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Ziad Hussein Abdel-Razzak

Full professor
Life & Earth Sciences department - Section I - Hadath
Speciality: Biology
Specific Speciality: cell and molecular biology applied to toxicology
Interests: Studying the scientific signs in the Holy Quran and Sunna
Skills: -wide array of cell biology techniques -wide array of molecular biology techniques -wide array of biochemistry techniques -MS office

Teaching 3 Taught Courses
(2014-2015) CBMB 511 - Metabolism and Pharmacogenomics of xenobiotic metabolizing enzymes

M2 Chemistry and Biology of Bioactive Molecules

(2014-2015) BioA 404 - Toxicology

M1 Environmental and life Sciences - Option : Animal

(2014-2015) BioA 405 - Toxicology Lab

M1 Environmental and life Sciences - Option : Animal

Education
2008: Training

CNRS-Lebanon
Chromatography

2003: Training

Lebanese University
Bioinformatics

1995 - 1996: English course

American University of Beirut
English

Upper 10%

1991 - 1995: PhD

Rennes-1 University
Biological Sciences

Tres honorable avec felicitations du jury

1991: Training

INSERM
ygiene and security of laboratory

1990 - 1991: DEA

Rennes-1 University
Cell and Molecular Biology Applied to health Sciences

Upper 5%

1989 - 1990: Maitrise

Rennes-1 University
Biochemistry

Upper 10%

1988 - 1989: Licence

Rennes-1 University
Biochemistry

Upper 10%

1985 - 1987: DEUG

Rouen University
Biology

Upper 20%

Publications 16 publications
Bachour-El Azzi P, Sharanek A, Burban A, Li R, Le Guével R, Abdel-Razzak Z, Stieger B, Guguen-Guillouzo C, Guillouzo A. Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG cells and primary human hepatocytes. Toxicol Sci. 2015

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HH) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In the present work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HH in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8-and 2.6- fold lower, than in SCHH and CCHH respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHH. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HH and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.

Bachour-El Azzi P, Sharanek A, Abdel-Razzak Z, Antherieu S, Al-Attrache H, Savary CC, Lepage S, Morel I, Labbe G, Guguen-Guillouzo C, Guillouzo A. Impact of inflammation on chlorpromazine-induced cytotoxicity and cholestatic features in HepaRG cells. 2014

Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24-hour exposure to proinflammatory cytokines interleukin-6 (IL-6) and IL-1β; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of cytochrome 3A4 (CYP3A4) and CYP1A2 transcripts, two major cytochrome P450 (P450) enzymes involved in its metabolism. Most effects of cotreatments with cytokines and CPZ were amplified or only observed after five daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na(+)-taurocholate cotransporting polypeptide mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by proinflammatory cytokines increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of P450 activities, could have important consequences if extrapolated to the in vivo situation.

Anthérieu S, Bachour-El Azzi P, Dumont J, Abdel-Razzak Z, Guguen-Guillouzo C, Fromenty B, Robin MA, Guillouzo A. Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells. 2013

Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+ -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. CONCLUSION: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes.

Bachour P, Yafawi R, Jaber F, Choueiri E, Abdel-Razzak Z. Effects of smoking, mother's age, body mass index, and parity number on lipid, protein, and secretory immunoglobulin A concentrations of human milk. 2012

AIM: This study investigated the effect of smoking, mother's age, body mass index (BMI), and parity number on density, lipids, proteins, and secreted immunoglobulin A (SIgA) of human milk. METHODS: Transitional and mature milk samples were collected from 23 nursing smoker mothers and 43 nursing nonsmoker mothers. Proteins, lipids, and SIgA concentrations were determined as well as the milk density and the general protein profile. RESULTS: Our investigation showed that the milk of smokers contained less lipids and proteins (statistically significant 26% and 12% decrease, respectively), whereas milk density was unchanged. SIgA concentration was 27% lower in milk from smokers, but the decrease was not statistically significant. The general protein profile showed no significant smoking-associated changes in the four identified proteins (β-casein, immunoglobulin A heavy chain, serum albumin, and lactoferrin). Mothers' age and residential area showed noticeable but statistically nonsignificant differences in some of the measured parameters. However, parity number, lactation stage, and BMI were associated with a significant modification of milk composition. Mature milk contained more lipids and less protein, whereas the increase of parity number was associated with an increase in lipid concentration. The group of overweight mothers showed lower milk protein concentration in comparison with the normal group. Multivariate analysis showed a statistically significant interaction effect of the variables (smoking, parity number, lactation stage, age, and BMI) on lipids and between some of them on proteins and SIgA. CONCLUSION: Our study showed that smoking was associated with lower milk lipid and protein concentrations and that the parity number and BMI were associated with a change in milk lipids and proteins content, respectively.

