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Cytokine-mediated down-regulation of CYP1A1 in Hepa1 cells
1Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
The activation of host defense mechanisms down-regulates microsomal cytochrome P450 in cell culture, humans, and animals. Investigation into various aspects of this effect using in vivo models has yet to define clearly the role that cytokines play in this phenomenon. The mechanism of down-regulation by immunostimulants, such as lipopolysaccharide (LPS), is explored with an in vitro model, utilizing a murine hepatoma (Hepa1) and a murine macrophage (IC-21) cell line. It is hypothesized that down-regulation of P450 activity by immunostimulants involves the activation of immune cells and the subsequent release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). The effects of immunostimulation on P450 activity are assessed by ethoxyresorufin O-dealkylase, an assay that measures CYP1A activity in Hepa1 cells. Initial studies demonstrated that LPS added directly to hepatoma cells had no effect on the levels of CYP1A1 activity. In contrast, a significant down-regulation in CYP1A1 activity occurred when hepatoma cells were incubated with monocyte conditioned medium obtained by incubating LPS with IC-21 cells. When pentoxifylline, a TNF-alpha synthesis inhibitor, was co-administered with LPS to macrophages, the down-regulation of CYP1A1 activity was prevented. The direct administration of murine recombinant TNF-alpha to hepatoma cells resulted in a down-regulation of CYP1A1 activity. These results implicated the release of TNF-alpha from macrophages as an important step in the down-regulation of CYP1A1 by LPS.
Insights
Host defense activation suppresses cytochrome P450. This study shows that tumor necrosis factor-alpha (TNF-alpha) released by macrophages mediates this suppression by lipopolysaccharide (LPS).
Area of Science:
- Pharmacology
- Immunology
- Biochemistry
Background:
- Host defense activation is known to reduce microsomal cytochrome P450 (CYP450) levels.
- The precise role of cytokines in this down-regulation remains unclear.
- Immunostimulants like lipopolysaccharide (LPS) trigger this effect, but the underlying mechanisms require further investigation.
Purpose of the Study:
- To elucidate the mechanism by which immunostimulants down-regulate CYP450 activity.
- To investigate the role of cytokines, specifically interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), in this process.
- To determine if TNF-alpha release from macrophages mediates LPS-induced CYP450 down-regulation.
Main Methods:
- Utilized an in vitro model with murine hepatoma (Hepa1) and macrophage (IC-21) cell lines.
- Assessed CYP1A activity using ethoxyresorufin O-dealkylase assay.
- Investigated the effects of LPS, macrophage-conditioned medium, pentoxifylline (a TNF-alpha inhibitor), and recombinant TNF-alpha on CYP1A1 activity.
Main Results:
- LPS alone did not affect CYP1A1 activity in hepatoma cells.
- Incubation of hepatoma cells with LPS-stimulated macrophage-conditioned medium significantly down-regulated CYP1A1 activity.
- Inhibition of TNF-alpha synthesis in macrophages prevented CYP1A1 down-regulation, and direct administration of TNF-alpha to hepatoma cells mimicked the effect.
Conclusions:
- The release of TNF-alpha from activated macrophages is a key mediator in the down-regulation of CYP1A1 activity by LPS.
- This study highlights the interplay between the immune system and drug metabolism pathways.
- Findings suggest a cytokine-driven mechanism for immune-mediated suppression of CYP450 enzymes.