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Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.
Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.
Assuntos
Testes de Toxicidade , Transcriptoma , Humanos , Medição de RiscoRESUMO
The understanding of how exogenous chemicals (xenobiotics) are metabolized, distributed, and eliminated is critical to determine the impact of the chemical and its metabolites to the (human) organism. This is part of the research and educational discipline ADMET (absorption, distribution, metabolism, elimination, and toxicity). Here, we review the work of Jan Commandeur and colleagues who have not only made a significant impact in understanding of phase I and phase II metabolism of several important compounds but also contributed greatly to the development of experimental techniques for the study of xenobiotic metabolism. Jan Commandeur's work has covered a broad area of research, such as the development of online screening methodologies, the use of a combination of enzyme mutagenesis and molecular modeling for structure-activity relationship (SAR) studies, and the development of novel probe substrates. This work is the bedrock of current activities and brings the field closer to personalized (cohort-based) pharmacology, toxicology, and hazard/risk assessment.
Assuntos
Xenobióticos , Humanos , Inativação Metabólica , Xenobióticos/metabolismoRESUMO
Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity. Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis. To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues. Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds. The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE "lipid accumulation". KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity. Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays. The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.
Assuntos
Rotas de Resultados Adversos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/toxicidade , Animais , Simulação por Computador , Fígado Gorduroso/induzido quimicamente , Perfilação da Expressão Gênica , Humanos , Peixe-ZebraRESUMO
Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC-MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.
Assuntos
Hidroquinonas/toxicidade , Oxirredução , Fenóis/toxicidade , Linhagem Celular Tumoral , Cromatografia Gasosa-Espectrometria de Massas , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Hidroquinonas/química , Fenóis/química , Reprodutibilidade dos TestesRESUMO
Aflatoxins are a group of mycotoxins that have major adverse effects on human health. Aflatoxin B1 (AFB1) is the most important aflatoxin and a potent carcinogen once converted into a DNA-reactive form by cytochrome P450 enzymes (CYP450). AFB1 biosynthesis involves the formation of Versicolorin A (VerA) which shares structural similarities with AFB1 and can be found in contaminated commodities, often co-occurring with AFB1. This study investigated and compared the toxicity of VerA and AFB1, alone or in combination, in HepG2 human liver cells. Our results show that both toxins have similar cytotoxic effects and are genotoxic although, unlike AFB1, the main genotoxic mechanism of VerA does not involve the formation of DNA double-strand breaks. Additionally, we show that VerA activates the aryl hydrocarbon receptor (AhR) and significantly induce the expression of the CYP450-1A1 (CYP1A1) while AFB1 did not induce AhR-dependent CYP1A1 activation. Combination of VerA with AFB1 resulted in enhanced genotoxic effects, suggesting that AhR-activation by VerA influences AFB1 genotoxicity by promoting its bioactivation by CYP450s to a highly DNA-reactive metabolite. Our results emphasize the need for expanding the toxicological knowledge regarding mycotoxin biosynthetic precursors to identify those who may pose, directly or indirectly, a threat to human health.
Assuntos
Aflatoxina B1/toxicidade , Antraquinonas/toxicidade , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mutagênicos/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sinergismo Farmacológico , Células Hep G2 , Humanos , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Polychlorinated dioxins and dibenzofurans (PCDD/Fs) are highly toxic contaminants that are strictly regulated and monitored in the environment and food to reduce human exposure. Recently, the increasing occurrence of polybrominated dioxins and dibenzofurans (PBDD/Fs) in the environment is raising concerns about the impact on human health by the combined exposure to chlorinated and brominated analogues of dioxins. Toxicological properties of PBDD/Fs relative to PCDD/Fs have not been firmly established, and brominated dioxins are not included in routine monitoring programs. In this study, we set out to determine human-relevant congener-specific potency values for a range of brominated and chlorinated dioxin congeners, based on their aryl hydrocarbon receptor (AhR)-mediated mode of toxic action. Transactivation of the AhR was measured using dioxin-responsive (DR) CALUX reporter gene assays. Because of known species-differences in dioxin-mediated toxicity, we developed and used a HepG2 human liver cell-based DR human CALUX assay that is a variant of the rodent-based DR CALUX. The assay was found to be highly inducible and stable, with low variations between independent measurements. Using both DR CALUX assays in an automated high-throughput mode we found that overall PBDD/Fs were as potent as PCDD/Fs in inducing AhR transactivation, but congener-specific differences were observed. We also observed species-specific differences in sensitivity and potency when comparing DR human REP values to those obtained in the rat-based DR CALUX. Finally, we observed significant differences between WHO-TEF values and DR human REP values, suggesting that actual WHO-TEF values may underestimate the hazards associated with exposure of humans to dioxins.
