Unknown,Transcriptomics,Genomics,Proteomics

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Critical Role of Sphingolipid Pathway Components in Murine Radiation-Induced Lung Injury: Protection by Sphingosine-1-Phosphate Analogues

ABSTRACT: Clinically significant radiation-induced lung injury (RILI) is associated with significant morbidity and mortality and a common toxicity in patients administered thoracic radiotherapy. While the molecular etiology of RILI is poorly understood, we previously characterized a murine model of RILI in which alterations in lung endothelial barrier integrity surfaced as a potentially important pathobiologic event. In these studies, inhibition of HMG-CoA reductase activity (simvastatin) reduced murine RILI-associated lung inflammation and vascular leak and attenuated radiation-induced dysregulation of sphingolipid metabolic pathway genes identified by genome-wide lung gene expression profiling. In the present study, we test the hypothesis that sphingolipid signaling components serve as important modulators of RILI pathobiology and novel therapeutic targets. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to cumulative sphingosine-1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage (BAL) fluid and lung tissue following 25 Gy exposure (6 weeks). Moreover, genetically-engineered mice with either targeted deletion of SphK1 (SphK1-/-), or with reduced expression of selective members of the S1P receptor family (S1PR1+/-, S1PR2-/-, S1PR3-/-,), exhibited marked susceptibility to RILI-mediated lung inflammation. Finally, we assessed the efficacy of three potent vascular barrier-protective S1P analogues FTY720 (FTY), fTysiponate (fTyS) and SEW-2871 (SEW) in attenuating indices of RILI. The phosphonate analogue, fTyS, and to a lesser degree SEW, exhibited significant attenuation of RILI and RILI-induced gene dysregulation compared to control RILI-challenged mice (6 weeks). In contrast, FTY failed to significantly alter physiologic or genomic changes compared to RILI-challenged controls. Together, these results support the targeting of sphingolipid components as a novel and effective therapeutic strategy in RILI. Four mice were treated with PBS as a control. Three mice were treated with (S)-FTY-phosphonate (0.1mg/kg) as a drug control. Three mice were treated with SEW-2871 (0.1mg/kg) as a drug control. Three mice were treated with FTY720 (0.1mg/kg) as a drug control. Three mice were treated with administered radiation (25 Gy) alone. Three mice were treated with both administered radiation (25 Gy) and (S)-FTY-phosphonate (0.1mg/kg). Three mice were treated with both administered radiation (25 Gy) and SEW-2871 (0.1mg/kg). Three mice were treated with both administered radiation (25 Gy) and FTY720 (0.1mg/kg).

ORGANISM(S): Mus musculus

SUBMITTER: Tong Zhou

PROVIDER: E-GEOD-25295 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Clinically significant radiation-induced lung injury (RILI) is associated with significant morbidity and mortality and a common toxicity in patients administered thoracic radiotherapy. While the molecular etiology of RILI is poorly understood, we previously characterized a murine model of RILI in which alterations in lung endothelial barrier integrity surfaced as a potentially important pathobiologic event. In these studies, inhibition of HMG-CoA reductase activity (simvastatin) reduced murine RILI-associated lung inflammation and vascular leak and attenuated radiation-induced dysregulation of sphingolipid metabolic pathway genes identified by genome-wide lung gene expression profiling. In the present study, we test the hypothesis that sphingolipid signaling components serve as important modulators of RILI pathobiology and novel therapeutic targets. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to cumulative sphingosine-1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage (BAL) fluid and lung tissue following 25 Gy exposure (6 weeks). Moreover, genetically-engineered mice with either targeted deletion of SphK1 (SphK1-/-), or with reduced expression of selective members of the S1P receptor family (S1PR1+/-, S1PR2-/-, S1PR3-/-,), exhibited marked susceptibility to RILI-mediated lung inflammation. Finally, we assessed the efficacy of three potent vascular barrier-protective S1P analogues FTY720 (FTY), fTysiponate (fTyS) and SEW-2871 (SEW) in attenuating indices of RILI. The phosphonate analogue, fTyS, and to a lesser degree SEW, exhibited significant attenuation of RILI and RILI-induced gene dysregulation compared to control RILI-challenged mice (6 weeks). In contrast, FTY failed to significantly alter physiologic or genomic changes compared to RILI-challenged controls. Together, these results support the targeting of sphingolipid components as a novel and effective therapeutic strategy in RILI.

