Evaluation of pancreatic δ- cells as a potential target site of graphene oxide toxicity in Japanese medaka (Oryzias latipes) fish.

In continuation to our previous investigations on graphene oxide (GO) as an endocrine disrupting chemical (EDC), in the present experiment, we have investigated endocrine pancreas of Japanese medaka adults focusing on δ-cells in the islet organs as an endpoint. Breeding pairs of adult male and female fish were exposed to 0 mg/L (control) or 20 mg/L GO by continuous immersion (IMR) for 96 h, or to 0 µg/g or 100 µg/g GO by a single intraperitoneal (IP) administration and depurated 21 days in a GO-free environment. Histological investigations indicated that the endocrine cells are concentrated in one large principal islet, and several small secondary islets scattered within the mesentery near the liver and intestine. The cells of the islet organ are in various shapes with basophilic nuclei and eosinophilic cytoplasm. Immunohistochemical evaluation using rabbit polyclonal antisomatostatin antibody indicated that immunoreactivity is localized either at the periphery or at the central region in principal islets, and throughout the secondary islets, and found to be enhanced in fish exposed to GO than controls. The soma of δ-cells exhibits neuron-like morphology and have filopodia like processes. Cell sorting as non-communicating δ-cells (NCDC), communicating cells (CC), and non- δ-cells (NDC) indicated that within an islet organ, the population of NDCC is found to be the least and NDC is the highest. Our data further indicated that GO-induced impairments in the islet organs of medaka pancreas are inconsistent and could be affected by the exposure roots as well as the sex of the fish.

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

Due to unique physicochemical properties and wide industrial and biomedical applications, graphene oxide (GO) is ubiquitous in the aquatic ecosystem. Using Japanese medaka (Oryzias latipes) fish as a model, we previously demonstrated minimal endocrine disrupting (ED) effects of GO on reproductive organs, and thyroids. Current study investigated the ED-effects of GO on the interrenal gland (IRG) of medaka. Breeding pairs of adult male and female fish were exposed to 0 mg/L (control) or 20 mg/L GO by continuous immersion for 96 h, or to 0 or 100 µg/g GO by intraperitoneal administration. Also, 1 day post-hatch (dph) larvae were exposed to different concentrations of GO (2.5-20 mg/L) for 96 h. IRG was evaluated by immunohistochemical techniques after 21 days depuration in adults and 6 weeks in larvae. IRG cells were counted and the nuclear area was measured in hematoxylin-eosin stained sections using ImageJ software. We found that IRG is distributed adjacent to the posterior cardinal vein and its branches within the head kidney. Columnar/oval shaped periodic acid-Schiff negative, tyrosine hydroxylase positive cells are arranged either in a single, or in groups, sometimes encircling a sinusoid, or in a straight chord, laying adjacent to the endothelium of the cardinal vein, and having eosinophilic cytoplasm with round/oval basophilic nuclei. GO effect on nuclei and cell population in IRG was inconsistent; depending on exposure route, sex, and/or age of the fish. Also, because of its high adsorptive property and sharp edges, GO probably agglomerated on IRG, and induced physical injury, and ED effects.

Graphene-Based Nanomaterials Toxicity in Fish.

Due to their unique physicochemical properties, graphene-based nanoparticles (GPNs) constitute one of the most promising types of nanomaterials used in biomedical research. GPNs have been used as polymeric conduits for nerve regeneration and carriers for targeted drug delivery and in the treatment of cancer via photothermal therapy. Moreover, they have been used as tracers to study the distribution of bioactive compounds used in healthcare. Due to their extensive use, GPN released into the environment would probably pose a threat to living organisms and ultimately to human health. Their accumulation in the aquatic environment creates problems to aquatic habitats as well as to food chains. Until now the potential toxic effects of GPN are not properly understood. Despite agglomeration and long persistence in the environment, GPNs are able to cross the cellular barriers successfully, entered into the cells, and are able to interact with almost all the cellular sites including the plasma membrane, cytoplasmic organelles, and nucleus. Their interaction with DNA creates more potential threats to both the genome and epigenome. In this brief review, we focused on fish, mainly zebrafish (Danio rerio), as a potential target animal of GPN toxicity in the aquatic ecosystem.

