Ann Oliver Cheek

Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds

The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17beta-estradiol (E2)-treated male fathead minnows (Pimephales promnelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a approximately 156 kDa protein in plasma from vitellogenic females and E2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E2 (10(-8), 10(-9) and 10(-10) M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml(-1) were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.

Environmental signaling: a biological context for endocrine disruption.

Endogenous and exogenous chemical signals have evolved as a means for organisms to respond to physical or biological stimuli in the environment. Sensitivity to these signals can make organisms vulnerable to inadvertent signals from xenobiotics. In this review we discuss how various chemicals can interact with steroid-like signaling pathways, especially estrogen. Numerous compounds have estrogenic activity, including steroids, phytoestrogens, and synthetic chemicals. We compare bioavailability, metabolism, interaction with receptors, and interaction with cell-signaling pathways among these three structurally diverse groups in order to understand how these chemicals influence physiological responses. Based on their mechanisms of action, chemical steroid mimics could plausibly be associated with recent adverse health trends in humans and animals.

Effects of Long-Term Hypoxia on Enzymes of Carbohydrate Metabolism in the Gulf Killifish, Fundulus grandis

SUMMARY The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l-1 dissolved oxygen (∼3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes.