Inflammatory stimulation of the liver leads to the induction of nitric oxide (NO) biosynthesis. Because NO binds to the catalytic heme moiety ofcytochromes P450 (CYPs), we investigated whether NO interferes with specific CYPdependent metabolic pathways. In a first experimental approach V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYPlAl and -1A2 were used. Incubation with the NO donors sodium nitroprusside and S-nitrosylacetylpenicillamine led to a concentration-dependent inhibition of all four CYP enzymes. CYPlAl was more sensitive to the inhibitory effect of NO than CYP1A2. In the second part of the study, endogenous NO synthesis was induced in rat hepatocytes by incubation with a mixture of cytokines and endotoxin. Concurrently, as NO production in hepatocytes increased within 24 hr, a decrease in CYPlAl-dependent benzo[a]pyrene turnover was observed to almost undetectable levels. The competitive inhibitor of NO synthesis, NGmonomethyl-L-arginine, was able to significantly restore CYPlAl activity in the presence of cytokines and endotoxin. Inhibition of hepatocellular CYP activity by NO was predominantly due to a direct effect on the enzymes. However, NO-dependent inhibition of CYP expression at a transcriptional level was also demonstrated. Our results indicate that inhibition of NO biosynthesis in patients suffering from systemic inflammatory response syndromes may help to restore biotransformation capacity of the liver.Hepatocellular dysfunction is a detrimental consequence of the metabolic response ofthe liver to prolonged inflammatory stimulation. Characteristic features are suppression of synthetic performance and a profound inhibition of xenobiotic biotransformation (1, 2). Therapy is not available because the molecular basis of these phenomena remains to be elucidated. However, a better understanding of the pathophysiology of the inflamed liver may result from the discovery of L-arginine-dependent nitric oxide (NO) production in hepatocytes and nonparenchymal liver cells (3, 4). As in many other cell types, an inducible NO synthase (iNOS) was identified in hepatocytes upon stimulation with cytokines and endotoxin (5, 6). Induction of iNOS was also observed in the course of parasitic infections, such as malaria (7). Recently, the gene encoding iNOS has been cloned from human hepatocytes in full length for heterologous expression (8).NO exerts a plethora of biologic functions (9). Many of these effects are based on modulation of enzyme activity through binding of NO to prosthetic iron complexes. In this context it is interesting that NO was used for years as a spin-label probe to investigate the role of heme groups in the catalytic centers of cytochrome P450 (CYP) enzymes (10). Consequently, it was demonstrated that NO inhibits CYPdependent reactions when microsomal preparations were exposed to NO (11). CYP enzymes, also referred to as microsomal monooxygenases, catalyze oxidative key reactions in the biotransformation of xenobiotics (12). Therefore, in...

A set of small nonpeptidic diaryl phosphonate inhibitors was prepared. Some of these inhibitors show potent and highly selective irreversible uPA inhibition. The biochemical and modeling data prove that the combination of a benzylguanidine moiety with a diaryl phosphonate ester results in optimized molecules for derivatizing the serine alcohol in the uPA active site. Selected compounds show significant antimetastatic effects in the BN-472 rat mammary carcinoma model. We report in this paper a preclinical proof of concept that selective, irreversible uPA inhibitors could be valuable in antimetastatic therapy.

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