Heterogeneity effects of nanoplastics and lead on zebrafish intestinal cells identified by single-cell sequencing

Plastic particles in water environment can adsorb heavy metals, leading to combined toxicity on aquatic organisms. However, current conclusions are mostly obtained based on cell population-average responses. Heterogeneity effects among cell populations in aquatic organisms remain unclear. This study firstly analyzed the basic toxic effects of 20 μg L^-1, 200 μg L^-1 100 nm polystyrene nanoplastics (PS-NPs), 50 μg L^-1 lead (Pb), and their combined exposures on zebrafish intestine. Results found that combined exposure of 200 μg L^-1 PS-NPs and 50 μg L^-1 Pb induced highest MDA, 8-OHdG, and TNF-α levels. Thus 200 μg L^-1 PS-NPs, 50 μg L^-1 Pb and their combined exposures were chosen to analyze the heterogeneity effects on zebrafish intestine cells by single-cell RNA sequencing. A total of 38,640 zebrafish intestinal cells were obtained and identified as seven cell populations, including enterocytes, macrophages, neutrophils, B cells, T cells, enteroendocrine cells, and goblet cells. 200 μg L^-1 PS-NPs exposure had the greatest influence on macrophages, while Pb exposure mostly influenced enterocytes. Results of MDA, 8-OHdG, and TNF-α analyses indicated that 20 μg L^-1 and 200 μg L^-1 PS-NPs increased the Pb toxicity. However, the scRNA-seq showed that the synergistic effects did not exist in most cell populations, except for goblet cells. Co-exposure of 200 μg L^-1 PS-NPs and Pb caused similar transcriptome profiles with 200 μg L^-1 PS-NPs exposure in macrophages, which changed immunological recognition and apoptosis processes. The Pb exposure influenced the macrophages by direct cytotoxicity. However, the Pb alone and combined exposures induced similar toxicities in the enterocytes, including the generation of oxidative stress and abnormality of lipid metabolism. This study shows the scRNA-seq is a powerful method to identify the target cell populations and corresponding toxic effects during combined exposure of pollutants.