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Triclosan (TCS) is a widely used antimicrobial, antifungal, and antiviral agent. To date, it has been reported that TCS can enter the human body and disrupt hormonal homeostasis. Therefore, the aim of our paper was to evaluate the impact of TCS on astrocytes, i.e. a crucial population of cells responsible for steroid hormone production. Our data showed that, in mouse primary astrocyte cultures, TCS can act as an endocrine disrupting chemical through destabilization of the production or secretion of progesterone (P 4), testosterone (T), and estradiol (E 2). TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, such as Cyp17a1 , 17β-Hsd, and Cyp19a1. Our data showed that a partial PPARγ agonist (honokiol) prevented changes in Cyp17a1 mRNA expression caused by TCS. Similarly, honokiol inhibited TCS-stimulated P 4 release. However, rosiglitazone (classic PPARγ agonist) or GW9662 (PPARγ antagonist) had a much stronger effect. Therefore, we believe that the changes observed in the P 4 , T, and E 2 levels are a result of dysregulation of the activity of the aforementioned enzymes, whose expression can be affected by TCS through a Pparγ-dependent pathway. TCS was found to decrease the aryl hydrocarbon receptor (AhR) and Sirtuin 3 protein levels, which may be the result of the activation of the these proteins. Since our study showed dysregulation of the production or secretion of neurosteroids in astrocytes, it can be concluded that TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed. [Display omitted] • TCS increases progesterone and testosterone release from astrocytes in vitro. • TCS decreases testosterone and estradiol production in astrocytes in vitro. • TCS decreases 17β-Hsd and Cyp19a1 mRNA expression in astrocytes in vitro. • TCS decreases AhR and Sirt3 protein expression in astrocytes in vitro. [ABSTRACT FROM AUTHOR]

There has been growing interest in the potential of vitamin D food fortification in Europe as a means of addressing low vitamin D status. The WHO-FAO suggest that choosing a suitable food vehicle and ensuring the combination of the food vehicle and the fortificant will be efficacious and effective are of key importance to a successful food fortification programme. Our key objective was to conduct a systematic review and meta-analysis to investigate the effect of various animal- and plant-based food vehicles fortified with vitamin D (as D 3 or D 2) on circulating 25-hydroxyvitamin D [25(OH)D] concentrations. A list of prioritised food vehicles was established and we searched PubMed, Embase, Scopus and Web of Science for randomised controlled trials (RCTs) which used these vehicles individually, and which met prespecified criteria. The searches identified 49 papers which described suitable RCTs using vitamin D-fortified bread/savoury biscuits (n = 5), orange juice (n = 5), UV-mushrooms (n = 8), cheese (n = 3), yogurt (n = 5), fluid milk (n = 13), powdered milk (n = 5), eggs (n = 2), edible oils (n = 4), or breakfast cereal (n = 1). No suitable RCTs were identified for rice, maize flour, butter, margarine or dairy spreads, plant-based milk or yogurt alternatives. Random-effects meta-analyses of each food vehicle individually indicated weighted mean differences (WMD) in 25(OH)D in the range ∼9–35 nmol/L (3–15 RCT arms, depending on vehicle), and all statistically significant (P < 0.01–0.0001), with the exception of UV-mushrooms (P = 0.06). Heterogeneity was variable (I 2 =33–99%, depending on vehicle), but subgroup analysis based on vitamer and dose reduced it in some instances. Sub-group analysis on the basis of whether the food vehicles were from plant-based or animal-based origin showed no significant difference in WMD (15.2 versus 15.9 nmol/L, respectively; P = 0.48). These results support the use of various animal- and plant-based food vehicles for vitamin D fortification to improve circulating 25(OH)D concentrations in populations. This work was registered with PROSPERO as CRD42023439883. • 49 RCTs using 8 vitamin D-fortified food vehicles identified in systematic review. • Individual meta-analyses of serum 25(OH)D for each food vehicle were performed. • WMD in serum 25(OH)D ranged from 9 to 35 nmol/L (P < 0.01-0.0001) for 7 food vehicles. • UV-mushrooms were exception with a borderline significant WMD of 8 nmol/L (P = 0.06). • WMD similar (P = 0.48) for plant- versus animal-based food vehicles for vitamin D. [ABSTRACT FROM AUTHOR]

Pentacyclic triterpenoids, particularly hopanoids, are organism-specific compounds and are generally considered as useful biomarkers that allow fingerprinting and classification of biological, environmental and geological samples. Simultaneous quantification of various hopanoids together with battery of related non-polar and low-molecular mass compounds may provide principal information for geochemical and environmental research focusing on both modern and ancient investigations. Target compounds can be derived from microbial biomass, water columns, sediments, coals, crude fossils or rocks. This create number of analytical problems due to different composition of the analytical matrix and interfering compounds and therefore, proper optimization of quantification protocols for such biomarkers is still the challenge. In this work we summarizing typical analytical protocols that were recently applied for quantification of hopanoids like compounds from different samples. Main steps including components of interest extraction, pre-purification, fractionation, derivatization and quantification involving gas (1D and 2D) as well as liquid separation techniques (liquid–liquid extraction, solid-phase extraction, planar and low resolution column chromatography, high-performance liquid chromatography) are described and discussed from practical point of view, mainly based on the experimental papers that were published within last two years, where significant increase in hopanoids research was noticed. The second aim of this review is to describe the latest research trends concerning determination of hopanoids and related low-molecular mass lipids analyzed in various samples including sediments, rocks, coals, crude oils and plant fossils as well as stromatolites and microbial biomass cultivated under different conditions. It has been found that majority of the most recent papers are based on uni- or bivariate approach for complex data analysis. Data interpretation involves number of physicochemical parameters and hopanoids quantities or given biomarkers mass ratios derived from high-throughput separation and detection systems, typically GC–MS and HPLC–MS. Based on quantitative data reported in recently published experimental works it has been demonstrated that multivariate data analysis using e.g. principal components computations may significantly extend our knowledge concerning proper biomarkers selection and samples classification by means of hopanoids and related non-polar compounds. [ABSTRACT FROM AUTHOR]

The human gut microbiome plays a crucial role in both health and disease. Metabolites in human faeces related to microbial activity might therefore be attractive surrogate markers to track changes of microbiota induced by diet or disease. The hyphenation of gas chromatography with triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad MS instruments. The versatility of gas chromatography-tandem mass spectrometry (GC–MS/MS) can be advantageously exploited in clinical laboratory medicine, e.g. for quantification of sterols in biological material. In this paper, we present the application of GC–MS/MS for determination of sterol components in human faeces. A serious problem of analysis of faeces is preanalytics. Uncontrolled degradation of metabolites during transport and storage of faeces before entering the clinical laboratory might occur. In our experiments we did not observe any increasing or decreasing concentration after storage of native faeces material even at room temperature. Furthermore, we answer the question of how personal metabolic responses with respect to sterols are and address the importance of sampling strategies. From a pilot study it is concluded that differentiation between high and low metabolizers is independent of the type of sampling and constant over several days. [ABSTRACT FROM AUTHOR]

• Hydroperoxy steroids derived from plants. • Hydroperoxy triterpenoids derived from plants. • Hydroperoxy steroids and triterpenoids produced by fungi. • Biological activity of steroids and triterpenoids derived from plant and fungi. Hydroperoxides (R-OOH) represent a small family of natural metabolites that have been isolated from higher plants, fungi, and marine organisms. This paper is devoted to the distribution of hydroperoxides in plants, fungi and terrestrial fungal endophytes and their biological activity. Hydroperoxides of plants demonstrate a wide range of biological activities however, antineoplastic and anti-ulcerative are most characteristic with confidence from 91 to 98 percent. For hydroperoxides from fungi, the dominant are antineoplastic and anti-hypercholesterolemic activities with confidence from 89 to 92 percent. Very interesting activity was found for some triterpenoid hydroperoxides, which is characterized as a treatment for the symptoms of dementia. The norlupane hydroperoxide shows activity for the treatment of dementia. It is interesting that the reliability of this activity was very high 97.2%. According to our preliminary data, the norlupane hydroperoxide is apparently the first natural metabolite that showed almost 100 percent activity for the treatment of dementia. However, to confirm these data requires practical and clinical experimental work. [ABSTRACT FROM AUTHOR]

