Effect of 11-Deoxycorticosterone in the Transcriptomic Response to Stress in Rainbow Trout Skeletal Muscle.

In aquaculture, many stressors can negatively affect growth in teleosts. It is believed that cortisol performs glucocorticoid and mineralocorticoid functions because teleosts do not synthesize aldosterone. However, recent data suggest that 11-deoxycorticosterone (DOC) released during stress events may be relevant to modulate the compensatory response. To understand how DOC modifies the skeletal muscle molecular response, we carried out a transcriptomic analysis. Rainbow trout (Oncorhynchus mykiss) were intraperitoneally treated with physiological doses of DOC in individuals pretreated with mifepristone (glucocorticoid receptor antagonist) or eplerenone (mineralocorticoid receptor antagonist). RNA was extracted from the skeletal muscles, and cDNA libraries were constructed from vehicle, DOC, mifepristone, mifepristone plus DOC, eplerenone, and eplerenone plus DOC groups. The RNA-seq analysis revealed 131 differentially expressed transcripts (DETs) induced by DOC with respect to the vehicle group, mainly associated with muscle contraction, sarcomere organization, and cell adhesion. In addition, a DOC versus mifepristone plus DOC analysis revealed 122 DETs related to muscle contraction, sarcomere organization, and skeletal muscle cell differentiation. In a DOC versus eplerenone plus DOC analysis, 133 DETs were associated with autophagosome assembly, circadian regulation of gene expression, and regulation of transcription from RNA pol II promoter. These analyses indicate that DOC has a relevant function in the stress response of skeletal muscles, whose action is differentially modulated by GR and MR and is complementary to cortisol.

The relative role of CYP3A4 and CYP3A5 in eplerenone metabolism.

Cytochrome P450 mediated metabolism is the rate-limiting step of elimination for many drugs. CYP3A4 is the most abundant hepatic isoform and CYP3A4/5 metabolize the largest fraction of drugs. Pharmacogenetic studies have not been able to characterize population variability in CYP3A4 activity because few variant alleles associated with aberrant enzyme activity have been found. Substrate probes such as midazolam and testosterone have been utilized in-vivo and in-vitro to determine catalytic activity of these enzymes, but they suffer from several limitations. Eplerenone, an aldosterone antagonist, is also metabolized by CYP3A enzymes, and it has the potential to be an excellent substrate probe for CYP3A4/5. Eplerenone's primary metabolite, 6 beta-hydroxyeplerenone is formed preferentially via CYP3A4, however, the relative contribution of CYP3A5 to the 21-hydroxyeplerenone metabolite formation is unknown. Through in-vitro microsomal incubations with recombinant CYP3A4 and CYP3A5 enzymes, we identified their relative contributions to 21-hydroxyeplerenone metabolism. The 21-hydroxy metabolite is formed preferentially via CYP3A5 Vmax/KM (3.3) versus CYP3A4 Vmax/KM (1.9). Based on these findings, eplerenone has the potential to serve as an in-vivo substrate probe for CYP3A4 by monitoring 6-beta-hydroxy metabolite formation as well as CYP3A4/5 by monitoring 21-hydroxy metabolite formation.