Alterations in the Mouse Mammary Gland Promoted by Exposure to the Neonicotinoid Imidacloprid

LEGUIZAMÓN M. AGUSTINA; ESPAÑOL A; BUJÁN S; PONTILLO C; CHIAPPINI F; LASAGNA M; COCCA C; RANDI A; MIRET NV

Congreso; Society of Environmental Toxicology and Chemistry (SETAC) Latin America 15th Biennial Meeting; 2023

Society of Environmental Toxicology and Chemistry (SETAC)

Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agriculture worldwide, posing great public health concern. IMI is an agonist of the nicotinic acetylcholine receptor (nAChR) and an endocrine disruptor, enhancing estradiol secretion in breast cancer cells. Since the mammary gland development is largely regulated by the endocrine system, we hypothesize that IMI exposure produces alterations in the mammary gland that favor tumorigenesis. We have previously found that IMI (10 μM) increases the migration ability of the mammary epithelial cell line NMuMG as well as the metalloproteases (MMP) 2 and 9 activity. Furthermore, IMI increases the expression of G protein-coupled estrogen receptor (GPER) at the same dose. Herein, we set out to delve into the IMI mechanism of action to promote NMuMG cell motility. In addition, we aimed to investigate whether the pesticide induces alterations in the mammary gland, so we exposed pre-pubertal BALB-c mice to IMI (0.01, 0.1 and 10 mg/kg/day) orally for 4 weeks. The whole mammary gland was mounted for morphological analysis, while hematoxylin-eosin stained tissue section were examined for histological studies.First, we analyzed the NMuMG cell migration (wound healing assay) and the MMP2 and 9 activity (gel zymography), in the presence or the absence of the GPER specific inhibitor, G15 (1 μM). Results showed that IMI (10 μM) stimulates both processes in a GPER-dependent manner (p‹0.001). Taking into account that α7-nAChR is expressed in the mammary gland, we studied its protein levels by western blot (WB) and found an enhancement at 10 μM (190% p‹ 0.001). Next, we analyzed c-Src activation, a kinase downstream of GPER and α7-nAChR, by WB. IMI (10 μM) increases c-Src phosphorylation after 1, 2 and 4 h of exposure (150%, 80% and 60% respectively, p‹ 0.01). In vivo assays showed IMI (10 mg/kg/day) to enhance ductal hyperplasia (140% p‹ 0.01) and the number of terminal end buds (TEBs, 140% p‹ 0.05). IMI (0.1 mg/kg/day)-treatment significantly induced ductal growth (46% p‹ 0.05), quantified as the distance between the lymph node and the mammary gland final edge, but reduced branch density (33% p‹ 0.05).Our findings show that IMI increases mammary epithelial cell motility trough GPER and alters mammary branching morphogenesis, likely leading to preneoplastic lesions and retaining TEBs. Taken together, these data support the notion that IMI may represent a risk factor for breast cancer development.