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Induction of cytochrome P4503a in vivo and in vitroWilliams, J. Andrew January 1995 (has links)
1. The induction of CYP3A enzymes was investigated using a range of structurally unrelated drugs using in vivo and in vitro models. Hepatic microsomal testosterone 6(3-hydroxylation, anti-CYP3A immunoblot analysis, and molecular biology approaches were utilised in the investigation. 2. Using the rat as an in vivo model, potent induction of CYP3A enzymes was observed after administration of the synthetic glucocorticoid dexamethasone (at 150mg.kg.day for 4 days) and pregnenolone 16?-carbonitrile (at 150mg.kg-1.day-1 for four days). However, no induction was observed after administration of rifampicin (at 50 g.kg-1.day-1 for 4 days, a dose which causes potent induction in the rabbit). 3. Investigations into the effects of drug exposure on testosterone 6?- hydroxylation in cultured female rat hepatocytes revealed a positive in vivo/in vitro correlation. Cultured cells were treated with the same drugs (at 50 M concentration) for 72hrs. Dexamethasone was shown to be more potent than PCN, and rifampicin again had no effect. Dexamethasone-mediated induction of testosterone 6?- hydroxylation was dose-dependent and was shown to be maximal after 72hrs exposure. 4. The presence of the differentiating agent dimethylsulphoxide at 2% (v/v) in the cultiure medium enhanced CYP3A induction by the synthetic steroids by approximately 100% (p 0.05). 5. The potent glucococorticoid antagonist RU 486 induced testosterone 60- hydroxylation 5-fold when administered at 50mg.kg-1.day-1 for 4 days. Induction of the CYP3A protein was confirmed by immunoblot analysis of liver microsomes. Administration of RU 486 at 50mg.kg-1.day-1 did not antagonise the induction of testosterone 6?-hydroxylatiomn by dexamethasone at 150mg.kg-1.day-1. 6. Dexamethasone (0.1 to 10 M) -mediated induction of testosterone 6(3- hydroxylation in cultured rat hepatocytes was attenuated in the presence of RU 486. It is not known whether this was due to effects on CYP3 A gene expression or inhibition of enzyme mediated activity at the active site of the enzyme. 7. The lipid lowering drug SK F 98016 (150mg.kg-1.day-1) induced testosterone 6?-hydroxylation 10-fold when administered at 150mg.kg-1.day-1 for 4 days. This was confirmed by immunoblot analysis. Co-administration of RU 486 with SK F 98016 attenuated induction of CYP3A-mediated enzyme activity. The mechanism of induction of the CYP3A genes by SK F 98016 may therefore involve 'steroidal' compounds, the action of which is antagonised by RU 486. The dexamethasone- mediated increase in spectrally determined cytochrome P450 levels was also attenuated after co-administration with RU 486. As CYP3A induction was not affected by co-administration of dexamethasone with the anti-glucocorticoid RU 486, this result suggests that the glucocorticoid receptor may be involved in the induction of other P450 genes. 8. Treatment of rat hepatocytes with SK F 98016 (50 M) for 72 hours did not result in an increase in testosterone 6?-hydroxylation. In fact testosterone 6?-, 16?- and 17-oxidation activities were reduced to 50% of the activities measured in untreated hepatocytes. This pointed to some P450 inhibitory potential of SK F 98016. Investigation of the inhibitory potential of SK F 98016 on testosterone 60- hydroxylation in hepatic microsomes from PCN-treated rats showed an inhibitory effect with an IC50 of 50 M. The inhibitory effect seen in hepatocytes is similar to the effects of exposure to clotrimazole (50 .M) for 72 hours where testosterone metabolism at the 60 and 17 positions were inhibited by >90%. 9. To investigate whether the lack of inducing effect of SK F 98016 was due to the very high lipophilicity and extensive partitioning into the cultured hepatocyte, therefore resulting in a non-physiological state, cultured hepatocytes were exposed to the same drugs with albumin (from bovine serum, at the concentration present in human blood-36g/litre) in the medium in attempt to encourage an equilibrium of drug concentration between the medium and the inside of the hepatocyte. No significant induction of testosterone 60-hydroxylation was observed in the presence of albumin.