Toxicity implications for early life stage Japanese medaka (Oryzias latipes) exposed to oxyfluorfen.

We investigated the potential toxic effects of Oxyfluorfen (OXY), an herbicide used in agriculture, on the embryo-larval development of Japanese medaka fish (Oryzias latipes). Embryos (1-day postfertilization) and larvae (2-day posthatch) were exposed to OXY (0.5-8 mg/L) for 96 h and evaluated for mortality and hatching on embryos, and the mortality and growth on larvae during depuration. It was observed that the embryo-mortality was inconsistently altered by OXY; only the 2 mg/L group showed significant reduction on embryo survivability. However, larval-mortality was concentration-dependent and OXY exposure induced scoliosis-like phenotypic features in the surviving larvae; the calculated LC50 was 5.238 mg/L. Our data further indicated that larval skeleton, both axial and appendicular, was the potential target site of OXY. Skeletal growth in larvae exposed to 2 mg/L was inhibited significantly until 1 week of depuration with regard to the linear lengths of neurocranium, Meckel's cartilage, caudal vertebrae (first 10) in the axial skeletons, and pectoral fin and urostyle in the appendicular skeletons. Moreover, the total protein content remained unaltered by OXY after 96 h exposure; while the RNA concentration was reduced significantly in larvae exposed to 2 mg/L. Expression analysis of several genes by quantitative real-time RT-PCR (RT-qPCR) showed significant upregulation of zic5, a zinc-finger type transcription regulator, at the transcription level. This study indicated that the scoliosis induced by OXY in Japanese medaka larvae was the result of stunted skeletal growth, probably because of deregulation of zinc-finger type transcription regulators, at the genomic level.

The anticancer potential of steroidal saponin, dioscin, isolated from wild yam (Dioscorea villosa) root extract in invasive human breast cancer cell line MDA-MB-231 in vitro.

Previously, we observed that wild yam (Dioscorea villosa) root extract (WYRE) was able to activate GATA3 in human breast cancer cells targeting epigenome. This study aimed to find out if dioscin (DS), a bioactive compound of WYRE, can modulate GATA3 functions and cellular invasion in human breast cancer cells. MCF-7 and MDA-MB-231 cells were treated in the absence/presence of various concentrations of DS and subjected to gene analysis by RT-qPCR, immunoblotting, and immunocytochemistry. We determined the ability of MDA-MB-231 cells to migrate into wound area and examined the effects of DS on cellular invasion using invasion assay. DS reduced cell viability of both cell lines in a concentration and time-dependent manner. GATA3 expression was enhanced by DS (5.76 µM) in MDA-MB-231 cells. DS (5.76 µM)-treated MDA-MB-231 cells exhibited the morphological characteristic of epithelial-like cells; mRNA expression of DNMT3A, TET2, TET3, ZFPM2 and E-cad were increased while TET1, VIM and MMP9 were decreased. Cellular invasion of MDA-MB-231 was reduced by 65 ± 5% in the presence of 5.76 µM DS. Our data suggested that DS-mediated pathway could promote GATA3 expression at transcription and translation levels. We propose that DS has potential to be used as an anti-invasive agent in breast cancer.

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.

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.

A systematic review of the evaluation of endocrine-disrupting chemicals in the Japanese medaka (Oryzias latipes) fish.

