Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes

Abstract

The testicular steroids androstenone (A), 17β-oestradiol (E2) and testosterone (T) were tested for their ability to alter CYP2E1 and CYP2A activity in porcine liver microsomes from male and female pigs. This is the first in vitro study indicating that sex steroids have a potential to modify microsomal CYP2E1 activity, the main skatole-metabolising enzyme. A and E2 exerted an inhibitory effect on CYP2E1 mediated hydroxylation of p-nitrophenol to p-nitrocatechol although the mechanism of this inhibition differed for these steroids. The inhibitory effect of A on CYP2E1, as determined by kinetic analysis, might be due to the competitive binding of A and p-nitrophenol to the same site of CYP2E1. Including E2 into the incubations resulted in decreased activities of CYP2E1 in male microsomes through a mixed mode of inhibition. Including pre-incubation steps eliminated this inhibition in male microsomes, and resulted in increased CYP2E1 activities in the microsomes from female pigs. Testosterone was ineffective as an inhibitor of either CYP2E1 or CYP2A activities. Overall, our findings indicate that A and E2 have the potential to modify the catalytic activities of porcine CYP2E1 in vitro. However, the significance of this modification for skatole metabolism in vivo is questionable.

Introduction

Skatole (3-methylindole) is a major contributor to a faecal-like odour in meat from entire male pigs. Skatole is produced in the large intestine of the pig and biotransformed in the liver via a two step process, phase I and phase II. The phase I step is responsible for the activation of the parent compound which typically introduces a hydroxyl group. This newly formed group then allows phase II enzymes to conjugate a variety of substrates. In general, the significance of this metabolism is to convert small lipophilic molecules into larger more water soluble compounds that are readily excreted. For skatole, conversion into more polar compounds is beneficial for its elimination. The majority of phase I skatole metabolism occurs through the cytochrome P450 system, a superfamily of heme-containing isoenzymes located within the endoplasmic reticulum in hepatocytes. Currently, cytochrome P450 (CYP) isoforms CYP2E1 and CYP2A are thought to be primarily responsible for the oxidative metabolism of skatole (Babol et al., 1998, Diaz and Squires, 2000a, Diaz and Squires, 2000b). Low activities of these enzymes in the liver may lead to decreased skatole metabolism and higher skatole accumulation in adipose tissue (Diaz and Squires, 2000b, Zamaratskaia et al., 2005a).

High skatole concentrations occur mainly in entire (un-castrated) male pigs, and not in either castrated males or female pigs. The mechanism responsible for the sex-related differences in skatole concentrations is not yet completely understood; however, it appears that liver metabolism extensively affects skatole accumulation in fat (Diaz and Squires, 2000b, Zamaratskaia et al., 2006). Entire male pigs expressed lower levels of hepatic CYP2E1 compared to that of castrates (Whittington et al., 2004), and had lower CYP2E1 activity compared to that of female pigs (Zamaratskaia et al., 2006). Interestingly, the differences in CYP2E1 activity between entire male and female pigs were observed only in mature pigs at 115 kg live weight, whereas the activities at 90 kg (pre-puberty) did not differ between the genders (Zamaratskaia et al., 2006).

Decreased metabolism and hepatic clearance of skatole in mature entire male pigs can be due to the presence of testicular steroids at puberty. In contrast to males of other species, entire male pigs produce high amounts of oestrogens (Claus and Hoffmann, 1980, Raeside and Renaud, 1983), which are positively correlated to skatole levels (Zamaratskaia et al., 2005b, Zamaratskaia et al., 2005c). However, the mechanism by which high levels of oestrogens might affect skatole accumulation remains unknown.

The hepatic metabolism of testicular steroids, similar to skatole, is dependent on cytochrome P450s. Inhibition or decreased metabolism of skatole can result from competition between skatole and steroids for the enzyme’s binding sites, modification of the enzyme by a reactive metabolite, or formation of a catalytically inactive complex between the enzyme and a steroid.

Androstenone, another boar taint compound, was shown to be involved in skatole metabolism either by direct enzyme inhibition (Babol et al., 1999) or by the inhibition of skatole-induced CYP2E1 expression (Doran et al., 2002, Whittington et al., 2004). Recently, Tambyrajah et al. (2004) also showed that androstenone decreases CYP2E1 promoter activity by inhibiting the binding of a transcription factor. Additionally, there is evidence that CYP2A is also strongly regulated by testicular steroids at the transcriptional level (Gillberg et al., 2006).

The aim of the present study was to further investigate the role of testicular steroids in hepatic skatole metabolism. For this purpose, the possibility of inhibition of CYP2E1 and CYP2A activities by androstenone (A), 17β-oestradiol (E2) and testosterone (T) was investigated by microsomal studies.