Fetal androgen exposure is a determinant of adult male metabolic health.

Androgen signalling is a critical driver of male development. Fetal steroid signalling can be dysregulated by a range of environmental insults and clinical conditions. We hypothesised that poor adult male health was partially attributable to aberrant androgen exposure during development. Testosterone was directly administered to developing male ovine fetuses to model excess prenatal androgenic overexposure associated with conditions such as polycystic ovary syndrome (PCOS). Such in utero androgen excess recreated the dyslipidaemia and hormonal profile observed in sons of PCOS patients. 1,084 of 15,134 and 408 of 2,766 quantifiable genes and proteins respectively, were altered in the liver during adolescence, attributable to fetal androgen excess. Furthermore, prenatal androgen excess predisposed to adolescent development of an intrahepatic cholestasis-like condition with attendant hypercholesterolaemia and an emergent pro-fibrotic, pro-oxidative stress gene and protein expression profile evident in both liver and circulation. We conclude that prenatal androgen excess is a previously unrecognised determinant of lifelong male metabolic health.

Urinary estrogens as a non-invasive biomarker of viable pregnancy in the giant panda (Ailuropoda melanoleuca).

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development.

Confirmation of ovulation from urinary progesterone analysis: assessment of two automated assay platforms.

Urinary concentrations of the major progesterone (P4) metabolite pregnanediol-3-glucuronide (PDG) are used to confirm ovulation. We aimed to determine whether automated immunoassay of urinary P4 was as efficacious as PDG to confirm ovulation. Daily urine samples from 20 cycles in 14 healthy women in whom ovulation was dated by ultrasound, and serial weekly samples from 21 women in whom ovulation was unknown were analysed. Daily samples were assayed by two automated P4 immunoassays (Roche Cobas and Abbott Architect) and PDG ELISA. Serial samples were assayed for P4 by Architect and PDG by ELISA. In women with detailed monitoring of ovulation, median (95% CI) luteal phase increase was greatest for PDG, 427% (261-661), 278% (187-354) for P4 Architect and least for P4 Cobas, 146% (130-191), p < 0.0001. Cobas P4 also showed marked inaccuracy in serial dilution. Similar ROC AUCs were observed for individual threshold values and two-sample percent rise analyses for P4 Architect and PDG (both >0.92). In serial samples classified as (an)ovulatory by PDG, P4 Architect gave ROC AUC 0.95 (95% CI 0.89 to 1.01), with sensitivity and specificity for confirmation of ovulation of 0.90 and 0.91 at a cutoff of 1.67 µmol/mol. Automated P4 may potentially be as efficacious as PDG ELISA but research from a range of clinical settings is required.

Complement C5a is present in CSF of human newborns and is elevated in association with preterm birth.

Neuroinflammation contributes to developmental brain injury associated with preterm birth, but the mediators that drive it are incompletely understood. Previous studies have shown that complement C5a is present and injurious in the brains of foetal mice exposed to preterm labour. Here, we demonstrate that C5a is present in the cerebrospinal fluid of newborn human infants and that levels are elevated in those born preterm. The difference is not explained by systemic infection. Complement activation in the neonatal brain and its role as a potential therapeutic target in preterm brain injury warrant further study. Activation in the neonatal brain and its role as a potential therapeutic target for preterm brain injury warrants further study.

Oxidative stress rather than triglyceride accumulation is a determinant of mitochondrial dysfunction in in vitro models of hepatic cellular steatosis.

BACKGROUND/AIMS: There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question as only a small proportion of individuals with steatosis progress to steatohepatitis. In this study, we focused on defining (i) the effects of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function (ii) the contributions of triglyceride, ROS and subsequent mitochondrial impairment on the metabolism of energy substrates. METHODS: Human hepatoblastoma C3A cells, were treated with various combinations of oleate, octanoate, lactate (L), pyruvate (P) and ammonia (N) acutely or for 72 h, before measurements of triglyceride concentration, cell respiration, ROS production, mitochondrial membrane potential, ketogenesis and gluconeogenesis, TCA cycle metabolite analysis and electron microscopy. RESULTS: Acutely, LPON treatment enhanced mitochondrial respiration and ROS formation. After 72 h, despite the similarities in triglyceride accumulation, LPON treatment, but not oleate, dramatically affected mitochondrial function as evidenced by decreased respiration, increased mitochondrial membrane potential and ROS formation with concomitant enhanced ketogenesis. By comparison, respiration and ROS formation remained unperturbed with oleate. Importantly, this was accompanied by an increased gluconeogenesis and ketogenesis. The addition of the antioxidant N-acetyl-L-cysteine prevented mitochondrial dysfunction and reversed metabolic changes seen with LPON, strongly suggesting ROS involvement in mediating mitochondrial impairment. CONCLUSIONS: Our data indicate that ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or reducing of ROS formation.

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody.

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11ßHSD type 2. To assess 11ß-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X-0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3 nM at 08.00 h and 5.1±3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22 nM that increased to 8.6±1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5 nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.

Effect of prolonged in vivo administration of progesterone in pregnancy on myometrial gene expression, peripheral blood leukocyte activation, and circulating steroid hormone levels.

OBJECTIVE: We aimed to investigate the effects of progesterone on gene expression and function of both myometrium and circulating leukocytes. METHODS: We recruited women participating in a randomized clinical trial of progesterone to prevent preterm delivery. These participants had a twin pregnancy and were managed in 1 of 2 tertiary referral centers. Participants were treated with progesterone (90 mg vaginally) or placebo from 24 to 34 weeks of pregnancy. The outcome measures were myometrial and leukocyte gene expression and expression of cell surface markers in circulating leukocytes, all quantified ex vivo. RESULTS: Prolonged in vivo administration of progesterone inhibited myometrial expression of connexins 26 and 43, endothelial nitric acid synthase (eNOS), and the prostaglandin receptor EP2 ex vivo. Administration of progesterone also increased numbers of circulating neutrophils while decreasing lymphocyte proportions and decreasing neutrophil CD11b expression. CONCLUSION: The observed effects of prolonged in vivo administration of progesterone will minimize the ability of the uterus to contract as a synctium and the ability of peripheral blood leukocytes to migrate into the myometrium during parturition. We suggest that these are putative mechanisms by which progesterone might prevent preterm birth in women at high risk.

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure.

BACKGROUND: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. METHOD: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. RESULTS: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. CONCLUSION: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Selenium and sulforaphane modify the expression of selenoenzymes in the human endothelial cell line EAhy926 and protect cells from oxidative damage.

OBJECTIVE: We examined the ability of sulforaphane and selenium to modify the expression of thioredoxin reductase (TR-1) and the glutathione peroxidases (GPX-1 and GPX-4) in EAhy926 cells. The effectiveness of these agents to protect cells against peroxidative damage was also assessed. METHODS: EAhy926 cells were supplemented with 40 nM of selenite and/or sulforaphane (10 microM) for 72 h and the expression of TR-1, GPX-1, and GPX-4 was assessed. Parallel cultures of selenium- and sulforaphane-treated cells were exposed to tertiary butyl hydroperoxide (t-BuOOH; 0-500 microM) for 20 h, and cell integrity was determined by the percentage of lactate dehydrogenase retained by the cellular layer. RESULTS: Selenite treatment increased the concentration of TR-1 (1.6 +/- 0.17 fold, P < 0.05), GPX-1 activity (8.2 +/- 1.08 fold, P < 0.001), and GPX-4 activity (3.1 +/- 0.25 fold, P < 0.001). Sulforaphane induced TR-1 expression in selenium-deficient cells (1.83 +/- 0.11 fold, P < 0.001) and selenium-supplemented cells (2.90 +/- 0.17 fold, P < 0.001) but had no inductive effect on GPX-1 or GPX-4. The combination of selenite and sulforaphane produced an increase in TR-1 expression that was significantly greater (P < 0.001) than that achieved when each agent was added alone. Selenium and sulforaphane acted in a synergistic manner to protect cells from damage caused by t-BuOOH. The susceptibility of cells to damage by t-BuOOH increased in this order: control > sulforaphane > selenite > selenite + sulforaphane (P < 0.0001). CONCLUSION: In endothelial cells, sulforaphane increases TR-1 but not GPX-1 and GPX-4 and in doing so confers protection against oxidative damage induced by lipid hydroperoxides. The results highlight the potential important role of TR-1 over the GPXs in protecting endothelial cells from oxidative cell damage. We also suggest that our results indicate a potential beneficial role for sulforaphane in protecting the vascular endothelium from oxidative damage.