Highlights from the article: The topic "metabolism" was covered by a paper indicating that the vitamin D compounds, 1 -hydroxyvitamin D SB 3 sb , 1 -hydroxyvitamin D SB 2 sb and 25-hydroxyvitamin D SB 3 sb (25(OH)D SB 3 sb ) lower cholesterol in vivo in hypercholesterolemic mice via the activation of 1,25(OH) SB 2 sb D SB 3 sb [[11]]. Notably this anti-infection response to vitamin D supplementation was most pronounced in individuals who were vitamin D-deficient (< 25 nmol/L serum 25(OH)D SB 3 sb ) before treatment, and in those who did not receive large bolus doses (>30,000 IU) of vitamin D supplementation [[18]]. Further modelling with different thresholds of 25OHD, considered country-specific UVB availability and the plasma 25(OH)D SB 3 sb response to vitamin D intake, showed how (bio) food fortification strategies may be applied to increase vitamin D status across the population and prevent vitamin D deficiency without reaching toxic levels of 25(OH)D SB 3 sb . The product, 3-epi-25(OH)D SB 3 sb , is generally the third most abundant form of vitamin D present in the serum after 25(OH)D SB 3 sb and vitamin D3. 3-epi-1 ,25-dihydroxyvitamin D3 has lower biological activity than 1,25(OH) SB 2 sb D SB 3 sb .

• Cyp27a1 is capable of metabolizing cholesterol to three vital regulatory oxysterols. • A new mitochondrial-initiated hepatic pathway of oxysterol/bile acid synthesis. • 24HC is metabolized to bile acids via both Cyp7b1 and an independent pathway. • Cyp7b1 is a key regulator of the levels of the Cyp27a1 generated oxysterols. • Oxysterols are key mediators of cholesterol and lipid homeostasis. The aim of this paper was to more completely study the mitochondrial CYP27A1 initiated acidic pathway of cholesterol metabolism. The mitochondrial CYP27A1 initiated pathway of cholesterol metabolism (acidic pathway) is known to synthesize two well-described vital regulators of cholesterol/lipid homeostasis, (25R)-26-hydroxycholesterol (26HC) and 25-hydroxycholesterol (25HC). Both 26HC and 25HC have been shown to be subsequently 7α-hydroxylated by Cyp7b1; reducing their regulatory abilities and furthering their metabolism to chenodeoxycholic acid (CDCA). Cholesterol delivery into the inner mitochondria membrane, where CYP27A1 is located, is considered the pathway's only rate-limiting step. To further explore the pathway, we increased cholesterol transport into mitochondrial CYP27A1 by selectively increased expression of the gene encoding the steroidogenic acute transport protein (StarD1). StarD1 overexpression led to an unanticipated marked down-regulation of oxysterol 7α-hydroxylase (Cyp7b1), a marked increase in 26HC, and the formation of a third vital regulatory oxysterol, 24(S)-hydroxycholesterol (24HC), in B6/129 mice livers. To explore the further metabolism of 24HC, as well as, 25HC and 26HC, characterizations of oxysterols and bile acids using three murine models (StarD1 overexpression, Cyp7b1−/−, Cyp27a1−/−) and human Hep G2 cells were conducted. This report describes the discovery of a new mitochondrial-initiated pathway of oxysterol/bile acid biosynthesis. Just as importantly, it provides evidence for CYP7B1 as a key regulator of three vital intracellular regulatory oxysterol levels. [ABSTRACT FROM AUTHOR]

Highlights • Low serum 25(OH)D is associated with bladder cancer risk. • Bladder epithelial cells express intact vitamin D signaling. • Bladder cells synthesize sufficient 1,25(OH) 2 D to stimulate a local immune response. • Induction of CAMP depends on stage of cancer cell differentiation. Abstract The development of some cancers is associated with vitamin D deficiency. We suggest that reduced conversion of 25-hydroxyvitamin D (25(OH)D) to 1,25-dihydroxyvitamin D (1,25(OH) 2 D) and the resulting modification of tissue specific immune responses may be key. Non-muscle-invasive bladder cancer is highly immunoresponsive and stimulation of an inflammatory response by intravesical bacillus Calmette-Guerin (BCG) treatment prevents recurrence. To assess the relationship between serum 25(OH)D and bladder cancer risk we conducted a systematic review. To test our hypothesis, the synthesis of 1,25(OH) 2 D by human bladder epithelial cell lines (T24/83 and RT4) was examined. Studies were identified from Medline, Web of Science, Embase and Cochrane library (limited to English language, humans and 1990–2018). After removal of duplicates, title and abstract review 6 full papers were appraised. Low vitamin D levels were associated with bladder cancer risk in 5/6 of the studies. Both cell lines express the vitamin D receptor, 25-hydroxyvitamin D 1α-hydroxylase (1α−OHase) and 24-hydroxylase (24−OHase) mRNA, which was induced by 1,25(OH) 2 D. 24−OHase mRNA was also increased by 25(OH)D indicating 1α−OHase activity. Both cell types expressed TLR1,2,4 and the TLR partners MyD88 and CD14mRNA. Cathelicidin mRNA was undetectable in both cell lines but was induced by 1,25(OH) 2 D and 25(OH)D in RT4 cells. The systematic review demonstrated that bladder cancer risk correlates with serum 25(OH)D levels. In addition, we have shown that transitional epithelial cells express functional vitamin D signaling and can synthesize sufficient 1,25(OH) 2 D to stimulate a local immune response. We suggest that in order to maintain optimal immune surveillance within the bladder adequate levels of serum 25(OH)D are required for direct synthesis of 1,25(OH) 2 D by bladder epithelial cells. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A-Ring fragment's synthesis started from cyclohexanone derived from quinic acid. • 19-Norcalcitriol analogs with pegylated 2-alkylidene groups were synthesized. • 2-Alkylidene chain is terminated by hydroxyl or 2-(pyridin-2′-yl)ethylamino group. • The affinities of the synthesized 19-norcalcitriol analogs to VDR were assessed. Abstract The results presented in this paper constitute an extension of our synthetic efforts focused on 19-norvitamin D compounds possessing elongated 2-alkylidene substituents. Based on our previous results, molecular modeling studies, and docking experiments, we selected a novel 19-norcalcitriol analog with long chain at C-2 containing several ether moieties and terminated by 2-(pyridin-2′-yl)ethylamino fragment. It was expected that such structural modification might allow binding of transition metal by the ligand, increase solubility of the formed complexes as well as improve their affinity to the VDR. For comparison, a 19-norcalcitriol analog was also obtained with the terminal hydroxyl group at its pegylated 2-alkylidene substituent. The synthesis of the target vitamin D compounds described in this work was performed using the Wittig-Horner approach. The respective A-ring phosphine oxide was obtained starting from the D -(-)-quinic acid and then coupled with the known Grundmann ketone. [ABSTRACT FROM AUTHOR]

Abstract: This paper is a continuation of our previous research focusing on development of micro-TLC methodology under temperature-controlled conditions. The main goal of present paper is to demonstrate separation and detection capability of micro-TLC technique involving simple analytical protocols without multi-steps sample pre-purification. One of the advantages of planar chromatography over its column counterpart is that each TLC run can be performed using non-previously used stationary phase. Therefore, it is possible to fractionate or separate complex samples characterized by heavy biological matrix loading. In present studies components of interest, mainly steroids, were isolated from biological samples like fish bile using single pre-treatment steps involving direct organic liquid extraction and/or deproteinization by freeze-drying method. Low-molecular mass compounds with polarity ranging from estetrol to progesterone derived from the environmental samples (lake water, untreated and treated sewage waters) were concentrated using optimized solid-phase extraction (SPE). Specific bands patterns for samples derived from surface water of the Middle Pomerania in northern part of Poland can be easily observed on obtained micro-TLC chromatograms. This approach can be useful as simple and non-expensive complementary method for fast control and screening of treated sewage water discharged by the municipal wastewater treatment plants. Moreover, our experimental results show the potential of micro-TLC as an efficient tool for retention measurements of a wide range of steroids under reversed-phase (RP) chromatographic conditions. These data can be used for further optimalization of SPE or HPLC systems working under RP conditions. Furthermore, we also demonstrated that micro-TLC based analytical approach can be applied as an effective method for the internal standard (IS) substance search. Generally, described methodology can be applied for fast fractionation or screening of the whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in biological or environmental samples. Due to low consumption of eluent (usually 0.3–1mL/run) mainly composed of water–alcohol binary mixtures, this method can be considered as environmentally friendly and green chemistry focused analytical tool, supplementary to analytical protocols involving column chromatography or planar micro-fluidic devices. [Copyright &y& Elsevier]