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Investigting the Cytoprotective Mechanisms of VIitamins B6 and B1 against Endogenous Toxin-induced Oxidative StressMehta, Rhea 10 January 2012 (has links)
Recent epidemiological evidence suggests that many chronic health disorders in the developed world are associated with endogenous toxins formed from the Western diet. The Western diet, which encompasses calorie dense foods, processed foods and increased quantities of red meat, can cause intracellular oxidative stress through increased formation of reactive oxygen species(ROS) and reactive carbonyl species (RCS). A number of micronutrients have been investigated for their protective capacity in in vitro and in vivo models of oxidative stress. This thesis investigated the cytotoxic targets of Fenton-mediated ROS and RCS and the subsequent protective mechanisms of vitamins B1 (thiamin) or B6 (pyridoxal, pyridoxamine or pyridoxine) in an isolated rat hepatocyte model. The approach was to use an “accelerated cytotoxicity mechanism screening” technique (ACMS) to develop an in vitro cell system that mimicked in vivo tissue cytotoxicity. Using this technique, we investigated the protective mechanisms of
vitamins B1 and/or B6 against the cytotoxic effects of two endogenous toxins associated with the Western diet: 1) RCS, as exemplified by glyoxal, a glucose/fructose autoxidation product and 2) biological ROS induced by exogenous iron. Firstly, we developed an understanding of the sequence of events contributing to glyoxal-induced oxidative stress, with a focus on protein
carbonylation. Next, we determined the mechanisms by which carbonyl scavenging drugs
(vitamin B6 included) protected against the intracellular targets of glyoxal-induced toxicity. Our results suggested that the agents used were cytoprotective by multiple mechanisms and glyoxal trapping was only observed when the agents were administered at concentrations equal to glyoxal. We also evaluated the protective capacity of vitamins B1 and B6 against iron-catalyzed
cytotoxicity and found that hepatocytes could be rescued from protein and DNA damage when vitamins B1 or B6 were added up to one hour after treatment with iron. The vitamins also varied in their primary mechanisms of protection. Our improved understanding of Western diet-derived endogenous toxins enabled us to identify and prioritize the specific inhibitory mechanisms of vitamins B1 or B6. The ability to delay, inhibit or reverse toxicity using multi-functional B1 or
B6 vitamins could prove useful as therapy to minimize oxidative stress in diet-induced chronic conditions.
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Investigting the Cytoprotective Mechanisms of VIitamins B6 and B1 against Endogenous Toxin-induced Oxidative StressMehta, Rhea 10 January 2012 (has links)
Recent epidemiological evidence suggests that many chronic health disorders in the developed world are associated with endogenous toxins formed from the Western diet. The Western diet, which encompasses calorie dense foods, processed foods and increased quantities of red meat, can cause intracellular oxidative stress through increased formation of reactive oxygen species(ROS) and reactive carbonyl species (RCS). A number of micronutrients have been investigated for their protective capacity in in vitro and in vivo models of oxidative stress. This thesis investigated the cytotoxic targets of Fenton-mediated ROS and RCS and the subsequent protective mechanisms of vitamins B1 (thiamin) or B6 (pyridoxal, pyridoxamine or pyridoxine) in an isolated rat hepatocyte model. The approach was to use an “accelerated cytotoxicity mechanism screening” technique (ACMS) to develop an in vitro cell system that mimicked in vivo tissue cytotoxicity. Using this technique, we investigated the protective mechanisms of
vitamins B1 and/or B6 against the cytotoxic effects of two endogenous toxins associated with the Western diet: 1) RCS, as exemplified by glyoxal, a glucose/fructose autoxidation product and 2) biological ROS induced by exogenous iron. Firstly, we developed an understanding of the sequence of events contributing to glyoxal-induced oxidative stress, with a focus on protein
carbonylation. Next, we determined the mechanisms by which carbonyl scavenging drugs
(vitamin B6 included) protected against the intracellular targets of glyoxal-induced toxicity. Our results suggested that the agents used were cytoprotective by multiple mechanisms and glyoxal trapping was only observed when the agents were administered at concentrations equal to glyoxal. We also evaluated the protective capacity of vitamins B1 and B6 against iron-catalyzed
cytotoxicity and found that hepatocytes could be rescued from protein and DNA damage when vitamins B1 or B6 were added up to one hour after treatment with iron. The vitamins also varied in their primary mechanisms of protection. Our improved understanding of Western diet-derived endogenous toxins enabled us to identify and prioritize the specific inhibitory mechanisms of vitamins B1 or B6. The ability to delay, inhibit or reverse toxicity using multi-functional B1 or
B6 vitamins could prove useful as therapy to minimize oxidative stress in diet-induced chronic conditions.