Darazy M, Balbaa M, Mugharbil A, Saeed H, Sidani H, Abdel-Razzak Z. CYP1A1, CYP2E1, and GSTM1 gene polymorphisms and susceptibility to colorectal and gastric cancer among Lebanese. 2010

Mutations in the genes encoding enzymes involved in the metabolism of chemical carcinogens can significantly affect the risk of cell transformation and cancer development. The resident Lebanese population has experienced a sharp increase in cancer incidence within the last few years. The relationship between gene polymorphisms of metabolic enzymes and gastrointestinal (GI) cancer incidence was not previously investigated. The aim of this study was to investigate the relationship between CYP1A1, CYP2E1, and GSTM1 gene polymorphisms and GI cancer incidence among Lebanese. Blood and/or paraffin-embedded biopsy samples were collected from patients and healthy controls. The genotypes were determined by polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism. The results of the present case-control study show that the studied Lebanese population generally resembles Caucasian populations with respect to the considered polymorphisms. Further, the GSTM1*0/*0 genotype is a significant risk factor for gastric (odds ratio = 4.1; 95% confidence interval: 1.2-14.5) and colorectal cancers (odds ratio = 3.8; 95% confidence interval: 1.7-8.5); on the other hand, CYP1A1*2A and CYP2E1*6 alone are not significantly associated with GI cancer development, although CYP1A1*2A was more frequent among patients. A remarkable and statistically significant 36.5-fold increase in the risk of gastric cancer was observed among patients with CYP1A1*2A/*2A combined with GSTM1*0/*0. The investigation of genetic risk factors and susceptibility gene polymorphisms in Lebanese is helpful for better understanding of GI cancer etiology.

Ziad ABDEL-RAZZAK Resume des activites de recherche pour l'obtention de l'HDR en biologie cellulaire et moleculaire appliquees a la toxicologie 2009

L’étude de la régulation de l’expression des enzymes du métabolisme des xénobiotiques (EMX) et de leur polymorphisme est importante du point de vue toxico-pharmacologique. Le modèle expérimental utilisé est celui de la culture d’hépatocytes et de lignées d’hépatome. L’expression des EMX, tels que les cytochromes P450 (CYP), est affectée par beaucoup de facteurs endogènes telles que les cytokines et exogènes tels que les hydrocarbures aromatiques polycycliques (HAP). Ce thème de recherche avait été initié à l’Inserm à Rennes (Pr Guillouzo) et ensuite poursuivie en collaboration avec l’Université de Paris 5 (Pr Barouki). Des résultats importants ont été obtenus et publiés concernant les effets inhibiteurs des cytokines de l’inflammation (IL1 bêta, IL6 et TNF alpha), et l’effet inducteur de l’IL4 ainsi que l’effet inhibiteur du TGF bêta. Les effets décrits concernent des enzymes d’intérêt toxicologique majeur tels que les CYP1A1, CYP2E1 et les glutathions transférases. Les séquences promotrices impliquées dans la réponse du CYP2E1 à l’IL4 et l’IL1 et la voie de transduction ont été élucidées. En collaboration avec l’Université Arabe de Beyrouth, trois projets en cours portent sur l’étude du polymorphisme des EMX et de son association avec la survenue de cancer au Liban. Ce thème, sur lequel trois étudiants en master travaillent, est justifié par le fait que beaucoup de substances procarcinogènes tels que les HAP et les nitrosamines sont activées par les cytochromes P450 tels que le CYP1A1 et le CYP2E1. Le contexte académique à l’Université Libanaise et l’intérêt porté à la santé publique, ont conduit à l’initiation de deux thèmes de recherche secondaires mais toujours relatifs à la toxicologie. Il s’agit de la détection des HAP dans les aliments, particulièrement le lait. Les HAP sont parmi les principales substances carcinogènes auxquelles les Libanais sont exposés. Dans le deuxième thème il est question de mettre en place des méthodes pour le contrôle génétique des produits alimentaires au Liban afin de soulever la question du contrôle de la propagation des OGM.

Abdel-Razzak Z, Garlatti M, Aggerbeck M, Barouki R. Determination of interleukin-4-responsive region in the human cytochrome P450 2E1 gene promoter. 2004

Cytochrome P450 2E1 (CYP2E1) gene expression is known to be induced by interleukin-4 (IL4) and repressed by inflammatory cytokines, such as interleukin-1beta3 (IL1beta3) in human hepatocytes. The mechanisms involved in these transcriptional regulations remain elusive. In order to study these mechanisms, various constructs of the human CYP2E1 promoter were prepared and transfected into the human HepG2 hepatoma cell line. Our findings revealed that an IL4-responsive region of 128bp (-671/-544) was required to mediate induction by IL4. IL1beta caused moderate but significant decrease of the promoter activity, which was abolished when the two cytokines were combined. The IL1beta inhibitory effect is mediated through a regulatory sequence independent of that of IL4. Furthermore, by using specific signaling pathway inhibitors, we demonstrated that IL4 activation required protein kinase C (PKC) activation. In addition, our results suggest that induction by IL4 was not dependent on a single binding site but rather on a complex region which includes putative binding sites for signal transducer and activator of transcription (STAT)6, activator protein (AP)-1, nuclear factor kappa-B (NFkappaB), nuclear factor of activated T cells (NFAT) and CCAAT enhancer binding protein (C/EBP). Electrophoretic mobility shift assays suggest that AP1 and NFAT transcription factors are able to bind to three sites in the IL4-responsive region.

Abdel-Razzak Z, Corcos L, Fautrel A, Guillouzo A. Interleukin-1 beta antagonizes phenobarbital induction of several major cytochromes P450 in adult rat hepatocytes in primary culture. 1995

We have investigated the effects of interleukin (IL)-1 beta and IL6 on expression and phenobarbital (PB) induction of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-deethylase (PROD) activities, as well as on mRNA levels of cytochromes P450 (CYP) 1A, 2B, 2C, 2E and 3A, in rat hepatocytes in primary culture. IL6 slightly antagonized PB-induced PROD activity. Strikingly, IL1 beta strongly inhibited basal EROD and PROD activities, and fully blocked their induction by PB in a dose-dependent fashion. Furthermore IL1 beta completely suppressed PB induction of all CYP mRNAs analyzed. Our results demonstrate that IL1 beta can suppress basal CYP activities, as well as PB-inducible expression of five CYP mRNAs in rat hepatocytes in primary culture.