Highlights • Androgenic signaling systems can evolve to support processes of behavioral adaptation. • This evolution can occur with regard to many molecular elements of androgenic signaling pathways. • Golden-collared manakinsare birds in which the androgenic system is evolutionarily modified to support courtship behavior. Abstract Sex steroids mediate the organization and activation of masculine reproductive phenotypes in diverse vertebrate taxa. However, the effects of sex steroid action in this context vary tremendously, in that steroid action influences reproductive physiology and behavior in markedly different ways (even among closely related species). This leads to the idea that the mechanisms underlying sex steroid action similarly differ across vertebrates in a manner that supports diversification of important sexual traits. Here, we highlight the Evolutionary Potential Hypothesis as a framework for understanding how androgen-dependent reproductive behavior evolves. This idea posits that the cellular mechanisms underlying androgenic action can independently evolve within a given target tissue to adjust the hormone's functional effects. The result is a seemingly endless number of permutations in androgenic signaling pathways that can be mapped onto the incredible diversity of reproductive phenotypes. One reason this hypothesis is important is because it shifts current thinking about the evolution of steroid-dependent traits away from an emphasis on circulating steroid levels and toward a focus on molecular mechanisms of hormone action. To this end, we also provide new empirical data suggesting that certain cellular modulators of androgen action—namely, the co-factors that dynamically adjust transcritpional effects of steroid action either up or down—are also substrates on which evolution can act. We then close the review with a detailed look at a case study in the golden-collared manakin (Manacus vitellinus). Work in this tropical bird shows how androgenic signaling systems are modified in specific parts of the skeletal muscle system to enhance motor performance necessary to produce acrobatic courtship displays. Altogether, this paper seeks to develop a platform to better understand how steroid action influences the evolution of complex animal behavior. [ABSTRACT FROM AUTHOR]

Highlights • We investigated the influence of the disordered NTD on distant domains of HaUsp. • The NTD determines the pattern of the protein-DNA interaction of full-length HaUsp. • The NTD stabilizes dimers of full-length HaUsp via a scorpion-like structure. Abstract Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NRs. [ABSTRACT FROM AUTHOR]

Abstract: The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the 13C/12C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the δ 13C values (‰) of androsterone, etiocholanolone, 5β-androstane-3α, 17β-diol (5β-diol), 5α-androstane-3α, 17β-diol (5α-diol) and 5β-pregnane-3α, 20α-diol (5β-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The δ values of 5β-diol and 5α-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5β-pdiol, which exceeded the WADA limit of 3‰. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred. [Copyright &y& Elsevier]

Abstract: Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. In contrast to tamoxifen, an antagonist of the estrogen receptor (ER), AIs have shown to be better tolerated along with decreased recurrence rates of the disease. Currently, three third-generation AIs are being used: exemestane, letrozole, and anastrozole. Our laboratory is attempting to understand several aspects of AI functionality. In this paper, we first review recent findings from our structure–function studies of aromatase as well as the molecular characterization of the interaction between AIs and aromatase. Based on these studies, we propose new evidence for the interaction of letrozole and exemestane with aromatase. In addition, we will discuss recent results generated from our AI-resistant cell lines. Our laboratory has generated MCF-7aro cells that are resistant to letrozole, anastrozole, exemestane, and tamoxifen. Basic functional characterization of aromatase and ERα in these resistant cell lines has been done and microarray analysis has been employed in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. The results generated so far suggest the presence of at least four types of resistant cell lines. Overall, the information presented in this paper supplements our understanding of AI function, and such information can be valuable for the development of treatment strategies against AI resistant breast cancers. [Copyright &y& Elsevier]

Glucocorticoid resistance is a problem in the treatment of many diseases. One possible factor involved in the modulation of a glucocorticoid response is the export of glucocorticoids out of the cell. It has been shown that multidrug resistance protein 1 (MDR1, ABCB1), a member of the ABC family, is capable of transporting some glucocorticoids. This paper uses a mouse cell line, LMCAT in which the glucocorticoid response can be modulated by inhibitors of multidrug resistance proteins. Glucocorticoids fall into three categories. Firstly, those that are transported by an Abcb1a/Abcb1b transporter and whose transport can be inhibited by inhibitors of ABCB1 activity. Functional Abcb1a/Abcb1b was detected by inhibition of rhodamine efflux by these drugs and mRNA for Abcb1a and Abcb1b were detected in these cells. Secondly, those that are not transported. Finally, those that are transported by an Abcc1a transporter. Calcein transport out of these cells was blocked by treatment with probenecid indicating a functional Abcc1a transporter. Abcc1a mRNA was also detected in these cells. Thus, this paper provides insight into the mechanisms of glucocorticoid transport in cells and demonstrates a diversity of two independent mechanisms of transport of glucocorticoids by Abcb1a/Abcb1b and Abcc1a with individual patterns of steroid specificity. [Copyright &y& Elsevier]

Insulin resistance (IR) is one of the significant aberrations in polycystic ovarian syndrome (PCOS), however is only observed in 70%–80% of obese PCOS and 20%–25% of lean PCOS. Hyperinsulinemia accompanies PCOS-IR along with hyperandrogenemia against normal insulin and androgen levels in PCOS-non insulin resistance (NIR). This could possibly be due to defects in the downstream signaling pathways. The study thus aims to unravel insulin and steroidogenic signaling pathways in luteinized granulosa cells isolated from PCOS-IR and NIR vs matched controls. Luteinized granulosa cells from 30 controls and 39 PCOS were classified for IR based on a novel method of down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. We evaluated expression of molecules involved in insulin, steroidogenic signaling and lipid metabolism in luteinized granulosa cells followed by analysis of estradiol, progesterone and testosterone in follicular fluid. Protein expression of INSR- β, pIRS (ser 307), PI(3)K, PKC-ζ, pAkt, ERK1/2, pP38MAPK and gene expression of IGF showed differential expression in the two groups. Increased protein expression of PPAR-γ was accompanied by up regulation in SREBP1c, FAS, CPT-1 and ACC-1 genes in PCOS-IR group. Expression of StAR, CYP19A1, 17 β- HSD and 3 β- HSD demonstrated significant decrease along with increase in CYP11A1, FSH-R and LH-R in both the groups. Follicular fluid testosterone increased and progesterone decreased in PCOS-IR group. This study shows how candidate molecules that were differentially expressed, aid in designing targeted therapy against the two phenotypes of PCOS. [ABSTRACT FROM AUTHOR]

The aim of this study is to determine and critically evaluate the plausible relationships of vitamin D with extra-skeletal tissues in humans. Severe vitamin D deficiency results in rickets in children and osteomalacia in adults; these beneficial effects in the musculoskeletal system and certain physiological functions are well understood. Nevertheless, mounting reports support additional beneficial effects of vitamin D, outside the musculoskeletal system. This review explores the recent advances in knowledge about the non-skeletal effects of vitamin D. Peer-reviewed papers were extracted from research databases using key words, to assess correlations between vitamin D and extra-skeletal diseases and conditions. As per the guidelines of the Preferred Reporting Items for Systematic Reviews (PRISMA); general interpretations of results are included; taking into consideration the broader evidence and implications. This review summarizes current knowledge of the effects of vitamin D status on extra-skeletal tissues with special attention given to relationships between vitamin D status and various diseases commonly affecting adults; the effects of intervention with vitamin D and exposure to sunlight. Evidence suggests that vitamin D facilitates the regulation of blood pressure; and cardiac; endothelial; and smooth muscle cell functions; playing an important role in cardiovascular protection. In addition; 1,25(OH) 2 D improves immunity; subdues inflammation; and reduces the incidence and severity of common cancers; autoimmune diseases and infectious diseases. Almost all adequately powered; epidemiological and biological studies that use; adequate doses of vitamin D supplementation in D-deficient populations have reported favorable outcomes. These studies have concluded that optimizing 25(OH)D status improves the functionality of bodily systems; reduces comorbidities; improves the quality of life; and increases survival. Although accumulating evidence supports biological associations of vitamin D sufficiency with improved physical and mental functions; no definitive evidence exists from well-designed; statistically powered; randomized controlled clinical trials. Nevertheless, most studies point to significant protective effects of vitamin D in humans when the minimum 25(OH)D serum level exceeds 30 ng/mL and is maintained throughout the year. [ABSTRACT FROM AUTHOR]

