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The Constitutional Court makes ruling on Cash Paymaster Services (CPS) contract with the South Africa Social Security Agency (Sassa) to pay social grants from April 1.
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The Constitutional Court makes ruling on Cash Paymaster Services (CPS) contract with the South Africa Social Security Agency (Sassa) to pay social grants from April 1.
This stream has ended. Click here for the verdict.
Allan Gray has been accused of benefiting from social grants crisis as they are investing in Net1, the parent company of CPS.
Zuma says he cannot be expected to act against Minister Bathabile Dlamini on social grants matter now before the ConCourt.
An investigation revealed that Net1 used a secret backdoor to get grant beneficiaries' information to its subsidiaries.
The Money Show's Bruce Whitfield interviews EWN's Stephen Grootes about the political ramifications of the Sassa debacle.
Minister of Social Development Bathabile Dlamini was absent from Parliament on Thursday afternoon.
Radebe says Bathabile Dlamini's involvement in an inter-ministerial task team to investigate Sassa 'not a conflict of interest'.
President Jacob Zuma will answer questions in Parliament for the first time in 2017, on matters including social grants payments.
The ANC postponed yesterday's meeting with AIC, and has until month-end to finalise the coalition agreement.
It's now up to the Contsitutional Court to decide whether Cash Paymaster Services (CPS) continues to deliver social grants.
Amabhungane says it has proof that Net1's subsidiaries use Sassa grantees personal data to sell them insurance and electricity.
EWN's Chip & Curtis take a dig at Minister Bathabile Dlamini and her side-kick Lumka Oliphant after Wednesday's ConCourt lashing.
Constitutional law expert says the Constitutional Court is likely to make ruling on social grants crisis within next two days.
Following a 7 hour sitting, the Constitutional Court is yet to make ruling over the mess at Sassa.
The former TRC commissioner responds to the absence of black legal counsel during ConCourt hearings on social grants saga.
Black Sash wants the ConCourt to have oversight over any interim measures implemented by Sassa and the Social Development Dept.
The Money Show's Bruce Whitfield interviews the underrated turnaround guru for this weekly "ShapeShifter" feature.
Andrew Breitenbach has asked the Constitutional Court to either extend the existing contract or allow an interim agreement.
Chief Justice Mogoeng berated Minister Bataibile Dlamini and Sassa, and Post Office says it can go ahead and pay social grants.
In Addis Ababa on Tuesday Dlamini-Zuma handed over her post as African Union chair to Moussa Faki Mahamat.
Social Development and the South African Social Security Agency explained why an interim contract with CPS should be allowed.
The finance minister says only a short term interim agreement may be reached between CPS and the Social Development Department.
Chief Justice's new Tuesday 3 pm deadline in place for Sassa CEOs and Social Development Minister to file affidavits.
Sassa and Social Development on Monday failed to meet the ConCourt's deadline to explain plans to pay social grants from April 1.
Allan Gray holds almost 16% of the share capital of Net1 on behalf of its clients. This investment has come under the spotlight.
Net1 UEPS owns the controversial social grants distributor Cash Paymaster Services. Bruce Whitfield interviews CIO Andrew Lapping.
Xolani Gwala says Constitutional Court's intervention on the social grants matter is decisive and important.
Scopa chair says it's still unclear what the plan is for 1 April, even though Minister Dlamini insists that grants will be paid.
Min. Bathabile Dlamini says she won't be drawn on how much a new deal with Cash Paymaster Services (CPS) will cost the taxpayer.
Social Development Minister finally answers questions on social grants payments before Parliament's Scopa but deal not signed yet.
Parliament's Scopa summoned Minister Bathabile Dlamini to explain plans to pay social grants from April 1.
The legality and terms of a new contract between CPS and Sassa should be the biggest concern in the grants mess, says Themba Godi.
Parliament's Standing Committee on Public Accounts chair says Minister Bathabile Dlamini deliberately failed the process.
Amabhungane investigative reporter Craig McKune reflects on the timeline of events which led Sassa to a legal contractor debacle.
With 13.5 percent of usable water left, Councilor Xanthea Limberg talks to Kieno Kammies about the City's plans.
Emmanuel Macron's history of how he met his wife has been met with some criticism.
Chris Malgas speaks about his experiences as a prison warder at the notorious Pollsmoor prison for four decades.
A new lifestyle market is set to open in Sea Point, offering a variety of food, art, fashion and entertainment seven days a week.


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Corn syrup Recipes
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Killer whales go on 'unprecedented' killing spree in Monterey Bay
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Monthly Archive for October, 2007
Revenge of the haunted toys
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As the really great and creative costume photos roll in, I think we ought to really take heed of what Peter said yesterday and remember that there's no need to feel inadequate. This isn't a …
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The Poop costume contest: Optimus Prime edition
It's not even Halloween Eve yet, and we've already received several excellent submissions for The Poop super-crafty Halloween costume contest. Courtesy Claire PittsMore than meets the eye … I …
Recall Madness! Vaya con Diego
cpsc.gov Welcome to the second edition of Recall Madness!, a consumer advocacy series from The Poop, designed to help you gauge how pissed off you should be as you wander through the vast wilderness …
Best and worst Bay Area pumpkin patches
Every year, around this time, I get really pissed off at a pumpkin patch. It's impossible not to get a cute picture of two kids in a wheelbarrow. Not the pumpkins specifically, but the corn maze …
A tribute to Lefty the Salesman
. The shafting of Grover has been a frequent topic on this blog. But playing Andrew Ridgeley to Elmo's George Michael isn't the worst fate for a Muppet. What about the beloved "Sesame Street" …
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Around the time the Night Stalker had people locking their doors and anonymous school kids fearing for their lives every night, my favorite aunt decided to tell me the story of the Zodiac Killer — …
The candy conundrum — how much is too much?
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My name is Russell Newquist. I am a software engineer, a martial artist, an author, an editor, a businessman and a blogger. I have a Bachelor of Arts degree in Philosophy and a Master of Science degree in Computer Science, but I'm technically a high school dropout. I also think that everything in this paragraph is pretty close to meaningless. I work for a really great small company in Huntsville, Alabama building really cool software. I'm the owner and head instructor of Madison Martial Arts Academy, which I opened in 2013 less to make money and more because I just really enjoy a good martial arts workout with friends. I'm the editor in chief of Silver Empire and also one of the published authors there. And, of course, there is this blog - and all of its predecessors. There's no particular reason you should trust anything I say any more than any other source. So read it, read other stuff, and think for your damn self - if our society hasn't yet over-educated you to the point that you've forgotten how.
There are no men like me. There is only me.
Silver Empire Authors
Here they are, in order from least to most popular: my top ten blog posts of 2016. Silver Empire calls for submission proved very popular, but they couldn't beat out scandal and sex appeal. Go figure!
By any metric, 2016 has been a great year for me. There were some hiccups, but altogether the good overwhelmed the bad. Here's a look back on the year.
2017 is looking to be an even better year.


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Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.
Citation: Harkitis P, Daskalopoulos EP, Malliou F, Lang MA, Marselos M, Fotopoulos A, et al. (2015) Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver. PLoS ONE 10(10): e0128708. https://doi.org/10.1371/journal.pone.0128708
Editor: Makoto Makishima, Nihon University School of Medicine, JAPAN
Received: July 7, 2014; Accepted: April 29, 2015; Published: October 14, 2015
Copyright: © 2015 Harkitis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: European Regional Development Fund- ERDF NSRF 2007-2013, Grant 346985/80753, MK and Greek Ministry of Development-GSRT (20%) PENED (03EΔ/957, MK.
