Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase

Abstract

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1–10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.

Introduction

Cytochrome P450 superfamily monooxygenases are important for the biotransformation of drugs and toxins. Numerous studies indicate that inflammation and infection suppress hepatic CYP levels [1] in various species including human, rat, and mouse. LPS has been used extensively as a model of sepsis to study the inhibitory effects of inflammation and infection on CYP activity and expression [2], [3], [4], [5].

During inflammatory responses such as that produced by LPS, cellular levels of nitric oxide increase greatly in many cell types due to the induction of inducible NOS (iNOS, NOS2) expression [6]. Several studies have suggested that the NO produced consequent to hepatic NOS2 induction mediates the suppression of hepatic CYP expression and activity caused by LPS treatment. In vitro treatments of rat liver microsomes or purified CYPs with NO, peroxynitrite, or donors of RNS inhibited the catalytic activities of CYP enzymes, a result hypothesized to be caused by reaction of the NO or derivative RNS with heme or amino acid residues on CYPs [7], [8], [9]. Inhibitors of NOS, such as N-methyl-l-arginine, N-nitro-l-arginine methyl ester, or aminoguanidine partially reverse the suppression of CYP mRNAs and proteins in primary rat hepatocytes or whole animals caused by LPS or cytokine treatment [10], [11], [12], [13].

Contrary evidence exists that NO is not required for the LPS-induced suppression of some CYPs. We showed that the suppression of several constitutively expressed CYPs caused by LPS treatment is NO-independent [14], [15]. Aminoguanidine inhibits the production of NO, but does not prevent the reduced hepatic expression of CYP2C11, 2E1, and 3A2 caused by LPS treatment in rats [15]. Similarly, NOS inhibitors failed to prevent the down-regulation of CYP2C11 mRNA or protein by LPS or interleukin-1β in cultured rat hepatocytes [14]. We also reported that LPS treatment causes equivalent suppression of CYP2C, 3A, and 2E1 mRNA and protein expression in both NOS2-null mice and wild-type mice, supporting the hypothesis that NO was not required for the LPS-induced suppression of these CYPs [16].

CYP2B1 and 2B2 are major PB-inducible CYPs in rat liver. Carlson and Billings [17] found that NOS inhibitors block the suppression of CYP2B proteins by a cytokine mixture in cultured rat primary hepatocytes, while an NO donor reduces CYP2B1/2 apoprotein levels, suggesting the involvement of NO in the suppression of CYP2B proteins. Khatsenko et al. [18] then reported that LPS treatment of rats inhibits the PB-induced expression of CYP2B1/2 mRNAs and proteins. They also concluded that NO participates in this effect of LPS, because it was attenuated by administration of an NOS inhibitor [18]. Further evidence that NO and RNS may play an important role in the regulation of CYP2B activity was provided by Roberts et al. [9], who found that the incubation of purified CYP2B1 protein with peroxynitrite resulted in the nitration of tyrosine residues on CYP2B1, which correlated with the loss of its enzymatic activity.

Recently, we found that the suppression of PB-inducible CYP2B1 expression by LPS treatment in rat hepatocytes occurs by dual mechanisms. Low concentrations of LPS cause an NO-independent suppression of CYP2B1 mRNA that leads to a slow down-regulation of the protein between 24 and 48 hr later [19]. At higher concentrations of LPS (10⁻⁷ g/mL and above), there is a rapid, NO-dependent suppression of CYP2B proteins that occurs within 6 hr, before there is a significant effect on the CYP2B1 mRNA [19].

The above studies showing that NO production is required for CYP2B mRNA and/or protein down-regulation by inflammatory stimuli relied on the use of NOS inhibitors to block NO production. Due to limitations of specificity inherent in the use of any drug, NOS inhibitors could potentially affect CYP expression in ways other than by inhibiting cellular NO production. The availability of NOS2-null mice allows the dissection of NOS2-dependent pathways in the absence of chemical inhibitors, and therefore we used this model to further investigate the role of NO in the in vivo regulation of CYP2B mRNAs and proteins by LPS. We report that the down-regulation of PB-induced CYP2B enzymes in mouse liver following LPS treatment occurs by a pretranslational mechanism in the physiologically relevant dose range, with no evidence for a rapid suppression of the CYP2B proteins. The effects were the same in wild-type and null animals, indicating that CYP2B suppression in mouse liver is not dependent upon NO production by NOS2.