Ziad ABDEL-RAZZAK Régulation de l'expression des cytochromes P450 par les cytokines dans les hépatocytes humains et de rat en culture primaire Rennes 1 University 1995

Langouet S, Corcos L, Abdel-Razzak Z, Loyer P, Ketterer B, Guillouzo A. Up-regulation of glutathione S-transferases alpha by interleukin 4 in human hepatocytes in primary culture. 1995

During inflammation and infection, overexpression of cytokines is associated with changes in cytochrome P450 (CYP) activities. The present study investigated the effect of cytokines on expression of the glutathione S-transferases (GST), phase II enzymes, involved in drug detoxication and in protection against lipid peroxidation. Human hepatocytes in primary culture were exposed to interleukin 6 (IL6), a proinflammatory cytokine and interleukin 4 (IL4) thought to be an anti-inflammatory cytokine and known to induce CYP2E1 specifically. After a three-day treatment, no reproducible effects of IL-6 could be demonstrated on either GSTA1 and/or A2 or M1 mRNA levels (GSTA1 and A2 were not discriminated by the cDNA probe). In contrast, GSTA1 and/or A2 mRNAs and GSTA1 and A2 proteins were reproducibly increased after IL4 treatment. This increase was blocked by alpha-amanitin, suggesting that active transcription is necessary and was associated with increased AP1 binding activities. These results provide evidences that IL4 exerts important effects on detoxifying hepatic drug metabolizing enzymes.

Guillouzo, A., S. Langouet, F. Morel, O. Fardel , Z. Abdel-Razzak and L. Corcos. he isolated human cells as a tool to predict in vivo metabolism of drugs. "Advances in drug metabolism in man" 1995

Abdel-Razzak Z, Corcos L, Fautrel A, Campion JP, Guillouzo A. Transforming growth factor-beta 1 down-regulates basal and polycyclic aromatic hydrocarbon-induced cytochromes P-450 1A1 and 1A2 in adult human hepatocytes in primary culture. 1994

The effects of interleukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-gamma, and transforming growth factor (TGF)-beta 1 on cytochrome P-450 (CYP) 1A expression and polycyclic aromatic hydrocarbon (PAH)-mediated induction in primary human hepatocyte cultures were determined. Most cytokines that were previously found to decrease basal CYP expression could counteract PAH induction of CYP1A mRNA and its associated ethoxyresorufin-O-deethylation (EROD) activity. IL-1 beta and TNF-alpha blocked 3-methylcholanthrene (3-MC)-induced EROD activity by up to 25 and 44%, respectively. IFN-alpha and IFN-gamma antagonized EROD induction by up to 61 and 70%, respectively. TGF-beta 1 proved to be the most effective cytokine, because 72 hr of treatment with 2 ng/ml TGF-beta 1 produced nearly 100% inhibition of 3-MC- and benzo(a)pyrene-induced CYP1A1 and CYP1A2 mRNAs and EROD activity. Treatment with cycloheximide in combination with 3-MC led to superinduction of CYP1A mRNA, under which conditions TGF-beta 1 did not block induction, suggesting the requirement for protein synthesis for the suppressive effect of the cytokine. In addition, TGF-beta 1 augmented AP-1-binding activity, suggesting that fos and/or jun protooncogene products could be implicated in the response. Our results demonstrate that IL-1 beta, TNF-alpha, and IFNs antagonized PAH-mediated induction of CYP1A gene expression in human hepatocytes. In addition, we report the finding of a novel effect of TGF-beta 1, which was able to prevent CYP1A1 and -1A2 induction by two different PAHs.

Abdel-Razzak, Z., L. Corcos and A. Guillouzo. Inhibition of cytochromes P-450 by cytokines in human hepatocyte cultures. "Cytochromes P450: Biochemistry, Biophysics and Molecular Biology". Lechner M.C. (Eds), John Libbey Eurotext, 1994

Abdel-Razzak, Z., P. Loyer, A. Fautrel, J.C. Gautier, L. Corcos, B. Turlin, P. Beaune, and A. Guillouzo. Cytokines down-regulate expression of major cytochrome P-450 enzymes in adult human hepatocytes in primary culture. 1993

Cytokines are thought to cause the depression of cytochrome P-450 (CYP)-associated drug metabolism in humans during inflammation and infection. We have examined the role of five cytokines, i.e., interleukin-1 beta, interleukin-4, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma, on the expression of CYP1A2, CYP2C, CYP2E1, CYP3A, and epoxide hydrolase in primary human hepatocyte cultures. Steady state P-450 and epoxide hydrolase mRNA levels, as well as ethoxyresorufin-O-deethylase and nifedipine oxidation activities, which are mainly supported by CYP1A1/1A2 and CYP3A, respectively, were measured. Interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha were found to be the most potent depressors of P-450 enzymes. After 3 days of treatment, both mRNA levels and enzyme activities were depressed, typically by at least 40%, whatever the cytokine and the enzyme considered. Interferon-gamma also suppressed CYP1A2 and CYP2E1 mRNA levels and ethoxyresorufin-O-deethylase activity but had no effect on CYP3A and epoxide hydrolase mRNAs. In addition, interleukin-4 had the opposite effect, compared with other cytokines, on CYP2E1 mRNA, which was increased up to 5-fold; ethoxyresorufin-O-deethylase and nifedipine oxidation activities were not significantly affected. These results provide the first demonstration that various cytokines act directly on human hepatocytes to affect expression of major P-450 genes and that a wide range of responses can be observed among the enzymes for a given cytokine, suggesting that different regulatory mechanisms may be involved.

