Altered protein S-glutathionylation depicts redox imbalance triggered by transition metal oxide nanoparticles in a breastfeeding system

Nanosafety has become a public concern following nanotechnology development. By now, attention has seldom been paid to breastfeeding system, which is constructed by mammary physiological structure and derived substances (endogenous or exogenous), cells, tissues, organs, and individuals (mother and child), connecting environment and organism, and spans across mother-child dyad. Thus, breastfeeding system is a center of nutrients transport and a unique window of toxic susceptibility in the mother-child dyad. We applied metabolomics combined with redox proteomics to depict how nanoparticles cause metabolic burden via their spontaneous redox cycling in lactating mammary glands. Two widely used nanoparticles [titanium dioxide (nTiO2) and zinc oxide (nZnO)] were exposed to lactating mice via intranasal administration. Biodistribution and biopersistence of nTiO2 and nZnO in mammary glands destroyed its structure, reflective of significantly reduced claudin-3 protein level by 32.1% (P < 0.01) and 47.8% (P < 0.01), and significantly increased apoptosis index by 85.7 (P < 0.01) and 100.3 (P < 0.01) fold change, respectively. Airway exposure of nTiO2 trended to reduced milk production by 22.7% (P = 0.06), while nZnO significantly reduced milk production by 33.0% (P < 0.01). Metabolomics analysis revealed a metabolic shift by nTiO2 or nZnO, such as increased glycolysis (nTiO2: fold enrichment = 3.31, P < 0.05; nZnO: fold enrichment = 3.68, P < 0.05), glutathione metabolism (nTiO2: fold enrichment = 5.57, P < 0.01; nZnO: fold enrichment = 4.43, P < 0.05), and fatty acid biosynthesis (nTiO2: fold enrichment = 3.52, P < 0.05; nZnO: fold enrichment = 3.51, P < 0.05) for tissue repair at expense of lower milk fat synthesis (35.7% reduction by nTiO2; 51.8% reduction by nZnO), and finally led to oxidative stress of mammary glands. The increased GSSG/GSH ratio (57.5% increase by nTiO2; 105% increase by nZnO) with nanoparticle exposure confirmed an alteration in the redox state and a metabolic shift in mammary glands. Redox proteomics showed that nanoparticles induced S-glutathionylation (SSG) modification at Cys sites of proteins in a nanoparticle type-dependent manner. The nTiO2 induced more protein SSG modification sites (nTiO2: 21; nZnO:16), whereas nZnO induced fewer protein SSG modification sites but at deeper SSG levels (26.6% higher in average of nZnO than that of nTiO2). In detail, SSG modification by nTiO2 was characterized by Ltf at Cys423 (25.3% increase), and Trf at Cys386;395;583 (42.3%, 42.3%, 22.8% increase) compared with control group. While, SSG modification by nZnO was characterized by Trfc at Cys365 (71.3% increase) and Fasn at Cys1010 (41.0% increase). The discovery of SSG-modified proteins under airway nanoparticle exposure further supplemented the oxidative stress index and mammary injury index, and deciphered precise mechanisms of nanotoxicity into a molecular level. The unique quantitative site-specific redox proteomics and metabolomics can serve as a new technique to identify nanotoxicity and provide deep insights into nanoparticle-triggered oxidative stress, contributing to a healthy breastfeeding environment.