BIOSENSOR-BASED METHOD FOR THE QUANTIFICATION OF VITELLOGENIN PRODUCED BY GRAY MULLET (MUGIL CEPHALUS) HEPATOCYTES EXPOSED TO ESTROGEN-LIKE COMPOUNDS

Vitellogenin (Vtg) has proven to be a sensitive biomarker for assessing the estrogenic potency of chemicals and the exposure of fish to estrogen-like compounds present in aquatic environments. In this regard, the gray mullet (Mugil cephalus) can be considered a key species for monitoring xenoestrogens due to its benthic feeding behavior, to its wide geographical distribution and also to its commercial value. The aim of this work was to develop an in vitro assay for measuring Vtg induction using cultured primary hepatocytes of gray mullet. Vtg production was measured by a rapid detection method based on the surface plasmon resonance (SPR) technique. Anti-Vtg was surface-blocked onto an IAsys plus optical biosensor and sensor optimisation was carried out using gray mullet Vtg at different concentrations. Limits of detection (LOD) and quantification (LOQ) of the proposed biosensor for Vtg were 0.2 and 0.7 nM, respectively. The antigenantibody complex was characterized by extremely high affinity (KD = 1.07 ± 0.14 nM), with a significant contribution of both association (kass) and dissociation kinetic parameters (kdiss). The developed immunosensor was tested with Vtg secreted into the culture medium from gray mullet hepatocytes exposed to estradiol-17 (E2) or 4 nonylphenol (4NP), a persistent and ubiquitous xenoestrogen. Results from our study demonstrated that gray mullet primary hepatocytes combined with a rapid biosensor-based method for quantification of Vtg could be absolutely useful for investigating the impact of environmental estrogens on fish. francesco.palermo@unicam.it