Experimental datasets on the immunohistological assessment of δ-cells in the islet organs of the endocrine pancreas of Japanese medaka (Oryzias latipes) fish exposed to graphene oxide

Abstract

The datasets of this article present the experimental parameters resulting from the assessment of δ-cells in the islet organs of the endocrine pancreas as a potential biomarker of endocrine disruption (ED) mediated by graphene oxide (GO), using Japanese medaka fish as the model. These datasets support the article "Evaluation of pancreatic δ-cells as a potential target site of graphene oxide toxicity in Japanese medaka (Oryzias latipes) fish". GO used in the experiments was either obtained from a commercial source or synthesized in the laboratory by us. GO was sonicated for 5 min in ice temperature before application. The experiments were conducted on reproductively active adult fish maintained as a breeding pair (one male and one female) in 500 ml balanced salt solution (BSS) either by immersion (IMR) in GO (20 mg/L) continuously for 96 h with the refreshing of media once in every 24 h, or by a single intraperitoneal (IP) administration of GO (100 µg/g) to both male and female partners. Control fish were maintained in BSS only (IMR experiment), or nanopure water (vehicle) was injected into the peritoneal cavity (IP experiment). The IP experimental fish were anesthetized in MS-222 (100 mg/L in BSS); the injected volume (0.5 µL/10 mg fish) never exceeds 50 µl/fish. After injection, the injected fish were allowed for recovery in clean BSS and after recovery both partners were transferred to 1 L glass jars with 500 mL BSS. During depuration, the media of the breeders refreshed once every 24 h and the eggs were collected. After 21 days, the survived fish were anaesthetized, and the trunk region was preserved in 4% paraformaldehyde in PBS (20 mM) containing 0.05% Tween 20. The phenotypic sex of adult fish was assessed externally by secondary sex characters (fin features) and internally by gonad (testis and ovary) histology. Once the location of pancreas was determined after HE stains, immunohistochemical technique was applied on next few slides using rabbit derived polyclonal antisomatostatin antibody as primary antibody and a commercial kit for colorimetric determination of δ-cells in the islet organs was used. Images were captured using an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. Using imagej software, a minimum 3 images of principal islets and one image of secondary islets were assessed. The immunoreactivity of δ-cells, due to neuron-like appearance and filopodia like processes, enabled us to separate them from other cell types found in the pancreatic islets of medaka. Based on immunoreactivity, we have classified islet cells into three categories; noncommunicating delta cells (NCDC), communicating cells (CC), and non-delta cells (NDC), and expressed as number of cells (NCDC/CC/NDC)/mm² of islet organs. The nuclear area (µm²) and the linear length of filopodia of NCDCs were also considered for evaluation. Numerical data were analysed by Kruskal-Wallis test followed by Mann-Whitney's test as post hoc test and presented as means ± SEM. Statistically significant differences were considered for p ≤ 0.05.

Keywords: Cell sorting; Delta cells; Endocrine disruption; Endocrine pancreas; Graphene oxide; Immersion; Intraperitoneal injection; Japanese medaka.