Toxicity and Biodistribution of Fragmented Polypropylene Microplastics in ICR Mice

Currently, polypropylene (PP) is used in various products, thus leading to high daily exposure in humans. Thus, it is necessary to evaluate the toxicological effects, biodistribution, and accumulation of PP microplastics in the human body. In this study, administration of two particle sizes of PP microplastics (approximately 5 and 10–50 µm) did not lead to any significant changes in several toxicological evaluation parameters, including body weight and pathological examination, compared with the control group in ICR mice. Therefore, the approximate lethal dose and no-observed-adverse-effect level of PP microplastics in ICR mice were established as ≥2000 mg/kg. Furthermore, we manufactured cyanine 5.5 carboxylic acid (Cy5.5-COOH)-labeled fragmented PP microplastics to monitor real-time in vivo biodistribution. After oral administration of the Cy5.5-COOH-labeled microplastics to the mice, most of the PP microplastics were detected in the gastrointestinal tract and observed to be out of the body after 24 h in IVIS Spectrum CT. Therefore, this study provides a new insight into the short-term toxicity, distribution, and accumulation of PP microplastics in mammals.

1. Introduction

Globally, plastic usage continues to increase [1]. This excessive use of plastic adds to environmental pollution, thus affecting humankind [2,3,4]. Plastic discarded in the environment is broken into small particles by physical and chemical environmental factors such as weathering, erosion, heat, and ultraviolet rays [5,6,7]. Plastic particles that are smaller than 5 mm are called microplastics [8]. Various microplastics such as polypropylene (PP) and polystyrene (PS) have been detected in the Central Atlantic Ocean in Morocco and the Northwestern Pacific Ocean [9,10]. There are reports of microplastics detected not only in the oceans but also in the soil and atmosphere of Iran [11,12]. Animals living in a microplastic-contaminated environment are exposed to microplastics through inhalation or ingestion [13]. There is much research on the detection of microplastics in living organisms. Microplastics have been detected in mussels, fishes in freshwater, and even in the gastrointestinal tracts of birds [14,15,16,17,18]. Moreover, there have been recent reports of multiple microplastics in human blood [19]. This evidence in the environment, organisms, and humans is directly related to the risk of microplastics.

Recently, a number of in vitro and in vivo studies have been published to confirm the potential risk of microplastics. Polyethylene (PE) and PS microplastics have been reported to interfere with cell viability, inflammation, and oxidative stress mechanisms. Interestingly, PS caused hepatotoxicity and impaired fat metabolism in liver organoids [20,21]. Additionally, in BEAS-2B, a human bronchial epithelial cell, an increase in cytotoxicity and reactive oxygen species production has been confirmed [22]. These cellular level studies suggest that microplastics primarily affect mechanisms associated with inflammation, oxidative stress, and physiological dysfunction of the system.

Microplastic studies on mammals are mainly conducted using rodents. Microplastics have been detected in the brain of mother mice after oral administration, and behavioral changes and increased autism-related factors in the fetus of mice have been reported [23]. It has been reported that microplastic administration affected flora–metabolite–cytokine axes regulation through hematopoietic system damage [24]. Recently, granulomatous inflammation has been observed in the lungs of mice during repeated oral administration of PE microplastics [25]. It has been reported that microplastic administration affects the expression of microglial differentiation markers along with the activation of NF-κB, thus regulating microglial immune activation in the mouse brain [26]. To assess the health risk of microplastics to the human body, research on microplastics using animals, especially mammals, is increasing. However, most studies are being conducted on specific systems. To overcome this limitation, it is necessary to evaluate the health risks of microplastics through toxicity and pathological analyses of various systems in vivo. In particular, further research on the biodistribution and accumulation of microplastics in vivo is needed. Substances that enter the body from the outside undergo the processes of absorption, distribution, metabolism, and excretion (ADME), and studies on ADME of microplastics are extremely rare [27]. The microplastics that are used to evaluate in vivo biodistribution are mainly spherical, which are labeled with fluorescent materials or tagged with radioactive isotopes [28,29,30]. Some studies have confirmed that spherical labeled or tagged microplastics move toward the liver, kidneys, and genital organs of mice [31,32]. However, microplastics that living organisms are exposed to from the environment have many more fragments or fibers than spherical ones. Therefore, it is necessary to check the biodistribution and accumulation levels by labeling fluorescent materials on microplastics in the form of fragments or fibers. Fluorescent dye labeling is a general strategy for studying the biodistribution of microplastics [33].

The combined swelling–diffusion (CSD) synthesis is a common method to prepare fluorescent nanoplastics and microplastics that entails the entrapment of fluorescent materials inside a microplastic by controlling the temperature or solvent combination [34]. The fluorescent dye used in this study, cyanine 5.5 carboxylic acid (Cy5.5-COOH), is commonly used in bioimaging and disease diagnosis due to its excellent spectral properties, including narrow absorption spectrum, and high sensitivity and stability [35]. PP plastics were chosen for this study because of their cost-effectiveness and common and wide usage in daily life [36,37]. They are used for various purposes, including microwave containers, label films for plastic bottles, and paper money [38,39,40]. There are higher chances of PP being exposed to the human body through the oral cavity as it is widely used in food containers and in various objects in everyday life including ropes, twine, tape, upholstery, clothing, camping equipment, etc. In this study, we evaluated the toxicity and biodistribution of two different particle sizes of fragmented PP microplastics in ICR mice. First, two suitable sizes of fragmented PP microplastics were prepared. The approximate lethal dose (ALD) and no-observed-adverse-effect level (NOAEL) were derived by performing a single and 4-week repeated toxicity test recommended by OECD guidelines using the prepared microplastics. Additionally, PP microplastics labeled with Cy5.5-COOH were used to study their biodistribution and accumulation in mice. From these results, we evaluated the toxicity and biodistribution of the PP microplastics in mice.