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From Mammalian Cell Culture to Aquatic Species: Deciphering the role of the Kynurenine-Tryptophan Ratio under Environmental Stress / Kynurenine-Tryptophan Ratio in Stress: Cells to SpeciesJamshed, Laiba January 2024 (has links)
Monitoring the impact of anthropogenic activities, particularly in industrial regions,
requires ecological screening tools and frameworks that provide a comprehensive
understanding of ecosystem responses to environmental changes. Biological indicators,
organisms like algae, insects, fish, and sentinel mammals, are critical for assessing
ecosystem health, particularly in areas of high industrial activity. The aim of this thesis was
to identify a cross-species biomarker that can assess organismal health and environmental
stress across various species, organs, and biological matrices.
A range of biological systems and signaling pathways related to xenobiotic metabolism,
energy homeostasis, immune responses, and stress adaptation were explored, leading to the
identification of the Tryptophan-Kynurenine Pathway, which consumes 60-90% of
tryptophan in vertebrates. Tryptophan and its metabolites play key roles in diverse
physiological processes, including cell growth and maintenance, immunity, disease states,
and the coordination of adaptive responses to environmental and dietary cues. This adaptive
response suggests that kynurenine-tryptophan ratio (KTR) may serve as a marker for
exposure to a variety of environmental stress conditions, including toxicants, nutrient
scarcity, predatory stress, and habitat loss—stressors that are prevalent in areas of high
industrial activity. In recent years, the KTR is increasingly recognized as a sensitive
biomarker in human diseases induced or exacerbated by stress; however, its role in
environmental exposure and wildlife health remains unexplored. This thesis explores the
question of whether KTR can be utilized as a cross-species biomarker for environmental
stress or environmental exposure to toxicants, particularly focusing on the Athabasca Oil
Sands Region (AOSR).
In vitro studies with mammalian hepatocytes exposed to polycyclic aromatic compounds
(PACs): benzo[a]pyrene (BaP), and a Bitumen Water Accommodated Fraction (BitWAF)
demonstrated that KTR increases were driven by elevated kynurenine levels, indicating
disruption of tryptophan metabolism via the aryl hydrocarbon receptor (AhR). Further
studies using acid extractable organics from Oil Sands Process-Affected Water (OSPW),
Naphthenic Acid Fraction Components (NAFCs) showed metabolic reprogramming,
including altered glucose and fatty acid uptake and mitochondrial dysfunction, mediated
through PPARα activation and upregulation of Tdo2, the enzyme responsible for
kynurenine production.
In vivo studies of longnose and white suckers from the AOSR were conducted to assess the
relationship between KTR and CYP1 enzyme activity (EROD). These studies revealed
species-specific responses, with an inverse correlation between KTR and EROD in
longnose suckers and a direct correlation in white suckers. These findings validate KTR as
a biomarker for environmental exposure in wildlife, with significant implications for
monitoring ecosystem health. Collectively, this work demonstrates the potential of KTR as
a novel biomarker for environmental toxicology, offering a valuable tool for assessing
organismal stress across species in response to environmental contaminants. / Thesis / Doctor of Philosophy (PhD) / Human activities, especially industrial operations, can significantly impact the
environment. To monitor these effects, scientists use various tools and organisms to assess
ecosystem health. This research introduces a new approach to measuring environmental
stress in wildlife by focusing on two key molecules: tryptophan and kynurenine. These
molecules are part of a conserved biological pathway that helps all organisms manage
stress, repair cells, adapt to their environment, and maintain overall health. Tryptophan, an
essential amino acid, is broken down into kynurenine, and the balance between them—
known as the kynurenine-tryptophan ratio (KTR)—can indicate the level of stress an
organism is experiencing.
This thesis investigates whether KTR can detect environmental stress caused by industrial
activity, particularly from petroleum-derived chemicals in the Athabasca Oil Sands Region
(AOSR). In laboratory experiments, mammalian liver cells were exposed to oil sands
compounds and complex mixtures from oil sands wastewater. These compounds changed
KTR, showing that the liver’s stress response was activated, and tryptophan metabolism
was disrupted. The study also found that these chemicals affected cellular energy use and
the way cells process fats and sugars. Furthermore, we examined fish species in the AOSR:
longnose and white suckers. Results showed that KTR varied depending on the species and
the location of exposure. In white suckers, KTR increased in response to stress, while in
longnose suckers, it decreased, indicating species-specific responses to environmental
changes.
Overall, our findings suggest that KTR could serve as a useful tool for measuring
environmental stress in different species and ecosystems, especially in areas affected by
anthropogenic or industrial activity. Understanding how KTR changes in response to
pollution can help scientists better monitor and protect wildlife and ecosystem health.
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