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1.
Mem Inst Oswaldo Cruz ; 115: e200303, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33146255

RESUMO

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.


Assuntos
Antiprotozoários , Resistência a Medicamentos/genética , Giardia lamblia , Metronidazol/farmacologia , Pró-Fármacos , Ativação Metabólica , Antiprotozoários/farmacologia , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/genética , Nucleotídeos
2.
PLoS One ; 15(9): e0239540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966316

RESUMO

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.


Assuntos
Aflatoxina B1/toxicidade , Hepatócitos/efeitos dos fármacos , Ativação Metabólica , Aflatoxina B1/administração & dosagem , Aflatoxina B1/farmacocinética , Animais , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/patologia , Hepatócitos/transplante , Humanos , Técnicas In Vitro , Dose Letal Mediana , Transplante de Fígado , Masculino , Camundongos , Camundongos SCID , RNA Interferente Pequeno/genética , Quimeras de Transplante , Vacúolos/efeitos dos fármacos , Vacúolos/patologia
3.
Toxicon ; 186: 4-11, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32687888

RESUMO

Pyrrolizidine alkaloids (PAs) are natural toxins found in about 3%-5% of flowering plants. Dehydropyrrolizidine alkaloids contain a double bond in 1, 2-position of the necine bases, including retronecine type PAs (RET-PAs) and their N-oxides (RET N-oxide-PAs), and otonecine type PAs (OTO-PAs), and are known for their significant hepatotoxicity. Most dehydropyrrolizidine alkaloids are metabolically activated by cytochrome P450 (CYP450) enzymes to generate active pyrroles, which further bind to proteins to form pyrrole-protein adducts (PPAs). Methods for predicting PA-induced liver injury are generally performed on in vitro models with extremely low activities of CYP450 enzymes, which is different from the situation in vivo. In this regard, primary cultured mouse hepatocytes, which showed comparable CYP450 activity with the in vivo models, were applied to illustrate the structure-toxicity relationship of 13 dehydropyrrolizidine alkaloids, namely, eight RET-PAs, three RET N-oxide-PAs, and two OTO-PAs. PA-induced cytotoxicity and PA-generated PPAs were analyzed in primary mouse hepatocytes treated with different PAs. Results showed that PA-induced toxicity was correlated with the amount of PA-generated PPAs. RET-PAs and OTO-PAs were generally more toxic than RET N-oxide-PAs and generated higher amount of PPAs. PPAs were utilized to evaluate the efficiency of metabolic activation and predict the toxic potencies of dehydropyrrolizidine alkaloids. The proposed model could be a new approach for toxicity evaluation and risk control of exposure to PAs.


Assuntos
Alcaloides de Pirrolizidina/toxicidade , Ativação Metabólica , Alcaloides , Animais , Hepatócitos , Camundongos , Proteínas/química , Alcaloides de Pirrolizidina/química
4.
Planta Med ; 86(10): 686-695, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32365393

RESUMO

Obacunone is one of the major bioactive constituents from Dictamni cortex, a traditional Chinese medicine widely used in China. Oral administration of obacunone or Dictamni cortex extract has been shown to cause liver injury in rats. Given that obacunone contains a furan ring, which is a structural alert, metabolic activation might be responsible for obacunone-induced liver injury. In this study, bioactivation pathways of obacunone in rat and human liver microsomes were investigated. Obacunone was first metabolized into cis-butene-1,4-dial, and then cis-butene-1,4-dial was captured by glutathione, N-acetyl-cysteine, and N-acetyl-lysine in the microsomal incubation system. A total of 13 adducts derived from the reaction of cis-butene-1,4-dial with glutathione and/or N-acetyl-lysine were detected and structurally identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. The major metabolite (M7) was identified to be the cyclic mono-glutathione conjugate of cis-butene-1,4-dial, which was detected in bile and urine of obacunone-treated rats. M9 and M10, obacunone-derived glutathione-cis-butene-1,4-dial-NAL conjugates, were detected in the microsomal incubations of obacunone fortified with glutathione and N-acetyl-lysine as trapping agents. M3 and M4, pyrroline-2-one derivatives, were also detected in microsomal incubations. Further phenotyping studies indicated that ketoconazole showed a strong inhibitory effect on the production of cis-butene-1,4-dial in a concentration-dependent manner. CYP3A4 was demonstrated to be the primary enzyme responsible for the bioactivation of obacunone by using individual recombinant human CYP450 enzymes. The current study provides an overview of CYP450-dominated bioactivation of obacunone and contributes to the understanding of the role of bioactivation in obacunone-induced liver injury.


