A Novel Mutation in the INSR Gene Causes Severe Insulin Resistance and Rabson-Mendenhall Syndrome in a Paraguayan Patient.

Rabson-Mendenhall syndrome (RMS) is a rare autosomal recessive disorder characterized by severe insulin resistance, resulting in early-onset diabetes mellitus. We report the first case of RMS in a Paraguayan patient. The patient is a 6-year-old girl who presented with hypertrichosis, acanthosis nigricans, nephrocalcinosis, and elevated levels of glucose and insulin that served as diagnostic indicators for RMS. Genetic testing by next-generation sequencing (NGS) revealed two pathogenic variants in exons 2 and 19 of the INSR gene: c.332G>T (p.Gly111Val) and c.3485C>T (p.Ala1162Val), in combined heterozygosis. The novel INSR c. 332G>T variant leads to the substitution of glycine to valine at position 111 in the protein, and multiple in silico software programs predicted it as pathogenic. The c.3485C>T variant leads to the substitution of alanine to valine at position 1162 in the protein previously described for insulin resistance and RMS. The management of RMS is particularly challenging in children, and the use of metformin is often limited by its side effects. The patient was managed with nutritional measures due to the early age of onset. This report expands the knowledge of RMS to the Paraguayan population and adds a novel pathogenic variant to the existing literature.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

Characterization of Two Novel Variants of the Steroidogenic Acute Regulatory Protein Identified in a Girl with Classic Lipoid Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) consists of several autosomal recessive disorders that inhibit steroid biosynthesis. We describe a case report diagnosed with adrenal insufficiency due to low adrenal steroids and adrenocorticotropic hormone excess due to lack of cortisol negative feedback signaling to the pituary gland. Genetic work up revealed two missense variants, p.Thr204Arg and p.Leu260Arg in the STAR gene, inherited by both parents (non-consanguineous). The StAR protein supports CYP11A1 enzyme to cleave the side chain of cholesterol and synthesize pregnenolone which is metabolized to all steroid hormones. We used bioinformatics to predict the impact of the variants on StAR activity and then we performed functional tests to characterize the two novel variants. In a cell system we tested the ability of variants to support cholesterol conversion to pregnenolone and measured their mRNA and protein expression. For both variants, we observed loss of StAR function, reduced protein expression and categorized them as pathogenic variants according to guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. These results fit the phenotype of the girl during diagnosis. This study characterizes two novel variants and expands the list of missense variants that cause CAH.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase.

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

Bioactivity of Curcumin on the Cytochrome P450 Enzymes of the Steroidogenic Pathway.

Turmeric, a popular ingredient in the cuisine of many Asian countries, comes from the roots of the Curcuma longa and is known for its use in Chinese and Ayurvedic medicine. Turmeric is rich in curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Curcuminoids have potent wound healing, anti-inflammatory, and anti-carcinogenic activities. While curcuminoids have been studied for many years, not much is known about their effects on steroid metabolism. Since many anti-cancer drugs target enzymes from the steroidogenic pathway, we tested the effect of curcuminoids on cytochrome P450 CYP17A1, CYP21A2, and CYP19A1 enzyme activities. When using 10 µg/ml of curcuminoids, both the 17α-hydroxylase as well as 17,20 lyase activities of CYP17A1 were reduced significantly. On the other hand, only a mild reduction in CYP21A2 activity was observed. Furthermore, CYP19A1 activity was also reduced up to ~20% of control when using 1-100 µg/ml of curcuminoids in a dose-dependent manner. Molecular docking studies confirmed that curcumin could dock onto the active sites of CYP17A1, CYP19A1, as well as CYP21A2. In CYP17A1 and CYP19A1, curcumin docked within 2.5 Å of central heme while in CYP21A2 the distance from heme was 3.4 Å, which is still in the same range or lower than distances of bound steroid substrates. These studies suggest that curcuminoids may cause inhibition of steroid metabolism, especially at higher dosages. Also, the recent popularity of turmeric powder as a dilatory supplement needs further evaluation for the effect of curcuminoids on steroid metabolism. The molecular structure of curcuminoids could be modified to generate better lead compounds with inhibitory effects on CYP17A1 and CYP19A1 for potential drugs against prostate cancer and breast cancer.

Cell-cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells.

FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell-cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1-Ig2-Ig3 or the Ig2-Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a ß-sheet, which is part of a larger ß-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell-cell fusion.

Specificity of anti-prostate cancer CYP17A1 inhibitors on androgen biosynthesis.

