Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC–MS/MS was developed. As a pre‐treatment procedure prior to LC–tandem mass spectrometry (LC–MS/MS) analysis, hydrolysis using β‐glucuronidase and solid‐phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 μm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal‐to‐noise ratio of 3 were 0.03–90 ng/mL. The developed novel LC–MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone‐dependent diseases.
Rationale: Parabens, the alkyl esters of 4-hydroxybenzoic acid, are a family of compounds widely used as preservatives in cosmetic products, including for children, and some are permitted in foods. Parabens are known to be weak endocrine disruptors because they interfere with the function of endogenous hormones through binding to estrogen receptors. Therefore, the levels of parabens in biological samples indicate endocrine-disruptive exposure. In particular, hair samples can provide information on accumulated exposure to parabens.Methods: For monitoring of long-term exposure to parabens, an improved analytical method for rapid and direct determination in hair sample was developed involving ultra-performance liquid chromatography-tandem mass spectrometry using on-line extraction. Five parabens (methyl-, ethyl-, propyl-, butyl-and benzylparaben) were separated within 10 min after incubation with 1 N HCl. Parabens were separated using a Waters BEH C 18 column (2.1 mm × 100 mm, 1.7 μm) and a mobile phase consisting of 10 mM ammonium acetate in water and acetonitrile with a gradient program at a flow rate of 300 μL/min. The analysis of the separated parabens was monitored with electrospray negative ionization tandem mass spectrometry. Results:The linearity of the method was demonstrated by r 2 ≥ 0.994. The limits of detection as defined by a signal-to-noise ratio of 3 were 1-5 ng/g. The mean concentration of the five parabens in hair of human subjects was measured to be 55.6 ± 24.3 to 136.9 ± 48.5 ng/g. Conclusions:The levels of parabens in hair samples may play an important role in understanding probable endocrine-disruptive exposure, and the described method could be used to evaluate and monitor long-term exposure to parabens as endocrine disruptors.
Epilepsy is one of the most common neurological disorders, and it is characterized by spontaneous seizures. In a previous study, we identified 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as a novel anti-epileptic agent in chemically- or genetically-induced epileptic zebrafish and mouse models. In this study, we investigated the anti-epileptic effects of GM-90432 through neurochemical profiling-based approach to understand the neuroprotective mechanism in a pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. GM-90432 effectively improved PTZ-induced epileptic behaviors via upregulation of 5-hydroxytryptamine, 17-β-estradiol, dihydrotestosterone, progesterone, 5α -dihydroprogesterone, and allopregnanolone levels, and downregulation of normetanephrine, gamma-aminobutyric acid, and cortisol levels in brain tissue. GM-90432 also had a protective effect against PTZ-induced oxidative stress and zebrafish death, suggesting that it exhibits biphasic neuroprotective effects via scavenging of reactive oxygen species and anti-epileptic activities in a zebrafish model. In conclusion, our results suggest that neurochemical profiling study could be used to better understand of anti-epileptic mechanism of GM-90432, potentially leading to new drug discovery and development of anti-seizure agents.
Valproic acid (VPA) is a known drug for treating epilepsy and mood disorders; however, it is not recommended for pregnant women because of its possible teratogenicity. VPA affects neurotransmission and gene expression through epigenetic mechanisms by acting as a histone deacetylase inhibitor and has been used to establish animal models of autism spectrum disorder (ASD). However, studies on the long-term effects of early exposure to VPA on glucocorticoid and neurosteroid synthesis in the brain are lacking. Therefore, this study aimed to investigate the long-term changes in metabolic alterations and gene expression regulation according to sex, using metabolic steroid profiling data from cerebral cortex samples of rats four weeks after VPA exposure (400 mg/kg). In neonatal VPA-exposed models, estradiol levels decreased, and cytochrome P450 19A1 gene (Cyp19a1) expression was reduced in the prepubertal male cortex. Progesterone and allopregnanolone levels decreased, and 3β-hydroxysteroid dehydrogenase 1 gene (Hsd3b1) expression was also downregulated in the prepubertal female cortex. Furthermore, cortisol levels increased, and mRNA expression of the nuclear receptor subfamily 3 group C member 1 gene (Nr3c1) was downregulated in the cortices of both sexes. Unlike the neonatal VPA-exposed models, although a decrease in progestin and estradiol levels was observed in females and males, respectively, no differences were observed in cortisol levels in the cortex tissues of 8-week-old adult rats administered VPA for four weeks. These results indicate that early environmental chemical exposure induces long-term neurosteroid metabolic effects in the brain, with differences according to sex.
Bisphenol F (BPF; 4,4′-dihydroxydiphenylmethane) is one of the most frequently used compounds in the manufacture of plastics and epoxy resins. Previous studies have demonstrated that BPF affects locomotor behavior, oxidative stress, and neurodevelopment in zebrafish. However, its neurotoxic effects are controversial, and the underlying mechanisms are unclear. In order to determine whether BPF affects the motor system, we exposed zebrafish embryos to BPF and assessed behavioral, histological, and neurochemical changes. Spontaneous locomotor behavior and startle response were significantly decreased in BPF-treated zebrafish larvae compared with control larvae. BPF induced motor degeneration and myelination defects in zebrafish larvae. In addition, embryonic exposure to BPF resulted in altered metabolic profiles of neurochemicals, including neurotransmitters and neurosteroids, which may impact locomotion and motor function. In conclusion, exposure to BPF has the potential to affect survival, motor axon length, locomotor activity, myelination, and neurochemical levels of zebrafish larvae.
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