Publications by authors named "Alexandre Marchand"

28 Publications

  • Page 1 of 1

EPO transgene detection in dried blood spots for antidoping application.

Drug Test Anal 2021 May 7. Epub 2021 May 7.

Analysis Department, Agence Française de Lutte contre le Dopage (AFLD), Châtenay-Malabry, France.

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.
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http://dx.doi.org/10.1002/dta.3059DOI Listing
May 2021

Detection of LongR -IGF-I, Des(1-3)-IGF-I, and R -IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes.

Drug Test Anal 2021 Feb 15. Epub 2021 Feb 15.

AFLD-Agence Française de Lutte contre le Dopage, Département des Analyses, Châtenay-Malabry, France.

Insulin-like growth factor-I (IGF-I) and its analogs LongR -IGF-I, Des(1-3)-IGF-I, and R -IGF-I are prohibited substances in sport. Although they were never approved for use in humans, they are readily available as black market products for bodybuilding and can be used to enhance physical performance. This study's aims were to validate a fast and sensitive detection method for IGF-I analogs and to evaluate their detectability after intramuscular administration in rats. The sample preparation consisted of an immunopurification on MSIA™ microcolumns using a polyclonal anti-human-IGF-I antibody. The target substances were then directly analyzed by nano-liquid chromatography coupled with high-resolution mass spectrometry. Abundant signs of lower quality, oxidized peptide forms were found in black market products, justifying the need to monitor at least both the native and mono-oxidized forms. The analytical performance of this method (linearity, carry over, detection limits, precision, specificity, recovery, and matrix effect) was studied by spiking the analogs into human serum. Following a single intramuscular administration (100 μg/kg) in rats, detection was evaluated up to 36 h after injection. While unchanged Des(1-3)-IGF-I and R -IGF-I were detected until 24 h after administration, LongR -IGF-I disappeared rapidly after 4 h. Des(1)-LongR -IGF-I, a new N-terminal Long-R -IGF-I degradation product, was detected in addition to Des(1-10)-LongR -IGF-I and Des(1-11)-LongR IGF-I: the latter was detected up to 16 h. The same products were found after in vitro incubation of the analogs in human whole blood, suggesting that observations in rats may be extrapolated to humans and that the validated method may be applicable to antidoping testing.
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http://dx.doi.org/10.1002/dta.3016DOI Listing
February 2021

Adaptation of Elecsys anti-severe acute respiratory syndrome coronavirus-2 immunoassay to dried blood spots: proof of concept.

Bioanalysis 2021 Feb 4;13(3):161-167. Epub 2021 Feb 4.

Analysis Department - Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro-92290 Châtenay-Malabry, France.

Several automated immunoassays have been validated on serum/plasma to evaluate the presence of significant levels of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies, signs of a present or past infection, but the use of dried blood spots (DBS) would facilitate sampling, shipping and storage. The aim of this project was to give proof of concept of the possibility to use of the automatized Elecsys anti-SARS-CoV-2 immunoassay with a volumetric DBS device. Linearity and correlation were satisfactory between volumetric DBS and plasma. A cut-off value was suggested and should be validated with more samples. this study strongly support the possibility to work with volumetric DBS instead of serum/plasma to test for anti-SARS-CoV-2 antibodies.
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http://dx.doi.org/10.4155/bio-2020-0318DOI Listing
February 2021

An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control.

Drug Test Anal 2020 Dec 2. Epub 2020 Dec 2.

Analysis Department, Agence Française de Lutte contre le Dopage (AFLD), Châtenay-Malabry, France.

