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.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with poor prognosis and limited treatment options. Since many patients present with metastatic disease and are thus ineligible for surgical resection, PDAC is almost ubiquitously fatal; new treatment options are therefore needed to combat this disease. A key hallmark of many cancers, including PDAC, is metabolic reprogramming and a shift towards a high glycolytic rate, known as the Warburg effect. This allows cancer cells to generate ATP in the face of hypoxia and to meet the increased metabolic requirements associated with rapid proliferation. We hypothesised that this shift towards glycolytic metabolism has important implications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining low [Ca2+]i and thus cell survival, is dependent on ATP to extrude cytosolic Ca2+. The relative contributions of mitochondrial vs glycolytic ATP in fuelling the PMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were therefore assessed. Moreover, the effects of numerous mechanistically distinct metabolic inhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated. Treatment with glycolytic inhibitors induced significant ATP depletion, PMCA inhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells, while mitochondrial inhibitors had no effect. Subsequently, these experiments were repeated on PDAC cells cultured in media formulated to "switch" their highly glycolytic phenotype back to one more reliant on mitochondrial metabolism. Culture in nominal glucose-free media supplemented with either galactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch in metabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis were significantly decreased, and in the case of cells cultured in KIC, oxidative phosphorylation rate was preserved (assessed using Seahorse XF technology). Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolytic inhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and [Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These data demonstrate that in PDAC cells exhibiting a high rate of glycolysis, glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thus is critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all found to associate with the plasma membrane in MIA PaCa-2 cells, possibly in a tyrosine phosphorylation-dependent manner. To investigate whether the dynamic membrane-association of glycolytic enzymes provides a privileged supply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitors was assessed on PMCA activity. However, while these inhibited PMCA activity, this occurred without accompanying global ATP depletion. These data indicate that glycolytic ATP is critical for the regulation of [Ca2+]i by the PMCA in PDAC, and that the glycolytic regulation of the PMCA may be an important therapeutic locus. However, further research is required to determine whether membrane-bound glycolytic enzymes regulate its activity.