Doaa Awad

Comparative Toxicology of Trypan Blue, Brilliant Blue G, and Their Combination Together with Polyethylene Glycol on Human Pigment Epithelial Cells

PURPOSE To determine the toxicity in ARPE-19 human retinal pigment epithelium cells of trypan blue (TB) at 0.15% and 0.25% concentration, brilliant blue G (BBG) at 0.025% and 0.05%, their combination, and the effect of the addition of 4% polyethyleneglycol (PEG), as an additive for increasing the density and thus improving the staining in internal limiting membrane removal, on the individual dyes and their combinations, and compare the toxicity of the dyes to that of clinically used preparations. METHODS Cells were exposed for 5 and for 30 minutes to the different preparations. Cell viability was measured with the WST-1 assay measuring intracellular dehydrogenase activity. RESULTS Solutions containing PEG with BBG (0.025%), TB (0.15%), and mixtures of BBG (0.025%) with TB (0.15% and 0.25%) were the least toxic of the preparations as well as preparations of BBG at 0.025% in phosphate-buffered saline solution, while TB at 0.25% in phosphate-buffered saline solution was the most toxic. The addition of PEG reduced the toxicity of preparations containing TB either alone or in combination with BBG. These results were seen only after an incubation for 30 minutes; for a 5-minute incubation, no toxicity was seen for any of the preparations. CONCLUSIONS For short incubation times, all dyes appear equally safe. For longer incubation times, TB preparations were more toxic than BBG preparations. The toxicity of TB was reduced by the addition of PEG. Further studies are required to determine the clinical impact of this finding.

Interaction of Na2B12H11SH with dimyristoyl phosphatidylcholine liposomes.

Previous investigations have revealed that the boron cluster compound Na2B12H11SH (BSH) is very potent in causing major structural rearrangements of and leakage from phosphatidylcholine liposomes. This somewhat unexpected finding is interesting from a fundamental point of view and may also constitute the basis of future important pharmaceutical/medical applications of BSH. In order to further explore the BSH-lipid interaction, we have studied the effects caused by BSH on dimyristoyl phosphatidylcholine (DMPC) liposomes. Cryo-transmission electron microscopy showed that BSH induces aggregation, membrane rupture and increasing wall thickness of the liposomes. Differential scanning calorimetry revealed a BSH dependent shift of the gel to liquid crystalline phase transition temperature of DMPC. The zeta potential of the liposomes decreases with increasing BSH concentrations, and an apparent dissociation constant of 0.23 mM was found. BSH caused leakage of liposome-encapsulated carboxyfluorescein; leakage was higher at 23 degrees C (near the phase transition temperature) than at 15 degrees C and 37 degrees C. It induced lipid mixing only at very high concentrations.

Valproic acid potentiates curcumin-mediated neuroprotection in lipopolysaccharide induced rats.

The etiology of neuroinflammation is complex and comprises multifactorial, involving both genetic and environmental factors during which diverse genetic and epigenetic modulations are implicated. Curcumin (Cur) and valproic acid (VPA), histone deacetylase 1 inhibitor, have neuroprotective effects. The present study was designed with an aim to investigate the ability of co-treatment of both compounds (Cur or VPA, 200 mg/kg) for 4 weeks to augment neuroprotection and enhance brain recovery from intra-peritoneal injection of (250 μg/kg) lipopolysaccharide-stimulated neuroinflammatory condition on rat brain cortex. Cortex activation and the effects of combined treatment and production of proinflammatory mediators, cyclooxygenase-2 (COX-2), APE1, and nitric oxide/inducible nitric oxide synthase (iNOS) were investigated. Neuroinflammation development was assessed by histological analyses and by investigating associated indices [β-secretase (BACE1), amyloid protein precursor (APP), presenilin (PSEN-1), and PSEN-2)]. Furthermore we measured the expression profile of lethal-7 (let-7) miRNAs members a, b, c, e, and f in all groups, a highly abundant regulator of gene expression in the CNS. Protein and mRNA levels of neuroinflammation markers COX-2, BACE1, APP, and iNOS were also attenuated by combined therapy. On the other hand, assessment of the indicated five let-7 members, showed distinct expression profile pattern in the different groups. Let-7 a, b, and c disappeared in the induced group, an effect that was partially suppressed by co-addition of either Cur or VPA. These data suggest that the combined treatment induced significantly the expression of the five members when compared to rats treated with Cur or VPA only as well as to self-recovery group, which indicates a possible benefit from the synergistic effect of Cur-VPA combination as therapeutic agents for neuroinflammation and its associated disorders. The mechanism elucidated here highlights the particular drug-induced expression profile of let-7 family as new targets for future pharmacological development.

