RESUMO
In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches.
Assuntos
Modelos Teóricos , Mutagênicos/toxicidade , Projetos de Pesquisa , Toxicologia/métodos , Animais , Simulação por Computador , Humanos , Testes de Mutagenicidade , Medição de RiscoRESUMO
The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.
Assuntos
Simulação por Computador , Testes de Toxicidade/métodos , Toxicologia/métodos , Animais , HumanosRESUMO
The goal of this investigation was to perform a comparative analysis on how accurately 11 routinely-used in silico programs correctly predicted the mutagenicity of test compounds that contained either bulky or electron-withdrawing substituents. To our knowledge this is the first study of its kind in the literature. Such substituents are common in many pharmaceutical agents so there is a significant need for reliable in silico programs to predict precisely whether they truly pose a risk for mutagenicity. The predictions from each program were compared to experimental data derived from the Ames II test, a rapid reverse mutagenicity assay with a high degree of agreement with the traditional Ames assay. Eleven in silico programs were evaluated and compared: Derek for Windows, Derek Nexus, Leadscope Model Applier (LSMA), LSMA featuring the in vitro microbial Escherichia coli-Salmonella typhimurium TA102 A-T Suite (LSMA+), TOPKAT, CAESAR, TEST, ChemSilico (±S9 suites), MC4PC and a novel DNA docking model. The presence of bulky or electron-withdrawing functional groups in the vicinity of a mutagenic toxicophore in the test compounds clearly affected the ability of each in silico model to predict non-mutagenicity correctly. This was because of an over reliance on the part of the programs to provide mutagenicity alerts when a particular toxicophore is present irrespective of the structural environment surrounding the toxicophore. From this investigation it can be concluded that these models provide a high degree of specificity (ranging from 71% to 100%) and are generally conservative in their predictions in terms of sensitivity (ranging from 5% t o 78%). These values are in general agreement with most other comparative studies in the literature. Interestingly, the DNA docking model was the most sensitive model evaluated, suggesting a potentially useful new mode of screening for mutagens. Another important finding was that the combination of a quantitative structure-activity relationship and an expert rules system appeared to offer little advantage in terms of sensitivity, despite of the requirement for such a screening paradigm under the ICH M7 regulatory guideline.
Assuntos
Simulação por Computador , Dano ao DNA , Modelos Biológicos , Mutagênicos/toxicidade , Bibliotecas de Moléculas Pequenas/toxicidade , DNA Bacteriano/química , DNA Bacteriano/genética , Transporte de Elétrons , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Mutagenicidade/métodos , Mutagênicos/química , Valor Preditivo dos Testes , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Sensibilidade e Especificidade , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Statistical-based and expert rule-based models built using public domain mutagenicity knowledge and data are routinely used for computational (Q)SAR assessments of pharmaceutical impurities in line with the approach recommended in the ICH M7 guideline. Knowledge from proprietary corporate mutagenicity databases could be used to increase the predictive performance for selected chemical classes as well as expand the applicability domain of these (Q)SAR models. This paper outlines a mechanism for sharing knowledge without the release of proprietary data. Primary aromatic amine mutagenicity was selected as a case study because this chemical class is often encountered in pharmaceutical impurity analysis and mutagenicity of aromatic amines is currently difficult to predict. As part of this analysis, a series of aromatic amine substructures were defined and the number of mutagenic and non-mutagenic examples for each chemical substructure calculated across a series of public and proprietary mutagenicity databases. This information was pooled across all sources to identify structural classes that activate or deactivate aromatic amine mutagenicity. This structure activity knowledge, in combination with newly released primary aromatic amine data, was incorporated into Leadscope's expert rule-based and statistical-based (Q)SAR models where increased predictive performance was demonstrated.
Assuntos
Aminas/toxicidade , Mineração de Dados/métodos , Bases de Conhecimento , Mutagênese , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Aminas/química , Aminas/classificação , Animais , Simulação por Computador , Bases de Dados Factuais , Humanos , Modelos Moleculares , Estrutura Molecular , Mutagênicos/química , Mutagênicos/classificação , Reconhecimento Automatizado de Padrão , Relação Quantitativa Estrutura-Atividade , Medição de RiscoRESUMO
The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.
