RESUMO
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) represent a severe spectrum of rare mucocutaneous reactions, primarily drug-induced and characterized by significant morbidity and mortality. These conditions manifest through extensive skin detachment, distinguishing them from other generalized skin eruptions. The rarity and severity of SJS/TEN underscore the importance of accurate diagnostic criteria and effective treatments, which are currently lacking consensus. This review proposes new diagnostic criteria to improve specificity and global applicability. Recent advancements in understanding the immunopathogenesis of SJS/TEN are explored, emphasizing the role of drug-specific T cell responses and HLA polymorphisms in disease onset. The review also addresses current therapeutic approaches, including controversies surrounding the use of immunosuppressive agents and the emerging role of TNF-α inhibitors. Novel therapeutic strategies targeting specific pathogenic mechanisms, such as necroptosis and specific immune cell pathways, are discussed. Furthermore, the development of new drugs based on these insights, including targeted monoclonal antibodies and inhibitors, are examined. The review concludes by advocating for more robust and coordinated efforts across multidisciplinary medical fields to develop effective treatments and diagnostic tools for SJS/TEN, with the aim of improving patient outcomes and understanding of the disease and its mechanisms.
RESUMO
Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.
Assuntos
Mitofagia , Neoplasias Pancreáticas , Proteínas Quinases , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Abacavir (ABC)-induced hypersensitivity (AHS) is strongly associated with human leukocyte antigen (HLA)-B*57 : 01 expression. Previous studies have demonstrated the feasibility of applying the HLA-transgenic mouse model in this context. ABC-induced adverse reactions were observed in HLA-B*57 : 01 transgenic (B*57 : 01-Tg) mice. Moreover, regulating immune tolerance could result in severe AHS that mimics symptoms observed in the clinical setting, which were modeled in CD4+ T cell-depleted programmed death-1 receptor (PD-1) knockout B*57 : 01-Tg (B*57 : 01-Tg/PD-1-/-) mice. Here, we aimed to examine whether thymus and activation-regulated chemokine (TARC)/CCL17 level can be used as a biomarker for AHS. Serum TARC levels increased in HLA-B*57 : 01-transgenic mice following oral administration of ABC; this increase was associated with the severity of skin toxicity. In ABC-fed CD4+ T cell-depleted B*57 : 01-Tg/PD-1-/- mice, TARC was detected in the epidermal keratinocytes of the ear. Skin toxicity was characterized by the infiltration of CD8+ T cells partially expressing C-C chemokine receptor type 4, which is the primary receptor for TARC. In vivo TARC neutralization effectively alleviated the symptoms of ear skin redness and blood vessel dilatation. Moreover, TARC neutralization suppressed the infiltration of CD8+ T cells to the ear skin but did not affect the ABC-induced adaptive immune response. Therefore, TARC was involved in ABC-induced skin toxicity and contributed to the recruitment of CD8+ T cells to skin. This evidence suggests that serum TARC level may be a functional biomarker for AHS.
Assuntos
Linfócitos T CD8-Positivos , Quimiocina CCL17 , Dermatite Atópica , Animais , Linfócitos T CD8-Positivos/imunologia , Quimiocina CCL17/genética , Quimiocinas , Ciclopropanos/efeitos adversos , Didesoxiadenosina/efeitos adversos , Didesoxiadenosina/análogos & derivados , Antígenos HLA-B/genética , Humanos , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1RESUMO
Mitochondrial injury contributes to severe drug-induced liver injury. Particularly, mitochondrial permeability transition (MPT) is thought to be relevant to cytolytic hepatitis. However, the mechanism of drug-induced MPT is unclear and prediction of MPT is not adequately evaluated in the preclinical stage. In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling. Herein, we investigated whether other drugs that induce MPT share similar properties as troglitazone, using isolated mitochondria from rat liver. Of the 22 test drugs examined, six drugs, including troglitazone, induced MPT and showed an uncoupling effect. Additionally, receiver operating characteristic analysis was conducted to predict the MPT potential from the respiratory control ratio, an indicator of uncoupling intensity. Results showed that 2.5 was the best threshold that exhibited high sensitivity (1.00) and high specificity (0.81), indicating that uncoupling was correlated with MPT potential. Activation of calcium-independent phospholipase A2 appeared to be involved in uncoupling-induced MPT. Furthermore, a strong relationship between MPT intensity and the uncoupling effect among similar compounds was confirmed. These results may help in predicting MPT potential using cultured cells and modifying the chemical structures of the drugs to reduce MPT risk.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hipoglicemiantes/toxicidade , Masculino , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/fisiologia , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , Troglitazona/toxicidadeRESUMO
Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments.
