A New LC-MS/MS Method for Simultaneous and Quantitative Detection of Bisphenol-A and Steroids in Target Tissues: A Power Tool to Characterize the Interference of Bisphenol-A Exposure on Steroid Levels

Abstract

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17β-Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0–83.3% for testicular tissue; 63.7–70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity.

Fetal-Perinatal Exposure to Bisphenol-A Affects Quality of Spermatozoa in Adulthood Mouse

“…On the contrary, the lower number of motile SPZ was realistically ascribable to a local accumulation and action of BPA in the epididymal caput region. In agreement, the differential accumulation of endocrine disruptors, including BPA, has been reported in different animal tissues [34] including the visceral fat mass, which in our exposure experimental model preferably accumulates BPA [35]. Interestingly, the gestational BPA exposure adversely affects spermatogenesis in adulthood mice.…”

The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation

“…In agreement with aromatase expression and physiological levels of testicular E 2 [33,54], results showed that CB1 gene deletion reduced intra-testicular estrogens levels. E 2 levels decreased bỹ 40% in CB1 -/vs.…”

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells

“…In animal models, the accumulation of BPA in foetuses and offspring during the foetal/neonatal period has been demonstrated to depend on the maternal exposure and to be mediated by the placenta or breast milk [ 11 , 12 ], as the efficient drug-metabolising systems of the mothers is slightly altered during pregnancy [ 13 , 14 , 15 ]. This accumulation occurs since the offspring acquire only late after birth the ability to metabolize BPA, quite late after birth [e.g., 21 days post partum ( dpp ) in rat] [ 16 ] thus introducing a large measure of uncertainty in any risk assessment that is exclusively based on dose-effect observations, not considering the real accumulation of BPA that also depends on variations in individual susceptibility [ 17 ]. Indeed, three hazard doses for BPA exposure were defined: tolerable daily intake (TDI; 50 μg/kg body weight (bw)/day, indicated as safe reference dose for human), no-observed-adverse-effect-level (NOAEL; 5 mg/kg bw/day), and lowest-observed-adverse-effect level (LOAEL; 50 mg/kg bw/day) [ 18 , 19 ].…”

“…More interesting is that both modulate either the progression of spermatogensis and the adiposity of epididymal fat (perigonadal epididymal withe tissue, pEWAT) [ 42 , 43 , 44 , 45 ], tracing a potential harmful effect of BPA on germ cells which could also result from the impairment of nearby pEWAT. Notably, BPA accumulates in fat tissue [ 1 , 12 , 17 , 46 , 47 , 48 , 49 ], it targets gene expression of enzymes metabolizing endocannabinoids [ 1 , 50 , 51 , 52 , 53 ] and steroids [ 54 , 55 ] and its accumulation in fat tissue is related to steroid levels [ 17 ].…”

“…Here, using tissues collected from the same batch of animals early exposed to BPA described above [ 17 , 36 ], we have characterized the estrogenic activity of BPA and verified the effects on testicular expression of ECS as a potential cause of impairment of spermatogenesis and sperm death in epididymal caput .…”