Mode of action hypothesis testing in chemical safety assessments using metabolomics as supporting evidence: Phenobarbital and cyclobutrifluram metabolomics profile comparison.

In long term rodent studies administering Cyclobutrifluram (TYMIRIUM® Technology), a new agrochemical, there was a slight elevation of incidence of hepatocellular carcinomas in male CD-1 mice that was within the historical control range but appeared to be dose responsive. Cyclobutrifluram's ability to activate mouse constitutive androstane receptor (CAR) mediated gene transcription was confirmed in vitro, therefore a 28-day dietary toxicity study was conducted in vivo in male CD-1 mice to assess the CAR activation mode of action hypothesis of Cyclobutrifluram along with phenobarbital, a known CAR activator. In addition to other end points comprehensive (polar and lipidomic) hybrid metabolomics analyses were performed on terminal plasma and liver samples following 2-, 7- and 28-days dietary exposure to cyclobutrifluram and phenobarbital. The data generation and quality assessments were performed in line with the principles of the MEtabolomics standaRds Initiative in Toxicology (MERIT).First the full annotated feature set was used to compare the metabolomic changes induced by the administration of the two test substances using Shared and Unique Structures plots. This gave a comprehensive overview of the similarity of the two effect profiles showing good correlation and demonstrated that no other, alternative effect signatures were detected. Then the phenobarbital induced differentially abundant metabolites were selected, compared to the literature and their direction of change was assessed in cyclobutrifluram profiles, finding good agreement. Both approaches concluded that the metabolomics data supports the CAR activation hypothesis. Comparison of the metabolomic effect profiles can be a line of evidence in mode of action hypothesis testing in the chemical risk assessment process.

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

Could be FOXO3a, miR-96-5p and miR-182-5p useful for Brazilian women with luminal A and triple negative breast cancers prognosis and target therapy?

FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

Phenobarbital in Nuclear Receptor Activation: An Update.

Phenobarbital (PB) is a commonly prescribed anti-epileptic drug that can also benefit newborns from hyperbilirubinemia. Being the first drug demonstrating hepatic induction of cytochrome P450 (CYP), PB has since been broadly used as a model compound to study xenobiotic-induced drug metabolism and clearance. Mechanistically, PB-mediated CYP induction is linked to a number of nuclear receptors, such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and estrogen receptor α, with CAR being the predominant regulator. Unlike prototypical agonistic ligands, PB-mediated activation of CAR does not involve direct binding with the receptor. Instead, dephosphorylation of threonine 38 in the DNA-binding domain of CAR was delineated as a key signaling event underlying PB-mediated indirect activation of CAR. Further studies revealed that such phosphorylation sites appear to be highly conserved among most human nuclear receptors. Interestingly, while PB is a pan-CAR activator in both animals and humans, PB activates human but not mouse PXR. The species-specific role of PB in gene regulation is a key determinant of its implication in xenobiotic metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In this review, we summarize the recent progress in our understanding of PB-provoked transactivation of nuclear receptors with a focus on CAR and PXR. SIGNIFICANCE STATEMENT: Extensive studies using PB as a research tool have significantly advanced our understanding of the molecular basis underlying nuclear receptor-mediated drug metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In particular, CAR has been established as a cell signaling-regulated nuclear receptor in addition to ligand-dependent functionality. This mini-review highlights the mechanisms by which PB transactivates CAR and PXR.

The induction and inhibition of UDP-glycosyltransferases in Haemonchus contortus and their role in the metabolism of albendazole.

Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research.

Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence-based screening assay.

Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Impact of Neonatal Activation of Nuclear Receptor CAR (Nr1i3) on Cyp2 Gene Expression in Adult Mouse Liver.

