An investigation into the effects of inorganic toxins and tryptophan metabolites on the forebrain cholinergic system and the pineal gland of the rat
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997
Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologies
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
Fungal and substrate-associated factors affecting lignocellulolytic mushroom cultivation on wood sources available in South African [i.e. Africa]
- Authors: Da Serra, Maria Fatima
- Date: 1997
- Subjects: Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4020 , http://hdl.handle.net/10962/d1004080 , Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Description: Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.
- Full Text:
- Date Issued: 1997
- Authors: Da Serra, Maria Fatima
- Date: 1997
- Subjects: Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4020 , http://hdl.handle.net/10962/d1004080 , Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Description: Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.
- Full Text:
- Date Issued: 1997
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:
- Date Issued: 1997
- 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:
- Date Issued: 1997
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:
- Date Issued: 1997
- 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:
- Date Issued: 1997
Regulation of tryptophan-2,3-dioxygenase and pineal indoleamines by selected tryptophan derivatives and antidepressants
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
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:
- Date Issued: 1997
- 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:
- Date Issued: 1997
- «
- ‹
- 1
- ›
- »