Progress 04/01/18 to 09/30/22
Outputs
Target Audience:Teams of all participating campuses (UA Little Rock, Texas A&M, UAMS) reached the target audience (federal regulatory agencies, agricultural industry, academia) through open data publishing (three published articles), conference presentations, talks for seminars and workshops, local media, and face-to-face meetings. During time of project PIs, postdoctoral scientist, graduate and undergraduate students made 5 poster and 12 oral presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project allowed two Ph.D. students (one from UA Little Rock and another from Texas A&M University) to perform research and collect data for PhD dissertations. Both students (Sudha Shanmugam (UALR) and Muhammad Anas (TAMU)) successfully defended their dissertations in 2020 and 2021 and got job offers. Additionally, a large group of students and one postdoctoral scientist (UAMS, UA Little Rock, Texas A&M University) got extensive training in metabolomics research, toxicology, and the detection of nanomaterials in plant/animal samples. Using created infrastructure Laboratory of Dr. Khodakovskaya organized training Workshops (2021, 2022) for minority students of historically black college Philander Smith College (Little Rock, AR) and provided oppotunity to learm methods of modern Plant Biology. How have the results been disseminated to communities of interest?During this project, results have been disseminated to communities of interest in a variety of ways. First of all, data were reported in three peer-reviewed articles published in the International Journal of Molecular Sciences (2021), ACS Applied Bio Materials (2021), ACS Nano (2022), and three Conference papers. Second, PIs and involved graduate students actively participated in and shared data at multiple International, National, and Regional Conferences including the 2019 6th Nano Today Conference (Portugal), 2020 PANNANO Conference (Brazil), Pharmacology Congress-2020 (Austria), 2019 Gordon Research Conference "Environmental Nanotechnology", 2018 and 2022 Gordon Research Conferences "Nanoscale Science and Engineering for Agriculture and Food Systems, USDA-NIFA Grantees Conferences, 9th and 10th Sustainable Nanotechnology Annual Conferences (SNO), 2021 Southeast Regional IDeA Conference, 2020 Experimental Biology Conference, 2021 Annual American Chemical Society (ACS) Conference, and 2019, 2020 UA Little Rock Student's Research & Creative Works Expo. Additionally, PIs directly reached stakeholders (Arkansas farmers, Arkansas community) by arranging face-to-face meetings and distributing information using State media and resources of Arkansas Research Alliance. For example, Arkansas Money & Politics advertised a project (https://www.armoneyandpolitics.com/the-story-of-arkansas-research/). This project was described in the magazine "Research in the Rock" in Fall 2021. PI was invited to give a talk to the government and academic institutions including Kennedy Space Center, Arkansast Resarch Academy, Idaho State University, University of Arkansas at Little Rock (Department of Biology). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Carbon-based nanomaterials (CBNs) already play a critical role in increasing plant productivity and food security in challenging climate conditions. Different plant products were developed with the help of CBNs used as growth regulators, sensors, carriers of chemicals/nucleic acids, or applied in food packaging. The safety of such products must be demonstrated before adding new technologies to the market. The establishment of links between the amount of nanoparticles absorbed by exposed plants and potential toxicity to animals and humans has been limited by the lack of quantitative methods permitting the assessment of CBNs inside plant and animal organs. As a result of this project, we created a new platform for a comprehensive investigation of the possible health risks of CBNs accumulated in the organs of exposed tomato plants. Using advanced methods of quantification of CBNs in biological samples (MIH, RF heating), we were able to link the amount of representative CBN type (multi-walled carbon nanotubes, CNT) absorbed by model plant organs (tomato fruits) with the amount of CNTs accumulated in organs of animals (mice model) which were consuming CNT-containing fruits. Combined with the assessment of in vivo toxicity and analysis of tomato metabolome (LC-MS) this comprehensive detection approach allowed us to evaluate the potential risks of the inclusion of CNT-contaminated plants into the food chain. Based on the data, we concluded that there is no measurable accumulation of CNTs in organs (liver, kidney, brain) of mice fed with CNT-contaminated tomato fruits, and there is no detectable toxicity associated with the metabolome of tomato fruits grown in presence of CNTs used as growth regulators. We documented that pure CNT can damage liver cells of mice only at the highest applied concentration (10 mg/kg) which is not achievable in fruits derived from plants exposed to CNT-based fertilizer. Established results suggested that the potential use of carbon-based nanomaterials as nanofertilizers, sensors, or carriers of chemicals/nucleic acids would pose low risks to consumers of agricultural plants. All suggested goals of the project were achieved. All required experiments were performed. Established data were published in ACS Nano (impact factor = 18) in 2022. Results were also shared by 5 poster and 12 oral presentations. Three patents related to topic of project were issued during time of project (2019, 2020, 2022). New patent application was submitted in 2022.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Sudha Shanmugam "Enhancement of abiotic stress tolerance in rice and soybean by biotechnological and nanotechnological approaches" 2020 - PhD Dissertation
