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Systems Biology of Cadmium: Effects on the Redox Proteome and Metabolome
Proteomics and Genomics 348 Utility of Toxicogenomics in Risk Assessment of Human Carcinogens: A Case Study on Benzo(a)pyrene 1
Nikolai L. Chepelev , Ivy D. Moffat1, Sarah Labib1, Julie Bourdon1, Byron Kuo1, Julie Buick1, France Lemieux1, Amal Malik1, Sabina Halappanavar1, and Carole L. Yauk1 1 Health Canada, Canada Regulatory agencies worldwide suffer from a backlog of potential chemical carcinogens in need of human health risk assessment. The current gold standard for cancer risk assessment is the 2year rodent cancer bioassay, which is extremely expensive, timeconsuming and uses large numbers of animals (up to 800 per bioassay). Toxicogenomics (the application of “omics” technologies to toxicology) has the potential to reduce the time and cost of cancer risk assessment by enabling more rapid identification of chemical modes of action, thus reducing the number of toxicity tests required, animal use, and exposure durations. However, the precise means by which toxicogenomics can be used in risk assessment is unclear. To address this, we evaluated the utility of toxicogenomics in the risk assessment of benzo(a)pyrene (BaP), a well-established rodent and human TM carcinogen. Adult B6C3F1 and Muta mice (which contain a transgenic mutation reporter gene) were exposed to carcinogenic doses of BaP via oral gavage for 3 or 28 days. Lung and liver gene expression was examined in conjunction with transgene mutant frequency and BaP-DNA adducts. Benchmark doses were calculated for individual gene expression changes and biological pathways in parallel with apical endpoints to determine the doses required to elicit significant responses. Gene expression profiles and dose-response modelling supported the known genotoxic mode of action of BaP, revealing that it triggers: i) activation of AhR signalling followed by ii) Nrf2-mediated oxidative stress response, and iii) DNA damage, repair and apoptosis. In the absence of apical data (DNA damage, mutations, and tumour incidence), toxicogenomics endpoints revealed the known mode of action of BaP using a short exposure period (< 4 weeks) and relatively few animals (70). Thus, we demonstrate the potential application of toxicogenomics to support and improve cancer risk assessment, assist in the prioritization and preliminary evaluation of potential chemical hazards, and enhance our knowledge of the mode of action of chemical carcinogens.
doi:10.1016/j.freeradbiomed.2012.10.387
349 Systems Biology of Cadmium: Effects on the Redox Proteome and Metabolome 1
Young-Mi Go , Michael Orr1, and Dean P. Jones1 1 Emory University Low-level Cadmium (Cd) exposure appears to contribute to severe human diseases affecting liver, kidney, lung and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Extensive cell biology and cell death studies implicate disruption of thiol systems and mitochondrial dysfunction as central components of toxicologic mechanisms; however, gene expression studies also show diverse and complex gene
expression responses, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated global effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria. Cd was administered to mice by intraperitoneal injection (10 mg/kg) and liver was studied at 6 h. No acute intoxication or organ pathology was detected at this time point, but the results showed that plasma and liver redox states of GSH/GSSG were significantly oxidized and protein thiol content was decreased. Consequently, acute Cd exposure induces global oxidation of thiol systems. To identify redoxsensitive liver proteins and gain insight into associated toxicological mechanism and functions, we analyzed redox states of liver mitochondrial proteins by redox proteomics using ICAT (isotope coded affinity tag)-combined mass spectrometry method. Redox proteomics provided data for 505 proteins with 933 cysteine-containing peptides. Of these, 23% (171 proteins with 217 peptides) were more oxidized more than 1.5 fold relative to controls. Bioinformatics analysis using ingenuity pathway analysis and GeneGo software showed that oxidized proteins function in pathways and network processes critical for mitochondrial function, e.g., energy production, lipid metabolism, and small molecule biochemistry. High-resolution metabolomics similarly showed global effects of Cd on liver metabolism. Principal component analysis showed clear separation according to Cd treatment, and metabolic pathways accounting for this separation overlapped with those identified by redox proteomics. Together, these results suggest that mitochondrial protein redox and metabolites are critical factors in Cd-induced hepatotoxicity. The results indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.
