Dalia Fouad

Role of nitric oxide in D-galactosamine-induced cell death and its protection by PGE1 in cultured hepatocytes.

Prostaglandin E(1) (PGE(1)) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE(1) protection against D-galactosamine (D-GalN) citotoxicity in cultured hepatocytes. PGE(1) was preadministered to D-GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite+nitrate (NO(x)) release, iNOS expression, and NF-kappaB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by D-GalN in hepatocytes. PGE(1) protection against D-GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by D-GalN. Nevertheless, iNOS inhibition showed that protection by PGE(1) was also mediated by NO. Low concentrations of a NO donor reduced D-GalN injury with a decrease in the extracellular NO(x) concentration. High concentrations of the NO donor enhanced NO(x) concentration and increased cell death by D-GalN. The present study suggests that low NO production induced by PGE(1) preadministration reduces D-GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.

PGE1‐induced NO reduces apoptosis by D‐galactosamine through attenuation of NF‐κB and NOS‐2 expression in rat hepatocytes

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. We have previously shown that PGE1 preadministration protects against NO‐dependent cell death induced by D‐galactosamine (D‐GalN) through a rapid increase of nuclear factor κB (NF‐κB) activity, inducible NO synthase (NOS‐2) expression, and NO production. The present study investigates whether PGE1‐induced NO was able to abolish NF‐κB activation, NOS‐2 expression, and apoptosis elicited by D‐GalN. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. PGE1 (1 μmol/L) was administered 2 hours before D‐GalN (5 mmol/L) in primary culture rat hepatocytes. PGE1 reduced inhibitor κBα degradation, NF‐κB activation, NOS‐2 expression, and apoptosis induced by D‐GalN. The administration of an inhibitor of NOS‐2 abolished the inhibitory effect of PGE1 on NF‐κB activation and NOS‐2 expression in D‐GalN–treated hepatocytes. Transfection studies using different plasmids corresponding to the NOS‐2 promoter region showed that D‐GalN and PGE1 regulate NOS‐2 expression through NF‐κB during the initial stage of hepatocyte treatment. PGE1 was able to reduce the promoter activity induced by D‐GalN. In addition, a NO donor reduced NOS‐2 promoter activity in transfected hepatocytes. In conclusion, administration of PGE1 to hepatocytes produces low levels of NO, which inhibits its own formation during D‐GalN–induced cell death through the attenuation of NF‐κB–dependent NOS‐2 expression. Therefore, a dual role for NO in PGE1‐treated D‐GalN–induced toxicity in hepatocytes is characterized by a rapid NO release that attenuates the late and proapoptotic NOS‐2 expression. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:1295–1303.)

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice.

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCEs)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCEs, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Role of NF-κB activation and nitric oxide expression during PGE1 protection against d-galactosamine-induced cell death in cultured rat hepatocytes

Abstract: Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. Nitric oxide (NO) mediates PGE1 protection against d‐galactosamine (d‐GalN)‐induced cell death. Nuclear factor kappa‐B (NF‐κB) plays a protective role in different experimental models of cell death. We investigated if NF‐κB was responsible for inducible nitric oxide synthase (iNOS) expression and cytoprotection induced by PGE1 against d‐GalN cell death in cultured hepatocytes. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. A kinetic study of cell death, NF‐κB activation, mRNA and protein iNOS expression, and NO production was carried in hepatocytes treated with d‐GalN (5 mM) in the presence or absence of PGE1 (1 μM) administered 2 h before the hepatotoxin. A proteasome inhibitor was used to evaluate the role of NF‐κB activation in our experimental conditions. PGE1 protection against d‐GalN‐induced cell death was associated with its capacity to rapidly enhance NF‐κB activation, mRNA and protein iNOS expression, and NO production in d‐GalN‐treated hepatocytes. The inhibition of NF‐κB activation abolished iNOS expression and cell protection by PGE1 in hepatocytes treated with the hepatotoxin. The present study shows that the cytoprotection by PGE1 against d‐GalN‐induced apoptosis was related to NF‐κB‐dependent iNOS expression.

Genomics, phylogeny and in silico analysis of mitochondrial glutathione S-transferase-kappa from the camel Camelus dromedarius.

