Research report
Modulation of cytochrome P450 by inflammation in astrocytes

https://doi.org/10.1016/S0006-8993(99)01261-5Get rights and content

Abstract

Activation of systemic host defense mechanisms results in the down-regulation of cytochrome P450 enzymes in the liver. This occurs for various induced and constitutive isoforms of cytochrome P450 in response to cytokines such as IFNs, IL-1, IL-6, and TNF-α, which are produced during infection. Although the levels of cytochrome P450 in brain regions are low, the enzymes are regionally distributed and may play a critical role in the activation or degradation of drugs and chemicals in localized areas. If activation of the immune response in the CNS by LPS modulates the activity of cytochrome P450 forms in the brain, this may alter normal metabolic pathways or contribute to drug or chemical toxicity. This hypothesis was addressed by examining the effect of LPS on a major cytochrome P450 form in isolated astrocytes obtained from newborn rats. These cells were shown to express CYP1A1/2 when induced by dibenz[a,h]anthracene (DBA) as determined by enzyme activity, immunohistochemistry, and Western blotting. The treatment of these cells with LPS significantly attenuated the activity of these enzymes but had no effect on CYP1A1/2 protein levels as determined by Western blotting. The lack of effect by detoxified LPS indicated the requirement of the lipid A region on LPS to stimulate this response. Pentoxifylline (PNTX) prevented the LPS evoked decrease in CYP1A1/2 activity suggesting that cytokine release was a required component of this effect in astrocytes. These results indicate that stimulation of the immune response by LPS in isolated astrocytes decreases CYP1A1/2 activity. The release of cytokines is implicated in this effect and thought to participate in the functional inhibition of the enzyme as no effect on CYP1A1/2 protein levels was observed.

Introduction

Cytochrome P450 is a heme-containing superfamily of enzymes that metabolize a broad spectrum of endogenous and exogenous compounds [12]. Although primarily found in the liver, cytochrome P450 enzymes are expressed in many different tissues such as the lungs, kidney, skin, adrenal cortex, and nasal epithelium [21]. In recent years, low levels of cytochrome P450 enzymes have been detected in brain tissue where the various isoforms are expressed in discrete regions of the brain 14, 21, 26, 27. These enzymes are likely to be involved in diverse CNS function including neurosteroid synthesis, metabolism of drugs, and the protection of brain tissue from toxins 2, 16.

Infection and inflammation can modulate levels of cytochrome P450 enzymes in the liver in vivo and in vitro leading to alterations in drug metabolism 6, 20, 24, 25. In the systemic system, the immune response to either infection or inflammation is characterized by the release of cytokines that is necessary for the observed effects on cytochrome P450 enzymes 11, 22. Administration of an immunogenic substance such as lipopolysaccharride (LPS) induces an inflammatory response in animals through the activation of macrophages and the subsequent release of cytokines [22]. LPS administration has been shown to modulate cytochrome P450 enzymes in the liver both in vivo and in vitro, an effect that relies on the production of cytokines from stimulated macrophages 11, 22.

Whether a similar phenomenon can occur with the cytochrome P450 enzymes in the brain is still under active investigation. In comparison to the systemic system, the immune response to a similar stimulus in brain is much slower and less intense. Microglia, the resident macrophages of the brain become activated, macrophage recruitment from the systemic system occurs after a 48-h delay, and the recruitment of neutrophils is low or absent [3]. Some preliminary reports have shown that stimulation of an immune response or ischemic injury in the CNS can alter levels of cytochrome P450 enzyme expression 30, 33. These results may be critically important in many diseases of the brain such as Parkinson's disease, Alzheimer's disease and multiple Sclerosis which are thought to involve an inflammatory component [13].

In this report, we demonstrate that the enzymatic activity of cytochrome P450 isoforms from the CYP1A family (CYP1A1 and 1A2) are modulated by LPS evoked inflammation in isolated astrocytes. Evidence is provided that cytokines participate in this response resulting in a functional type of inhibition of the enzyme.

Section snippets

Reagents

Dibenz[a,h]anthracene, Escherichia coli lipopolysaccharide (regular and detoxified; serotype: 0127:B8), pentoxifylline (PNTX), ethoxyresorufin, anti-goat IgG conjugated to FITC, anti-mouse conjugated to TRITC, and anti-goat IgG POD conjugate were purchased from Sigma-Aldrich Chemical (St. Louis, MO). The monoclonal anti-rat CYP1A1 antibody was purchased from Gentest (Woburn, MA) and known to cross-react with CYP1A2. Anti-rat GFAP monoclonal antibody was obtained from Boehringer-Mannheim (Laval,

Astrocyte cultures

Immunohistochemical studies using specific antibodies were carried out to confirm that isolated cells were astrocytes, demonstrate the absence of neurons, and to demonstrate that isolated astrocytes expressed CYP1A1/2 protein. Immunoreactivity to an antibody directed against GFAP, a specific astrocyte marker, indicated that the majority of isolated cells were astrocytes (Fig. 1A,C). When cells were probed with an antibody against neurofilament 160 no staining was observed demonstrating the lack

Discussion

DBA induced the cytochrome P450 forms CYP1A1 and 1A2 in cultured astrocytes as demonstrated by the immunohistochemical studies, an enzymatic activity assay (EROD), and Western blot analysis of protein levels. This treatment provided sufficient levels of enzyme to determine if LPS could modulate the activity of these isoforms. The expression of CYP1A1/2 have been shown to be distributed in discreet areas of the brain including the striatum, hypothalamus, globus pallidus (CYP1A1), and olfactory

Acknowledgements

This work was supported by a grant from the Medical Research Council of Canada.

References (33)

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