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti-EPO antibodies. In addition to IEF-PAGE, the SDS-PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW). However, to adapt to new generations of rEPOs exhibiting higher MW, which were not well detected after SDS-PAGE, sodium lauroyl sarcosinate (SAR) is now used instead of sodium dodecyl sulfate (SDS) for the initial EPO testing procedure on doping control samples. The SAR-PAGE method is nevertheless expensive as it requires frequent buffer preparations using highly purified sarkosyl powder. In addition, this reagent needs to be handled with care due to acute toxicity by inhalation. The aim of this work was to improve the SDS-PAGE method by increasing its sensitivity and transfer of high-MW rEPOs. First, using a biotinylated primary anti-EPO antibody and avoiding the use of a secondary antibody increased the general sensitivity of both SDS-PAGE and SAR-PAGE to all rEPOs about four-fold. Then, by changing the buffer system during the protein transfer, with a CAPS buffer and a discontinuous buffer transfer system, high-MW rEPOs, EPO-Fc and CERA were transferred with higher efficiency and detected with high sensitivity. This optimized SDS-PAGE protocol could be adopted by anti-doping laboratories as an alternative to SAR-PAGE.
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http://dx.doi.org/10.1002/dta.2601DOI Listing
December 2020

Evaluation of erythropoietin biosimilars Epotin™, Hemax® and Jimaixin™ by electrophoretic methods used for doping control analysis and specific N-glycan analysis revealed structural differences from original epoetin alfa drug Eprex®.

J Pharm Biomed Anal 2021 Feb 5;194:113750. Epub 2020 Nov 5.

Analyses Department, Agence Française de Lutte contre le Dopage (AFLD), Châtenay-Malabry, France. Electronic address:

Recombinant human erythropoietin (rEPO) biosimilars are copies of epoetin drugs developed after the first patents ended. However differences in the process of production can result in small structural differences when compared to the reference product. Differences in N-glycosylation profiles are of particular importance for rEPOs, because they can drastically impact the half-life in circulation and activity. Changes of structure can also impact electrophoretic profiles that are used to reveal the presence of a rEPO in a doping control sample. In this study three not well characterized biosimilars were evaluated (Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria). As these products could be used for doping, first their EPO profiles were determined using the antidoping methods (electrophoretic separation by the charge (isolectric focusing, IEF-PAGE) or the molecular weight (SDS-PAGE) and specific EPO immunodetection). Compared to the original epoetin alfa Eprex®, it revealed more basic isoforms for Epotin™ and Jimaixin™ after IEF-PAGE and a slightly lower molecular weight after SDS-PAGE in particular for Hemax®. To better understand the reason for these differences, EPO specific N-glycans were evaluated using two complementary approaches: MALDI-TOF mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. All three biosimilars presented a significant decrease in the major glycan forms of Eprex® along with an increase in less complex forms. Jimaixin™ and Epotin™ presented also a lower amount of fully sialylated forms. HILIC method also showed that O-acetylation level of sialic acid residues might vary from one rEPO to the other.
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http://dx.doi.org/10.1016/j.jpba.2020.113750DOI Listing
February 2021

Detection of a nonerythropoietic erythropoietin, Neuro-EPO, in blood after intranasal administration in rat.

Drug Test Anal 2020 Nov 9;12(11-12):1605-1613. Epub 2020 Sep 9.

Analysis Department, French Anti-Doping Agency (AFLD), Châtenay-Malabry, France.

Nonerythropoietic erythropoietins (EPOs) are investigated for their high antioxidant properties. A new drug candidate under clinical investigation to treat brain diseases is Neuro-EPO, produced by selecting EPO isoforms with low sialic acid content. Intranasal administration allows to bypass the blood-brain barrier to get a fast and concentrated delivery to the brain. The aims of this project were to characterize Neuro-EPO with anti-doping methods used to detect conventional recombinant EPOs (isoelectric focusing [IEF] and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and to evaluate the window of detection of Neuro-EPO in brain and blood (plasma) after a single intranasal administration in rats. Neuro-EPO drug analyzed by IEF-PAGE presented a very basic profile completely detected only when using a 2-8 or 2-10 pH gradient instead of the conventional 2-6 pH gradient. Its profile consisted in six main bands that did not interfere with endogenous EPO profile from human or rat. After SDS-PAGE, a broad band was detected for Neuro-EPO in the same area as endogenous EPO, making Neuro-EPO identification very difficult by this approach. Therefore, IEF was the method for identification chosen after administration in rats. Neuro-EPO was clearly identified in blood 2 and 6 h after the delivery. Fainter signals were obtained between 12 and 48 h, but some characteristic very basic bands remained detectable. Surprisingly, brain extracts did not show the presence of Neuro-EPO even 2 h after administration, indicating a fast degradation or elimination from the brain to the bloodstream. This experiment indicated that detection of Neuro-EPO after intranasal delivery should be possible for a few days.
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http://dx.doi.org/10.1002/dta.2924DOI Listing
November 2020

Improved detection methods significantly increase the detection window for EPO microdoses.