Assuntos
Aldeídos , Microssomos Hepáticos , Ativação Metabólica , Animais , Benzoxepinas , China , Cromatografia Líquida de Alta Pressão , Glutationa , Humanos , Limoninas , Ratos
5.
Toxicology ; 440: 152478, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32437779

RESUMO

Thiazoles are biologically active aromatic heterocyclic rings occurring frequently in natural products and drugs. These molecules undergo typically harmless elimination; however, a hepatotoxic response can occur due to multistep bioactivation of the thiazole to generate a reactive thioamide. A basis for those differences in outcomes remains unknown. A textbook example is the high hepatotoxicity observed for sudoxicam in contrast to the relative safe use and marketability of meloxicam, which differs in structure from sudoxicam by the addition of a single methyl group. Both drugs undergo bioactivation, but meloxicam exhibits an additional detoxification pathway due to hydroxylation of the methyl group. We hypothesized that thiazole bioactivation efficiency is similar between sudoxicam and meloxicam due to the methyl group being a weak electron donator, and thus, the relevance of bioactivation depends on the competing detoxification pathway. For a rapid analysis, we modeled epoxidation of sudoxicam derivatives to investigate the impact of substituents on thiazole bioactivation. As expected, electron donating groups increased the likelihood for epoxidation with a minimal effect for the methyl group, but model predictions did not extrapolate well among all types of substituents. Through analytical methods, we measured steady-state kinetics for metabolic bioactivation of sudoxicam and meloxicam by human liver microsomes. Sudoxicam bioactivation was 6-fold more efficient than that for meloxicam, yet meloxicam showed a 6-fold higher efficiency of detoxification than bioactivation. Overall, sudoxicam bioactivation was 15-fold more likely than meloxicam considering all metabolic clearance pathways. Kinetic differences likely arise from different enzymes catalyzing respective metabolic pathways based on phenotyping studies. Rather than simply providing an alternative detoxification pathway, the meloxicam methyl group suppressed the bioactivation reaction. These findings indicate the impact of thiazole substituents on bioactivation is more complex than previously thought and likely contributes to the unpredictability of their toxic potential.


Assuntos
Meloxicam/metabolismo , Tiazinas/metabolismo , Ativação Metabólica , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Elétrons , Compostos de Epóxi/metabolismo , Humanos , Hidroxilação , Técnicas In Vitro , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Tiazóis/metabolismo
6.
Chem Biol Interact ; 322: 109053, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198085

RESUMO

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 µM and 0.62 min-1, respectively. The calculated kobs at 10 µM was 0.29 min-1, above the 0.02 min-1 risk level. After incubation with NOT at 10 µM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.


Assuntos
Cumarínicos/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Ativação Metabólica , Apiaceae/química , Apiaceae/metabolismo , Cumarínicos/química , Citocromo P-450 CYP2D6/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
7.
Toxicology ; 437: 152438, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32199159

RESUMO

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with human carcinogenicity. Many lower chlorinated and non-dioxin-like PCBs have been observed to be mutagenic following activation by human CYP2E1, while activation of dioxin-like (DL-) PCBs by this enzyme has never been evidenced. In this study, each DL-PCB was analyzed by molecular docking to human CYP2E1 protein for predicting a substrate interaction. All compounds demonstrated high affinities with the active site of human CYP2E1, binding energy being -8.7 ∼ -9.7 kcal/mol. However, most compounds demonstrated ligand-heme distances as ≥ 6.8 Å, while the values for 2,3,3',4,4'- (PCB 105) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) were 5.3 and 5.4 Å, respectively (valid for electron transfer). Experimentally, both PCB 105 and 118 induced micronuclei in a V79-derived cell line engineered for expression of human CYP2E1 at low micromolar concentrations, while inactive or weakly positive in V79-Mz control cells; these effects were blocked or reduced by 1-aminobenzotriazole, a suicide CYP inhibitor. However, DL-PCBs 77, 81 and 126 were all negative in both cell lines. In a human hepatoma (C3A) cell line, PCB 105 and 118 induced micronuclei marginally, while with ethanol pretreatment (to stabilize CYP2E1) both compounds induced micronuclei efficiently, and co-exposure to trans-1,2-dichloroethylene (a selective CYP2E1 inhibitor) led to clearly negative results with both compounds. Finally, both PCB 105 and 118 induced PIG-A gene mutations in C3A cells, which was blocked by trans-1,2-dichloroethylene. In summary, in silico and experimental results consistently suggest that DL- PCBs 105 and 118 may be activated by human CYP2E1 for mutagenic activities.