Japanese medaka (Oryzias latipes) is an acceptable small laboratory fish model for the evaluation and assessment of endocrine-disrupting chemicals (EDCs) found in the environment. In this research, we used this fish as a potential tool for the identification of EDCs that have a significant impact on human health. We conducted an electronic search in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (https://scholar.google.com/) using the search terms, Japanese medaka, Oryzias latipes, and endocrine disruptions, and sorted 205 articles consisting of 128 chemicals that showed potential effects on estrogen-androgen-thyroid-steroidogenesis (EATS) pathways of Japanese medaka. From these chemicals, 14 compounds, namely, 17ß-estradiol (E2), ethinylestradiol (EE2), tamoxifen (TAM), 11-ketotestosterone (11-KT), 17ß-trenbolone (TRB), flutamide (FLU), vinclozolin (VIN), triiodothyronine (T3), perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA), terephthalic acid (TPA), trifloxystrobin (TRF), ketoconazole (KTC), and prochloraz (PCZ), were selected as references and used for the identification of apical endpoints within the EATS modalities. Among these endpoints, during classification, priorities are given to sex reversal (masculinization of females and feminization of males), gonad histology (testis-ova or ovotestis), secondary sex characteristics (anal fin papillae of males), plasma and liver vitellogenin (VTG) contents in males, swim bladder inflation during larval development, hepatic vitellogenin (vtg) and choriogenin (chg) genes in the liver of males, and several genes, including estrogen-androgen-thyroid receptors in the hypothalamus-pituitary-gonad/thyroid axis (HPG/T). After reviewing 205 articles, we identified 108 (52.68%), 46 (22.43%), 19 (9.26%), 22 (17.18%), and 26 (12.68%) papers that represented studies on estrogen endocrine disruptors (EEDs), androgen endocrine disruptors (AEDs), thyroid endocrine disruptors (TEDs), and/or steroidogenesis modulators (MOS), respectively. Most importantly, among 128 EDCs, 32 (25%), 22 (17.18%), 15 (11.8%), and 14 (10.93%) chemicals were classified as EEDs, AEDs, TEDs, and MOS, respectively. We also identified 43 (33.59%) chemicals as high-priority candidates for tier 2 tests, and 13 chemicals (10.15%) show enough potential to be considered EDCs without any further tier-based studies. Although our literature search was unable to identify the EATS targets of 45 chemicals (35%) studied in 60 (29.26%) of the 205 articles, our approach has sufficient potential to further move the laboratory-based research data on Japanese medaka for applications in regulatory risk assessments in humans.

Epigenetic events associated with breast cancer and their prevention by dietary components targeting the epigenome.

Aberrant epigenetic alterations in the genome such as DNA methylation and chromatin remodeling play a significant role in breast cancer development. Since epigenetic alterations are considered to be more easily reversible compared to genetic changes, epigenetic therapy is potentially very useful in reversing some of these defects. Methylation of CpG islands is an important component of the epigenetic code, and a number of genes become abnormally methylated in breast cancer patients. Currently, several epigenetic-based synthetic drugs that can reduce DNA hypermethylation and histone deacetylation are undergoing preclinical and clinical trials. However, these chemicals are generally very toxic and do not have gene specificity. Epidemiological studies have shown that Asian women are less prone to breast cancer due to their high consumption of soy food than the Caucasian women of western countries. Moreover, complementary/and or alternative medicines are commonly used by Asian populations which are rich in bioactive ingredients known to be chemopreventive against tumorigenesis in general. Examples of such agents include dietary polyphenols, (-)-epigallocatechin-3-gallate (EGCG) from green tea, genistein from soybean, isothiocyanates from plant foods, curcumin from turmeric, resveratrol from grapes, and sulforaphane from cruciferous vegetables. These bioactive components are able to modulate epigenetic events, and their epigenetic targets are known to be associated with breast cancer prevention and therapy. This approach could facilitate the discovery and development of novel drugs for the treatment of breast cancer. In this brief review, we will summarize the epigenetic events associated with breast cancer and the potential of some of these bioactive dietary components to modulate these events and thus afford new therapeutic or preventive approaches.

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.

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.

Histological and Histochemical Evaluation of the Effects of Graphene Oxide on Thyroid Follicles and Gas Gland of Japanese Medaka (Oryzias latipes) Larvae.