Competing interests: The authors have declared that no competing interests exist.
CYP1A1, CYP1A2 and CYP1B1 belong to a battery of genes that are transcriptionally activated by the aromatic hydrocarbon receptor [1]. More than 90% of known chemical carcinogens, including aromatic amines and polycyclic aromatic hydrocarbons (PAH)s, are substrates of these cytochromes [2–8], and their metabolism often results in the formation of active carcinogenic metabolites [9,10]. Benzo[a]pyrene (B[a]P) is the major PAH component in cigarette smoke and environmental mixtures, such as coal tar and diesel exhaust condensate and is found in the heavily polluted air of urban and industrial areas, in water and heavily cooked food [11]. B[a]P is partly metabolized by CYP1A isozymes to an electrophilic reactive intermediate that covalently binds to DNA and initiates carcinogenesis [3,5]. In addition, B[a]P, acts as a ligand of the AhR and as an inducer of the CYP1 enzymes. The dual role of B[a]P as an inducer of CYP1A1/2 and CYP1B1 and as a pre-carcinogenic substrate for these cytochromes, indicates that B[a]P and related compounds constitute a particularly important group of toxicants able to enhance their own metabolic activation and carcinogenicity [12].
Previous studies have shown that psychological stress and adrenergic receptor (AR)-linked pathways can regulate the expression of cytochrome P450 enzymes [13–18]. Specifically, restraint stress up-regulated CYP1A2 in the murine and rat liver [13,19,20], and AR-agonists or antagonists, and drugs modifying central and peripheral catecholaminergic activity, have a strong impact on the expression of constitutive and B[a]P-induced CYP1A1/2 expression [13]. These results suggest a strong regulatory role of stress and related adrenergic signalling pathways in the regulation of both constitutive and B[a]P induced CYP1A1/2 expression [13,21].
Dopaminergic systems play also significant roles in the regulation of several CYP isozymes catalyzing the metabolism of the majority of prescribed drugs [21–23]. In particular, inhibition of dopamine D2-receptors markedly repressed hepatic CYP2C11, CYP2D1/2, CYP2E1 and CYP3A1/2 expression in rats [22,23]. In this regulatory loop the role of insulin/PI3K/AKT signalling pathway is critical [24].
The D2-dopaminergic receptor-mediated CYP regulation is potentially highly significant as a wide array of drugs, prescribed for a variety of diseases, such as psychosis, depression, bipolar disorder and Parkinson's disease, exert their effects mainly via D2-dopaminergic receptor-linked pathways [25]. These drugs acting as either D2-receptor-agonists or antagonists can modify the activity of several hormonal pathways including the insulin/PI3K/AKT signalling pathway thus influencing the expression of various drug metabolizing cytochromes. This effect may lead to significant drug-drug interactions and may influence the outcome of pharmacotherapy and drug toxicity [18,26,27].
The aim of this study was to investigate the role of D2-dopaminergic receptor- related pathways in the regulation of cytochrome CYP1A1, CYP1A2 and CYP1B1 in the liver. For this purpose, rats were treated with selective D2-antagonists and exposed to either B[a]P or the vehicle alone [22]. The findings indicated the critical role of dopamine D2-receptors in the regulation of the constitutive and B[a]P-induced expression of these cytochromes, and suggest that drugs binding to dopamine D2-receptors may modify the toxicity of environmental pollutants and pre-carcinogens interfering with their metabolism.
Materials and Methods
Adult male inbred Wistar rats (Kuo/Ioa/rr) 3 months old (weighing 250–300g) were used for this study. All animals were housed in groups of 5 and maintained in plastic cages (Makrolon) with wood chip bedding, under a constant temperature (20°C) and a 12h light/dark cycle. Food (the standard rodent chow) and tap water was available ad libitum. All in vivo animal experiments and in vitro experiments employing primary hepatocytes isolated from rats were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical School of the University of Ioannina, and the study has been carried out in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23), revised 1996 and with the Guiding Principles in the Use of Animals in Toxicology, the adopted by the Society of Toxicology in 1989. Efforts were made in order to minimize the number of animals used and reduce their suffering.
The following drugs have been used in this study: Sulpiride (Sigma, USA.); benzo[a]pyrene (Sigma- Aldrich, USA) and L-741,626 (SID 50104688 in PubChem; Sigma-Aldrich, USA).
Experimental procedure
Groups of five animals each received the selective dopamine D2-receptor antagonist, sulpiride (12mg/kg b.w., s.c.; SULP) or the vehicle (normal saline). Alternatively, the highly selective dopamine D2-receptor antagonist, L-741,626 (1.5mg/kg b.w., i.p.) was also administered in a group of rats. The drugs were administered twice daily and for four consecutive days (totally 7 injections in each treatment group). In parallel to SULP treatment, the animals received either olive oil or benzo[a]pyrene (10mg/kg b.w., i.p.) once daily for three consecutive days. The last dose of B[a]P was administered 24 hours before sacrifice.
At the end of the experiment, two hours after the last drug treatment, the animals were sacrificed by decapitation and trunk blood was collected for hormonal determinations. The samples were kept at -20°C until assayed. Simultaneously, the brains were rapidly removed and the dissected hypothalamus was immediately frozen in liquid nitrogen and stored at -80°C until assayed. Parts of the livers were also taken for microsome isolation, total RNA, nuclear and cytosol extraction and were kept at -80°C until analyzed.
Neurochemical analysis
Dopaminergic activity in the hypothalamus was assessed by measuring DA, DOPAC and HVA concentrations. Hypothalamic DA turnover ratio was determined by calculating the rates of DOPAC over DA levels in this brain region. The DOPAC/DA turnover ratio was used as an index of the DA turnover rate, which reflects the dopaminergic activity including the release and/or metabolism of DA, because evidence suggests that dopaminergic activity is better evaluated with DOPAC/DA than with tissue DA, DOPAC and HVA levels [28,29]. For the determination of DA and its metabolites concentration in the hypothalamus, the high performance liquid chromatography with electrochemical detection (HPLC-EC) was used, as previously described [30–32], with some minor modifications. Briefly, the samples were weighed and homogenized for 20 s with a sonicator in ice cold 0.2 N perchloric acid (HClO4). The homogenate was centrifuged at 13,000 rpm at 4°C for 15 min and the supernatant was divided into two portions. The measured compounds were DOPAC and HVA. An aliquot of 200 μl was transferred to an eppendorf tube containing 20 mg activated alumina and was used to extract dopamine (DA) and DOPAC from the homogenate prior to HPLC detection. The following operating conditions were used: column type: Apex-C-18 ODS 5μ (Jones) reverse phase column; mobile phase: 0.1 M sodium acetate (CH3-COONa), 0.1 M citric acid (C6H8O7.H2O), 2.7 x 10−4 M octyl sulphate (C8H17O4; Sna), with 25% methanol (v/v); flowrate: 0.6 ml/min; detector: electrochemical detector (Shimadzu, Japan) maintained at 0.75 V. Data were acquired on a PC-compatible computer using BAS-5 interface. Standard curves were constructed using 8 points between 0.625 and 80 pg/10 μl for dopamine, 23.4–3000 pg/10 μl for DOPAC and 15.62–2000 pg/10 μl for HVA. Correlation coefficients (r) of >0.98 were obtained for all curves. The working standard solutions were kept at -80°C and 10 μl of the standard solution was injected in the beginning of the analysis and between biological samples.
