Heat-induced hepatotoxicity accompanying hyperthermic liver perfusion was studied in the isolated, haemoglobin-free perfused rat liver. Trypan blue uptake, a sensitive indicator of cell death, was used to examine the relationship between the efflux of oxidized glutathione (oxidative stress), the appearance of cytosolic enzymes in the perfusate and cell death. Livers were perfused at 37, 42, 42.5 and 43°C. The efflux of total glutathione (GSH) and oxidized glutathione (GSSG) increased with time and temperature. Differences between temperature groups were significant for both parameters for 37 versus 42, 42.5 and 43°C (p < 0.05). Temperature-related differences in GSH levels appeared at 15 min for 37 versus 42 °C and in GSSG levels at 30 min for 37 versus 42 and 42.5°C. Biliary excretion of total GSH increased from 72 nmol at 37°C to 144 nmol at 42°C, 160 nmol at 42.5°C and 124 nmol at 43°C, which was significant for 37 versus 42 and 42.5°C (p < 0.05). The release of allantoin into the perfusate, a measure of purine catabolism and flux through xanthine oxidase, was increased at 42, 42.5 and 43°C compared to 37°C (p < 0.05). Liver injury was assessed by measuring the release of asportate aminotransferase (AST) and lactate dehydrogenase (LDH) and uptake of trypan blue after perfusion at each temperature. There was a pronounced release of LDH and AST into the perfusate after 60 min of perfusion at 42, 42.5 and 43°C, the levels of which were significantly different from the 37°C mean level. There was no uptake of trypan blue after 60 min perfusion at 37°C. Perfusion at 42, 42.5 and 43°C resulted in the uptake of trypan blue in the pericentral areas, but the dye uptake was significant (p < 0.05) compared to 37°C at 42.5 and 43°C only. These data show that heat-induced pericentral cell death is minimal after 60 min at 42-43°C, and that the biochemical processes which occurred during this period suggest 'oxidative stress' as a causative factor in hyperthermic hepatotoxicity. In addition, this liver toxicity is probably related to xanthine oxidase activity or the depletion of GSH as the initiating event which leads to lipid peroxidation and cellular damage.

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Oxidative stress contributes to inadequate in vitro maturation of porcine oocytes which leads to a failure of successful fertilization and embryo development. Therefore, the overall objective of this research was to characterize the mechanisms of oxidative stress in maturing oocytes and determine how oocytes alleviate oxidative stress with the assistance of supplemental antioxidants. A preliminary study was conducted to evaluate the effects of glutathione (GSH), N-acetyl-cysteine (NAC), and N-acetyl-cysteine-amide (NACA) supplemented to the maturation medium on intracellular GSH concentrations, nuclear maturation, fertilization success and embryo development. Antioxidants GSH, NAC and NACA (1.0 mM) were supplemented to the media during oocyte maturation. Intracellular GSH concentrations were recorded at 48 h of maturation and nuclear maturation and fertilization were analyzed 12 h after IVF. Embryo development was analyzed at 48 h and 144 h after IVF or intracytoplasmic sperm injection (ICSI). Supplementation of antioxidants had no effect on intracellular levels of GSH, nuclear maturation or fertilization traits. Blastocyst formation for NAC (35.0 ± 7.4%) and NACA (40.0 ± 7.4%) supplementation were higher (P < 0.05) than the control (20.0 ± 7.4%) and GSH supplemented (20 ± 7.4%) oocytes. The same pattern was seen for ICSI-derived embryos: blastocyst formation for NAC (22.0 ± 5.9%) and NACA (25.0 ± 4.6%) supplementation were higher (P < 0.05) than the un-supplemented (10.0 ± 6.0%) oocytes. There were no differences between NAC and NACA supplementation and there were no differences between the cleavage rates for any of the treatment groups. These results indicate that supplementing 1.0 mM of NAC or NACA to the oocyte maturation medium and the ICSI medium increased the percentage of viable embryos reaching the blastocyst stage of development, and could warrant further investigation. The next study was conducted to evaluate the effects of different concentrations of NAC supplemented to the maturation medium on embryo development. Comparisons of significant concentrations of NAC and NACA on embryo development were evaluated for nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0 mM) were supplemented to maturing oocytes and embryo development was analyzed at 48 h and 144 h post-fertilization. There were no differences between cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mM NAC (56.5 ± 9.2%) was significantly higher (P < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization when comparing 1.5 mM NAC and 1.5 mM NACA supplementation to the maturation media. There was no difference between cleavage rates of 1.5 NAC and 1.5 mM NACA supplementation to the maturation media. Blastocyst formation for 1.5 mM NAC (44.4 ± 4.7%) and 1.5 mM NACA (46.2 ± 3.4%) supplementation were significantly higher (P < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mM of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development and could be used during the oxidative stress experiments. In the final study, the mechanisms of oxidative stress in maturing oocytes were studied in addition to evaluating the effects of antioxidant supplementation to the media. This study focused on superoxide dismutase (SOD), GSH peroxidase, catalase and intracellular GSH concentrations with respect to DNA fragmentation evaluated using the single cell Comet assay. Results indicate that when SOD was inhibited, the GSH peroxide levels and length of DNA migration significantly increased (P < 0.05). Catalase levels significantly decreased (P < 0.05) and intracellular GSH remained unchanged. When GSH peroxidase was inhibited, the SOD levels and catalase levels significantly decreased (P < 0.05) but the intracellular GSH and DNA migration length significantly increased (P < 0.05). The supplementation of 1.5 mM NAC and 1.5 mM NACA had multiple effects on the enzyme levels. Specifically, supplementation of 1.5 mM NAC or 1.5 mM NACA significantly decreased (P < 0.05) the length of DNA migration when other enzymes were inhibited compared to no antioxidant supplementation. These results indicate that antioxidant supplementation may alleviate the free radicals associated with oxidative stress in the maturing porcine oocyte. In conclusion, supplementing the antioxidants NAC or NACA to the oocyte maturation media does not have negative effects on IVF or embryo culture. Supplementation of NACA increases the number of oocytes reaching the blastocyst stage of development. Glutathione, SOD, catalase, and GSH peroxidase are all required to be functional during oocyte development to alleviate oxidative stress on the oocyte. Antioxidants enhance the enzyme activity during oocyte maturation and may even contribute to protecting the oocyte when enzyme activity is impaired. / Ph. D.

