Experimental data sets on the evaluation of graphene oxide as a thyroid endocrine disruptor and a modulator of gas gland cells in Japanese medaka (Oryzias latipes) larvae at the onset of maturity

Abstract

This article presents the experimental datasets obtained from the histological/histochemical studies of endocrine disrupting effects of graphene oxide (GO) on thyroid follicles and gas gland (GG) cells of Japanese medaka larvae at the onset of maturity. The experiment was conducted on one day-post hatch (dph) starved fries (orange-red variety) immersed in different concentrations of GO (2.5-20.0 mg/L) and no GO (controls) in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). After treatment, larvae were maintained in balanced salt solution (BSS) with food and allowed depuration for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS 222 and their total lengths (mm) and weights (mg) were measured, and they were then cut into three small pieces (head, trunk, and tail). Head and trunk regions were fixed in 4% PFA in 20 mM PBS for 48 h at room temperature and the post-anal tail was preserved in TRI reagent and kept at −20 °C until analysis. Tissues in 4% PFA were used for cutting 5µm thick paraffin sections in a manual rotary microtome. Sections of head regions were evaluated for thyroid follicles after hematoxylin-eosin (HE) or Periodic acid-Schiff (PAS) staining. Trunk sections were used for swim bladder (SB) inflation studies and for phenotypic sex (ovary and testis) of the larvae after HE staining. Genetic sex assessment was made from tail DNA by genotyping Y chromosome-specific male sex-determining gene dmy . Digital images were captured by using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 microscope with DP22 camera and CellSens software. Images of thyroid follicles and GG cells were analyzed using imagej software. HE stained histological sections of thyroid follicles near the heart and branchial regions were captured and the area (µm 2 ) of individual follicles (minimum 3) available in the entire section were measured. The heights of thyrocytes (µm) were determined directly. Manual counting of GG cells was made from the digital images captured in several regions of the SB avoiding blood cells and other cells which have indistinct nucleus and pale cytoplasm; results were expressed as the number of GG cells/mm 2 . Data were analyzed by GraphPad prism version 7.04. For normally distributed data, one-way ANOVA followed by post-hoc Tukey's test or unpaired parametric “t” test including Welch's correction was used. Otherwise, Kruskal-Wallis test followed by nonparametric Mann-Whitney's test as a post hoc test was used. Data were expressed as means ±SEM and the level of significance was set at p < 0.05.

Histopathological evaluation of the interrenal gland (adrenal homolog) of Japanese medaka (Oryzias latipes) exposed to graphene oxide

“…From our histopathological evaluation of the gonadal structures and thyroid follicles, we found that the damages observed after GO administration was mainly due to aggregation/agglomeration of GO on or within the organs rather than directly acting as an EDC. 18,19,22,23 Our other investigation of GO effect on the sex reversal (SR) of medaka larvae showed no apparent EDC effect. 20,21 In the present study, we have extended our investigations on the histopathology of the IRG of medaka adults and larvae exposed to different concentrations/doses of GO either by a single intraperitoneal (IP) injection or by 96 h continuous immersion (IMR).…”

“…As the secondary sexual features were yet to develop in the larvae on 47th dph, the sex of the larvae was determined by phenotypic (histological examination of the gonads either as testis or ovary) and genotyping (XY and XX) methods. [20][21][22][23] We have observed that 27% male phenotypes instead of XY genotypes, were XX (8 XX larvae have testis out of 29 larvae with phenotypic testis), and we considered these larvae as sex-reversed during our previous studies on gonads 20,21 and thyroids. 22,23 In the present study, we are unable to track the IRG of all the experimental larvae.…”

“…[20][21][22][23] We have observed that 27% male phenotypes instead of XY genotypes, were XX (8 XX larvae have testis out of 29 larvae with phenotypic testis), and we considered these larvae as sex-reversed during our previous studies on gonads 20,21 and thyroids. 22,23 In the present study, we are unable to track the IRG of all the experimental larvae. Therefore, the larvae having testis as gonad are considered as male phenotypes (testis) and the larvae genotyped XX were considered as females, irrespective of the presence of testis or ovary.…”

“…16 Our laboratory has been evaluating GO, as a potential EDC using Japanese medaka fish (Oryzias latipes), as our experimental model. [17][18][19][20][21][22][23] We have demonstrated that the males of medaka fish are more sensitive to GO than the females. From our histopathological evaluation of the gonadal structures and thyroid follicles, we found that the damages observed after GO administration was mainly due to aggregation/agglomeration of GO on or within the organs rather than directly acting as an EDC.…”

“…Previously we exposed 1 dph medaka fries (Iwamatsu Stage 40,1st fry stage) to different concentrations (2.5, 5, 10, 20 mg/L in ERM) of GO (Sigma-Aldrich, St. Louis, MO) for 96 h continuously by IMR and assessed the EDC activities of GO on gonads (testis and ovary) and thyroids after 6 weeks post-treatment. [20][21][22][23] In this study, we have evaluated the IRG of some of these larvae by histological/ histochemical methods. Due to technical problems, we were unable to track the IRG of all the larvae we used during our previous experiments.…”