Isolation of microsomes
Microsomal fractions were prepared by homogenization of rat liver samples in ice-cold homogenization buffer (0.15 M KCl, 10 mM K2EDTA, 1mM Dithiothreitol, pH 7.4). The homogenates were centrifuged at 9,000 g (4°C) for 20 min. The upper phase was transferred carefully into new vials and was centrifuged for 70 min at 96,552 g (4°C). After removal of the liquid phase, followed washing of the microsomal pellet: re-suspension in ice-cold homogenization buffer, homogenization and centrifugation for 60 min at 96,552 g (4°C). The washed microsomal pellet was re-suspended in ice-cold storage buffer (K2HPO4/KH2PO4 pH 7.4, 1 mM K2EDTA, 0.1 mM dithiothreitol, 20% glycerol) and stored at -80°C until assayed [33].
Assessment of hepatic EROD and MROD activities
In the microsomes of rat livers the CYP1A1/2-dependent activities were determined. Microsomal protein content was determined by the method of Lowry et al. [34].
Ethoxyresorufin 7-deethylase activity (EROD) was measured fluorometrically in rat liver microsomes, using 7-ethoxyresorufin as substrate in order to assess cytochrome CYP1A1-dependent activity [35].
Methoxyresorufin 7-demethylase activity (MROD), which is mainly catalyzed by cytochrome CYP1A2, was determined fluorometrically in the rat liver microsomes according to Burke and Mayer [35].
Total P450 content was determined from CO-differential spectra of dithionite-reduced samples following the method, which was described by Omura and Sato [36]
Primary hepatocyte cultures
Primary hepatocytes were isolated from rats and used in cultures according to the method of Klaunig et al. [37], [22]. In brief, as previously described by Daskalopoulos et al. [22], primary hepatocytes were isolated from rats weighing 250–300 g using a two-step collagenase perfusion method. They were suspended in William's Medium E (Gibco) containing 1% L-glutamine (PAA) and 1% penicillin/streptomycin. The cells were counted in a Neubauer cell chamber and plated at a density of 1 x 105 cells per well, in 3.8 square centimeter diameter collagen type I coated dish (BIOCOAT, Cell Environment, Becton Dickinson Labware, UK). The viability of the isolated hepatocytes was checked with trypan blue dye 0.4% exclusion and only cells with viability higher than 85% just before plating were used. Hepatocytes were cultured at 37°C for 24 hours under an atmosphere of humidified 5% CO2, in order to allow them to adhere to the wells. Time and dose response experiments started 24 hours later. Primary hepatocyte cultures were treated either with SULP (10 or 25μM) or insulin (1μM) [8], in combination with wortmannin (1μM), an inhibitor of the PI3K/AKT signalling pathway. Wortmannin was added 30 min prior to insulin. Primary hepatocytes were also cultured in the presence of B[a]P at doses raging between 1 and 10μM. Either SULP or insulin were also added in the cell cultures 30 min prior to B[a]P. Time response experiments were conducted with SULP treatment of primary hepatocytes ranging between 1 and 24 hours. These in vitro experiments employing primary hepatocytes were approved by the Institutional Animal Care and Use Committee of the Medical School of the University of Ioannina.
Western blot analysis
Immunoblot analysis of the cytochrome CYPs, STAT5b and FOXO1 apoprotein levels was carried out using microsomes (CYPs) and nuclear extracts or cytosol of liver samples, respectively. For the preparation of the nuclear extracts and cytosol the NE-PER nuclear extraction kit (Pierce, Rockford, IL) was used. The content of the phosphorylated AKT and p70S6K was determined by western blot in total cellular proteins, extracted from the liver using RIPA buffer supplemented with protease inhibitors, PMSF (10μM), BGP (50μM) and NaF (50μM). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL). Proteins were subjected to SDS-PAGE gel electrophoresis and immunoblotting using the following antibodies: rat polyclonal CYP1A1 and CYP1A2 IgGs (they were kindly donated by Dr Ronald Wolf and Colin J. Henderson, London, UK), rat monoclonal total STAT5a/b IgG (Santa Cruz Biotechnology) and rabbit monoclonal p-STAT5b IgG (Tyr 694, Cell Signalling Technology), rabbit polyclonal p-p70S6K IgG (Thr 389), rabbit polyclonal total p70S6K IgG (Cell Signalling Technology), rabbit polyclonal p-FOXO1 (Ser 256) and total FOXO1 IgGs (Santa Cruz Biotechnology), as well as rabbit polyclonal p-AKT (Ser 473), total AKT IgGs (Santa Cruz Biotechnology) and rabbit monoclonal p-mTOR IgG (Ser2448, Cell Signalling) were also used. Secondary antibodies, conjugated with horseradish peroxidase (Santa Cruz Biotechnology) were used and the proteins were detected using a chemiluminescence detection kit (ECL, Amersham, GE Healtcare). Immunoblotting with GAPDH (Santa Cruz Biotechnology) and anti-mouse IgG horseradish peroxidase conjugated secondary antibody, were used as loading control. The membranes were developed by chemiluminescence using the Phototope-HRP Detection Kit for Western blotting (Biolabs INC, New England) and exposed to film.
Quantitative real-time PCR
The TRIzol reagent (Invitrogen) following the manufacturer's protocol was used for the isolation of total RNA from liver tissue and primary hepatocytes. A spectrophotometric method was used for the determination of total RNA concentration in each sample. Quantitative real-time reverse transcriptase PCR (qPCR) was performed with cDNA generated from 1 μg total RNA with a SuperScript II reverse transcriptase kit (Invitrogen). The sequences of the forward and reverse gene-specific primers, which were used are shown in S1 Table. For the real-time PCR reactions the SYBR Green PCR master mix was used (Applied Biosystems, Warrington, UK). These reactions were carried out using the Thermal Cycler Real-Time Detection System C1000 (BioRad, Italy). Relative mRNA expression levels were normalized to β-actin (QuantiTect Primer Assay, Qiagen) and values were quantified using the comparative threshold cycle method.
Hormonal determinations
The GH serum levels were assessed using the rat growth hormone RIA kit (Millipore, MA, USA). The detection limit was 0.5 ng/ml and the intra-assay coefficient of variation was 10%. Prolactin (PRL) serum concentration was determined using the rat prolactin RIA kit (MP Biomedicals Europe, France) and the detection limit was 0.5 ng/ml. Serum thyroid hormone concentrations were determined using the Dynatest T3, Dynatest T4 and Dynatest TSH kits (Brahms, Germany). The normal ranges were 80–200 ng/dl (Dynatest T3), 4.5–12 μg/dl (Dynatest T4) and 0.4–4 mg/ml (Dynatest TSH), respectively. The insulin levels were measured using an EIA kit (Mercodia Rat ELISA kit for insulin, Uppsala, Sweden). The detection limit was 3.3ng/ml and the intra-assay coefficient of variation was 3.1%. The blood glucose levels were measured with a commercially available kit (Merck, Germany) using the technique of glucose oxidase (Trinder, 1969).