According to the binding mode of 14- epi -1α,25-dihydroxy-19-nortachysterol in the ligand binding domain of human vitamin D receptor (hVDR), i.e ., 5,6- and 7,8- s - trans configuration that was shown by X-ray co-crystallographic analysis, 7,8- cis -locked 1α,25(OH) 2 D 3 analogs were synthesized. In this paper, the synthesis and biological activity of 2α- and 2β-(3-hydroxypropyl)-7,8- cis -14- epi -1α,25-dihydroxy-19-norvitamin D 3 are reported. The A-ring and CD-ring precursors for the Julia-Kociensky coupling reaction to create a diene system of the target molecules were prepared using our original methods. hVDR binding affinity and osteocalcin promoter transactivation activity of the new 7,8- cis -14- epi -vitamin D 3 analogs were evaluated. Interestingly, the 2β-substituted 7,8- cis -analog was a better binder for hVDR than the 2α-isomeric counterpart. [ABSTRACT FROM AUTHOR]

Within Europe, dark-skinned ethnic groups have been shown to be at much increased risk of vitamin D deficiency compared to their white counterparts. Increasing the dietary supply of vitamin D is potentially the only modifiable environmental component that can be used to prevent vitamin D deficiency among dark-skinned ethnic groups living at high latitude. Empirical data to support development of such strategies is largely lacking. This paper presents the development and validation of an integrated model that may be adapted within the UK population to design fortification strategies for vitamin D, for application in both white and black and Asian minority ethnic (BAME) population groups. Using a step-wise approach, models based on available ultraviolet B (UVB) data, hours of sunlight and two key components (the dose-response of serum 25-hydroxyvitamin D [25(OH)D] to UVB in white and BAME persons and the dose-response of 25(OH)D to vitamin D) were used to predict changes population serum 25(OH)D concentrations throughout the year, stratified by ethnicity, 'via increases' in dietary intake arising from food fortification simulations. The integrated model successfully predicted measured average wintertime 25(OH)D concentrations in addition to the prevalence of serum 25(OH)D <30 nmol/L in adult white and BAME individuals (18–70 y) in the UK-based National Diet and Nutrition Survey both separately (21.7% and 49.3% predicted versus 20.2% and 50.5% measured, for white and BAME, respectively) and when combined at UK population-relevant proportions of 97% white and 7% BAME (23.2% predicted versus 23.1% measured). Thus this integrated model presents a viable approach to estimating changes in the population concentrations of 25(OH)D that may arise from various dietary fortification approaches. [ABSTRACT FROM AUTHOR]

Studies in vitro have demonstrated a key molecular role for 1,25-dihydroxyvitamin D (1,25D) in skeletal muscle function, with vitamin D -deficiency (low serum 25-hydroxyvitamin D, 25D) being associated with muscle pain and weakness. Despite this, an understanding of the overall role of vitamin D in muscle health (particularly the impact of vitamin D -related genetic variants) has yet to be fully resolved, relative to more well-studied targets such as the skeleton. Thus, we aimed to review existing studies that have investigated relationships between skeletal muscle function and single nucleotide polymorphisms (SNPs) within vitamin D -related genes. A systematic review of papers published between January 2000 and June 2022 on PubMed, EMBASE and Web of Science pertaining to association between functionally relevant vitamin D receptor genetic variants and variants within genes of the vitamin D pathway and skeletal muscle function/outcomes was performed. 21 articles were included in the review for final analysis, of which 20 only studied genetic variation of the VDR gene. Of the included articles, 81 % solely included participants aged ≥ 50 years and of the 9 studies that did not only include White individuals, only 2 included Black participants. Within the vitamin D system, the VDR gene is the primary gene of which associations between polymorphisms and muscle function have been investigated. VDR polymorphisms have been significantly associated with muscle phenotypes in two or more studies. Of note A1012G was significantly associated with higher handgrip strength, but the results for other SNPs were notably variable between studies. While the lack of definitive evidence and study heterogeneity makes it difficult to draw conclusions, the findings of this review highlight a need for improvements with regards to the use of more diverse study populations, i.e., inclusion of Black individuals and other people of colour, and expanding research scope beyond the VDR gene. • The role of the vitamin D system in skeletal muscle health (particularly the impact of genetic variance)is unclear. • We reviewed studies exploring relationships between skeletal muscle function and SNPs of vitamin D-related genes. • VDR is the main gene of the vitamin D system in which SNPs have been investigated in relation to skeletal muscle function. • Variability in results, methodologies, and population demographics across studies makes it difficult to draw conclusions. • Future studies should expand research scope beyond VDR gene and be conducted in more diverse study populations. [ABSTRACT FROM AUTHOR]

Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group “Sulfated Steroids in Reproduction” was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction. [ABSTRACT FROM AUTHOR]

As a continuation of our efforts directed to the structure-activity relationship studies of vitamin D compounds, we present in this paper the synthesis of new analogues of 1α,25-(OH) 2 D 3 characterized by numerous structural modifications, especially a cleaved D ring. Total synthesis of the CD fragment required for the construction of the target vitamins was based on the Stork approach. The structure of the key intermediate – bicyclic hydroxy lactone – was established by crystallographic and electronic circular dichroism (ECD) spectral analysis. Following the attachment of the hydroxyalkyl side chain, the formed D-seco Grundmann ketone was subjected to Wittig-Horner coupling with the corresponding A-ring phosphine oxides providing two desired D-seco analogues of 19-nor-1α,25-(OH) 2 D 3 , one without a substituent at C-2 and the other possessing a 2-exomethylene group. Both compounds were biologically tested and the latter was found to be more active in in vitro tests. Despite so many structural changes introduced in its structure, the biological activity of the 2-methylene analogue approached that of the natural hormone. The synthesized D-seco vitamins, however, proved to be inactive on bone and intestine in vivo . [ABSTRACT FROM AUTHOR]

The presence of the vertebrate steroids, testosterone (T) and 17β-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel ( Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([ 3 H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24 h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5 μg L −1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [ 3 H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex. [ABSTRACT FROM AUTHOR]

Adrenocortical carcinoma (ACC) is a rare, often fatal disease, that may be seen sporadically or with hereditary predisposition syndromes. Patients with ACC are usually girls under the age of seven who present signs of excess production of adrenal glucocorticoids and androgens, with the diagnosis being confirmed by imaging. Here we reproduce and examine what we believe to be the first autopsy case report of a child with ACC, reported by Dr. Henry Sampson in Philosophical Transactions , published by The Royal Society of London in 1697. The paper describes the autopsy of a girl with severe virilization and profound signs of Cushing syndrome who died at age six, strongly suggesting ACC. She apparently had extensive pulmonary metastases, and may have had liver involvement. The report indicates her disease arose from her left kidney and there is no indication of an adrenal origin, perhaps because the adrenal gland was not generally known as a separate organ at that time. This classic example of an early case report is particularly instructive in the context of medical knowledge and understanding in the 17th century compared to current knowledge. [ABSTRACT FROM AUTHOR]

1,25-dihydroxyvitamin D 3 (1,25D), a steroid hormone which regulates calcium/phosphate homeostasis, has a broad spectrum of anti-cancer activities, including differentiation of acute myeloid leukemia (AML) cells. In order to avoid undesirable side effects such as hypercalcemia, low-calcemic analogues should be produced for therapeutic purposes. In this paper, we describe biological activities of double-point modified analogues of vitamin D 2 and we compare them to 1,25D and to paricalcitol, the drug used to treat secondary hyperparathyroidism. In vivo , our new analogues have lower calcemic effects, and lower toxicity in comparison to 1,25D. They have enhanced pro-differentiating and transcription-inducing activities in AML cells. Interestingly, differentiation effects do not correlate with the affinities of the analogues to the vitamin D receptor (VDR). [ABSTRACT FROM AUTHOR]

The Department of Nutrition for Health and Development of the World Health Organization (WHO) in collaboration with the Executive Committee of the 18th Vitamin D Workshop (VDW), organised a joint symposium on the prevention and consequences of vitamin D deficiency in pregnant women and children, convening experts on vitamin D, clinicians and policy-makers. The overall aim was to identify priority areas for research and to discuss the need for global options for policy, with a focus on the prevention of rickets in infants and children and vitamin D deficiency in pregnant women. The scope and purpose were: (i) to present the WHO research strategy for health, addressing vitamin D-related public health problems and the process for the development of evidence-informed guidelines in general and how vitamin D interventions in diverse populations could be prioritised; (ii) to provide an overview of vitamin D status in children and pregnant and lactating women across the world; (iii) to review the health risks associated with vitamin D deficiency in children and in pregnant women and their offspring; (iv) to understand the aetiology of vitamin D deficiency in pregnant women and children; (v) to identify and interpret biomarkers to assess vitamin D status and to consider possible clinical and biochemical screening tools for determining the prevalence of nutritional rickets in at risk groups or communities; and (vi) to provide an overview of policies and recommendations on vitamin D across the world. The format of the symposium was a composite of comprehensive scientific presentations and a panel debate with international experts on WHO guidelines, nutritional rickets, nutritional policy and consequences of vitamin D deficiency during pregnancy. This paper summarizes the content and outcomes of the panel debate. [ABSTRACT FROM AUTHOR]

As part of the World Cancer Research Fund/American Institute for Cancer Research (WCRF–AICR) Continuous Update project we performed a systematic review of prospective studies with data for both measured or predicted 25(OH)D concentration and kidney cancer risk. PubMed was searched from inception until 1st December 2014 using WCRF/AICR search criteria. The search identified 4 papers suitable for inclusion, reporting data from three prospective cohort studies, one nested case-control study and the Vitamin D Pooling Project of Rarer Cancers (8 nested case-control studies). Summary effect sizes could not be computed due to incompatibility between studies. All studies except the Pooling Project suggested a reduced risk of kidney cancer by 19–40% with higher or adequate vitamin D status,. However, these estimates only reached statistical significance in one cohort (Copenhagen City Heart Study; CCHS, HR = 0.75 (0.58 to 0.96)). In the European Prospective Investigation into Cancer and Nutrition (EPIC) study, a significant reduction in risk by 18% was seen when using combined matched and non-matched controls OR = 0.82 (0.68, 0.99), but not when using only matched controls (OR = 0.81 (0.65, 1.00). Pooled (but not single cohort) data for predicted 25(OH)D from the Nurses' Health Study (NHS) and Health Professionals Follow-up Study (HPFS) showed a statistically significant reduction in risk by 37% (HR = 0.63 (0.44, 0.91)). There is no clear explanation for the inconsistency of results between studies, but reasons may include prevalence of smoking or other study population characteristics. Methods for assessing circulating 25(OH)D levels and control for confounders including seasonality or hypertension do not seem explanatory. [ABSTRACT FROM AUTHOR]

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2–FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1–3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1–3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D -induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation. • Mutations affecting genes encoding fibroblast growth factor receptors are seen in many malignancies. • We overexpressed fibroblast growth factor receptors in acute myeloid leukemia cells. • Vitamin D receptor gene expression and activity was increased in the transfected cells. • 1,25-dihydroxyvitamin D -induced cell differentiation was enhanced in the transfected cells. • Overexpression of fibroblast growth factor receptor in bone cancer cells did not lead to the above effects. [ABSTRACT FROM AUTHOR]

It is predicted that around 20% of the worlds population will be age 60 or above by 2050. Prevalence of cognitive decline and dementia is high in older adults and modifiable dietary factors may be able to reduce risk for these conditions. Phytoestrogens are bioactive plant chemicals found in soy, which have a similarity in structure to natural estradiol (the most abundant circulating estrogen). This structural likeness enables phytoestrogens to interact with estrogen receptors in the brain, potentially affecting cognition. However, findings in this domain are largely inconsistent, with approximately 50% of studies showing positive effects of phytoestrogens on cognition and the other half resulting in null/negative findings. This paper provides an updated review of the relationship between consumption of phytoestrogens and risk for cognitive decline and/or dementia. In particular, possible mediators were identified to explain discrepant findings and for consideration in future research. A case can be made for a link between phytoestrogen consumption, thyroid status and cognition in older age, although current findings in this area are very limited. Evidence suggests that inter-individual variants that can affect phytoestrogen bioavailability (and thus cognitive outcome) include age and ability to breakdown ingested phytoestrogens into their bioactive metabolites. Factors of the study design that must be taken into account are type of soy product, dosage, frequency of dietary intake and type of cognitive test used. Guidelines regarding optimal phytoestrogen dosage and frequency of intake are yet to be determined. [ABSTRACT FROM AUTHOR]

Background Brain sex differences are found in nearly every region of the brain and fundamental to sexually dimorphic behaviors as well as disorders of the brain and behavior. These differences are organized during gestation and early adolescence and detectable prior to puberty. Endocrine disrupting compounds (EDCs) interfere with hormone action and are thus prenatal exposure is hypothesized to disrupt the formation of sex differences, and contribute to the increased prevalence of pediatric neuropsychiatric disorders that present with a sex bias. Objective Available evidence for the ability of EDCs to impact the emergence of brain sex differences in the rodent brain was reviewed here, with a focus on effects detected at or before puberty. Methods The peer-reviewed literature was searched using PubMed, and all relevant papers published by January 31, 2015 were incorporated. Endpoints of interest included molecular cellular and neuroanatomical effects. Studies on behavioral endpoints were not included because numerous reviews of that literature are available. Results The hypothalamus was found to be particularly affected by estrogenic EDCs in a sex, time, and exposure dependent manner. The hippocampus also appears vulnerable to endocrine disruption by BPA and PCBs although there is little evidence from the pre-pubertal literature to make any conclusions about sex-specific effects. Gestational EDC exposure can alter fetal neurogenesis and gene expression throughout the brain including the cortex and cerebellum. The available literature primarily focuses on a few, well characterized EDCs, but little data is available for emerging contaminants. Conclusion The developmental EDC exposure literature demonstrates evidence of altered neurodevelopment as early as fetal life, with sex specific effects observed throughout the brain even before puberty. [ABSTRACT FROM AUTHOR]

This introductory chapter provides an overview of the levels and sites at which endocrine disruptors (EDs) affect steroid actions. In contrast to the special issue of Journal of Steroid Biochemistry and Molecular Biology published three years ago and devoted to EDs as such, this paper focuses on steroids. We tried to point to more recent findings and opened questions. EDs interfere with steroid biosynthesis and metabolism either as inhibitors of relevant enzymes, or at the level of their expression. Particular attention was paid to enzymes metabolizing steroid hormones to biologically active products in target cells, such as aromatase, 5α-reductase and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases. An important target for EDs is also steroid acute regulatory protein (StAR), responsible for steroid precursor trafficking to mitochondria. EDs influence receptor-mediated steroid actions at both genomic and non-genomic levels. The remarkable differences in response to various steroid-receptor ligands led to a more detailed investigation of events following steroid/disruptor binding to the receptors and to the mapping of the signaling cascades and nuclear factors involved. A virtual screening of a large array of EDs with steroid receptors, known as in silico methods ( computer simulation), is another promising approach for studying quantitative structure activity relationships and docking. New data may be expected on the effect of EDs on steroid hormone binding to selective plasma transport proteins, namely transcortin and sex hormone-binding globulin. Little information is available so far on the effects of EDs on the major hypothalamo–pituitary–adrenal/gonadal axes, of which the kisspeptin/GPR54 system is of particular importance. Kisspeptins act as stimulators for hormone-induced gonadotropin secretion and their expression is regulated by sex steroids via a feed-back mechanism. Kisspeptin is now believed to be one of the key factors triggering puberty in mammals, and various EDs affect its expression and function. Finally, advances in analytics of EDs, especially those persisting in the environment, in various body fluids (plasma, urine, seminal fluid, and follicular fluid) are mentioned. Surprisingly, relatively scarce information is available on the simultaneous determination of EDs and steroids in the same biological material. This article is part of a Special Issue entitled ‘Endocrine disruptors & steroids’. [ABSTRACT FROM AUTHOR]

