The effects of 17-α-ethinylestradiol (EE2) on molecular signaling cascades in mummichog (Fundulus heteroclitus).

Exposures to ≤10 ng/L of 17-α-ethinylestradiol (EE2) will reduce or shut down egg production in freshwater fish models, while mummichog (Fundulus heteroclitus), an estuarine species, are able to produce eggs at EE2 concentrations >3000 ng/L. The objective of this study was to gain mechanistic insight into how mummichog are able to produce eggs during exposures to high EE2. Mummichog were exposed to 0, 50 or 250 ng/L of EE2 for 14 d. There were no changes in gonadosomatic index, liversomatic index, gonad development, or plasma estradiol levels after exposure to EE2. However, testosterone significantly decreased with EE2 exposures (50, 250 ng/L). Microarray analysis in the liver revealed that cell processes associated with lipids were affected by EE2 at the transcriptome level. Based on the transcriptomics data, we hypothesize that mummichog are able to maintain lipid transport and uptake into the ovary and this may be associated with apolipoproteins, facilitating normal oocyte development. Novel gene regulatory networks for protein modification targets were also constructed to learn more about the potential roles of estrogens in the teleost liver. Although post-translational modifications (PTMs) are important regulatory mechanisms, the roles of PTMs in protein regulation in fish and the susceptibility of PTMs to aquatic pollutants are largely unexplored and may offer novel insight into mechanisms of endocrine disruption.

Assessment of the potential of the rock gunnel (Pholis gunnellus) along the Atlantic coast of Canada as a species for monitoring the reproductive impacts of contaminant exposures.

Evaluating the impacts of point source discharges on fish species in estuarine environments can be challenging because of a paucity of resident species. We evaluated the biology of rock gunnel (Pholis gunnellus) at three relatively uncontaminated sites in the Bay of Fundy, along the Atlantic coast of Canada. Rock gunnel are seasonally resident (April to November) in tide pools, but little was known about their life history in Atlantic Canada or their potential for use for monitoring environmental quality. Fish were collected between April and November, and ranged from 2.46 g-15.2g in weight and 97 mm-170 mm in length, with a maximum age of 7 years. Both males and females were similar in size, and both reached sexual maturity at a size of 5.5 g. Organ weights and condition indices of fish were stable from spring when they returned from offshore (April to May) until late summer (August to September), but fall fish (October to November) had slightly larger gonads, livers and condition indices. Rock gunnel may be a useful indicator to provide insight into local impacts of point sources over a short time period. However, they do not provide adequate information on reproductive development and performance since they are not exposed to onshore contaminants during the periods of gonadal development that have most commonly found to be sensitive to anthropogenic stressors.

Effects of metal mining effluent on Atlantic salmon (Salmo salar) and slimy sculpin (Cottus cognatus): using artificial streams to assess existing effects and predict future consequences.

In the summer of 2000, the effects of metal mine discharge on fish growth and exercise performance were assessed at a Zn-Pb-Cu mine in New Brunswick, Canada. Juvenile Atlantic salmon (Salmo salar) were exposed to 0%, 20%, and 80% treated metal mine effluent in a mobile, fish-only artificial stream system. Fish were fed commercial salmon pellets throughout the study. Young-of-the-year slimy sculpins (Cottus cognatus) were exposed to the same treatments in a multitrophic level, modular artificial stream system or mesocosm, in which the fish were dependent on seeded algae and invertebrates for nutrition. Treatment concentrations were chosen to represent existing discharge dilutions (80%) and a scenario of reduced effluent discharge (20%) as predicted upon mine closure (scheduled for 2008). Al, Ba, B, Fe, Mn, Sr, Tl, Ti, and Zn increased in a concentration-dependent fashion across the three treatments. Salmon body burdens of Ba, Cd, Li, Cu, Mn, Se, Sr, and Zn were increased in the 80% treatment, while Tl increased across all treatment levels. Mortalities and depressions in growth in both fish species paralleled treatment concentrations (80%>20%>0%). Salmon liver weight was significantly greater in fish exposed to 20% and 80% effluent in a concentration-dependent fashion. Exercise performance in fish, as assessed by the ability to recover from forced exercise, showed little effect of treatment. The contamination of the receiving environment by mine discharges has led to loss of fish, making it impossible to study the system in situ. However, the use of the artificial stream systems enabled us to assess effects of present conditions on fish, as well as the potential impacts of mine reclamation. The 20% discharge predicted following mine reclamation is potentially favourable for the reinstitution of native fishes into the system.