Assuntos
Aldo-Ceto Redutases/antagonistas & inibidores , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Aldeído Redutase , Aldo-Ceto Redutases/metabolismo , Aldo-Ceto Redutases/fisiologia , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glucose/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Rodanina/análogos & derivados , Rodanina/farmacologia , Rodanina/uso terapêutico , Tiazolidinas/farmacologia , Tiazolidinas/uso terapêuticoRESUMO
Bone homeostasis is maintained by bone remodeling, which involves continuous bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation of bone turnover, caused by osteoclast overactivation, causes destructive bone diseases. However, the mechanisms underlying the maintenance of osteoclast differentiation and activation are unclear. Herein, we examined the role of autophagy in the maintenance of osteoclast differentiation and maturation. We used in vitro and in vivo assays to evaluate relationships between mitochondrial activity and autophagy during osteoclast differentiation and maturation. Our results indicate that autophagy was enhanced during osteoclast differentiation and maturation, and autophagic activity was positively correlated with osteoclast activity and survival. Maintenance of mitochondrial function, which is critical during osteoclast differentiation and maturation, was controlled by autophagy. Continuous exposure of osteoclasts to glucocorticoids upregulated autophagic processes. Treatment with the autophagic inhibitor chloroquine suppressed prolonged survival of activated osteoclasts and attenuated excessive osteoclast activity. Our study shows that autophagy-dependent mitochondrial function plays an important role in osteoclast differentiation and maturation. Elucidating the mechanisms regulating autophagic activity in osteoclasts, and developing bone-tissue-specific inhibitors of autophagy, will lead to improved understanding of the pathologies involved in destructive bone diseases.
Assuntos
Autofagia , Mitocôndrias/metabolismo , Osteoclastos/citologia , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismoRESUMO
Various types of transgenic mice carrying either class I or II human leukocyte antigen (HLA) molecules are readily available, and reports describing their use in a variety of studies have been published for more than 30 years. Examples of their use include the discovery of HLA-specific antigens against viral infection as well as the reproduction of HLA-mediated autoimmune diseases for the development of therapeutic strategies. Recently, HLA transgenic mice have been used to reproduce HLA-mediated idiosyncratic drug toxicity (IDT), a rare and unpredictable adverse drug reaction that can result in death. For example, abacavir-induced IDT has successfully been reproduced in HLA-B*57:01 transgenic mice. Several reports using HLA transgenic mice for IDT have proven the utility of this concept for the evaluation of IDT using various HLA allele combinations and drugs. It has become apparent that such models may be a valuable tool to investigate the mechanisms underlying HLA-mediated IDT. This review summarizes the latest findings in the area of HLA transgenic mouse models and discusses the current challenges that must be overcome to maximize the potential of this unique animal model.