The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Peninsula. For most of its life, this species is exposed to both intrinsic and extrinsic genotoxic factors that cause gross DNA alterations in many organisms. GST enzymes constitute an important supergene family involved in protection against the deleterious effects of oxidative stress and xenobiotics. Cloning the camel mitochondrial GST kappa (GSTK) gene and comparing its structural similarities with different species may aid in understanding its evolutionary relics. We cloned the camel GSTK using RT-PCR. This yielded an open reading frame of 678 nucleotides, encoding a protein of 226 amino acid residues. In a comparative analysis, the cloned GSTK was used to screen orthologues from different organisms. Phylogenetic analysis demonstrated that the camel GSTK apparently evolved from an ancestral GSTK gene that predated the appearance of vertebrates, and it grouped with pig, cattle, dog, horse, human and monkey GSTKs. The calculated molecular weight of the translated ORF was 25.52 kDa and the isoelectric point was 8.4. The deduced cGSTK sequence exhibited high identity with many mammals, such as Bactrian camel (99.55%), pig, cattle and human (>74%), and lower identity with other unrelated organisms, such as frog (Xenopus tropicalis, 61%), chicken (Gallus gallus, 57%), salmon (Salmo salar, 49%), sponge (Amphimedon queenslandica, 46%), tick (Amblyomma maculatum, 45%) and roundworm (Caenorhabditis elegans, 33%). A 3D structure was built based on the crystal structure of the human and rat enzymes. The levels of cGSTK expression in five camel tissues were examined via real-time PCR. The highest level of cGSTK transcripts was found in the camel liver, followed by the testis, spleen, kidney and lung.

Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius

Superoxide dismutase (SOD) is the first line of defense against oxidative stress induced by endogenous and/or exogenous factors and thus helps in maintaining the cellular integrity. Its activity is related to many diseases; so, it is of importance to study the structure and expression of SOD gene in an animal naturally exposed most of its life to the direct sunlight as a cause of oxidative stress. Arabian camel (one humped camel, Camelus dromedarius) is adapted to the widely varying desert climatic conditions that extremely changes during daily life in the Arabian Gulf. Studying the cSOD1 in C. dromedarius could help understand the impact of exposure to direct sunlight and desert life on the health status of such mammal. The full coding region of a putative CuZnSOD gene of C. dromedarius (cSOD1) was amplified by reverse transcription PCR and cloned for the first time (gene bank accession number for nucleotides and amino acids are JF758876 and AEF32527, respectively). The cDNA sequencing revealed an open reading frame of 459 nucleotides encoding a protein of 153 amino acids which is equal to the coding region of SOD1 gene and protein from many organisms. The calculated molecular weight and isoelectric point of cSOD1 was 15.7 kDa and 6.2, respectively. The level of expression of cSOD1 in different camel tissues (liver, kidney, spleen, lung and testis) was examined using Real Time-PCR. The highest level of cSOD1 transcript was found in the camel liver (represented as 100%) followed by testis (45%), kidney (13%), lung (11%) and spleen (10%), using 18S ribosomal subunit as endogenous control. The deduced amino acid sequence exhibited high similarity with Cebus apella (90%), Sus scrofa (88%), Cavia porcellus (88%), Mus musculus (88%), Macaca mulatta (87%), Pan troglodytes (87%), Homo sapiens (87%), Canis familiaris (86%), Bos taurus (86%), Pongo abelii (85%) and Equus caballus (82%). Phylogenetic analysis revealed that cSOD1 is grouped together with S. scrofa. The predicted 3D structure of cSOD1 showed high similarity with the human and bovine CuZnSOD homologues. The Root-mean-square deviation (rmsd) between cSOD1/hSOD1 and cSOD1/bSOD1 superimposed structure pairs were 0.557 and 0.425 A. The Q-score of cSOD1-hSOD1 and cSOD1-bSOD1 were 0.948 and 0.961, respectively.

Renin–angiotensin system gene polymorphisms among Saudi patients with coronary artery disease

BackgroundThe polymorphisms in the components of the renin-angiotensin system (RAS) are important in the development and progression of coronary artery disease (CAD) in some individuals. Our objectives in the present investigation were to determine whether three RAS polymorphisms, angiotensin-converting enzyme insertion/deletion (ACE I/D), angiotensin receptor II (Ang II AT2 - C3123A) and angiotensinogen (AGT-M235T), are associated with CAD in the Saudi population. We recruited 225 subjects with angiographically confirmed CAD who had identical ethnic backgrounds and 110 control subjects. The polymerase chain reaction-restriction fragment length polymorphisms (RFLP) technique was used to detect polymorphisms in the RAS gene.ResultsWithin the CAD group, for the ACE I/D genotype, DD was found in 64.4%, 26.3% carried the ID genotype, and 9.3% carried the II genotype. Within the control group, the DD genotype was found in 56.4%, 23.6% carried the ID genotype, and 20% carried the II genotype. The odds ratio (OR) of the ACE DD vs II genotype with a 95% confidence interval (CI) was 2.45 (1.26-4.78), with p = 0.008. For the Ang II AT2 receptor C3123A genotype, within the CAD group, CC was found in 39.6%, 17.8% carried the CA genotype, and 42.6% carried the AA genotype. Within the control group, CC was found in 39.1%, 60.9% carried the CA genotype, and there was an absence of the AA genotype. The OR of the Ang II AT2 receptor C3123A CC vs AA genotypes (95% CI) was 0.01, with p = 0.0001. A significant association with CAD was shown. For the AGT-M235T genotype, within the CAD group, MM was found in 24.0%, 43.6% carried the MT genotype and 32.4% carried the TT genotype. Within the control group, MM was found in 26.4%, 45.5% carried the TT genotype and 28.2% carried the MT genotype. The OR of MM vs TT (95% CI) was 0.79 (0.43 to 1.46), which was insignificant.ConclusionsThere is an association between the ACE I/D and Ang II AT2 receptor C3123A polymorphisms and CAD, however, no association was detected between the AGT M235T polymorphism and CAD in the Saudi population.