Drug Test Anal 2021 Jan 9;13(1):101-112. Epub 2020 Aug 9.

Analysis Department - Agence Française de Lutte contre le Dopage (AFLD), Châtenay-Malabry, France.

To reproduce a potential doping scenario, a 2 week administration of recombinant erythropoietin (rEPO) microdoses alone or in combination with growth hormone (GH) microdoses (three times a week) was performed on healthy and athletic male subjects. The aim of this study was to evaluate the identification capability of rEPO in samples obtained during and post treatment. Detection was tested in urine and blood using the antidoping techniques for rEPO detection (iso-electric focusing (IEF)-, sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and for some urine samples the sarcosyl (SAR)-PAGE method) with some improvements: for blood samples, instead of a simple concentration step, immuno-extraction of EPO was performed for all urines to limit protein contamination that can affect migration. In addition, elution buffer modifications also improved the quality of migration. The use of a recently validated biotinylated anti-EPO antibody simplified the protocols, allowing a single transfer step instead of a double-blot even by IEF with a lowered background. The criteria for suspicious blood and urine samples by IEF were also re-evaluated. While endogenous EPO was not decreased over the course of the study, EPO microdoses were detectable in blood and urine between 24 h and 72 h after an administration. Detection in urine in combination with SDS-PAGE was the most sensitive combination for prolonged detection (100% identification after 48 h, 91% after 72 h), slightly better than IEF. Urine samples also tested by SAR-PAGE indicated a similar sensitivity of detection to SDS-PAGE. GH co-administration had no impact on rEPO elimination/detection.
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http://dx.doi.org/10.1002/dta.2904DOI Listing
January 2021

Use of capillary dried blood for quantification of intact IGF-I by LC-HRMS for antidoping analysis.

Bioanalysis 2020 Jun 1;12(11):737-752. Epub 2020 Jun 1.

AFLD - Agence Française de Lutte contre le Dopage, Département des analyses, Châtenay-Malabry, France.

 IGF-I is used as a biomarker to detect Growth Hormone doping in athletes' blood samples. Our aim was to develop and validate a fast, high-throughput and accurate quantification of intact IGF-I from volumetric absorptive microsampling (VAMS) dried blood using LC coupled to high resolution mass spectrometry (LC-HRMS). IGF-I was extracted from the VAMS, released from its binding proteins, concentrated using microelution SPE and analyzed by LC-HRMS. The method was successfully validated in accordance with the World Anti-Doping Agency's requirements. Subsequently, IGF-I measurements from capillary dried blood and serum were compared. The combination of VAMS, microelution SPE and LC-HRMS is a promising strategy applicable to IGF-I quantification in athletes' samples.
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http://dx.doi.org/10.4155/bio-2020-0013DOI Listing
June 2020

Applying metabolomics to detect growth hormone administration in athletes: Proof of concept.

Drug Test Anal 2020 Jul 19;12(7):887-899. Epub 2020 Apr 19.

Département des analyses, Agence Française de Lutte contre le Dopage (AFLD), Châtenay-Malabry, France.

Growth hormone (GH), an endogenous peptide regulating anabolism and lipolysis in humans, is known to be abused by athletes to improve their performance. Despite the development of two distinct screening methods, few positive cases have been reported by the antidoping authorities, probably due to the quick turnover of GH and the masking effects of age, ethnicity, and sex. Apart from growth regulation, GH is known to affect several metabolic pathways in humans including ketosis, amino-acid uptake, and protein breakdown. It is reasonable to imagine observing its markers of effects through the leading tool on metabolism study, metabolomics. In this proof-of-concept study, a cohort of well-trained volunteers was split in two equal groups and administered with micro-doses of EPO or EPO + GH every second day for 2 weeks. Urine and plasma samples were collected before, during, and after treatment and analyzed using metabolomics and lipidomics approaches. The results show that, by applying a direct discriminant analysis on the treated groups, it is possible to distinguish the treatments, and to use this difference to classify them correctly. High intragroup variability is observed, due to the subject-specific effect of the hormones. Through time 0 centering the data, a longitudinally tracking of the group was performed and a higher difference was observed between the groups, including a perfect classification of the samples before and after the treatments.
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http://dx.doi.org/10.1002/dta.2798DOI Listing
July 2020

The case of the EPO-poisoned syringe.

