Aromatisation of steroids in the bivalve Mytilus trossulus

Abstract

In this study, we demonstrated the presence of the enzymatic complex able to perform aromatization (estrogen synthesis) in both, the microsomal and mitochondrial fractions of gills and gonads from Mytilus trossulus. Based on in vitro experiments, we highlighted the importance of temperature as the limiting factor of aromatisation efficiency (AE) in mussels. After testing range of temperatures (4-23 °C), the highest AE was found during incubation at 8 °C and pH 7.6 (41.66 pmol/h/mg protein in gills and 58.37 pmol/h/mg protein in gonads). The results were confirmed during field studies where the most efficient aromatisation occurred in bivalves collected in spring while the least effective in those collected in winter. During in vitro studies, AE turned out to be more intensive in female gonads than in male gonads. The process was also more intensive in mitochondrial fraction than in microsomal one (62.97 pmol/h/mg protein in male gills and 73.94 pmol/h/mg protein in female gonads). Enzymatic complex (aromatase-like enzyme) catalysing aromatisation in mussels was found to be insensitive to inhibitory effect of selective inhibitors of mammalian aromatase such as letrozole and anastrazole, suggesting its different structure from vertebrate aromatase. Further in vivo studies using ¹³C-labeled steroids at 8 °C temperature window confirmed that bivalves are able to uptake testosterone and androstenedione from the ambient environment and metabolise them to estrone and 17β-estradiol thus confirming endogenous estrogen' synthesis.

Keywords: Aromatase-like enzyme; Aromatisation of steroids; Bivalves; Estrogen syntheis; Temperature.

Figure 1. Identification of ¹³C₃-labeled steroids by LC-MS/MS.

Positive ion chromatogram of steroid standards (A) was compared to chromatogram obtained from extract of M. trossulus exposed to Testosterone-2,3,4-¹³C₃ over 24 h (B). Abbreviations: D, Dexamethasone (internal standard); A-¹³C₃, 4-Androstene-3,17-dione-2,3,4-¹³C₃; T-¹³C₃, Testosterone-2,3,4-¹³C₃; E1-¹³C₃, Estrone-2,3,4-¹³C₃; 17βE2-¹³C₃, 17β-estradiol-2,3,4-¹³C₃.

Figure 2. Aromatization efficiency in microsomal fraction isolated from gills (black column) and gonads (gray column) of M. trossulus (A) temperature dependent efficiency, (B) pH dependent efficiency.

Data presented as mean ± SD (n = 5).

Figure 3. Seasonal differences in AE in microsomes isolated from gills (black column) and gonads (gray column) of M. trossulus.

Data presented as mean ± SD (n = 3). Significant differences with respect to the “May 2012” groups indicated by *^,#p < 0.05 (Kruskal–Wallis ANOVA).

Figure 4. Sex-related aromatization efficiency in microsomes and mitochondria isolated from gills (black column) and gonads (gray column) of M. trossulus.

Data presented as mean ± SD (n = 3); *p < 0.05 compared “gill” and “gonads” groups (Kruskal–Wallis ANOVA), ^#p < 0.05 compared “female” and “males” groups (Kruskal–Wallis ANOVA) and ^&p < 0.05 compared “microsomes” and “mitochondria” groups (Kruskal–Wallis ANOVA).

Figure 5. Effect of inhibitors on aromatization efficiency in microsomes isolated from gonads of M. trossulus.

The effect of letrozole is presented in black column, anastrozole in gray column and ketoconazole in white column. Data presented AE in relation to control (black line marks: AE without inhibitors = 39.04 ± 7.34 pmol/h/mg of protein). Data presented as mean ± SD (n = 3). Significant differences indicated by *p < 0.05 (Kruskal–Wallis ANOVA).

Figure 6. Levels of A-¹³C₃, 4-Androstene-3,17-dione-2,3,4-¹³C₃; T-¹³C₃, Testosterone-2,3,4-¹³C₃; E1-¹³C₃.

E1-¹³C₃, Estrone-2,3,4-¹³C₃ and 17βE2-¹³C₃, 17β-estradiol-2,3,4-¹³C₃ in M. trossulus exposed to 4-Androstene-3,17-dione-2,3,4-¹³C₃ (black column) and Testosterone-2,3,4-¹³C₃ (gray column). Data presented as mean ± SD (n = 10).