Assuntos
Antivirais/toxicidade , Modelos Animais de Doenças , Antígenos HLA/genética , Camundongos Transgênicos , Viroses/tratamento farmacológico , Alelos , Animais , Antivirais/uso terapêutico , Didesoxinucleosídeos/uso terapêutico , Didesoxinucleosídeos/toxicidade , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , CamundongosRESUMO
Drug-induced liver injury (DILI) can cause hepatic failure and result in drug withdrawal from the market. It has host-related and compound-dependent mechanisms. Preclinical prediction of DILI risk is very challenging and safety assessments based on animals inadequately forecast human DILI risk. In contrast, human-derived in vitro cell culture-based models could improve DILI risk prediction accuracy. Here, we developed and validated an innovative method to assess DILI risk associated with various compounds. Fifty-four marketed and withdrawn drugs classified as DILI risks of "most concern", "less concern", and "no concern" were tested using a combination of four assays addressing mitochondrial injury, intrahepatic lipid accumulation, inhibition of bile canalicular network formation, and bile acid accumulation. Using the inhibitory potencies of the drugs evaluated in these in vitro tests, an algorithm with the highest available DILI risk prediction power was built by artificial neural network (ANN) analysis. It had an overall forecasting accuracy of 73%. We excluded the intrahepatic lipid accumulation assay to avoid overfitting. The accuracy of the algorithm in terms of predicting DILI risks was 62% when it was constructed by ANN but only 49% when it was built by the point-added scoring method. The final algorithm based on three assays made no DILI risk prediction errors such as "most concern " instead of "no concern" and vice-versa. Our mechanistic approach may accurately predict DILI risks associated with numerous candidate drugs.
Assuntos
Bioensaio/métodos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Valor Preditivo dos Testes , Algoritmos , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Linhagem Celular , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Dose Máxima Tolerável , Mitocôndrias/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
The interaction of human leukocyte antigen (HLA) with specific drugs is associated with delayed-type hypersensitivity reactions, which cause severe cutaneous toxicity. Such interactions induce structural alterations in HLA complexes via several different mechanisms such as the hapten theory, p-i concept, and altered peptide repertoire model, leading to the activation of cytotoxic T cells. To date, comprehensive detection of such structural alterations in preclinical studies has been difficult. Here, we evaluated structural alterations in HLA complexes focusing on the interaction between the HLA-B*57 : 01 allele and abacavir (an anti-human immunodeficiency virus drug), representing a model of abacavir hypersensitivity syndrome induced by changes in the peptide repertoire on the HLA molecule. We employed a phage display method using a commercially available antibody library to screen specific phage antibodies able to recognize HLA-B*57 : 01. The affinity of selected phage antibodies increased because of structural alterations in HLA-B*57 : 01 following exposure to abacavir, indicating that specific phage antibodies can identify drug-mediated structural changes in HLA complexes. We also identified an unreported structural change in HLA-B*57 : 01 using the phage display method, whereby abacavir increased the expression of peptide-deficient HLA-B*57 : 01 on the cell surface. These results suggest that phage display technology is a useful method for detecting structural changes in HLA complexes. This technology represents a potential novel strategy for predicting HLA-associated hypersensitivity reactions by drugs in pre-clinical studies.
Assuntos
Fármacos Anti-HIV/farmacologia , Didesoxinucleosídeos/farmacologia , Antígenos HLA-B/química , Anticorpos/imunologia , Técnicas de Visualização da Superfície Celular , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Células HeLa , HumanosRESUMO
Early hepatocyte death occurs in most liver injury cases and triggers liver inflammation, which in combination with other risk factors leads to the development of liver disease. However, the pathogenesis of early phase hepatocyte death remains poorly understood. Here, C57BL/6J mice were treated with the hepatotoxic drug flucloxacillin (FLUX) and the toll-like receptor 9 agonist CpG oligodeoxynucleotide (ODN) to reproduce the early phase of drug-induced hepatotoxicity and investigate its pathogenesis. C57BL/6J mice were treated with FLUX (100 mg/kg, gavage) alone or in combination with ODN (40 µg/mouse, intraperitoneally). Plasma alanine aminotransferase (ALT) level was measured as a marker of hepatotoxicity. FLUX or ODN alone was insufficient to induce ALT elevation, whereas combination treatment with