Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

Molecular, cellular, and physiological processes within an organism are set to occur at specific times throughout the day. The timing of these processes is under control of a biological clock. Nearly all organisms on Earth have biological clocks, ranging from unicellular bacteria and fungi to multicellular plants, insects, reptiles, fish, birds, and mammals. The biological clock is an endogenous time-keeping mechanism that generates the onset of many processes and coordinates the phases of processes over 24 hours. While the biological clock allows these organisms to maintain roughly 24-hour, or circadian, timing in daily processes, many organisms have the ability to set their clocks, or entrain them, to changes in light. In mammals, the suprachiasmatic nucleus (SCN) is the master biological clock that entrains daily physiological and behavioral rhythms to the appropriate times of day and night. The SCN is located in the hypothalamus and contains thousands of neurons and glia that function in coordinating system-level physiological rhythms that are entrained to environmental light cues. Many of these neurons and glia are individual circadian oscillators, and the cellular mechanisms that couple them into ensemble oscillations are emerging. Adenosine triphosphate (ATP) is a transmitter involved in local communication among astrocytes and between astrocytes and neurons. ATP released from astrocytes may play a role in SCN cellular communication and synchrony. Extracellular ATP accumulated rhythmically in the rat SCN in vivo, and ATP released from rat SCN astrocytes in vitro was rhythmic, with a periodicity near 24 hours. ATP released from mouse SCN astrocytes was circadian, and disruption of the molecular clock abolished rhythmic extracellular ATP accumulation. SCN astrocyte cultures with disrupted molecular clocks also had marked reductions in total ATP accumulation compared to SCN astrocyte cultures with functional biological clocks. Furthermore, ATP-induced calcium transients were rhythmic, and this rhythmic purinergic sensitivity was abolished in clock mutant astrocytes. Pharmacological blockade of purinergic signaling, with antagonists of both the P2X7 and P2Y1 receptors, led to a gradual reduction in the amplitude of coordinated ATP accumulation over three days. These purinergic receptor antagonists, as expected, led to a reduction in calcium responses of SCN astrocytes to ATP and led to a dampening of clock gene expression rhythms as determined by PER2::LUC bioluminescence reporting in SCN astrocytes. These data demonstrate that astrocytes of the mammalian SCN rhythmically release ATP and are rhythmically sensitive to ATP in a manner dependent on their intrinsic molecular clock. Ensemble rhythmicity of SCN astrocytes is, in turn, dependent on that rhythmic purinergic signaling via both P2X and P2Y classes of ATP receptors. These results are indicative of a functional role for ATP accumulation within the SCN, with astrocytes releasing ATP every 24 hours for continual signaling onto astrocytes and neurons to maintain daily coordinated synchrony of the clocks in these cells.