Assuntos
Citocromo P-450 CYP2E1/metabolismo , Proteínas de Membrana/genética , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Simulação de Acoplamento Molecular , Mutação , Bifenilos Policlorados/toxicidade , Ativação Metabólica , Animais , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Domínio Catalítico , Cricetulus , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Ligação Proteica , Conformação Proteica
8.
Toxicol Lett ; 323: 48-56, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32017980

RESUMO

Traditional Chinese medicine (TCM) has become a crucial cause of drug-induced liver injury (DILI). Differ from chemical medicines, TCM feature more complex and mostly indefinite components. This review aimed to clarify the classification, underlying mechanisms and targets of the risk components in TCM-induced liver injury to further guide the secure application of TCM. Relevant studies or articles published on the PubMed database from January 2008 to December 2019 were searched. Based on the different chemical structures of the risk ingredients in TCM, they are divided into alkaloids, glycosides, toxic proteins, terpenoids and lactones, anthraquinones, and heavy metals. According to whether drug metabolism is activated or hepatocytes are directly attacked during TCM-induced liver injury, the high-risk substances can be classified into metabolic activation, non-metabolic activation, and mixed types. Mechanisms of the hepatotoxic ingredients in TCM-induced hepatotoxicity, including cytochrome P450 (CYP450) induction, mitochondrial dysfunction, oxidative damage, apoptosis, and idiosyncratic reaction, were also summarized. The targets involved in the risk ingredient-induced hepatocellular injury mainly include metabolic enzymes, nuclear receptors, transporters, and signaling pathways. Our periodic review and summary on the risk signals of TCM-induced liver injury must be beneficial to the integrated analysis on the multi-component, multi-target, and multi-effect characteristics of TCM-induced hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Medicina Tradicional Chinesa/efeitos adversos , Ativação Metabólica , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/classificação , Sistema Enzimático do Citocromo P-450/fisiologia , Humanos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Risco , Transdução de Sinais/efeitos dos fármacos
9.
Environ Mol Mutagen ; 61(4): 408-432, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32039521

RESUMO

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.


Assuntos
Ativação Metabólica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Animais , Anisomicina/toxicidade , Brefeldina A/toxicidade , Linhagem Celular , Cicloeximida/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Histonas/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Proteína Supressora de Tumor p53/genética
10.
Food Chem Toxicol ; 138: 111188, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32045649

RESUMO

Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug candidates, reduce drug development time, expenses and the number of test animals. Currently, human hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells are not constantly available and lose their metabolic activity in culture. To solve these problems many different approaches have been developed in the past decades. The aim of this review is to present these approaches and to discuss the possibilities and limitations as well as future opportunities and directions.


Assuntos
Técnicas de Cultura de Células , Doença Hepática Induzida por Substâncias e Drogas/terapia , Hepatócitos/efeitos dos fármacos , Ativação Metabólica , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Técnicas de Cocultura , Meios de Cultura/química , Desenvolvimento de Medicamentos , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Falência Hepática Aguda/complicações , Falência Hepática Aguda/diagnóstico , Modelos Animais , Esferoides Celulares , Tecidos Suporte
11.
Am J Physiol Endocrinol Metab ; 318(4): E579-E585, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101030

