Performing Department
(N/A)
Non Technical Summary
Micro-nanoplastics (MNPs) are increasingly found in natural environments and in food and beverages consumed by humans. Recent studies have shown that MNPs can adsorb to plant surfaces and enter plant tissues, and that the presence of MNPs in soil adversely affects plant health and also alter plant uptake of other chemical environmental pollutants (EPs). Humans ingest an estimaged 5 g (one credit card) of MNPs per week and ingested MNPs can be taken up by the intestine and distributed to all organs and tissues throughout the body. The purpose of this project is to better understand the implications of MNP exposures on food crops and the transmission of MNPs and associated EPs to humans through ingestion of the edible portions of crops. We will create several relevant model MNPs for testing using degradation platforms that simulate incineration and environmental degradation of waste plastic. We will then systematically assess the interactions of these model MNPs with toxic EPs, including a typical toxic heavy metal (arsenic), a heavily used pesticide (boscalid), and a common perfluoroalkyl/polyfluoroalkyl substance (PFOS). To allow quantification of MNPs in our plant tissue and cellular samples the project includes the development of a "pyrolysis gas chromatography-mass spectrometry (Py-GCMS)" method that will allow robust quantitative analysis of MNPs in our plant and cellular samples; We will then assess the responses to and uptake of MNPs and MNP effects on EP uptake in model plants (lettuce and wheat) and further assess the uptake of MNPs and impact of MNPs on EP uptake by the human small intestine, using a cellular small intestinal epithelial model. This project will provide important data on the environmental health implications of MNPs, which will help to avoid significant adverse effects in the future and to identify policy measures that should be adopted to curb plastics pollution.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
Proposed Research: In this project we will develop model environmentally relevant micro-nano-plastics using state of the art life cycle material degradation platforms. We also study MNPs as carriers of other EPs to evaluate their potential to increase the bioavailability of inorganic and organic pollutants in plants and in the human intestine. We hypothesize that the effects of MNPs on bioavailability of EPs are dependent on both the size and polymer of the MNP. The two model polymers to be studied (PVC and PE) are of particular relevance in agriculture, since PVC is a plastic material widely used to construct hydroponic systems, while films of PE are vastly utilized as covers in open fields to insulate soil.The project consists of three interconnected AIMs:Aim 1: Synthesis, Characterization, and Environmental Pollutant (EP) Interactions of Model Environmental MNPs. A major roadblock in bioactivity studies of MNPs is the lack of environmentally relevant MNPs, which has limited previous studies to primary MNPs such as polystyrene beads. Such simplistic model MNPs are not environmentally relevant in terms of physicochemical and morphological properties and the associated bioactivity data are of limited utility for risk assessment. We will use existing life cycle material degradation platforms at NAMC to generate environmentally relevant reference MNPs of two highly produced polymers commonly used in agricultural applications, polyethylene (PE) and polyvinyl chloride (PVC). These platforms include: 1) A thermal degradation system that will be used to simulate municipal incineration of plastics; and 2) A combined cryomilling and UV/photo-ageing platform that will be used to simulate photo-oxidative and mechanical degradation of plastics in the environment. These model MNPs will be fully characterized using state-of-the-art analytical methods available in our labs. We will also assess the sorption and concentration of EPs by the model MNPs from water containing environmentally relevant concentrations of a model PFAS (PFOS), a model pesticide (boscalid), and toxic metals (As, Cr, Pb).Aim 2: Assessment of Accumulation and Impact of MNPs and Sorbed EPs in Plants Under Hydroponic and Greenhouse Soil Conditions. We will study environmentally relevant MNPs and size-effects on the bioavailability of inorganic (As, Cr, Pb) and organic (boscalid and PFOS) compounds. Plants