Modulation of estrogen synthesis through activation of protein kinase A in H295R cells by extracts of estuary sediments

Abstract

Sediments from two estuaries within Liaodong Bay, China, were examined for the effects on steroidogenesis using H295R human adrenocortical carcinoma cells. Total extracts (TE) isolated from sediments by Soxhlet extraction were separated into three fractions (F1, F2, and F3) using Florisil columns. After exposing H295R cells to each TE and fractions, the expressions of six steroidogenic genes (cytochrome P450 cholesterol side-chain cleavage [CYP11A], 3β-hydroxysteroid dehydrogenase type 1 [3β-HSD1], 3β-hydroxysteroid dehydrogenase type 2 [3β-HSD2], cytochrome P450 17-hydroxylase/17-20 lyase [CYP17], cytochrome P450 aromatase [CYP19], 17β-hydroxysteroid dehydrogenase [17β-HSD]), and the production of six steroid hormones (progesterone [PGT], 17-hydroxyprogesterone [17-HPT], testosterone [TTR], androstenedione [ADD], estrone [E1], and 17β-estradiol [17β-E2]) were measured. The gene expressions of CYP11A, CYP17, 3β-HSD2, and CYP19, and hormone productions of PGT, 17-HPT, TTR, ADD, E1, and 17β-E2 were significantly increased after exposure to F3 extracts from the Daliao River. In particular, greater concentrations of E1 (up to 48-fold) and 17β-E2 (up to 20-fold), as well as up-regulation of CYP19 gene expression (up to tenfold), were caused by exposure to the F3 fraction from the Daliao River, but not from the Daling River. Insight into the mechanism of action was obtained by use of principal component analysis (PCA), the results of which were consistent with unidentified constituents in F3 from the Daliao River activating the protein kinase A (PKA) pathway. This hypothesis was confirmed by reversal of the effects caused by F3 through a co-exposure of a PKA inhibitor (H89) and F3 extract. The H89 down-regulated CYP19 messenger RNA (mRNA) expression with concomitant lesser production of E1 and 17β-E2 in the co-exposure group, indicating unidentified constituents that could modulate estrogen synthesis, primarily through a mechanism of PKA activation.