RESUMO

Defining the host receptors and metabolic consequences of bacterial components can help explain how the microbiome influences metabolic diseases. Bacterial peptidoglycans that activate nucleotide-binding oligomerization domain-containing (NOD)1 worsen glucose control, whereas NOD2 activation improves glycemia. Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is required for innate immunity instigated by NOD1 and NOD2. The role of RIPK2 in the divergent effects of NOD1 versus NOD2 on blood glucose was unknown. We found that whole body deletion of RIPK2 negated all effects of NOD1 or NOD2 activation on blood glucose during an acute, low level endotoxin challenge in mice. It was known that NOD1 in hematopoietic cells participates in insulin resistance and metabolic inflammation in obese mice. It was unknown if RIPK2 in hematopoietic cells is required for the glucose-lowering and anti-inflammatory effects of NOD2 activation. We hypothesized that RIPK2 in nonhematopoietic cells dictated the glycemic effects of NOD2 activation. We found that whole body deletion of RIPK2 prevented the glucose-lowering effects of repeated NOD2 activation that were evident during a glucose tolerance test (GTT) in high-fat diet (HFD)-fed wild-type (WT) mice. NOD2 activation lowered glucose during a GTT and lowered adipose tissue inflammation in mice with RIPK2 deleted in hematopoietic cells. We conclude that RIPK2 in nonhematopoietic cells mediates the glucose lowering and anti-inflammatory effects of NOD2-activating postbiotics. We propose a model where lipopolysaccharides and NOD1 ligands synergize in hematopoietic cells to promote insulin resistance but NOD2 activation in nonhematopoietic cells promotes RIPK2-dependent immune tolerance and lowering of inflammation and insulin resistance.


Assuntos
Glicemia/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Microbiota , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Ativação Metabólica , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica , Teste de Tolerância a Glucose , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética
12.
PLoS Biol ; 18(1): e3000612, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31986134

RESUMO

Antibiotic resistance increasingly limits the success of antibiotic treatments, and physicians require new ways to achieve efficient treatment despite resistance. Resistance mechanisms against a specific antibiotic class frequently confer increased susceptibility to other antibiotic classes, a phenomenon designated collateral sensitivity (CS). An informed switch of antibiotic may thus enable the efficient treatment of resistant strains. CS occurs in many pathogens, but the mechanisms that generate hypersusceptibility are largely unknown. We identified several molecular mechanisms of CS against the antibiotic nitrofurantoin (NIT). Mutants that are resistant against tigecycline (tetracycline), mecillinam (ß-lactam), and protamine (antimicrobial peptide) all show CS against NIT. Their hypersusceptibility is explained by the overexpression of nitroreductase enzymes combined with increased drug uptake rates, or increased drug toxicity. Increased toxicity occurs through interference of the native drug-response system for NIT, the SOS response, with growth. A mechanistic understanding of CS will help to develop drug switches that combat resistance.


Assuntos
Sensibilidade Colateral a Medicamentos/genética , Nitrofurantoína/farmacologia , Ativação Metabólica/efeitos dos fármacos , Ativação Metabólica/genética , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação/efeitos dos fármacos , Nitrofurantoína/farmacocinética , Organismos Geneticamente Modificados , Pró-Fármacos/farmacocinética , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
13.
Eur J Immunol ; 50(1): 130-137, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434164

RESUMO

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.


Assuntos
Adjuvantes Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Inosina Pranobex/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Ativação Metabólica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo
14.
Arthritis Rheumatol ; 72(3): 386-395, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31562704

RESUMO

Autoimmune diseases are characterized by dysregulated immune tolerance to self and inflammatory damage to tissues and organs. The development of inflammation involves multiple innate and adaptive immune pathways. Inflammasomes are multimeric cytosolic protein complexes that form to mediate host immune responses upon recognizing pathogen- or damage-associated molecular patterns via pattern-recognition receptors (PRRs). The accelerating pace of inflammasome research has demonstrated important roles for inflammasome activation in many pathologic conditions, including infectious, metabolic, autoinflammatory, and autoimmune diseases. The inflammasome generally comprises a PRR, procaspase 1, and an adaptor molecule connecting the PRR and procaspase 1. Upon inflammasome activation, procaspase 1 becomes active caspase 1 that converts pro-interleukin-1ß (proIL-1ß) and proIL-18 into mature and active IL-1ß and IL-18, respectively. The cytokines IL-1ß and IL-18 have multipotent effects on immune and nonimmune cells and induce and promote systemic and local inflammatory responses. Human studies have shown increased levels of these cytokines, altered activation of inflammasome-related molecules, and/or the presence of inflammasome activators in rheumatic diseases, including systemic lupus erythematosus, rheumatoid arthritis, crystal-induced arthropathies, and Sjögren's syndrome. Such changes are found in the primary target organs, such as the kidneys, joints, and salivary glands, as well as in the cardiovascular system. In animal models of rheumatic diseases, inflammation and tissue damage improve upon genetic or pharmacologic targeting of the inflammasome, supporting its pathogenic role. Herein, we review the clinicopathologic significance and therapeutic targeting of inflammasome activation in rheumatic diseases and related conditions based on recent findings.


