Regulation of 3β-hydroxysteroid dehydrogenase and sulphotransferase 2A1 gene expression in primary porcine hepatocytes by selected sex-steroids and plant secondary metabolites from chicory (Cichorium intybus L.) and wormwood (Artemisia sp.)

Abstract

In pigs the endogenously produced compound androstenone is metabolised in the liver in two steps by 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulphotransferase 2A1 (SULT2A1). The present study investigated the effect of selected sex-steroids (0.01-1 μM androstenone, testosterone and estradiol), skatole (1-100 μM) and secondary plant metabolites (1-100 μM) on the expression of 3β-HSD and SULT2A1 mRNA. Additionally the effect of a global methanolic extract of dried chicory root was investigated and compared to previous obtained in vivo effects. Primary hepatocytes were isolated from the livers of piglets (crossbreed: Landrace×Yorkshire and Duroc) and cultured for 24h before treatment for an additionally 24h. RNA was isolated from the hepatocytes and specific gene expression determined by RT-PCR using TaqMan probes. The investigated sex-steroids had no effect on the mRNA expression of 3β-HSD and SULT2A1, while skatole decreased the content of SULT2A1 30% compared to control. Of the investigated secondary plant metabolites artemisinin and scoparone (found in Artemisia sp.) lowered the content of SULT2A1 by 20 and 30% compared to control, respectively. Moreover, we tested three secondary plant metabolites (lactucin, esculetin and esculin) found in chicory root. Lactucin increased the mRNA content of both 3β-HSD and SULT2A1 by 200% compared to control. An extract of chicory root was shown to decrease the expression of both 3β-HSD and SULT2A1. It is concluded that the gene expression of enzymes with importance for androstenone metabolism is regulated by secondary plant metabolites in a complex manner.

Keywords: 3β Hydroxysteroid dehydrogenase; 3β-HSD; 4-(2-Hydroxyethyl)piperazine-1-ethanesulphonic acid; ANOVA; AhR; BSA; CAR; Ct; DHEA; DMSO; DPBS; Dulbecco's phosphate buffered saline; EGTA; FCS; GAPDH; Gene regulation; HEPES; PCR; PXR; Primary porcine hepatocytes; RT-PCR; SEM; SQL; SULT2A1; Secondary plant metabolites; Sex-steroids; WME; Williams E medium; XR; analysis of variance; aryl hydrocarbon receptor; bovine serum albumin; cDNA; complementary DNA; constitutive androstane receptor; dehydroepiandrosterone; dimethyl sulphoxide; ethylene glycol tetraacetic acid; foetal calf serum; glyceraldehyde 3-phosphate dehydrogenase; mRNA; messenger RNA; pregnane X receptor; reverse transcriptase polymerase chain reaction; sesquiterpene lactone; standard error of the mean; sulphotransferase 2A1; threshold cycle; xenobiotic receptor.