The orteronel, abiraterone and galeterone, which were developed to treat castration resistant prostate cancer, inhibit 17,20 lyase activity but little is known about their effects on adrenal androgen biosynthesis. We studied the effect of several inhibitors and found that orteronel was selective towards 17,20 lyase activity than abiraterone and galeterone. Gene expression analysis showed that galeterone altered the expression of HSD3B2 but orteronel did not change the expression of HSD3B2, CYP17A1 and AKR1C3. The CYP19A1 activity was not inhibited except by compound IV which lowered activity by 23%. Surprisingly abiraterone caused complete blockade of CYP21A2 activity. Analysis of steroid metabolome by gas chromatography - mass spectrometry revealed changes in steroid levels caused by different inhibitors. We can conclude that orteronel is a highly specific inhibitor of 17,20 lyase activity. The discovery of these specific drug actions on steroidogenic enzyme activities would be valuable for understanding the regulation of androgens.

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria.

Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3% of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16% of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30% of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.

Mutations and polymorphisms in the human argininosuccinate lyase (ASL) gene.

Argininosuccinate lyase deficiency (ASLD) is caused by a defect of the urea cycle enzyme argininosuccinate lyase (ASL) encoded by the ASL gene. Patients often present early after birth with hyperammonemia but can also manifest outside the neonatal period mainly triggered by excessive protein catabolism. Clinical courses comprise asymptomatic individuals who only excrete the biochemical marker, argininosuccinic acid, in urine, and patients who succumb to their first hyperammonemic decompensation. Some patients without any hyperammonemia develop severe neurological disease. Here, we are providing an update on the molecular basis of ASLD by collecting all published (n = 67) as well as novel mutations (n = 67) of the ASL gene. We compile data on all 160 different genotypes ever identified in 223 ASLD patients, including clinical courses whenever available. Finally, we are presenting structural considerations focusing on the relevance of mutations for ASL homotetramer formation. ASLD can be considered as a panethnic disease with only single founder mutations identified in the Finnish (c.299T>C, p.Ile100Thr) and Arab (c.1060C>T, p.Gln354*) population. Most mutations are private with only few genotypes recurring in unrelated patients. The majority of mutations are missense changes including some with more frequent occurrence such as p.Arg12Gln, p.Ile100Thr, p.Val178Met, p.Arg186Trp, p.Glu189Gly, p.Gln286Arg, and p.Arg385Cys.

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation.

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.

STAR splicing mutations cause the severe phenotype of lipoid congenital adrenal hyperplasia: insights from a novel splice mutation and review of reported cases.

OBJECTIVE: The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only. PATIENT: A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycaemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals, and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone. DESIGN, METHODS AND RESULTS: Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4-6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5-7 which are essential for StAR-cholesterol interaction. CONCLUSIONS: STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype.

Innovative Delivery Systems for Curcumin: Exploring Nanosized and Conventional Formulations.

Curcumin, a polyphenol with a rich history spanning two centuries, has emerged as a promising therapeutic agent targeting multiple signaling pathways and exhibiting cellular-level activities that contribute to its diverse health benefits. Extensive preclinical and clinical studies have demonstrated its ability to enhance the therapeutic potential of various bioactive compounds. While its reported therapeutic advantages are manifold, predominantly attributed to its antioxidant and anti-inflammatory properties, its efficacy is hindered by poor bioavailability stemming from inadequate absorption, rapid metabolism, and elimination. To address this challenge, nanodelivery systems have emerged as a promising approach, offering enhanced solubility, biocompatibility, and therapeutic effects for curcumin. We have analyzed the knowledge on curcumin nanoencapsulation and its synergistic effects with other compounds, extracted from electronic databases. We discuss the pharmacokinetic profile of curcumin, current advancements in nanoencapsulation techniques, and the combined effects of curcumin with other agents across various disorders. By unifying existing knowledge, this analysis intends to provide insights into the potential of nanoencapsulation technologies to overcome constraints associated with curcumin treatments, emphasizing the importance of combinatorial approaches in improving therapeutic efficacy. Finally, this compilation of study data aims to inform and inspire future research into encapsulating drugs with poor pharmacokinetic characteristics and investigating innovative drug combinations to improve bioavailability and therapeutic outcomes.

Clinical spectrum of human STAR variants and their genotype-phenotype correlation.

Biallelic variants of steroidogenic acute regulatory protein (STAR/STARD1) may cause primary adrenal insufficiency and 46,XY disorder of sex development. STAR plays a pivotal role in transporting cholesterol into mitochondria where cholesterol serves as an essential substrate for initiating steroid biosynthesis by its conversion to pregnenolone. Generally, loss-of-function mutations of STAR cause the classic form of lipoid congenital adrenal hyperplasia (LCAH) where steroidogenesis of the adrenal cortex and the gonads is severely affected. By contrast, partial activity of STAR causes a less severe phenotype, the non-classic LCAH, which is characterized by later onset and initial manifestation with isolated adrenal insufficiency only. Disease-causing STAR variants are very rare. Numerous variants of all types have been described worldwide. Prevailing variants have been reported from Japan and Korea and in some population clusters where STAR is more common. Genotype-phenotype correlation is pretty good for STAR variants. While the exact mechanisms of cholesterol transport into mitochondria for steroidogenesis are still under investigation, the important role of STAR in this process is evident by inactivating STAR variants causing LCAH. The mechanism of disease with STAR deficiency is best described by a two-hit model: the first hit relates to impaired cholesterol import into mitochondria and thus lack of substrate for all steroid hormone biosynthesis; the second hit then relates to massive cytoplasmic lipid overload (evidenced by typically enlarged and fatty adrenal glands) leading to cell death and organ destruction. This review summarizes phenotype and genotype characteristics of human STAR variants found through the ClinVar database.