The metabolism of steroids and retinoids has been studied in detail for a long time, as these compounds are involved in a broad spectrum of physiological processes. Many enzymes participating in the conversion of such compounds are members of the short-chain dehydrogenase/reductase (SDR) superfamily. Despite great effort, there still remain a number of poorly characterized SDR proteins. According to various bioinformatics predictions, many of these proteins may play a role in the metabolism of steroids and retinoids. Dehydrogenase/reductase (SDR family) member 7 (DHRS7) is one such protein. In a previous study, we determined DHRS7 to be an integral membrane protein of the endoplasmic reticulum facing the lumen which has shown at least in vitro NADPH-dependent reducing activity toward several eobiotics and xenobiotics bearing a carbonyl moiety. In the present paper pure DHRS7 was used for a more detailed study of both substrate screening and an analysis of kinetics parameters of the physiologically important substrates androstene-3,17-dione, cortisone and all- trans -retinal. Expression patterns of DHRS7 at the mRNA as well as protein level were determined in a panel of various human tissue samples, a procedure that has enabled the first estimation of the possible biological function of this enzyme. DHRS7 is expressed in tissues such as prostate, adrenal glands, liver or intestine, where its activity could be well exploited. Preliminary indications show that DHRS7 exhibits dual substrate specificity recognizing not only steroids but also retinoids as potential substrates and could be important in the metabolism of these signalling molecules. [ABSTRACT FROM AUTHOR]

Evidence accumulated in recent years suggests that the systemic treatment with neuroactive steroids, or the pharmacological modulation of its production by brain cells, represent therapeutic options to promote neuroprotection. However, new findings, which are reviewed in this paper, suggest that the factors to be considered for the design of possible therapies based on neuroactive steroids are more complex than previously thought. Thus, although as recently reported, the nervous system regulates neuroactive steroid synthesis and metabolism in adaptation to modifications in peripheral steroidogenesis, the neuroactive steroid levels in the brain do not fully reflect its levels in plasma. Even, in some cases, neuroactive steroid level modifications occurring in the nervous tissues, under physiological and pathological conditions, are in the opposite direction than in the periphery. This suggests that the systemic treatment with these molecules may have unexpected outcomes on neural steroid levels. In addition, the multiple metabolic pathways and signaling mechanisms of neuroactive steroids, which may change from one brain region to another, together with the existence of regional and sex differences in its neural levels are additional sources of complexity that should be clarified. This complexity in the levels and actions of these molecules may explain why in some cases these molecules have detrimental rather than beneficial actions for the nervous system. This article is part of a Special Issue entitled ‘Steroid Perspectives’. [ABSTRACT FROM AUTHOR]

Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism studies of ATD in horses, with an objective of identifying the target metabolites with the longest detection time for controlling ATD abuse. In vitro metabolism studies of ATD were performed using homogenized horse liver. ATD was found to be extensively metabolized, and its metabolites could not be easily characterized by gas chromatography/mass spectrometry (GC/MS) due to insufficient sensitivity. Liquid chromatography/high resolution mass spectrometry (LC/HRMS) was therefore employed for the identification of in vitro metabolites. The major biotransformations observed were combinations of reduction of the olefin groups and/or the keto group at either C3 or C17 position. In addition, mono-hydroxylation in the D-ring was observed along with reduction of the olefin groups and/or the keto group at C17 position. Fourteen in vitro metabolites, including two epimers of androsta-1,4,6-trien-17-ol-3-one (M1a, M1b), androsta-4,6-diene-3,17-dione (M2), boldione (M3), androsta-4,6-diene-17β-ol-3-one (M4), androsta-4,6-diene-3-ol-17-one (M5), boldenone and epi-boldenone (M6a, M6b), four stereoisomers of hydroxylated androsta-1,4,6-trien-17-ol-3-one (M7a to M7d), and two epimers of androsta-1,4-diene-16α,17-diol (M8a, M8b), were identified. The identities of all metabolites, except M1a, M5, M7a to M7d, were confirmed by matching with authentic reference standards using LC/HRMS. For the in vivo metabolism studies, two thoroughbred geldings were each administered with 800 mg of ATD by stomach tubing. ATD, and twelve out of the fourteen in vitro metabolites, including M1a, M1b, M2, M4, M5, M6, M7a to M7d, M8a and M8b, were detected in post-administration urine. Two additional urinary metabolites, namely stereoisomers of hydroxylated androsta-4,6-dien-17-ol-3-one (M9a, M9b), were tentatively identified by mass spectral interpretation. Elevated level of testosterone was also observed. In post-administration blood samples, only the parent drug, M1b and M2 were identified. This study showed that the detection of ATD administration would be best achieved by either monitoring the metabolites M1b (androsta-1,4,6-trien-17β-ol-3-one) or M4 (both excreted as sulfate conjugates) in urine, which could be detected for up to a maximum of 77 h post-administration. The analyte of choice for plasma is M1b, which could be detected for up to 28 h post administration. [ABSTRACT FROM AUTHOR]

Memory formation and utilization is a complex process involving several brain structures in conjunction as the hippocampus, the amygdala and the adjacent cortical areas, usually defined as medial temporal lobe structures (MTL). The memory processes depend on the formation and modulation of synaptic connectivity affecting synaptic strength, synaptic plasticity and synaptic consolidation. The basic neurocognitive mechanisms of learning and memory are shortly recalled in the initial section of this paper. The effect of sex hormones (estrogens, androgens and progesterone) and of adrenocortical steroids on several aspects of memory processes are then analyzed on the basis of animal and human studies. A specific attention has been devoted to the different types of steroid receptors (membrane or nuclear) involved and on local metabolic transformations when required. The review is concluded by a short excursus on the steroid activated epigenetic mechanisms involved in memory formation. [ABSTRACT FROM AUTHOR]

Over the last decade a convergent body of evidence has emerged from epidemiology, animal experiments and clinical trials which links low vitamin D status with a range of adverse neuropsychiatric outcomes. This research demonstrates that the timing of exposure to low vitamin D influences the nature of brain phenotypes, as exposures during gestation versus adulthood result in different phenotypes. With respect to early life exposures, there is robust evidence from rodent experiments indicating that transient developmental vitamin D (DVD) deficiency is associated with changes in brain structure, neurochemistry, gene and protein expression and behavior. In particular, DVD deficiency is associated with alterations in the dopaminergic neurotransmitter systems. In contrast, recently published animal experiments indicate that adult vitamin D (AVD) deficiency is associated with more subtle neurochemical and behavioral phenotypes. This paper explores key issues that need to be addressed in future research. There is a need to define the timing and duration of the ‘critical window’ during which low vitamin D status is associated with differential and adverse brain outcomes. We discuss the role for ‘two-hit hypotheses’, which propose that adult vitamin D deficiency leaves the brain more vulnerable to secondary adverse exposures, and thus may exacerbate disease progression. Finally, we explore the evidence implicating a role for vitamin D in rapid, non-genomic mechanisms that may involve L-type calcium channels and brain function. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’. [ABSTRACT FROM AUTHOR]

In this paper we tested how oxysterols influence on fusion process between viral lipid envelope and host cells membranes. For this purpose, the Zika virus was selected, while dendritic cell (DC) and neural cell (NC) membranes were chosen as target membranes. The investigated systems were modeled as multicomponent Langmuir monolayers and characterized using surface manometry and imaging in micro- (Brewster angle microscopy, BAM) and nanoscale (Atomic Force Microscopy, AFM) to monitor local heterogeneity. The fusion process was conducted by mixing viral and host cell membranes devoid and in the presence of oxysterols: 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) as representatives of chain- and ring-oxidized oxysterols, respectively. Our results show that oxysterols hinder the fusion with host cell membranes by modifying their biophysical properties. Moreover, oxysterols applied to an already infected membrane reverse the changes caused by the infection. It could therefore be concluded that oxysterols may display antiviral activity in two ways: they prevent the healthy membrane from viral infection by blocking the fusion process; and protect already infected membrane from pathological changes induced by the virus. • Oxysterols' antiviral activity was investigated on the example of ZIKA virus. • Langmuir monolayer technique was used to model virus and hosts membranes. • Fusion was conducted by mixing viral and host membranes without and with oxysterols 25-OH and 7β-OH. • Oxysterols block the fusion with host membranes by modifying film properties. • Oxysterols can reverse the changes caused by the infection. [ABSTRACT FROM AUTHOR]

