Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

Impurity profile study of dutasteride.

During the process development of dutasteride in the laboratory analysis showed some impurity peaks in HPLC ranging from 0.05 to 0.1%. The same samples were analyzed by LCMS method and identified peak at m/z 508 (desmethyl dutasteride), 530 (dihydro dutasteride) and 528 (isomer of dutasteride). These impurities were synthesized individually and characterized based on the spectroscopic data (HPLC, IR, NMR and MS). The structures of these impurities were 17beta-N-[2,5-bis(trifluoromethyl) phenyl]carbamoyl-3-hydroxyl-4-azaestra-1,3,5,7,9-pentaene (desmethyl of dutasteride 2), 17beta-N-[2,5-bis(trifluoromethyl)phenyl]carbamoyl-4-aza-5alpha-androstane-3-one (dihydro impurity of dutasteride 3), and 17beta-N-[2,5-bis(trifluoromethyl) phenyl] carbamoyl-4-aza-5beta-androst-1-ene-3-one (beta-isomer of dutasteride 4), respectively. The formation and synthesis of dutasteride impurities are discussed.

13C-NMR study of 4-azasteroids in solution and solid state.

A group of biologically active 4-azasteroids was studied by 13C-NMR spectroscopy in solution and in the solid phase. A full assignment of signals in the spectra of samples in chloroform was performed for thirteen 4-azasteroids using two-dimensional techniques. Substituent and steric effects of a nitrogen atom, and their influence on chemical shifts of the neighboring carbon atoms are discussed. CP MAS spectra were obtained for five 4-azasteroids including finasteride. The spectra confirmed polymorphism of the latter compound. In addition to the polymorphic forms that are already known, a new molecular complex of finasteride with dioxane is reported.

Syntheses and ultraviolet absorption of aza-steroids.

A series of aza-steroids was synthesized containing chromophoric groups, such as a, beta-unsaturated ketones and doubly unsaturated conjugated and monoconjugated heteroannular dienones with the heteroatom in the D ring at the 20 and 17a positions. The effect of an appositely placed electronegative center in these molecules upon their ultraviolet absorptions was studied. In order to obtain a basis for the observed spectral changes, the corresponding carbocyclic compounds were also synthesized. The maximum absorptions of the aza-steroids were hypsochromically shifted relative to the carbocyclic compounds in all cases (n=8). The displacement caused by the electronegative center on absorption maximum of the chromophores is, however, highly dependent on its location within the molecules.

[Molecular and crystallographic structure of D-11-aza-19-nortestosterone]. / Molekuliarnaia i kristallicheskaia struktura D-11-aza-19-nortestosterona.

Crystal and molecular structure of D-11-aza-19-nortestosterone monohydrate C17H25NO2.H2O (a 8,288(2), b 12,433(2), c 7,570(2) A, beta 90,25(1) degrees; space group P2(1), R 8.3%) has been determined by X-ray analysis. Its comparison with the molecular structure of D-19-nortestosterone showed that the decrease in the hormonal activity upon 11-aza-substitution may be due to difference in chemical properties of imino and methylene groups.

Mode of action of human pharmaceuticals in fish: the effects of the 5-alpha-reductase inhibitor, dutasteride, on reproduction as a case study.

In recent years, a growing number of human pharmaceuticals have been detected in the aquatic environment, generally at low concentrations (sub-ng/L-low µg/L). In most cases, these compounds are characterised by highly specific modes of action, and the evolutionary conservation of drug targets in wildlife species suggests the possibility that pharmaceuticals present in the environment may cause toxicological effects by acting through the same targets as they do in humans. Our research addressed the question of whether or not dutasteride, a pharmaceutical used to treat benign prostatic hyperplasia, may cause adverse effects in a teleost fish, the fathead minnow (Pimephales promelas), by inhibiting the activity of both isoforms of 5α-reductase (5αR), the enzyme that converts testosterone into dihydrotestosterone (DHT). Mammalian pharmacological and toxicological information were used to guide the experimental design and the selection of relevant endpoints, according to the so-called "read-across approach", suggesting that dutasteride may affect male fertility and steroid hormone dynamics. Therefore, a 21-day reproduction study was conducted to determine the effects of dutasteride (10, 32 and 100 µg/L) on fish reproduction. Exposure to dutasteride significantly reduced fecundity of fish and affected several aspects of reproductive endocrine functions in both males and females. However, none of the observed adverse effects occurred at concentrations of exposure lower than 32 µg/L; this, together with the low volume of drug prescribed every year (10.34 kg in the UK in 2011), and the extremely low predicted environmental concentration (0.03 ng/L), suggest that, at present, the potential presence of dutasteride in the environment does not represent a threat to wild fish populations.

Estimation of glycoalkaloids as solasodine in Solanum laciniatum.

Solasodine is isolated from Solanum laciniatum leaves by a rapid, one-step procedure which involves extraction and hydrolysis of the glycoalkaloids. The resulting steroidal aglycone is directly estimated by the formation of a colored complex with bromocresol green. The method is applicable to concentrations of 25-125 mug solasodine/7ml.

Low level determination of a novel 4-azasteroid and its carboxylic acid metabolite in human plasma and semen using high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

Compound I (4.7beta-dimethyl-4-azacholestan-3-one, MK-0386) is a potent 5alpha-reductase type 1 (5alphaR1) inhibitor. Sensitive (0.2 ng/ml), specific and separate assays have been developed and validated for the analysis of I and its carboxylic acid metabolite (II) in human semen and plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. After liquid-liquid extraction of the analytes from biological matrix, the extracts were chromatographed on a short (50 mm) analytical column during analysis of I, and on a longer (150 mm) column with a weaker mobile phase during the analysis of II. This additional chromatographic separation was required to separate II from a secondary metabolite present in post-dose plasma samples interfering with the quantification of II. The MS-MS detection was performed on a Sciex API III Plus tandem mass spectrometer using the heated nebulizer probe. Monitoring the parent-->product ion combinations of m/z 416-->114 and 404-->114, in the multiple reaction monitoring (MRM) mode, after chromatographic separation, allowed quantification of both analytes. The standard curve in plasma was linear in the concentration range of 0.2 to 200 ng/ml for both I and II with correlation coefficients greater than 0.99 and coefficients of variation of less than 15% for replicate (n=5) analysis at all concentrations within the standard curve range. For the semen assay the linear range for determination of I was from 0.2 to 50 ng/ml. These assays were applied to support a number of clinical studies with I and their validity and long-term performance was confirmed during analyses of clinical samples from these studies. The need for careful assessment of the specificity of MS-MS assays in post-dose biological fluid samples in the presence of metabolites was emphasized.