Assuntos
Ativação Metabólica/imunologia , Doenças Autoimunes/imunologia , Inflamassomos/imunologia , Caspase 1/imunologia , Humanos , Pesquisa Médica Translacional
15.
J Pharm Biomed Anal ; 177: 112876, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31525575

RESUMO

Flavonoids-enriched extract from Scutellaria baicalensis roots (FESR) ameliorated influenza A virus (IAV) induced acute lung injury (ALI) in mice by inhibiting the excessive activation of complement system in vivo. However, FESR had no anti-complementary activity in vitro. In order to reveal the effective materials of FESR for the treatment of IAV-induced ALI, the present research explored the metabolic process of FESR both in nomal and IAV infected mice by the method of UHPLC-ESI-LTQ/MS, as well as the metabolic activating mechanism. The results showed that the inactive flavonoid glycosides of FESR were partly metabolized into anti-complementary aglycones in vivo, mainly including 5,7,4'-trihydroxy-8-methoxy-flavone, norwogonin, baicalein, wogonin, oroxylin A and chrysin. Moreover, compared with the normal mice, IAV-induced ALI mice exhibited more efficient on producing and absorbing these active metabolites, with AUC0-t and Cmax in plasma and concentrations in lungs and intestines markedly elevated in the IAV treated groups (P <  0.05). Interestingly, the intestinal bacteria from IAV-induced ALI mice showed stronger ß-glucuronidase activity and also had higher efficiency on transforming FESR to the flavonoid aglycones. These findings suggested that the anti-complementary aglycones produced by metabolic activation in vivo should be the potential effective materials of FESR against IAV infections, and intestinal bacteria might play an important role on the higher bioavailability of FESR in IAV infected mice. Additionally, the animals under the pathological state are more suitable for the metabolic study of traditional Chinese medicine.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Inativadores do Complemento/farmacocinética , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/farmacocinética , Influenza Humana/tratamento farmacológico , Scutellaria baicalensis/química , Ativação Metabólica , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/virologia , Animais , Inativadores do Complemento/administração & dosagem , Inativadores do Complemento/química , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Flavonoides/administração & dosagem , Microbioma Gastrointestinal/fisiologia , Glucuronidase/metabolismo , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Influenza Humana/metabolismo , Influenza Humana/virologia , Pulmão/patologia , Camundongos , Raízes de Plantas/química , Organismos Livres de Patógenos Específicos
16.
Xenobiotica ; 50(3): 261-269, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31173561

RESUMO

1. We investigated the structure-activity relationship of 31 kinds of synthesized atorvastatin esters, thioesters, amides and lactone, selected as prodrug models, for metabolic activation by microsomes and hydrolases.2. The susceptibility to human carboxylesterase 1 (hCES1) was influenced not only by the size of the acyl group and alkoxy group but also by the degree of steric crowding around the alkoxy group.3. The susceptibility to human carboxylesterase 2 (hCES2) increased with a decrease in electron density around the alkoxy group of the substrate.4. Lactone was specifically hydrolyzed by paraoxonase 3 (PON3).5. These findings should be useful in prodrug design for controlling metabolic activation.


Assuntos
Atorvastatina/metabolismo , Hidrolases/metabolismo , Ativação Metabólica , Carboxilesterase , Hidrolases de Éster Carboxílico , Microssomos Hepáticos/metabolismo , Pró-Fármacos , Relação Estrutura-Atividade , Especificidade por Substrato
17.
Rapid Commun Mass Spectrom ; 34(7): e8661, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31732995