Inhibition of NF-ĸB, Bcl-2 and COX-2 Gene Expression by an Extract of Eruca sativa Seeds during Rat Mammary Gland Carcinogenesis

The effect of Eruca sativa seed extract (SE) on nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) gene expression levels was investigated in rat mammary gland carcinogenesis induced by 7,12 dimethylbenz(α)anthracene (DMBA). DMBA increased NF-κB, COX-2 and Bcl-2 gene expression levels and lipid peroxidation (LP), while, decreased glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities and total antioxidant concentration (TAC) compared to the control group. After DMBA administration, SE treatment reduced NF-κB, COX-2 and Bcl-2 gene expression levels and LP. Hence, SE treatment reduced inflammation and cell proliferation, while increasing apoptosis, GST and SOD activities and TAC. Analysis revealed that SE has high concentrations of total flavonoids, triterpenoids, alkaloids and polyphenolic compounds such as gallic, chlorogenic, caffeic, 3,4-dicaffeoyl quinic, 3,5-dicaffeoyl quinic, tannic, cinnamic acids, catechin and phloridzin. These findings indicate that SE may be considered a promising natural product from cruciferous vegetables against breast cancer, especially given its high antioxidant properties.

Detection of New Point Mutations of BRCA1 and BRCA2 in Breast Cancer Patients

This study included 20 selected female patients with breast cancer, 30 of their female relatives (sisters and daughters), and 10 healthy females as a control group. Genomic DNA was extracted from peripheral blood lymphocytes of all the subjects, and the polymerase chain reaction was carried out using specific primers for BRCA1 (exons 2 and 8) and BRCA2 (exons 9, 11, and 21). The mutations were detected using a single-strand conformation polymorphism assay and heteroduplex analysis. Finally, the sample variants and their controls were sequenced. Mutations were detected in 44% of the study population, with 18% found in the BRCA1 gene and 26% attributed to BRCA2. Five sequence variants were identified, including two frameshift mutations, one nonsense mutation, and two missense mutations. Therefore, we conclude that germline mutations in two major genes, BRCA1 and BRCA2, may have an important influence on the predisposition and development of familial breast cancer.

Effect of Cd, Zn and Hg complexes of barbituric acid and thiouracil on rat brain monoamine oxidase-B (in vitro).

Metal pyrimidine complexes (MPCs) including cadmium-barbiturate (Cd-BA), zinc-barbiturate (Zn-BA), cadmium-thiouracil (Cd-TU) and mercury-thiouracil (Hg-TU) were prepared and their analysis was carried out. These MPCs were evaluated as monoamine oxidase-B (MAO-B) inhibitors. Rat brain MAO-B was inhibited (in vitro) by Cd-BA, Zn-BA, Cd-TU and Hg-TU complexes. The inhibition of MAO-B by these complexes was time and concentration dependent. The values of IC50 of Zn-BA, Cd-BA, Hg-TU and Cd-TU were 10.2, 15.8, 16.2 and 20.4 nM, respectively. The effect of different substrate concentrations in the absence and in the presence of MPCs was determined. Lineweaver-Burk plots were plotted and the values of apparent Michaelis constant (Km), maximum velocity (Vmax), the dissociation constant of enzyme inhibitor complex (Ki) and the percent of inhibition (i%) were calculated. The data showed that the inhibition of MAO-B by all studied MPCs was the non-competitive type. The sequence of inhibition zone was: Zn-BA>Cd-BA and Hg-TU>Cd-TU affected by the chemistry of both the metal and the ligand. Otherwise, the results of the present study showed that the inhibition of MAO-B by all MPCs was fully reversible. The data showed that the presence of Cd-BA, Zn-BA, Cd-TU and Hg-TU complexes changed the optimum temperature and pH of MAO-B.

Combination between Taxol-Encapsulated Liposomes and Eruca sativa Seed Extract Suppresses Mammary Tumors in Female Rats Induced by 7,12 Dimethylbenz(α)anthracene

Taxol (paclitaxel) is a powerful anti-cancer drug widely used against several types of malignant tumors. Because Taxol may exert several side effects, a variety of formulations have been developed. One of these features liposomes, regarded as one of the most promising drug carriers, biocompatible and best able to reduce drug toxicity without changing efficacy against tumor cells. Eruca sativa seed extract (SE) is considered a promising natural product from cruciferous vegetables against breast cancer, increasing chemotherapeutic and eliminating harmful side effects. The effects of Taxol-encapsulated liposomes (T) alone and in combination between Eruca sativa seed extract on nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) gene expression levels were investigated in rat mammary gland carcinogenesis induced by 7,12 dimethylbenz(α) anthracene (DMBA) using qRT-PCR. The results showed that DMBA increased NF-κB, COX-2 and Bcl-2 gene expression levels and lipid peroxidation (LP), while decreasing glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities and total antioxidant concentration (TAC) compared to the control group. T and T-SE treatment reduced NF-κB, COX-2 and Bcl-2 gene expression levels and LP. Hence, T and T-SE treatment appeared to reduce inflammation and cell proliferation, while increasing apoptosis, GST and SOD activities and TAC.

Hemorrhage in mouse tumors induced by dodecaborate cluster lipids intended for boron neutron capture therapy.

The potential of boron-containing lipids with three different structures, which were intended for use in boron neutron capture therapy, was investigated. All three types of boron lipids contained the anionic dodecaborate cluster as the headgroup. Their effects on two different tumor models in mice following intravenous injection were tested; for this, liposomes with boron lipid, distearoyl phosphatidylcholine, and cholesterol as helper lipids, and containing a polyethylene glycol lipid for steric protection, were administered intravenously into tumor-bearing mice (C3H mice for SCCVII squamous cell carcinoma and BALB/c mice for CT26/WT colon carcinoma). With the exception of one lipid (B-THF-14), the lipids were well tolerated, and no other animal was lost due to systemic toxicity. The lipid which led to death was not found to be much more toxic in cell culture than the other boron lipids. All of the lipids that were well tolerated showed hemorrhage in both tumor models within a few hours after administration. The hemorrhage could be seen by in vivo magnetic resonance and histology, and was found to occur within a few hours. The degree of hemorrhage depended on the amount of boron administered and on the tumor model. The observed unwanted effect of the lipids precludes their use in boron neutron capture therapy.

Toxicity and phototoxicity in human ARPE-19 retinal pigment epithelium cells of dyes commonly used in retinal surgery:

Purpose: To compare, for the first time, systematically the toxicity and phototoxicity of dyes and dye combinations used in vitreoretinal surgery. The dyes were trypan blue, brilliant blue G, trypan blue + brilliant blue G, indocyanine green, bromophenol blue, bromophenol blue + brilliant blue G, and acid violet 17, in clinically used concentrations. Methods: Human ARPE retinal pigment epithelium cells were exposed to the dyes for 30 min. For phototoxicity, the cells were exposed for 15 min to high-intensity light from a light emitting diode source with an intensity similar to surgical conditions. Toxicity was assayed either directly after exposure to either dye alone or dye and light, or with a delay of 24 h. Results: None of the dyes or their combinations was toxic when cells were exposed to them at ambient light. Acid violet led to a reduction viability by 90% already immediately after light exposure. Bromophenol blue and its combination with brilliant blue G showed strong phototoxicity (reduction of viability by 83%) when assayed with delay. Indocyanine green with different agents to adjust osmolarity (balanced salt solution, glucose, and mannitol) was not found to be toxic. Conclusion: The strong immediate phototoxicity of acid violet reflects its clinical toxicity. Bromophenol blue might also be disadvantageous for patient outcome because of its delayed phototoxicity. The other dyes (trypan blue, brilliant blue g, and indocyanine green) were not found to be toxic neither with exposure to ambient light nor after exposure to light of intensities used in surgery.