Loyer P, Ilyin G, Abdel Razzak Z, Banchereau J, Dezier JF, Campion JP, Guguen-Guillouzo C, Guillouzo A Interleukin 4 inhibits the production of some acute-phase proteins by human hepatocytes in primary culture. 1993

Interleukin 4 (IL4) has been shown to exhibit anti-inflammatory effects by inhibiting the secretion by monocytes of proinflammatory cytokines such as interleukin 1 (IL1), interleukin 6 (IL6), and tumor necrosis factor (TNF) and by inducing the secretion of the IL1 receptor antagonist. We investigated the role of this cytokine on the production of acute-phase proteins in primary human hepatocyte cultures. Cells were exposed to either IL4 and/or IL6, the most potent mediator of hepatic acute phase proteins. IL4 led to decreased production of haptoglobin, C-reactive protein and albumin while alpha 1-antitrypsin and fibrinogen remained unaffected. These inhibitory effects of IL4 were also observed at the mRNA level. In addition, IL4 inhibited the IL6-induced production of haptoglobin although it had no effect on the induced C-reactive protein and fibrinogen. Our results demonstrate that IL4 can affect the production of a subset of acute-phase proteins by human hepatocytes and can antagonize some of the effects of IL6. These observations reinforce the notion that IL4 can be considered as an anti-inflammatory cytokine.

Guillouzo, A., P. Loyer, Z. Abdel-Razzak, F. Delers, A. Fautrel and C. Guguen-Guillouzo. expression of acute phase proteins in cultured liver cells. "Cellular and Molecular Aspects of Cirrhosis". John Libbey Eurotext 1992

Supervision 12 Supervised Students
Gene polymorphism of CYP1A1, CYP2E1 and GSTM1 and their potential association with gastrointestinal cancer risk in the Lebanese population

Mohamad DARAZY
Master 2 practical research project see article: http://www.ncbi.nlm.nih.gov/pubmed/21385088

Mutations in genes of xenobiotics metabolizing enzymes (XMEs), particularly those involved in the metabolism of chemical carcinogens, can significantly affect the risk of cell transformation and cancer development. Carcinogen metabolism is catalyzed by various families of XMEs including cytochrome P450 (CYP) family members and glutathione S-transferases (GSTs). The CYPs primarily activate carcinogens in phase-I, while GSTs catalyze their detoxification in phase-II. Accordingly, it is expected that individuals with high CYP activity and low GST levels are at a high risk of cancer development. Genetic polymorphism is one of the causes of variation in XME activity and several mutations in XME genes have been shown to be associated with increased cancer risk in different populations. In Lebanon, the relationship between XME gene polymorphism and cancer incidence was not investigated. The aim of the present study was to conduct a case-control study to test any potential association between genetic polymorphisms of phase-I (CYP1A1 and CYP2E1) and phase-II (GSTM1) genes and the incidence of GI cancer in the Lebanese population. Blood and/or paraffin-embedded biopsy samples were collected from GI Lebanese cancer patients and healthy controls. After DNA extraction, the genotypes were determined by PCR and PCR-RFLP. The Fisher’s exact test and the odds ratio with 95% CI were used to test the strength of association between the different polymorphic alleles and the occurrence of GI cancers. Our results show that, among Lebanese, the GSTM1-null genotype is a risk factor for GI cancers (CRC and GC, P < 0.05), whereas the variant CYP1A1 Msp I positive allele (6235T→C) and CYP2E1 Dra I negative allele (7632T→A) are not associated with GI cancer development, and the frequencies of both alleles is lower than in other tested populations (<10% and <5% respectively). The investigation of genetic risk factors and susceptibility gene polymorphisms in the Lebanese population is helpful for better GI cancer screening, prevention, and treatment.

Gene Polymorphisms of Glutathione S-Transferase M1 and its Possible Association with Breast Cancer in Lebanese Population

Zahraa HIJAZI
Master 2 practical research project

The aim of the present study was to conduct a case-control study to test any potential association between the CYP1A1, GSTM1 and NAT2 polymorphisms and an increased or decreased risk for breast cancer in the Lebanese population. To do so, blood samples from eighty two breast cancer patients and another thundered samples from healthy women (control), were collected from many hospitals found in different regions in Lebanon including Beirut, South, and Al-Bikaa. The practical work started by DNA extraction from all samples followed by gel electrophoresis. Then specific regions of CYP1A1, GSTM1, and NAT2 were amplified using PCR. However, the genetic polymorphism and the genotypes of the above mentioned enzymes were carried out using the allele-specific PCR products and RFLP. In order to study the distribution of the different alleles among patients and controls, the obtained results were analyzed using statistical analysis by MedCalc program.