doi:10.1016/j.freeradbiomed.2012.10.388
350 Role of JNK-Mediated Phosphorylation in Mitochondrial Dysfunction and Liver Injury 1
Sehwan Jang , Yuan Gao2, Mohamed A Abdelmegeed1, Atrayee Banerjee1, Li-Rong Yu2, and B J Song1 1 National Institute on Alcohol Abuse and Alcoholism (NIAAA), 2 NIH, Food and Drug Administration (FDA) Background: Despite the well-established role of JNK in cell-death pathway, the target proteins of JNK and their roles are poorly understood. Thus we aimed to study the role of JNK and its target proteins in CCl4-mediated acute liver damage. Methods: Male mice were injected with a single dose of CCl4 (50 mg/kg, ip, n=5/group) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other activities were measured in CCl4-exposed mice without or with the JNK inhibitor SU3327, which blocked the JNK. Phosphorylated mitochondrial proteins from CCl4-exposed liver tissues were purified by metal-affinity column and their identities determined by mass-spectrometry analysis. Results: JNK was rapidly activated within 1 h accompanied by markedly increased phosphorylation of many mitochondrial proteins between 1- and 8-h while liver damage was maximal at 24-h post-CCl4 injection. Prior treatment with the JNK inhibitor SU3327 significantly reduced CCl4-mediated liver damage and phosphorylation of mitochondrial proteins in CCl4-exposed mice, suggesting a causal relationship between phosphorylation and liver damage. Mass-spectral data verified that more than 100 mitochondrial proteins including aldehyde dehydrogenase, NADHubiquinone oxidoreductase, Į-ketoglutarate dehydrogenase, etc, were phosphorylated in CCl4-exposed mice. Immunoprecipitation followed by immunoblot with the specific anti-phospho-Ser-Pro antibody revealed that the proteins listed above were
SFRBM 2012
S143
Nikolai L. Chepelev , Ivy D. Moffat1, Sarah Labib1, Julie Bourdon1, Byron Kuo1, Julie Buick1, France Lemieux1, Amal Malik1, Sabina Halappanavar1, and Carole L. Yauk1 1 Health Canada, Canada Regulatory agencies worldwide suffer from a backlog of potential chemical carcinogens in need of human health risk assessment. The current gold standard for cancer risk assessment is the 2year rodent cancer bioassay, which is extremely expensive, timeconsuming and uses large numbers of animals (up to 800 per bioassay). Toxicogenomics (the application of “omics” technologies to toxicology) has the potential to reduce the time and cost of cancer risk assessment by enabling more rapid identification of chemical modes of action, thus reducing the number of toxicity tests required, animal use, and exposure durations. However, the precise means by which toxicogenomics can be used in risk assessment is unclear. To address this, we evaluated the utility of toxicogenomics in the risk assessment of benzo(a)pyrene (BaP), a well-established rodent and human TM carcinogen. Adult B6C3F1 and Muta mice (which contain a transgenic mutation reporter gene) were exposed to carcinogenic doses of BaP via oral gavage for 3 or 28 days. Lung and liver gene expression was examined in conjunction with transgene mutant frequency and BaP-DNA adducts. Benchmark doses were calculated for individual gene expression changes and biological pathways in parallel with apical endpoints to determine the doses required to elicit significant responses. Gene expression profiles and dose-response modelling supported the known genotoxic mode of action of BaP, revealing that it triggers: i) activation of AhR signalling followed by ii) Nrf2-mediated oxidative stress response, and iii) DNA damage, repair and apoptosis. In the absence of apical data (DNA damage, mutations, and tumour incidence), toxicogenomics endpoints revealed the known mode of action of BaP using a short exposure period (< 4 weeks) and relatively few animals (70). Thus, we demonstrate the potential application of toxicogenomics to support and improve cancer risk assessment, assist in the prioritization and preliminary evaluation of potential chemical hazards, and enhance our knowledge of the mode of action of chemical carcinogens.