L'ATP és una molècula molt carregada elèctricament important per a l'obtenció de l'energia necessària per l'activitat cel·lular. A més a més, també actua com a molècula de senyalització cel·lular. Normalment, la secreció controlada d'ATP té lloc a través de l'exocitosi de grànuls o vesícules. Tot i això, en algunes cèl·lules, altres mecanismes poden controlar l'alliberació d'ATP. En aquesta tesi s'han estudiat dos d'aquests mecanismes: l'alliberació d'ATP a través de la proteïna CD39 i l'alliberació d'ATP a través de connexines.La CD39, o NTPDasa 1, és una proteïna que hidrolitza nucleòtids trifosfat i difosfat fins a nucleòtids monofosfat. Resultats anteriors del nostre laboratori suggerien que, aplicant polsos hiperpolaritzants en la membrana dels oòcits de Xenopus, l'ectoenzim NTPDasa1 podia esdevenir permeable per l'ATP. Continuant amb l'estudi d'aquesta proteïna, es va voler determinar també si estava relacionada amb l'alliberació d'ATP induïda per estrès hipertònic descrita en oòcits. Per portar a terme aquests estudis, era necessari aconseguir un oligonucleòtid antisentit contra la CD39 dels oòcits de Xenopus. Per tant, el nostre primer objectiu va ser sintetitzar i validar aquest oligonucleòtid, per tal de poder-lo utilitzar posteriorment a l'hora de fer els experiments. Mitjançant el disseny de dos oligonucleòtids antisentit per dita proteïna es va mesurar l'alliberació d'ATP en grups d'oòcits injectats amb els oligonucleòtids i en grups control, després de ser sotmesos a un xoc hiperosmòtic. Al mateix temps, per a comprovar l'efectivitat dels oligonucleòtids injectats, es va mesurar l'activitat ATPàsica en aquests oòcits. Es va observar com l'antisentit per la CD39 reduïa de manera significativa l'alliberació d'ATP i el corrent iònic després de 13 minuts d'exposició hipertònica.Els hemicanals, formats per connexines, és un altre dels possibles mecanismes de regulació de l'alliberació d'ATP. En les gap junctions, les connexines uneixen el citoplasma de dues cèl·lules adjacents establint un canal intercel·lular que permet el pas d'ions i molècules més petites d'1 KDa. Es va estudiar aquest mecanisme en oòcits de Xenopus, estudiant la Cx38, un tipus específic de connexina endògena dels oòcits, i la Cx32, expressada en els oòcits de manera heteròloga. En els experiments amb la Cx38, vam observar que solucions lliures d'ions divalents activaven un corrent d'entrada en els oòcits que era inhibit per octanol i àcid flufenàmic, dos inhibidors de les gap junctions. Aquest corrent sensible a calci, depenia de l'expressió de Cx38: disminuïa en oòcits injectats amb un oligonucleòtid antisentit per la Cx38 (ASCx38) i augmentava en oòcits que sobrexpressaven la Cx38. A més, l'activació dels hemicanals de Cx38 induïa l'alliberació d'ATP, la qual era inhibida per l'àcid flufenàmic, l'octanol i l'ASCx38 i augmentada en la sobrexpressió de Cx38. Aquests resultats suggerien que l'activació dels hemicanals era responsable de l'alliberació d'ATP en els oòcits de Xenopus.D'altra banda, vam investigar l'alliberació d'ATP a través dels hemicanals formats per Cx32 expressada en oòcits. Aquesta proteïna s'expressa en la majoria d'òrgans humans, però, particularment, mutacions de la Cx32 en les cèl·lules de Schwann produeixen la malaltia de Charcot-Marie-Tooth lligada al cromosoma X (CMTX). En oòcits que expressen la Cx32, vam combinar l'ús de tècniques de fixació de voltatge per registrar els corrents iònics generats pels hemicanals i la reacció bioluminiscent de la luciferina-luciferasa per mesurar l'alliberació d'ATP. Els hemicanals formats per la Cx32 eren estimulats mitjançant polsos despolaritzants generant un corrent iònic de sortida. Després de l'estimulació, en el període de repolarització, hi havia un corrent de cua que coincidia amb el moment d'alliberació d'ATP. L'àrea d'aquest corrent tenia una relació lineal amb la quantitat d'ATP alliberada. El conjunt de resultats d'aquesta tesi ens suggereixen que, tant les connexines com la CD39, participen en l'alliberació d'ATP a través de la membrana plasmàtica. / ATP is an electrically charged molecule required to obtain the energy necessary for cellular activity; in addition, it is an intercellular signaling molecule. Usually, the controlled secretion of ATP occurs through the exocytosis of granules and vesicles. However, in some cells, other mechanisms control ATP release. Two of these mechanisms have been studied in this thesis: ATP release through CD39 and ATP release through connexin hemichannels. CD39, or NTPDasa 1, is a protein that hydrolyze nucleotide triphosphates and nucleotide diphosphates to nucleotide monophosfates. To study its relationship with ATP release we have synthetized an antisense oligonucleotide against Xenopus oocyte CD39. Our results indicate that the antisense against CD39 significanty reduce ATP release and also the ionic current due to the activation of CD39 by hypertonic solution. It has been suggested that hemichannels, formed by connexins, may be an alternative pathway for ATP release. In gap junctions, connexins link the cytoplasm of two adjacent cells by establishing an intercellular channel. We have investigated the release of ATP from Xenopus oocytes through hemichannels formed by connexin 38 (Cx38), an endogenous, specific type of connexin, and by connexin32 (Cx32) heterologously expressed in Xenopus oocytes. In Xenopus oocytes, calcium-free solution reversibly activates an inward current that is inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it's decreased in oocytes injected with an antisense oligonucleotide against Cx38 (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of Cx38 also induce the release of ATP, which is inhibited by flufenamic acid, octanol and ASCx38 and increased by Cx38 overexpression. Connexin 32 is expressed in most of human organs but, particularly, Cx32 mutations present in Schwann cells produce X-linked Charcot-Marie-Tooth disease. In oocytes expressing Cx32, application of depolarizing pulses to positive potentials induce outward hemichannel currents that become inward during the repolarization phase. The release of ATP occurred during the repolarization period and the amount of ATP released correlate with the area of the tail current.

El ATP es una molécula ampliamente conocida por su papel en muchas funciones como la homeostasis celular, el mantenimiento de gradientes iónicos, el mantenimiento del pH en gránulos secretores, el almacenamiento energético, regulador de la interacción actina-miosina, etc. Además, el ATP puede actuar como molécula señalizadora a través de los receptores purinérgicos P2. De receptores P2 hay de dos tipos, los P2X, que son ionotrópicos, y los P2Y que son metabotrópicos. Los primeros son una familia de canales iónicos permeables a cationes que se abren cuando se les une el ATP. Los segundos son receptores acoplados a proteínas G, y la unión del ATP desencadena diferentes reacciones metabólicas. Ambos tipos de receptores se han relacionado con diferentes patologías.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 92-107.

Diss. Växjö : Växjö universitet, 2007.