Statistical analysis
The data were expressed as means±SE and were analysed using one-way analysis of variance (ANOVA) followed by multiple comparisons with Bonferoni's and Tuckey's list honest significant difference methods. The significance level for all analyses was set at probability of less than or equal to 0.05.
SULP, a dopamine D2- receptor antagonist, reduced total P450 content in the rat liver and prevented the B[a]P-induced increase in it (Fig 1A). These effects suggest that one or more hepatic Cytochrome P450 isoforms are down-regulated following blockade of the dopamine D2-receptor-linked signalling pathways.
(A) Assessment of sulpiride effect on total P450 content in the liver of rats treated with either normal saline or benzo[a]pyrene. (B) Assessment of sulpiride effect on hepatic CYP1B1 mRNA expression following treatment with either normal saline or benzo[a]pyrene. Bonferroni's correction and Tukey post-hoc tests took place in the comparisons of the data presented here. (C vs SULP, C vs B[a]P and B[a]P vs (B[a]P+SULP)). C: controls treated with normal saline; SULP: sulpiride (dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil; *P<0.05, **P<0.01, #P<0.005, ***P<0.001.
Blockade of D2-dopaminergic receptors down-regulates CYP1A1/2 and CYP1B1
Treatment of rats with the selective D2-antagonist SULP, markedly repressed constitutive CYP1A1 and CYP1A2 expression in the liver (Figs 2 and 3). This effect was evident at mRNA, apoprotein and enzyme activity (EROD and MROD) levels and was almost identical for both cytochromes. Furthermore, SULP markedly restricted the hepatic B[a]P-induced EROD and MROD activities (Figs 2 and 3). D2-receptor blockade also repressed the constitutive and B[a]P-induced CYP1B1 mRNA and protein expression in the rat liver (Fig 1B). These findings suggest that the D2-receptor-related signalling pathways potentially interfere with the mechanism of AhR activation.
Assessments of the effects induced by the selective dopamine D2-antagonist sulpiride (SULP) on CYP1A1 relative mRNA expression by using quantitative PCR assays, on CYP1A1 apoprotein levels by using Western blotting, and on CYP1A1-catalyzed EROD activity by using a fluorometric assay. Bonferroni's correction and Tukey post-hoc tests took place in the comparisons of the data presented here (C vs SULP, C vs B[a]P and B[a]P vs (B[a]P+SULP)). Numbers in the western blot captures, correspond to the lower lanes and indicate the relative CYP1A apoprotein expression following treatment compared to the control expression level that was set at 1. The antibody used against CYP1A1 is not highly specific and recognizes CYP1A2 as well. C: controls treated with normal saline; SULP: sulpiride (dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil; *P<0.05, **P<0.01, ***P<0.001.
Assessments of the effects induced by the selective dopamine D2-antagonist sulpiride (SULP) on CYP1A2 relative mRNA expression by using quantitative PCR assays, on CYP1A2 apoprotein levels by using Western blotting, and on CYP1A2-catalyzed MROD activity by using a fluorometric assay. Bonferroni's correction and Tukey post-hoc tests took place in the comparisons of the data presented here (C vs SULP, C vs B[a]P and B[a]P vs (B[a]P+SULP)). Numbers in the western blot captures indicate the relative CYP1A apoprotein expression following treatment compared to the control expression level that was set at 1. C: controls treated with normal saline; SULP: sulpiride (dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil; *P<0.05, **P<0.01, ***P<0.001.
In order to ensure that the effect of SULP on CYP1A1, CYP1A2 and CYP1B1 regulation is mediated by D2-dopaminergic receptors, animals were treated with the highly selective dopamine D2-antagonist, L-741,626. The data confirmed that blockade of D2-dopaminergic receptors results in significant repression of CYP1A1, CYP1A2 and CYP1B1 mRNA expression (P<0.001, Fig 4), indicating that the down-regulating effect of SULP is, indeed, mediated by inhibition of dopamine D2-receptors.
Assessments of the effects induced by the selective dopamine D2-antagonist L-741,626 on CYP1A1, CYP1A2 and CYP1B1 relative mRNA expression by using quantitative PCR assays. Bonferroni's test took place in the comparisons of the data presented here (C vs L-741,626). C: controls treated with normal saline; L-741,626: highly selective dopamine D2-antagonist; ***P<0.001.
In vivo inhibition of dopamine D2-receptors down-regulates the hepatic AhR-dependent CYP regulation system
D2-receptor blockade with SULP resulted in repression of AhR, heat shock protein 90 (HSP90) and aryl hydrocarbon receptor nuclear translocator (ARNT) in the liver of rats exposed to B[a]P (Fig 5). In contrast, SULP further increased the B[a]P-induced AhR repressor (AhRR) mRNA expression (Fig 5). Interestingly, SULP did not affect ARNT and AhRR constitutive mRNA expression, whereas up-regulated HSP90 basal expression (Fig 5).
Assessments of the effects induced by the selective dopamine D2-antagonist sulpiride on aryl hydrocarbon receptor [1], aryl hydrocarbon receptor repressor (AhRR), aryl hydrocarbon receptor nuclear translocator (ARNT) and heat shock protein 90 (HSP90) relative mRNA expression by using quantitative PCR assays. Bonferroni's correction and Tukey post-hoc tests took place in the comparisons of the data presented here. In particular, comparisons of data (Relative ARNT and AhRR mRNA levels) between the group of controls (C) and SULP took place using the Bonferroni's and Tukey tests and no difference was found. (C vs SULP, C vs B[a]P and B[a]P vs (B[a]P+SULP)). C: controls treated with normal saline; SULP: sulpiride (dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil; *P<0.05, **P<0.01, #P<0.005, ***P<0.001.
Role of the insulin/PI3K/AKT/FOXO1 pathway, in the D2-receptor-mediated down- regulation of CYP1A and CYP1B
In order to elucidate the mechanism underlying the down-regulation of CYP1 enzymes following inhibition of D2-receptors, in vitro experiments were employed using primary hepatocyte cultures. Time- and dose-response experiments were conducted for the determination of the optimal conditions for the treatment of primary hepatocytes with SULP (S1 and S2 Figs). It was found that treatment of hepatocytes with SULP did not alter the constitutive and B[a]P-induced expression of CYP1A1/2 and CYP1B1 (Fig 6). The findings of this in vitro study, where SULP affected directly the hepatocytes, indicated that the down-regulation of the CYP1 genes observed following the in vivo SULP administration appears to involve primarily extrahepatic D2-receptor linked pathways.
Assessments of SULP effects on CYP1A1, CYP1A2 and CYP1B1 relative mRNA expression in primary hepatocytes by using quantitative PCR assays. The role of insulin in the regulation of the above mentioned CYPs was also assessed in primary hepatocyte cultures treated either with insulin (1μM, 24hr) alone or in combination with the inhibitor of the PI3K signalling pathway, wortmannin (1μM, 24hr) [76]. Controls were treated with dimethylsulfoxide (DMSO); SULP: sulpiride (selective dopamine D2-antagonist); INS: insulin; WORT: wortmannin; B[a]P: benzo[a]pyrene; ***P<0.001. Comparisons of the relative CYP1A1, CYP1A2, CYP1B1, AhR and ARNT mRNA expression between DMSO- and INS-, as well as between DMSO- and (INS+WORT)-treated hepatocytes were done using the Bonferroni's test and were performed in both, constitutive and B[a]P-induced states. No differences were found between DMSO- and (INS+WORT)-treated hepatocytes, indicating that wortmannin has completely blocked the repressive effect of insulin on both, constitutive and B[a]P-induced mRNA expression of the above mentioned genes.