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Sajedeh Rezaei Cherati, Muhammad Anas, Shijie Liu, Sudha Shanmugam, Kamal Pandey, Steven Angtuaco, Randal S. Shelton, Aida N. Khalfaoui, Alena V. Savenka, Erin B. Porter, Todd Fite, Huaixuan Cao, Micah J. Green, Alexei G Basnakian, Khodakovskaya M Comprehensive risk assessment of carbon nanotubes used for agricultural applications , ACS Nano 2022, https://doi.org/10.1021/acsnano.2c02201
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Rezaei Cherati S, Shanmugam S, Pandey K, Khodakovskaya M* Whole-transcriptome responses to environmental stresses in agricultural and industrial crops treated with carbon-based nanomaterials, ACS Applied Bio Materials 4(5):4292-4301 |https://doi.org/10.1021/acsabm.1c00108,
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Moore CL, Savenka AV, Basnakian AG TUNEL assay: a powerful tool for evaluation of kidney injury. Int J Mol Sci, 2021, 22, 412. doi.org/10.3390/ijms22010412
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Muhammad Anas Radiofrequency heating of carbon-based nanomaterial films - PhD dissertation
|
Progress 04/01/21 to 03/31/22
Outputs
Target Audience:Teams from all participating campuses (UALR, UAMS, TAMU) prepared a joint manuscript combining data of all objectives. The manuscript will be submitted in January 2022 to Nature Nanotechnology. Publication of all generated data in 2022 will allow us to reach out target audience. Also, established data were already disseminated to the community of inyerest by the publication of abstracts (online) and oral presentations for the 2021 Southeast Regional IDeA Conference, 2021 ACS Conference, 10th SNO Conference, 2021 USDA Grantees Conference, 2021 Arkansas NASA EPSCoR conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four graduate students, one postdoctoral scientist, and one undergraduate student worked on project during year 4. All trainees got extensive experience in "omics" research, toxicology, and detection of nanomaterials in plant samples. Ph.D. student from TAMU successfully defended his dissertation in 2021 (adviser is Co-PI of project Dr. Green). How have the results been disseminated to communities of interest?During the fourth year results of the project were shared with the community, colleagues, and stakeholders by presentations at multiple Conferences/Meetings including the 2021 USDA Grantees Conferences (September 9, 2021-ZOOM), 10th Conference of Sustainable Nanotechnology Organization Conference (November 5, 2021-ZOOM), 2021 American Chemical Society Conference (August 22, 2021-ZOOM), 2021 Arkansas NASA EPSCoR Conference (April 15, 2021-ZOOM). At all four attended ZOOM conferences, PI gave oral presentations. PI worked also as Chair for sessions of two conferences (Food/Agriculture for SNO 2021 and biological session for Arkansas Space Grant Virtual Consortium Symposium). Additionally, PI discussed the commercialization of established results and contacts with the industry with leaders of Arkansas Research Alliance (ARA). ARA invests in research that stimulates innovation and commercialization in Arkansas. What do you plan to do during the next reporting period to accomplish the goals?Due to COVID-associated delay, we have requested NCE for additional 6 months. During the additional time, we are planning to complete analysis for all performed experiments and publish data in the high-ranked journal.
Impacts
What was accomplished under these goals?
The first objective of the project was directed to the evaluation of the effects of MWCNT absorbed by plants during nanofertilization on total plant metabolome. LC-MS analysis of organs collected from tomato plants grown in presence of MWCNT was performed during years 1-3. No potentially toxic compounds were identified in analysis of lists of up-regulated compounds in tomato organs collected from CNT-treated plants. During the additional year (year 4), two major potentially toxic compounds of tomato (tomatine, atropine) were individually analyzed in different organs of MWCNT-treated plants by HPLC (laboratory of Dr. Khodakovskaya, UALR). Overall, we can conclude that absorbed CNT significantly affected the total metabolome of exposed tomato plants. However, this modification did not result in the upregulation of known toxic compounds in tomatoes. The second objective of the project was focused on the quantification of absorbed MWCNT in organs of mice involved in prolonged in vivo experiments (6 months). Quantification of nanomaterials accumulated by organs of orally exposed animals is a challenging task. Our attempts to quantify MWCNT inside the liver, brain, and kidney were not successful during year 3. But during the year 4 (reporting period) group of Dr. Green (TAMU) resolved technological difficulties associated with the RF heating method and results were established. Dried mice organs were collected at the end of the experiment, ground, and analyzed by RF heating. It was found that the differences between the CNT-containing diets and control diets were not significant and the MWCNT contents corresponding to the heart rates were below the detectable threshold associated with established calibration curve. Similar findings were obtained for other animal organs such as the brain and kidneys. The third objective of the project was to evaluate the potential toxicity of tomato fruits contaminated with CNT using a mouse model. During the reported period toxicity of fruits collected from CNT-exposed tomato plants to mice was assessed by TUNEL assay. Visualization of MWCNT inside animal organs was performed using TEM. Chronic feeding of mice with dried CNT-containing tomato fruits was performed for 6 months (laboratory of Dr. Banakian, UAMS). The experiment was started with 48 CD-1 male mice, which were divided into 4 groups: Diet A (mice fed with powdered regular mouse chow (PRMC); Diet B (mice fed with 95% PRMC + 5% dried control tomato fruit powder (TFP); Diet C (mice fed with 95% PRM + 5% control TFP and added pure CNTs in concentration that was same with the amount of CNT in fruits collected from hydroponically growth tomato plants (25 µg/g) and Diet D (mice fed with 95% PRMC + 5% CNT-grown TFP). The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, the most sensitive and universal assay for irreversible cell death was used for assessment of the structural toxicity of the nanoparticles (cell death). TUNEL confirmed the absence of structural damage in the liver and TEM images of livers collected from animals received Diets C, D looked like control and had no MWCNT visual inclusions. Established data were in a good correlation with data on blood markers associated with damage of organs. It was found early (year 3 of award) that all 14 tested blood markers, were within normal ranges for Diets C, D and did not reveal any statistical difference compared to the controls (Diets A, B) at all-time points. TUNEL assay supported our previous observation of liver damage in animals that received the highest dose of pure MWCNT (10 mg/kg) through gavage by indication of liver cell death at applied dose. A significant portion of the time associated with reporting period (year 4) was dedicated to the preparation of the manuscript combining all data generated for three objectives. The manuscript is currently in completed form and will be submitted for publication in January 2022. Overall conclusion: Our experiments provide evidence that the low amount of CNT accumulated in tomato fruits (25 µg/g of dry fruit) as a result of nanofertilization is insufficient to cause toxicity during prolonged oral administration of CNT-contaminated fruits. Significantly higher amounts of CNT are linked with the accumulation of CNT in animal organs and toxicity to cells.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Moore CL, Savenka AV, Basnakian AG TUNEL assay: a powerful tool for evaluation of kidney injury. Int J Mol Sci, 2021, 22, 412. doi.org/10.3390/ijms22010412
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Braman N, Brents L, Savenka AV, Shelton RS, Fite T, Apostolov EO, Moore CL, Hinson J, Basnakian AG (2021) Application of TUNEL assay for mechanistic evaluation of kidney and liver injury. 2021 Southeast Regional IDeA Conference, November 12-14, 2021, San Juan, Puerto Rico; Abstract Book, p45
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Muhammad Anas Radiofrequency heating of carbon-based nanomaterial films , Aug 2021 Ph.D. Dissertation
|
Progress 04/01/20 to 03/31/21
Outputs
Target Audience:Generated data were presented at the Experimental Biology Meeting (San Diego, CA, April 4-7, 2020), Pharmacology Congress-2020 (May 4-5, Vienna, Austria), The second Pan-American Nanotechnology Conference (PANNANO, Aguas de Lindoia, Brazil, March 5, 2020), 9th Conference of Sustainable Nanotechnology Organization (DC, United States, November 13, 2020) and 2020 UA Little Rock Student Research & Creative Works Expo. PD, Co-PDs of the project, and graduate students presented results to participants of Conferences representing the academic community, government, regulatory agencies, and industry. PD of the project made two oral presentations for International Conferences. Ph.D. students who were involved in the project had a chance to present established results during the UA Little Rock Student Expo, 9th Conference of Sustainable Nanotechnology Organization (SNO), and PANNANO 2020. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three graduate students (one graduate student per participating campus), two postdoctoral scientists, and two undergraduate students were involved in research activities for the project's objectives. One Ph.D. student who worked on the project during the second year of the award moved to a postdoctoral position at the laboratory of PD (Dr. Khodakovskaya) during the third year. Students got extensive experience in new methods and techniques of Bioengineering, Toxicology and Plant Biochemistry. Graduate students and postdoctoral scientists who were involved in the project presented established results at several International Conferences. Ph.D. student of UA Little Rock Ms. Sajedeh Rezaei Cherati won the Second Award in Poster Competition at the 9th Conference of Sustainable Nanotechnology Organization Conference (November 12-13, 2020 - Virtual Conference). She presented data related to objective 1 of the project. How have the results been disseminated to communities of interest?At this time, we are working on the preparation of the manuscript that will combine all results that were generated during years 1- 3 of the project. The manuscript will be submitted next reporting year. Partially, established results were disseminated to the community of interest by the publication of abstracts (on-line) forExperimental Biology Meeting, San Diego, CA, April 4-7, 2020 and Pharmacology Congress-2020, May 4-5, Vienna, Austria. What do you plan to do during the next reporting period to accomplish the goals?Plans for the no-cost extension year: For the first objective, we will perform an HPLC analysis of potentially toxic metabolites of tomato (tomatine, atropine) that were up-regulated in samples collected from CNT-exposed tomato plants according to LC-MS analysis. For the second objective, we plan to do an in-depth analysis of the collected heating response data on the mouse organs. If needed, we will also repeat the experiments and measure data for other organs to draw firm conclusions regarding CNT quantification. We will also investigate the possibility of creating positive control samples in lab-based on the direct addition of CNTs to negative control samples. For the third objective, we plan to further characterize the potential liver injury by using more sensitive structural toxicity markers, such as TUNEL assay combined with immunohistochemistry for caspase-dependent apoptosis (active caspase-3 or caspase-activated DNase), caspase-independent apoptosis (endonuclease G), proliferation (Ki67 or PCNA), and fibrosis (collagen IV). Overall, we plan to prepare and submit a solid manuscript that will combine all the established data during no-cost extension year.
Impacts
What was accomplished under these goals?