• The aromatase reaction, converting androgens into estrogens, occurs in three steps, each requiring NADPH and O 2. • In Step 1 , a 19-hydroxy intermediate is produced, which in Step 2 , is converted into a 19-oxo derivative presumably via a gem - diol intermediate with the stereospecific loss of H Re. • In Step 3 , a scission of the C-10-C-19 bond occurs releasing C-19 as formic acid (HCOOH) and incorporating an atom of oxygen from O 2, • The finding of Step3 has been questioned and the present paper deals with this issue. Our previous studies, using precursors for two classes of estrogens, estrone and estriol, have highlighted the following facets of aromatase. The overall reaction, converting androgens into estrogens, occurs in three steps, each requiring NADPH and O 2. In Step 1 , a 19-hydroxy intermediate is produced, which in Step 2 , is converted into a 19-oxo derivative via a gem - diol intermediate with the stereospecific loss of H Re. In Step 3 , a scission of the C-10-C-19 bond occurs releasing C-19 as formic acid (HCOOH) and incorporating an atom of oxygen from O 2, The other oxygen atom of formic acid is derived from the hydroxyl group introduced in Step 1 . These experiments were performed using the classical placental microsomal system. Our findings were confirmed and extended by (the late) Caspi's group. However, incorporation of oxygen in Step 3 , has been challenged in a subsequent study using a soluble reconstituted system. The latter authors have implied the superiority of their system over the microsomal preparation. However, several assumptions under pinning their own work were derived from the use of placental microsomes. Furthermore, the authors have not considered that when a previous work is challenged it needs to be repeated under the conditions described in the original publication. [ABSTRACT FROM AUTHOR]

Abstract: Although breast cancer incidence in the US is highest for women of European ancestry (EA), women of African ancestry (AA) have higher incidence of cancer diagnosed before age 40 and tumors with more aggressive features (high grade and negative for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2)), which precludes targeted therapies and leads to poorer outcomes. It is unclear what underlies these disparities. It has been hypothesized that dark skin with high melanin content is the ancestral skin color of origin, with adaptation to northern environs resulting in lighter skin. Although intense sunlight in sub-Saharan Africa may compensate for low sun absorption through skin, an urban or western lifestyle may result in less synthesis of vitamin D with higher skin pigmentation. Laboratory and preclinical data indicate that vitamin D is involved in preventing breast carcinogenesis and progression. Vitamin D receptor (VDR) knock-out mice are more likely to develop tumors that are ER-negative, and we have shown that serum levels of 25OHD are lowest among EA women with triple-negative tumors (negative for ER, PR and HER2); and among non-cancer patients, vitamin D levels are lower in AAs than in EAs. Thus, it is plausible to hypothesize that low vitamin D levels could be associated with the higher prevalence of more aggressive tumors among AA women. In this paper, we review the current literature on vitamin D and aggressive breast cancer subtypes, discuss vitamin D in AA women from a perspective of evolution and adaption, and examine the potential role of vitamin D in cancer racial disparities. We present our recently published data showing two single nucleotide polymorphisms in vitamin D catabolic enzyme CYP24A1 associated with higher risk of estrogen ER-negative risk in AA than in EA women. The relationship of vitamin D with breast cancer risk may be subtype-specific, with emerging evidence of stronger effects of vitamin D for more aggressive breast cancer, particularly in women of African ancestry. [Copyright &y& Elsevier]

Abstract: Up to the present, numerous vitamin D derivatives have been synthesized, but most of them have straight side chains, and there are few publications described about in vitro and in vivo evaluations on bone by vitamin D derivatives. In our previous paper, we reported the synthesis of various C-2 substituted vitamin D derivatives (2b–2i) with a 2,2-dimethylcyclopentanone unit in the CD-ring side chains, and that the derivatives have strong activity for enhancing bone growth. On the basis of results, this time, we report the synthesis of 2α-substituted vitamin D3 derivatives with chiral cyclopentanone (3–6 and 12–16). These derivatives were obtained by Pd-coupling reaction with A-ring precursor and CD-rings precursor. We evaluated novel derivatives in vitro assays, for affinities for VDR and transactivation assays by human osteosarcoma (HOS) cells. In this research, we demonstrated that some novel vitamin D derivatives (12-MP, 13-MP, 15-MP and 16-LP) have strong transactivation activities in spite of lower affinity for VDR than 1. In addition, we also demonstrated that these derivatives have strong activities for enhancing bone growth using OVX therapeutic rats. This article is part of a Special Issue entitled ‘Vitamin D Workshop’. [Copyright &y& Elsevier]

Abstract: Interconversion of hormonally active cortisol (F) into the corresponding inactive 11-keto form, cortisone (E), is catalyzed by 11beta-hydroxysteroid dehydrogenases (11β-HSDs). With a view to estimating in vivo activities of some 11β-HSD isoforms, the measurement of urinary F and E and their tetrahydro metabolites (tetrahydrocortisol, THF, allotetrahydrocortisol, ATHF, tetrahydrocortisone, THE) has been suggested. The basic knowledge of THF, ATHF and THE levels in farm cattle is limited. Therefore the aim of this study was first to optimize a simple and quick method to determine F and E tetrahydro-metabolites in bovine urine by HPLC–mass spectrometry with electrospray ionization (HPLC–ESI-MS) and then to apply the method to real urine of bovines treated with prednisolone. The samples underwent filtration, deconjugation, solid-phase extraction (SPE) and the relevant analytes were measured by HPLC–ESI-MS. The method described in this paper is simple and efficient, featuring good linearity (up to 0.996) and reproducibility (6.8–12.5%, CV). Especially, good LODs were obtained, from 1.63 to 2.67ppb, depending on the analyte. The chromatographic conditions were optimized in order to obtain a resolution which would allow to simultaneously measure two diastereoisomers, i.e. THF and ATHF. In our study, ATHF turns out to be below the detection limit, while for 18 samples tested the contents of examinated metabolites were as followed: THF (12.5±4.8ppb), THE (10.9±5.5ppb), F (11.6±3.3ppb) and E (5.0±2.2ppb). When the method was applied to the subject treated with prednisolone a major increase in the concentration of tetrahydro metabolites was observed before the slaughter, mainly due to stress conditions; prednisolone treatment, most presumably, influenced the 11β-HSD activity, as indicated by the decrease in the F/E ratio. This work may provide a useful methodological contribution to the future definition of F, E, THF, ATHF and THE urinary baseline values in order to obtain indirect evaluations of HSDs activity in farm cattle and possible applications in screenings for suspected abuse of synthetic corticosteroids in bovines. [Copyright &y& Elsevier]

Abstract: Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ1-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ4-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism. [Copyright &y& Elsevier]

Abstract: Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D3 (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells. [Copyright &y& Elsevier]

Abstract: Progesterone affects lipid metabolism in adipose tissue and influences fat distribution in human. The aim of the study was to analyze the effect of progesterone on rat body and fat mass and on expression of genes encoding adipokines involved in the regulation of energy homeostasis. The results presented here indicate that progesterone administration to females caused increase in body and inguinal white adipose tissue mass. The increase of inguinal white adipose tissue mass is associated with the hypertrophy of adipocyte. The same dose of progesterone caused increase of its circulating concentration in males, however it barely reached the value observed in non-treated control females and did not have any effect on body and fat mass. The elevated circulating progesterone concentration was associated with an approximately 6- and 2-fold increase of leptin and resistin mRNA level respectively, and 2-fold decrease of adiponectin mRNA level only in inguinal white adipose tissue of females. RU 486, specific antagonist of progesterone receptor, abolished the effect of progesterone on the adipokine mRNA level in inguinal adipose tissue. In males, the elevated circulating progesterone concentration showed no effects on leptin, resistin or adiponectin mRNA level in inguinal, retroperitoneal or epididymal adipose tissue. Moreover, the results presented in this paper demonstrate a relatively high level of progesterone receptor mRNA in inguinal white adipose tissue of females, which was down-regulated in response to progesterone administration. In retroperitoneal adipose tissue of control females progesterone receptor mRNA level was approximately 3-fold lower as compared to inguinal adipose tissue. In inguinal, epididymal and retroperitoneal white adipose tissue of males progesterone receptor mRNA was hardly detected. Our results suggest that depot- and sex-dependent responsiveness of adipose tissue to the pharmacological dose of progesterone is controlled by both circulating concentration of progesterone and the white adipose tissue progesterone receptor level. [Copyright &y& Elsevier]

