Hazem K. Ghneim

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1−1) were added. Results indicated that ascorbate at 100 µmol 1−1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1−1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1−1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright

Biochemical Markers of Oxidative Stress in Saudi Women with Recurrent Miscarriage

This study was undertaken to investigate the antioxidant/oxidant status in recurrent miscarriage patients. Antioxidants including glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR), reduced glutathione (GSH) and selenium (Se), as well as the oxidants hydrogen peroxide (H2O2), oxidised glutathione (GSSG) and lipid peroxidation were assayed in plasma, whole blood and placental tissue of non-pregnant women (NP), healthy pregnant women (HP), and recurrent miscarriage (RM) patients. Results indicated that all antioxidant activities and levels in plasma and whole blood of HP women were consistently moderately lower, and much more significantly lower in RM patients when both were compared to those seen in NP women (P<0.05 and P<0.001, respectively). Furthermore, whereas plasma antioxidant activities and levels were significantly lower in RM patients, those of whole blood and placental tissue were much more significantly lower when compared with HP women (P<0.001). Concurrent with these findings there were consistent increases of equal statistical significance and magnitude in the levels of all investigated oxidants assayed in all samples when compared in between subjects of the study as indicated above. Data thus illustrated a distinct shift in favor of oxidative reactions and reactive oxygen species (ROS) generation, and very significant decreases in the GSH/GSSG ratios in whole blood and placental tissue of RM patients when compared to HP and NP women (P<0.001). The above noted oxidative stress could have been a major causative factor of recurrent miscarriage.

Enzymatic Variations Related to Glucose and Glycogen Catabolism in Serially Subcultured Human Fibroblasts

The specific activities of some glucose and glycogen degradative enzymes were studied in serially subcultured fibroblasts harvested at the same state of growth and replication. A continuous increase i

The effect of micronutrients on superoxide dismutase in senescent fibroblasts

The specific activities of zinc/copper (Zn/Cu)‐superoxide dismutase (SOD‐1) and manganese (Mn)‐superoxide dismutase (SOD‐2) were assayed in young passage 5 fibroblasts and in serially subcultured cells that were characterized as senescent at passages 15–35. SOD‐1 and SOD‐2 activities did not significantly change in senescent and young cells cultured in either routine medium [minimum essential medium 1 (MEM1)], or in Zn, Cu and Mn supplemented medium (MEM2) containing normal human plasma levels of the cations. SOD‐1 and SOD‐2 activities, however, underwent parallel progressive significant activity increases in senescent passage 20 and 25 cells, which peaked in value in passage 30 and 35 cells subcultured in supplemented medium (MEM3) containing triple human plasma levels of the cations. Concurrently, superoxide radical generation rates underwent progressive significant increases in senescent passage 15–25 cells, which peaked in value in passage 30 and 35 cells subcultured in MEM1 or MEM2. These rates, however, were significantly lowered in senescent cells subcultured in MEM3. We infer that it was only possible to significantly stimulate SOD‐1 and SOD‐2 activities in senescent MEM3 cultured cells enabling them to combat oxidative stress. Copyright

Expression profiling of selected microRNA signatures in plasma and tissues of Saudi colorectal cancer patients by qPCR

MicroRNAs (miRNAs or miRs) have been advocated as potentially robust and highly stable biomarkers of diverse disease conditions including cancer. The primary aim of this study was two-fold: i) to profile the expression levels of selected mature miRNA signature genes, such as miR-145, miR-195, miR-29 and miR-92, in a paired-study design of 20 colorectal cancer (CRC) tissues from patients versus adjacent neoplasm-free mucosal tissues employing reverse transcription-quantitative polymerase chain reaction; and ii) to examine their expression level in the plasma of the same CRC patients in relation to the age-matched plasma of healthy controls. Statistically significant (P<0.01) increases in miR-29 (2.5) and miR-92 (2.6) were observed in CRC tissues compared with adjacent neoplasm-free mucosal tissues. Profiling of CRC plasma samples showed that the expression levels of circulating miR-29 and miR-92 were significantly higher (P<0.01) than in the age-matched normal plasma. By contrast, miR-145 and miR-195 exhibited significant (P<0.05) decreases in their mean expression levels in CRC tissue samples in relation to the normal tissues. The mean expression levels of miR-145 and miR-195 were significantly lower (P<0.05) in CRC plasma than the healthy controls. Distinct stage-dependent changes in the expression level of the four miRNA gene profiles were observed between stages II and IV plasma of CRC patients relative to the control plasma. Taken together, the results clearly reflect a similar trend for the four miRNA expression levels in tissue and plasma as well as the positive correlation in the levels of miRNAs in tissues and plasma. These findings may be useful to clarify the molecular mechanisms underlying colorectal carcinogenesis and to underscore the potential of the investigated miRNAs as novel early diagnostic biomarkers of CRC.

Superoxide dismutase activity and gene expression levels in Saudi women with recurrent miscarriage

The antioxidant activities of superoxide dismutase 1 (SOD1) and SOD2, as well as the levels of the oxidant superoxide anion (SOA) and the micronutrients zinc (Zn), copper (Cu) and manganese (Mn), were assayed in plasma, whole blood and placental tissue of non-pregnant (NP), healthy pregnant (HP) women and recurrent miscarriage (RM) patients. The results showed that SOD1 and SOD2 activities and the levels of Zn, Cu and Mn in plasma and whole blood of HP women were slightly, but significantly lower, and even more significantly decreased in RM patients compared to those observed in NP women (P<0.05 and P<0.0001, respectively). Additionally, whereas plasma SOD1 and SOD2 activities and Zn, Cu and Mn levels were significantly lower in RM patients, those of whole blood and placental tissue were significantly lower when compared to HP women (P<0.001 and P<0.0001, respectively). Concurrently, there were consistent increases of equal magnitude and statistical significance in SOA levels in all the assayed samples as identified by a comparison between the subjects. The findings thus supported oxidative metabolism and excessive reactive oxygen species generation. The resultant oxidative stress, identified in whole blood and placental tissues of RM patients, may have been a primary cause of RM. Dietary supplementation of Zn, Cu and Mn may be beneficial to these patients pre- and post-conception.

Effect of selenium supplementation on glutathione peroxidase and catalase activities in senescent cultured human fibroblasts.

Aims: To investigate the effect of senescence and selenium supplementation on glutathione peroxidase (cGPx) and catalase (CAT) activities, and concurrent hydrogen peroxide (H2O2) generation in subcultured human fibroblasts. Methods: cGPx and CAT activities and H2O2 levels were assayed in presenescent passage 5 and 10 cells, and in senescent passage 20, 25, 30 and 35 cells cultured in routine medium (MEM1) and supplemented media MEM2 and MEM3 containing normal and triple human plasma levels of Se, respectively. Senescent cells were identified by studying their growth and replication states, and by monitoring their activity of key glucose and glycogen degradative enzymes. Results: cGPx activity showed moderate increases in senescent cells at passages 20–35 subcultured in MEM1 or MEM2. This activity underwent highly significant progressive increases in the same senescent cells subcultured in MEM3. In contrast, CAT activity showed progressive, highly significant increases in senescent cells at passages 20–35 regardless of the culture medium type. Concurrent H2O2 generation was significantly increased in passage 15–25 cells and peaked to higher levels in passage 30 and 35 cells cultured in MEM1 or MEM2. These rates, however, were significantly reduced in senescent passage 20–35 cells cultured in MEM3. Conclusions: The highest cGPx activity and coupled lower H2O2 generation were achieved in senescent cells cultured in MEM3.

The Effect of Crude and Purified Cerastes Vipera Venom Protein Fractions on Respiratory Chain Function in Cultured Human Fibroblasts

This study was conducted to investigate the effect of crude and venom protein fractions of Cerastes vipera on the major enzymatic components of the respiratory chain in cultured human fibroblasts. Cerastes vipera crude venom was fractionated into seven protein fractions by using 8% preparative native polyacrylamide gel electro-phoresis. The effect of crude venom and purified venom fractions (F1-F7) on respiratory chain function in cultured human fibroblasts was investigated. Confluent fibroblast cultures were incubated with crude venom and venom protein fractions for a fixed period of three hours at 37°C. Crude venom and venom protein fractions 2, 4, 5 and 6 significantly increased the production of lactate as compared to control fibroblasts. The production of pyruvate was markedly decreased in all these cell lines. The ratio of lactate/pyruvate was increased in the cells and in the culture medium by crude venom and venom protein fractions 2, 4, 5 and 6. On the other hand fractions 1,3 and 7 exhibited no effect in relation to lactate and pyruvate production. Similarly the crude venom protein and venom protein fractions 2, 4, 5 and 6 significantly lowered the activity of the major enzymatic component of the respiratory chain in fibroblast mitochondria. In conclusion it is apparent that the crude and venom protein fractions 2, 4, 5 and 6 resulted in a significant lowering of the mitochondrial respiratory chain activity in cultured human fibroblasts.

The effect of Echis coloratus venom on biochemical and molecular markers of the antioxidant capacity in human fibroblasts.

ABSTRACT This study was undertaken to examine the activities and levels of major antioxidants/oxidants in cultured human fibroblasts incubated with a sublethal dose of Echis coloratus venom (EcV). Glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities and gene expression levels as well as reduced glutathione (GSH) levels, and the concurrent hydrogen peroxide (H2O2), superoxide anions (SOA), lipid peroxides (LPO) and oxidized glutathione (GSSG) generation rates were assayed in fibroblast cultures and sonicates incubated with 0.5 µg ml–1 medium EcV for 4 h at 37°C. Data indicated that the activities of all antioxidant enzymes were significantly decreased and their corresponding transcripts downregulated in EcV-incubated cells compared to controls (p < 0.001). In contrast, there were parallel equally significant increases in H2O2, SOA and LPO generation rates in venom-incubated cells compared to controls (p < 0.001). Additionally, GSH levels were significantly decreased and those of GSSG were equally significantly increased in venom-incubated cultures compared to controls (p < 0.001) leading to a lowered GSH/GSSG ratio. In conclusion, incubation of fibroblast cultures with EcV resulted in a shift towards oxidative metabolism causing severe OS. This correlated with significant downregulation in the expression levels of all investigated antioxidant genes.

Ascorbate ameliorates Echis coloratus venom‑induced oxidative stress in human fibroblasts

Reports related to the effects of Echis coloratus venom (EcV) on the antioxidant capacity of human tissues is very scarce. The present study was undertaken to investigate the activities and gene expression levels of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT), as well as the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the generation rates of superoxide anions (SOA), hydrogen peroxide (H2O2) and lipid peroxides (LPO) in cultured human fibroblasts incubated with EcV, ascorbate (Asc) and EcV plus Asc at concentrations and incubation periods that maintained cell viability. Results indicated that the activities of all antioxidant enzymes and their corresponding transcripts underwent highly significant decreases and downregulation in EcV-treated cultures (0.5 µg/ml medium for 4 h) compared to venom-free controls (P<0.001). Additionally, there were concurrent equally significant increases in SOA, H2O2 and LPO generation rates in the venom-incubated cultures compared to controls (P<0.001). Results also indicated very significant decreases and parallel equally significant increases in GSH and GSSG levels respectively in the envenomed cultures compared to controls (P<0.001) leading to a drastically lower GSH/GSSG ratio. However, further incubation of the EcV-treated cultures with Asc (400 µM for 12 h) restored the activities and levels of all investigated parameters including the expression levels of the antioxidant genes to control venom-free values. It is concluded that Asc acted to neutralize the increased reactive oxygen species generation, thus ameliorating the EcV-induced oxidative stress and alleviating the downregulation of antioxidant genes.