The findings coming from the in vivo study indicate that the pancreatic β-cell D2-receptor/insulin-related pathway potentially has a central role in the SULP-mediated repressive effect on the constitutive and B[a]P-induced CYP1A1, CYP1A2 and CYP1B1 expression. It is well documented that SULP by blockade of pancreatic islet β-cell D2-receptors increases insulin release (Fig 7), an effect that was confirmed by the present study (Table 1; [22]). It was also found that treatment of rats with SULP activated the insulin/PI3K/AKT/FOXO1 signalling pathway in the liver (Fig 7). Specifically, SULP increased AKT phosphorylation that subsequently activated FOXO1β in the nucleus, thus leading to its translocation into the cytoplasm, and termination of CYP1 gene transcription at a constitutive level (Fig 8). This hypothesis is also supported by the finding that treatment of primary hepatocytes with insulin strongly repressed CYP1A1, CYP1A2, CYP1B1, AhR and ARNT mRNA expression, effects that were completely prevented by wortmannin, a PI3K inhibitor, able to block the insulin/PI3K/AKT/FOXO1 signalling pathway (Fig 6), [38]. SULP also activated AKT and in turn, increased FOXO1β phosphorylation in the nucleus of hepatocytes of B[a]P-exposed rats, indicating an activation of the PI3K/AKT signalling pathway (Fig 8). In vitro treatment of primary hepatocytes with insulin (it is released in vivo following SULP treatment, [22]), resulted in a strong restriction of CYP1 inducibility by B[a]P, an effect that was prevented by wortmannin, a PI3K inhibitor (Fig 6).
Dopamine stimulates D2-ARs on pancreatic β-cells and restricts the release of insulin in response to increased plasma glucose levels [61]. In contrast, blockade of D2-dopaminergic receptors by sulpiride, increases insulin release [22], which in turn, stimulates insulin receptors (IR) in hepatocyte plasma membranes, an effect resulting in the phosphorylation of the Insulin Receptor Substrate (IRS) at different docking sites, where the phosphatidylinositol 3-kinase (PI3K) binds. Activated PI3K converts phosphatidylinositol biphosphate to phosphatidylinositol triphosphate, which subsequently activates protein kinase B (AKT). Upon activation AKT phosphorylates the transcription factor forkhead box O1 (FOXO1), which then translocates into the cytoplasm thus terminating CYP1A1, CYP1A2 and CYP1B1 gene transcription [22,75].
Western blotting showing the SULP-mediated AKT phosphorylation that activated FOXO1 in the nucleus, which in turn translocated into the cytoplasm (reduced FOXO1β phosphorylation in the nucleus and increased FOXO1β phosphorylation in the cytoplasm). Numbers in the western blot captures indicate the relative FOXO1β, AKT and mTOR phosphorylation level following treatment compared to the control level that was set at 1. C: controls treated with normal saline; SULP: sulpiride (selective dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil.
Taken together these data indicate that the mechanism of SULP-mediated down-regulation of CYP1A and CYP1B constitutive expression potentially involves activation of the hepatic insulin/PI3K/AKT/FOXO1β signalling pathway. This activation, however, may not apply for the SULP-mediated restriction of the CYP1 inducibility by B[a]P. It is possible that factors, other than FOXO1β, downstream to the PI3K/AKT signalling pathway are involved and hold predominant roles in the SULP-mediated repression of CYP1 inducibility.
Based on the fact that there is a cross-talk between mTOR/HIF1a and the AhR-linked signalling pathway [39–41], the effect of SULP on mTOR phosphorylation and HIF1a expression was investigated. As mentioned above, D2-receptor blockade with SULP activated the PI3K/AKT signalling pathway in non exposed to B[a]P livers (Fig 8), an activation also seen at the level of mTOR (a down-stream element in this pathway). It should be noted though, that SULP did not alter the level of mTOR phosphorylation in the B[a]P-exposed livers (Fig 8). Furthermore, this D2-receptor antagonist did not alter both, constitutive and B[a]P-induced HIF1a mRNA expression in the rat liver (S3 Fig). Apparently, this cross-talk is not critically involved in the SULP-mediated down-regulation of CYP1 inducibility by B[a]P.
Dopamine D2-receptor blockade inactivates the GH/STAT5b signalling pathway
It is well established that dopamine stimulates the secretion of growth hormone (GH) from the anterior-pituitary lobe [42], which has a down- regulating effect on hepatic CYP1A expression via the Jak2/STAT5b pathway [43]. In the present experimental setting, SULP reduced serum GH levels (Table 1) and consequently, STA5b phosphorylation both, in B[a]P-exposed and non-exposed rats (Fig 9). Based on these findings a CYP1A and CYP1B up-regulation should be expected. However, the opposite is true indicating that the pancreatic D2-receptor-insulin-PI3K/AKT/FOXO1 pathway plays a dominant role overriding that of the GH/STAT5b pathway.
Western blotting showing the SULP-mediated suppression of STAT5b phosphorylation. Numbers in the western blot captures indicate the STAT5b phoshorylation level following treatment compared to the control level that was set at 1. Lanes C: control; SULP: sulpiride (selective dopamine D2-antagonist); B[a]P: benzo[a]pyrene; OIL: olive oil.
Effect of dopamine D2-receptor blockade on neurochemical and hormone levels
The role of hormones, such as GH, thyroid hormones, PRL and glucocorticoids in the regulation of CYP1A and CYP1B isozymes is well documented [26,44–48]. The secretion of these hormones is under a central noradrenergic and dopaminergic control [42]. Therefore, the levels of norepinephrine (NE) and dopamine (DA) were determined in the hypothalamus, the brain site where the corresponding hormone releasing factors are released from [42].
SULP reduced NE content in the hypothalamus of both, B[a]P-exposed and non-exposed rats (P<0.05 and P<0.01, respectively, Table 2). Hypothalamic DA levels were also reduced by SULP only in the non-exposed to B[a]P rats (P<0.05, Table 2), whereas DA turnover ratio was increased in this group of treatment (P<0.01, Table 2), indicating an increased dopaminergic activity in the hypothalamus.
SULP reduced serum T3, T4, GH and corticosterone levels both, in B[a]P-exposed and non-exposed rats, but the drug increased PRL and insulin concentration in these rats (Table 1). Interestingly, B[a]P suppressed serum T3 and T4 levels (Table 1).
Previous studies have shown that central and peripheral catecholaminergic systems play a critical role in the regulation of several CYPs that are involved in the metabolism of the majority of prescribed drugs and toxicants. In particular, noradrenergic systems hold prevalent roles in CYP regulation [13,18,20–22,26,49]. Accumulating evidence indicates that dopaminergic systems and mainly those related to dopamine D2-receptors, are also involved [22,23]. Specifically, inhibition of D2-dopaminergic receptors with either sulpiride or L,741,626 led to a robust down-regulation of CYP2C, CYP2D, CYP2E1, and CYP3A in the rat liver [22,23]. The present study has focused on the role of dopamine D2-receptor-linked pathways in the regulation of constitutive and B[a]P-induced CYP1A1/2 and CYP1B1 expression. It should be noted that dopamine D2-receptors are targets of drugs used in the treatment of various neurodegenerative and psychopathological disorders, such as the Parkinson's disease, depression and psychosis [50–52]. The drugs prescribed in these diseases are either D2-agonists or antagonists that can influence the functional efficiency of the dopaminergic system by mimicking, blocking, or modifying the sensitivity of D2-receptors to dopamine.