Assuntos
Testes de Carcinogenicidade/métodos , Dano ao DNA , Mineração de Dados/métodos , Mutagênese , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Toxicologia/métodos , Animais , Testes de Carcinogenicidade/normas , Simulação por Computador , Bases de Dados Factuais , Fidelidade a Diretrizes , Guias como Assunto , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Mutagenicidade/normas , Mutagênicos/química , Mutagênicos/classificação , Formulação de Políticas , Relação Quantitativa Estrutura-Atividade , Medição de Risco , Toxicologia/legislação & jurisprudência , Toxicologia/normasRESUMO
Cytochrome P450 2D6 (CYP2D6) is a polymorphic enzyme responsible for metabolizing approximately 25% of all drugs. CYP2D6 is highly expressed in the brain and plays a role as the major CYP in the metabolism of numerous brain-penetrant drugs, including antipsychotics and antidepressants. CYP2D6 activity and inhibition have been associated with numerous undesirable effects in patients, such as bioactivation, drug-associated suicidality and prolongation of the QTc interval. Several in silico tools have been developed in recent years to assist safety assessment scientists in predicting the structural identity of CYP2D6-derived metabolites. The first goal of this study was to perform a comparative evaluation on the ability of four commonly used in silico tools (MetaSite, StarDrop, SMARTCyp and RS-WebPredictor) to correctly predict the CYP2D6-derived site of metabolism (SOM) for 141 compounds, including 10 derived from the Genentech small molecule library. The second goal was to evaluate if a bioactivation prediction model, based on an indicator of chemical reactivity (ELUMO-EHOMO) and electrostatic potential, could correctly predict five representative compounds known to be bioactivated by CYP2D6. Such a model would be of great utility in safety assessment since unforeseen toxicities of CYP2D6 substrates may in part be due to bioactivation mechanisms. The third and final goal was to investigate whether molecular docking, using the crystal structure of human CYP2D6, had the potential to compliment or improve the results obtained from the four SOM in silico programs.
Assuntos
Citocromo P-450 CYP2D6/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/enzimologia , Simulação de Acoplamento Molecular , Ativação Metabólica , Sítios de Ligação , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Humanos , Polimorfismo Genético , Ligação Proteica , Conformação Proteica , Medição de Risco , Fatores de Risco , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Tienilic acid (TA) is selectively oxidized at the C-5 position of the thiophene ring by the human liver enzyme cytochrome P450 2C9 (CYP2C9). This oxidation is mediated by the proximal positioning of the thiophene over the heme iron, which is proposed to be coordinated by an interaction of the TA carboxylic acid to a cationic binding pocket in the enzyme active site. In this study, we investigated how chemical modification of TA influences the bioactivation by CYP2C9. For this investigation, nine analogs of TA were chosen with substitutions on either side of the molecule. We tested three parameters, including CYP2C9 inhibition, metabolic profiling, and in silico docking. Of the 10 compounds tested, only two (TA and a noncarboxyl analog) resulted in competitive and time-dependent inhibition of CYP2C9. Metabolic profiling revealed a trend in which substitution of the carboxylate with nonionizable functional groups resulted in metabolic switching from oxidation of the aromatic ring to dealkylation reactions at the opposite side of the structure. The in silico modeling predicted an opposite binding orientation to that of TA for many analogs, including the 3-thenoyl regio-isomer analog, which contradicts previous models. Together these data show that disrupting interactions with the cationic binding pocket of CYP2C9 will impact the sites of metabolism and inhibition of the enzyme.
Assuntos
Citocromo P-450 CYP2C9/metabolismo , Ticrinafeno/metabolismo , Cátions , Simulação por Computador , Diuréticos/metabolismo , Diuréticos/farmacocinética , Humanos , Técnicas In Vitro , Especificidade por Substrato , Ticrinafeno/farmacocinética , Uricosúricos/metabolismo , Uricosúricos/farmacocinéticaRESUMO
1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with (S)-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and (S)-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with (S)-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [(S)-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of (S)-warfarin (20 µM) were 0.0826 and 0.273 min(-1) for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 µM)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of (S)-warfarin.
