Plasma Steroid Research Articles

An extension of the RITUX-ERAH study, multicenter randomized clinical trial comparing rituximab to placebo in acute antibody-mediated rejection after renal transplantation.

The treatment of active antibody-mediated rejection (ABMR) is still a matter of debate, the place of rituximab remaining controversial. The French multicenter double-blind RITUX-ERAH study included 38 patients with ABMR in the first year of renal transplantation. All patients received plasma exchanges, intravenous immunoglobulins, and corticosteroids and were randomly assigned rituximab or placebo infusion at day 5. Additional rituximab infusions were allowed. In the intention-to-treat analysis, 12-month graft survival and renal function were not different between the rituximab and placebo groups. Long-term data are needed to conclude. Evaluation of the 7-year outcomes of the RITUX-ERAH study patients according to the rituximab or placebo treatment received. Eleven patients received placebo and 27 at least one infusion of rituximab. Seven years after ABMR, death-censored kidney allograft survival and renal function were not different between the groups. The evolution of anti-HLA sensitization was similar. There was no statistically significant difference in the incidence of infectious or neoplastic complications, but to be noted, seven cancers developed in six patients treated with rituximab (mean period of 44months post-ABMR). In this cohort, there was no benefit 7years after ABMR of rituximab in addition to plasma exchanges, intravenous immunoglobulins, and steroids.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150ng/mL and from 0.020 to 0.500ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Human DENND1A.V2 Drives Cyp17a1 Expression and Androgen Production in Mouse Ovaries and Adrenals.

The DENND1A locus is associated with polycystic ovary syndrome (PCOS), a disorder characterized by androgen excess. Theca cells from ovaries of PCOS women have elevated levels of a DENND1A splice variant (DENND1A.V2). Forced expression of this variant in normal theca cells increases androgen biosynthesis and CYP17A1 expression, whereas knockdown of the transcript in PCOS theca cells reduced androgen production and CYP17A1 mRNA. We attempted to create a murine model of PCOS by expressing hDENND1A.V2 using standard transgenic approaches. There is no DENND1A.V2 protein equivalent in mice, and the murine Dennd1a gene is essential for viability since Dennd1a knockout mice are embryonically lethal, suggesting that Dennd1a is developmentally critical. Three different hDENND1A.V2 transgenic mice lines were created using CMV, Lhcgr, and TetOn promoters. The hDENND1A.V2 mice expressed hDENND1A.V2 transcripts. While hDENND1A.V2 protein was not detectable by Western blot analyses, appropriate hDENND1A.V2 immunohistochemical staining was observed. Corresponding Cyp17a1 mRNA levels were elevated in ovaries and adrenals of CMV transgenic mice, as were plasma steroid production by theca interstitial cells isolated from transgenic ovaries. Even though the impact of robust hDENND1A.V2 expression could not be characterized, our findings are consistent with the notion that elevated hDENND1A.V2 has a role in the hyperandrogenemia of PCOS.

Pharmacomicrobiomics of Hepatic Drug Processing Genes During Pregnancy

At present, little is known about how gut microbiome and pregnancy interplay to regulate expression or the activities of drug processing genes (DPGs). The objective of this study was to investigate the effects of gut microbiome on host hepatic DPGs during pregnancy using conventional (CV) and germ‐free (GF) mice. Four groups of C57BL/6 female mice were used: CV non‐pregnant (CVNP, n=6), GF non‐pregnant (GFNP, n=6), CV pregnant (CVP, n=5), and GF pregnant (GFP, n=5) mice. Hepatic transcriptome and proteome for DPGs were profiled using multi‐omics approach. Plasma bile acids and steroid hormone levels were determined using LC‐MS/MS. Cyp activities were measured using hepatic microsome incubations. Overall, RNA‐seq analysis revealed a similar trend of gene regulation by pregnancy, but different magnitudes of change in hepatic DPGs, in CV and GF mice. Of note, the Cyp3a11 mRNA levels were decreased 1.7‐fold and 3.2‐fold by pregnancy in CV and GF mice, respectively. On the contrary, the Cyp3a11 protein abundance was increased 1.2‐fold and 1.6‐fold by pregnancy in CV and GF mice, respectively. The Cyp3a activity was also significantly induced by pregnancy in CV and GF mice; however, the magnitude of induction in CV mice was >5‐fold greater than that in GF mice. Plasma bile acid and steroid hormone levels were significantly impacted by microbiome and pregnancy‐status, respectively, which may contribute to the differential effects of pregnancy in CV and GF mice. This is the first study to demonstrate that the gut microbiome may play a role in altering hepatic drug disposition in pregnancy.Support or Funding InformationThis project was supported by the University of Washington Drug Metabolism, Transport and Pharmacogenomics Research Program (DMTPR)

Ouabain and Marinobufagenin: Physiological Effects on Human Epithelial and Endothelial Cells.

Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 μM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 μM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 μM completely suppressed DNA synthesis. MBG at the concentration of 0.1 μM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 μM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 μM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 μM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.

Sex Differences in Metabolic Responses to Chronic Immobilization Stress in Rats

Abstract Background Responses to stress could range from homeostatic variations to life-threatening effects depending on the type, timing and severity of the applied stimulus. Gender is accompanied by variations in oxidative stress, which causes the development of metabolic diseases. Under physiological conditions, females were found to be less susceptible to oxidative stress. Aim of work: This study was conducted to highlight the sex differences in the metabolic responses to chronic immobilization stress in rats, and to elucidate the possible underlying mechanisms. Materials and Methods Forty adult Wistar rats of both sexes; were randomly divided into 2 main groups: control group and stressed group, each of which was further subdivided into male and female groups. Stressed groups were exposed to chronic immobilization for 4 weeks. All rats were subjected to determination of body mass index, visceral fat weight, absolute and relative weights of liver and pancreas, plasma glucose, glucose uptake by diaphragm, glucose output by kidneys, lipid profile and plasma levels of insulin, leptin and sex hormones, malodialdehyde (MDA), total antioxidant capacity (TAC) and nitrite. HOMA-B and HOMA-IR were calculated. Also, caspase 3 was assessed in pancreas by immunohistochemistry. Results Stressed male rats showed lower BMI, higher relative liver weight, dyslipidemia, fasting hyperglycemia, higher glucose output by kidneys, lower glucose uptake by diaphragm, HOMA-B and plasma levels of insulin, testosterone and TAC and higher plasma estrogen and MDA levels compared to the control male group. Compared to the control female group, stressed female rate exhibited hyperglycemia, hypoinsulinemia, dyslipidemia, oxidative stress, higher plasma sex hormones. Compared to stressed male rats, stressed female group showed significantly higher BMI percentage change and plasma levels of TAC, estrogen and testosterone, but significantly lower absolute liver weight, glucose output by the kidneys, plasma levels of total cholesterol and triglycerides, and atherogenic index. Conclusion Chronic immobilization stress imposes greater hyperglycemic and hyperlipidemic status in males than in females. The altered sex hormonal pattern and lowered antioxidant defences could be contributory mechanisms

In vivo blockade of ovarian sympathetic activity by Neosaxitoxin prevents polycystic ovary in rats.

A high sympathetic tone is observed in the development and maintenance of the polycystic ovary (PCO) phenotype in rats. Neosaxitoxin (NeoSTX) specifically blocks neuronal voltage-dependent Na+ channels, and we studied the capacity of NeoSTX administered into the ovary to block sympathetic nerves and PCO phenotype that is induced by estradiol valerate (EV). The toxin was administered with a minipump inserted into the bursal cavity using two protocols: (1) the same day as EV administration and (2) 30 days after EV to block the final step of cyst development and maintenance of the condition. We studied the estrous cycling activity, follicular morphology, steroid plasma levels, and norepinephrine concentration. NeoSTX administered together with EV decreased NA intraovarian levels that were induced by EV, increased the number of corpora lutea, decreased the number of follicular cyst found after EV administration, and decreased the previously increased testosterone plasma levels induced by the PCO phenotype. Estrous cycling activity also recovered. NeoSTX applied after 30 days of EV administration showed near recovery of ovary function, suggesting that there is a specific window in which follicular development could be protected from cystic development. In addition, plasma testosterone levels decreased while those of progesterone increased. Our data strongly suggest that chronic inhibition of sympathetic nerves by a locally applied long-lasting toxin is a new tool to manage the polycystic phenotype in the rat and could be applied to other mammals depending on sympathetic nerve activity.