Assuntos
Dioxinas , Dibenzodioxinas Policloradas , Animais , Dibenzofuranos , Dibenzofuranos Policlorados , Dioxinas/toxicidade , Genes Reporter , Humanos , Dibenzodioxinas Policloradas/toxicidade , Ratos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação TranscricionalRESUMO
In this paper, we investigated the possible presence of endocrine disrupting chemicals (EDCs) based on measuring the total estrogenic and androgenic activity in human milk samples. We used specific bioassays for analysis of the endocrine activity of estrogens and estrogen-like EDCs and androgens and androgen-like EDCs and developed a separation method to evaluate the contribution from natural hormones in comparison to that of EDCs to total endocrine activities. We extracted ten random samples originating from the Norwegian HUMIS biobank of human milk and analyzed their agonistic or antagonistic activity using the ERα- and AR CALUX® bioassays. The study showed antagonistic activity towards the androgen receptor in 8 out of 10 of the assessed human milk samples, while 2 out of 10 samples showed agonistic activity for the ERα. Further investigations demonstrated anti-androgenic activity in the polar fraction of 9 out of 10 samples while no apolar extracts scored positive. The culprit chemicals causing the measured antagonistic activity in AR CALUX was investigated through liquid chromatography fractionation coupled to bioanalysis and non-target screening involving UHPLC-Q-TOF-MS/MS, using a pooled polar extract. The analysis revealed that the measured anti-androgenic biological activity could not be explained by the presence of endogenous hormones nor their metabolites. We have demonstrated that human milk of Norwegian mothers contained anti-androgenic activity which is most likely associated with the presence of anthropogenic polar EDCs without direct interferences from natural sex hormones. These findings warrant a larger scale investigation into endocrine biological activity in human milk, as well as exploring the chemical sources of the activity and their potential effects on health of the developing infant.
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Disruptores Endócrinos , Poluentes Químicos da Água , Disruptores Endócrinos/análise , Disruptores Endócrinos/toxicidade , Estrogênios/análise , Hormônios Esteroides Gonadais , Humanos , Leite Humano/química , Receptores Androgênicos , Espectrometria de Massas em Tandem , Poluentes Químicos da Água/análiseRESUMO
Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
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Documentação , Processamento Eletrônico de Dados/legislação & jurisprudência , Regulamentação Governamental , Testes de Toxicidade , Toxicologia/legislação & jurisprudência , Animais , Células Cultivadas , Europa (Continente) , Humanos , Formulação de Políticas , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Terminologia como Assunto , Peixe-Zebra/embriologiaRESUMO
Three dipolar aprotic solvents were designed to possess high dipolarity and low toxicity: N,N,N',N'-tetrabutylsuccindiamide (TBSA), N,N'-diethyl-N,N'-dibutylsuccindiamide (EBSA), and N,N'-dimethyl-N,N'-dibutylsuccindiamide (MBSA). They were synthesized catalytically by using a K60 silica catalyst in a solventless system. Their water immiscibility stands out as an unusual and useful property for dipolar aprotic solvents. They were tested in a model Heck reaction, metal-organic framework syntheses, and a selection of polymer solubility experiments in which their performances were found to be comparable to traditional solvents. Furthermore, MBSA was found to be suitable for the production of an industrially relevant membrane from polyethersulfone. An integrated approach involving inâ silico analysis based on available experimental information, prediction model outcomes and read across data, as well as a panel of inâ vitro reporter gene assays covering a broad range of toxicological endpoints was used to assess toxicity. These inâ silico and inâ vitro tests suggested no alarming indications of toxicity in the new solvents.
RESUMO
In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .
RESUMO
Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.
Assuntos
Testes de Toxicidade/métodos , Animais , Estudos de Avaliação como Assunto , Humanos , Organização para a Cooperação e Desenvolvimento Econômico , Reprodutibilidade dos Testes , Projetos de Pesquisa , Testes de Toxicidade/normasRESUMO
Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the 'migration-inhibition of NCC (cMINC)' assay, a large number of toxicity mechanisms and protein targets can be covered. However, the key events that lead to the adverse effects of a given chemical or group of related compounds are hard to elucidate. To address this issue, we explored here, whether the establishment of two overlapping structure-activity relationships (SAR)-linking chemical structure on the one hand to a phenotypic test outcome, and on the other hand to a mechanistic endpoint-was useful as strategy to identify relevant toxicity mechanisms. For this purpose, we chose polychlorinated biphenyls (PCB) as a large group of related, but still toxicologically and physicochemically diverse structures. We obtained concentration-dependent data for 26 PCBs in the cMINC assay. Moreover, the test chemicals were evaluated by a new high-content imaging method for their effect on cellular re-distribution of connexin43 and for their capacity to inhibit gap junctions. Non-planar PCBs inhibited NCC migration. The potency (1-10 µM) correlated with the number of ortho-chlorine substituents; non-ortho-chloro (planar) PCBs were non-toxic. The toxicity to NCC partially correlated with gap junction inhibition, while it fully correlated (p < 0.0004) with connexin43 cellular re-distribution. Thus, our double-SAR strategy revealed a mechanistic step tightly linked to NCC toxicity of PCBs. Connexin43 patterns in NCC may be explored as a new endpoint relevant to developmental toxicity screening.