Assuntos
Triazóis/farmacologia , Varfarina/farmacologia , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2C9 , Diclofenaco/administração & dosagem , Diclofenaco/química , Diclofenaco/farmacologia , Feminino , Humanos , Técnicas In Vitro , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Especificidade por Substrato , Espectrometria de Massas em Tandem , Fatores de Tempo , Triazóis/administração & dosagem , Triazóis/química , Varfarina/administração & dosagem , Varfarina/químicaRESUMO
Microphysiological systems (MPS) are designed to recapitulate aspects of tissue/organ physiology in vivo, thereby providing potential value in safety and efficacy assessments of FDA-regulated products and regulatory decision-making. While there have been significant advances in the development, use, and proposals of qualification criteria for human organ MPS, there remains a gap in the development using animal tissues. Animal MPS may be of value in many areas including the study of zoonotic diseases, assessment of the safety and efficacy of animal therapeutics, and possibly reduction of the use of animals in regulatory submissions for animal therapeutics. In addition, the development of MPS from various animal species enables comparison to animal in vivo data. This comparison, while not always critical for all contexts of use, could help gain confidence in the use and application of human MPS data for regulatory decision-making and for the potential identification of species-specific effects. The use of animal MPS is consistent with the replacement, reduction, and refinement (3Rs) principles of animal use by identifying toxic compounds before conducting in vivo studies and identifying the appropriate species for testing.
Microphysiological systems (MPS) mimic aspects of organs in humans or animals. These systems may provide information useful for FDA-regulated products. While there have been significant advances in the development of MPS made from human cells, there remains a gap in the development of MPS using animal cells. FDA believes animal MPS may be of value in many areas including the study of diseases transmitted from animals to humans, assessment of the safety and efficacy of animal drugs, and reduction of the use of animals in regulatory submissions. The development of animal MPS enables comparison to data from studies conducted in animals. This comparison provides confidence in the use of human MPS data for regulatory decision-making. The use of animal MPS is consistent with the 3Rs principles of animal use by allowing identification of toxic compounds before conducting animal studies and by helping select the appropriate species for further testing.
RESUMO
Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.
Assuntos
Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Aleatória , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
Electrostatic potential (ESP) is a useful physicochemical property of a molecule that provides insights into inter- and intramolecular associations, as well as prediction of likely sites of electrophilic and nucleophilic metabolic attack. Knowledge of sites of metabolic attack is of paramount importance in DMPK research since drugs frequently fail in clinical trials due to the formation of bioactivated metabolites which are often difficult to measure experimentally due to their reactive nature and relatively short half-lives. Computational chemistry methods have proven invaluable in recent years as a means to predict and study bioactivated metabolites without the need for chemical syntheses, or testing on experimental animals. Additional molecular properties (heat of formation, heat of solvation and E(LUMO) - E(HOMO)) are discussed in this paper as complementary indicators of the behavior of metabolites in vivo. Five diverse examples are presented (acetaminophen, aniline/phenylamines, imidacloprid, nefazodone and vinyl chloride) which illustrate the utility of this multidimensional approach in predicting bioactivation, and in each case the predicted data agreed with experimental data described in the scientific literature. A further example of the usefulness of calculating ESP, in combination with the molecular properties mentioned above, is provided by an examination of the use of these parameters in providing an explanation for the sites of nucleophilic attack of the nucleic acid cytosine. Exploration of sites of nucleophilic attack of nucleic acids is important as adducts of DNA have the potential to result in mutagenesis.
