Cytotoxic and epigenetic effect of benzodiazepine delora-zepam on embryonic development of X.laevis

Delorazepam is a benzodiazepine (BZD), a class of psychotropic drugs used for the treat-ment of anxiety and sleep disorder and, for at least twenty years, underestimated water pollutants. Indeed, they are released into the wastewater but not degraded during sewage treatment, therefore accumulating in effluent waters at concentrations ranging from ng/L to µg/L, interfering with a wide range of non-target organisms. An increasing number of stu-dies reported the harmful effects of BZDs on adults' behaviour and physiology, but little information is available regarding developing organisms, exposed since early stages. For this reason, we investigated the possible cytotoxic and epigenetic activity of delorazepam, a benzodiazepine with high half-life and commonly purchased in Italy, at three concentrati-ons (environmental, 1 μg/L; and near future, 5 and 10 μg/L) using Xenopus laevis embryos, amphibian model species at high risk of exposure to water contaminants. Through the con-ventional FETAX test, embryos were exposed to the drug until stage 45/46, and then they were collected for morphological and ultrastructural investigations, for Luminometric Methylation Assay (LUMA) and RAMAN spectroscopy. Results revealed a teratogenic and cytotoxic activity of delorazepam on several embryonic tissues, indicative of interference with the developing nervous and motor system, and a dose-dependent epigenetic effect. Data were confirmed by RAMAN spectroscopy which also revealed a biochemical difference in treated embryos. The resulting condition significantly impairs embryos’ development and threatens their survival. Similar effects should be ex-pected also in embryos belonging to other aquatic species though so far, they have not been considered targets for benzodiazepines.