The present findings indicated that blockade of dopamine D2-receptors markedly repressed both, the constitutive and B[a]P-induced expression of CYP1A1/2 and CYP1B1. It is well documented that AhR, a member of the basic helix-loop-helix/PER-ARNT-SIM family of DNA-binding proteins, is a determinant transcription factor that regulates both, constitutive and B[a]P-induced CYP1A1/2 and CYP1B1 expression[53]. Transcriptional activation of these CYP isozymes requires firstly binding of the ligand, such as B[a]P, to AhR, which is associated with HSP90 in the cytoplasm. Upon ligand binding the complex translocates into the nucleus where HSP90 dissociates. The ligand-AhR complex then forms a heterodimer with the ARNT [54] and interacts with the xenobiotic responsive elements at the promoters of the CYP1A1/2 and CYP1B1 genes, thus inducing their transcription [54,55]. Therefore, the findings of this study indicate that the reduction of CYP1A1/2 and CYP1B1 inducibility by B[a]P that was detected following D2-receptor blockade with SULP, and that of AhR, HSP90 and ARNT, indicates a mechanism that profoundly includes impaired transcription of the AhR responsive genes [56]. In support of this hypothesis is the fact that SULP further increased the B[a]P-induced AhRR expression. It is well established that contrary to what applies for the induced CYP1 gene expression, the induced levels of AhRR inhibit the AhR function by competing with it in forming a heterodimer with ARNT thus compromising its XRE binding activity [57].
The fact that SULP further enhanced the B[a]P-induced AhRR expression, instead of suppressing it, as in the case of other AhR target genes, confirms previous observations that the outcome of AhR-AhRR interactions is more complex than suggested by a simple AhR-induced, AhRR-mediated feedback model, which is based on the ability of AhR to transactivate the AhRR gene, and the ability of AhRR to repress AhR activity, at least as defined by CYP1 induction. In particular, it has been reported that the ligand-activated AhR may not transactivate AhRR in all tissues and under some circumstances, may actually repress AhRR transcription, thereby maximizing AhR activity [58].
It should be noted that AhRR, HSP90 and ARNT do not hold essential roles in the SULP-mediated repression of CYP1A and CYP1B constitutive expression, because the drug did not affect basal ARNT and AhRR expressions, whereas increased that of HSP90. Profoundly, SULP along with AhR, repressed some of the numerous ancillary factors that are involved in the activation of the AhR transcriptional complex that regulates constitutive CYP1 expression [59]. Finally, it appears that while AhR regulates both, the constitutive and the induced CYP1 expression, the mechanism mediating the SULP repressive effect on each expression state is not identical. There may be differences at the level of AhR interaction with other transcription factors, or potentially at some upstream signalling pathways, or at the AhR affinity level in binding with the respective gene promoters.
The role of dopamine D2-receptor-linked pathways in the down-regulation of hepatic CYP1A1/2 and CYP1B1 was confirmed with another, highly selective D2-antagonist, the L-741,626, which also repressed their expression. This D2-receptor mediated effect is probably indirect, because treatment of primary hepatocyte cultures with SULP had no effect on the expression of CYP1A1/2 and CYP1B1. It is therefore, hypothesized that the repressive effect on CYP1A1/2 and CYP1B1, which is observed when SULP is administered in vivo, is the outcome of the drug's effects on central and peripheral dopaminergic and hormonal systems that have an impact on hepatic signalling pathways regulating these CYP genes [22].
Our results suggest a role for the hepatic insulin/PI3K/AKT/FOXO1 signalling pathway in the SULP-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression. It has been previously shown that inhibition of pancreatic D2-dopaminergic receptors with SULP triggers insulin secretion from pancreatic islet β-cells. This effect leads to activation of the insulin/PI3K/AKT signalling pathway in the hepatocytes [22] and ultimately, to phosphorylation of the nuclear factor FOXO1β and its subsequent translocation into the cytoplasm and termination of CYP1A1, CYP1A2 and CYP1B1 transcription. Upstream to the insulin/PI3K/AKT/FOXO1 pathway, dopamine is a significant regulatory factor that restricts the release of insulin in response to increased plasma glucose levels (Fig 7). Dopamine, exerts its negative control in insulin release via stimulation of pancreatic D2-receptors expressed in islet β-cell membranes [60]. In contrast, blockade of these receptors with SULP increases insulin release (Fig 7), [23], which in turn, activates the hepatic insulin/PI3K/AKT/FOXO1 signalling pathway, thus exerting a negative regulatory control on several P450s [22,60–64]. This hypothesis is also supported by the findings of an in vitro study. Treatment of primary hepatocyte cultures with insulin resulted in a strong down-regulation of CYP1A1, CYP1A2 and CYP1B1 via activation of the PI3K/AKT signalling pathway, because pre-treatment of these cells with wortmannin, a PI3K inhibitor, prevented the repressive effect of insulin on these cytochromes. The PI3K/AKT signalling pathway may also participate at least in part, in the SULP-mediated reduction of CYP1A1/2 and CYP1B1 inducibility by B[a]P. This hypothesis is based on the fact that SULP increased AKT and FOXO1β phosphorylation in the liver of B[a]P-exposed rats, indicating an activation of the insulin/PI3K/AKT signalling pathway. Furthermore, insulin represses CYP1 inducibility by B[a]P in primary heatocytes and wortmannin completely blocked this effect. The in vivo and in vitro findings indicate that profoundly, some distinct downstream elements in the insulin/PI3K/AKT signalling pathway participate in the SULP-mediated repression of CYP1A1/2 and CYP1B1 inducibility by B[a]P than those regulating the repression of CYP1 constitutive expression. However, further investigation involving potentially Chip assays is needed in order to completely clarify the involvement of PI3K/AKT signalling pathway in CYP1A and CYP1B regulation by SULP.
Previous studies reported a cross-talk between the AhR and HIF1a signalling pathways [39]. It is well established that activation of the PI3K/AKT/mTOR-related pathway up-regulates HIF1a [39,41], which in turn, inactivates AhR, thus repressing CYP1A inducibility [40]. It should be taken also into account the fact that the impact of this cross-talk depends on ARNT availability, which is an essential element in both, AhR and HIF1a signalling pathways [39] and SULP repressed the B[a]P-induced ARNT expression. The present study also indicated that SULP activated mTOR in the liver of non exposed to B[a]P rats, but had no effect on the B[a]P-exposed livers. In addition, SULP did not affect HIF1a inducibility by B[a]P. Combined these data indicate that the HIF1a/mTOR and AhR cross-talk did not have any critical role in the SULP-mediated suppression of the CYP1A1/2 and CYP1B1 inducibility by B[a]P.
In the D2-receptor related regulatory system, the cross-talk between the AhR- and insulin/PI3K/AKT-linked signalling pathways apparently, holds significant roles in CYP1 regulation [65–67]. In light of this interaction, it is suggested that the SULP-induced repression of CYP1A1/2 and CYP1B1, could be attributed, at least in part, to the insulin-induced up-regulation of the carbohydrate-responsive element-binding protein (ChREBP) in the liver. This factor exerts a negative control on ARNT/HIF-1β, an essential element in the AhR regulatory system [65].
