic and pyrrole-like) metabolites. Here we investigated the metabolism of selected PAs (echimidine, europine, lasiocarpine, lycopsamine, retrorsine, and senecionine) in rat hepatocytes in primary culture and in human CYP3A4-transfected HepG2 cells. The open-chained diesters echimidine and lasiocarpine and the cyclic diester senecionine were extensively metabolized in rat hepatocytes into a broad spectrum of products released into the medium. A large portion of unidentified, possibly irreversibly bound, products remained in the cells while detectable amounts of reactive and other metabolites were found in the incubation media. In HepG2-CYP3A4 cells, lasiocarpine was more extensively metabolized than echimidine and senecionine which also gave rise to the release of pyrrolic metabolites. In human cells, no pyrrolic metabolites were detected in retrorsine or lycopsamine incubations, while no such metabolites were detected from europine in both cell types. Other types of metabolic changes comprised modifications such as side chain demethylation or oxygenation reactions like the formation of N-oxides. The latter, considered as a detoxification step, was a major pathway with cyclic diesters, was less distinctive for echimidine and lycopsamine and almost negligible for lasiocarpine and europine. Our data are in agreement with previously published cyto- and genotoxicity findings and suggests that the metabolic pattern may contribute substantially to the specific toxic potency of a certain congener. In addition, marked differences were found for certain congeners between rat hepatocytes and transfected human HepG2 cells, whereby a high level of bioactivation was found for lasiocarpine, whereas a very low level of bioactivation was observed for monoesters, in particular in human cells.
Assuntos
Hepatócitos/efeitos dos fármacos , Alcaloides de Pirrolizidina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Humanos , Masculino , Estrutura Molecular , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
Pyrrolizidine alkaloids (PA) exert their toxic effects only after bioactivation. Although their toxicity has already been studied and metabolic pathways including important metabolites were described, the quantification of the latter revealed a large unknown portion of the metabolized PA. In this study, the qualitative and quantitative metabolite profiles of structurally different PAs in rat and human liver microsomes were investigated. Between five metabolites for europine and up to 48 metabolites for lasiocarpine were detected. Proposals for the chemical structure of each metabolite were derived based on fragmentation patterns using high-resolution mass spectrometry. The metabolite profiles of the diester PAs showed a relatively good agreement between both species. The metabolic reactions were summarized into three groups: dehydrogenation, oxygenation, and shortening of necic acid(s). While dehydrogenation of the necine base is considered as bioactivation, both other routes are considered as detoxification steps. The most abundant changes found for open chained diesters were dealkylations, while the major metabolic pathway for cyclic diesters was oxygenation especially at the nitrogen atom. In addition, all diester PAs formed several dehydrogenation products, via the insertion of a second double bond in the necine base, including the formation of glutathione conjugates. In rat liver microsomes, all investigated PAs formed dehydropyrrolizidine metabolites with the highest amount formed by lasiocarpine, whereas in human liver microsomes, these metabolites could only be detected for diesters. Our findings demonstrate that an extensive analysis of PA metabolism can provide the basis for a better understanding of PA toxicity and support future risk assessment.
Assuntos
Microssomos Hepáticos/metabolismo , Alcaloides de Pirrolizidina/análise , Alcaloides de Pirrolizidina/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Alcaloides de Pirrolizidina/química , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 µM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration-response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration-response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.
Assuntos
Hepatócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênese , Mutagênicos/toxicidade , Alcaloides de Pirrolizidina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Relação Dose-Resposta a Droga , Indução Enzimática , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Masculino , Testes para Micronúcleos , Estrutura Molecular , Ratos Sprague-Dawley , Medição de Risco , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Relação Estrutura-AtividadeRESUMO
In the original publication, the starting point in time for the three feeding trials.
RESUMO
In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.