FLUX and ODN increased ALT levels 24 h after FLUX treatment and upregulated Fas ligand in natural killer T (NKT) cells and Fas in hepatocytes. FLUX induced mitochondrial permeability transition (MPT), and pretreatment with ODN sensitized mitochondria to FLUX-induced MPT. The increase in ALT levels induced by ODN and FLUX co-treatment was suppressed in Fas ligand (gld/gld)-deficient mice and in mice deficient in a component of MPT pore opening (cyclophilin D-knockout mice). These results suggested that ODN activated the Fas/Fas ligand-mediated pathway in NKT cells and hepatocytes, which may predispose to FLUX-induced mitochondrial dysfunction and lead to early phase hepatocyte apoptosis. Taken together, these findings elucidate a potentially novel mechanism underlying drug-induced early phase hepatocyte death related to the Fas/Fas ligand death receptor pathway and mitochondrial dysfunction.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Ligante Fas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Doenças Mitocondriais/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/agonistas , Alanina Transaminase/sangue , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Proteína Ligante Fas/genética , Feminino , Hepatócitos/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Doenças Mitocondriais/induzido quimicamente , OligodesoxirribonucleotídeosRESUMO
Tyrosine kinase inhibitors (TKI), including imatinib (IM), improve the outcome of CML therapy. However, TKI treatment is long-term and can induce resistance to TKI, which often leads to a poor clinical outcome in CML patients. Here, we examined the effect of continuous IM exposure on intracellular energy metabolism in K562 cells, a human Philadelphia chromosome-positive CML cell line, and its subsequent sensitivity to anti-cancer agents. Contrary to our expectations, we found that continuous IM exposure increased sensitivity to TKI. Cancer energy metabolism, characterized by abnormal glycolysis, is linked to cancer cell survival. Interestingly, glycolytic activity was suppressed by continuous exposure to IM, and autophagy increased to maintain cell viability by compensating for glycolytic suppression. Notably, increased sensitivity to TKI was not caused by glycolytic inhibition but by altered intracellular signaling, causing glycolytic suppression and increased autophagy, as evidenced by suppression of p70 S6 kinase 1 (S6K1) and activation of AMP-activated protein kinase (AMPK). Using another human CML cell line (KCL22 cells) and BCR/ABL+ Ba/F3 cells (mimicking Philadelphia chromosome-positive CML cells) confirmed that suppressing S6K1 and activating AMPK increased sensitivity to TKI. Furthermore, suppressing S6K1 and activating AMPK had a synergistic anti-cancer effect by inhibiting autophagy in the presence of TKI. The present study provides new insight into the importance of signaling pathways that affect cellular energy metabolism, and suggests that co-treatment with agents that disrupt energy metabolic signaling (using S6K1 suppressors and AMPK activators) plus blockade of autophagy may be strategies for TKI-based CML therapy.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.
Assuntos
Benzobromarona/análogos & derivados , Benzobromarona/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Uricosúricos/química , Uricosúricos/metabolismo , Ativação Metabólica , Animais , Benzobromarona/farmacologia , Benzobromarona/toxicidade , Técnicas de Química Sintética , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ratos , Ácido Úrico/metabolismo , Uricosúricos/farmacologia , Uricosúricos/toxicidadeRESUMO
Immune-mediated idiosyncratic drug toxicity (IDT) is a rare adverse drug reaction, potentially resulting in death. Although genome-wide association studies suggest that the occurrence of immune-mediated IDT is strongly associated with specific human leukocyte antigen (HLA) allotypes, these associations have not yet been prospectively demonstrated. In this study, we focused on HLA-B*57:01 and abacavir (ABC)-induced immune-mediated IDT, and constructed transgenic mice carrying chimeric HLA-B*57:01 (B*57:01-Tg) to determine if this in vivo model may be useful for evaluating immune-mediated IDT. Local lymph node assay (LLNA) results demonstrated that percentages of BrdU+, IL-2+, and IFN-γ+ in CD8+ T cells of ABC (50 mg/kg/day)-applied B*57:01-Tg mice were significantly higher than those in littermates (LMs), resulting in the infiltration of inflammatory cells into the ear. These immune responses were not observed in B*57:03-Tg mice (negative control). Furthermore, oral administration of 1% (v/v) ABC significantly increased the percentage of CD44highCD62Llow CD8+ memory T cells in lymph nodes and spleen derived from B*57:01-Tg mice, but not in those from B*57:03-Tg mice and LMs. These results suggest that B*57:01-Tg mice potentially enable the reproduction and evaluation of HLA-B*57:01 and ABC-induced immune-mediated IDT.