Drug Test Anal 2020 May 23;12(5):637-640. Epub 2020 Jan 23.

Analysis Department - Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro, Châtenay-Malabry, 92290, France.

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http://dx.doi.org/10.1002/dta.2757DOI Listing
May 2020

Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods.

Drug Test Anal 2019 Nov 16;11(11-12):1698-1713. Epub 2019 Aug 16.

Laboratoire de Biophysique et Bio-Analyses, Institut des BiomoléculesMax Mousseron (IBMM),  UMR 5247,CNRS-ENSCM-Université Montpellier, France.

The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2-week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum. While the rEPO microdose regimen was associated with a significant increase in reticulocytes, no clear elevation in hemoglobin concentration (HGB) was observed. Using a correction by plasma volume did not reveal more effects of EPO on HGB. Our results did not show any additional effect when the GH microdoses were co-administered. In addition, no clear increase in VO2max was observed after treatment, with an elevation in only half the subjects in both groups (EPO and EPO+GH). A clear effect of GH on insulin-like growth factor I (IGF-I) was seen but it was lower on procollagen III amino-terminal propeptide (P-III-NP). GH detection using the direct isoform test identified only one subject 24 hours after receiving GH. The GH biomarker test combining IGF-I and P-III-NP was not able to detect the GH administration. However, a longitudinal follow-up of the intraindividual variations showed a significant increase in IGF-I 24 and 48 hours after GH administration in most subjects, while the effect of GH microdoses on P-III-NP was less straightforward.
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http://dx.doi.org/10.1002/dta.2674DOI Listing
November 2019

Inhibition of miR-223 Expression Using a Sponge Strategy Decreases Restenosis in Rat Injured Carotids.

Curr Vasc Pharmacol 2020 ;18(5):507-516

INSERM U1088, CURS, CHU Amiens Picardie, Avenue Rene Laennec, Salouel, F-80054, Amiens, France.

Objective: Restenosis is a frequent complication of angioplasty. It consists of a neointimal hyperplasia resulting from progression and migration of vascular smooth muscle cells (VSMC) into the vessel lumen. microRNA miR-223 has recently been shown to be involved in cardiovascular diseases including atherosclerosis, vascular calcification and arterial thrombosis. In this study, our aim was to assess the impact of miR-223 modulation on restenosis in a rat model of carotid artery after balloon injury.

Methods: The over and down-expression of miR-223 was induced by adenoviral vectors, containing either a pre-miR-223 sequence allowing artificial miR-223 expression or a sponge sequence, trapping the native microRNA, respectively. Restenosis was quantified on stained rat carotid sections.

Results: In vitro, three mRNA (Myocyte Enhancer Factor 2C (MEF2C), Ras homolog gene family, member B (RhoB) and Nuclear factor 1 A-type (NFIA)) reported as miR-223 direct targets and known to be implicated in VSMC differentiation and contractility were studied by RT-qPCR. Our findings showed that down-expression of miR-223 significantly reduced neointimal hyperplasia by 44% in carotids, and was associated with a 2-3-fold overexpression of MEF2C, RhoB and NFIA in a murine monocyte macrophage cell line, RAW 264.7 cells.

Conclusion: Down-regulating miR-223 could be a potential therapeutic approach to prevent restenosis after angioplasty.
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http://dx.doi.org/10.2174/1570161117666190705141152DOI Listing
December 2020

Detection of Hypoxia-Regulated MicroRNAs in Blood as Potential Biomarkers of HIF Stabilizer Molidustat.

Microrna 2019 ;8(3):189-197

Analysis Department ‒ Agence Francaise de Lutte Contre le Dopage (AFLD), 143 Avenue Roger Salengro, 92290 Chatenay- Malabry, France.

