Terence P. N. Talorete

Induction of growth inhibition and differentiation of human leukemia HL-60 cells by a tunisian gerboui olive leaf extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.

Inhibitory Effect of Fulvic Acid Extracted from Canadian Sphagnum Peat on Chemical Mediator Release by RBL-2H3 and KU812 Cells

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and 13C-nuclear magnetic resonance (13C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001–10.0 μg/ml of CP-FA and determined the β-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca2+] i level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001–10.0 μg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the β-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca2+] i level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.

Stress response of mammalian cells incubated with landfill leachate

Environmental contamination from landfill leachate has been linked to disturbances in human health. Often, however, only global parameters, such as dissolved organic content, chemical oxygen demand, and 5-d biological oxygen demand, are used to evaluate wastewater quality. In the present study, we determined leachate cytotoxicity and stress response of leachate-treated mammalian cells using in vitro bioassays and other molecular techniques. The modified E-screen assay using human breast cancer MCF-7 cells was used to determine the estrogenic potential and/or cytotoxicity of water samples from two solid-waste landfills in Tunisia. The cytotoxicity mechanism of the leachate was determined by DNA fragmentation and lactate dehydrogenase assays. The stress response of heat shock protein (HSP) 47-positive Chinese hamster ovary cells treated with leachate also was determined. Proteomics analyses and real-time reverse transcription-polymerase chain reaction were used to determine and confirm the enhanced expressions of certain stress-related proteins. Results showed that the leachate samples generally did not have estrogenic activity. Instead, they were cytotoxic toward MCF-7 cells, and the cytotoxicity was by necrosis during the early stages of incubation. Leachate also enhanced the expression of HSP and various stress-related proteins, such as heterogeneous nuclear ribonucleoprotein E1, phosphoglycerate mutase, and nuclear matrix protein 200, in MCF-7 cells. These can be considered as survival mechanisms against leachate-induced cytotoxicity.

Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor

Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 μM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 β-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.

Alkylphenolic compounds and their effect on the injury rate, survival and acetylcholinesterase activity of the rat neuronal cell line PC12

Most studies on hormonally active agents or endocrine disruptors have been limited to polychlorinated biphenyls and dioxins. In this paper, we report results of in vitro studies on the effects of alkylphenolic compounds, namely, n-pentylphenol, n-hexylphenol, n-heptylphenol, n-octylphenol, and n-nonylphenol, on the injury rate, survival, and acetylcholinesterase activity of the rat pheochromocytoma cell line PC12. Results using the lactate dehydrogenase cytotoxicity assay to determine cell injury rate reveal that the alkylphenols mentioned did not induce cell necrosis beyond 30%, even at concentrations as high as 300 μM in a 15-min incubation period. Exposing the cells to alkylphenols for 4 hr and testing for DNA fragmentation showed that nonylphenol and octylphenol also did not induce apoptosis, even at concentrations as high as 500 and 100 μM, respectively. However, incubating the cells with the alkylphenols for 24 hr significantly inhibited acetylcholinesterase activity at concentrations as low as 0.8 μM, with n-octylphenol showing the most significant effect Since it is believed that human exposure to nonylphenol from drinking water is around 0.7 μg day-1 and that these compounds can accumulate in adipose tissue, this finding may implicate alkylphenols in neurological and behavioral disturbances in both animals and humans.

Protein Expression by Human Intestinal Epithelial Cells in Response to Wastewater Constituents

The effects of wastewater constituents, nonylphenol and lipopolysaccharide (LPS) on the intestinal epithelial Caco-2 cells, a human intestinal epithelial cell line derived from a human colon carcinoma, were evaluated. Results of proteomics analysis showed that galectin-3, a galactose-specific lectin, glutathione S-transferase A2 subunit and peroxiredoxin-1 were overexpressed by nonylphenol-treated cells while Hsp90b was overexpressed by LPS-treated cells. From the results of proteomics analysis of human intestinal Caco-2 cells treated with wastewater constituents, we found that the overexpression of specific proteins can be used as biomarkers for the risk assessment of water and wastewater.

Leaf Extracts from Tunisian Olive Cultivars Induce Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells

Recently a lot of interest is given to the beneficial actions and healthy effects of olive leaves. Thus, this work is an investigation of the antiproliferative activities and other effects of olive leaf extracts (OLEs) from seven principal Tunisian olive varieties on the human promyelocytic cell line HL60.Three in vitro assays were performed for this study: MTT assay to evaluate the cytotoxicity effect of OLE on HL60 cells, double staining esterase assay and nitroblue tetrazolium reduction assay for assessment of cell differentiation. We find that all OLEs exert a cytotoxic effect on HL60 cells which is confirmed by some morphological changes. Despite their apoptotic effect, ethanol extract of Tunisian olive leaves induced differentiation of HL60 cells into granulocytes. The differentiation-inducing activity of G extract was more remarkable than those of the other extracts (NBT reduction greater than 90%). To identify the bioactive components of OLEs the cytotoxicity of oleuropein and luteolin, two major phenols of olive leaves, were tested, nevertheless, no statistically significant difference was observed versus control. These results suggest that OLEs probably contain unknown bioactive components that are present in relatively high amounts in the G sample and are responsible for the cell differentiation, or possibly, cell differentiation is the result of synergism between compounds present in OLEs.

Capsaicin-enhanced ribosomal protein P2 expression and recovery of tight junction permeability in human intestinal CACO-2 cells

On the basis of transepithelial electrical resistance measurements, we momentary decrease in tight-junction (TJ) permeability followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein of relative molecular mass of 14 kDa was found to be expressed higher in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyse revealed that the protein expressed significantly by capsaicin treatment was the ribosomal protein P2 was and its cDNA sequence was identical to that found in the human genome database. An increase in amount of cellular filamentous actin (F- actin) was shown after 8 h of incubation with capsaicin. It is reported that ribosomal protein P2 can activate elongation factor 2, which stabilizes F- actin filaments, and that the deploymerization of F-actin was associated with the decrement in TJ permeability. Consequently, these results suggest that ribosomal protein P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal Caco-2 cells.

Phytoestrogens Enhance the Acetylcholinesterase Activity of the Rat Pheochromocytoma Cell Line PC12 by Binding to the Estrogen Receptor

Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 µM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. On the other hand, incubating the PC12 cells in increasing concentrations of 17 s-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activity in PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.

Two-Dimensional Electrophoresis of Proteins from Human Intestinal Caco-2 Cells Exposed to the Known Xenoestrogen Nonylphenol

As plastics continue to replace traditional building materials worldwide, the production of alkylphenolic compounds, esp. nonylphenol, is expected to increase at a steady pace (Lorenc et al., 1992). Nonylphenol is used to harden PVC plastic and is also a stabilizer of polystyrene containers that are used to hold beverages such as milk and juices. This compound is also a degradation product of detergents and household cleaners, and as such, is found in wastewater effluents. Human exposure to alkylphenols, esp. nonylphenol from drinking water, is around 0.7 μg day−1 (Weeks et al., 1996).