Assuntos
Didesoxinucleosídeos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Antígenos HLA-B/imunologia , Testes de Toxicidade/métodos , Administração Oral , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Didesoxinucleosídeos/administração & dosagem , Didesoxinucleosídeos/toxicidade , Antígenos HLA-B/genética , Humanos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Most cancer cells predominantly produce energy by glycolysis, even in the presence of adequate oxygen. Therefore, inhibition of glycolysis is a promising cancer treatment target. Recently, it has been recognized that to conduct thorough treatment of cancer, comprehensive understanding of cancer metabolism is essential, not only focusing on glycolysis. Here, we investigated the supporting mechanism of autophagy, which is a catabolic process that recycles intracellular components, for energy supply in the glycolysis-inhibited condition. Autophagy is thought to be highly activated in cancers and to promote their growth or progression by adapting to the harsh surrounding microenvironment. We found that cancer cells positively promoted autophagy to overcome the energy shortage from glycolysis by maintaining mitochondrial activity for ATP production essential for survival. Conclusively, autophagy plays a role in determining whether cancer cells live or die, and autophagic ability in cancer cells is a promising target for therapy.
Assuntos
Autofagia , Sobrevivência Celular , Glucose/metabolismo , Glicólise , Leucemia/metabolismo , Leucemia/patologia , Metabolismo Energético , Humanos , Células Jurkat , Mitocôndrias/metabolismoRESUMO
Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Células Cultivadas , Galactose/farmacologia , Hepatócitos/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Masculino , Oxigênio/farmacologia , Consumo de Oxigênio , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Rotenona , TransferrinaRESUMO
Drug-induced liver injury (DILI) is of concern to the pharmaceutical industry, and reliable preclinical screens are required. Previously, we established an in vitro bile acid-dependent hepatotoxicity assay that mimics cholestatic DILI in vivo. Here, we confirmed that this assay can predict cholestatic DILI in clinical situations by comparing in vitro cytotoxicity data with in vivo risk. For 38 drugs, the frequencies of abnormal increases in serum alkaline phosphatase (ALP), transaminases, gamma glutamyltranspeptidase (γGT), and bilirubin were collected from interview forms. Drugs with frequencies of serum marker increases higher than 1% were classified as high DILI risk compounds. In vitro cytotoxicity was assessed by monitoring lactate dehydrogenase release from rat and human sandwich-cultured hepatocytes (SCRHs and SCHHs) incubated with the test drugs (50 µM) for 24 hours in the absence or presence of a bile acids mixture. Receiver operating characteristic analyses gave optimal cutoff toxicity values of 19.5% and 9.2% for ALP and transaminases in SCRHs, respectively. Using this cutoff, high- and low-risk drugs were separated with 65.4-78.6% sensitivity and 66.7-79.2% specificity. Good separation was also achieved using SCHHs. In conclusion, cholestatic DILI risk can be successfully predicted using a sandwich-cultured hepatocyte-based assay.
Assuntos
Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestase/metabolismo , Hepatócitos/metabolismo , Preparações Farmacêuticas/metabolismo , Adulto , Idoso , Animais , Células Cultivadas , Colestase/induzido quimicamente , Feminino , Previsões , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Fatores de RiscoRESUMO
The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.
