Bioaccumulation of heavy metals by the yeast S. cerevisiae and the bioremediation of industrial waste water
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
Metabolic responses in melanoma cells to combined nutrient supplementation
- Authors: Midgley, Nicola-Ann
- Date: 1997
- Subjects: Melanoma Tumors -- Growth
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4036 , http://hdl.handle.net/10962/d1004096
- Description: This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.
- Full Text:
- Authors: Midgley, Nicola-Ann
- Date: 1997
- Subjects: Melanoma Tumors -- Growth
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4036 , http://hdl.handle.net/10962/d1004096
- Description: This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.
- Full Text:
Vitamin E supplementation and secondary metabolites interactions and effects on melanoma growth
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- «
- ‹
- 1
- ›
- »