Research paperEnhanced reproductive toxicities induced by phthalates contaminated microplastics in male mice (Mus musculus)
Graphical Abstract
Introduction
Due to the ubiquitous occurrence and notorious environmental ecological risks, particularly in aquatic environment, microplastics (MPs) have raised worldwide attention (Ivar do Sul and Costa, 2014, Wright et al., 2013, Law and Thompson, 2014). For example, MPs have been frequently detected in different water bodies with concentrations range from 1.0 × 10−2 to 1.0 × 108 particles/m3 (Ivar do Sul and Costa, 2014, Koelmans et al., 2019, Mason et al., 2018). Simultaneously, various of adverse effects have been identified in aquatic organisms after exposure to MPs (Ivar do Sul and Costa, 2014, Anbumani and Kakkar, 2018). Compared with aquatic organisms, the study on the health risks of MPs to terrestrial animals is still in its infancy (de Souza Machado et al., 2018), although increasing evidences have presented that MPs could be ingested by human and other terrestrial animals via multiple ways (i.e. drinking, earing, or breathing) (Wright and Kelly, 2017). Previous studies have shown that MPs could accumulate not only in human intestinal epithelial cells (Wu et al., 2019), but also in the gut, liver, and kidney of mouse (Deng et al., 2017a, Jin et al., 2019). A few toxic effects involved in gene expression and histopathology changes have been observed in terrestrial animals after exposed to MPs (Deng et al., 2020a). For example, MPs could not only affect the expression of genes related to oxidative stress, immune response, and mucin secretion in mouse gut (Deng et al., 2020b), but also affect the expression of genes related to transport, synthesis, and signaling of bile acids in mouse liver (Lu et al., 2018). Moreover, the metabolites related to oxidative stress and lipid metabolism in the serum and liver of mouse (Deng et al., 2017a, Lu et al., 2018), as well as pathological changes in the liver and gut were also identified after exposure to MPs (Deng et al., 2017a, Deng et al., 2020b, Jin et al., 2019). These findings suggest that MPs have the potential to induce the enterotoxicity and hepatotoxicity in terrestrial mammals.
The above adverse effects on mice indicated widespread health risks of MPs, however, little attention has been paid to the reproductive toxicity of MPs on terrestrial mammals. Previous studies suggested that micron-size particles are difficult to enter the productive tissues of mice (Lan and Yang, 2012). In particularly, due to the protection of blood-testis barrier (BTB), few MPs can penetrate into the testis (Lan and Yang, 2012). However, two recent studies have identified that MPs (≤10 µm) could enter the testis of mouse and rat, and reduce the sperm quality and testosterone level (Jin et al., 2020, Amereh et al., 2020). Moreover, it has also been reported that MPs could induce inflammation and abnormal testicular spermatogenesis in mouse testis (Hou et al., 2020). Thus, the potential reproductive toxicity of MPs should not be ignored. Nevertheless, the information about the potential reproductive toxic effects induced by MPs is still rare.
MPs can adsorb pollutants from surrounding environments and carry these pollutants into aquatic organisms (Engler, 2012, Ziccardi et al., 2016). However, whether MPs could induce significant accumulation of pollutants in tissues is still controversial (Ziccardi et al., 2016). For instance, nano-size MPs could enhance bioaccumulation of phenanthrene-derived residues in daphnid body, whereas no enhanced bioaccumulation was induced by micron-size MPs (Ma et al., 2016). In addition, more complex body structures of terrestrial mammals would increase the uncertainty of this controversial issue. Therefore, the distribution of toxic chemicals in aquatic animals transported by MPs were not suitable for directly applying to the terrestrial mammals. Meanwhile, increasing studies are conducted to explore the consequent toxicities of co-exposure to MPs and other pollutants (Browne et al., 2013, Qiao et al., 2019). Although most of these studies demonstrated that MPs could enhance the toxicity of pollutants (Ivar do Sul and Costa, 2014), some other studies presented that no significant changes were induced after the co-exposure comparing with individual exposure of MPs and pollutants (Tang et al., 2020, Oliveira et al., 2013). These inconsistent conclusions may be ascribed to the distribution of MPs in tissues which deserve profound investigation.
Recently, we have found that polyethylene (PE) MPs (45–53 µm) could transport phthalate esters (PAEs) into mouse gut and induce aggravated adverse effects (Deng et al., 2020b). In addition, MPs with relatively small particle sizes could enter to multiple mammal tissues, such as liver and testis, via endocytosis or persorption (Wright and Kelly, 2017). Moreover, smaller size of MPs have larger specific surface area and have potential to transport more PAEs into tissues (Liu et al., 2018). As notorious environmental endocrine disrupters, PAEs can cause reproductive toxicities in male animals (Meeker et al., 2007). Therefore, PAE-contaminated MPs are very likely to cause serious reproductive toxicity.