The first objective of the project was directed to the evaluation of the effects of CNT absorption by plants on total plant metabolome. The goal was left unchanged. Experimental tasks planned for the third year for the first objective were fully performed. During the 3rd year of the award, we have run and analyzed tomato samples collected from plants grown in soil supplemented with MWCNT (the second type of MWCNT treatment) using LC-MS. The total metabolome of leaves, flowers, fruits, roots, shoots generated from tomato plants incubated in soil medium (control samples) and tomato plants are grown in soil supplemented with multi-walled carbon nanotubes (MWCNT) was fully analyzed by already established methodology. Mass detection, building chromatograms, and deconvolution were performed using MZmine software. Further statistical, functional, and integrative analysis of generated metabolomics data was done through MetaboAnalyst software. As a result, for each tomato organ we have created lists of metabolites that were differentially regulated in response to the treatment of plants with MWCNT. Metabolomics pathways that were affected significantly by MWCNT exposure were identified by using resources of the KEGG database. Additionally, we have identified toxic tomato compounds that were up-regulated in organs collected from plants exposed to CNT through soil or hydroponics. Such metabolites will be subject to detailed HPLC analysis during the addition no-cost extension year. The second objective of the project was focused on the quantification of the amounts of absorbed MWCNT in mouse organs. The goal was left unchanged. Co-PD of the project (Dr.Basnakian) provided organs of mice fed with fruits from tomato plants grown in presence of CNT. Detection of CNT inside organs was carried out using two parallel methods: radio frequency heating (RF) and microwave-induced heating (MIH) in the laboratory of Co-PD Dr. Green: Radio frequency heating of thin aanimal organ slices: Thin slices (30 μm thickness) of liver, heart, and kidney from mice fed with different diets were supplied for carbon nanotube (CNT) distribution analysis via radio frequency (RF) heating method. A fringing-field RF applicator with two parallel copper traces spaced 4 mm apart was fabricated to couple RF fields with the slices. RF signal generator and a power amplifier were used to create RF fields with a frequency of 135 MHz and 30 W. The heating was monitored using a Forward-Looking Infrared (FLIR) camera and steady-state thermal images were captured during heating. Thermal images of organ slices did not show any hot spots that could be indicative of CNT presence in these slices. Organ slices from mice fed with control diets showed a lower average heating response relative to that from mouse fed with CNT-containing diets; however, the spread in the data was large enough that the data were inconclusive. Microwave-induced heating of whole animal organs: Several of the whole organs of mice fed with different diets were analyzed using the microwave-induced heating (MIH) method for CNT presence. The livers, kidneys, and brains (stored at -80 °C) were freeze-dried for at least 72 hours to remove the water from the organs. After freeze-drying, the organs were grinded using a mortar and pestle and kept under vacuum before MIH testing. During the MIH testing, samples were exposed to microwaves (2.45 GHz) at a power of 30 W for 10 s. A K-type thermocouple was used to measure the temperature rise over time. The differences in the temperature rise observed for mice organs fed with different diets were obtained for CNT detection analysis. Statistical significance between the temperature difference observed between organs from mice fed with control and the CNT-containing diets was tested with a p-test. It was determined that the average temperature difference for livers of mice fed with CNT-containing diets (fruits + powder CNT; fruits collected from plants cultivated in presence of CNT) was substantial compared to the control diets (no CNT). We did not see similar trends for kidneys and the brain. The third objective of the project was to evaluate the potential toxicity of tomato fruits/leaves with accumulated CNTs using a mouse model. The goal was left unchanged. We hypothesized that manmade CNTs will not have significant negative effects on animals (mice) at the low doses that can be accumulated in plant organs during the application of CNTs as plant growth regulators. This study was performed by the team led by Co-PD, Dr. Alexei Basnakian (University of Arkansas for Medical Sciences). The animal studies were performed after approval from the Animal Care and Use Committee of the Central Arkansas Veteran's Healthcare System (CAVHS, Little Rock, AR). CD-1 mice (6-8 weeks old) were purchased from the Jackson Laboratory. For consistency of the experiments, only male mice were used. During the previous 2 years, we have tested the potential target organs and assess potential acute toxicity using pure CNTs fed orally by gastric gavage with water. The experimental groups have received CNTs as a single dose in the range of concentrations. Our data indicated that a single oral gavage of CNTs induced some liver injury only at the highest used dose of 10 mg/kg as evident by an elevation of a sensitive liver toxicity marker, serum alanine transaminase (ALT), and TUNEL-positivity indicative of liver cell death. Because the concentration of CNTs in tomato fruits is expected to be 2-3 orders of magnitude lower than 10 mg/kg, it was concluded the concentrations of CNTs found in tomato fruits are likely to be non-toxic. Our further analysis included the application of dark-field microscopy (DFM) for (a) the colocalization between CNTs and TUNEL-positive cells and (b) the development of a method to quantify CNTs burden in liver tissue. Our data indicated that the DFM method designed for particle