Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ).
Selective serotonin reuptake inhibitors are used as first line treatment in major depressive disorder. However, selective serotonin reuptake inhibitors have also been associated with sexual disorders, abnormalities, and sexual dysfunction, although mechanisms are unclear. The aim of this project was to investigate the possible endocrine disrupting effect of sertraline (SER) on sex steroid production in male rats exposed to 3 therapeutically realistic doses of SER 1.25, 5, and 20 mg/kg/day. To achieve this, we analyzed all the major steroids in testis, adrenals, brain, and plasma using Liquid chromatography tandem mass spectrometry. Furthermore, we investigated the potential effects on gene expression on the major genes involved in testicular, adrenal and brain steroidogenesis using quantitative PCR. Moreover, plasma luteinizing hormone (LH) levels were analyzed. We observed significant reduction in steroid production, in particular on the testicular Δ-4 axis and on the adrenal CYP17-hydroxylase axis. Effects in brain and plasma were less pronounced. Testicular gene transcription was also significantly down-regulated except for the CYP19 (aromatase) gene. In contrast, no effects on the adrenal gene expression were observed, except for an up-regulation of the CYP17. Plasma LH and LH/TS were increased, in particular in the lowest exposure group, indicating some degree of compensatory hypogonadism. In conclusion, this study demonstrates extensive endocrine disruption during SER exposure in male rats, both directly on steroid production in major endocrine tissues, but also indirectly by affecting gene expression. Furthermore, increased LH levels may augment decreased sex steroid production, in particular testosterone production, inducing a state of compensatory hypogonadism.
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