Perinatal exposure to environmental chemicals is proposed to reprogram development and alter disease susceptibility later in life. Supporting this, neonatal activation of the nuclear receptor constitutive androstane receptor (CAR) (Nr1i3) by TCPOBOP was previously reported to induce persistent expression of mouse hepatic Cyp2 genes into adulthood, and was attributed to long-term epigenetic memory of the early life exposure. Here, we confirm that the same high-dose neonatal TCPOBOP exposure studied previously (3 mg/kg, 15x ED50) does indeed induce prolonged (12 weeks) increases in hepatic Cyp2 expression; however, we show that the persistence of expression can be fully explained by the persistence of residual TCPOBOP in liver tissue. When the long-term presence of TCPOBOP in tissue was eliminated by decreasing the neonatal TCPOBOP dose 22-fold (0.67× ED50), strong neonatal increases in hepatic Cyp2 expression were still obtained but did not persist into adulthood. Furthermore, the neonatal ED50-range TCPOBOP exposure did not sensitize mice to a subsequent, low-dose TCPOBOP treatment. In contrast, neonatal treatment with phenobarbital, a short half-life (t1/2 = 8 h) agonist of CAR and PXR (Nr1i2), induced high-level neonatal activation of Cyp2 genes and also altered their responsiveness to low-dose phenobarbital exposure at adulthood by either increasing (Cyp2b10) or decreasing (Cyp2c55) expression. Thus, neonatal xenobiotic exposure can reprogram hepatic Cyp2 genes and alter their responsiveness to exposures later in life. These findings highlight the need to carefully consider xenobiotic dose, half-life, and persistence in tissue when evaluating the long-term effects of early life environmental chemical exposures.

Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health.

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.

Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

Quantitative protein profiling of phenobarbital-induced drug metabolizing enzymes in rat liver by liquid chromatography mass spectrometry using formalin-fixed paraffin-embedded samples.

Administration of a compound can induce drug-metabolizing enzymes (DMEs) in the liver. DME induction can affect various parameters in toxicology studies. Therefore, evaluation of DME induction is important for interpreting test compound-induced biological responses. Several methods such as measurement of hepatic microsomal DME activity using substrates, electron microscopy, or immunohistochemistry have been used; however, these methods are limited in throughput and specificity or are not quantitative. Liquid chromatography mass spectrometry (LC/MS)-based protein analysis can detect and quantify multiple proteins simultaneously per assay. Studies have shown that formalin-fixed paraffin-embedded (FFPE) samples, which are routinely collected in toxicology studies, can be used for LC/MS-based protein analysis. To validate the utility of LC/MS using FFPE samples for quantitative evaluation of DME induction, we treated rats with a DME inducer, phenobarbital, and compared the protein expression levels of 13 phase-I and 11 phase-II DMEs between FFPE and fresh frozen hepatic samples using LC/MS. A good correlation between data from FFPE and frozen samples was obtained after analysis. In FFPE and frozen samples, the expression of 6 phase-I and 8 phase-II DMEs showed a similar significant increase and a prominent rise in Cyp2b2 and Cyp3a1 levels. In addition, LC/MS data were consistent with the measurement of microsomal DME activities. These results suggest that LC/MS-based protein expression analysis using FFPE samples is as effective as that using frozen samples for detecting DME induction.

A molecular trap inside microtubules probes luminal access by soluble proteins.

The uniquely hollow structure of microtubules (MTs) confers characteristic mechanical and biological properties. Although most regulatory processes take place at the outer surface, molecular events inside MTs, such as α-tubulin acetylation, also play a critical role. However, how regulatory proteins reach the site of action remains obscure. To assess luminal accessibility, we first identified luminally positioned residues of ß-tubulin that can be fused to a protein of interest. We then developed a chemically inducible technique with which cytosolic proteins can be rapidly trapped at the lumen of intact MTs in cells. A luminal trapping assay revealed that soluble proteins of moderate size can enter the lumen via diffusion through openings at the MT ends and sides. Additionally, proteins forming a complex with tubulins can be incorporated to the lumen through the plus ends. Our approach may not only illuminate this understudied territory, but may also help understand its roles in MT-mediated functions.

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors.

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

Acute and chronic treatment with moclobemide, a reversible MAO-inhibitor, potentiates the antielectroshock activity of conventional antiepileptic drugs in mice.

