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Suppression of flavin-containing monooxygenase by overproduced nitric oxide in rat liver
C S Park1, H M Baek, W G Chung
1Department of Pharmacology and Toxicology, Medicinal Toxicology Research Center, College of Medicine, Inha University, Inchon, Korea.
Abstract:
Effects of excessive nitric oxide (NO) produced in vivo by an i.p. injection of bacterial lipopolysaccharide (LPS) on hepatic microsomal drug oxidation catalyzed by flavin-containing monooxygenase (FMO) were determined. At 6 and 24 h after the LPS injection, liver microsomes were isolated and FMO activities were determined by using FMO substrates like thiobenzamide, trimethylamine, N,N-dimethylaniline, and imipramine. Liver microsomal FMO activities of LPS-treated rats were decreased significantly for all these substrates. Microsomal content of FMO1 (the major form in rat liver) in LPS-treated rats as determined by immunoblotting, was severely decreased as well. In support of this, hepatic content of FMO1 mRNA was decreased by 43.6 to 67.3%. However, the hepatic content of inducible NO synthase (iNOS) mRNA was increased by 2.6- to 5.4-fold and the plasma nitrite/nitrate concentration was increased by about 30-fold in the LPS-treated rats. When this overproduction of NO in the LPS-treated rats was inhibited in vivo by a single or repeat doses of either a general NOS inhibitor N(G)-nitro-L-arginine or a specific iNOS inhibitor aminoguanidine, the FMO1 mRNA levels were not severely depressed (70-85% of the control level). Attendant with the reduction of plasma nitrite/nitrate concentration by single and repeated doses of NOS inhibitors, activity and content of FMO1 in liver microsomes isolated from these NOS inhibitor cotreated rats were restored partially (in single-dose inhibitors) or completely (in repeat doses). In contrast to these NO-mediated in vivo suppressive effects on the mRNA and enzyme contents of FMO1 as well as the FMO activity, the NO generated in vitro from sodium nitroprusside did not inhibit the FMO activities present in microsomes of rat and rabbit liver as well as those present in rabbit kidney and lung. Combined, the excessive NO produced in vivo (caused by the LPS-dependent induction of iNOS) suppresses the FMO1 mRNA and enzyme contents as well as the FMO activities without any direct in vitro effect on the activities of premade FMO enzyme. These findings suggest that NO is an important mediator involved in the suppression of FMO1 activity in vivo. Thus, together with the previously reported suppression on the cytochrome P-450 activities, the overproduced NO in the liver caused by induction of iNOS under conditions of endotoxemia or sepsis suppresses FMO and appears to be responsible for the decreased drug oxidation function observed generally under conditions of systemic bacterial or viral infections.
Insights
Excessive nitric oxide (NO) production in vivo suppresses flavin-containing monooxygenase 1 (FMO1) activity and levels by decreasing FMO1 mRNA. Inhibiting NO production restores FMO1 function, indicating NO mediates this suppression.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Bacterial lipopolysaccharide (LPS) induces excessive nitric oxide (NO) production in vivo.
- Hepatic microsomal drug oxidation is catalyzed by flavin-containing monooxygenase (FMO).
- The role of LPS-induced NO in modulating FMO activity is not fully understood.
Purpose of the Study:
- To determine the effects of excessive in vivo NO production on hepatic microsomal FMO activity.
- To investigate the impact of LPS-induced NO on FMO1 mRNA and protein levels.
- To elucidate the mechanism by which NO influences FMO activity.
Main Methods:
- Rats were injected with LPS to induce NO production.
- Liver microsomes were isolated at 6 and 24 hours post-injection.
- FMO activities were measured using specific substrates.
- FMO1 mRNA and protein levels were quantified by RT-PCR and immunoblotting, respectively.
- Nitric oxide synthase (NOS) inhibitors were used to block NO production.
Main Results:
- LPS injection significantly decreased hepatic microsomal FMO activities for multiple substrates.
- FMO1 mRNA and protein levels were significantly reduced in LPS-treated rats.
- LPS treatment increased hepatic inducible NO synthase (iNOS) mRNA and plasma nitrite/nitrate levels.
- Inhibition of NO production with NOS inhibitors partially or completely restored FMO1 mRNA, protein, and activity.
- In vitro NO did not directly inhibit FMO activity, suggesting an indirect in vivo mechanism.
Conclusions:
- Excessive in vivo NO production, induced by LPS, suppresses FMO1 mRNA and enzyme content, leading to decreased FMO activity.
- NO acts as a mediator in the suppression of FMO1 activity in vivo.
- These findings suggest that NO-induced suppression of FMO and cytochrome P-450 contributes to impaired drug oxidation during infections like sepsis.