toxicology has only limited potential for measurement of nanoparticle toxicity and that some mouse liver cells are capable of accumulating MWCNTs without inducing immediate cell death. During the 3d year of the study, we tested the toxicity of tomato fruits from plants grown in hydroponics supplemented with CNTs. The experiment was started with 48 CD-1 male mice, which were divided into 4 groups (n=12/group): control fed with powdered regular mouse chow (PRMC), control fed with 95% PRMC/5% dried control tomato fruit powder (TFP), mice fed with 95% PRMC/5% TFP and added CNTs (positive control), and mice fed with 95% PRMC/5% CNT-grown TFP. All mice were fed ad libitum. Blood (200 µL) was collected via retro-orbital puncture under isoflurane anesthesia to monitor toxicity at the baseline, as well as at 1 month, 3 months, and 6 months of feeding, and subjected to the 14 organ function assays described above. The following tissues will be harvested: liver, kidney, spleen, lung, heart, aorta, intestine, pancreas, brain, bladder, skeletal muscle, skin, subcutaneous adipose tissue, and bone. Tissues will be fixed in 10% buffered formalin and processed to determine CNT distribution and structural organ damage. Our main conclusion is that no signs of organ toxicity were noticed in assessed organs. Overall Conclusion: All planned experiments (year N3) for each objective were fully performed. Our experiments performed in years 1-3 for each objective allowed us to achieve all goals. LC-MC experiments were finalized. Data established in the vivo experiments indicated that a single oral gavage of CNTs induced mild and transient liver injury only at the highest used dose of 10 mg/kg. In vivo experiments with feeding mice, up to 6 months with CNT-containing tomato fruits showed the absence of toxicity of tomato fruits contaminated with CNT. We were able to detect the presence of CNT inside of organs of animals fed with CNT-contaminated tomato fruits.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Angtuaco S, Savenka A, Fite T, Liu S, Khodakovskaya M, Basnakian AG (2020) Qualitative Assessment of Carbon Nanotube Toxicity to Mouse Liver using Dark-field Microscopy. Experimental Biology Meeting, San Diego, CA, April 4⿿7, 2020 - https://faseb.onlinelibrary.wiley.com/doi/10.1096/fasebj.2020.34.s1.04954
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Liu S, Angtuaco S, Savenka A, Fite T, Khodakovskaya M, Basnakian AG (2020) Evaluation of microscopy-based approaches for the assessment of nanomaterial toxicity. Pharmacology Congress-2020, May 4-5, Vienna, Austria - https://www.longdom.com/cme-pharmacology/scientific-program
|
Progress 04/01/19 to 03/31/20
Outputs
Target Audience:Generated data were presented at the 6th Nano Today Conference (June 17, 2019, Lisbon, Portugal), 8th Sustainable Nanotechnology Organization Conference (November 7, 2019, San Diego, CA, USA), Gordon Research Conference (Environmental Nanotechnology, June 3, 2019, Newry, ME, USA) and USDA-NIFA Grantees Conference (May 20, 2019, Vanderbilt University, TN, USA). PD of the project presented results to participants of Conferences representing the academic community, government (USDA-NIFA, NSF, NASA) regulatory agencies (FDA) and industry (agricultural and nanotechnological sectors). Additionally, generated data were used for educational activities. PD made three invited oral presentations for Graduate Students of the University of Arkansas at Little Rock, Idaho State University (Biological Seminars) and Seminar for Research Staff of Kennedy Space Center (October 28, 2019, NASA, Merrit Island, FL, USA). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four Graduate students, one postdoctoral scientist and two undergraduate students at three campuses (University of Arkansas at Little Rock, Texas A&M University and University of Arkansas for Medical Sciences) were involved in research activities described for each objective. Students got extensive training in a variety of methods and techniques and developed new skills. How have the results been disseminated to communities of interest?At this time, the final results of the study have not been obtained. We are in the process of the completion of the most critical experiments (in vivo studies). Therefore, it will be premature to distribute the intermediate findings in open sources. We expect that data will be disseminated in the communities of interest during the third year of award. What do you plan to do during the next reporting period to accomplish the goals?For the first objective, we will perform LC-MS analyses of tomato organs collected from plants grown in soil supplemented with MWCNT (the second type of MWCNT treatment). LC-MS data will be analyzed using the already established methodology. For the second objective, we will use the MIH method for quantification exact amount of MWCNT in organs of animals used in in vivo studies. It will allow us to link the absorption of MWCNT by animal organs with used MWCNT concentration and any possible toxicity of MWCNT to animals. The RF heating method will be used to create maps of MWCNT distribution in animal organ samples. For the third objective, we will finalize the structural damage studies (whole in vivo experiment), total analysis of toxicity, visualization of MWCNT inside animal organs (TEM, DFM). We are planning to prepare and submit a manuscript that will combine all results established during whole project. Additionally, we are planing to present generated data at the number of International Conferences: PANNANO 2020 Conference (March 4-7, 2020, Sao Paulo, Brazil); Gordon Research Conference - Nanoscale Science and Engineering for Agriculture and Food Systems (June 21-26, 2020, NH, USA); 9th SNO Conference (October 4-6, 2020, Denver, CO, USA), Experimental Biology 2020 Conference (April 4-7, 2020, San Diego, CA).
Impacts
What was accomplished under these goals?