Assuntos
Ração Animal/normas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Alimentos Geneticamente Modificados , Glicina/análogos & derivados , Herbicidas/toxicidade , Plantas Geneticamente Modificadas/efeitos dos fármacos , Zea mays , Animais , Testes de Carcinogenicidade , Resistência a Medicamentos/genética , Feminino , Glicina/toxicidade , Masculino , Plantas Geneticamente Modificadas/genética , Ratos Wistar , Testes de Toxicidade Crônica , Testes de Toxicidade Subcrônica , Zea mays/efeitos dos fármacos , Zea mays/genética , GlifosatoRESUMO
Pyrrolizidine alkaloids (PA) are secondary metabolites of certain flowering plants. The ingestion of PAs may result in acute and chronic effects in man and livestock with hepatotoxicity, mutagenicity, and carcinogenicity being identified as predominant effects. Several hundred PAs sharing the diol pyrrolizidine as a core structure are formed by plants. Although many congeners may cause adverse effects, differences in the toxic potency have been detected in animal tests. It is generally accepted that PAs themselves are biologically and toxicologically inactive and require metabolic activation. Consequently, a strong relationship between activating metabolism and toxicity can be expected. Concerning PA susceptibility, marked differences between species were reported with a comparatively high susceptibility in horses, while goat and sheep seem to be almost resistant. Therefore, we investigated the in vitro degradation rate of four frequently occurring PAs by liver enzymes present in S9 fractions from human, pig, cow, horse, rat, rabbit, goat, and sheep liver. Unexpectedly, almost no metabolic degradation of any PA was observed for susceptible species such as human, pig, horse, or cow. If the formation of toxic metabolites represents a crucial bioactivation step, the found inverse conversion rates of PAs compared to the known susceptibility require further investigation.
Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Alcaloides de Pirrolizidina/farmacocinética , Animais , Biotransformação , Bovinos , Feminino , Cabras , Cavalos , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Alcaloides de Pirrolizidina/toxicidade , Coelhos , Ratos , Ovinos , Especificidade da Espécie , SuínosRESUMO
Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Cinurenina/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Comunicação Autócrina , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/imunologia , Humanos , Cinurenina/imunologia , Cinurenina/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Comunicação Parácrina , Receptores de Hidrocarboneto Arílico/imunologia , Triptofano/metabolismo , Triptofano Oxigenase/deficiência , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismoRESUMO
α-, ß- and γ-asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae, Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and ß-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and ß-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic γ-asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and ß-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and ß-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic γ-asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH-γ-asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic γ-asarone.
Assuntos
Anisóis/toxicidade , Compostos de Epóxi/toxicidade , Mutagênicos/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Ativação Metabólica , Derivados de Alilbenzenos , Animais , Anisóis/química , Anisóis/metabolismo , Anisóis/farmacologia , Arilsulfotransferase , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Compostos de Epóxi/farmacologia , Hidroxilação , Isomerismo , Microssomos Hepáticos/metabolismo , Testes de Mutagenicidade , RatosRESUMO
ß-Asarone (1) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound 1 is found in several plants, e.g., Acorus calamus or Asarum europaeum. Compound 1-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although 1 has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic allylic phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. In vivo experiments suggested that this pathway does not play a major role in the carcinogenicity of the propenylic compound 1 as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rats, bovines, porcines, and humans using (1)H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of 1 presumably yielded (Z)-asarone-1',2'-epoxide (8a) which instantly was hydrolyzed to the corresponding erythro- and threo-configurated diols (9b, 9a) and the ketone 2,4,5-trimethoxyphenylacetone (13). This was the main metabolic pathway in the metabolism of 1 in all investigated liver microsomes. Hydroxylation of the side chain of 1 led to the formation of three alcohols at total yields of less than 30%: 1'-hydroxyasarone (2), (E)- and (Z)-3'-hydroxyasarone (4 and 6), with 6 being the mainly formed alcohol and 2 being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of 4 and 6 were further oxidized to the corresponding carbonyl compounds (E)- and (Z)-3'-oxoasarone (5, 7). 1'-Oxoasarone (3) was probably also formed in incubations with 1 but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-O-demethylated metabolites of 1 were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of 1. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71-75% of 1 will be metabolized via epoxidation, 21-15% via hydroxylation (and further oxidation), and 8-10% via demethylation at lower as well as higher concentrations of 1, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of 1 are the ultimate carcinogens formed from 1.