Background: The recent development of drugs that stabilize HIFalpha, called HIF stabilizers, offers a new strategy for treating anemia. Although these drugs are still in clinical trials, misuse for doping has already begun. Identifying the biomarkers of HIF stabilizers would therefore help in detecting this drug misuse by athletes.

Objective: Our aim was twofold: to determine whether hypoxamiRs, the microRNAs associated with the cellular response to hypoxia, are potential biomarkers of HIF stabilizers in blood and whether the response to treatment with an HIF stabilizer differs from the response to a hypoxic environment.

Method: Rats were treated for 6 days with either a placebo or 2mg/kg of Molidustat, an HIF stabilizer, or they were put under hypoxia (10% oxygen) for the same length of time. Plasma samples were analyzed before, during and 48 hours after the treatments.

Results: EPO concentration increased significantly in plasma during hypoxia and Molidustat treatment and showed a negative retro-control 2 days after the end of the treatments. On the contrary, circulating levels of VEGF were not modified. Among the hypoxamiRs tested, miR-130a and miR-21 were significantly increased during Molidustat treatment and miR-21 was still increased 48 hours after treatment end.

Conclusion: Although using these microRNAs as biomarkers seems unlikely due to other possible factors of regulation, this study provides the first identification of a specific effect of HIF stabilizers on microRNAs. Further investigations are needed to better understand the possible consequences of such regulation.
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http://dx.doi.org/10.2174/2211536608666190117170317DOI Listing
July 2020

Combined immuno-purification and detection of recombinant erythropoietins and activin receptor type II-Fc fusion proteins by isoelectric focusing for application in doping control.

Drug Test Anal 2019 Jan 28;11(1):168-172. Epub 2018 Aug 28.

Analysis Department, Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro-92290, Châtenay-Malabry, France.

Iso-electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti-Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs - luspatercept and sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre-loading and gel conditions, and a single detection of all rEPOs and ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII-Fc.
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http://dx.doi.org/10.1002/dta.2476DOI Listing
January 2019

miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice.

Biochim Biophys Acta 2016 04 13;1862(4):611-621. Epub 2016 Jan 13.

Institute for Cardiometabolism and Nutrition (ICAN), Paris F-75013, France; INSERM UMR-S 1166, Paris F-75013, France; Sorbonne Universités, Université Pierre et Marie Curie -UPMC Univ Paris 06, UMR-S 1166, Paris F-75013, France. Electronic address:

We identified murine miR-322, orthologous to human miR-424, as a new regulator of insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11week-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.
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http://dx.doi.org/10.1016/j.bbadis.2016.01.010DOI Listing
April 2016

Emergence of Orai3 activity during cardiac hypertrophy.

Cardiovasc Res 2015 Mar 11;105(3):248-59. Epub 2014 Sep 11.

Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1166, ICAN, F-75005 Paris, France INSERM, UMR_S 1166, ICAN, F-75005 Paris, France

Aims: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes.

Methods And Results: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current.

Conclusion: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.
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http://dx.doi.org/10.1093/cvr/cvu207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351368PMC
March 2015

miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat.

Cardiovasc Res 2013 Jun 26;98(3):458-68. Epub 2013 Feb 26.

INSERM UMRS 956, Faculté de Médecine Pierre et Marie Curie, 91 boulevard de l'Hôpital, 75634, Paris Cedex 13, France.

Aims: Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action.

Methods And Results: MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis.

Conclusion: Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.
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http://dx.doi.org/10.1093/cvr/cvt045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656613PMC
June 2013

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation.

Am J Physiol Cell Physiol 2012 Nov 26;303(10):C1104-14. Epub 2012 Sep 26.

INSERM, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche UMR_S 956, Faculté de Médecine Pitié-Salpétrière, Paris, France.

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with β-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.
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http://dx.doi.org/10.1152/ajpcell.00051.2012DOI Listing
November 2012

miR-421 and miR-30c inhibit SERPINE 1 gene expression in human endothelial cells.

PLoS One 2012 31;7(8):e44532. Epub 2012 Aug 31.