Investigation of CYP1A2 Gene Polymorphism and its Association with Gastrointestinal Cancer

Mariam SWAID
Master 2 practical research project

Gastrointestinal (GI) cancer in general and gastric and colorectal cancers in particular has been associated with increase of environmental heterocyclic amine (HCAs) carcinogens and polycyclic aromatic hydrocarbons (PAHs). CYP1A2 enzyme is among the major phase I metabolizing enzyme responsible for initiating genotoxicity of these compounds. Over 15 polymorphic variant of CYP1A2 gene have been identified. However, CYP1A2*1F has been reported to be more frequent in Caucasian and Asian populations. Such polymorphisms could alter HCAs and PAHs metabolism and therefore it may influence GI cancer susceptibility. The aim of this study was to investigate this polymorphism for CYP1A2 gene (CYP1A2*1F) and its possible association with GI cancer in a Lebanese population. DNA samples of 156 blood and paraffin-embedded tissues samples (96 patients and 60 controls) were used. 79 samples (35 patients and 44 controls) were genotyped for -163C>A (CYP1A2*1F) polymorphism using PCR-RFLP. In patients group, the percentages distribution of the three genotypes was 20% for the homozygous wild type (CYP 1A2*1A/CYP1A2*1A), 70% for the heterozygous (CYP1A1*1A/CYP1A2*1F) and 10% for the homozygous mutant type (CYP1A2*1F/CYP1A2*1F). Where as in controls group, 10% were homozygous wild type, 40% were heterozygous and 50% were homozygous mutant. The alleles percentages for the wild allele (CYP1A2*1A) and mutant one (CYP1A2*1F) were 45% and 55% respectively in patients while 70% and 30% respectively in controls. The difference was statistically significant between patients and controls (OR=0.1, 95% CI=0.02-0.5, p <0.05 for CYP1A2*1F/*1F genotype and OR=0.35, 95% CI=0.18-0.68, p<0.05 for CYP1A2*1F allele). CYP1A2*1F was less associated with GI cancer patients. Therefore, the absence of CYP1A2*1F polymorphic variant could be a cause for GI cancer development. Due to the inefficiency of restriction enzyme activity, these results require confirmation by further investigations of this polymorphism on additional samples using new restriction enzyme.

Investigation of CYP17, CYP19 and SRD5A2 Polymorphisms and Their Association with Prostate Cancer in a Lebanese population

Ahmad SHARANEK
Master 2 practical research project

There has been accumulating evidences that endogenous levels of androgens and estrogens are associated with the development of prostate cancer. Therefore, genes involved in the biosynthesis, activation, metabolism and degradation of sex steroids represent important candidates for affecting the development and progression of prostate cancer. In addition the strikingly divergent incidence of prostate cancer across different ethnic/racial groups may come in part from differences in the prevalence of common inherited genetic variations in genes coding for enzymes involved in the synthesis and metabolism of androgens and estrogens. Among these genes CYP17, SRD5A2 and CYP19 represent strong candidates for affecting prostate cancer. CYP17 gene encodes the cytochrome P450c17α-enzyme, which is the rate limiting enzyme in androgen biosynthesis. The T to C polymorphism in the 5’promoter region (denoted T27C or A1 and A2 alleles, respectively) has been associated with prostate cancer. However, contradictory data exists concerning the risk allele. SRD5A2 gene encodes the steroid 5α-reductase type 2 enzymes which irreversibly converts testosterone into the most potent prostatic androgen, DHT. One polymorphism in the SRD5A2 gene in which a valine residue at codon 89 is replaced with leucine (Val 89 Leu) as a result of substitution G/C at nucleotide 296 may be associated with increased risk of prostate cancer. The CYP19 gene encodes the enzyme aromatase that catalyzes the irreversible conversion of testosterone to estradiol. A tetranucleotide (TTTA)n simple tandem repeat polymorphism (STRP) in intron 4 of CYP19 was reported to be positively associated with prostate cancer. In this study, we aimed to investigate the polymorphisms in these three loci and their possible association with prostate cancer in a Lebanese population. Methods: Blood samples from both prostate cancer patients and men controls aged over 50ys have been collected; the genomic DNA was extracted using the phenol-chloroform extraction technique. PCR-RFLP was used for the CYP17 and SRD5A2 genotyping assays where MspAI and RsaI restriction enzymes were used, respectively. However PCR where used to identify the STRP in the CYP19 gene. Preliminary results: To date CYP17 polymorphism was genotyped in 13 patient samples and 12 men controls. Even though we found a clear difference in the percentage of A1/A1 and A1/A2 genotypes between the cases and controls, statistical analysis showed no significant association between prostate cancer risk and the genotypes A1/A1 nor A1/A2 (OR=0.53, 95% CI =0.09 – 2.97, p= 0.67), to complete this work, we plan to collect more patient and control samples to give strength to the statistical analysis and make a strong conclusion. Regarding CYP19 and SRD5A2, we encountered some technical problems that prevented the genotyping and we are working to solve these problems.

Effets du tabagisme sur la composition du lait maternel.

Pamela BACHOUR
Master 2 practical research project see article: http://www.ncbi.nlm.nih.gov/pubmed/22166069