A comparative study of functional outcome between platelet rich plasma injection versus steroid infiltration in plantar fasciitis

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Plantar fasciitis (PF) is considered as degenerative tendinopathies. Repeated micro trauma is the major etiology of these diseases. Autologous platelet rich plasma (PRP) injections are becoming more popular in the treatment of enthesopathies like PF. The growth factors in PRP cause tissue healing. We compared the result of injecting intra-lesional autologous PRP injections versus steroid infiltration in chronic PF.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; A prospective, interventional and analytic comparative study was done and 81 patients (120 heels) were included in this study and were followed up for 6 months. We assessed the outcome of each patient using visual analog score (VAS) and foot and ankle disability index (FADI) on follow-up at 1, 3, and 6 months. &lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; In our study, female preponderance was seen. Left side was more common as compared to right side. Unilateral PF is more common than bilateral. The difference with in the individual group at baseline and at 1,3 and 6 months was statistically highly significant in terms of VAS and FADI (p=0.0001) But the difference in the between the two groups was insignificant for VAS and FADI at 1, 3 and 6 months.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; In our study, as there is no significant difference in VAS and FADI score between corticosteroid injection group and PRP injection group at 1, 3 and 6 months follow up. So, it’s reasonable to conclude that both are equally effective in PF. But as PRP injection comes out to be more time consuming and more costly, corticosteroid seems to be more efficient, cost and time wise. Hence, the latter should be a better choice. &lt;/p&gt;

SUCCESSFUL THERAPEUTIC PLASMA EXCHANGE IN POST-OPERATIVE MYASTHENIC CRISIS IN ELDERLY WOMAN

Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disorder that causes weakness of skeletal muscles responsible for breathing and moving parts of the body such as arms and legs. Surgical removal of thymoma may be associated with post-operative myasthenic crisis (POMC). Here, we report the case of a 70-year-old woman treated for squamous cell carcinoma of the uterine cervix, after a span of 11 years, presented with clinical features suggestive of MG. Investigations revealed type AB thymoma. The patient developed POMC, 3 days after the surgical excision procedure. The patient was subsequently treated with therapeutic plasma exchange (TPE), anticholinesterases, and steroids. The TPE in this elderly woman was not associated with any complications and was discharged in complete stable remission. Earlier studies show that many cases have immediate benefits from plasma exchange in MG, particularly MG crisis. TPE is a disease-modifying therapy for MG crisis.

Steroid Hormones in Reproduction and Roles of GnRH-a in Gonadal Maturation of Marine Fish: A Review

Reproduction in teleosts is regulated by a series of hormones including gonadotropin-releasing hormones (GnRHs), gonadotropins (GTHs) and steroid hormones. To contribute better understanding of steroid hormones in reproduction and GnRH-a in gonadal maturation, this literature review is concerned with the changes of steroid hormone levels in relation with sex inversion, reproductive behavior and gonadal development as well as the application of GnRH-a for inducing maturation of marine fish. The results revealed that in many species of teleost, steroid hormones E2, 11-11-KT and DHP are abundantly produced in gonadal tissues under the control of pituitary gonadotropins, and are essential for critical steps of gametogenesis. Plasma steroid levels have been used as indicators for both of the sex of the fish and its stage in the seasonal reproductive cycle, particularly with regard to induction of spawning. Determination of plasma steroid levels in relation with the sexual status of the gonads over several reproductive seasons might provide valuable information on the mechanisms of sex inversion in ambisexual fish species. In addition, changes of plasma steroid levels in correlation with gonadal development, number of spawning, fecundity, were described clearly in many marine species. The review also indicated that exogenous administration of GnRH-a triggered for final maturation of brood stock of some teleosts. In summary E2, T, 11-KT and C21 steroids are in relation with sex inversion, reproductive processes and GnRH-a is successful for inducing gonadal maturation in some fish species.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited. Here, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders. Using LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing's syndrome (SCS) and polycystic ovary syndrome (PCOS) - 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased. The LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.

Plasma Estradiol and Testosterone Levels and Ischemic Stroke in Postmenopausal Women.

