Diethyl phosphate disrupts hypothalamus-pituitary-adrenal axis endocrine hormones via nuclear receptors GR and Nur77: Integration of evidences from in vivo, in vitro and in silico approaches

Abstract

Plenty of population epidemiology and cohort studies have found dialkyl phosphates (DAPs) in the urine were related to endocrine hormone disorders. However, we did not know whether these effects were caused by parent organophosphorus pesticides (OPs) or metabolite DAPs, especially the non-specific metabolite diethyl phosphate (DEP), which was the metabolic end product of most widely used diethyl OPs. In this study, animal experiments (in vivo), cell experiments (in vitro), small molecule-protein binding interaction experiments and computer molecular simulations (in silico) were used to explore the disturbing effects and molecular mechanisms of DEP on the hypothalamic-pituitary-adrenal (HPA) axis endocrine hormones. The animal experiments showed that chronic DEP exposure significantly disturbed the serum contents of HPA axis hormones in adult male rats. The target genes of glucocorticoid receptor (GR) in rat liver, including 11β-hsd1 and Pepck1 and PEPCK protein expressions, were down-regulated. Moreover, the gluconeogenic abilities of rats were impaired. However, it did not affect the expression of GR in the rat hypothalamus. These results indicated that the physiological functions of glucocorticoids and GR were damaged. Furthermore, spectroscopy experiments, cell experiments, molecular docking and molecular dynamics simulations also suggested that DEP can bind to nuclear receptors GR and Nur77, affecting their transcription factor functions, and the transcriptional expression levels of their downstream target genes were reduced. The biosynthesis and secretion of adrenocorticotropic hormone and glucocorticoids were blocked. Therefore, DEP can inhibit the production and physiological functions of HPA axis endocrine hormones by disrupting these related proteins and antagonizing nuclear receptors. These results were considered to provide a theoretical basis for strictly controlling the residue limits of OPs and their metabolites in foods, agricultural products and the environment. They also revealed new targets for evaluating the toxicities and risks of pesticide metabolites.