Experimental data-sets on the histopathological and immunohistological assessment of the Interrenal gland (adrenal homolog) 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 adrenal gland as a potential biomarker of endocrine disruption mediated by graphene oxide (GO), a nanocarbon, using Japanese medaka fish as the model. These data sets support the article “Histopathological evaluation of the interrenal gland (adrenal homolog) of Japanese medaka (Oryzias latipes) exposed to graphene oxide”. 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 in GO (20 mg/L in BSS) continuously for 96 h with refreshing of media once in every 24 h or by a single intraperitoneal (IP) injection of GO (100 µg/g) to both male and female fish. The experimental fish were allowed breeding and assessed after 21 days post-treatment. Moreover, one day-post hatch (dph) Japanese medaka fries (orange-red variety) were exposed to different concentrations of GO (2.5–20 mg/L) by immersion in embryo-rearing medium (ERM) for 96 h (1–5 dph) with refreshing of media every 24h. Food was given to the adults, however, the larvae remained fasting during the GO-exposure (0–5 dph) period. Control adults and larvae were identically maintained either in BSS (adults) or ERM (larvae), with no GO. After treatment, both adults and the larvae were maintained in BSS with feeding in a GO-free environment. After 21 days post-treatment, adults, and after six weeks post-treatment, larvae, were anaesthetized in MS-222, and the trunk region was preserved in 4% paraformaldehyde in PBS (20 mM) containing 0.05% Tween 20. Evaluation of interrenal gland (IRG) in kidneys were made in 5 µm thick sections stained on haematoxylin-eosin (HE). The phenotypic sex of adults was assessed by secondary sex characters (fin features) and gonad (testis and ovary) histology; in larvae, phenotypic sex was determined by gonad histology and the genotypic sex by genotyping dmy gene. The location of IRG in the kidney were determined by immunohistochemical technique using rabbit polyclonal antityrosine hydroxylase antibody as primary antibody. The digital images of sections were captured using an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. Using imagej software, a minimum of 3 images of kidney consisting IRG were assessed for cell (separated as dark and pale stained nucleus after HE staining) sorting (cells/ mm2) and also measured the nuclear area (µm2). Counting of IRG cells, lined between the cardinal vein and the interstitial cells in the kidneys, were limited to maximum three layers in a given area. Numerical data, presented as means ± SEM, were analysed by one-way ANOVA followed by post-hoc Tukey's multiple comparison test or unpaired parametric ‘t’ test including Welch's correction, if distributed normally; or by Kruskal-Wallis test followed by Mann-Whitney's test as post hoc test, if the data did not meet the criteria of using a parametric test. Statistically significant difference were considered for p ≤ 0.05. The collected data on IRG of Japanese medaka fish will be used for the assessment of GO as an EDC disposed in the environment.