Background and Purpose- Although exogenous hormone therapy (HT) use has been associated with increased risk of ischemic stroke in postmenopausal women, it remains unknown whether sex hormone levels contribute to ischemic stroke risk. We aimed to estimate associations between plasma sex hormone levels and ischemic stroke risk, by HT status, in a nested case-control study of postmenopausal women from the NHS (Nurses' Health Study). Methods- Women with confirmed incident ischemic stroke (n=419) were matched with controls (n=419) by age, HT use, and other factors. Plasma estradiol and testosterone levels were measured using liquid chromatography tandem mass spectrometry; SHBG (sex hormone-binding globulin) was assayed by electrochemiluminescence immunoassay. Associations of total and free estradiol and testosterone, the estradiol/testosterone ratio, and SHBG with ischemic stroke were estimated using conditional logistic regressions stratified by HT status with adjustment for matching and cardiovascular risk factors. Results- Current HT users had different hormone profiles from never/past users. No clear linear trends were observed between estradiol (total or free) levels or the estradiol/testosterone ratio and ischemic stroke risk among either current users (Ptrend>0.1) or never/past users (Ptrend>0.6). For both current and never/past users, the associations between some of the sex hormones and ischemic stroke differed by body mass index categories (Pinteraction≤0.04). For women with a body mass index <25 kg/m2, a higher estradiol/testosterone ratio was associated with significantly elevated ischemic stroke risk among current users (Ptrend=0.01), and higher levels of total and free estradiol were significantly associated with higher ischemic stroke risk among never/past users (Ptrend≤0.04). Testosterone and SHBG were not associated with ischemic stroke in either current or never/past users. Conclusions- Our findings do not support a role of sex hormone levels in mediating ischemic stroke risk among postmenopausal women. Replications in additional larger studies are required.

Newer Immunotherapies for the Treatment of Acute Neuromuscular Disease in the Critical Care Unit.

PURPOSE OF REVIEW: In this review, we discuss current treatment options for commonly encountered neuromuscular disorders in intensive care units. We will discuss epidemiology, pathophysiology, and acute and chronic treatment options for myasthenia gravis, Guillain-Barré syndrome, West Nile virus, Botulism, and amyotrophic lateral sclerosis. Eculizumab is the newest immunomodulator therapy approved by the Food and Drug Administration in treatment of myasthenia gravis, shown to improve long-term functional outcomes. Edaravone is the newest therapy in management of amyotrophic lateral sclerosis, shown to slow functional deterioration. Efgartigimod showed great promise in a phase 2 safety and efficacy trial in the treatment of stable generalized myasthenia gravis. Eculizumab was found to be safe in a small phase 2 trial for use in Guillain-Barré syndrome. Currently, therapies such as plasma exchange, intravenous immunoglobulins, and steroids remain the mainstay of treatment in the ICU for many neuromuscular disorders. While there are some newer immunotherapies available, few have been studied in the acute setting. However, with the advent of new immunotherapies and biologics, changes in these approaches may be on the horizon.

Acute Statin Administration Reduces Levels of Steroid Hormone Precursors.

Cholesterol-lowering statin drugs are used by approximately 25% of US adults 45 years of age and older and frequency of use is even higher among the elderly. Cholesterol provides the substrate for steroid hormone synthesis and its intracellular concentrations are tightly regulated. Our aim was to evaluate whether statin use acutely changes the circulating levels of cortisol, other glucocorticoid precursor molecules and their metabolites. Fourteen subjects not taking statins were administered a single oral dose (2 mg) of pitavastatin. Blood samples collected at baseline and 24 h post-treatment were analyzed for plasma cholesterol and steroid hormone profile. A parallel study in mice entailed the administration of atorvastatin (10 mg/kg) via orogastric delivery for three consecutive days. Cholesterol and corticosterone levels were quantified at baseline and at 1-day and 1-week post-treatment. Several precursor molecules in the steroidogenic pathway (corticosterone, cortisone, and 11-deoxycortisol) were significantly decreased 24 h after administration of a single dose of pitavastatin in human study subjects. Their circulating cholesterol concentrations were unchanged. In mice, there were no significant differences in serum cholesterol or corticosterone at 1-day or 1-week post-treatment compared to both pre-treatment baseline levels and control group levels. We conclude that acute dysregulation of the production of certain glucocorticoid precursor molecules was observed after a single treatment with a lipophilic statin drug. This may be of clinical relevance for individuals with underlying or subclinical adrenal insufficiency.

