Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Particulate matter 10 induces oxidative stress and apoptosis in rhesus macaques skin fibroblast.

Background: Particulate matter (PM) is a major air pollutant that affects human health worldwide. PM can pass through the skin barrier, thus causing skin diseases such as heat rash, allergic reaction, infection, or inflammation. However, only a few studies have been conducted on the cytotoxic effects of PM exposure on large-scale animals. Therefore, herein, we investigated whether and how PM affects rhesus macaque skin fibroblasts. Methods: Rhesus macaque skin fibroblasts were treated with various concentrations of PM10 (1, 5, 10, 50, and 100 µg/mL) and incubated for 24, 48, and 72 h. Then, cell viability assay, TUNEL assay, and qRT-PCR were performed on the treated cells. Further, the reactive oxygen species, glutathione, and cathepsin B levels were determined. The MTT assay revealed that PM10 (>50 µg/mL) proportionately reduced the cell proliferation rate. Results: PM10 treatment increased TUNEL-positive cell numbers, following the pro-apoptosis-associated genes (CASP3 and BAX) and tumor suppressor gene TP53 were significantly upregulated. PM10 treatment induced reactive oxidative stress. Cathepsin B intensity was increased, whereas GSH intensity was decreased. The mRNA expression levels of antioxidant enzyme-related genes (CAT, GPX1 and GPX3) were significantly upregulated. Furthermore, PM10 reduced the mitochondrial membrane potential. The mRNA expression of mitochondrial complex genes, such as NDUFA1, NDUFA2, NDUFAC2, NDUFS4, and ATP5H were also significantly upregulated. In conclusion, these results showed that PM10 triggers apoptosis and mitochondrial damage, thus inducing ROS accumulation. These findings provide potential information on the cytotoxic effects of PM10 treatment and help to understand the mechanism of air pollution-induced skin diseases.

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine ß-casein gene locus.

OBJECTIVE: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine ß-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). METHODS: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine ß-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). RESULTS: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. CONCLUSION: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.