will be grown in hydroponics and soil and exposed to MNPs generated in Aim 1, in the presence or absence of EPs. Lettuce and wheat will be used as model monocot and dicot species, and our system will evaluate effects in both hydroponic and greenhouse soil systems for each. Environmentally relevant concentrations of MNPs and EPs will be used to derive dose-response relationships. In addition, the NJIT team will develop a pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) method for the robust analysis and quantification of secondary MNPs as part of this Aim.Aim 3: Assessment of Uptake and Translocation/Bioavailability of MNPs and Sorbed EPs in a Triculture Small Intestinal Epithelium. We will use a three-phase (oral, gastric, small intestinal) simulated digestion coupled with a transwell triculture cellular model of the small intestinal epithelium to assess the health effects of MNP ingestion. We will assess intestinal epithelial cellular uptake and translocation of the model MNPs alone, as well as the effect of MNPs on the bioaccessibility and bioavailability of EPs. Cellular and molecular mechanisms that could underlie or lead to impaired intestinal boundary function and increased EP absorption in the presence of MNPs will be assessed
Project Methods
Aim 1: MNP Synthesis and Characterization:Incinerated MNPs will be generated using our INEXS system with standard protocols routinely used in our lab, size fractionated using the Compact Cascade Impactor (CCI), and extracted in water.Cryomilled and UV-aged MNPs: A Retsch Mixer Mill MM 400 will be used to produce cryomilled particles. The resulting powder will be sieved in Retsch AS 200 fractionator to collect the PM3.0 fraction. This will then be UV/photo-aged using a Q-SUN Xe-1 Xenon Test Chamber system to reproduce 10 y of environmental exposure using ISO and ASTM standard protocols.Characterization of MNPs: Surface elemental analysis will be by XPS, surface groups by FTIR, density by pycnometry, SSA and porosity by BET, size, morphology, and elemental composition SEM/TEM with EDX, size distribution by MALD and DLS, and zeta potential by ELS. Characterization of MNP-EP Interactions: MNPs will be incubated 48 h in EP cocktail; Sorption will be determined from concentrations of EPs measured in filtrates and residues of the mixtures. EPs will include PFOS at 15 ng/L, Boscalid at 150 ppm, and Cr, As, and Pb, at their EPA-established MCLs. MNPs will be suspended in Milli-Q® water, and spiked with EPs. MNPs will be added at 10 and 100 mg/mL and reacted for 48 h. Suspensions will be filtered at 3 kDa and EPs measured in filtrates/residues to determine partition coefficients. Nano ITC will be used to determine MNP-EP affinity, stoichiometry, and thermodynamics. Metals will be quantified by ICP-MS and PFAS and boscalid by LC-MS.Aim2:Plant Studies:Lettuce and wheat will be grown in standard greenhouse conditions. Seeds will be germinated in germination paper for hydroponic studies (500 mL amber bottles) or directly in pots with 500 g of agricultural loam. MNPs will be mixed with the EPs at 0, 5, 25, and 50 mg/L in the hydroponics system and 0, 10, 50 and 200 mg/kg in soil; As, Cr, Pb, and boscalid will be added at 0.5 mg/L each in the hydroponic system and 1 mg/kg each in soil, and PFOS will be added at 5 µg/L in the hydroponic and 50 µg/kg in the soil system. Controls will include plants without EPs, plants with MNPs only, and plants with EPs only. The following analyses will be conducted to assess phytotoxicity and crop health/nutritional status as a function of MNP and/or EP exposure, and uptake of MNPs and EPs by plant tissues:Wet/dry biomass of root, shoot/stem, leaf, and grain tissues will be measured at 50% of life cycle.Transpiration and photosynthetic potential will be measured using a PhotosynQ system every 2 w during exposure.Total chlorophyll and pigment content will be determined at harvest by standard CAES protocols.Oxidative stress response in root/shoot tissue will be assessed by lipid peroxidation (malondialdehyde formation). Total ROS, superoxide dismutase, catalase, peroxidase, glutathione reductase, ascorbate peroxidase, protein, amino acids and glutathione will be determined by CAES standard assays.Element content of plant tissues will be determined by ICP-OES or ICP-MS after acid digestion. Nitrogen will be quantified on a Leco Nitrogen Determinator. 10 macro/micronutrients will be measured using a standard CAES analysis protocol.Boscalid and PFOS content will be determined using methods developed under the CAES ISO/IEC 17025:2017 food safety program.Molecular analysis: RNA will be isolated using the Qiagen RNAeasy mini Plant RNA isolation kit and converted to cDNA library using commercial kits. Regulation of candidate genes will be studied by qPCR.MNP concentrations in plant tissues will be analyzed by Py-GC-MS.Visualization and localization of MNPs in plant tissue sections will be performed using Enhanced Darkfield Hyperspectral Microscopy (EDHM).Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) Method for Quantification of MNPs in Environmental and Biological Samples:A Thermo Single Quadrupole Mass spectrometer ISQ LT model and Trace 1310 Gas chromatograph will be retrofitted with a Shimadzu pyrolyzer or GC-MS 2020NX with EGA/PY-3030D Multi-Shot Pyrolysis and autosampler option.Verifying effectiveness of peroxide digestion: Produce will be purchased from local markets. Labware containing plastics orinorganic carbonwill be avoided to minimize contamination. Containers and equipment will be pre-washed with UPLC-MS Grade water and then acetone. Produce will be washed with UPLC-MS Grade water and blended with a plastic-free blender. For soil samples, we will follow USDA and EPA sampling guidelines. Initial testing will be done using Standard Reference Materials from NIST.Quantifying MNPs in samples by Py-GC-MS: We will employ double-shot pyrolysis, first placing the sample in a pyrolyzer unit at 100 oC and heating to 300 oC at a rate of 50 oC /min to desorb unpolymerized monomers or additives, then performing second-shot pyrolysis at a higher temperature range to determine target polymer concentrations. For quality control, we will perform test analyses of procedural blanks, spiked blanks, replicate samples, and spiked produce samples along with the original samples.Aim3:Simulated Digestion:Simulated digestion: Water suspensions of MNPs at 50, 250, and 1000 µg/mL will undergo sequential simulated oral, gastric, and small intestinal digestion using standard procedures in the Demokritou lab.MNP biotransformations: Size distributions of particles in each phase will be obtained using MALD and NTA. Morphology and surface bio-transformations will be assessed by SEM/TEM with XPS.MNP Uptake and Translocation of MNPs and EPs: Triculture epithelium will be grown on 24 mm 3.0 µm pore transwell inserts as per standard procedures in our lab.Exposures: Digestas of MNPs will be applied to the apical chambers of transwells while fresh media is applied to the basolateral chambers before 4 or 24 h incubation. Assessment of MNP uptake and translocation: MNPs will be quantified in cell lysates and basolateral media by Py-GC-MS (described in Aim 2) to determine uptake and translocation.Assessment of EP bioavailability: EPs will be quantified in cell lysates and basolateral fluid by LC-MS and ICP-MS to determine EP bioavailability with and without MNPs.Visualization/Localization of MNPs in tricultures will be performed using EDHM.Mechanisms of MNP uptake and translocation: Digestas of 250 µg/mL MNPs will be applied to the apical chamber of triculture transwells after treatment with inhibitors or siRNA knockdown, and incubated for 4 or 24 h, with MNP uptake/translocation assessed as described above.Barrier Function will be assessed by introducing fluorescent 3 and 10 kDa dextran to the apical transwell chamber and measuring fluorescence in the basolateral chamber after 4 h.Membrane Damage will be assessed by measuring LDH in apical fluid using a standard colorimetric assay.Oxidative Stress: ROS will be assessed after 4 and 24 h exposure using CellROX Green.Inflammation: Apical fluid samples after 24 h exposures will be analyzed for cyto/chemokines using a Human Primary Cyto/chemokine 41-Plex Panel.Inhibitor Studies: Tricultures will be treated with inhibitors at concentrations and for durations optimized in an intial pilot study. Tricultures will then be exposed to MNP/MNP-EP digestas and uptake and translocation assessed after 4 and 24 h.Knockdown Studies: We will employ Silencer Select siRNAs (Thermo). Cells will be transfected using Lipofectamine RNAiMAX three days prior to seeding transwells. Knockdowns will be verified by western blot. Uptake/translocation of each knockdown and control will be assessed after 4 and 24 h.