Stimulation of GH secretion from the anterior pituitary lobe by dopamine is well documented [42]. STAT5b, is the major GH pulse-activated transcription factor, which is involved in the regulation of several P450s in the liver [43,68]. Therefore, the possible involvement of the GH/STAT5b pathway in the SULP-mediated repression of CYP1A and CYP1B1 was assessed. Treatment with SULP resulted in decreased serum GH levels and inactivation of the GH/STAT5b signalling pathway. Thyroid hormone levels were also decreased by SULP. Based on the fact that both, GH and thyroid hormones, hold a negative control on CYP1A regulation [43,69,70], it is assumed that the SULP-induced repression of CYP1A and CYP1B1 is not mediated by inactivation of the GH/STAT5b- and thyroid hormone-related pathways.
Other regulatory pathways are potentially involved including PRL, as SULP increased serum PRL concentration, which has a down-regulating effect on various P450 isoforms [69,71]. Glucocorticoid-related pathways could be also involved, as SULP reduced serum corticosterone concentration, which is a positive regulator of CYP1A1/2 and CYP1B1 in the rat liver [72].
In conclusion, inhibition of dopamine D2-receptors results in significant repression of constitutive and B[a]P-induced CYP1A1, CYP1A2 and CYP1B1 expression in the rat liver. The activation of the AhR-related regulatory system was reduced following D2-inhibition, indicating a cross-talk between the D2-receptor- and AhR-regulatory pathways. Previous studies indicated that D2-receptor-linked signalling pathways potentially cross-talk also with a broad array of cellular transcription systems that regulate various P450 genes encoding isozymes that metabolize a plethora of prescribed drugs and toxicants [22], indicating that the down-regulating effect of D2-receptor inhibition is not specific for the AhR-related regulatory system. The down-regulating effect of SULP on constitutive CYP1A and CYP1B expression appears to be mediated by activation of the insulin/PI3K/AKT pathway, which though has a less significant contribution in the SULP-mediated reduction of CYP1 inducibility by B[a]P. The PRL-activated negative regulatory pathway and inactivation of the glucocorticoid-linked up-regulating pathway may also contribute in the SULP-mediated down-regulation of CYP1A and CYP1B. As dopamine D2-receptors serve as targets for various prescribed drugs, patients following a relative treatment may have altered drug toxicity and efficacy outcomes due to reduced enzymatic activity when exposed to substrates of the CYP1A1/2 and the CYP1B1. These findings potentially indicate that the regulatory pathways involving D2-receptors could be considered as targets of the pharmaceutical strategy for the protection of individuals heavily exposed to environmental toxicants and pre-carcinogens that are activated upon CYP1A- and CYP1B-catalyzed metabolism. It should be noted though that carcinogenesis is a complex and multi-factorial process that involves various regulatory systems, and not only those related to CYP1A1/2 and CYP1B1 enzymes [73,74]. Therefore, further investigation is needed in order to clarify the outcome of D2-receptor inhibition in carcinogenesis, such as assessment of the B[a]P-DNA-adduct formation in the liver and other tissues.
Supporting Information
S1 Fig. Time response variation in CYP1 gene expression following sulpiride.
Alterations in CYP1A1, CYP1A2 and CYP1B1 relative mRNA expression following sulpiride (SULP, 10μM) exposure of primary hepatocytes for different time periods was assessed. Control cells were treated with DMSO. SULP1: incubation of hepatocytes with sulpiride for 1 hr; SULP6: incubation of hepatocytes with sulpiride for 6 hr; SULP12: incubation of hepatocytes with sulpiride for 12 hr; SULP24: incubation of hepatocytes with sulpiride for 24 hr; ***P<0.001.
S2 Fig. Dose response variation in CYP1 gene expression following sulpiride.
Alterations in CYP1A1, CYP1A2 and CYP1B1 relative mRNA expression following exposure of primary hepatocytes with either 10μM or 25μM sulpiride (SULP) for 24 hr. Control cells were treated with DMSO.
S3 Fig. Sulpiride effect on constitutive and B[a]P-induced HIF1a mRNA expression in the rat liver.
S1 Table. Nucleotide sequences of the primers used.
These primers were used for the quantitation of the mRNA levels of the genes tested using q-PCR.
This research project has been co-financed by 1) the European Union (European Regional Development Fund- ERDF) and Greek national funds through the Operational Program "THESSALY- MAINLAND GREECE AND EPIRUS-2007-2013" of the National Strategic Reference Framework (NSRF 2007–2013, Grant 346985/80753) and 2) the E.U.-European Social Fund (80%) and the Greek Ministry of Development-GSRT (20%) PENED (03EΔ/957). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions
Conceived and designed the experiments: MK. Performed the experiments: PH EPD FM MK AF GA. Analyzed the data: MK AF MM. Contributed reagents/materials/analysis tools: MK ML MM AF. Wrote the paper: MK ML.
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text:
© Getty Images
Sen. Ted CruzTed CruzWarren builds her brand with 2020 down the road Trump wall faces skepticism on border No Congress members along Mexico border support funding Trump's wall MORE (Texas) is firing back at Sen. John McCain (R-Ariz.) for suggesting there are questions about Cruz's eligibility to run for president.
Cruz brushed off McCain's suggestion that it was "worth looking into" the issue, saying the 2008 GOP nominee is simply trying to help Sen. Marco RubioMarco RubioTop Trump officials push border wall as government shutdown looms Rubio defends Trump: 'This whole flip-flop thing is a political thing' Rubio: Shutdown would have 'catastrophic impact' on global affairs MORE (R-Fla.) win the Republican nomination.
"I think it is no surprise to anybody that John McCain is going to be supporting Marco Rubio in this election," Cruz told Bloomberg's Mark Halperin on "With All Due Respect."
"It's no surprise at all that he's trying to do what he can to help the candidate that he's favoring who he thinks shares policy positions with him," Cruz added.
McCain, who faced similar eligibility questions during his 2008 bid, argued during a local radio program in Arizona on Wednesday that there is a difference between him and Cruz.
Cruz was born in Canada to an American mother. McCain was born on a U.S. military base in Panama.
"So I think there is a question. I am not a Constitutional scholar on that, but I think it's worth looking into. I don't think it's illegitimate to look into it," McCain said.
Cruz has argued that he is eligible for the presidency and a "natural born citizen" as stipulated by the Constitution after rival Donald Trump questioned his eligibility on Tuesday.
McCain had endorsed Sen. Lindsey Graham (R-S.C.) for president, but Graham dropped out of the race last month after failing to gain traction in the polls.
Cruz and McCain have clashed frequently in the Senate, with McCain once calling Cruz and Sen. Rand Paul (R-Ky.) "wacko birds."


text:
"Great products but very troubled company because of highly leveraged purchase from Thomson and poor product decisions"
- Work/Life Balance
- Culture & Values
- Career Opportunities
- Comp & Benefits
- Senior Management
Helpful (8)
I worked at Cengage full-time (More than 5 years)
Best in class products, industry leadership in the library reference space and some niche areas on the textbook side.
Too much debt and bad management decisions have caused a major upheaval. Constant reorganization starting in 2010 left many good employees jobless while, for the most part, the upper-middle management folks have snaked their way into new positions within the company. Just because you claim to be a "digital" company doesn't make it so.
