The effects of methimazole on development of the fathead minnow, Pimephales promelas, from embryo to adult.

The importance of thyroid hormones in regulating early developmental processes of many amphibian and fish species is well known, but the impacts of exposure to disrupters of thyroid homeostasis during the embryo-larval-juvenile transitions are unclear. To investigate these impacts, fathead minnows, Pimephales promelas, were exposed to a model thyroid axis disrupter, methimazole, an inhibitor of thyroid hormone synthesis, at control (0), 32, 100, and 320 mug/l, starting at <24-h postfertilization, for 28, 56, and 83/84 days postfertilization (dpf). Thyroid disruption was evident at 28 dpf, when survival was significantly reduced by 32 or 100 mug/l methimazole concomitant with a reduced thyroxine (T(4)) content. However, the T(3) content of these fish was similar to that of control fish, and body mass was unaffected (as in all groups), suggesting compensatory mechanisms overcame reduced T(4) synthesis. At the highest concentration of methimazole (320 mug/l), activation of feedback mechanisms on the hypothalamic-pituitary-thyroid axis was suggested by the normal T(4) content after 28 dpf exposure to methimazole, although triiodothyronine (T(3)) content of these fish was significantly reduced. The generally less pronounced disruption of thyroid hormone homeostasis after 56 days exposure to methimazole also suggests compensatory mechanisms in juvenile/adult fish that may regulate T(4) content, despite exposure to methimazole at 32 or 100 mug/l (in fish held in 320 mug/l methimazole, the T(4) content was significantly higher than in controls). Whole body T(3) content at 56 dpf was significantly depressed only in fish held in 100 mug/l methimazole. By 83/84 dpf, length, body mass, and thyroid hormone concentrations were similar in all experimental groups and controls, indicating that adult fish may achieve regulation of their thyroid axis despite prolonged exposures to thyroid disruptors throughout early development.

Effects of ammonium perchlorate on thyroid function in developing fathead minnows, Pimephales promelas.

Perchlorate is a known environmental contaminant, largely due to widespread military use as a propellant. Perchlorate acts pharmacologically as a competitive inhibitor of thyroidal iodide uptake in mammals, but the impacts of perchlorate contamination in aquatic ecosystems and, in particular, the effects on fish are unclear. Our studies aimed to investigate the effects of concentrations of ammonium perchlorate that can occur in the environment (1, 10, and 100 mg/L) on the development of fathead minnows, Pimephales promelas. For these studies, exposures started with embryos of < 24-hr postfertilization and were terminated after 28 days. Serial sectioning of thyroid follicles showed thyroid hyperplasia with increased follicular epithelial cell height and reduced colloid in all groups of fish that had been exposed to perchlorate for 28 days, compared with control fish. Whole-body thyroxine (T4) content (a measure of total circulating T4 in fish exposed to 100 mg/L perchlorate was elevated compared with the T4 content of control fish, but 3,5,3-triiodothyronine (T3) content was not significantly affected in any exposure group. Despite the apparent regulation of T3, after 28 days of exposure to ammonium perchlorate, fish exposed to the two higher levels (10 and 100 mg/L) were developmentally retarded, with a lack of scales and poor pigmentation, and significantly lower wet weight and standard length than were control fish. Our study indicates that environmental levels of ammonium perchlorate affect thyroid function in fish and that in the early life stages these effects may be associated with developmental retardation.

Physiological responses to acute temperature increase in European eels Anguilla anguilla infected with Anguillicola crassus.

The swimbladder parasite, Anguillicola crassus has infected, and spread rapidly, through European eel Anguilla anguilla (L.) populations over the past 20 to 25 yr. Our aim in the present studies was to elucidate whether the presence of A. crassus in these eels alters their rapid physiological responses to an acute temperature increase, compared to the response of uninfected fish. Both infected and uninfected fish showed significant increases in plasma cortisol after 2 h at a raised environmental temperature with increased plasma glucose after 6 h. However, infected eels exhibited a slight lag in glucose mobilisation, which may be due to the metabolic cost of harbouring a sanguiverous parasite. Both infected and uninfected fish showed a significant increase in haematocrit after 6 h of temperature elevation, but only uninfected fish exhibited a significant increase in haemoglobin at this point. However, there were no significant changes in mean erythrocyte haemoglobin concentration in either group. Our results suggest that acute temperature increase alone is unlikely to cause significant mortality of A. crassus-infected European eels; however, the effects of chronic increases in temperature in combination with other factors such as toxicants and hypoxia requires examination.

Angiotensin I-converting enzyme-like activity in tissues from the Atlantic hagfish (Myxine glutinosa) and detection of immunoreactive plasma angiotensins.

Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His-Leu min(-1) mg protein(-1), respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10(-5) M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8+/-11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin-angiotensin system in hagfishes, the earliest evolved group of craniates.