Le lait maternel est l’aliment le plus adapté à la nutrition des nouveau-nés puisqu’il répond aux besoins de développement et de croissance des nourrissons en fonction de leur âge. Il assure également aux nouveau-nés une immunité passive avant la maturation de leurs systèmes immunitaires. Le tabagisme qui a beaucoup d’effets néfastes sur la santé, est un facteur qui pourrait avoir un effet sur le lait maternel en terme de quantité et de qualité. Le but de notre projet est d’étudier les effets du tabagisme sur la composition du lait maternel en lipides et en protéines. Nous avons collecté 66 échantillons de lait humain dont 23 sont issus de mères fumeuses et 43 de mères non-fumeuses. Nous avons mesuré la densité des échantillons de lait et nous n’avons pas trouvé de différence statistiquement significative associé au tabagisme. Le dosage des lipides est réalisé par extraction liquide-liquide suivie d’une gravimétrie ainsi que par analyse spectrophotométrique. La teneur du lait en protéines est mesurée par la méthode de Bradford. Le profil protéique de chacun des échantillons est déterminé par électrophorèse sur gel de SDS-polyacrylamide suivie d’une identification des protéines majoritaires du lait selon leurs poids moléculaire apparent et d’une densitométrie sur les photographies des gels. La moyenne des concentrations des lipides dans le lait des mères fumeuses est inferieure à celle des laits des mères non fumeuses. Cette diminution (15% selon la méthode de gravimétrie, 33% selon la méthode de spectrophotométrie) est statistiquement significative (t-test à variance égale, p ˂ 0.05). De même, la concentration de protéines dans le lait des mères fumeuses est inferieure de 12.4% par rapport aux non-fumeuses, cette diminution est statistiquement significative (t-test à variance égale, p ˂ 0.05). L’analyse du profil protéique et la densitométrie ne montre pas de différence statistiquement significative entre les moyennes de densités optiques (fumeuses/non-fumeuses) correspondant à la caséine-β, à la chaîne lourde des IgA, à l’albumine sérique, et à la lactoferrine. Notre étude montre que le tabagisme est associé avec une diminution des concentrations des lipides et des protéines dans le lait maternel. Le lait des mères fumeuses a une valeur nutritive inferieure par rapport au lait des mères non-fumeuses suite à sa moindre teneur en lipides et en protéines.

Induction d’une cholestase par la chlorpromazine dans les cellules hépatiques humaines HepaRG : Etude des mécanismes impliqués et de l’influence d’un stress inflammatoire

Pamela BACHOUR
PhD student in co-tutelle

Drug-induced liver injury is the major cause of drug withdrawal during development and marketing process. The most common manifestation of adverse drug reactions is cholestasis, which results from alteration of bile flow. Adverse drug reactions are usually classified either as dose-dependent and reproducible (intrinsic) or unpredictable (idiosyncratic) occurring only in certain susceptible patients as observed with chlorpromazine (CPZ), a neuroleptic drug. Our work aimed to induce cholestasis with this drug and to study the mechanisms involved in the presence or absence of an inflammatory stress using differentiated HepaRG liver cells derived from a human cholangio-hepatocarcinoma as an experimental model. We firstly validated this cell model by demonstrating that the major canalicular and basolateral influx and efflux transporters are localized to the appropriate membrane domains, and that the bile canaliculi are functional and closed as in sandwich-cultured human hepatocytes, the reference model. Treatment with CPZ at a high concentration (50µM) induces, as early as 15min, generation of oxidative stress which is associated with altered mitochondrial membrane potential, disruption of the pericanalicular F-actin cytoskeleton distribution and inhibition of canalicular efflux of taurocholic acid. After 24-hour treatment with CPZ, mRNA expression of the two main canalicular bile transporters, BSEP and MDR3, and of the main influx transporter, NTCP, was decreased. By contrast, expression of MRP4 mRNA, a basolateral transporter, was increased. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Inflammation is considered as a factor of susceptibility to idiosycratic hepatotoxicity. We investigated whether in an inflammatory stress induced by IL-6 and IL-1β, cytotoxic and cholestatic effects of CPZ are exacerbated. After a 24 hour pre-treatment by either pro-inflammatory cytokines, HepaRG cells were co-exposed to 20μM CPZ for 1 to 5 days. Although cytokines have induced inflammatory stress and inhibited the metabolism of CPZ and transcripts of CYP3A4 and CYP1A2, two main CYPs involved in the metabolism of this drug, the modulation of cytotoxic and cholestatic effects of CPZ was limited, even after 5 daily treatments. Increased cytotoxicity by 20 %, amplification of NTCP mRNA and activity inhibition and deregulation of the expression of other genes associated with cholestasis, were observed in CPZ- and cytokine-co-treated cells. Altogether, our results show that it is possible to induce in vitro cholestasis using HepaRG cells and that CPZ-induced cholestasis depends on the generation of oxidative stress. They also show that the certain susceptibility factors may be investigated.

Etude de la toxicite du diclofenac et de la trovafloxacine dans differents modeles in vitro.

Houssein Al-ATTRACHE
PhD student

Beaucoup de médicaments peuvent causer des lésions hépatiques qui peuvent être dose-dépendantes ou non dose-dépendantes (idiosyncratiques). La toxicité de ces derniers dépend de plusieurs facteurs, parmi eux le stress inflammatoire. Ceci a été montré pour plusieurs médicaments comme la ranitidine, la chlorpromazine, le diclofenac (dic), la trovafloxacine (TVX) (Buchweitz et al, 2002; Deng et al, 2006; Luyendyk et al, 2003; Waring et al, 2006). Le but de notre projet est d’étudier les mécanismes de toxicité du dic (antiinflammatoire) et de la TVX (antibiotique) seuls ou en présence de cytokines dans le modèle cellulaire hépatique humain HepaRG et de voir s’il y a un effet des cellules immunes sur ces mécanismes par co-culture des cellules HepaRG avec des macrophages et d’autres cellules immunes. Après induction d’un statut inflammatoire dans notre modèle HepaRG par traitement avec des cytokines inflammatoires (TNF-α, IL-6, IL-1β) pendant 24 heures suivi par un co-traitement dic-cytokines ou TVX-cytokines pendant 24 heures, nous avons trouvé que le co-traitement des cellules avec chacune des molécules+cytokine aggrave leur toxicité et active l’activité de la caspase 3 (marqueur d’apoptose). Nous avons confirmé que les cytokines induisent des marqueurs inflammatoires (CRP et IL8), inhibent des enzymes de la phase I (CYP3A4, CYP2C9, CYP1A2, CYP2B6) et de la phase II (UGT2B7) et que le co-traitement inhibe des gènes de transporteurs des acides biliaires (BSEP, NTCP) et induit des gènes du stress oxydant (HO1 et MnSOD). La comparaison des effets des médicaments+cytokines sur les cellules HepaRG différenciées et non différenciées (proliférantes et n’ayant pas d’activité de biotransformation) montre que l’interaction entre l’inflammation et les médicaments se fait au moins partiellement au niveau de leurs métabolismes via l’inhibition des enzymes de phase I et II. Des expériences en cours sur le mécanisme de leur toxicité permettront de voir l’implication éventuelle des voies extrinsèques et intrinsèques de l’apoptose avec détermination du rôle des ROS, GSK3β et JNK. La co-culture des cellules HepaRG avec des cellules immunes aidera à comprendre l’importance de leur interaction dans la toxicité induite par ces médicaments.

