Imidacloprid actions in the mouse mammary gland structure and epithelial mammary cells. Role of GPER and nAChR.

The neonicotinoid imidacloprid (IMI) is used worldwide for insect control and represents a potential risk to populations, due to its potential action as an endocrine disruptor (ED). IMI binds to the postsynaptic nicotinic acetylcholine receptor (nAChR), whereas other neonicotinoids can activate the G protein-coupled estrogen receptor (GPER). Here, we examined the IMI effect on the mammary gland in a peripuberal model where female mice were exposed to IMI (0.01, 0.1 and 10 mg/kg/day). Alterations observed in the mammary morphology included an increase in transverse ductal growth at 0.1 and 10 mg/kg/day and a reduction in branch density at 0.1 mg/kg/day. Furthermore, 10 mg/kg/day IMI increased the number of terminal end buds (TEBs), induced ductal hyperplasia and epithelial cell proliferation. In mammary epithelial NMuMG cells, IMI (0.01-10 μM) showed no cytotoxic effect but boosted clonogenic capacity at 0.1 and 1 μM. Western blot findings revealed that 1 and 10 μM IMI rises GPER and aromatase expression, but declines progesterone receptor levels at all assayed doses. IMI (10 μM) also increased α7-nAChR expression at 24 h and induced c-Src phosphorylation after 1 h of treatment. Finally, 10 μM IMI promoted cell migration and the proteolytic activity of metalloproteinase (MMP)-2 and -9 through GPER, α7-nAChR, and c-Src. In summary, IMI promotes preneoplastic lesions and TEBs retention and increases mammary epithelial cell motility through GPER and α7-nAChR. Our results support the hypothesis that IMI acts as an ED, affecting mammary gland structure.