Molecular cloning and 3D structure modeling of APEX1, DNA base excision repair enzyme from the Camel, Camelus dromedarius.

The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, producing the highly mutagenic apurinic/apyrimidinicsites (AP sites). Therefore, recognition of AP sites is fundamental to cell/organism survival. In the present work, the full coding sequence of a putative cAPEX1 gene was amplified for the first time from C. dromedarius by RT-PCR and cloned (NCBI accession number are HM209828 and ADJ96599 for nucleotides and amino acids, respectively). cDNA sequencing was deduced to be 1041 nucleotides, of which 954 nucleotides encode a protein of 318 amino acids, similar to the coding region of the APEX1 gene and the protein from many other species. The calculated molecular weight and isoelectric point of cAPEX1 using Bioinformatics tools was 35.5 kDa and 8.11, respectively. The relative expressions of cAPEX1 in camel kidney, spleen, lung and testis were examined using qPCR and compared with that of the liver using a 18S ribosomal subunit as endogenous control. The highest level of cAPEX1 transcript was found in the testis; 325% higher than the liver, followed by spleen (87%), kidney (20%) and lung (5%), respectively. The cAPEX1 is 94%–97% similar to their mammalian counterparts. Phylogenetic analysis revealed that cAPEX1 is grouped together with that of S. scrofa. The predicted 3D structure of cAPEX1 has similar folds and topology with the human (hAPEX1). The root-mean-square deviation (rmsd) between cAPEX1 and hAPEX1 was 0.582 and the Q-score was 0.939.

Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius

Reactive oxygen species (ROS) oxidize the guanine base in the DNA to 8-oxoguanine (8-oxoG). This lesion, if left unrepaired, causes the transversion of G:C pair to T:A following replication. 8-oxoG is targeted by one of the DNA glycosylases, namely OGG1. Arabian camel (one humped camel, Camelus dromedarius ) is adapted to live in desert climate conditions under direct exposure to endogenous and exogenous ROS-producing conditions, among of them the sunlight. In the recent study, partial sequence of camel OGG-1 gene was cloned and analyzed for the first time. A DNA fragment of 567 bases was amplified by reverse transcription PCR. It is equivalent to about 55% from the coding region of the known transcript of many organisms. The level of expression of OGG-1 in different camel tissues (liver, kidney, spleen, lung and testis) was examined using real time-PCR. The highest level of OGG-1 transcript was found in the camel liver (represented as 100%) followed by testis (85%), spleen (78%), kidney (37%) and lung (3%) using 18S ribosomal subunit as endogenous control. The obtained cDNA sequence of OGG-1 showed high similarity with Ailuropoda melanoleuca (86%), Sus scrofa (86%), Canis familiaris (85%), Bos taurus (85%), Macaca mulatta (85%), Homo sapiens (84%), Pan troglodytes (84%) and Pongo abelii (82%). Keywords: Camelus dromedarius , cloning, OGG1, gene expression, DNA glycosylase

Biochemical Characterization of the Detoxifying Enzyme Glutathione Transferase P1-1 from the Camel Camelus Dromedarius

Glutathione transferase (GST, EC 2.5.1.18) is a primary line of defense against toxicities of electrophile compounds and oxidative stress and therefore is involved in stress-response and cell detoxification. In the present study, we investigated the catalytic and structural properties of the glutathione transferase (GST) isoenzyme P1-1 from Camelus dromedarius (CdGSTP1-1). Recombinant CdGSTP1-1 was produced in Escherichia coli BL21(DE3) and purified to electrophoretic homogeneity. Kinetic analysis revealed that CdGSTP1-1 displays broad substrate specificity and shows high activity towards halogenated aryl-compounds, isothiocyanates and hydroperoxides. Computation analysis and structural comparison of the catalytic and ligand binding sites of CdGSTP1-1 with other pi class GSTs allowed the identification of major structural variations that affect the active site pocket and the catalytic mechanism., Affinity labeling and kinetic inhibition studies identified key regions that form the ligandin-binding site (L-site) and gave further insights into the mechanism of non-substrate ligand recognition. The results of the present study provide new information into camelid detoxifying mechanism and new knowledge into the diversity and complex enzymatic functions of GST superfamily.