Toxicogenomic effects of nano- and bulk TiO2 particles in the soil nematode Carnorhabditis elegans
ABSTRACT: The toxicity and toxicogenomics of selected anatase and rutile nanoparticles (NP) and bulk titanium dioxide (TiO2) particles were evaluated in the soil nematode Caenorhabditis elegans. Results indicated that bulk or nano-TiO2 particles were slightly toxic to soil nematode C. elegans, as measured by reproduction EC50 values ranging from 4 to 32 mg/L. Whole-genome microarray results indicated that the regulation of glutathione-S-transferase gst-3, cytochrome P450 cypp33-c11, stress resistance regulator scl-1, oxidoreductase wah-1, and embryonic development pod-2 genes were significantly affected by nano-sized and bulk TiO2 particles. More specifically, it was determined that anatase particles exerted a greater effect on metabolic pathways, whereas rutile particles had a greater effect on developmental processes. The up-regulation of the pod-2 gene corroborated the phenotypic effect observed in the reproduction test. Our results demonstrated that C. elegans is a good genomic model for nano-TiO2 toxicity assessment. Overall design: TiO2 genomic experiments were performed using three separate biological replicates for each concentration.
INSTRUMENT(S): Agilent-020186 C.elegans v2 (Name as ID)
Project description:The nematode C. elegans was exposed to TiO2 nanoparticles (NPs) to evaluate the ecotoxicity of TiO2 nanoparticles. We used the DNA microarray method to understand changes in gene expression after the exposure to TIO2 NPs. We identified various genes involved in metal detoxification as well as in regulating worm development. Overall design: We employ the model organism C. elegans (with a fully sequenced genome) to effectively study the toxicology of TiO2 NPs (i. e., anatase) under dark conditions.
Project description:Little progress has been made in studying the toxicity of realistic 'non-pristine' forms of nanoparticles that presents in real soil environment. It is presently unkown whether the transformed nanoparticles in realistic environment exerts an adverse effect to rhizobium-legume symbiosis on molecular level. We used microarray to investigate the toxicogenomic responses of the model legume Medicago truncatula following 30 days exposure to three different types of biosolids (control biosolids (control BS), a mixture of Ag, ZnO and TiO2 manufactured nanomaterials added biosolids (Nano BS) and a corresponding bulk metals added biosolids (Bulk BS) ) amended soil that were aged for 6 months prior to exposure in pot experiment. Overall design: Our Genechip® Medicago Genome Array is designed specially to monitor gene expression in Medicago truncatula, Medicago sativa, and the symbiotic organism Sinorhizobium meliloti. For our study, RNA were extracted from shoots and roots of Medicago truncatula that exposure to control, Bulk and Nano BS treatments for 30 days, and used for all hybridization on Affymetrix microarray. The objective of our study is to investigate the molecular mechanisms of toxicity of Nano BS in comparison with their counterpart Bulk BS treatment, using a commercial Medicago truncatula microarrays.
Project description:Little progress has been made in studying the toxicity of realistic 'non-pristine' forms of nanoparticles that presents in real soil environment. It is presently unkown whether the transformed nanoparticles in realistic environment exerts an adverse effect to rhizobium-legume symbiosis on molecular level. We used microarray to investigate the toxicogenomic responses of the model legume Medicago truncatula following 30 days exposure to three different types of biosolids (control biosolids (control BS), a mixture of Ag, ZnO and TiO2 manufactured nanomaterials added biosolids (Nano BS) and a corresponding bulk metals added biosolids (Bulk BS) ) amended soil that were aged for 6 months prior to exposure in pot experiment. Our Genechip® Medicago Genome Array is designed specially to monitor gene expression in Medicago truncatula, Medicago sativa, and the symbiotic organism Sinorhizobium meliloti. For our study, RNA were extracted from shoots and roots of Medicago truncatula that exposure to control, Bulk and Nano BS treatments for 30 days, and used for all hybridization on Affymetrix microarray. The objective of our study is to investigate the molecular mechanisms of toxicity of Nano BS in comparison with their counterpart Bulk BS treatment, using a commercial Medicago truncatula microarrays.
2015-07-01 | E-GEOD-64788 | ArrayExpress
Project description:Toxicogenomic effects of nano- and bulk TiO2 particles in the soil nematode Carnorhabditis elegans using juglone as a positive control for oxidative stress
Project description:In order to evaluate the identification of genes and pathways, the global gene expression profiles were assessed in response to UV, TiO2 and UV+TiO2 on nematode, Caenorhabditis elegans. We performed whole genome DNA microarray experiments using age synchronized young adult C. elegans population exposed to UV, TiO2 and UV+TiO2 for 24h. We used whole genome microarrays to screen for global changed in C. elegans transcription profiles and with subsequent quantitative analysis conducted on selected genes. Young adults C. elegans were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Gene expression profiling of the human keratinocytes cell line (HaCaT) exposure to ultrafine, fine, and submicron TiO2 particles were employed to gain insights into the molecular events. Overall design: Three types of bulk TiO2 anatase particles, ST-01(average primary particle size= 7nm, specific surface area = 316m2/g), ST-21(20nm and 66m2/g), and ST-41(200nm and 10m2/g) used in this study were purchased from Ishihara Sangyo Kaisha Ltd., Japan. TiO2 particles were dispersed and size-fractionated in Dulbecco’s modified Eagle’s medium (DMEM) . TiO2 particles was dispersed in heat-inactivated fetal bovine serum (FBS) and centrifuged at 16,000 x g for 20 min. Removing the supernatant, the pellet was washed with DMEM, and centrifuged at 16,000 x g for 20 min. The supernatant was removed and dispersed in fresh DMEM with 10 % FBS medium and antibiotic/antifungal-agent (100 units/ml of penicillin, 100 μg/ml of streptomycin, and 250 ng/ml of amphotericin B) defined as DMEM-FBS medium, and centrifuged at 8,000 x g for 20 min. Removing the supernatant, the pellet was vortexed with fresh DMEM-FBS medium and centrifuged at 4,000 x g for 20min. The supernatant of 4,000 x g fraction was collected. Continuously, the centrifugal force was reduced to 2,000 x g, 1,000 x g and 500 x g. Each supernatant of 2,000 x g, 1,000 x g, and 500 x g fraction was collected. The 500 x g fraction supernatant was 0.8 µm membrane-filtered. The concentrations and the secondary average diameter of TiO2 particles in the supernatant fractions were individually characterized using X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscopy (TEM) at 120 kV. The samples were defined as T-7 (ST-01, 4,000 x g fraction), T-20 (ST-21, 1,000 x g fraction), and T-200 (ST-41, 500 x g fraction), and were utilized for the following DNA microarray analysis. Human keratinocyte cells (HaCaT) purchased from the German Cancer Research Center (DKFZ) were cultured in DMEM-FBS medium at 37 °C in a humidified atmosphere of 5% CO2 and 95% air The passage numbers 2–10 were used in the experiments, and cells were seeded at a density of approximately 2 x 10^5 cells/ml. All experiments were performed in triplicate (n=3).