Project description:Background: Microvascular injury and increased vascular leakage are prominent features of the radiation-induced lung injury (RILI) which follows cancerâ??associated thoracic irradiation. The mechanisms of RILI are incompletely understood and therapeutic strategies to limit RILI are currently unavailable. We established a murine model of radiation pneumonitis in order to assess mechanism-based therapies for RILI-induced inflammation and vascular barrier dysfunction. Based on prior studies, we investigated the therapeutic potential of simvastatin as a vascular barrier protective agent in RILI. Methods: C57BL6/J mice receiving single dose exposure to 18, 20, 22, or 25 Gy, (n=10/group) were temporally assessed (4-12 weeks) for cellular and biochemical indices of injury present in both bronchoalveolar lavage (BAL) and lung tissues (cytokines, tyrosine nitrosylated proteins, leukocytes, extravasation of Evans blue dye or EBD, BAL albumin, histology). In specific experiments, irradiated mice (25Gy) received simvastatin (10 mg/kg) via intraperitoneal injection three times a week (pre and post irradiation) for 2- 6 weeks post irradiation. Results. Acute RILI evolved in a dose- and time-dependent fashion. Mice irradiated with 25Gy exhibited modest increases in BAL leukocytes but significant increases in BAL IL-6 (p=0.03) and TNF-a (p=0.01) at 4 weeks compared to controls. Increases in BAL nitrotyrosine content peaked at 6 weeks (p=0.03) and was accompanied by marked nitrotyrosine immunostaining in lung tissues. Indices of increase lung vascular permeability such as EBD extravasation, BAL protein and BAL albumin significantly increased over time beginning at 6 weeks (p>0.002 all) with histological evidence of severe edema formation and airway inflammation. Simvastatin- treated irradiated mice were noted to exhibit marked attenuation of vascular leak with significantly decreased BAL protein (p=0.01) and inflammatory cell infiltration (50% reduction). Conclusion: Simvastatin is a potentially important therapeutic strategy to limit RILI and may influence radiation associated morbidity and mortality. We used microarrays to detail the global programme of gene expression induced by radiation in Wild type and the protection of SIMVA Experiment Overall Design: animals were treated by Vehical, Radiation (25Gy), SIMVA(10mg/kg), or both.

