Different In Vitro Systems Affect CYPIA1 Activity in Response to 2,3,7,8-Tetrachlorodibenzo- p-dioxin

Abstract

The induction of cytochrome P450IA1 (CYP1A1) activity in human hepatoma cell lines has been used as a model response for exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). CYP1A1 induction is mediated by the intracellular Ah receptor. We have designed a cell culture analogue system to evaluate the effect of low dose continuous exposures to TCDD such that the responses can be compared with traditional static in vitro exposures. The two-compartment model is designed to mimic aspects of time-dependent dose exposure in humans and consists of a ‘liver’ compartment with HepG2 cells and an ‘other tissues’ compartment. Exposure of microcarrier-attached HepG2 cells in spinner flask to a single dose of TCDD resulted in an EC 50 for ethoxyresorufin- o-deethylase (EROD) activity induction of 1.49 n m. Using a one-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of 0.69 n m TCDD. Finally, using a two-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of about 0.05 n m. Parallel studies using 3H-TCDD were performed to determine the amount of cell associated 3H-TCDD and subsequently to estimate the amount of bound Ah receptor. CYPIA1 mediated EROD activities in both cell lines correlated with the estimated amount of bound Ah receptor. To illustrate how these systems might alter estimates of risk, the data were evaluated to estimate the TCDD level which would increase CYPIA1 mediated EROD activity to 0.01% of the maximal response. The two-compartment reactor data yielded an estimate of allowable TCDD exposure of 1×10 −5 n m for an acceptable ‘risk’ level of 0.01%. In contrast, values were estimated as 4×10 −4 n m from the one compartment reactor and 4×10 −3 n m from spinner flasks. This work demonstrates the importance of design of the in vitro system on risk assessment.