Graphene oxide (GO) has become a topic of increasing concern for its environmental and health risks. However, studies on the potential toxic effects of GO, especially as an endocrine disrupting chemical (EDC), are very limited. In the present study we have used Japanese medaka fish as a model to assess the endocrine disruption potential of GO by evaluating its toxic and histopathologic effects on thyroid follicles and the gas gland (GG) of medaka larvae. One day post-hatch (dph) starved medaka fries were exposed to GO (2.5, 5.0, 10.0, and 20 mg/L) for 96 h, followed by 6 weeks depuration in a GO-free environment with feeding. Larvae were sacrificed and histopathological evaluation of thyroid follicles and the GG cells were done microscopically. Different sizes of spherical/oval shape thyroid follicles containing PAS positive colloids, surrounded by single-layered squamous/cuboidal epithelium, were found to be scattered predominantly throughout the pharyngeal region near the ventral aorta. We have apparently observed a sex-specific difference in the follicular size and thyrocytes height and a non-linear effect of GO exposure on the larvae on 47th day post hatch (dph). The GG is composed of large uniform epithelial cells with eosinophilic cytoplasm. Like thyroids, our studies on GG cells indicate a sex-specific difference and GO exposure non-linearly reduced the GG cell numbers in males and females as well as in XY and XX genotypes. Our data further confirm that sex effect should be carefully considered while assessing the toxicity of EDCs on the thyroid gland.

Potential utility of natural products as regulators of breast cancer-associated aromatase promoters.

Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.4 in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7) aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs) are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for specific hormone-dependent breast cancer.

Sex-reversal and Histopathological Assessment of Potential Endocrine-Disrupting Effects of Graphene Oxide on Japanese medaka (Oryzias latipes) Larvae.

Sex-ratio is considered as an end point during endocrine disrupting chemicals (EDCs) evaluation. Many fish species including Japanese medaka have XX/XY sex determination mechanism, however, sex reversal (SR) can be induced by external and genetic factors. SR imposed an imbalance in natural sex ratio of a population living in any ecosystem. Considering SR as an end point, we aimed to investigate the potential EDC effects of graphene oxide (GO), a nanocarbon, using Japanese medaka as a model. One-day post-hatch (dph) medaka fries were exposed to GO (2.5, 5.0, 10.0 and 20 mg/L) for 96 h without food, followed by 6 weeks depuration in a GO-free environment with feeding. Phenotypic sex was determined by gonad histology; genotypic sex by genotyping Y-chromosome-specific male sex determining gene, dmy. Our data indicated testes in both XY and XX genotypes, while ovaries were only in XX females. Histopathology of XY and XX testis showed isogenic spermatocysts with active spermatogenesis. Distribution of spermatocytes (SPTs), not the spermatogonium (SPGs), showed enhancement in XY than XX testis. Female phenotypes had single ovary, either in stage 0 or 1. Ovo-testis/testis-ova were absent in XX or XY gonads. GO (2.5-20 mg/L) had inconsistent concentration-dependent effect in both SPGs and SPTs; however, no effect on ovarian follicles. Despite genotypic differences (XY/XX), in the histopathology/histochemistry of liver and kidneys GO effects was found to be minimum. Taken together, present study showed spontaneous induction of SR in some XX genotypes; however, exposure of fasting fries to GO had no apparent EDC effects.

Experimental data-sets on sex reversal and histopathological assessment of potential endocrine disrupting effects of graphene oxide on Japanese medaka (Oryzias latipes) larvae at the onset of maturity.

The datasets of this article present the experimental parameters resulting from the assessment of sex reversal (SR) as a biomarker of endocrine disrupting effect of graphene oxide (GO), together with the histopathological assessment of ovary, testis, liver and kidneys of medaka larvae. These data sets support the published article "Sex-reversal and histopathological assessment of potential endocrine-disrupting effect of graphene oxide on Japanese medaka (Oryzias larvae) larvae." The experiments were conducted on one day-post hatch (dph) Japanese medaka fries (orange-red variety) exposed to different concentrations of GO (2.5-20 mg/L) by immersion in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). No food was given during the GO-exposure period. Controls (no GO) were identically maintained in ERM. After treatment, the larvae were maintained in balanced salt solution (BSS) with feeding and allowed to grow for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS-222, and the total length (mm) and body weight (mg) were recorded. For histopathological and phenotypic sex assessments, after sacrifice, the body excluding post-anal tail was preserved in 4% paraformaldehyde containing 0.05% Tween 20; ovary, testis, liver and kidneys were evaluated in 5 µm thick sections stained on haematoxylin eosin (HE) following OECD guidelines. The photomicrographs of sections were made using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. A minimum 3 images of gonads in different regions were further analysed by imagej software and used for counting spermatogonia (SPG) and spermatocytes (SPT) in testis as well as perinucleolar (PNO) and cortical alveolar (CAO) oocytes in ovary. Data were expressed as number of SPG or SPT/mm2 testis and % CAO or PNO in an ovary. Preserved tail in TRI reagent was used for genomic DNA extraction and the genetic sex was assessed by genotyping Y chromosome-specific male sex-determining gene dmy. Two different sets of buffers and primers were used and the reactions were conducted in a thermal cycler. The amplified products were separated in 2% agarose gel containing 0.01% ethidium bromide. The gels were viewed on an UV illuminator and the genotypes were identified by visual inspection. The first primer set amplified a 355 bp product for XY genotypes and no amplification for XX. The second set of primers amplified two products; one at 1249 bp and another at 986 bp for XY, and one product at 1249 bp for XX. Experimental data were expressed as means ± SD or SEM, analysed either by one-way analysis of variance (ANOVA) followed by post-hoc Tukey's multiple comparison test or unpaired parametric 't' test including Welch's correction, if distributed normally (lengths and weights), or by Kruskal-Wallis test followed by Man-Whitney's test as post hoc test, if data (stromal follicles in ovary and SPGs and SPTs in testis) did not meet the criteria of using a parametric test. Statistically significant difference were considered for p ≤ 0.05.

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.

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 (µm2) 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/mm2. 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.

Teratogenic effects of blue cohosh (Caulophyllum thalictroides) in Japanese medaka (Oryzias latipes) are probably mediated through GATA2/EDN1 signaling pathway.

Blue cohosh (Caulophyllum thalictroides) (BC) has been used widely to induce labor and to treat other uterine conditions. However, the safety and effectiveness of this herbal product has not yet been evaluated by the US Food and Drug Administration (FDA). Several conflicting reports indicated that the root extract of BC is a teratogen and, by some unknown mechanisms, is able to induce cardiovascular malfunctions in new-born babies. To understand the mechanism, we have used Japanese medaka (Oryzias latipes) embryo-larval development as the experimental model and the methanolic extract of BC root as the teratogen. The embryo mortality, hatching efficiency, and morphological abnormalities in craniofacial and cardiovascular systems are considered for the evaluation of BC toxicity. Our results indicate that BC is able to disrupt cardiovascular and craniofacial cartilage development in medaka embryo in a dose and developmental stage-specific manner. Moreover, embryos in precirculation are to some extent more resistant to BC than ones with circulation. By using subtractive hybridization, we have observed that gata2 mRNA was differentially expressed in the circulating embryos after BC treatment. As GATA-binding sequences are required for the expression of the endothelin1 (edn1) gene and edn1 expressed in blood vessels and craniofacial cartilages, we have extended our investigations to edn1 gene expression regulation by BC. We found that edn1, furin1, and endothelin receptor A (ednrA) genes are developmentally regulated; endothelin converting enzyme mRNA (ece1) maintained a steady-state level throughout development. Circulating medaka embryos (3 days post fertilization, dpf) exposed to BC (10 microg/mL) for 48 h have increased levels of gata2, ece1, and preproenodthelin (preproedn1) mRNA contents; however, other mRNAs (furin and ednrA) remained unaltered. Therefore, the enhanced expression of gata2 mRNA followed by ece1 and preproedn1 mRNA by BC might be able to induce vasoconstriction and cardiovascular defects and disrupt craniofacial cartilages in medaka embryos. We conclude that cardiovascular and craniofacial defects in medaka embryogenesis by BC are probably mediated through a GATA2-EDN1 signaling pathway.

Experimental datasets on the characterization of graphene oxide and its reproductive and developmental effects on Japanese medaka (Oryzias latipes) fish.