Pharmacogenomics of poor drug metabolism in greyhounds: Canine P450 oxidoreductase genetic variation, breed heterogeneity, and functional characterization.

Greyhounds metabolize cytochrome P450 (CYP) 2B11 substrates more slowly than other dog breeds. However, CYP2B11 gene variants associated with decreased CYP2B11 expression do not fully explain reduced CYP2B11 activity in this breed. P450 oxidoreductase (POR) is an essential redox partner for all CYPs. POR protein variants can enhance or repress CYP enzyme function in a CYP isoform and substrate dependent manner. The study objectives were to identify POR protein variants in greyhounds and determine their effect on coexpressed CYP2B11 and CYP2D15 enzyme function. Gene sequencing identified two missense variants (Glu315Gln and Asp570Glu) forming four alleles, POR-H1 (reference), POR-H2 (570Glu), POR-H3 (315Gln, 570Glu) and POR-H4 (315Gln). Out of 68 dog breeds surveyed, POR-H2 was widely distributed across multiple breeds, while POR-H3 was largely restricted to greyhounds and Scottish deerhounds (35% allele frequencies), and POR-H4 was rare. Three-dimensional protein structure modelling indicated significant effects of Glu315Gln (but not Asp570Glu) on protein flexibility through loss of a salt bridge between Glu315 and Arg519. Recombinant POR-H1 (reference) and each POR variant (H2-H4) were expressed alone or with CYP2B11 or CYP2D15 in insect cells. No substantial effects on POR protein expression or enzyme activity (cytochrome c reduction) were observed for any POR variant (versus POR-H1) when expressed alone or with CYP2B11 or CYP2D15. Furthermore, there were no effects on CYP2B11 or CYP2D15 protein expression, or on CYP2D15 enzyme kinetics by any POR variant (versus POR-H1). However, Vmax values for 7-benzyloxyresorufin, propofol and bupropion oxidation by CYP2B11 were significantly reduced by coexpression with POR-H3 (by 34-37%) and POR-H4 (by 65-72%) compared with POR-H1. Km values were unaffected. Our results indicate that the Glu315Gln mutation (common to POR-H3 and POR-H4) reduces CYP2B11 enzyme function without affecting at least one other major canine hepatic P450 (CYP2D15). Additional in vivo studies are warranted to confirm these findings.

Effect of Essential Oil Components on the Activity of Steroidogenic Cytochrome P450.

Endocrine-disrupting chemicals (EDCs) may impact the development of prostate cancer (PCa) by altering the steroid metabolism. Although their exact mechanism of action in controlling tumor growth is not known, EDCs may inhibit steroidogenic enzymes such as CYP17A1 or CYP19A1 which are involved in the production of androgens or estrogens. High levels of circulating androgens are linked to PCa in men and Polycystic Ovary Syndrome (PCOS) in women. Essential oils or their metabolites, like lavender oil and tea tree oil, have been reported to act as potential EDCs and contribute towards sex steroid imbalance in cases of prepubertal gynecomastia in boys and premature thelarche in girls due to the exposure to lavender-based fragrances. We screened a range of EO components to determine their effects on CYP17A1 and CYP19A1. Computational docking was performed to predict the binding of essential oils with CYP17A1 and CYP19A1. Functional assays were performed using the radiolabeled substrates or Liquid Chromatography-High-Resolution Mass Spectrometry and cell viability assays were carried out in LNCaP cells. Many of the tested compounds bind close to the active site of CYP17A1, and (+)-Cedrol had the best binding with CYP17A1 and CYP19A1. Eucalyptol, Dihydro-ß-Ionone, and (-)-α-pinene showed 20% to 40% inhibition of dehydroepiandrosterone production; and some compounds also effected CYP19A1. Extensive use of these essential oils in various beauty and hygiene products is common, but only limited knowledge about their potential detrimental side effects exists. Our results suggest that prolonged exposure to some of these essential oils may result in steroid imbalances. On the other hand, due to their effect on lowering androgen output and ability to bind at the active site of steroidogenic cytochrome P450s, these compounds may provide design ideas for novel compounds against hyperandrogenic disorders such as PCa and PCOS.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

OBJECTIVE: Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. DESIGN: Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. METHODS: On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. RESULTS: The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. CONCLUSION: Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.