RESUMO

RATIONALE: Erianin, a bioactive component isolated from Dctidrobium chrysotoxum Lindl, was demonstrated to have many biological properties relevant to cancer prevention and therapy. However, the metabolic profiles of erianin remain unknown. This study was carried out to investigate the metabolic profiles of erianin in rats and humans. METHODS: Erianin was orally administered to rats at a single dose of 50 mg/kg. Urine and bile samples were collected. For in vitro metabolism, erianin was co-incubated with rat or human hepatocytes at 37°C for 2 h. The samples from incubations and rat were analyzed by liquid chromatography combined with electrospray ionization high-resolution mass spectrometry. The data were processed by MetWorks software. The structures of the metabolites were proposed by comparing the mass spectra with that of the parent compound. RESULTS: A total of twenty-four metabolites were detected in vitro and in vivo, including seven phase I and eighteen phase II metabolites. The phase I metabolic pathways of erianin were hydroxylation, demethylation and dehydrogenation. Erianin undergoes metabolic activation to form reactive metabolites quinoid intermediates, which were further trapped by glutathione (GSH) or N-acetylcysteine. The phase II metabolic pathways were glucuronidation, glutathione and N-acetylcysteine conjugation. CONCLUSIONS: The present study provides an overview pertaining to the in vitro and in vivo metabolic profiles of erianin, which is indispensable for us to understand the efficacy and safety of erianin, as well as the herbal medicine D. chrysotoxum.


Assuntos
Bibenzilas/metabolismo , Bibenzilas/urina , Fenol/metabolismo , Fenol/urina , Ativação Metabólica , Animais , Bibenzilas/análise , Bile/química , Bile/metabolismo , Linhagem Celular , Cromatografia Líquida , Hepatócitos/metabolismo , Humanos , Redes e Vias Metabólicas , Fenol/análise , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
18.
Eur J Pharm Sci ; 143: 105195, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31852629

RESUMO

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5'-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Piperazinas/farmacocinética , para-Aminobenzoatos/farmacocinética , Ativação Metabólica , Animais , Cães , Feminino , Glucuronídeos/metabolismo , Glutationa/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Piperazinas/sangue , Piperazinas/urina , Ratos Sprague-Dawley , para-Aminobenzoatos/sangue , para-Aminobenzoatos/urina
19.
Chem Res Toxicol ; 32(12): 2488-2498, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31799839

RESUMO

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.


Assuntos
Amidas/toxicidade , Hidrocarboneto de Aril Hidroxilases/metabolismo , Furanos/toxicidade , Ativação Metabólica , Amidas/síntese química , Amidas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/química , Domínio Catalítico , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/metabolismo , Inibidores das Enzimas do Citocromo P-450/toxicidade , Furanos/síntese química , Furanos/metabolismo , Células Hep G2 , Humanos , Hidroxilação , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Coelhos , Relação Estrutura-Atividade , Terpenos/química , Terpenos/toxicidade
20.
Sci Rep ; 9(1): 15901, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685846

RESUMO

Cardiovascular disease (CVD) remains the leading cause of death in chronic kidney disease (CKD) patients despite treatment of traditional risk factors, suggesting that non-traditional CVD risk factors are involved. Trimethylamine-N-oxide (TMAO) correlates with atherosclerosis burden in CKD patients and may be a non-traditional CVD risk factor. Serum TMAO concentrations are significantly increased in CKD patients, which may be due in part to increased hepatic flavin monooxygenase (FMO)-mediated TMAO formation. The objective of this work was to elucidate the mechanism of increased FMO activity in CKD. In this study, FMO enzyme activity experiments were conducted in vitro with liver microsomes isolated from experimental CKD and control rats. Trimethylamine was used as a probe substrate to assess FMO activity. The FMO activator octylamine and human uremic serum were evaluated. FMO gene and protein expression were also determined. FMO-mediated TMAO formation was increased in CKD versus control. Although gene and protein expression of FMO were not changed, metabolic activation elicited by octylamine and human uremic serum increased FMO-mediated TMAO formation. The findings suggest that metabolic activation of FMO-mediated TMAO formation is a novel mechanism that contributes to increased TMAO formation in CKD and represents a therapeutic target to reduce TMAO exposure and CVD.


Assuntos
Metilaminas/metabolismo , Oxigenases de Função Mista/metabolismo , Insuficiência Renal Crônica/patologia , Ativação Metabólica , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Modelos Animais de Doenças , Cinética , Masculino , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/metabolismo , Especificidade por Substrato
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