The first objective of the project was directed to the evaluation of the effects of CNT absorption by plants on total plant metabolome. Experimental tasks planned for the second year for the first objective were fully performed. First, The LC-MS analysis of tomato samples collected during the first year of the project was finalized. Thus, the total metabolome of leaves, flowers, fruits, roots, shoots generated from 10-weeks-old tomato plants incubated in regular hydroponics medium (control samples) and tomato plants are grown in hydroponics medium supplemented with multi-walled carbon nanotubes (MWCNT). Mass detection, building chromatograms, and deconvolution were performed using MZmine software. Further statistical, functional and integrative analysis of generated metabolomics data was done through MetaboAnalyst software. As a result of the performed analysis, for each tomato organ we have created lists of metabolites that were differentially regulated in response to treatment of plants with MWCNT. Metabolomics pathways that were affected significantly by MWCNT exposure were identified by using resources of the KEGG database. Second, a group of PD generated and collected frozen and dry samples (leaves, flowers, fruits, roots, shoots) from tomato plants grown in soil supplemented with MWCNT (the second type of treatment required by reviewers). The metabolome of frozen samples will be fully analyzed by LC-MS during the third year of the award. Dry samples of organs collected from soil experiments were sent to Co-PD (Dr. Green) for quantification of the amounts of absorbed MWCNT in organs of exposed plans. The second objective of the project was focused on quantification of the amounts of absorbed MWCNT in organs of exposed plans. PD of the project (Dr. Khodakovskaya) provided flowers, fruits, roots, leaves and roots of tomato plants grown in soil (the second type of "nano"- treatment) to Co-PD Dr. Green (Texas A&A University). Group of Dr. Green performed the thermal mapping of carbon nanotubes (MWCNTs) in provided plant organs via scanning over radio-frequency (RF) fields. We also used our previously developed method Microwave-Induced Heating (MIH) to determine CNT content in plant organs collected from tomato plants grown in soil supplemented with MWCNT. Previously generated calibration curves relating to ΔT and MWCNT content were used and adjusted as appropriate for these samples. The intercept is calculated using the response of the control sample for each organ. The MWCNT content per mass of sample (ng/mg sample) is finally determined by first comparing the temperature rise of the sample to the calibration curve for the total amount of MWCNTs (ng) and then dividing by the sample mass (mg) used for analysis. As a result, exact concentrations of MWCNT (ng/mg of the sample) were calculated for wide range organs (fruits, leaves, shoots, roots) harvested from MWCNT-exposed tomato plants grown in soil. Such investigation allowed us to determine organs that absorbed MWCNT in highest dose and compare level of absorption of MWCNT by organs collected in from plants grown in MWCNT-hydroponics (data were generated in the first year of award) with absorption of MWCNT by organs collected in from plants grown in soil system supplemented with MWCNT (data generated in the second year of award). Thus, we have addressed the suggestion of reviewers to investigate how amounts of absorbed carbon-based nanomaterials (CBNs) are linked to the method of delivery of CBNs to plants. The third objective of the project was to evaluate the potential toxicity of tomato fruits/leaves with accumulated CNTs using a mouse model. Activities proposed for Objective 3 during the second year of the project has been completed. All animal studies were performed after approval from the Animal Care and Use Committee of the Central Arkansas Veteran's Healthcare System (CAVHS, Little Rock, AR). Tomato fruits collected from plants grown in hydroponics medium supplemented with MWCNTs were provided by Dr. Khodakovskaya for further in vivo experiments. The exact concentration of absorbed MWCNT was in MWCNT-contaminated fruits was calculated by Co-PD (Dr. Green) during the previous year. In vivo studies were performed by the team led by Co-PD, Dr. Basnakian. The experiment was started with 48 CD-1 make mice, which were divided into 4 groups (n=12/group): control fed with powdered regular mouse chow (PRMC), control fed with 95% PRMC/5% dried control tomato fruit powder (TFP), mice fed with 95% PRMC/5% TFP and added CNTs (positive control), and mice fed with 95% PRMC/5% CNT-grown TFP. All mice were fed ad libitum. Blood (200 µL) was collected via retro-orbital puncture under isoflurane anesthesia to monitor toxicity at the baseline and at 1 month of feeding and subjected to the 14 organ function assays described above. So far, no signs of organ toxicity are noticed. The experiment will be continued for at least 3 more months. Four months from the beginning of the experiment, the mice will be euthanized and blood and tissue will be collected. We will harvest liver, kidney, spleen, lung, heart, aorta, intestine, pancreas, brain, bladder, skeletal muscle, skin, subcutaneous adipose tissue, and bone. Tissues will be fixed in 10% buffered formalin and processed to determine CNT distribution and structural organ damage. Blood chemistry markers will be measured to determine organ toxicity as well. In addition to measurements of functional damage to organs, for toxicology analyses, it is equally important to develop methods for quantitative assessment of the CNT load in animal tissues. Therefore, our further analysis included the application of dark-field microscopy (DFM) for (a) the colocalization between CNTs and TUNEL-positive cells and (b) development of a method to quantify CNTs burden in liver tissue. DFM clearly showed some elevation of particulate material in the liver with occasional association with TUNEL-positive cells. DFM did not seem to allow precise quantification of nanoparticle aggregates due to the low sensitivity of DFM at the concentration observed in the liver. However, we discovered that CNT gavage increased the percentage of nuclei colocalized with DF objects by systematically counting the number of liver cell nuclei in contact with dark-field (DF) objects. TUNEL-positive nuclei were photographed, analyzed at 100-fold magnification and sorted by CNT treatment category. The process was repeated for DAPI-stained nuclei to serve as a control. Our results indicated that the rate of colocalization between dark-field objects and nuclei appears to correlate directly with CNT-gavage treatment. Another important observation, confirmed by electron microscopy, was that there were many alive TUNEL-negative cells containing CNTs. Overall, generated data indicated that the DFM method designed for particle toxicology has only limited potential for measurement of nanoparticle toxicity and that some mouse liver cells are capable of accumulating MWCNTs without inducing immediate cell death. Overall Conclusion: All planned experiments (year N2) for each objective were fully performed. The metabolomics studies of tomato plants exposed to MWCNT through the hydroponics system were completed. The determination of concentrations of MWCNT inside different organs of tomato plants was finalized. The toxicological in vivo experiment is in progress but the experiment will be fully completed during the third year. Established results of in vivo experiment from the 2nd year so far confirmed that MWCNT-grown tomato fruits are non-toxic to mice after 1 month of feeding. We also found a latent form of MWCNTs present in mouse liver cells after pure MWCNT consumption at a high dose that is not associated with toxicity.
