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Methods for controlled preparation and dosing of microplastic fragments in bioassays.

ABSTRACT: Microplastic fragments (microfragments) are among the most abundant microplastic shapes found in marine ecosystems throughout the world. Due to their limited commercial availability, microfragments are rarely used in laboratory experiments. Here a novel method of microfragment production has been developed and validated. Polyethylene and polypropylene plastic stock (2 and 3 mm thick respectively) was ground using a cryomill, washed, and rinsed through a stack of sieves. Microfragments were prepared at three distinct size classes (53-150, 150-300, 300-1000 μm) and were confirmed to be accurate and consistent in size. Employing a novel ice cap dosing technique, microfragments were accurately dosed into experimental vials while excluding headspace, facilitating particle suspension without the aid of chemical surfactants. A proof of principle ingestion experiment confirmed the bioavailability of 53-150 μm polyethylene microfragments to brine shrimp Artemia sp. Together, these methods provide a controlled way to produce and dose microplastic fragments for experimental and analytical research.

SUBMITTER: Boettcher H

PROVIDER: S-EPMC10063547 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Methods for controlled preparation and dosing of microplastic fragments in bioassays.

Boettcher Hayden H Kukulka Tobias T Cohen Jonathan H JH

Scientific reports 20230330 1

Microplastic fragments (microfragments) are among the most abundant microplastic shapes found in marine ecosystems throughout the world. Due to their limited commercial availability, microfragments are rarely used in laboratory experiments. Here a novel method of microfragment production has been developed and validated. Polyethylene and polypropylene plastic stock (2 and 3 mm thick respectively) was ground using a cryomill, washed, and rinsed through a stack of sieves. Microfragments were prepa ...[more]

PMID: 36997607

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Project description:Polypropylene (PP), polystyrene (PS), and polyethylene (PE) plastics are commonly used in household items such as electronic housings, food packaging, bottles, bags, toys, and roofing membranes. The presence of inhalable microplastics in indoor air has become a topic of concern as many people spent extended periods of time indoors during the COVID-19 pandemic lockdown restrictions, however, the toxic effects on the respiratory system are not properly understood. We examined the toxicity of PP, PS, and PE microplastic fragments in the pulmonary system of C57BL/6 mice. For 14 days, mice were intratracheally instilled 5 mg/kg PP, PS, and PE daily. The number of inflammatory cells such as macrophages, neutrophils, and eosinophils in the bronchoalveolar lavage fluid (BALF) of PS-instilled mice was significantly higher than that in the vehicle control (VC). The levels of inflammatory cytokines and chemokines in BALF of PS-instilled mice increased compared to the VC. However, the inflammatory responses in PP- and PE-stimulated mice were not significantly different from those in the VC group. We observed elevated protein levels of toll-like receptor (TLR) 2 in the lung tissue of PP-instilled mice and TLR4 in the lung tissue of PS-instilled mice compared with those to the VC, while TLR1, TLR5, and TLR6 protein levels remained unchanged. Phosphorylation of nuclear factor kappa B (NF-κB) and IĸB-α increased significantly in PS-instilled mice compared with that in VC. Furthermore, Nucleotide‑binding oligomerization domain‑like receptor family pyrin domain‑containing 3 (NLRP3) inflammasome components including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and Caspase-1 in the lung tissue of PS-instilled mice increased compared with that in the VC, but not in PP- and PE-instilled mice. These results suggest that PS microplastic fragment stimulation induces pulmonary inflammation due to NF-ĸB and NLRP3 inflammasome activation by the TLR4 pathway.Supplementary informationThe online version contains supplementary material available at 10.1007/s43188-023-00224-x.

Project description:Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results: We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion: RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. Keywords: Whole Blood, Protocol Variation, Expression Profiling, Microarray, globin, PAXGene, PNA

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Methods for controlled preparation and dosing of microplastic fragments in bioassays.

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Methods for controlled preparation and dosing of microplastic fragments in bioassays.

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