Assuntos
Benzobromarona/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ativação Metabólica , Animais , Benzobromarona/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Cinética , Fígado/metabolismo , Fígado/patologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Dilatação Mitocondrial/efeitos dos fármacos , NADP/metabolismo , Ratos Sprague-DawleyRESUMO
PURPOSE: Drug transfer into milk is of concern due to the unnecessary exposure of infants to drugs. Proposed prediction methods for such transfer assume only passive drug diffusion across the mammary epithelium. This study reorganized data from the literature to assess the contribution of carrier-mediated transport to drug transfer into milk, and to improve the predictability thereof. METHODS: Milk-to-plasma drug concentration ratios (M/Ps) in humans were exhaustively collected from the literature and converted into observed unbound concentration ratios (M/Punbound,obs). The ratios were also predicted based on passive diffusion across the mammary epithelium (M/Punbound,pred). An in vitro transport assay was performed for selected drugs in breast cancer resistance protein (BCRP)-expressing cell monolayers. RESULTS: M/Punbound,obs and M/Punbound,pred values were compared for 166 drugs. M/Punbound,obs values were 1.5 times or more higher than M/Punbound,pred values for as many as 13 out of 16 known BCRP substrates, reconfirming BCRP as the predominant transporter contributing to secretory transfer of drugs into milk. Predictability of M/P values for selected BCRP substrates and non-substrates was improved by considering in vitro-evaluated BCRP-mediated transport relative to passive diffusion alone. CONCLUSIONS: The current analysis improved the predictability of drug transfer into milk, particularly for BCRP substrates, based on an exhaustive data overhaul followed by focused in vitro transport experimentation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Leite Humano/metabolismo , Proteínas de Neoplasias/metabolismo , Preparações Farmacêuticas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Algoritmos , Animais , Antineoplásicos/farmacocinética , Mama/metabolismo , Difusão , Cães , Epitélio/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Células Madin Darby de Rim CaninoRESUMO
PURPOSE: To investigate the role of organic cation transporters (Octs) and multidrug and toxin extrusion protein 1 (Mate1) in the disposition of thiamine. METHODS: The uptake of [(3)H]thiamine was determined in Oct1-, Oct2-, and Oct3-expressing HEK293 cells and freshly isolated hepatocytes. A pharmacokinetic study of thiamine-d3 following intravenous infusion (1 and 100 nmol/min/kg) was conducted in male Oct1/2(+/+) and Oct1/2(-/-) mice. A MATE inhibitor, pyrimethamine, (5 mg/kg) was administered intravenously. The plasma and breast milk concentrations of thiamine were determined in female mice. RESULTS: Thiamine is a substrate of Oct1 and Oct2, but not Oct3. Oct1/2 defect caused a significant reduction in the uptake of [(3)H]thiamine by hepatocytes in vitro, and elevated the plasma thiamine concentration by 5.8-fold in vivo. The plasma clearance of thiamine-d3 was significantly decreased in Oct1/2(-/-) mice. At the higher infusion rate of 100 nmol/min/kg thiamine-d3, Oct1/2 defect or pyrimethamine-treatment caused a significant reduction in the renal clearance of thiamine-d3. The total thiamine and thiamine-d3 concentrations were moderately reduced in the intestine of Oct1/2(-/-) mice but were unchanged in the kidney, liver, or brain. The milk-to-plasma concentration ratio of thiamine was decreased by 28-fold in the Oct1/2(-/-) mice. CONCLUSIONS: Oct1 is possibly responsible for the plasma clearance of thiamine via tissue uptake and for milk secretion. Oct1/2 and Mate1 are involved in the renal tubular secretion of thiamine.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Hepatócitos/metabolismo , Leite/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Tiamina/farmacocinética , Animais , Transporte Biológico , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/genética , Cromatografia Líquida , Células HEK293 , Humanos , Lactação , Masculino , Taxa de Depuração Metabólica , Camundongos Knockout , Proteínas de Transporte de Cátions Orgânicos/genética , Espectrometria de Massas em Tandem , Tiamina/sangue , Tiamina/metabolismo , Distribuição Tecidual , TransfecçãoRESUMO
1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions. 2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague-Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs. 3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17ß-glucuronide (E217ßG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides. 4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.