The datasets of this article present the experimental parameters resulting from the synthesis and characterization of graphene oxide (GO) using scanning and transmission electron microscopy (SEM, TEM) and spectrophotometric (FTIR, AFM, EDX) methods, and the assessment of its toxicological and endocrine-disrupting effects on the Japanese medaka fish by acute toxicity testing, and histopathological evaluations. These datasets support the article "Reproductive and Developmental Effects of Graphene Oxide on Japanese Medaka (Oryzias latipes)". GO synthesis was performed following the modified Hummer's method. Its particle diameter and zeta potential were determined using Zeta Sizer Nano ZS analyzer, and characterized by SEM and TEM. After 5 min sonication in water, GO (25-200 µg/g) was injected intraperitoneally to the reproductively active male and female fish maintained as a breeding pair (one male, one female) in 500 mL balanced salt solution (BSS) in glass jars under standard laboratory conditions (25±1 °C; 16L:8D light cycle). The control fish were injected with water. The maximum volume of the injected material is 1 µL/10 mg body weight. To avoid movement, during injection the fish were briefly anesthetized in MS 222 (100 mg/L) and after injection transferred to BSS for recovery. LD50 values of GO related to fish mortality were determined from the linear regression analysis using a software program. Reproductive activities (fecundity) were determined by daily collection of eggs 7 days before and 21 days after injection from a breeding pair and expressed as percent eggs laid every day post-injection relative to the average (mean of 7 days) eggs laid prior to injection. Developmental abnormalities of the embryos were assessed by culturing the collected fertilized eggs in ERM for a maximum period of 14 day-post fertilization (dpf). The fish that survived after 21days post-injection were sacrificed and the entire fish excluding post-anal tail were cut into three small pieces and fixed in 4% paraformaldehyde containing 0.05% Tween 20. Histopathological evaluations of gonads (ovary and testis), liver, and kidneys were made in 5 µm thick sections stained mainly on hematoxylin and eosin (HE) following the guidelines published by OECD. The Photomicrographs of the sections were made using Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or in Nikon Eclipse 50i microscope attached to Nikon DS-Fi1 camera. Four types of follicles in the stromal compartments of the ovary, perinucleolar (PNO), cortical alveolar (CAO), early vitellogenic (EVO) and late vitellogenic (LVO) were considered as differentiating, and the post ovulatory and atretic follicles were considered as degenerating follicles, and counted in an entire section made through four different regions (anterior, upper middle, lower middle, and anal) of the ovary. The follicular data were expressed as percent follicles (individual follicles or differentiating or degenerating) or as the ratio of differentiating and degenerating follicles found in that particular region of the ovary. The data were analyzed either by one- or two-way ANOVA followed by post-hoc Tukey's multiple comparison test and expressed as means ±SEM.

Assessment of reproductive and developmental effects of graphene oxide on Japanese medaka (Oryzias latipes).

Due to its unique properties, graphene oxide (GO) has potential for biomedical and electronic applications, however environmental contamination including aquatic ecosystem is inevitable. Moreover, potential risks of GO in aquatic life are inadequately explored. Present study was designed to evaluate GO as an endocrine disrupting chemical (EDC) using the model Japanese medaka (Oryzias latipes). GO was injected intraperitoneally (25-200 µg/g) once to breeding pairs and continued pair breeding an additional 21 days. Eggs laid were analyzed for fecundity and the fertilized eggs were evaluated for developmental abnormalities including hatching. Histopathological evaluation of gonads, liver, and kidneys was made 21 days post-injection. LD50 was found to be sex-dependent. Fecundity tended to reduce in a dose-dependent manner during early post-injection days; however, the overall evaluation showed no significant difference. The hatchability of embryos was reduced significantly in the 200 µg/g group; edema (yolk and cardiovascular) and embryo-mortality remained unaltered. Histopathological assessment identified black particles, probably agglomerated GO, in the gonads of GO-treated fish. However, folliculogenesis in stromal compartments of ovary and the composition of germinal elements in testis remained almost unaltered. Moreover, granulosa and Leydig cells morphology did not indicate any significant EDC-related effects. Although liver and kidney histopathology did not show GO as an EDC, some GO-treated fish accumulated proteinaceous fluid in hepatic vessels and induced hyperplasia in interstitial lymphoid cells (HIL) located in kidneys. GO agglomerated in medaka gonads after 21-days post-injection. However, gonad histopathology including granulosa and Leydig cells alterations were associated with GO toxicity rather than EDC effects.

