A. Forbes Howie

Dual action of sulforaphane in the regulation of thioredoxin reductase and thioredoxin in human HepG2 and Caco-2 cells.

We have previously demonstrated that sulforaphane is a potent inducer for thioredoxin reductase in HepG2 and MCF-7 cells (Zhang et al. Carcinogenesis 2003, 24, 497-503; Wang et al. J. Agric. Food Chem. 2005, 53, 1417-1421). In this study, we have shown that sulforaphane is not only an inducer for thioredoxin reductase but also an inducer for its substrate, thioredoxin in HepG2, and undifferentiated Caco-2 cells. Sulforaphane acts at two levels in the regulation of thioredoxin reductase/thioredoxin system by the upregulation of the expression of both the enzyme and the substrate. In human hepatoma HepG2 cells, sulforaphane induced thioredoxin reductase mRNA and protein by 4- and 2-fold, respectively, whereas thioredoxin mRNA was induced 2.9-fold and thioredoxin protein was unchanged in whole cell extracts, but an increase in nuclear accumulation (1.8-fold) was observed. Moreover, the induction of thioredoxin reductase was found faster than that of thioredoxin. The effects of PI3K and MAPK kinase inhibitors, LY294002, PD98059, SP600125, and SB202190, have been investigated on the sulforaphane-induced expression of thioredoxin reductase and thioredoxin. PD98059 abrogates the sulforaphane-induced thioredoxin reductase at both mRNA and protein levels in HepG2 cells, although other inhibitors were found less effective. However, both PD98059 and LY294002 significantly decrease thioredoxin mRNA expression in HepG2 cells. None of the inhibitors tested were able to modulate the level of expression of either thioredoxin reductase mRNA or protein in Caco-2 cells suggesting that there are cell-specific responses to sulforaphane. In summary, the dietary isothiocyanate, sulforaphane, is important in the regulation of thioredoxin reductase/thioredoxin redox system in cells.

A highly-sensitive anti-Müllerian hormone assay improves analysis of ovarian function following chemotherapy for early breast cancer☆

Aim Anti-Müllerian hormone (AMH) shows promise as a biomarker of the ovarian reserve but current assays are insufficiently sensitive to allow assessment of this post-chemotherapy in most women. We have assessed a new highly sensitive AMH assay (Ansh picoAMH) in the evaluation of ovarian activity in women with very low ovarian reserve after chemotherapy. Methods A prospective cohort and an independent validation cohort of premenopausal women with early breast cancer (eBC) were recruited at the time of diagnosis (combined n = 98), and ovarian reserve markers 2–5 years later following chemotherapy were assessed in relation to menstrual activity. Results The picoAMH assay had a limit of detection of 7.5 pg/ml. AMH clearly distinguished women with ongoing menses from those with amenorrhoea at 2 years after diagnosis (mean 522 ± 169 versus 8.9 ± 1.3 pg/ml, P < 0.0001) with high predictive value for continuing menses or amenorrhoea for the subsequent 3 years. AMH was detectable in more women than using a previous assay (P = 0.004). Other markers of the ovarian reserve (follicle-stimulating hormone (FSH), inhibin B) were also of discriminatory value but to lesser extents. This finding was validated in a second, independent cohort of women treated for eBC. Conclusion The 10-fold increased assay sensitivity showed very clear distinction between groups based on ovarian activity with excellent prediction of future menses or amenorrhoea. This will improve assessment of post-chemotherapy ovarian function in women and may aid treatment decisions.

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs) - integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications.

Synergy between broccoli sprout extract and selenium in the upregulation of thioredoxin reductase in human hepatocytes.

Dietary isothiocyanates and selenium (Se) can up-regulate thioredoxin reductase 1 (TR1) in cultured human HepG2 and MCF-7 cells [Zhang et al. (2003). Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation. Carcinogenesis, 24, 497-503; Wang et al. (2005). Sulforaphane, erucin and iberin up-regulate thioredoxin reductase expression in human MCF-7 cells. Journal of Agricultural and Food Chemistry, 53, 1417-1421] at both the protein and mRNA levels. In this study, broccoli sprout extract (a rich source of the isothiocyanates sulforaphane and iberin) and Se interacted synergistically to induce TR1 in immortalised human hepatocytes. Broccoli sprout extracts containing 1.6, 4 and 8μM isothiocyanates were tested for their ability to induce TR1 at the protein and mRNA level. Although induction of TR1 mRNA by broccoli sprout extract (1.6-8μM) was only 1.7-2.2-fold, co-treatment with Se (0.2-1μM) enhanced the expression of TR1 mRNA (3.0-3.3-fold). Moreover, broccoli sprout extract induced the cellular concentration of TR1 and TR enzymatic activity, an induction that was augmented by Se addition. Thus, broccoli sprout extract (8μM) and Se induced cellular TR1 concentration and enzymatic activity 3.7- and 5-fold respectively, whereas, Se or broccoli sprout extract alone produced an induction of only approximately 2-fold. These data suggest that dietary isothiocyanates from broccoli sprouts and Se are important agents in the regulation of redox status in human liver cells. The synergistic effect between isothiocyanates and Se at physiologically-relevant concentrations on the induction of TR1 may play an important role in protection against oxidative stress.

Ischemic Preconditioning in the Liver Is Independent of Regulatory T Cell Activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.

Comparison of Bioenergetic Activity of Primary Porcine Hepatocytes Cultured in Four Different Media

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were Williams E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 × 1010; viability 87 ± 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 × 106 viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis, ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. Williams E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.

Functional characterisation of the H365Y mutation of the 21-hydroxylase gene in congenital adrenal hyperplasia

The study subject was a 13 day-old boy admitted to hospital, with weight loss since birth. He presented with the vomiting and hypotension that are classical features of congenital adrenal hyperplasia (CAH). The most common type of CAH is an autosomal recessive disorder caused by mutations in the 21-hydroxylase (CYP21A2) gene. To examine the CYP21A2 gene, gene-specific PCR was carried out, followed by sequencing. The baby was shown to be a compound heterozygote H365Y/R356W for two CYP21A2 gene mutations each inherited from a different parent. One of the mutations has not previously been functionally characterised. The mutations were reconstructed in an expression plasmid and characterised in vitro after transient transfection into human embryonic kidney (HEK293T) and hepatoblastoma (C3A) cell lines followed by measurement of enzyme activity. The CYP21A2 H365Y mutant exhibited minimal 21-hydroxylase activity to convert 17-hydroxyprogesterone to 11-deoxycortisol or progesterone to 11-deoxycorticosterone. Western immunoblotting indicated that the H365Y enzyme was produced in more variable amounts than wild type; in particular, the H365Y mutant protein may be unstable and/or subject to a more rapid degradation by the human proteosome as well as catalytically inefficient. The double mutant genotype with a severe mutation on each allele is compatible with the clinical presentation.

Placental hormone profiles as predictors of preterm birth in twin pregnancy: A prospective cohort study

Objective The objective of the study was to analyse placental hormone profiles in twin pregnancies to determine if they could be used to predict preterm birth. Study design Progesterone, estradiol, estriol and corticotropin-releasing hormone were measured using competitive immunoassay and radioimmunoassay in serum and saliva samples of 98 women with twin pregnancies,at 3 or more gestational timepoints. Hormone profiles throughout gestation were compared between very preterm (<34 weeks; n = 8), preterm (<37 weeks; n = 40) and term (37+ weeks; n = 50) deliveries. Results No significant differences were found between preterm and term deliveries in either absolute hormone concentrations or ratios. Estimated hormone concentrations and ratios at 26 weeks did not appear to predict preterm delivery. Salivary and serum hormone concentrations were generally poorly correlated. Conclusion Our results suggest that serial progesterone, estradiol, estriol and corticotropin-releasing hormone measurements in saliva and serum are not robust biomarkers for preterm birth in twin pregnancies.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal’s changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson’s correlation between creatinine and USpG was high (r = 0.805–0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13–86.93 and 31.61–306.45 ng/mL (USpG correction) versus 2.33–31.20 and 36.36–249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35–3.21 and 0.85–6.80 ng/mL (USpG correction) versus 0.52–10.31 and 2.10–272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Serum, milk, saliva and urine progesterone and estradiol profiles in crossbred (Zebu x Holstein Friesian) dairy cattle

Effective breeding management is a crucial tool to enhance the reproductive and productive performance of dairy cattle. To achieve this, regular monitoring of their reproductive status is important. The aims of this study were to monitor the reproductive status of crossbred dairy cattle based on progesterone (P4) concentration in serum, whole milk, saliva and urine, and estradiol in serum using quantitative laboratory ELISA tests, and to document hormonal profiles at different reproductive stages. Three hundred and thirty-six (336) crossbred (Holstein Friesian x Zebu) dairy cattle (232 cows and 104 heifers) from 47 dairy farms owned by smallholder or organized commercial dairy farmers were included in the study. Matched blood, milk and saliva samples were collected twice at 11-day intervals at afternoon milking, while urine was collected just once. Serum was separated within 48 hr of blood collection. All samples were stored at -80oC until assayed. In both cows and heifers, P4 was higher during pregnancy than in other reproductive conditions. Estradiol was higher during estrus than at other reproductive stages. Estradiol levels were significantly higher (p<0.05) in estrus cows compared with heifers. The mean level of P4 in serum strongly correlated with the value in milk (r = 0.6368, P< 0.0001), however there was no correlation in P4 concentration between serum and saliva or between P4 in serum and in urine. Hence, determination of P4 in serum and milk using an ELISA test is a reliable and precise method to monitor reproductive status in crossbred dairy cattle. *Correspondence to: Mekonnin AB, The University of Edinburgh, College of Medicine and Veterinary Medicine, MRC Centre for Reproductive Health, QMRI, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK; E-mail: alemselambnh@yahoo.com