Single Cell Analysis of Switch-Like Induction of CYP1A1 in Liver Cell Lines

Abstract

The shape of the dose-response curve may vary depending on whether one examines response at a population or a single cell level. Populations of cells may exhibit a graded response whereas single cell responses may have threshold or switch-like behavior. Studies in vivo and in vitro using primary hepatocyte cultures have shown that induction of CYP1A1 in the liver exhibits switch-like behavior in response to PCB 126 (3,3',4,4',5-pentachlorobiphenyl). The goal of the present study was to determine if two liver cell lines (H4IIE rat hepatoma and Hepa 1c1c7 mouse hepatoma) also show switch-like behavior and develop experimental models for studying mechanisms of these switch-like responses. Both cell lines were analyzed via concentration-response and time-course studies using quantitative real-time PCR, revealing a sigmoidal concentration-response curve for CYP1A1 mRNA induction at the population level. To study CYP1A1 protein induction on a single cell level, flow cytometry was employed. In both cell lines the distribution of fluorescence increased with increasing concentrations of PCB 126. The switch behavior was more pronounced in the H4IIE cells than in the Hepa 1c1c7 cells, exhibiting a well-defined shift of induction from the "off" to the "on" state. The concentration-response curve at the single cell level appeared more switch-like with two populations of cells-basal levels and maximally induced. Immunocytochemistry studies of individual cells also support these conclusions. Our data support the hypothesis that PCB 126 induces CYP1A1 in a switch-like fashion in H4IIE rat hepatoma cells. These cells can now be used to study the mechanism of the biological switch.

Dioxin and Related Compounds

“…These results indicated that CYP1A1 induction was switch-like, and once the threshold was triggered, the induction was graded. Similar results were later reported in H4IIE rat hepatoma cells (Broccardo et al 2004). This phenomenon was described as "a hybrid switching module where a switch works in concert with a rheostat, much like a dimmer on a light in a home" (French et al 2004), or simply as a "dimmer switch" (Wilson 2004).…”

Carcinogenic risks of dioxin: Mechanistic considerations

“…The model therefore suggested that the dose-response for induction of CYP1A1 and CYP1A2 by TCDD is highly nonlinear at low doses. This Wnding is substantiated by recent data demonstrating a switch-like behavior of CYP1A1 induction in response to PCB-126 when determined on a single cell level in cell lines (Broccardo et al, 2004).…”

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a Trichoplusia ni cell line

“…Higher concentrations up to 0.005% (w/v) did give a greater degree of induction but led to very high levels of mortality (Wang and Hobbs 1995). The reason might be that the response of the single cell in the population to insecticide is different at various concentrations (Broccardo et al 2004); some induced at lower concentrations may have died at the higher concentration of insecticide; the detoxifying enzymes (P450s) decreased with increasing insecticide concentration, so their ability to detoxify the cells declined, and more cells then died.…”