Endocrine Disruptors and Estrogens in Human Prostatic Tissue.

Endocrine disruptors (EDs) are ubiquitous substances both in the environment and everyday products that interfere with the hormonal system. Growing evidence demonstrates their adverse effects on the organism, including the reproductive system and the prostate, owing to their (anti)estrogenic or antiandrogenic effects. Since EDs can interact with steroid hormone actions on-site, understanding the levels of intraprostatic EDs in conjunction with steroids may hold particular significance. The aim of this study was to develop and validate a method for determining estrogens, various groups of EDs (bisphenols, parabens, oxybenzone and nonylphenol) and phytoestrogens in their unconjugated and conjugated forms in prostate tissue by liquid chromatography-tandem mass spectrometry, and subsequently analyze 20 human prostate tissue samples. The method enabled 20 compounds to be analyzed: estrogens (estrone, estradiol, estriol), bisphenols (bisphenol A- BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methyl-, ethyl-, propyl-, butyl-, benzyl- paraben), oxybenzone, nonylphenol and phytoestrogens (daidzein, genistein, equol) with LLOQs between 0.017-2.86 pg/mg of tissue. The most frequently detected EDs in prostate tissues were propylparaben (conjugated and unconjugated forms in 100 % of tissues), methylparaben (unconjugated in 45 % and conjugated in 100 %), ethylparaben (unconjugated in 25 % and conjugated in 100 % BPA (unconjugated in 35 % and conjugated in 60 % and oxybenzone (both forms in 45 % To the best of our knowledge, this is the first study detecting EDs, phytoestrogens and estriol conjugate (E3C) in the prostate. E3C was the most abundant estrogen in prostatic tissue. This highlights the need for further explorations into estrogen metabolism within the prostate.

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma.

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Steroid diagnostics of 21st century in the light of their new roles and analytical tools.

The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First we focus on specific aspects arising from the pre-analytical phase of steroid determination that need to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17alpha-hydroxy-pregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.

[Detection of DNA specific for Mycobacterium tuberculosis in archeological material using the polymerase chain reaction]. / Detekce DNA specifické pro Mycobacterium tuberculosis v archeologických materiálech metodou polymerázové retezové reakce.

The paleopathological diagnosis of bone tuberculosis in archeological findings may be confirmed by the polymerase chain reaction (PCR). If the M. tuberculosis-specific DNA fragment is amplified, then the presence of this microorganism in the sample is demonstrated. The pilot study presented investigated whether our molecular biology laboratory can collaborate with anthropologists in paleopathological analyses and to verify the use of the commercial diagnostic kit Cleanmix (Talent, Italy), for DNA isolation from archeological samples. The results were compared with the conclusions of anthropologists. Successful amplification of specific DNA fragments was achieved in a specimen from the period of the 13th to 15th century. The specimen consists of four thoracic vertebrae modified by osseous tuberculosis (gibbus). The PCR result was also positive in a five-year-old femur sample of a patient with chronic pulmonary tuberculosis. All other specimens of various ages but without macroscopic symptoms of osseous tuberculosis, were PCR negative. These results suggest that it is possible to detect former infections with pathogenic microorganisms in archeological bones find.

An interesting case of extreme microcephaly.

The collections of the Museum in the Department of Anatomy, Faculty of Medicine, J. E. Purkyne University in Brno, include a skull of about 25 to 30-year-old individual with extreme microcephaly that has been so far described rarely in the morphological literature. The skull capacity is 355 cm3, which is only 26.1% of the skull capacity in a "normal" individual. While the facial skeleton is reduced only by 10-15% if compared with the norm, the cerebral part is striking by its extraordinarily small dimensions (smaller by 30-40% in comparison with the norm), particularly in the area of the frontal bone squama. The size of the skull is characterized best by the values of the basic metric measurements: maximal length of the skull 122 mm (norm: 172 mm), maximal breadth of the skull 94 mm (norm: 140 mm), height of the skull 96 mm (norm: 130 mm), circumference of the skull through the glabella 351 mm (norm: 510 mm). Foramen occipitale magnum is shifted strikingly to the dorsal direction. The cause of the microcephaly described cannot be explained explicitly just on the basis of the findings in the skull. For finding the actual cause a number of other data should be known. Authors' hypothesis that the problem is primary microcephaly has been supported even by roentgenological finding.