Publications
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Progress 04/01/18 to 03/31/19
Outputs
Target Audience:Generated data were presented at the Gordon Research Conference "Nanoscale Science and Engineering for Agriculture and Food" (June 6-8, 2018; Mount Holyoke, MA). PD of the project presented results to Conference participants representing regulatory agencies (FDA) and industry (agricultural and nanotechnological sectors). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three graduate students-trainees in three campuses (University of Arkansas at Little Rock, Texas A&M and University of Arkansas for Medical Sciences) were involved in research activities described for each objective. Graduate students got training in a variety of methods and techniques and developed new skills. How have the results been disseminated to communities of interest?At this time, the final results of the study have not been obtained. Therefore it will be premature to distribute the intermediate findings. As soon as they are reached, the data will be disseminated in the communities of interest. What do you plan to do during the next reporting period to accomplish the goals?For the first objective, we expect to perform metabolome analysis of different tomato organs harvested in Year 1 by LC-MS. We will analyses established data and confirm generated results by HPLC. For the second objective, we will continue with the task of quantifying the number of carbon nanotubes (CNTs) in different organs of CNT exposed plants. Our initial tests show that we are able to create maps of CNT distribution in flower, leaf, and root samples using our newly developed radio frequency (RF) heating method. We are also able to calibrate our previously developed Microwave-Induced Heating (MIH) method for different plant organs and can detect CNTs up to ng CNTs/mg of sample in fruits, leaves, shoots, and roots of CNT exposed plants. For year 2, we will use our methods to analyze the organs of plants grown in hydroponic and soil environments. This will allow us to compare differences in CNT distribution and content from different plant growth scenarios. First, we will use the newly developed RF heating method to create a map of carbon nanotube (CNT) distribution in the plant samples. Then, we will use our MIH method to determine the CNT content in the samples. Additionally, methods calibrated for appropriate animal samples and applied to animal samples from Objective 3. For the third objective, we are going to perform protocol 2 (gavage with CNT tomato samples). We will collect organ and blood samples and perform analysis of functional toxicity, and preliminary analysis of structural damage. We are planning to present generated data at two international Conferences: "6th Nano Today Conference" (Lisbon, Portugal - June 2019) and Gordon Research Conference "Environmental Nanotechnology" (Jordan Hotel at Sunday River in Newry ME United States - June 2019).
Impacts
What was accomplished under these goals?
Carbon-based nanomaterials can be used to accelerate seed germination and plant growth. The major goal of this project is the evaluation of a potential environmental risk of the accumulation of carbon nanomaterials delivered to the environment when used as regulators of plant growth or as waste. Here, we were able to measure the exact amount of carbon-nanotubes that exposed plants (tomato) can accumulate in different organs. As the first stage of testing toxicity of plant organs contaminated with carbon nanotubes, we applied a different concentration of pure carbon nanotubes to experimental animals (mice). Our data indicated that a single oral dose of carbon-nanotubes induced some liver injury only at the highest used dose of 10 mg/kg. Because the concentration of carbon nanotubes in exposed tomato fruits is much less than 10 mg/kg, it was concluded the concentrations of carbon nanotubes found in tomato organs are likely to be non-toxic to animals. We expect that generated data will be a very important source of information for community, regulatory agencies and the agricultural industry. Accomplishments: The first objective of the project was directed to the evaluation of the effects of CNT absorption by plants on total plant metabolome. The goal was left unchanged. Experimental tasks planned for the first year for the first objective were fully performed. As a critical step for the whole project, efforts of the laboratory of Dr. Khodakovskaya (University of Arkansas at Little Rock) were focused on establishment large amount of control and CNT-exposed tomato plants for all three objectives. Thus, we run two hydroponics experiments in parallel for 4 months, collected control and CNT-exposed organs, established frozen and dried samples of leaves, flowers, fruits, roots, shoots. Dried samples were sent to the laboratory of Dr. Green for detection of the exact amount of CNT inside organs. Frozen samples were used for the preparation of samples for metabolomics (LC-MS). Currently, we are in process of analysis of established samples by upgraded LC-MS system (University of Arkansas at Little Rock). The second objective of the project was focused on quantification of the amounts of absorbed carbon-based nanomaterials in organs of exposed plans. The goal was left unchanged. PD of the project (Dr. Khodakovskaya) provided flowers, fruits, roots, leaves and roots of CNT-exposed tomato plants to Co-PD Dr. Micah Green (Texas A&M University). Group of Dr. Green