doi:10.1016/j.freeradbiomed.2012.10.387
349 Systems Biology of Cadmium: Effects on the Redox Proteome and Metabolome 1
Young-Mi Go , Michael Orr1, and Dean P. Jones1 1 Emory University Low-level Cadmium (Cd) exposure appears to contribute to severe human diseases affecting liver, kidney, lung and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Extensive cell biology and cell death studies implicate disruption of thiol systems and mitochondrial dysfunction as central components of toxicologic mechanisms; however, gene expression studies also show diverse and complex gene
expression responses, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated global effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria. Cd was administered to mice by intraperitoneal injection (10 mg/kg) and liver was studied at 6 h. No acute intoxication or organ pathology was detected at this time point, but the results showed that plasma and liver redox states of GSH/GSSG were significantly oxidized and protein thiol content was decreased. Consequently, acute Cd exposure induces global oxidation of thiol systems. To identify redoxsensitive liver proteins and gain insight into associated toxicological mechanism and functions, we analyzed redox states of liver mitochondrial proteins by redox proteomics using ICAT (isotope coded affinity tag)-combined mass spectrometry method. Redox proteomics provided data for 505 proteins with 933 cysteine-containing peptides. Of these, 23% (171 proteins with 217 peptides) were more oxidized more than 1.5 fold relative to controls. Bioinformatics analysis using ingenuity pathway analysis and GeneGo software showed that oxidized proteins function in pathways and network processes critical for mitochondrial function, e.g., energy production, lipid metabolism, and small molecule biochemistry. High-resolution metabolomics similarly showed global effects of Cd on liver metabolism. Principal component analysis showed clear separation according to Cd treatment, and metabolic pathways accounting for this separation overlapped with those identified by redox proteomics. Together, these results suggest that mitochondrial protein redox and metabolites are critical factors in Cd-induced hepatotoxicity. The results indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.
doi:10.1016/j.freeradbiomed.2012.10.388
350 Role of JNK-Mediated Phosphorylation in Mitochondrial Dysfunction and Liver Injury 1
Sehwan Jang , Yuan Gao2, Mohamed A Abdelmegeed1, Atrayee Banerjee1, Li-Rong Yu2, and B J Song1 1 National Institute on Alcohol Abuse and Alcoholism (NIAAA), 2 NIH, Food and Drug Administration (FDA) Background: Despite the well-established role of JNK in cell-death pathway, the target proteins of JNK and their roles are poorly understood. Thus we aimed to study the role of JNK and its target proteins in CCl4-mediated acute liver damage. Methods: Male mice were injected with a single dose of CCl4 (50 mg/kg, ip, n=5/group) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other activities were measured in CCl4-exposed mice without or with the JNK inhibitor SU3327, which blocked the JNK. Phosphorylated mitochondrial proteins from CCl4-exposed liver tissues were purified by metal-affinity column and their identities determined by mass-spectrometry analysis. Results: JNK was rapidly activated within 1 h accompanied by markedly increased phosphorylation of many mitochondrial proteins between 1- and 8-h while liver damage was maximal at 24-h post-CCl4 injection. Prior treatment with the JNK inhibitor SU3327 significantly reduced CCl4-mediated liver damage and phosphorylation of mitochondrial proteins in CCl4-exposed mice, suggesting a causal relationship between phosphorylation and liver damage. Mass-spectral data verified that more than 100 mitochondrial proteins including aldehyde dehydrogenase, NADHubiquinone oxidoreductase, Į-ketoglutarate dehydrogenase, etc, were phosphorylated in CCl4-exposed mice. Immunoprecipitation followed by immunoblot with the specific anti-phospho-Ser-Pro antibody revealed that the proteins listed above were
SFRBM 2012
S143