Project description:The primary goal of toxicology and safety testing is to identify agents that have the potential to cause adverse effects in humans. Unfortunately, many of these tests have not changed significantly in the past 30 years and most are inefficient, costly, and rely heavily on the use of animals. The rodent cancer bioassay is one of these safety tests and was originally established as a screen to identify potential carcinogens that would be further analyzed in human epidemiological studies. Today, the rodent cancer bioassay has evolved into the primary means to determine the carcinogenic potential of a chemical and generate quantitative information on dose-response behavior in chemical risk assessments. Due to the resource-intensive nature of these studies, each bioassay costs $2 to $4 million and takes over three years to complete. Over the past 30 years, only 1,468 chemicals have been tested in a rodent cancer bioassay. By comparison, approximately 9,000 chemicals are used by industry in quantities greater than 10,000 lbs and nearly 90,000 chemicals have been inventoried by the U.S. Environmental Protection Agency as part of the Toxic Substances Control Act. Given the disparity between the number of chemicals tested in a rodent cancer bioassay and the number of chemicals used by industry, a more efficient and economical system of identifying chemical carcinogens needs to be developed. Experiment Overall Design: Five-week old female B6C3F1 mice were exposed for 13 weeks in the following dose groups: 1) 1,5-Naphthalenediamine, CAS No. 2243-62-1, feed, 2000 ppm, positive lung carcinogen; 2) 2,3-Benzofuran, CAS No. 271-89-6, gavage, 240 mg/kg, positive lung carcinogen; 3) 2,2-Bis(bromomethyl)-1,3-propanediol, CAS No. 3296-90-0, feed, 1250 ppm, positive lung carcinogen; 4) N-Methylolacrylamide, CAS No. 924-42-5, gavage (water), 50 mg/kg, positive lung carcinogen; 5) 1,2-Dibromoethane, CAS No. 106-93-4, gavage (corn oil), 62 mg/kg, positive lung carcinogen; 6) Coumarin, CAS No. 91-64-5, gavage (corn oil), 200 mg/kg, positive lung carcinogen; 7) Benzene, CAS No. 71-43-2, gavage (corn oil), 100 mg/kg, positive lung carcinogen; 8) N-(1-naphthyl)ethylenediamine dihydrochloride, CAS No. 1465-25-4, feed, 2000 ppm, negative lung carcinogen; 9) Pentachloronitrobenzene, CAS No. 82-68-8, feed, 8187 ppm, negative lung carcinogen; 10) 4-Nitroanthranilic acid, CAS No. 619-17-0, feed, 10000 ppm, negative lung carcinogen; 11) 2-Chloromethylpyridine hydrochloride, CAS No. 6959-47-3, gavage (water), 250 mg/kg, negative lung carcinogen; 12) Diazinon, CAS No. 333-41-5, feed, 200 ppm, negative lung carcinogen; 13) Malathion, CAS No. 121-75-5, feed, 16000 ppm, negative lung carcinogen; 14) Corn oil control, gavage; 15) Water control, gavage; 16) Rodent diet control, feed. Feed animals were exposed 7 days/week and gavage animals were exposed 5 days/week (5 ml/kg). After 13 weeks, animals were euthanized and lungs were collected. The right lobe was used for microarray analysis. Microarray analysis was performed on the lungs of three to four mice per treatment group.

Project description:Although various mechanisms have been inferred for combinatorial actions of multiple carcinogens, these mechanisms have not been well demonstrated in experimental carcinogenesis models. We evaluated mammary carcinogenesis initiated by combined exposure to various doses of radiation and chemical carcinogens. Female rats at 7 weeks of age were M-NM-3-irradiated (0.2M-bM-^@M-^S2 Gy) and/or exposed to 1-methyl-1-nitrosourea (20 or 40 mg/kg, single intraperitoneal injection) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (40 mg/kg/day by gavage for 10 days) and were observed until 50 weeks of age. The incidence of mammary carcinoma increased steadily as a function of radiation dose in the absence of chemicals; mathematical analysis supported an additive increase when radiation was combined with a chemical carcinogen, irrespective of the chemical species and its dose. Hras mutations were characteristic of carcinomas that developed after chemical carcinogen treatments and were overrepresented in carcinomas induced by the combination of radiation and MNU (but not PhIP), indicating an interaction of radiation and MNU at the level of initiation. The expression profiles of seven classifier genes, previously shown to distinguish two classes of rat mammary carcinomas, categorized almost all examined carcinomas that developed after individual or combined treatments with radiation (1 Gy) and chemicals as belonging to a single class; more comprehensive screening using microarrays and a separate test sample set failed to identify differences in gene expression profiles among these carcinomas. These results suggest that a complex, multilevel interaction underlies the combinatorial action of radiation and chemical carcinogens in the experimental model. Mammary cancers were from untreated rats (n = 3) and rats treated with radiation (1 Gy; n = 4), MNU (40 mg/kg; H-rasM-bM-^@M-^Smutated cancers, n = 5; H-rasM-bM-^@M-^Snonmutated cancers, n = 4), PhIP (H-rasM-bM-^@M-^Smutated, n = 1; H-rasM-bM-^@M-^Snonmutated, n = 3), radiation 1 Gy plus MNU (40 mg/kg; H-rasM-bM-^@M-^Smutated, n = 5; H-rasM-bM-^@M-^Snonmutated, n = 4) and radiation 1 Gy plus PhIP (H-rasM-bM-^@M-^Snonmutated , n = 4). Normal mammary tissues were from untreated rats (n = 3).

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Critical Role of Sphingolipid Pathway Components in Murine Radiation-Induced Lung Injury: Protection by Sphingosine-1-Phosphate Analogues

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