The endocrine disruption potential of betamethasone using Japanese medaka as a fish model

The presence of synthetic glucocorticosteroids (GCs) in surface water and their potential endocrine disruption (ED) activity at environmental concentrations has not been fully investigated. Synthetic GCs may interfere with endogenous receptors within the hypothalamic-pituitary-gonadal (HPG) axis and disruptions of this pathway can result in decreased reproduction and/or adverse developmental effects. Betamethasone, a synthetic GC, has been on the market in the United States since the 1980's. The Pharmaceutical Assessment and Transport Evaluation model estimated betamethasone concentrations to be <0.6 ng/L in 95% of all U.S. surface waters. Concentrations of 0.01, 0.1, and 1.0 µg betamethasone/L were used in a two-generation fish full life cycle study with Japanese medaka. Gross endpoints, secondary sexual characteristics, and vitellogenin expression were evaluated. The highest concentration at which ED outcomes are not anticipated was determined to be 0.1 µg/L. The ratio of the predicted environmental concentration to the no effect concentration for ED is less than one, indicating no risk to aquatic life from environmentally relevant concentrations of betamethasone.     

Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity

Two vitelline envelope precursors (choriogenin H: Chg H; choriogenin L: Chg L) and an egg yolk precursor (vitellogenin B: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be approximately 215 kDa and approximately 69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to approximately 51 kDa and approximately 44 kDa, respectively. The mass of intact VgB was approximately 530 kDa and resolved into a polypeptide of approximately 185 kDa in SDS-PAGE. Specific antisera were raised against each purified protein and specific immunoassays were developed. When Chg H, Chg L and VgB were induced in the serum of immature mullet by injection with various doses of estradiol-17beta (E(2)), VgB exhibited the most sensitive response exhibiting high variation in its induced levels. The variation in induced levels of Chg H and L was relatively minimal although induction required higher doses of E(2) than with VgB. Serum samples obtained from immature mullet populations collected from their natural habitat exhibited similar profiles in the levels of these proteins. The present study suggests that the utilization of multiple biomarkers holds great importance for the reliable and accurate evaluation of estrogenic activity in aquatic environments.     

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

Autoantibodies as potential biomarkers for nasopharyngeal carcinoma

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients' sera with phage-expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage-expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA-1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor-associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.     

Chimeric RNAs as potential biomarkers for tumor diagnosis

Cancers claim millions of lives each year. Early detection that can enable a higher chance of cure is of paramount importance to cancer patients. However, diagnostic tools for many forms of tumors have been lacking. Over the last few years, studies of chimeric RNAs as biomarkers have emerged. Numerous reports using bioinformatics and screening methodologies have described more than 30,000 expressed sequence tags (EST) or cDNA sequences as putative chimeric RNAs. While cancer cells have been well known to contain fusion genes derived from chromosomal translocations, rearrangements or deletions, recent studies suggest that trans-splicing in cells may be another source of chimeric RNA production. Unlike cis-splicing, trans-splicing takes place between two pre-mRNA molecules, which are in most cases derived from two different genes, generating a chimeric non-co-linear RNA. It is possible that trans-splicing occurs in normal cells at high frequencies but the resulting chimeric RNAs exist only at low levels. However the levels of certain RNA chimeras may be elevated in cancers, leading to the formation of fusion genes. In light of the fact that chimeric RNAs have been shown to be overrepresented in various tumors, studies of the mechanisms that produce chimeric RNAs and identification of signature RNA chimeras as biomarkers present an opportunity for the development of diagnoses for early tumor detection. [BMB reports 2012; 45(3): 133-140].     

Synthesis of DEHP metabolites as biomarkers for GC-MS evaluation of phthalates as endocrine disrupters

Phthalates are used primarily as plasticizers to make polyvinyl chloride (PVC) soft and flexible. In recent years the phthalate esters have attracted increasing attention as environmental and biomedical pollutants and, because of their toxicological characteristics. In particular, they are more and more recognized as endocrine disrupters. In this context, we describe herein an efficient synthetic pathway leading to a series of metabolites of di(2-ethylhexyl) phthalate (DEHP). Mono(2-ethylhexenyl) phthalate was used as starting material to obtain these products in good yield, large scale and GC-MS purity. The metabolites of DEHP were synthesized, for the first time, as biomarkers to verify their quantitative determination in human urine and serum by GC-MS analysis for studying the exposure to phthalates and establishing reference values.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.     

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.     

Proteomic detection of nitroproteins as potential biomarkers for cardiovascular disease

Increased levels of reactive oxygen and nitrogen species are linked to many human diseases and can be formed as an indirect result of the disease process. The accumulation of specific nitroproteins which correlate with pathological processes suggests that nitration of protein tyrosine represents a dynamic and selective process, rather than a random event. Indeed, in numerous clinical disorders associated with an upregulation in oxidative stress, tyrosine nitration has been limited to certain cell types and to selective sites of injury. Additionally, proteomic studies show that only certain proteins are nitrated in selective tissue extracts. A growing list of nitrated proteins link the negative effects of protein nitration with their accumulation in a wide variety of diseases related to oxidation. Nitration of tyrosine has been demonstrated in diverse proteins such as cytochrome c, actin, histone, superoxide dismutase, α-synuclein, albumin, and angiotensin II. In vitro and in vivo aspects of redox-proteomics of specific nitroproteins that could be relevant to biomarker analysis and understanding of cardiovascular disease mechanism will be discussed within this review.     

MicroRNA expression aberration as potential peripheral blood biomarkers for schizophrenia

Since brain tissue is not readily accessible, a new focus in search of biomarkers for schizophrenia is blood-based expression profiling of non-protein coding genes such as microRNAs (miRNAs), which regulate gene expression by inhibiting the translation of messenger RNAs. This study aimed to identify potential miRNA signature for schizophrenia by comparing genome-wide miRNA expression profiles in patients with schizophrenia vs. healthy controls. A genome-wide miRNA expression profiling was performed using a Taqman array of 365 human miRNAs in the mononuclear leukocytes of a learning set of 30 cases and 30 controls. The discriminating performance of potential biomarkers was validated in an independent testing set of 60 cases and 30 controls. The expression levels of the miRNA signature were then evaluated for their correlation with the patients'' clinical symptoms, neurocognitive performances, and neurophysiological functions. A seven-miRNA signature (hsa-miR-34a, miR-449a, miR-564, miR-432, miR-548d, miR-572 and miR-652) was derived from a supervised classification with internal cross-validation, with an area under the curve (AUC) of receiver operating characteristics of 93%. The putative signature was then validated in the testing set, with an AUC of 85%. Among these miRNAs, miR-34a was differentially expressed between cases and controls in both the learning (P = 0.005) and the testing set (P = 0.002). These miRNAs were differentially correlated with patients'' negative symptoms, neurocognitive performance scores, and event-related potentials. The results indicated that the mononuclear leukocyte-based miRNA profiling is a feasible way to identify biomarkers for schizophrenia, and the seven-miRNA signature warrants further investigation.     

Purification and establishment of ELISA for vitellogenin of Japanese sardine (Sardinops melanostictus)

Two female-specific serum proteins (FSSPs) were detected immunologically in estradiol-treated Japanese sardine Sardinops melanostictus. The major FSSP was demonstrated to be a high molecular estradiol-inducible glycolipophosphoprotein with an immunological relation to a major yolk protein, and was suggested to be vitellogenin (VTG). VTG was purified using negative immunoaffinity chromatography. The isolated VTG was used for raising the specific antiserum against VTG. A homologous enzyme-linked immunosorbent assay (ELISA) was developed using the antiserum and the isolated VTG. The sensitivity range of the ELISA was 44 ng/ml to 2670 ng/ml of VTG concentration for the conditions used in our investigation.     

The concept of endocrine disruption and human health

In Europe, endocrine disruptors (EDs) have been defined as substances foreign to the body that have deleterious effects on the individuals or their descendants, due to changes in endocrine function. In the United States, EDs have been described as exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of the natural ligands responsible for maintaining homeostasis and regulating body development. These two definitions are complementary, but both indicate that the effects induced by EDs probably involve mechanisms relating in some way to hormonal homeostasis and action. EDs are generally described as substances with anti-oestrogenic, oestrogenic, anti-androgenic or androgenic effects. More recently, other targets have been evidenced such as the thyroid and immune system. Many different EDs are present in the various compartments of the environment (air, water and land) and in foods (of plant and animal origin). They may originate from food packaging, combustion products, plant health treatments, detergents and the chemical industry in general. In addition to the potential effects of these compounds on adults, the sensitivity of embryos and fetuses to many of the xenobiotic compounds likely to cross the placenta has raised considerable concern and led to major research efforts. With the exception of the clearly established links between diethylstilbestrol, reproductive health abnormalities and cancers, very little is known for certain about the effects of EDs on human health. Given the lack of available data, current concerns about the possible involvement of EDs in the increase in the incidence of breast cancer, and possibly of endometriosis and early puberty in girls, remain hypothetical. Conversely, the deterioration in male reproductive health is at the heart of preoccupations and progress in analyses of the relationship between EDs and human health. This literature review aims to describe the current state of knowledge about endocrine disruption, focusing in particular on the problem of food contaminants.