Project description:Rationale: Maternal immune responses can promote allergy development in offspring. Pilot data show that neonates of mother mice exposed during pregnancy to air pollution particles have increased allergic susceptibility. Objective: We investigated whether inflammatory response to titanium dioxide (TiO2) particles earlier considered immunologically ‘inert’ is enhanced during pregnancy. Methods: Pregnant BALB/c mice (or non-pregnant controls) received particle suspensions intranasally at day 14 of pregnancy. Lung inflammatory responses were evaluated 19 and 48 h after exposure. Results: Pregnant mice showed robust and persistent acute inflammatory responses after exposure to TiO2, while non-pregnant females had the expected minimal responses. Genomic profiling identified genes differentially expressed in pregnant lungs exposed to TiO2. Neonates of mothers exposed to TiO2 (but not PBS) developed increased susceptibility to allergens. Conclusion: Pregnancy enhances lung inflammatory responses to otherwise relatively innocuous inert particles. Keywords: Particles exposure, pregnancy vs normal Overall design: To test whether pregnancy alters the normally minimal inflammatory response to ‘inert’ particles, we have administered TiO2 and DEP suspensions (50 ug/mouse) or PBS solution by intranasal insufflation to normal or pregnant E14 mice (see Figure 1B). The mice were subjected to pathologic analysis 19 or 48 hrs later. Respirable-size TiO2 particles were generously provided by Dr. Ian Gilmour (US EPA). Particle samples were baked at 165 0C for 3 h to eliminate endotoxin, aliquoted and stored frozen at – 80 0C. Particle suspensions (50 ug in 50 uL) or PBS solution (vehicle) were administered by single intranasal insufflation of pregnant or normal Balb/c mice under light halothane anesthesia Total lung RNA extraction and isolation was performed 19 hrs after exposure using a Qiagen RNAeasy Mini kit according to manufacturer’s instructions (Qiagen, Valencia, CA). RNA purity and quality were analyzed by Agilent Bioanalyzer 2100 scan. The hybridization was carried out at the Harvard Partners Genomic Center Microarray facility (Cambridge, MA) using the Affymetrix GeneChip ® platform and Affymetrix mouse 430 2.0 chips (Affymetrix, Santa Clara, CA). 4 samples were analyzed in each of 4 groups: Normal PBS, Normal TiO2, Pregnant PBS and Pregnant TiO2, for a total of 16 samples.
Project description:Pulmonary exposure to high doses of nanoparticles (NP) leads to well characterized lung toxicity in addition to long-term NP retention. However, pulmonary NP accumulation and toxicity following low dose exposures are not well described. In the present study we sought to: (1) investigate particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO2) and (2) determine the effects of long-term particle accumulation on pulmonary systems. Female C57BL/6 mice were exposed to rutile nano-TiO2 (primary size of 20.6 nm and surface area of 107.7 m2/g) via single intratracheal instillations of 18, 54 and 162 µg/mouse and sampled 1, 3 and 28 days post-exposure. The deposition of nano-TiO2 in the lungs was assessed using Nanoscale Hyperspectral Microscope. DNA microarrays, pathway-specific real-time RT-PCR (qPCR) and gene-specific qPCR arrays, and tissue protein analyses were employed to characterize pulmonary responses. Hyperspectral mapping showed dose-dependent retention of nano-TiO2 in the lungs up to 28 days post-exposure time. Retention did not correlate with the extent of inflammatory neutrophil influx into the lungs. DNA microarray analysis showed altered expression of approximately 3000 genes across all treatment groups (±1.3 fold; p<0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein levels. Although the low dose exposure failed to induce observable inflammation, significant changes in the expression of genes and proteins associated with inflammation were observed. Moreover, diminished (or absent) neutrophil influx in the low and medium dose groups was correlated with negative regulation of genes associated with ion homeostasis and muscle regulation. Gene expression changes for several inflammatory mediators have previously been noted in mice exposed to the same nano-TiO2 via inhalation. Our results suggest that retention of nano-TiO2 in the absence of inflammation and effective clearance can perturb calcium and ion homeostasis, and affect smooth muscle activities over time. This experiment consists of three different dosages of TiO2, e.g., low (18 ug), medium (54 ug) and high (162 ug), and one control. There are 3 time points for each treatment and control group, e.g., day 1, day 3 and day 28. Each dose or time point has 5-6 biological replicates. There are total 65 samples (arrays)