Assuntos
Química Farmacêutica/métodos , Mutagênese , Acetaminofen/química , Compostos de Anilina/química , Simulação por Computador , Citosina/química , DNA/química , Desenho de Fármacos , Temperatura Alta , Imidazóis/química , Modelos Moleculares , Neonicotinoides , Nitrocompostos/química , Piperazinas , Solubilidade , Eletricidade Estática , Triazóis/química , Cloreto de Vinil/química , Água/químicaRESUMO
1. Here we report on the mechanism of ribose conjugation, through NADH as a cofactor, of a pyrazole-containing compound (PT). Incubation of PT in rat liver microsomes supplemented with NADPâº/H, NADâº/H, and ß-nicotinamide mononucleotide (NMN) resulted in complete conjugation to the adenine dinucleotide phosphate conjugate (ADP-C), adenine dinucleotide conjugate (AD-C), and 5-phosphoribose conjugate (Rib-C1), respectively. In hepatocytes, PT predominantly formed three ribose conjugates: Rib-C1, the ribose conjugate (Rib-C2), and the carboxylic acid of Rib-C2 (Rib-C3). 2. Phosphatase inhibitors were added to hepatocyte incubations. AD-C was detected in this reaction, which suggests that one of the major pathways for the formation of the ribose conjugates is through NADâº/H. When AD-C was incubated with phosphatase, Rib-C1 and Rib-C2 formed. 3. To understand the in vivo relevance of this metabolic pathway, rats were dosed with PT and Rib-C2 was found in the urine. 4. Structure-activity relationship shows that replacement of the distal thiazole group in the PT to a phenyl group abolishes this conjugation. Three amino acid residues in the active site preferentially interact with the sulfur atom in the thiazole of PT. 5. In summary, PT forms direct AD-C in hepatocytes, which is further hydrolyzed by phosphatase to give ribose conjugates.
Assuntos
Fígado/metabolismo , Pirazóis/metabolismo , Ribose/metabolismo , Roedores/metabolismo , ADP Ribose Transferases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Animais , Domínio Catalítico , Cromatografia por Troca Iônica , Hepatócitos/enzimologia , Humanos , Hidrólise , Masculino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Ribose/urina , Frações Subcelulares/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance.
Assuntos
Arabidopsis/imunologia , Ascomicetos/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanidinas/farmacologia , Imidazóis/farmacologia , Inseticidas/farmacologia , Nitrocompostos/farmacologia , Ácido Salicílico/imunologia , Tiazóis/farmacologia , Arabidopsis/microbiologia , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica de Plantas/imunologia , Guanidinas/química , Guanidinas/imunologia , Imidazóis/química , Imidazóis/imunologia , Inseticidas/química , Inseticidas/imunologia , Espectrometria de Massas , Estrutura Molecular , Neonicotinoides , Nitrocompostos/química , Nitrocompostos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/metabolismo , Tiazóis/química , Tiazóis/imunologiaRESUMO
Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.
Assuntos
Aldeído Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Malato Desidrogenase/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Monoterpenos/farmacologia , Albumina Sérica/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído-Desidrogenase Mitocondrial , Animais , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Fígado/química , Malato Desidrogenase/antagonistas & inibidores , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.
Assuntos
Compostos de Anilina/metabolismo , Glutationa/metabolismo , Halogenação , Desintoxicação Metabólica Fase II , Microssomos Hepáticos/metabolismo , Compostos de Anilina/química , Cromatografia Líquida , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , NADP/metabolismo , Relação Estrutura-AtividadeRESUMO
Widespread public and scientific interest in promoting the care and well-being of animals used for toxicity testing has given rise to improvements in animal welfare practices and views over time, as well as laws and regulations that support means to reduce, refine, and replace animal use (known as the 3Rs) in certain toxicity studies. One way these regulations continue to achieve their aim is by promoting the research, development, and application of alternative testing approaches to characterize potential toxicities either without animals or with minimal use. An important example of an alternative approach is the use of computational toxicology models. Along with the potential capacity to reduce or replace the use of animals for the assessment of particular toxicological endpoints, computational models offer several advantages compared to in vitro and in vivo approaches, including cost-effectiveness, rapid availability of results, and the ability to fully standardize procedures. Pharmaceutical research incorporating the use of computational models has increased steadily over the past 15 years, likely driven by the motivation of companies to screen out toxic compounds in the early stages of development. Models are currently available to aid in the prediction of several important toxicological endpoints, including mutagenicity, carcinogenicity, eye irritation, hepatotoxicity, and skin sensitization, albeit with varying degrees of success. This review serves to introduce the concepts of computational toxicology and evaluate their role in the safety assessment of compounds, while also highlighting the application of in silico methods in the support of the goal and vision of the 3Rs.