Etude de l’association entre le polymorphisme des gènes des CYP1A1 et CYP2E1 et le cancer de la prostate chez un échantillon de la population Libanaise

Ghina RAMMAL
Master 2 practical research project

Le cancer de la prostate est le premier cancer masculin dans le monde. Malgré une incidence en croissance importante pendant cette dernière décennie, les causes de la genèse d’un tel cancer restent mal connues. Plusieurs facteurs de risques sont incriminés dont l’âge, les antécédents familiaux et l’origine ethnique ainsi qu’une variété de facteurs individuels (habitudes alimentaires, tabagisme, alcoolisme…). Certains cytochromes P450 (CYP) jouent un rôle essentiel dans le développement du cancer et cela par métabolisation et bio-activation de pro-carcinogène comme la benzo[a]pyrène. Les activité des CYP présentent une forte variation interindividuelle due aux polymorphismes des gènes, mais aussi à de diverses facteurs. Une mutation 3801 T>C dans la région 3’ non codante engendre un site de restriction par MspI du gène CYP1A1 engendrant un phénotype fortement inductible de l’enzyme. La mutation du site 7632 T>A dans l’intron 6 dy CYP2E1 induit la disparition d’un site de restriction par DraI. Le but de cette étude est de trouver une éventuelle association entre l’allèle m2 du CYP1A1 et l’allèle C du CYP2E1 et le risque de cancer de la prostate chez la population libanaise. Nous avons collecté 40 échantillons de sang de patients atteints de cancer de la prostate et 43 échantillons de contrôles. Après collection des échantillons de sang, l’ADN a été extrait et l’amplification des fragments d’ADN d’intérêts a été effectuée par PCR. Ensuite le génotypage est effectué par RFLP. Les résultats obtenus sont préliminaires à cause du petit nombre d’échantillons analysés concrètement parmi ceux collectés. Pour le CYP1A1 les résultats ne sont pas statistiquement significatifs alors que le test statistique du CYP2E1 n’a pas été effectué. Toutefois les résultats suggèrent une éventuelle association entre le génotype DD et le cancer de la prostate. Cette étude doit être continuée en analysant la totalité des échantillons et généralisée en considérant tous les facteurs génétiques et environnementaux pouvant affecter le risque de cancer de la prostate.

Etude de l’association des Polymorphismes des Gènes du CYP1A1 et du CYP2E1 avec le cancer de la prostate chez un échantillon de la population libanaise

Houssein Al-ATTRACHE
Master 2 practical research project

Le cancer de la prostate est une maladie qui touche principalement les hommes âgés de plus de 50 ans. Dans de nombreuses régions du monde, il est le plus fréquent cancer chez l'homme, parfois (et selon les sources) il est le deuxième cancer chez l'homme après le cancer des poumons. Le cancer de prostate représente un des cancers les plus répandus au Liban. Plusieurs facteurs déterminent l’étiologie du cancer de prostate. Il y a des facteurs environnementaux et relatifs au mode de vie, et le facteur âge en plus de facteurs héréditaires. En effet, le risque de survenue de ce cancer dépend du polymorphisme de certains gènes dont ceux codant pour les enzymes impliquées dans le métabolisme des agents cancérigènes. Parmi ces enzymes il y a le CYP1A1 et le CYP2E1 qui jouent un rôle clef dans le métabolisme de pro carcinogènes. L’association du polymorphisme de ces gènes avec le cancer de prostate a été peu étudiée à l’échelle internationale et aucune étude n’avait été réalisée sur la population Libanaise. Le but de notre projet est d’étudier l’association entre le polymorphisme des CYP1A1*2A (allèle m2, 6235T˃C, rs4646903) et CYP2E1*6 (7632T˃A, rs6413432) et le cancer de prostate chez un échantillon de la population du Liban. Nous avons collecté l’ADN d’individus contrôles (n= 121) et de patients atteints de cancer de prostate (n= 52). Le génotypage des contrôles et des patients a été réalisé par PCR-RFLP et les fréquences alléliques et génotypiques déterminées et comparées entre patients et contrôles. Nous avons trouvé que l’allèle m2 (CYP1A1*2A) est significativement associé au cancer de prostate augmentant le risque de ce cancer d’un facteur de 2.7. Les génotypes CYP1A1 m1/m2 (CYP1A1 wt/*2A) et CYP1A1 m2/m2 (CYP1A1*2A/*2A) sont aussi significativement associés au cancer de prostate augmentant le risque de ce cancer d’un facteur 2.7. Aucune association n’a été trouvée dans notre étude entre les différents génotypes du CYP1E1 (CYP2E1*6) et le cancer dans de la prostate.

