Time- and concentration-dependent induction of CYP1A1 and CYP1A2 in precision-cut rat liver slices incubated in dynamic organ culture in the presenceof 2,3,7,8-tetrachlorodibenzo-p-dioxin

In a previous 24-h study, precision-cut rat liver slices were validated as a useful in vitro model for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2,3,7, 8-tetrachlorodibenzo-p -dioxin (TCDD), Further assessment of the utility of this model was accompl ished by initially exposing rat liver slices to medium containing TCDD (0.0 1 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-f ree medium. The slices remained viable throughout the incubation period wit h an intracellular potassium content varying from 45.2 +/- 2.3 mu mol/g at 48 h to 50.0 +/- 1.6 mu mol/g at 72 h, In TCDD-exposed slices, CYP1A1 prote in and its respective enzymatic activity, the O-deethylation of ethoxyresor ufin (EROD), significantly increased with time over the 96-h incubation per iod, with EROD activity increasing from 63.6 +/- 14.2 at 24 h to 905 +/- 29 1 pmol/mg/min at 96 h, Under identical incubation conditions, but in the ab sence of TCDD, the EROD activity for the control liver slices ranged from 1 4.3 +/- 4.3 to 44.9 +/- 11.9 pmol/min/mg. Conversely, the level of CYP1A2 p rotein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentrati on-effect relationship at 96 h was characterized by incubating rat liver sl ices for the initial 24 h in medium containing TCDD at concentrations rangi ng from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displ aying greater than 100-fold induction compared to control (0 nM TCDD). Immu nohistochemical localization of CYP1A1 protein within liver slices supporte d the time- and concentration-dependent induction of EROD activity by TCDD, The induction of CYP1A1 was initially observed to be centrilobular, with i ncreased expression due to both elevated CYP1A1 within cells and the recrui tment of additional cells expressing CYP1A1 throughout the entire liver sli ce. Additionally, the immunohistochemical analysis of the liver slices demo nstrated the conservation of tissue architecture following up to 96 h of in cubation in dynamic organ culture and provided further evidence for mainten ance of tissue viability, In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver), In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and m inimally enhanced CYP1A2 expression, Moreover, extending the incubation per iod to 96 h resulted in in vitro induction profiles for CYP1A1 and CYP1A2 t hat were qualitatively and quantitatively similar to that previously observ ed following in vivo exposure to TCDD (Drahushuk et al,, Toxicol. Appl, Pha rmacol. 140, 393-403, 1996). 1999 Academic Press.