RESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Luciferases/genética , Plasmídeos/genética , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Elementos de Resposta/genética , Animais , Bioensaio , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Células Clonais , Monitoramento Ambiental/métodos , Indução Enzimática , Humanos , Luciferases/biossíntese , Camundongos , Valor Preditivo dos Testes , Ratos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Reprodutibilidade dos Testes , Elementos de Resposta/efeitos dos fármacos , Especificidade da Espécie , Transcrição Gênica/efeitos dos fármacos , Transfecção , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Analysis of the Ah Receptor Signal Transduction Pathway (Michael S. Denison, Jane M. Rohers, S. Renee Rushing. Carol L. Jones, Selwyna C. Tetangico, and Sharon Heath-Pagliuso, University of California, Davis, California).The protocols in this unit will allow researchers to detect the Ah receptor and characterize its functional activities (i.e., ligand binding, transformation and DNA binding, and gene expression) in their biological test system and to use these methods to detect chemical and biochemical events that affect this signaling system.