performed the thermal mapping of carbon nanotubes (CNTs) in provided plant organs via scanning over radio-frequency (RF) fields. We applied our newly developed RF heating method to create a map of carbon nanotube (CNT) distribution in different plant organs. Generated results show that there are high amounts of CNTs in the roots compared to flowers and leaves because of higher maximum heating temperatures. We also used our previously developed method Microwave-Induced Heating to determine CNT content in plant organs. Previously generated calibration curves relating ΔT and CNT content were used and adjusted as appropriate for these samples. The intercept is calculated using the response of the control sample for each organ. As result, exact concentrations of CNT (ng/mg of the sample) were calculated for wide range organs (fruits, leaves, shoots, roots) harvested from CNT-exposed tomato plants. As expected the highest concentration of CNT was found in roots. The third objective of the project was to evaluate the potential toxicity of tomato fruits/leaves with accumulated CNTs using a mouse model. The goal was left unchanged. We hypothesized that manmade CNTs will not have significant negative effects on animals (mice) at the low doses that can be accumulated in plant organs during the application of CNTs as plant growth regulators. The Objective 3 of the proposal planned for the first year has been completed. These included the performance of protocol 1 (gavage with pure CNTs), organ and blood collection, analysis of systemic functional toxicity based on 14 blood markers, and preliminary analysis of structural damage based on TUNEL analysis. This study was performed by the team led by Co-PD, Dr. Alexei Basnakian, a Professor of Toxicology and Director of the DNA Damage and Toxicology Core Center at the University of Arkansas for Medical Sciences (UAMS). The animal studies were performed after approval from the Animal Care and Use Committee of the Central Arkansas Veteran's Healthcare System (CAVHS, Little Rock, AR). CD-1 mice (6-8 weeks old) were purchased from the Jackson Laboratory. For consistency of the experiments, only male mice were used. We have tested the potential target organs and assess potential acute toxicity using pure CNTs fed orally by gastric gavage with water (0.2 ml). The following experimental groups will receive CNTs as a single dose in the range of concentrations above and below the ones described in the above study (n=12 per group): (1) 0 mg/kg (negative control); (2) 0.001 mg/kg; (3) 0.01 mg/kg; (4) 0.1 mg/kg; (5) 1 mg/kg; (6) 10 mg/kg. In total, 72 mice were used in this protocol. The animals were euthanized 24 and 72 hours later (n=6/time point), and blood and organs were collected. The following organs were harvested and fixed in formalin: liver, kidney, spleen, lung, heart, aorta, intestine, pancreas, brain, bladder, skeletal muscle, skin, subcutaneous adipose tissue, and bone. The endpoints consisted of (1) assessment of systemic toxicity by measuring the function of organs; and (2) assessment of structural damage to organs. To monitor the function of organs, 100 µl blood were analyzed using the VetScan VS2 instrument (Abaxis, Union City, CA) by 14 parameters of the Comprehensive Diagnostic Profile - including alanine aminotransferase, albumin, alkaline phosphatase, amylase, calcium, creatinine, globulin, glucose, phosphorus, potassium, sodium, total bilirubin, total protein, and blood urea nitrogen - to quantitatively assess functions of the liver, kidney, heart, intestine, pancreas, and other organs that could be affected by the CNTs. To assess the structural toxicity of the nanoparticles, we used the TUNEL assay. Our data indicated that a single oral gavage of CNTs induced some liver injury only at the highest used dose of 10 mg/kg as evident by an elevation of a sensitive liver toxicity marker, serum alanine transaminase (ALT) and TUNEL-positivity indicative of liver cell death. Our another unexpected finding was that the lowest used concentration there was another peak of ALT elevation. However, this one was transient and was not associated with any significant liver cell death measured by TUNEL assay. It was suggestive of a transient liver cell dysfunction rather than actual; organ damage. Because the concentration of CNTs in tomato fruits is expected to be 2-3 orders of magnitude lower than 10 mg/kg, it was concluded the concentrations of CNTs found in tomato fruits are likely to be non-toxic. Tomato organs collected from plants grown in medium supplemented with CNT were provided by Dr. Khodakovskaya for further in vivo experiments. Overall Conclusion: All planned experiments (year N1) for each objective were fully performed. We have established plant samples for a whole project using hydroponics system supplemented with CNT. The exact amount of CNT inside of CNT-exposed plant organs was estimated and a map of distribution in CNT inside plant organs was generated. We monitored toxicity of pure CNT in a wide range of concentrations using mouse model system. Data established in the vivo experiments indicated that a single oral gavage of CNTs induced mild and transient liver injury only at the highest used dose of 10 mg/kg. Therefore, the concentrations of CNTs found in different tomato organs are likely to be non-toxic. We can conclude that we have achieved significant progress toward the goals of projects.
Publications
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