Oral Co-Exposures to zinc oxide nanoparticles and CdCl₂ induced maternal-fetal pollutant transfer and embryotoxicity by damaging placental barriers

Highlights

• Co-exposure of ZnO NPs/Cd²⁺ during organogenesis enhanced the maternal-fetal transfer of both pollutants.

• Combined pollutants aggravated embryotoxicity after organogenesis exposures.

• Placental tight junction disruption was attributed to the enhanced maternal-fetal transfer and embryotoxicity.

Abstract

Synergistic toxicity from multiple environmental pollutants poses greater threat to humans, especially to susceptible pregnant population. Here we evaluated combined toxicity from environment pollutants zinc oxide nanoparticles (ZnO NPs) and cadmium chloride (CdCl₂) using two pregnant mice models established by oral administration during peri-implantation or organogenesis period. We found that exposures to combined pollutants only at organogenesis stage induced higher fetal deformity rate compared to co-exposures at peri-implantation stage. We further discovered that surface charge of ZnO NPs were modified after Cd²⁺ adsorption and the resulting nanoadducts caused more severe damages in placental barriers by causing shed endothelial cells and decreased expressions of tight junction proteins ZO1, occludin, claudin-4 and claudin-8. These cellular and molecular events enhanced maternal-fetal transfer of both pollutants and aggravated embryotoxicity. Our findings help elucidate synergistic embryotoxicity by nanoparticle/pollutant adducts and establish proper safety criteria for pregnant population in an era that nanotechnology-based products are widely used.

Introduction

Broad applications of nanotechnology and commercialization of nanomaterial-containing consumer products have led to an increased environmental release of nanoparticles. For example, zinc oxide nanoparticles (ZnO NPs) have been widely used as sunscreens (Osmond and McCall, 2010) that can easily release into water environment. Uptake of ZnO particles, or dissolved Zn from the particles may lead to an elevated Zn level in the internal organs while the absorption, distribution, metabolism and excretion (ADME) of nanoparticles is not well understood. Due to their small size and large surface areas, ZnO NPs adsorb various environmental pollutants (Deng et al., 2017). Environmental ZnO NP concentrations in wastewater treatment plant effluent were as high as 45 μg/L (Sun et al., 2014). ZnO NP concentrations in surface water were found to be as high as 74 μg/L in US (Abbott Chalew et al., 2013) and 1.84 μg/L in Singapore (Majedi et al., 2012). With increasing production volume and applications of ZnO NPs, the environmental concentration of ZnO NPs will be inevitably increased (Zhang et al., 2017). Currently drinking water treatment facilities are not equipped to remove nanomaterials. The risk of oral exposures to such nanoparticle/pollutant in human has becoming eminent. Cd²⁺ is one of the most toxic heavily metal pollutants in environmental waters. Oral exposures to Cd²⁺ may cause injuries in respiratory system, liver, kidney, skeleton, reproductive system, and even cause cancer (Satarug et al., 2010). The reported Cd concentrations in industrial wastewater is high as 3707 μg/L in Saudi Arabia (Al Hamouz et al., 2017) and 970 μg/L in India (Chand et al., 2015). The Cd²⁺ concentration in surface water is as high as 14 μg/L in Singapore (Chai et al., 2017). Interactions of nanoparticles with heavy metal ions may increase bioaccumulation and toxicity of metal ions via a role as carriers, such as Si NPs/Cd²⁺ (Guo et al., 2013) and ZnO NPs/Pb²⁺ (Jia et al., 2017a) co-exposure in mice.

Nanoparticles or nanoparticle/pollutant adducts exposures may induce adverse outcomes to various physiological systems (Bai et al., 2010; Zhang et al., 2014), especially in susceptible populations such as newborn (Zhang et al., 2015a), aged (Wei et al., 2016), lactating (Zhang et al., 2015b), and overweight (Jia et al., 2017a, 2017b) populations. Pregnant females are highly vulnerable because many critical developmental processes are involved during pregnancy such as implantation (Cha et al., 2012), placental formation (Watson and Cross, 2005), and fetal development (Hawkins et al., 2018; Pietroiusti et al., 2011; Yamashita et al., 2011). Knowing that nanoparticles affect the bioaccumulation and toxicity of co-existing contaminants in cells, aquatic organisms and healthy adult mammals (Deng et al., 2017), it is imperative to find out whether pregnant population can tolerate nanoparticle/pollutant adducts at a concentration tolerable to normal people.

The blood-placental barrier plays a crucial role in maintaining a normal fetal development process, while tight junction maintains the integrity of blood-placental barrier. The down-regulations of tight junction-related proteins zonula occludens-1 (ZO1), occludin, or transmembrane proteins claudins disrupt the tight junction in placenta. Previous investigations have shown that relocation and alteration of tight junction proteins were associated with dysfunction of blood-placental barrier (Mutlu et al., 2011; Wang et al., 2012). However, the regulation of tight junction underlying the placental barrier when met with nanoparticles is still poorly understood.

Using ZnO NP/CdCl₂ adducts as an example, in this work we investigated their effects on pregnant ICR mice with oral exposures at either peri-implantation stage or organogenesis stage. This work aims at elucidating the embryotoxicity caused by coexposure of ZnO NPs and Cd²⁺ and the underlying mechanisms. We hypothesize that oral exposures of nanoadducts at organogenesis stage increased maternal-fetal transfer of both ZnO NPs and CdCl₂, damaged placental barrier by perturbing the integrity of tight junctions, and aggravated embryotoxicity. Although no immediate risk at most current environmental concentrations of ZnO NPs/CdCl₂, our results raised special safety concerns on pregnant population under situations such as long-term exposures or pollution accidents.