BACKGROUND: Due to enhancing serotonergic and noradrenergic neurotransmission, moclobemide may influence seizure phenomena. In this study, we examined the effect of both acute and chronic treatment with moclobemide on seizures and the action of first-generation antiepileptic drugs: valproate, carbamazepine, phenobarbital and phenytoin. METHODS: The effect of moclobemide on seizures was assessed in the electroconvulsive threshold test, while its influence on antiepileptic drugs was estimated in the maximal electroshock test in mice. Undesired effects were evaluated in the chimney test (motor impairment) and step-through passive-avoidance task (long-term memory deficits). Finally, brain concentrations of antiepileptics were determined by fluorescence polarization immunoassay. RESULTS: Given acutely, moclobemide at 62.5 and 75 mg/kg increased the electroconvulsive threshold. In contrast, chronic treatment with moclobemide up to 75 mg/kg did not influence this parameter. Acute moclobemide applied at subthreshold doses (up to 50 mg/kg) enhanced the antielectroshock effects of carbamazepine, valproate and phenobarbital. Chronic moclobemide (37.5-75 mg/kg) increased the action of all four antiepileptic drugs. All revealed interactions, except these between moclobemide and phenobarbital, seem to have pharmacokinetic nature, because the antidepressant drug, either in acute or in chronic treatment, increased the brain concentrations of respective antiepileptic drugs. In terms of undesired neurotoxic effects, acute and chronic moclobemide, antiepileptic drugs, and their combinations did not produce significant motor or long-term memory impairment. CONCLUSIONS: Acute and chronic therapy with moclobemide can increase the effectiveness of some antiepileptic drugs against the maximal electroshock test. In mice, this effect was, at least partially, due to pharmacokinetic interactions. So far as the results of experimental studies can be transferred to clinical conditions, moclobemide seems safe for the application in patients with epilepsy and depression. Possibly, in the case of certain antiepileptic drugs combined with moclobemide, their doses should be adjusted downwards.

Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Notch activation in the mouse mammary luminal lineage leads to ductal hyperplasia and altered partitioning of luminal cell subtypes.

Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.

Distinct roles for luminal acidification in apical protein sorting and trafficking in zebrafish.

Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis.

Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.

Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.

Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

Rabbit dehydrogenase/reductase SDR family member 11 (DHRS11): Its identity with acetohexamide reductase with broad substrate specificity and inhibitor sensitivity, different from human DHRS11.

Human dehydrogenase/reductase SDR family member 11 (DHRS11) has been recently reported to be an NADP+-dependent 3(17)ß-hydroxysteroid dehydrogenase, and its orthologs are predicted in genomic analyses of various animals. Among them, the amino acid sequence of predicted rabbit DHRS11 shares 92% identity with that of human DHRS11 and matches peptide sequences (composed of total 87 amino acids) of rabbit heart acetohexamide reductase (RHAR) previously reported. However, the physiological role of RHAR remains unknown, because its known substrates are only acetohexamide and 1,4-naphthoquinone. To elucidate whether the two rabbit enzymes are identical, we have isolated the cDNA for rabbit DHRS11, which was abundantly detected in the brain, heart, kidney and intestine by RT-PCR. The recombinant rabbit DHRS11 reduced acetohexamide and 1,4-naphthoquinone, and was inhibited by tolbutamide and phenobarbital (RHAR-specific inhibitors), demonstrating its identity with RHAR. Rabbit DHRS11 also reduced α-dicarbonyl compounds, aldehydes and aromatic ketones (acetylbenzenes and acetylpyridines), and exhibited 3(17)ß-hydroxysteroid dehydrogenase activity. It was competitively inhibited not only by tolbutamide and phenobarbital, but also more potently by several non-steroidal anti-inflammatory drugs such as diclofenac and sulindac. The broad substrate specificity and inhibitor sensitivity were different from those of human DHRS11, which did not reduce aliphatic aldehydes and aromatic ketones despite its higher 3(17)ß-hydroxysteroid dehydrogenase activity, and was insensitive to tolbutamide, phenobarbital and diclofenac. The site-directed mutagenesis of Thr163 and Val200 in human DHRS11 to the corresponding residues (Gly and Leu, respectively) in rabbit DHRS11 suggested that these residues are pertinent to the differences in properties of rabbit and human DHRS11s.

Successful treatment of seizure disorder by evaluating phenobarbital clearance in a paediatric patient undergoing continuous haemodiafiltration.

WHAT IS KNOWN AND OBJECTIVE: Determination of phenobarbital (PB) dosing during continuous haemodiafiltration (CHDF) requires evaluation of both the patient's own clearance and CHDF clearance. CASE DESCRIPTION: Systemic clearance of PB in a 9-year-old female patient with Dravet syndrome, who was undergoing CHDF, was calculated by the Sawchuk-Zaske method, and clearance by CHDF was calculated as the difference between PB levels in influx and efflux, adjusted for the blood purification conditions. Dosage adjustment produced seizure control. WHAT IS NEW AND CONCLUSION: Adjusting PB dose by evaluating the patient's own PB clearance as well as that by CHDF resulted in accurate control of PB level and a reduction in the occurrence of seizures.