Investigations of acute effects of polystyrene and polyvinyl chloride micro- and nanoplastics in an advanced in vitro triple culture model of the healthy and inflamed intestine

Highlights

• Differences were seen in relation to the models' health status.

• During inflammation, PVC caused increased IL-1β levels and loss of epithelial cells.

• PS-NH₂ nanoparticles induced cytotoxic effects in healthy state.

• PS nanoparticles did not cause any effects in healthy or inflamed state.

Abstract

The continuous degradation of plastic waste in the environment leads to the generation of micro- and nanoplastic fragments and particles. Due to the ubiquitous presence of plastic particles in natural habitats as well as in food, beverages and tap water, oral exposure of the human population with plastic particles occurs worldwide. We investigated acute toxicological effects of polystyrene (PS) and polyvinyl chloride (PVC) micro- and nanoparticles in an advanced in vitro triple culture model (Caco-2/HT29-MTX-E12/THP-1) mimicking the healthy and inflamed human intestine to study the effect of inflammatory processes on plastic particle toxicity. We monitored barrier integrity, cytotoxicity, cell layer integrity, DNA damage, the release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and mucus distribution after 24 h of particle exposure. In addition, we investigated cytotoxicity, DNA damage and IL-1β release in monocultures of the three cell lines. Amine-modified polystyrene nanoparticles (PS–NH₂) served as a positive control for particle-induced toxicity. No acute effects in the investigated endpoints were observed in the model of the healthy intestine after PS or PVC exposure. However, during active inflammatory processes, exposure to PVC particles was found to augment the release of IL-1β and to cause a loss of epithelial cells. Our results suggest that prevalent intestinal inflammation might be an important factor to consider when assessing the hazard of ingested micro- and nanoplastic particles.

Introduction

Environmental pollution with mismanaged waste is a major problem of our modern society. About 10% of discarded waste is plastic, which accumulates in the environment due to its highly persistent nature (Barnes et al., 2009). Borrelle et al. (2020) estimated that up to 23 million metric tons of plastic waste entered aquatic ecosystems in 2016. Although plastics are generally appreciated for their resistance to degradation, environmental factors like UV radiation and mechanical abrasion can cause the fragmentation of plastic polymers into smaller particles known as micro- and nanoplastics (Andrady, 2012; Julienne et al., 2019). These terms describe a large, heterogeneous group of plastic fragments of different chemical composition in size ranges below 5 mm or 1000 nm, respectively (Vianello et al., 2013; Gigault et al., 2016; Lambert and Wagner, 2016). The continuous degradation and accumulation of plastic nanoparticles might increase the health hazard concerning plastic particles, due to the higher biological availability and reactivity of nanoparticles compared to microparticles (Park et al., 2011; Bouwmeester et al., 2015). Through uptake by aquatic and terrestrial organisms, plastic particles are introduced into the food chain. As reviewed by Barboza et al. (2018), many studies showed the presence of microplastic particles in various fish and shellfish. Furthermore, contamination with microplastics was demonstrated in other foods and beverages (Kosuth et al., 2018), honey (Liebezeit and Liebezeit, 2015) and salt (Karami et al., 2017). Most importantly, plastic particles were found in drinking water of various sources (Marsden et al., 2019; Mintenig et al., 2019; Shruti et al., 2020; Tong et al., 2020; Zhang et al., 2020) and Schwabl et al. (2019) reported the presence of microplastics in human stool samples collected from eight individuals from Europe and Asia. This has led to the reasonable assumption that oral exposure of the human population with plastic particles is present worldwide. Recent animal studies in mice and zebrafish have identified the gastrointestinal tract (GIT) as a possible target organ for plastic particle-induced toxicity, linking the oral uptake of microplastics with intestinal inflammation, microbial dysbiosis and reduced mucus secretion (Lu et al., 2018; Li et al., 2019; Qiao et al., 2019).

Inflammatory bowel diseases (IBD), namely Crohn's disease and ulcerative colitis, are modern western diseases with an estimated prevalence of 0.3% in Europe and 1.3% in the US (Burisch et al., 2013; Dahlhamer et al., 2016). IBD is marked by a chronically inflamed GIT and is thought to result from a multi-factorial interaction involving genetics, diet and microbial dysbiosis, yet its precise etiology is still unknown (Cho and Brant, 2011). IBD is characterized by increased levels of pro-inflammatory cytokines (Neurath, 2014), cytotoxicity (Kappeler and Mueller, 2000), oxidative DNA damage (Pereira et al., 2016), impairment of intestinal barrier integrity (Maloy and Powrie, 2011), the decline of goblet cells (Strugala et al., 2008) and an increased risk of colon cancer (Kim and Chang, 2014).

Only limited studies are available regarding toxicological effects of micro- and nanoplastics in the GIT, and a prevalent intestinal inflammation appears to be an overlooked potential risk factor. Major advancements in the development of intestinal in vitro models and multifactorial testing strategies provide highly advanced model systems of the human GIT and sophisticated approaches for nanosafety research (Kämpfer et al., 2020a). Still, the majority of in vitro studies investigating intestinal plastic particle toxicity is restricted to simple single cell line experiments (Thubagere and Reinhard, 2010; Forte et al., 2016; Riebeling et al., 2018; Wu et al., 2020; Yan et al., 2020). On the other hand, studies using advanced in vitro co-culture models of the GIT focused on the transfer across the gastrointestinal barrier (Walczak et al., 2015; Abdelkhaliq et al., 2018) or cellular uptake (Stock et al., 2019) instead of intestinal toxicity. Most of these studies exclusively used polystyrene (PS) as model particles and some were carried out in the context of polymer-based nanoparticle drug delivery instead of nanoplastic hazard assessment.

To address this knowledge gap regarding human health risks, we investigated the acute hazards of micro- and nanoplastics in the context of inflammation. We used an advanced in vitro triple culture model of the healthy and inflamed intestine, which we recently developed (Kämpfer et al., 2020b), to investigate possible effects of PS and polyvinyl chloride (PVC) micro- and nanoplastic particles on the healthy or inflamed gut. The model is composed of the human cell lines Caco-2, as a model of enterocytes, HT29-MTX-E12, as a model of mucus-producing goblet cells, and PMA-differentiated THP-1 cells representing macrophages. To mimic an intestinal inflammation, the THP-1 cells were activated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ).