Gene-specific effects of inflammatory cytokines on cytochrome P450 2C, 2B6 and 3A4 mRNA levels in human hepatocytes

Abstract

Cytochromes P450 (P450s) are down-regulated in hepatocytes in response to inflammation and infection. This effect has been extensively studied in animal models, but significantly less is known about responses in humans and even less about responses in the absence of inducing agents. This article focuses on the effects of bacterial lipopolysaccaride (LPS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF), interferon gamma (IFN), transforming growth factor-beta (TGF) and interleukin-1 beta (IL-1) on expression of CYP2B6 and the CYP2C mRNAs in human hepatocytes. These effects were compared with responses of the better studied and more abundant CYP3A4. CYP3A4 and CYP2C8 were down-regulated by all cytokine treatments. CYP2C18, which is expressed at very low levels in liver, was unaffected by cytokine treatments. The other CYP2Cs and CYP2B6 showed cytokine-specific effects. CYP2C9 and CYP2C19 showed almost identical response patterns, being down-regulated by IL-6 and TGF but not significantly affected by LPS, TNF, IFN, or IL-1. CYP2B6 mRNA responded only to IL-6 and IFN. IL-6 down-regulated the mRNAs of all P450s studied. Western blot analysis of P450 protein expression supported the mRNA data to a large extent, although some inconsistencies were observed. Our results show that human CYP2C8, 2C9, 2C18, 2C19, 2B6, and 3A4 responses to inflammation are independently regulated and indicate that this fine control may have a critical effect on human drug responses in disease states.

Fig 1

Effects of cytokines on CYP mRNA expression in human hepatocytes. Cells were treated with phosphate-buffered saline (1 μl/ml, Control), lipopolysaccharide (LPS, 10 μg/ml), interleukin-6 (IL-6, 10ng/ml)), tumor necrosis factor-α (TNF, 10ng/ml)), interferon-γ (IFN, 10ng/ml)), transforming growth factor-β (TGF, 10ng/ml) or interleukin-1 (IL-1, 5ng/ml) for 24 h and mRNA levels of (A) CYP2C8, (B) CYP2C9, (C) CYP2C18, (D) CYP2C19, (E) CYP3A4 and (F) CYP2B6 were determined as described in Methods. All treatments were carried out in triplicate for each individual and the results are the means ± S.E.M. of 9 subjects in each group. Significant differences compared with control are denoted by *=p < 0.05 and **= p < 0.005.

Fig 2

Regulation of human CYP 2C and 3A protein expression by cytokines. Cells from patient HH1294 or HH1315 were cultured in T25 flasks, and treated with phosphate-buffered saline (1 μl/ml, Control), lipopolysaccharide (LPS, 10 μg/ml), interleukin-6 (IL-6, 10ng/ml)), tumor necrosis factor-α (TNF, 10ng/ml)), interferon-γ (IFN, 10ng/ml)), tumor growth factor-β (TGF, 10ng/ml) or interleukin-1 (IL-1, 5ng/ml) for 24 or 40 hr, respectively. Cell lysates were prepared and Western blot assays for (A) CYP2C9 and (B) CYP3A4 were performed as described in Methods. Lanes marked 2C9 and 2C19 were loaded with 1 μg of insect cell microsomes (Supersomes) expressing the respective CYPs. In each section, the upper panels show Western blots of HH1315 (40 h treatment) probed for CYP2C9 or CYP3A4. Blots reprobed with antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are displayed under the respective CYP blots. In the lower panels, relative levels of CYP proteins were quantified, normalized to GAPDH to account for any loading or transfer discrepancies, and plotted as a percentage of the control group mean. Data represent the means ± S.E.M. of three independent samples for each group. Significant differences compared with control are denoted by *=p < 0.05 and **= p < 0.005.

Fig 3

Regulation of human CYP 2B6 protein expression by cytokines. Cells from patient HH1294 or HH1315 were cultured in T25 flasks, and treated with phosphate-buffered saline (1 μl/ml, Control), lipopolysaccharide (LPS, 10 μg/ml), interleukin-6 (IL-6, 10ng/ml)), tumor necrosis factor-α (TNF, 10ng/ml)), interferon-γ (IFN, 10ng/ml)), tumor growth factor-β (TGF, 10ng/ml) or interleukin-1 (IL-1, 5ng/ml) for 24 or 40 hr, respectively. Cell lysates were prepared and Western blot assays for CYP2B6 were performed as described in Methods. Upper panel, Western blots of HH1315 (40 h treatment) probed for CYP2B6. Blots reprobed with antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are displayed under the CYP blots. Lower panel, relative levels of CYP2B6 proteins were quantified, normalized to GAPDH to account for any loading or transfer discrepancies, and plotted as a percentage of the control group mean. Data represent the means ± S.E.M. of three independent samples for each group. Significant differences compared with control are denoted by *=p < 0.05.