Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata

Coprostanol (5 beta (H)-cholestan-3 beta ol) is a reduced metabolite of cho lesterol produced by micro-organisms found in the intestinal tract of mamma ls. This substance abounds in urban effluents and is accumulated by organis ms living in the vicinity of municipal effluent outfalls. In an earlier stu dy, freshwater mussels exposed to contaminated river water for 62 days accu mulated large quantities of coprostanol (Cop) in their soft tissues (16 mug /g dry wt.). Moreover, these mussels were found to have elevated levels of vitellin in their hemolymphs, suggesting estrogenic effects. Although munic ipal wastewaters are known to contain other estrogenic compounds capable of inducing Vn synthesis in mussels, the estrogenic potential of coprostanol was singled out for examination. To this end, mussels were first injected w ith concentrations of coprostanol via the abductor muscle route, and allowe d to stand in aerated water for 72 h at 15 degreesC. The levels of Vn in mu ssel hemolymph were assayed using the organic alkali-labile phosphate metho d. A competitive estradiol-binding assay was then devised to measure the ab ility of coprostanol to compete in the binding of fluorescein-labeled estra diol-albumin to cytosolic proteins. Coprostanol partially reversed the bind ing of labeled estradiol-albumin to cytosolic proteins with an EC50 of 1 mM , In addition, injections of coprostanol and estradiol-17 beta led to incre ased levels of vitellins in the hemolymph of treated mussels. Moreover, inc ubation of cop in gonad homogenate extracts in the presence of NADPH led to the formation of two compounds, as determined by high-performance thin-lay er chromatography. One of these compounds appears to be the C17 oxidation p roduct of coprostanol, whose polarity is similar to that of estradiol. The results present evidence that coprostanol is estrogenic to freshwater musse ls. (C) 2001 Elsevier Science Ltd. All rights reserved.