Dioxins and related compounds induce cytochrome P4501A group enzymes (CYP1As) through binding with arylhydrocarbon receptor (AhR) in the first step of their toxic effect. We developed a sensitive bioassy system for dioxin-like toxicity by determination of CYP1As levels in bovine and chicken primary cultured hepatocytes for both gene expression and enzyme activity. Bovine and chicken hepatocytes were cultured primarily maintaining biotransformation function. After 24 hours of culture, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) was added and the hepatocytes were incubated for a further 24 hours. Then RT-PCR and ethoxyresorufin-O-deethylase (EROD) activity assay were performed to determine levels of CYP1As mRNA expression and enzyme activity of CYP1As products, respectively. RT-PCR primers were constructed from the partial sequences of bovine CYP1A1 that we obtained by cloning and from the known sequences of chicken CYP1A4 and CYP 1A5. Bovine CYP1A1 was responsive to very low levels of TCDD (from 100 fM). EROD activity were also inducible in a dose-dependent manner. Chicken CYP1A4 and CYP1A5 gene expressions and EROD activities were inducible with very low amounts of TCDD (from 1 pM) in a dose-dependent manner. This bioassay system is useful to detect dioxin-like toxicity because of its sensitivity. (Toxico-Biochemistry Section, Department of Safety Research TEL +81-29-838-7821)
Reference:
- Guruge et al. (2001) Organohalogen Compounds 53:251-254.
- Yamanaka et al. (2001) Organohalogen Compounds 53:255-257.