Abstract: The relationships between the parameters of metabolic syndrome and non-aromatizable metabolites of testosterone have been discussed in literature. Some papers describe these metabolites as one of the possible causes of male-type obesity. On the contrary, other studies show a protective influence of dihydrotestosterone on visceral obesity. The aim of this study to analyse the relationship between anthropometric parameters, lipid spectrum, glycemia and the level of endogenous testosterone and dihydrotestosterone, and to compare the effects of these androgens. Our population-based study involved 232 healthy men ranging from 20 to 78 years with BMI 18 to 39kg/m2. Serum testosterone, dihydrotestosterone and sex hormone binding globulin SHBG levels, lipid spectrum, glucose metabolism parameters were measured and the oral glucose tolerance test was carried out in all subjects. Their anthropometric parameters (weight, height, waist, hips, waist-to-hip ratio, 14 skin folds) and body composition parameters were determined and calculated by the Antropo program. Multiple regression analysis showed a correlation between hormonal levels, esp. of testosterone and dihydrotestosterone, and the anthropometric data, lipid spectrum and parameters of glucose regulation. Low testosterone and/or dihydrotestosterone was correlated to a higher body-mass index, fat content, waist diameter, total-, HDL-, LDL-cholesterol and triglycerides, fasting glucose, insulin resistance and lower muscle and bone mass. In addition, statistical analysis using multivariate regression with reduction in dimensionality did not discover any striking difference between aromatizable and non-aromatizable androgens in their association to lipid and glucose metabolism parameters in healthy, normosthenic men. In conclusion, the association of endogenous testosterone and dihydrotestosterone to anthropometric data, lipid spectrum and insulin sensitivity are of the same quality; however, the effect of the circulating levels of dihydrotestosterone is quantitatively smaller. [Copyright &y& Elsevier]

Abstract: CCR5 and CXCR4 play an important role in the establishment of HIV infection and disease progression. Caucasian people exposed to HIV but uninfected (EU) present a deletion of 32bp in CCR5 that has not been reported in EU Hispanics from Latin America. Therefore, other factors besides mutations should be involved in this phenomenon. Studies in healthy women have shown that sex hormones such as progesterone (P) can modulate CCR5/CXCR4 expression through an unknown mechanism. The aim of this paper was to determine the role of P in the regulation of CCR5 and CXCR4 in peripheral blood mononuclear cells (PBMCs) of HIV-1 infected and EU women. We analyzed HIV-1-infected women with stable highly active antiretroviral therapy (HAART) with CD4+ cell counts <400/mm3 or diminution of 20%, EU and HIV-1 seronegative healthy controls. 5×106 PBMCs, from HIV-1 infected women, EU women and HIV-1 seronegative healthy controls were cultured and incubated with P (10 or 100nM), RU486 (P antagonist, 1μM) or P (100nM)+RU486 (1μM). CCR5/CXCR4 content was determined by Western blot. Densitometry data were analyzed using Mann–Whitney test. We found that CCR5 content was reduced by P in all groups. In contrast, CXCR4 content was increased by P in healthy controls and in HIV-1 infected women. Interestingly, CXCR4 content was reduced by P in EU. RU486 did not block P effects in any group. These findings suggest that P should participate in the acquisition and progression of HIV-1 infection by modulating CCR5 and CXCR4 expression. P could contribute to the resistance acquisition of HIV by EU through the down-regulation of both coreceptors. [Copyright &y& Elsevier]

Abstract: Classically, the estrogen signaling system has two core components: cytochrome P450 aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact with the regulatory region of target genes to mediate the biological effects of estrogen. While the importance of estrogens for regulation of reproduction, development and physiology has been well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g., the amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules and pathways. To address this question, this paper briefly reviews relevant earlier studies that help to illuminate the history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates. We then present new analyses of amphioxus aromatase and ER sequence and function, including an in silico model of the amphioxus aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with vertebrates (9 coding exons) and moderate sequence conservation (40% amino acid identity with human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated docking of androstenedione in comparison to the human aromatase shows that the substrate binding site is conserved and predicts that androstenedione could be a substrate for amphioxus CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and key residues of the ligand binding domain. Consistent with results from other laboratories, amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence of estradiol, 4-hydroxytamoxifen, diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene, the amphioxus “steroid receptor” (SR), can be activated by estrogens and that amphioxus ER can repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue, suggesting an ancient paracrine/autocrine signaling role, but it is not yet known how their expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a role in mediating any biological effects. Functional studies are clearly needed to link emerging bioinformatics and in vitro molecular biology results with organismal physiology to develop an understanding of the evolution of estrogen signaling. This article is part of a Special Issue entitled ‘Marine organisms’. [Copyright &y& Elsevier]

Abstract: Concerning temporal trends in human reproductive health has prompted concern about the role of environmentally mediated risk factors. The population is exposed to chemicals present in air, water, food and in a variety of consumer and personal care products, subsequently multiple chemicals are found human populations around the globe. Recent reviews find that endocrine disrupting chemicals (EDCs) can adversely affect reproductive and developmental health. However, there are still many knowledge gaps. This paper reviews some of the key scientific concepts relevant to integrating information from human epidemiologic and model organisms to understand the relationship between EDC exposure and adverse human health effects. Additionally, areas of new insights which influence the interpretation of the science are briefly reviewed, including: enhanced understanding of toxicity pathways; importance of timing of exposure; contribution of multiple chemical exposures; and low dose effects. Two cases are presented, thyroid disrupting chemicals and anti-androgens chemicals, which illustrate how our knowledge of the relationship between EDCs and adverse human health effects is strengthened and data gaps reduced when we integrate findings from animal and human studies. [Copyright &y& Elsevier]

Abstract: cRNA microarray and real-time PCR (qPCR) studies from our lab identified five Cell Cycle Pathway (CCP) genes (CCNA2, CCNE2, CDC25A, CDKN1B, and PLK-1) as targets for luteolin in PC-3 prostate cancer cells [Shoulars et al., J. Steroid Biochem. Mol. Biol. 118 (2010) 41–50]. In this paper, Ingenuity Pathway Analysis of the microarray data identified 7 luteolin-regulated genes (EGFR, c-Fos, SOS, GRB2, JNK1, MKK4 and RasGAP) in the Epidermal Growth Factor Signaling Pathway (EGFSP) potentially involved in luteolin regulation of CCP genes and cell proliferation. To address these possibilities, we compared the response profiles (RNA and protein) of these EGFSP and CCP genes to luteolin and gefitinib by real-time PCR (qPCR) and Western blot analyses. Luteolin and gefitinib are known antagonists of EGFR-associated tyrosine protein kinase. Thus, the response profiles of EGFR regulated EGFSP or CCP genes should be very similar if genes in both pathways are controlled through this common mechanism of action. Treatment of PC-3 cell with luteolin for 24h caused a 4-fold stimulation of c-Fos gene expression, significant inhibition (p <0.001) of the CCP genes and G2/M arrest. Treatment of PC-3 cells with gefitinib also inhibited most of the CCP genes in a fashion similar to that of luteolin, however, the EGFR antagonist inhibited c-Fos gene expression, stimulated CDKN1B (p27) and arrested the cells in G0/G1. Thus, although the response patterns of most of the CCP genes to luteolin or gefitinib were similar, the effects of the two compounds on EGFSP gene expression and cell cycle arrest were clearly different. Combination studies revealed that the response of EGFSP genes to luteolin was not affected by gefitinib, even though the two compounds were additive with respect to their abilities to inhibit CCNA2, CCNE2, CDC25A and PCNA. These findings suggest that luteolin and gefitinib regulate CCP gene expression through a common mechanism involving EGFR-associated tyrosine kinase. Conversely, luteolin regulates PC-3 cell proliferation through an EGFR-tyrosine kinase independent mechanism(s), likely involving the epigenetic control of gene EGFSP gene expression through histone H4 binding interactions resulting in the upregulation of c-Fos and p21 gene expression. [Copyright &y& Elsevier]