Characterization of the rates of testosterone metabolism to various products and of glutathione transferase and sulfotransferase activities in rat intestine and comparison to the corresponding hepatic and renal drug-metabolizing enzymes

Abstract

Metabolism of testosterone to various products (catalyzed by several different CYP isozymes) and the activities of phenol sulfotransferase (pST) and glutathione transferase (GST) in S9 fractions prepared from the mucosa of the duodenum, jejunum, ileum, caecum and upper and lower colon of male Sprague–Dawley rats were determined and compared to the corresponding hepatic and renal activities.

Incubation of the S9 fraction prepared from the jejunum with testosterone and NADPH resulted in the formation of 2α-, 6α-, 6β- and 16α-hydroxytestosterone and androstenedione at rates that were 1.6, 24, 1.3, 0.6 and 1.3%, respectively, of the corresponding hepatic values. The production of 2α-hydroxytestosterone was catalyzed only by the preparations from the duodenum and jejunum; whereas 6α-, 6β- and 16α-hydroxytestosterone and androstenedione were produced in all regions of the intestine. In the case of the rat kidney, the rates of formation of the different testosterone metabolites were between 0.6 and 35% of the corresponding liver activity.

The activity of glutathione transferase was approximately 12–26% of the corresponding hepatic activity throughout the intestine. The highest activity of phenol sulfotransferase was observed in the lower colon (almost 6% of the liver activity) and the lowest activity in the duodenum (1%). The renal activities of GST and pST were 70 and 1%, respectively, of the corresponding liver values.

In summary, the metabolism of testosterone and the activities of GST and pST in rat intestine are generally low to very low in comparison to the corresponding activities in rat liver. In most cases, these activities are present throughout the entire intestine and not restricted to a particular portion(s) of this organ.

Introduction

Drug-metabolizing enzymes are expressed at high levels in the liver and metabolism of xenobiotics in this organ has been characterized extensively. However, extrahepatic tissues may also play an important role in drug metabolism. For instance, the gastric mucosa has first contact with orally ingested xenobiotics and cytochrome P450 (CYP) isozymes in enterocytes exhibit a substantial capacity for drug metabolism [1], [2].

Many of the metabolites produced by the CYP system are electrophilic, highly reactive and thereby toxic to the cell. These products can both be deactivated and rendered more hydrophilic for excretion by phase II conjugating enzymes. Consequently, characterization of phase I and II drug-metabolizing systems in extrahepatic tissues is essential for understanding tissue-specific toxic and genotoxic effects of xenobiotics.

In the case of extrahepatic tissues, xenobiotic-metabolizing enzymes are often quantitated at the protein level or, in tissues with low expression of these proteins, by determination of mRNA employing the polymerase chain reaction. However, not all protein molecules thus detected are enzymatically active nor is the level of mRNA proportional to the amount of active enzyme present. Thus, direct measurement of enzyme activities is desirable whenever possible.

The pattern of testosterone hydroxylation is commonly used to characterize phase I metabolism by different forms of cytochrome P450 in the rat liver [3] (Table 1). The phase II conjugating enzyme sulfotransferase is also present in several isoforms and catalyzes the sulfation of xenobiotics, certain endogenous compounds and/or their metabolites. Phenol sulfotransferase (pST) activity is often assayed employing 1-naphthol as substrate [4], [5]. By conjugating electrophilic compounds with glutathione, the glutathione transferases (GST) generally render these electrophiles considerably less harmful and more hydrophilic. GST activity is routinely measured utilizing 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate [6].

Many investigators have demonstrated the presence of the cytochrome P450 system, as well as of phase II enzymes in the mammalian intestine. However, these studies are often restricted to a particular portion of the intestine and thus do not reveal the distribution of xenobiotic metabolism throughout this entire organ. Our aim here was to determine the activities of testosterone hydroxylases, phenol sulfotransferase and glutathione transferase in all different regions of the rat intestine and to then compare these activities with the corresponding values for the rat liver and kidney, which have been more extensively characterized.