Étude de l’interaction entre la chlorpromazine et le benzo-a-pyrène ou l’éthanol sur le métabolisme de la chlorpromazine et les mécanismes de sa toxicité.

Jinane TRAD
Master 2 practical research project

Cholestasis is a liver disease known by the accumulation of bile acids in hepatocytes. It is either genetically acquired or induced by drugs during their metabolism in the liver, disrupting synthesis enzymes and transport systems of bile acids. Chlorpromazine (CPZ), an antipsychotic drug used in acute and chronic psychosis, induces intrahepatic cholestasis, but it is sometimes accompanied by ethanol intake and / or cigarettes. To investigate whether these drugs interfere with CPZ in cholestasis, differentiated human HepaRG cells and undifferentiated human HepG2 cells were treated by the CPZ alone, CPZ with Benzo (a) pyrene (BaP) (a highly toxic substance present in cigarettes), the CPZ with ethanol, and CPZ with rifampicin (RIF) (an antibiotic which induces CPZ metabolism enzymes, CYPs). After 24 and 48 hours of treatment, the MTT assay was performed to quantify cell viability. This test was accompanied by microscopy to examine cell morphology during toxicity. Protein determination was made as to check whether it refers to intracellular proteins or metabolites of CPZ. The test was also followed by the HPLC analysis which quantifies the CPZ and its metabolites that are produced by cells. After these tests, a small (but little understood) effect of drugs was noticed on the toxicity of CPZ. This effect needs to be confirmed pending future experiments.

Investigation of chlorpromazine toxicity on Saccharomyces cerevisae

Houssam ALSIBAII
Master 2 practical research project

Toxicological effects of chlorpromazine (CPZ) and Sodium Salicylate (SS) on yeast (Sacchromyces cerevisiae) were investigated. Different concentrations of CPZ and SS were used to treat yeast cells in their exponential growth phase. The results showed that 50 µM and 100 µM concentrations of CPZ are extremely toxic preventing growth of S. cerevisiae, whereas 100 mM concentration of SS is non significantly toxic. Moreover, 65% and 32% of cells are viable at concentrations of 50 µM and 100 µM of CPZ. Furthermore, the activity of superoxide dismutase (SOD) in S. cerevisiae was significantly induced by CPZ, whereas the highest activity occurs at a concentration of 100 µM CPZ .On the other hand, a non-significant difference of SOD activity was obtained upon using SS. Total protein measurement was done and showed an increase that may be attributed to accumulation of protein in S. cerevisiae cells treated with CPZ. SS treated cells showed a decreased protein content in comparison with control. In conclusion, CPZ decreases yeast growth causing high rate of cell death and induces SOD activity, probably as a result of an oxidative stress. SS showed no significant toxicity based on the measured endpoints.

Etude de la toxicite de la chlropromazine et du BaP in vitro

Katia Sayyed
Master 2 student

La chlorpromazine est un médicament antipsychotique largement utilisé. Chez certains patients il induit une toxicité idiosyncratique (toxicité imprévisible non dépendante de la dose) au niveau du foie. Plus précisément, il induit une cholestase hépatique (altération de la synthèse et la sécrétion des acides biliaires) se traduisant parfois par une nécrose des hépatocytes. Cette cholestase se déclenche spontanément à un moment donné du traitement avec une dose supposée non toxique chez la plupart des personnes (1). Cette forme de toxicité induite par la chlorpromazine est médiée en partie par la génération d’un stress oxydant dans les cellules de type hépatique in vitro (2) altérant ainsi les fonctions de synthèse et de sécrétion des acides biliaires. Bien que les facteurs génétiques et immunologiques puissent régir cet effet idiosyncratique, l’inflammation est un facteur susceptible de promouvoir cette toxicité (3). L’exposition des patients aux polluants de la fumée de tabac (hydrocarbure aromatiques polycycliques, HAP, la nicotine,…) pourrait contribuer au développement de cette toxicité étant donné que certains HAP interfèrent et dérégulent le système immunitaire (4,5), ainsi que l’expression des gènes de synthèse/métabolisme et de transport des acides biliaires et induisent un stress oxydant (6). De plus, certains HAP pourraient interagir avec la toxicité de la chlorpromazine puisqu’ils modifient l’expression des gènes impliqués dans son métabolisme et son élimination (6). Nos résultats préliminaires sont encourageants à cet égard. Basée sur la bibliographie et sur nos données préliminaires, notre hypothèse est que les polluants de la fumée de tabac interagissent avec la toxicité de la chlorpromazine. L’objectif de ce projet est d’étudier si ces polluants (HAP et/ou la nicotine,…) affectent la toxicité induite par la chlorpromazine, particulièrement la cholestase. Des cultures de cellules de type hépatique in vitro seront traitées par diverse concentrations du médicament et des HAP et/ou la nicotine. En fin de traitement, certains paramètres de toxicité seront mesurés (par exemple MTT) ainsi que l’expression de certains gènes, particulièrement des gènes connus pour jouer un rôle dans la cholestase comme le BSEP, le NTCP, le CYP8B1,... et des gènes de métabolisme de la chlorpromazine comme le CYP1A2, et des gènes du stress oxydant (exemple le MnSOD). L’extrapolation des résultats de ce projet devraient prévoir si les patients traités avec la chlorpromazine et exposés à la fumée de tabac (à d’autres sources d’HAP), sont plus susceptibles au développement de l’effet toxique idiosyncratique de la chlorpromazine. L’application directe de ce projet est clinique, il s’agit de prendre les précautions nécessaires lors de la prescription de ce médicament chez des patients fumeurs passif ou act

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