RESUMO
Metabolically induced drug-toxicity is a major cause of drug failure late in drug optimization phases. Accordingly, in vitro metabolic profiling of compounds is being introduced at earlier stages of the drug discovery pipeline. An increasingly common method to obtain these profiles is through overexpression of key CYP450 metabolic enzymes in immortalized liver cells, to generate competent hepatocyte surrogates. Enhanced cytotoxicity is presumed to be due to toxic metabolite production via the overexpressed enzyme. However, metabolically induced toxicity is a complex multi-parameter phenomenon and the potential background contribution to metabolism arising from the use of liver cells which endogenously express CYP450 isoforms is consistently overlooked. In this study, we sought to reduce the potential background interference by applying this methodology in kidney-derived HEK293 cells which lack endogenous CYP450 expression. Overexpression of CYP3A4 resulted in increased HEK293 proliferation, while exposure to four compounds with reported metabolically induced cytotoxicity in liver-derived cells overexpressing CYP3A4 resulted in no increase in cytotoxicity. Our results indicate that overexpression of a single CYP450 isoform in hepatic cell lines may not be a reliable method to discriminate which enzymes are responsible for metabolic induced cytotoxicity.
Assuntos
Clorpromazina/toxicidade , Citocromo P-450 CYP3A/metabolismo , Células Epiteliais/efeitos dos fármacos , Labetalol/toxicidade , Propranolol/toxicidade , Rosiglitazona/toxicidade , Ativação Metabólica , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/metabolismo , Citocromo P-450 CYP3A/genética , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Células HEK293 , Humanos , Labetalol/metabolismo , Propranolol/metabolismo , Medição de Risco , Rosiglitazona/metabolismo , Especificidade por Substrato , Testes de ToxicidadeRESUMO
We conducted an experimental database analysis to determine the expression of 61 CD4+ Th subset regulators in human and murine tissues, cells, and in T-regulatory cells (Treg) in physiological and pathological conditions. We made the following significant findings: (1) adipose tissues of diabetic patients with insulin resistance upregulated various Th effector subset regulators; (2) in skin biopsy from patients with psoriasis, and in blood cells from patients with lupus, effector Th subset regulators were more upregulated than downregulated; (3) in rosiglitazone induced failing hearts in ApoE-deficient (KO) mice, various Th subset regulators were upregulated rather than downregulated; (4) aortic endothelial cells activated by proatherogenic stimuli secrete several Th subset-promoting cytokines; (5) in Treg from follicular Th (Tfh)-transcription factor (TF) Bcl6 KO mice, various Th subset regulators were upregulated; whereas in Treg from Th2-TF GATA3 KO mice and HDAC6 KO mice, various Th subset regulators were downregulated, suggesting that Bcl6 inhibits, GATA3 and HDAC6 promote, Treg plasticity; and (6) GATA3 KO, and Bcl6 KO Treg upregulated MHC II molecules and T cell co-stimulation receptors, suggesting that GATA3 and BCL6 inhibit Treg from becoming novel APC-Treg. Our data implies that while HDAC6 and Bcl6 are important regulators of Treg plasticity, GATA3 determine the fate of plastic Tregby controlling whether it will convert in to either Th1-Treg or APC-T-reg. Our results have provided novel insights on Treg plasticity into APC-Treg and Th1-Treg, and new therapeutic targets in metabolic diseases, autoimmune diseases, and inflammatory disorders.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Plasticidade Celular/imunologia , Fator de Transcrição GATA3/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Aorta/citologia , Aorta/metabolismo , Células Endoteliais/metabolismo , Fator de Transcrição GATA3/genética , Desacetilase 6 de Histona/genética , Humanos , Resistência à Insulina/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout para ApoE , Proteínas Proto-Oncogênicas c-bcl-6/genética , Psoríase/imunologia , Rosiglitazona/toxicidade , Linfócitos T Reguladores/citologia , Células Th1/citologiaRESUMO
The purpose of this study was to establish a 2-stage model of urinary bladder carcinogenesis in male Sprague-Dawley rats to identify tumor promoters. In phase 1 of the study, rats ( n = 170) were administered 100 mg/kg of the tumor initiator, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), twice weekly by oral gavage (po) for a period of 6 weeks. Phase 2 consisted of dividing rats into 4 groups ( n = 40 per group) and administering one of the following for 26 weeks to identify putative tumor promoters: (1) vehicle po, (2) 25 mg/kg/day rosiglitazone po, (3) 5% dietary sodium l-ascorbate, and (4) 3% dietary uracil. Rats were necropsied after 7.5 months, and urinary bladders were evaluated by histopathology. BBN/vehicle treatments induced the development of urothelial hyperplasia (83%) and papillomas (15%) but no transitional cell carcinomas (TCCs). Rosiglitazone increased the incidence and severity of papillomas (93%) and resulted in TCC in 10% of treated rats. Uracil was the most effective tumor promoter in our study and increased the incidence of papillomas (90%) and TCC (74%). Sodium ascorbate decreased the incidence of urothelial hyperplasia (63%) and did not increase the incidence of urothelial papillomas or TCC. These data confirm the capacity of our 2-stage model to identify urinary bladder tumor promoters.