UMR_S 956, INSERM, Paris, France.

In this work, we assessed whether SERPINE1 expression could be under the influence of microRNAs (miRNAs) predicted to bind the SERPINE1 3'UTR region. We specifically focused on the 3'UTR region harboring a common polymorphism, rs1050955, that have been found associated to SERPINE1 monocyte expression, and investigated whether the presence of different alleles at rs1050955 could modify the miRNAs binding efficiency and affect PAI-1 protein levels. We demonstrated that, in human umbilical vein endothelial cells, both miR-421 and miR-30c directly interacted with PAI-1 mRNA to inhibit the expression of the associated protein. However, these inhibitory mechanisms were independent on the allele present at the rs1050955 locus. We further showed that miR-421 levels correlated with PAI-1 activity in the plasma sample of 40 patients with venous thrombosis. Our results strongly suggest that the regulation of PAI-1 molecule could be under the influence of several miRNAs whose measurement in the plasma of patients could be envisaged as a biomarker for inflammatory and thrombotic disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044532PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432110PMC
February 2013

Calcium signaling in vascular smooth muscle cells: from physiology to pathology.

Adv Exp Med Biol 2012 ;740:795-810

Inserm UMRS 956/ Université Pierre et Marie Curie Paris6, 91 boulevard de l'hôpital, 75013 Paris, France.

Cyclic variations in calcium (Ca(2+)) concentrations, through a process called excitation-contraction coupling, allow regulation of vascular smooth muscle cells contractility and thus modulation of vascular tone and blood pressure. As a second messenger, Ca(2+) also activates signaling cascades leading to transcription factors activation in a process called excitation-transcription coupling. Furthermore, recent evidences indicate an interaction between post-transcriptional regulation by microRNAs (miRNAs) and Ca(2+) signaling. All these actors, which are frequently altered in vascular diseases, will be reviewed here.
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http://dx.doi.org/10.1007/978-94-007-2888-2_35DOI Listing
September 2012

Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion.

Proc Natl Acad Sci U S A 2011 Aug 25;108(32):13258-63. Epub 2011 Jul 25.

Institut de la Santé et de la Recherche Médicale, U1046, F-34295 Montpellier, France.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.
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http://dx.doi.org/10.1073/pnas.1100286108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156220PMC
August 2011

The Wnt/beta-catenin pathway is activated during advanced arterial aging in humans.

Aging Cell 2011 Apr 29;10(2):220-32. Epub 2010 Dec 29.

INSERM UMRS_956; UPMC Univ Paris 06, 91 boulevard de l'Hôpital, Paris Cedex 13, France.

Aging is the main risk factor for cardiovascular diseases, but the associated molecular mechanisms are poorly understood. The Wnt signaling pathway was shown to be induced during aging in muscle and in the skin, but the regulation and role of Wnt signaling in the aged vessel have not yet been addressed. While screening for age-related changes in gene expression in the intima/media of human mammary arteries, we observed that the expression of frizzled 4 (Fzd4), a Wnt receptor, and of several targets of the Wnt/β-catenin/TCF signaling pathway [Wnt-inducible secreted protein 1 (WISP1), versican, osteopontin (SPP1), insulin-like growth factor binding protein 2 (IGFBP-2), and p21] were modified with age, suggesting an activation of the Wnt/β-catenin pathway. In contrast, we did not observe any regulation of forkhead transcription factor (FoxO) target genes. Beta-catenin-activating phosphorylation at position Ser675 was increased in aging mammary arteries, confirming the activation of this pathway. We confirmed in vitro that Wnt3a or Wnt1 treatment of human vascular smooth muscle cells (VSMCs) induced β-catenin phosphorylation at Ser675 and WISP1, SPP1, and IGFBP-2 expression. In vitro, Wnt treatment induced proliferation and cyclin D1 expression in VSMC from young (6 weeks old) rats but not in cells from older rats (8 months old), even though low-density lipoprotein receptor-related protein 6 and β-catenin phosphorylation, and β-catenin nuclear translocation demonstrated β-catenin activation in both cell types. Beta-catenin silencing demonstrated that Wnt induction of cyclin D1 expression is β-catenin dependent. Altogether, our data show that the Wnt/β-catenin/TCF pathway is activated in aging human mammary artery cells, but fails to induce the proliferation of aging vascular cells.
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http://dx.doi.org/10.1111/j.1474-9726.2010.00661.xDOI Listing
April 2011

2,3,7,8-tetrachlorodibenzo-p-dioxin counteracts the p53 response to a genotoxicant by upregulating expression of the metastasis marker agr2 in the hepatocarcinoma cell line HepG2.