A Case of Acute Liver Failure Associated with HELLP Syndrome which was Successfully Treated by Plasma Exchange and Steroid Pulse Treatment

A Case of Acute Liver Failure Associated with HELLP Syndrome which was Successfully Treated by Plasma Exchange and Steroid Pulse Treatment

Reproductive toxicity and estrogen activity in Japanese medaka (Oryzias latipes) exposed to environmentally relevant concentrations of octocrylene

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 μg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 μg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 μg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 μg/L OC (p < 0.05). In addition, the levels of fshβ and lhβ in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erβ and cyp19β in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 μg/L OC and upregulation of star and hsd3β was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

Precocious maturation in male tiger pufferfish Takifugu rubripes: genetics and endocrinology

Testes of the tiger pufferfish Takifugu rubripes are a delicacy in Japan, and selective breeding for a male precocious phenotype, i.e., with early initiation of testes development, is desirable. However, it is unknown if precocious gonad development in this species is under genetic control. Here, we investigated genetic involvement in precociousness by using progeny tests with sires from two cultured populations, including a family line anecdotally known for its precociousness, and a wild population. Progeny derived from the “precocious” line consistently had greater testes weight than that from the other lines, even after accounting for effects of body weight, which indicates that precociousness is truly heritable. We also compared chronological changes in plasma steroid hormones between progenies sired by males from the precocious line and a wild population, and found that the precocious family line had higher levels of plasma estradiol-17β (E2) prior to the initiation of testicular development. Our findings suggest that selective breeding for testes precociousness in the tiger pufferfish is feasible, and that plasma E2 may be an indicator of this phenotype, which would allow for phenotype evaluation without the need to sacrifice specimens.

Contaminant levels and endocrine disruptive effects in Clarias gariepinus exposed to simulated leachate from a solid waste dumpsite in Calabar, Nigeria

Solid waste dumpsites (SWDs) and landfills are significant sources of emerging contaminants to terrestrial and aquatic ecosystems. We have studied the endocrine disruptive effects of simulated leachate from a solid waste dumpsite in Calabar, Nigeria. Juvenile C. gariepinus were exposed to simulated leachate, diluted to 0:0 (control), 1:10, 1:50, 1:100 for 3, 7 and 14 days. In addition, 17β-estradiol (E2: 100 μg/L)-exposed positive control group was included. Hepatic transcripts for the genes encoding vitellogenin (vtg), estrogen receptor-α (er-α), and aromatase (cyp19a1) were analyzed by real-time PCR. Protein expression for Vtg and Cyp19 were measured by immunoblotting and plasma steroid hormones (testosterone: T and E2) were measured using enzyme immunoassay (EIA). Soil samples from the dumpsite were analyzed for selected group of contaminants showing that DEHP was the only detected phthalate ester (PE) at 1300 ± 400 ng/g. Further, perfluoroalkyl substances (PFASs) such as PFBS, PFOS, PFHxA, PFOA, PFNA, PFDA, PFUnDA and PFDoDA were detected in the soil samples from the dumpsite. We observed significant and apparent concentration-dependent increases in mRNA (vtg, er-α, and cyp19a1) and their corresponding functional protein products, after exposure to the simulated leachates. Further, the simulated leachate produced concentration-specific changes in plasma E2 and T levels. In general, the estrogenic endocrine and reproductive alterations in the exposed fish may directly be attributed to the PFASs and DEHP detected at the dumpsites. However, in addition to PFASs and DEHP, there could be other estrogenic contaminants in the leachate. Given the rapid utilization, for residential purposes, and increases in human settlement in areas around the Lemna SWDs, this study provides a direct cause-and-effect evidence of the potential contaminants at the dumpsite with significant environmental and human health consequences.

Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency

21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative.We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35).The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids.Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient’s classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles.Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.

A predictive model to diagnose pregnancy in guanacos (Lama guanicoe) using non-invasive methods

Pregnancy status is usually not included in ecological studies because it is difficult to evaluate. The use of non-invasive methods to determine pregnancy, without physically restraining individuals, would enable pregnancy to be included in population studies. In this study, we evaluated sex steroid hormones in plasma and fecal samples from pregnant and non-pregnant females to develop a pregnancy predictive model for guanacos (Lama guanicoe (Müller, 1776)). Samples were obtained during live-shearing management (i.e., capture, shear, and release) of guanacos. Enzyme immunoassays were used to evaluate progesterone (P4) and estradiol (E2) concentrations in plasma and pregnanediol glucuronides (PdG) and conjugated estrogens (EC) in feces. Mean hormonal and fecal metabolite concentrations were significantly higher in pregnant females than in non-pregnant females. A linear relationship was found between each hormone and its fecal metabolite. Finally, hormonal data were combined with an independent source of pregnancy diagnosis such as abdominal ballottement to develop a logistic regression model to diagnose pregnancy in non-handled individuals. The use of predictive models and non-invasive methods might be suitable to incorporate pregnancy information in large-scale population studies on guanaco and other free-ranging ungulates.