Advice to Management
Declare Bankruptcy, sell off the profitable divisions that have strong reoccurring revenue to companies that can maximize their potential
Helpful (3)
I worked at Cengage full-time (More than 10 years)
The company was one of the best in the industry and that has now changed! The management was at one time the best in the industry. There was pride, and respect that came with being a success at Cengage Learning.People make the company!
Too much change all at once now everyone is in fear of their future and the companies as well , The sharks a circling the tank.
Advice to Management
Your sales force is the driving force and don't forget this!
Helpful (1)
I worked at Cengage full-time (More than 10 years)
Decent base pay w/ good incentive compensation if you make your number. Great benefits package...sales reps get a car w/ maintenance and gas all paid for, along with medical, 401(k), etc. Very competitive textbook offerings in most disciplines.
Too many different technology offerings with no consistent brand. Financially unstable, which has lead to mass layoffs. Long and non-traditional hours, so lots of nights and weekends make it hard to get that work/life balance.
Advice to Management
Reinvent yourself by changing the business model. Trying to convert everything from print to digital offerings is only half the battle. Need to market and sell directly to students.


text:
Lemon Apple Butter
|4 Pound (About 12 Medium)
|2 Cup (32 tbs)
|1 , grated (Peeled)
1. Wash apples; cut into eighths.
2. In a large saucepan, place apples and add little water; cook until apples are tender.
3. In a food mill, process cooked apples.
4. Return processed apples to the saucepan and add sugar and grated lemon peel; cook until mixture is thick.
5. Into hot sterilized jars, ladle mixture leaving 1/8 inch empty on top.
6. Using a clean and dry kitchen towel, wipe the rim of jar and adjust lid.
7. Into a canner of boiling water, place the jars and process for 5 minutes.
8. Remove jars and place over a clean towel.
9. Let the jars cool down and seal completely until they are self-sealing type.
10. Spread Lemon Apple Butter over bread and serve.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 2503 Calories from Fat 27
% Daily Value*
Total Fat 3 g5%
Saturated Fat 0.53 g2.7%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 19.9 mg0.8%
Total Carbohydrates 657 g218.9%
Dietary Fiber 46.3 g185.1%
Sugars 588.1 g
Protein 5 g10.8%
Vitamin A 19.9% Vitamin C 213.5%
Calcium 14.8% Iron 14.6%
*Based on a 2000 Calorie diet


text:
Cannot connect to certain computers via IP address using WMI.
All SAM versions
This issue is caused by an improper configuration or bad record on the DNS server. Microsoft uses DNS reverse lookups when trying to connect through WMI using IP addresses.
Add a period, (.) to the end of the IP address when making any query. This forces WMI to go through DNS resolution.
In the SolarWinds.APM.Probes.dll.config file:
[WmiSettings Retries="3"
Retries - number of retries when trying to connect using WMI.
Replaces old [add key="WMI.Monitor.Retries" value="3"/]
WaitToRetry - interval between retry attemps.
Replaces old <add key="WMI.Monitor.WaitToRetry" value="10"> </add>
Default - use IP address
OnlyAutoCorrected - use only IP address + "." (if IP address is IPv4 address)
BothAutoCorrectedFirst - try to use AutoCorrected version first and then Default version
BothDefaultFirst - try to use Default version first and then AutoCorrected version
If "true," then the Reverse DNS Lookup is executed and the log file contains IP address list of target machine to check if we are connecting to correct machine. You can find following message in the log file:
2012-02-23 12:36:43,766 [STP Pool:4 Thread #0] [C0] DEBUG SolarWinds.APM.Probes.WMI.WMIHelper - List of all IP addresses for '10.140.126.27': 2001:10:140:126:d083:e915:7c6d:48db;10.140.126.27


text:
Stilnovo (Manufacturer)
Satin glass pendant.
Place of Origin
Date of Manufacture
circa 1950s
Excellent. Rewired. E27 60w maximum bulb..
38 in. H
97 cm H
Dealer Location
Los Angeles, CA
Number of Items
Reference Number
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text:
Good news for employers can be found in a study by Hewitt Associates that reports that health insurance costs increased by 9.2 percent this year. That's the first single-digit increase since 2000. The report contained not-so-good news for employees. Their contributions to health care premiums increased 65 percent, from $877 per individual in 2002 to $1,444 this year.... Humana has agreed to pay $40 million (as well as an additional $18 million in legal fees to the plaintiffs) in a settlement of a class-action lawsuit filed by more than 700,000 physicians. The suit says that Humana delayed or denied payment of claims for medical services rendered by the doctors. Humana is the latest of the 10 health insurers named in the suit to reach a settlement.... Do as I say, not as I do? Medical malpractice claims paid by insurers and hospitals on behalf of doctors in their employ must be reported to the National Practitioner Data Bank. Now, however, a report by the Office of Inspector General says that between 1997 and 2004, agencies under the Department of Health and Human Services failed to report 474 instances in which they paid medical malpractice claims. HHS Inspector General Daniel Levinson sums up the problem by noting that "underreporting of the department's own medical malpractice cases lessens the usefulness of the National Practitioner Data Bank and undermines departmental efforts to regulate private and public sector compliance." Indeed.
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The future of biosimilars in this country is nothing if not uncertain. Most immediately, the U.S. Supreme Court is hearing a case that will determine the timing of the 180-day waiting period before a biosimilar can go on the market. But there are larger and longer-term issues at play as well.
While coupons help individual consumers, they are also having a major impact on the insurance industry and anyone responsible for paying health care bills. Insurers and pharmacy benefit managers complain that they foil formularies and other pricing strategies designed to steer consumers to less-expensive drugs.
The hard truth is that telehealth's future—its size, its contours—will depend a lot on what payers will be willing to pay for. Currently, commercial plans cover only a limited number of services. In addition, research suggests that there may be quality and utilization problems.
Insurers should consider covering new drug-delivery devices that can improve outcomes while lowering disease-specific pharmacy and long-term overall health care costs. Managing these devices in the pharmacy benefit will consolidate volume-based purchasing and capitalize on PBM strategies for improving adherence.
Basaglar is coming on the scene during tumultuous times for insulin products. Manufacturers are under attack for price hikes. There are allegations of backroom rebate deals. And a class-action lawsuit has been brought on behalf of uninsured patients, charging insulin makers with setting artificially high prices.
Evaluating the quality of telemedicine care is about as easy as evaluating the quality of health care, period, and researchers are still ironing out the methodological kinks. That may be one reason research results are all over the place. This article involved reviewing nine such studies, and the findings are a mixed bag.
The results can be tragic. Patients with addictions are unlikely to wait the hours or days it takes health insurers to approve the medications they need. Insurers are changing their practices, but not without some outside pressure.


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Been busy working today...I need to take a break! lol Here are a few goodies I added to my shop today! Yay!
15% off with code LUVMOM now thru Mother's Day!
~Be beautiful, inspired, and blessed!
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Following you on GFC and coming over from Blog It Forward Tuesdays. Love the peacock earrings and did a little write up about them on my blog. Keep up the excellent work.
Christine Senter
Thanks so much for stopping by Christine!
Glad you like the earrings the design just popped into my head! I though a mom who loves peacock feathers would love these!
Checking out your site now!
~Take care!
Hello lovelies! Thanks so much for stopping by my blog!
I hope it uplifts and inspires you! You comments are very appreciated!