Toxicol Sci 2010 Jun 18;115(2):501-12. Epub 2010 Mar 18.

INSERM UMR-S 747, 75006 Paris, France.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that binds the aryl hydrocarbon receptor (AhR), a transcription factor that triggers various biological responses. In this study, we show that TCDD treatment counteracts the p53 activation (phosphorylation and acetylation) elicited by a genotoxic compound, etoposide, in the human hepatocarcinoma cell line HepG2 and we delineated the mechanisms of this interaction. Using small interfering RNA knockdown experiments, we found that the newly described metastasis marker, anterior gradient-2 (AGR2), is involved in this effect. Both AGR2 messenger RNA (mRNA) and protein levels were increased (sixfold and fourfold, respectively) by TCDD treatment, and this effect was mediated by the AhR receptor. The half-life of AGR2 mRNA was unchanged by TCDD treatment. Analysis of the promoter of the AGR2 gene revealed three putative xenobiotic-responsive elements (XREs) in the proximal 3.5-kb promoter. Transient transfection of HepG2 cells by the Gaussia luciferase reporter gene driven by various deleted and mutated fragments of the promoter indicated that only the most proximal XRE was active. Binding of the AhR to the endogenous AGR2 promoter was also triggered by TCDD treatment. These results suggest that AhR ligands such as TCDD might contribute to tumor progression by inhibiting p53 regulation (phosphorylation and acetylation) triggered by genotoxicants via the increased expression of the metastasis marker AGR2.
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http://dx.doi.org/10.1093/toxsci/kfq082DOI Listing
June 2010

Role of Paris PM(2.5) components in the pro-inflammatory response induced in airway epithelial cells.

Toxicology 2009 Jul 19;261(3):126-35. Epub 2009 May 19.

Univ Paris Diderot-Paris 7, Unit of Functional and Adaptive Biology (BFA) EAC CNRS 7059, Laboratory of Molecular and Cellular Responses to Xenobiotics, 5 rue Thomas Mann, Case 7073, 75205 Paris Cedex 13, France.

Particulate matter (PM) is suspected to play a role in environmentally-induced pathologies. Due to its complex composition, the contribution of each PM components to PM-induced biological effects remains unclear. Four samples of Paris PM(2.5) having different polyaromatic hydrocarbons and metals contents were compared with each other and with their respective aqueous and organic extracts used alone or in combination. The four PM(2.5) samples similarly induced granulocyte macrophage-colony stimulating factor (GM-CSF) release, a pro-inflammatory cytokine, by human bronchial epithelial cells. It results from the activation of upstream signalling pathways and the modulation of the cellular redox state that is different according to PM(2.5) samples. The PM-aqueous extracts contained soluble metals involved in hydroxyl radical production in abiotic conditions. However they slightly contributed to the intracellular reactive oxygen species production and GM-CSF release by comparison with organic extracts. Organic compounds transactivated the xenobiotic responsive element (XRE) and antioxidant responsive element (ARE), leading to increased cytochrome P450 1A1 expression and NADPH-quinone oxydoreductase-1 expression respectively but to different extend according to PM samples underlying differences in their bioavailability. Our study underlines that chemical composition of particles per se is insufficient to predict cellular effects and that the interaction and the bioavailability of the various components were critical.
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http://dx.doi.org/10.1016/j.tox.2009.05.007DOI Listing
July 2009

Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release.

J Biol Chem 2008 May 20;283(21):14867-82. Epub 2008 Mar 20.

Commissariat à l'Energie Atomique (CEA), Institut de Biologie et de Technologies de Saclay, SBSM, URA CNRS 2096, Laboratoire de Recherche Associé, 17V University of Paris-Sud, Gif sur Yvette, France.