Activation of the newly discovered cyclostome renin-angiotensin system in the river lamprey Lampetra fluviatilis.

This study describes the first investigations of the physiological signals involved in activating the newly discovered cyclostome renin-angiotensin system (RAS) and its role in the river lamprey Lampetra fluviatilis. Experimental manipulation showed that volume depletion (removal of 40% blood volume) rapidly activated the RAS of lampreys acclimated to water at 576 mOsm kg(-1) (21 p.p.t.), significantly increasing plasma angiotensin concentrations after 30 min and 60 min. In agreement with these results, a rapid change in environmental salinity (758 mOsm kg(-1) to freshwater (FW) and FW to 605 mOsm kg(-1)), resulted in a rapid decrease and increase in plasma [angiotensin], respectively. Intraperitoneal (i.p.) injection of FW-acclimated river lampreys with 1% body mass by volume of nominally isosmotic saline (120 mmol l(-1) NaCl; 233 mOsm kg(-1)) resulted in a significant decrease in the plasma angiotensin concentration within 15 min. In contrast, i.p. injection of hyperosmotic saline (4 mol l(-1) NaCl) at 1% body mass by volume, which significantly increased plasma osmolality, had no significant effect on plasma [angiotensin], suggesting that volume/pressure receptors and osmoreceptors interact in regulating the lamprey RAS. These results indicate an important role for volume/pressor receptors, as in teleosts, but with an additional osmoreceptor mechanism, such that circulatory [angiotensin] is determined by interaction of volume/pressure and osmoreceptors and their relative sensitivities. The volume/pressure sensitivity is in keeping with the recent evidence of a vasoconstrictor action of homologous lamprey angiotensin and provides evidence that the fundamental role of the RAS in maintaining volume and pressure is an ancient function conserved over 500 million years of vertebrate evolution.

Developmental changes of thyroid hormones in the fathead minnow, Pimephales promelas.

The fathead minnow is widely used in ecotoxicological studies and such investigations have begun to focus on potential disruption of the thyroid axis. However, normal levels of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) and their developmental patterns are unknown. To provide these baseline data, radioimmunoassays were developed and validated for analysis of T4 and T3 after extraction from plasma or whole fish. Female fish showed consistently higher plasma levels of T4 than male fish. Analysis of thyroid hormones during development showed a significant rise in both T4 and T3 during the pre-hatch period, indicating embryonic production of both thyroid hormones. After hatching, whole-body content of both T4 and T3 significantly increased in early development, peaking at 16 days post-hatch (dph) and 9 dph, respectively, when T4 reached 32.88 +/- 3.30 ng g(-1) body weight and T3 reached 24.17 +/- 3.15 ng g(-1) body weight. Thyroid hormones subsequently declined to a low plateau in later development with approximately 5 ng g(-1) body weight T4 and 2 ng g(-1) body weight T3. These data suggest a prominent role for thyroid hormones in early developmental processes when we predict that the ecotoxicological effects of thyroid disrupters will be most significant.

Angiotensin converting enzyme-like activity in tissues from the river lamprey or lampern, Lampetra fluviatilis, acclimated to freshwater and seawater.

Angiotensin converting enzyme (ACE) or kininase II is a dipeptidyl-carboxypeptidase that converts angiotensin I (Ang I) to angiotensin II (Ang II) in the renin-angiotensin system (RAS) and inactivates bradykinin in the kallikrein-kinin system (KKS). Angiotensin converting enzyme-like activity (ACELA) has been demonstrated in a wide range of vertebrates, and only in lampreys is a lack of ACELA still suggested. Though long controversial, a lamprey RAS has recently been identified by isolation and sequencing of lamprey Ang I and the measurement of circulating plasma angiotensins. We therefore re-investigated the presence of ACE in tissues from the river lamprey or lampern, Lampetra fluviatilis, using a highly sensitive fluorimetric assay. Significant detection of ACELA was found in a wide range of lamprey tissues (brain, gill, gonad, gut, heart, liver, skeletal muscle, skin, kidney, and plasma). The mammalian ACE inhibitor captopril at 10(-5)M was an effective, but variable inhibitor of the ACELA found in most lamprey tissues. The brain contained the highest ACELA, while kidney (including urinary duct), skin, gonads, and heart only contained very low ACELA. In most tissues, ACELA was similar in lampreys acclimated to freshwater (FW) and seawater (SW). However, gut ACELA was significantly higher in lampreys acclimated to SW than in FW-acclimated lampreys. Liver, skin, and gonad ACELA was significantly lower in lampreys acclimated to SW than in FW lampreys. Male and female lampreys acclimated to FW showed similar ACELA in all tissues except the kidney (including the urinary duct), where ACELA was significantly higher in male than in female lampreys. These results indicate that ACELA, a component of the RAS and KKS, is present in tissues from one of the earliest evolved groups of vertebrates.