Assuntos
Crista Neural/efeitos dos fármacos , Bifenilos Policlorados/química , Bifenilos Policlorados/toxicidade , Relação Estrutura-Atividade , Animais , Disponibilidade Biológica , Movimento Celular/efeitos dos fármacos , Conexina 43/metabolismo , Junções Comunicantes/efeitos dos fármacos , Humanos , Camundongos , Células NIH 3T3 , Crista Neural/citologia , Bifenilos Policlorados/farmacocinética , Imagem com Lapso de TempoRESUMO
Identification and monitoring of so-called endocrine-disrupting compounds has received ample attention; both the OECD and the United States Environmental Protection Agency (US EPA) have designed tiered testing approaches, involving in vitro bioassays to prioritize and partly replace traditional animal experiments. Since the estrogen (ER) and androgen (AR) receptor are frequent targets of endocrine disrupting chemicals, bioassays detecting interaction with these receptors have a high potential to be of use in risk assessment of endocrine active compounds. However, in many bioassays in vivo hepatic metabolism is not accounted for, which hampers extrapolation to the in vivo situation. In the present study, we have developed a metabolic module using rat liver S9 as an add-on to human cell-based reporter gene assays. The method was applied to reporter gene assays for detection of (anti-) estrogens and (anti-) androgens, but can be extended to cell-based reporter gene assays covering a variety of endpoints related to endocrine disruption.
Assuntos
Antagonistas de Androgênios/toxicidade , Disruptores Endócrinos/toxicidade , Antagonistas de Estrogênios/toxicidade , Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Microssomos Hepáticos/enzimologia , Alternativas aos Testes com Animais , Animais , Linhagem Celular , Receptor alfa de Estrogênio/genética , Humanos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , TransfecçãoRESUMO
Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.
Assuntos
Alternativas aos Testes com Animais , Teratogênicos/toxicidade , Testes de Toxicidade/métodos , Animais , Linhagem Celular , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Genes Reporter , Humanos , Camundongos , Receptores de Estrogênio/metabolismo , Reprodução , Teratogênicos/classificação , Teratogênicos/farmacocinética , Toxicocinética , Peixe-Zebra/embriologiaRESUMO
Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.
Assuntos
Antineoplásicos Alquilantes/metabolismo , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Ifosfamida/metabolismo , Mutação , Ativação Metabólica , Antineoplásicos Alquilantes/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclofosfamida/farmacologia , Relação Dose-Resposta a Droga , Genótipo , Humanos , Hidroxilação , Ifosfamida/farmacologia , Cinética , Microssomos Hepáticos/enzimologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , FenótipoRESUMO
BACKGROUND: The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S](2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB. CONCLUSIONS/SIGNIFICANCE: This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.
Assuntos
Escherichia coli/enzimologia , Fumarato Hidratase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Fumarato Hidratase/química , Proteínas Ferro-Enxofre/química , Isoenzimas , Cinética , Dados de Sequência Molecular , Ácido Oxaloacético/metabolismo , Oxirredução , Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Tartaratos/metabolismoRESUMO
The hyperthermophilic archaeon Pyrococcus furiosus is a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely related Pyrococcus horikoshii and Pyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on the Pyr. furiosus genome. In the present study, these three genes were heterologously overexpressed in Escherichia coli to enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe-4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe-S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe-4S]-cluster-containing fumarate hydratases exemplified by MmcBC from Pelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase in Pyr. furiosus has yet to be established.
Assuntos
Aconitato Hidratase/metabolismo , Fumarato Hidratase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Pyrococcus furiosus/enzimologia , Aconitato Hidratase/química , Aconitato Hidratase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico , Escherichia coli/metabolismo , Fumarato Hidratase/química , Fumarato Hidratase/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Dados de Sequência Molecular , Pyrococcus furiosus/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Análise EspectralRESUMO
Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Modelos Moleculares , Mutação , Simulação por Computador , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Humanos , Ligantes , Preparações Farmacêuticas/metabolismo , Conformação ProteicaRESUMO
Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/metabolismo , Mutação , Preparações Farmacêuticas/metabolismo , Acetaminofen/análogos & derivados , Acetaminofen/química , Acetaminofen/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biotransformação , Catálise , Clozapina/análogos & derivados , Clozapina/química , Clozapina/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Diclofenaco/análogos & derivados , Diclofenaco/química , Diclofenaco/metabolismo , Glutationa/química , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Estrutura Molecular , NADPH-Ferri-Hemoproteína Redutase , Preparações Farmacêuticas/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches to calculate the free energy differences have been investigated and were compared to the experimental binding data. From the differences between calculations based on forward and backward processes and the closure of thermodynamic cycles, it was clear that not all simulations converged sufficiently. The approach that calculates the free energies of exchanging R-propranolol with S-propranolol in the F483A mutant relative to the exchange free energy in WT CYP2D6 accurately reproduced the experimental binding data. Careful inspection of the end-points of the MD simulations involved in this approach, allowed for a molecular interpretation of the observed differences.