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.
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http://dx.doi.org/10.1074/jbc.M710165200DOI Listing
May 2008

Stabilization of IGFBP-1 mRNA by ethanol in hepatoma cells involves the JNK pathway.

J Hepatol 2007 Nov 27;47(5):691-8. Epub 2007 Jun 27.

INSERM U747, Laboratoire de Pharmacologie, Toxicologie et Signalisation Cellulaire, Paris F-75006, France.

Background/aims: Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates cell growth and metabolism in a variety of physiopathological conditions. The aim of this study was to determine the molecular mechanisms involved in IGFBP-1 upregulation by ethanol.

Methods: We studied IGFBP-1 regulation by ethanol at the protein, mRNA and gene promoter levels in the human hepatocarcinoma cell line, HepG2, which does not express significantly ethanol-metabolizing enzymes.

Results: Ethanol (35-150mM) induced the IGFBP-1 mRNA and protein up to 5-fold in a dose-dependent manner. A similar effect was observed using primary cultures of human hepatocytes. Various inhibitors of ethanol metabolism and the antioxidant N-acetylcysteine did not prevent ethanol effects. While ethanol did not modify the IGFBP-1 gene promoter activity, it elicited a 2- to 3-fold increase in IGFBP-1 mRNA half-life and this stabilization required the 5' and the 3' untranslated mRNA region. Ethanol triggered a rapid activation of c-Jun N-terminal Kinase (JNK) in HepG2 cells and IGFBP-1 induction was significantly decreased by a specific inhibitor of JNK.

Conclusions: This study reveals a novel pathway of gene regulation by alcohol which involves the activation of JNK and the consequent mRNA stabilization.
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http://dx.doi.org/10.1016/j.jhep.2007.05.018DOI Listing
November 2007

Endoplasmic reticulum stress induction of insulin-like growth factor-binding protein-1 involves ATF4.

J Biol Chem 2006 Jul 10;281(28):19124-33. Epub 2006 May 10.

INSERM UMR-S 747, Université Paris-Descartes, 45 Rue des Saints-Pères, 75270 Paris, France.

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.
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http://dx.doi.org/10.1074/jbc.M602157200DOI Listing
July 2006

Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells.

Am J Physiol Lung Cell Mol Physiol 2003 Sep 2;285(3):L671-9. Epub 2003 May 2.

Laboratoire de Cytophysiologie et Toxicologie cellulaire, Université Paris 7 - Denis Diderot, Tour 53-54, 3e étage, case courrier 7073, 2 place Jussieu, 75251 Paris cedex 05, France.

Diesel exhaust particles (DEP) induce a proinflammatory response in human bronchial epithelial cells (16HBE) characterized by the release of proinflammatory cytokines after activation of transduction pathways involving MAPK and the transcription factor NF-kappaB. Because cellular effects induced by DEP are prevented by antioxidants, they could be mediated by reactive oxygen species (ROS). Using fluorescent probes, we detected ROS production in bronchial and nasal epithelial cells exposed to native DEP, organic extracts of DEP (OE-DEP), or several polyaromatic hydrocarbons. Carbon black particles mimicking the inorganic part of DEP did not increase ROS production. DEP and OE-DEP also induced the expression of genes for phase I [cytochrome P-450 1A1 (CYP1A1)] and phase II [NADPH quinone oxidoreductase-1 (NQO-1)] xenobiotic metabolization enzymes, suggesting that DEP-adsorbed organic compounds become bioavailable, activate transcription, and are metabolized since the CYP1A1 enzymatic activity is increased. Because NQO-1 gene induction is reduced by antioxidants, it could be related to the ROS generated by DEP, most likely through the activation of the stress-sensitive Nrf2 transcription factor. Indeed, DEP induced the translocation of Nrf2 to the nucleus and increased protein nuclear binding to the antioxidant responsive element. In conclusion, we show that DEP-organic compounds generate an oxidative stress, activate the Nrf2 transcription factor, and increase the expression of genes for phase I and II metabolization enzymes.
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http://dx.doi.org/10.1152/ajplung.00419.2002DOI Listing
September 2003