Assuntos
Anisóis/metabolismo , Carcinógenos/metabolismo , Microssomos Hepáticos/metabolismo , Derivados de Alilbenzenos , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Ratos , Ratos Wistar , RoedoresRESUMO
Consensus toxicity factors (CTFs) were developed as a novel approach to establish toxicity factors for risk assessment of dioxin-like compounds (DLCs). Eighteen polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and biphenyls (PCBs) with assigned World Health Organization toxic equivalency factors (WHO-TEFs) and two additional PCBs were screened in 17 human and rodent bioassays to assess their induction of aryl hydrocarbon receptor-related responses. For each bioassay and compound, relative effect potency values (REPs) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin were calculated and analyzed. The responses in the human and rodent cell bioassays generally differed. Most notably, the human cell models responded only weakly to PCBs, with 3,3',4,4',5-pentachlorobiphenyl (PCB126) being the only PCB that frequently evoked sufficiently strong responses in human cells to permit us to calculate REP values. Calculated REPs for PCB126 were more than 30 times lower than the WHO-TEF value for PCB126. CTFs were calculated using score and loading vectors from a principal component analysis to establish the ranking of the compounds and, by rescaling, also to provide numerical differences between the different congeners corresponding to the TEF scheme. The CTFs were based on rat and human bioassay data and indicated a significant deviation for PCBs but also for certain PCDD/Fs from the WHO-TEF values. The human CTFs for 2,3,4,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, and 1,2,3,4,7,8,9-heptachlorodibenzofuran were up to 10 times greater than their WHO-TEF values. Quantitative structure-activity relationship models were used to predict CTFs for untested WHO-TEF compounds, suggesting that the WHO-TEF value for 1,2,3,7,8-pentachlorodibenzofuran could be underestimated by an order of magnitude for both human and rodent models. Our results indicate that the CTF approach provides a powerful tool for condensing data from batteries of screening tests using compounds with similar mechanisms of action, which can be used to improve risk assessment of DLCs.
Assuntos
Benzofuranos/toxicidade , Bifenilos Policlorados/toxicidade , Dibenzodioxinas Policloradas/análogos & derivados , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Benzofuranos/química , Simulação por Computador , Dibenzofuranos Policlorados , Humanos , Técnicas In Vitro , Bifenilos Policlorados/química , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/toxicidade , Relação Quantitativa Estrutura-Atividade , Ratos , RoedoresRESUMO
Methyleugenol--a natural constituent of herbs and spices--is hepatocarcinogenic in rodent models. It can form DNA adducts after side-chain hydroxylation and sulfation. We previously demonstrated that human sulfotransferases (SULTs) 1A1 and 1A2 as well as mouse Sult1a1, expressed in Salmonella target strains, are able to activate 1'-hydroxymethyleugenol (1'-OH-ME) and 3'-hydroxymethylisoeugenol (3'-OH-MIE) to mutagens. Now we investigated the role of these enzymes in the formation of hepatic DNA adducts by methyleugenol in the mouse in vivo. We used FVB/N mice [wild-type (wt)] and genetically modified strains in this background: Sult1a1 knockout (ko), transgenic for human SULT1A1/2 (tg) and the combination of both modifications (ko-tg). Methyleugenol (50mg/kg body mass) formed 23, 735, 3770 and 4500 N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adducts per 10(8) 2'-deoxyribonucleosides (dN) in ko, wt, ko-tg and tg mice, respectively. The corresponding values for an equimolar dose of 1'-OH-ME were 12, 1490, 12 400 and 13 300 per 10(8) dN. Similar relative levels were observed for the minor adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Thus, the adduct formation by both compounds was nearly completely dependent on the presence of SULT1A enzymes, with human SULT1A1/2 producing stronger effects than mouse Sult1a1. Moreover, a dose of 0.05 mg/kg methyleugenol (one-fourth of the estimated average daily exposure of humans) was sufficient to form detectable adducts in humanized (ko-tg) mice. Although 3'-OH-MIE was equally mutagenic to 1'-OH-ME in Salmonella strains expressing human SULT1A1 or 1A2, it only formed 0.14% of hepatic adducts in ko-tg mice compared with an equimolar dose of 1'-OH-ME, suggesting an important role of detoxifying pathways for this isomer in vivo.
Assuntos
Arilsulfotransferase/genética , Adutos de DNA , Eugenol/análogos & derivados , Fígado/efeitos dos fármacos , Animais , Sequência de Bases , Primers do DNA , Relação Dose-Resposta a Droga , Eugenol/metabolismo , Eugenol/farmacologia , Feminino , Humanos , Limite de Detecção , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da PolimeraseRESUMO
Chemical structure data and corresponding measured bioactivities of compounds are nowadays easily available from public and commercial databases. However, these databases contain heterogeneous data from different laboratories determined under different protocols and, in addition, sometimes even erroneous entries. In this study, we evaluated the use of data from bioactivity databases for the generation of high quality in silico models for off-target mediated toxicity as a decision support in early drug discovery and crop-protection research. We chose human acetylcholinesterase (hAChE) inhibition as an exemplary end point for our case study. A standardized and thorough quality management routine for input data consisting of more than 2,200 chemical entities from bioactivity databases was established. This procedure finally enables the development of predictive QSAR models based on heterogeneous in vitro data from multiple laboratories. An extended applicability domain approach was used, and regression results were refined by an error estimation routine. Subsequent classification augmented by special consideration of borderline candidates leads to high accuracies in external validation achieving correct predictive classification of 96%. The standardized process described herein is implemented as a (semi)automated workflow and thus easily transferable to other off-targets and assay readouts.
Assuntos
Modelos Teóricos , Algoritmos , Inteligência Artificial , Simulação por ComputadorRESUMO
The EFSA Scientific Committee has updated its 2010 Guidance on risk-benefit assessment (RBA) of foods. The update addresses methodological developments and regulatory needs. While it retains the stepwise RBA approach, it provides additional methods for complex assessments, such as multiple chemical hazards and all relevant health effects impacting different population subgroups. The updated guidance includes approaches for systematic identification, prioritisation and selection of hazardous and beneficial food components. It also offers updates relevant to characterising adverse and beneficial effects, such as measures of effect size and dose-response modelling. The guidance expands options for characterising risks and benefits, incorporating variability, uncertainty, severity categorisation and ranking of different (beneficial or adverse) effects. The impact of different types of health effects is assessed qualitatively or quantitatively, depending on the problem formulation, scope of the RBA question and data availability. The integration of risks and benefits often involves value-based judgements and should ideally be performed with the risk-benefit manager. Metrics such as Disability-Adjusted Life Years (DALYs) and Quality-Adjusted Life Years (QALYs) can be used. Additional approaches are presented, such as probability of all relevant effects and/or effects of given severities and their integration using severity weight functions. The update includes practical guidance on reporting results, interpreting outcomes and communicating the outcome of an RBA, considering consumer perspectives and responses to advice.
RESUMO
The European Commission asked EFSA to update its 2011 risk assessment on tetrabromobisphenol A (TBBPA) and five derivatives in food. Neurotoxicity and carcinogenicity were considered as the critical effects of TBBPA in rodent studies. The available evidence indicates that the carcinogenicity of TBBPA occurs via non-genotoxic mechanisms. Taking into account the new data, the CONTAM Panel considered it appropriate to set a tolerable daily intake (TDI). Based on decreased interest in social interaction in male mice, a lowest observed adverse effect level (LOAEL) of 0.2 mg/kg body weight (bw) per day was identified and selected as the reference point for the risk characterisation. Applying the default uncertainty factor of 100 for inter- and intraspecies variability, and a factor of 3 to extrapolate from the LOAEL to NOAEL, a TDI for TBBPA of 0.7 µg/kg bw per day was established. Around 2100 analytical results for TBBPA in food were used to estimate dietary exposure for the European population. The most important contributors to the chronic dietary LB exposure to TBBPA were fish and seafood, meat and meat products and milk and dairy products. The exposure estimates to TBBPA were all below the TDI, including those estimated for breastfed and formula-fed infants. Accounting for the uncertainties affecting the assessment, the CONTAM Panel concluded with 90%-95% certainty that the current dietary exposure to TBBPA does not raise a health concern for any of the population groups considered. There were insufficient data on the toxicity of any of the TBBPA derivatives to derive reference points, or to allow a comparison with TBBPA that would support assignment to an assessment group for the purposes of combined risk assessment.