In this study, we propose the hypothesis that the distribution of MPs in tissues would affect the bioaccumulation and reproductive toxicity of PAEs in mice. To prove our hypothesis, commercial polyethylene (PE) particles with a size range of 0.4–5 µm were chosen as model MPs. MPs with this size range can enter the circulatory system through the digestive tract (Lee et al., 2013, Jeong et al., 2016). PE-MPs are frequently detected in the environment and have a high adsorption capacity for organic pollutants (Ivar do Sul and Costa, 2014, Bakir et al., 2012). The mixture of four PAEs, including di-(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), diethyl phthalate (DEP) and dimethyl phthalate (DMP), and DEHP were selected as target pollutants because of their reproductive and development toxicities on male mammals (USEPA, 2007, USEPA, 2014) and their accumulations in mouse gut (Deng et al., 2020b). MPs pre-adsorbed with DEHP or PAE mixture, termed as DEHP- or MIX-contaminated MPs, were used for the exposure experiments. Male CD-1 mice were chosen as a typical terrestrial mammal model. The adsorption of PAE on MPs, bioaccumulation of PAE and distribution of MPs in tissues were determined. Furthermore, indicators of sperm physiological, spermatogenesis, and oxidative stress, as well as transcriptomic profiles of testis were analyzed to evaluate the potential reproductive toxicity of PAE-contaminated MPs male mice.
Section snippets
MPs and PAEs
Virgin PE MPs spheres with a size range of 0.4–5 µm were purchased from Cospheric (Santa Barbara, CA, USA). The morphology and size of MPs was confirmed by a Scanning Electron Microscopy (SEM, Hitachi, Tokyo, Japan) and a Laser Scattering Particle Size Distribution Analyzer (LSPSDA, Horiba, Tokyo, Japan). Additionally, the composition of MPs was confirmed by Fourier Transform Infrared Spectroscopy (FTIR, Nicolet, Waltham, MA, USA). The standards of DEHP, DBP, DEP, and DMP were purchased from
MPs characterization
According to SEM analysis, the MPs used in this study were spheres (Fig. S1), and 95% of MPs ranged from 0.4 to 3.2 µm with one major peak at 1.4 µm and 24.50% of MPs were smaller than 1 µm (Fig. S2). FTIR spectroscopy confirmed that the polymer type of MPs was polyethylene (Fig. S3). These MPs met the design requirements of this study.
DEHP- and MIX-contaminated MPs
After incubation, DEHP and PAE mixture was respectively detected on the MPs, indicating that the DEHP- and MIX-contaminated MPs have been successfully prepared (
Discussion
Here we show that MPs with a size range of 0.4–5 µm could induce significant accumulation of PAEs in the gut and liver of mice. More importantly, compared with MPs and PAEs alone, enhanced reproductive toxicities including sperm physiological alterations and spermatogenesis disorders were observed in the testis after exposure to PAE-contaminated MPs. This is the first suitably controlled experimental evidence showing that MPs could transport PAEs into multiple tissues of mice and induce
Conclusion
In this study, we investigated the bioaccumulation of PAEs and reproductive toxicity due to contaminated MPs exposure. Compared with virgin MPs and PAE alone, PAE-contaminated MPs notably increased the accumulation of PAEs in the gut and liver of mice, while no significantly increase in accumulation of PAEs was observed in the testis. At the meanwhile, PAE-contaminated MPs caused enhanced reproductive toxicities including sperm physiological alterations, spermatogenesis disorders and oxidative
CRediT authorship contribution statement
Yongfeng Deng and Yan Zhang designed the research. Yongfeng Deng and Zehua Yan performed the experiments. Yongfeng Deng, Ruqin Shen and Yan Zhang analyzed the data. Yongfeng and Yan Zhang wrote the paper. Yichao Huang and Hongqiang Ren edited the language.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by National Natural Science Foundation of China (21777068, 21507058, and 21906066), and China Postdoctoral Science Foundation (No. 2019M653280).
Author contributions
Y.F.D. and Y.Z. designed the research. Y.F.D., Z.H.Y., and R.Q.S performed the experiments. Y.F.D. and Y.Z, analyzed the data. Y.F.D., Y.Z., Y.C.H and R.H.Q. wrote the paper.
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ORCID ID: 0000–0002-4762–6639.