Ethanol teratogenesis in Japanese medaka: effects at the cellular level.

The adverse effects of alcohol on the developing humans represent a spectrum of structural and neurobehavioral abnormalities, most appropriately termed as fetal alcohol spectrum disorder (FASD). The mechanism by which ethanol induces FASD is unknown. Human studies of FASD are very limited due to ethical constraints; however, several animal models from nematodes to mammals are utilized to understand the molecular mechanism of this disorder. We have used Japanese medaka (Oryzias latipes) embryo-larval development as a unique non-mammalian model to study the molecular mechanism of FASD. Fertilized medaka eggs were exposed to ethanol (0-400 mM) for 48 h post fertilization (hpf) and then maintained in regular embryo rearing medium without ethanol. Viable embryos were harvested on 0, 2, 4 and 6 day post fertilization (dpf) and analyzed for DNA, RNA and protein contents of the embryos. By applying semi-quantitative RT-PCR (rRT-PCR) and quantitative real-time RT-PCR (qRT-PCR), RNA samples were further analyzed for seven transcription factors, emx2, en2, iro3, otx2, shh, wnt1 and zic5 which are expressed in the neural tube of medaka embryo during early phase of development. RNA and protein contents of the embryos were significantly reduced by ethanol at 400 mM dose on 4 and 6 dpf compared to the control (no ethanol), and 100 mM ethanol treated embryos. However, significant reduction of DNA was observed only in 4 dpf embryos. Total protein contents of yolk remained unaltered after ethanol treatment. Expression pattern of emx2, en2, iro3, otx2, shh, wnt1, and zic5 mRNAs were found to be developmentally regulated, however, remained unaltered after ethanol treatment. It is therefore concluded that alteration of nucleic acid and protein contents of medaka embryo by ethanol could be used as an indicator of embryonic growth retardation which might be the result of disruption of specific gene function during development.

Anti-aromatase activity of the constituents from damiana (Turnera diffusa).

ETHNOPHARMACOLOGICAL RELEVANCE: Damiana (Turnera diffusa Willd. Ex Schult) has traditionally been used as an herbal aphrodisiac. AIM OF THE STUDY: The study was aimed to investigate the anti-aromatse activity and the estrogenic activity of the constituents isolated from Turnera diffusa. MATERIALS AND METHODS: The methanolic extract and 24 compounds isolated from the leaves of Turnera diffusa were evaluated for aromatase activity by using a tritiated-water release assay and for estrogenic activity by using yeast estrogen screen (YES) assay. RESULTS: The methanolic extract demonstrated a dose-dependent inhibitory activity of the aromatase enzyme with the IC(50) value of 63.1 microg/ml. Among the 24 tested compounds, pinocembrin and acacetin showed the most potent inhibition with IC(50) values of 10.8 and 18.7 microM, respectively. Estrogenic activity was also observed in the extract and three compounds including apigenin 7-glucoside, Z-echinacin and pinocembrin with EC(50) values of 10, 20 and 67 microuM, respectively. CONCLUSIONS: The extract of Turnera diffusa and two isolated compounds pinocembrin and acacetin could significantly suppress aromatase activity. Moreover, apigenin 7-glucoside, Z-echinacin and pinocembrin showed estrogenic activity.

A Review on Graphene-Based Nanomaterials in Biomedical Applications and Risks in Environment and Health.

Graphene-based nanomaterials (GBNs) have attracted increasing interests of the scientific community due to their unique physicochemical properties and their applications in biotechnology, biomedicine, bioengineering, disease diagnosis and therapy. Although a large amount of researches have been conducted on these novel nanomaterials, limited comprehensive reviews are published on their biomedical applications and potential environmental and human health effects. The present research aimed at addressing this knowledge gap by examining and discussing: (1) the history, synthesis, structural properties and recent developments of GBNs for biomedical applications; (2) GBNs uses as therapeutics, drug/gene delivery and antibacterial materials; (3) GBNs applications in tissue engineering and in research as biosensors and bioimaging materials; and (4) GBNs potential environmental effects and human health risks. It also discussed the perspectives and challenges associated with the biomedical applications of GBNs.