Assuntos
Alternativas ao Uso de Animais , Testes de Toxicidade/métodos , Bem-Estar do Animal , Animais , PesquisaRESUMO
The chemoselective functionalization of a range of dihaloaromatics with methyl, cyclopropyl, and higher alkyl Grignard reagents via iron-catalyzed cross-coupling is described. The site selectivity of C-X (X = halogen) activation is determined by factors such as the position of the halogen on the ring, the solvent, and the nucleophile. A one-pot protocol for the chemoselective synthesis of mixed dialkyl heterocycles is achieved solely employing iron catalysis.
Assuntos
Alcanos/química , Reagentes de Ligações Cruzadas/química , Halogênios/química , Hidrocarbonetos Halogenados/química , Ferro/química , Catálise , Estrutura MolecularRESUMO
Neonicotinoids not only control insect pests but also sometimes independently alter plant growth and response to stress. We find that imidacloprid, thiacloprid, acetamiprid, thiamethoxam, and clothianidin but not nitenpyram and dinotefuran induce foliar lesions and peroxidative damage in soybean ( Glycine max ) seedlings assayed with the 3,3'-diaminobenzidine stain. The chloropyridinyl-carboxylic acid (COOH) but not the -carboxaldehyde (CHO) metabolites induce peroxidative damage but in a different pattern. Surprisingly, the chlorothiazolyl -CHO and -COOH metabolites induce chlorosis but no clear superimposable peroxidative damage or cell death. Four metallo-oxidases known to modulate reactive oxygen species were not sensitive in vitro to the parent neonicotinoid itself but were to several CHO and COOH metabolites and related compounds, with a sensitivity order of CHO > COOH and tyrosinase > xanthine oxidase and aldehyde oxidase > catalase. Although metallo-oxidase inhibition does not correlate overall with lesion formation, it may play an as yet unknown role in plant response to neonicotinoids.
Assuntos
Inibidores Enzimáticos/farmacologia , Glycine max/efeitos dos fármacos , Inseticidas/farmacologia , Nitrocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Proteínas de Plantas/antagonistas & inibidores , Regulação para Baixo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Glycine max/enzimologia , Glycine max/metabolismoRESUMO
Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19, and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19, and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than it does that of TMX. Imidacloprid (IMI), CLO, and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI, and 4-hydroxythiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid, and CLO metabolism in vivo in mice, elevating brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites.
Assuntos
Aldeído Oxidase/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Inseticidas/metabolismo , Nitrocompostos/metabolismo , Aldeído Oxidase/química , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Guanidinas/química , Guanidinas/metabolismo , Humanos , Imidazóis/química , Imidazóis/metabolismo , Inseticidas/química , Cinética , Masculino , Microssomos Hepáticos/química , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Neonicotinoides , Nitrocompostos/química , Oxazinas/química , Oxazinas/metabolismo , Especificidade por Substrato , Tiametoxam , Tiazóis/química , Tiazóis/metabolismoRESUMO
The metabolism of seven commercial neonicotinoid insecticides was compared in spinach seedlings (Spinacia oleracea) using HPLC-DAD and LC-MSD to analyze the large number and great variety of metabolites. The parent neonicotinoid levels in the foliage following hydroponic treatment varied from differences in uptake and persistence. The metabolic reactions included nitro reduction, cyano hydrolysis, demethylation, sulfoxidation, imidazolidine and thiazolidine hydroxylation and olefin formation, oxadiazine hydroxylation and ring opening, and chloropyridinyl dechlorination. The identified phase I plant metabolites were generally the same as those in mammals, but the phase II metabolites differed in the conjugating moieties. Novel plant metabolites were various neonicotinoid-derived O- and N-glucosides and -gentiobiosides and nine amino acid conjugates of chloropyridinylcarboxylic acid. Metabolites known to be active on nicotinic acetylcholine receptors included the desnitro- and descyanoguanidines and olefin derivatives. The findings highlight both metabolites common to several neonicotinoids and those that are compound specific.