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Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

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Patterns of physiological electrical activity in the central nervous system (CNS) cause longlasting changes in gene expression that promote neuronal survival. These changes can be mediated by signalling pathways activated by Ca2+ influx through synaptic N-methyl DAspartate receptors (NMDARs). Identification and study of these, and other neuroprotective signalling pathways of the CNS, is invaluable; as this may one day lead to therapeutic strategies against the deleterious effects of CNS injury or degeneration. The data presented in this thesis focuses on activity-dependent neuroprotection and how it interacts with other signalling pathways to protect against apoptotic and oxidative insults. A previously unobserved role of activity-dependent neuroprotection in mediating the effects of the neuropeptide PACAP is demonstrated. By promoting cAMP/PKA signalling PACAP triggers neuronal firing activity, which is essential for the neuroprotective effects mediated by PACAP. This firing activity cooperates with direct signalling by PKA in promoting longlasting CREB-mediated gene expression. The molecular events associated with PACAP mediated stimulation of CRE-dependent gene expression are presented. Investigation of the control of neuronal antioxidant defences by neuronal activity, both on its own and in cooperation with astrocyte-derived support, was also investigated. Neuronal activity is demonstrated to strongly increase the capacity of the antioxidant glutathione (GSH) system, through a program of coordinated transcriptional events. The utilisation, biosynthesis and recycling of GSH is enhanced in neurons, leading to increased resistance against oxidative insults. Since several GSH pathway enzyme genes are regulated by the transcription factor Nrf2, the ability of CDDO-F3, a small molecule activator of Nrf2, to mimic the effect of firing activity on neuronal GSH levels was examined. CDDO-F3 sustains neuronal GSH levels and confers neuroprotection against oxidative insult. These actions are dependent on the presence of astrocytes; whereas Nrf2 mediated regulation of GSH pathway genes is essentially inactive in neurons. Neuronal activity and activation of the astrocytic Nrf2 pathway can cooperate, maintaining neuronal GSH levels and protecting neurons against strong oxidative insults. Collectively this work expands our knowledge as to the molecular mechanisms of activity-dependent neuroprotection, and how such signals may synergise with other protective pathways to promote neuronal health.

Inhibition of disulfide bond formation in Escherichia coli implicates an intricate collaboration of proteins which comprise the glutathione and thioredoxin reducing pathways. Bioengineers have successfully engineered E. coli possessing mutated reducing pathways that promote, rather than inhibit, disulfide bond formation in the cytoplasm. The transcriptome of six such mutant E. coli strains have been characterized using Microarray technology. We find that all mutant strains, exhibit a unique response to oxidative stress, not observed in wild type. Statistical analyses revealed the expression of more than 200 genes that are affected by mutations within the reducing pathways. Significantly up-regulated biological processes include cysteine biosynthesis, histidine biosynthesis, NADH Dehydrogenase I biosynthesis, sugar catabolic processes, and activation of stress responses . The second part of this work describes the construction of an E. coli strain that promotes the complete conversion of glutathione into its seemingly dormant derivative, glutathionylspermidine. This engineered strain can be used in assays designed to evaluate the effectiveness of glutathionylspermidine as a substitute for glutathione and, hopefully, allude to its true metabolic function. / text

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