Effects of water-borne 4-nonylphenol and 17beta-estradiol exposures during parr-smolt transformation on growth and plasma IGF-I of Atlantic salmon (Salmo salar L.).

4-Nonylphenol (4-NP) is an endocrine disrupting substance (EDS) capable of mimicking the action of 17beta-estradiol (E2). It has been hypothesized that 4-NP in a pesticide formulation is linked to historical declines in Canadian Atlantic salmon (Salmo salar L.) populations, with effects being related to exposure during parr-smolt transformation (PST). To test this hypothesis, Atlantic salmon smolts were exposed to pulse-doses of water-borne 4-NP (20 ug/l), sustained doses of water-borne E2 (100 ng/l) (positive control), or ethanol vehicle (negative control) in mid-May during the final stages of PST. Individually tagged smolts were then sampled at three times (June, July and October) to monitor subsequent growth in sea water and plasma insulin-like growth factor I (IGF-I) concentrations. Smolt weights and plasma IGF-I concentrations were both affected by E2 and 4-NP. The effects of E2 and 4-NP on mean smolt weights were most prominent in July and October [E2 (*98.1 +/- 2.8, *242.3 +/- 10.6 g), 4-NP (*102.1 +/- 3.1, 255.7 +/- 9.5 g), controls (112.5 +/- 2.8, 282.3 +/- 8.8 g)] (P < 0.05), while their effects on mean plasma IGF-I concentrations were most prominent in June and October [E2 (15.0 +/- 1.9, 28.4 +/- 1.8 ng/ml), 4-NP (*14.8 +/- 1.9, *21.6 +/- 1.7 ng/ml), controls (20.0 +/- 1.1, 31.1 +/- 2.0 ng/ml)] (P < 0.05). Additionally, results suggest that the mechanisms of action of E2 and 4-NP involve disruption in the GH/IGF-I axis, and that they may be different from each other. The effects of E2 and 4-NP on growth and plasma IGF-I concentrations observed in this study are ecologically significant because they evoke concerns for successful growth and survival of wild salmon smolts exposed to low levels of estrogenic substances that may occur from current discharges into rivers supporting sea-run salmon stocks.

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

Identification and treatment of a waste stream at a bleached-kraft pulp mill that depresses a sex steroid in the mummichog (Fundulus heteroclitus).

Changes to indicators of reproductive performance have been documented in fish exposed to some bleached kraft pulp mill effluents (BKPMEs). However, responses are not consistent across mill types or processes. It is not clear where the sources of the effects are within mills, what the causative compounds are, or what process changes are effective to remove these sources. Our previous studies suggested that condensates were a source of compounds that reduced plasma testosterone in the mummichog (Fundulus heteroclitus). Results also suggested that reverse osmosis (RO) treatment of condensates removed this source. The objective of this study was to use a toxicity identification evaluation approach and expose mummichog to various waste streams in the laboratory to identify the effluent source that depressed plasma testosterone in the mummichog and the effects of RO treatment. In 7- and 21-d exposures, mummichog were exposed to dilutions of RO feed condensate, RO permeate, combined mill effluent (CME), and final effluent. Results confirmed that condensates depressed plasma testosterone in mummichog. Chemical characterization of the condensate indicated that plant phytosterols were likely not the responsible compounds. Results also confirmed that RO treatment removed the potential of the condensates to depress plasma testosterone in mummichog at environmentally relevant concentrations of final mill effluent (1%).

The phytoestrogen beta-sitosterol alters the reproductive endocrine status of goldfish.

There is a growing awareness that chemicals in the environment may function as hormone mimics and affect endocrine function in wildlife. In this study, the effects of beta-sitosterol, a phytoestrogen present in high concentration in bleached kraft pulp mill effluent (BKME), on reproductive fitness of goldfish were investigated. Plasma reproductive hormone levels were measured in male and female goldfish on Day 4 following two intraperitoneal injections of beta-sitosterol or an oxidized sitosterol preparation. In some experiments, plasma hormone levels were also measured after fish were injected with Ovaprim, which contains a superactive analog of salmon GnRH and the dopamine receptor antagonist domperidone and leads to increased secretion of gonadotropin (GtH)-II (LH-type GtH). Plasma testosterone (T) and 11-ketotestosterone levels in males and T and 17 beta-estradiol levels in females were significantly decreased in beta-sitosterol-treated fish on Day 4 and 24 hr after an injection of Ovaprim. Plasma GtH-II levels were elevated in male fish treated with beta-sitosterol on Day 4 and further increased in response to Ovaprim, suggesting that reduced plasma steroid levels were not due to effects on pituitary function. In other studies, testes pieces from beta-sitosterol-treated goldfish produced reduced levels of T and pregnenolone in vitro both basally and in response to the GtH-II agonist human chorionic gonadotropin (hCG) when compared to the testes from control fish. Basal and hCG-stimulated pregnenolone and hCG-stimulated T were reduced in follicles from beta-sitosterol-treated fish; however, basal T production was not different from controls. These results suggest that beta-sitosterol reduces the gonadal steroid biosynthetic capacity through effects on cholesterol availability or the activity of the side chain cleavage enzyme P450SCC. These findings raise the possibility that beta-sitosterol could be a contributing factor to the reproductive dysfunction observed in fish exposed to BKME.

Thyroid hormone deiodinase systems in salmonids, and their involvement in the regulation of thyroidal status.

The trout thyroid secretes L-thyroxine (T4) which undergoes enzymatic deiodination in liver and other tissues. Based on mammalian studies, T4 outer-ring deiodination (ORD) or T4 inner-ring deiodination (IRD) could generate respectively 3,5,3'-triiodo-L-thyronine (T3) or 3,3',5'-T3(rT3), while subsequent T3ORD or T3IRD could generate respectively 3,5-diiodo-L-thyronine (T2) or 3,3'-T2, and rT3ORD or rT3IRD could generate respectively 3,3'-T2 or 3',5'-T2. In practice, T4 in trout undergoes hepatic ORD to produce T3 but negligible IRD to produce rT3, and T3 in turn undergoes negligible ORD but modest IRD to produce 3,3'-T2. T4ORD, which is particularly important in converting T4 to the biologically more potent T3, also occurs in gill, muscle and kidney. At least two isozymes are involved: i) a high-affinity, propylthiouracil (PTU)-sensitive T4ORD which displays ping-pong kinetics, requires thiol as a cofactor, and is present in liver, gill and muscle, and ii) a low-affinity, PTU-insensitive T4ORD with sequential kinetics with a thiol cofactor, and is present in liver and kidney. Receptor-bound T3 is derived primarily from the plasma for kidney, mainly from intracellular sources for gill and about equally from both plasma and intracellular sources for liver. Thus, the high-affinity T4ORD may produce T3 for local intracellular use while the low-affinity 5'-monodeiodinase may produce T3 for systemic use. T4ORD activity responds to nutritional factors and the physiologic state of the fish. Furthermore, T3 administered orally for either 6 weeks or 24h reduces the functional level (Vmax) of hepatic T4ORD, and T3 added to isolated hepatocytes also reduces activity, indicating direct T3 autoregulation of T4ORD to maintain hepatocyte T3 homeostasis. However, T3 administration also induces T4IRD to produce biologically inactive rT3 and induces T3IRD to produce 3,3'-T2. Thus, the trout liver has several iodothyronine deiodinase systems which in a coordinated manner regulate tissue T3 homeostasis in the face of a T3 challenge. It does this by decreasing formation of T3 itself, by diverting T4 substrate to biologically inactive rT3 and by increasing the degradation of T3. These deiodinases differ in many respects from any mammalian counterparts.

Properties of T4 5'-deiodinating systems in various tissues of the rainbow trout, Oncorhynchus mykiss.

L-Thyroxine (T4) 5'-monodeiodinase (5'D) activity was examined in the microsomal fractions of liver, kidney, gill, white skeletal muscle, and red blood cells (RBC) of fed rainbow trout held in freshwater at 12 degrees. Two distinct 5'D systems were established and were examined at low (0.08-1.3 nM) or high (1.6-25 nM) T4 substrate ranges. The low substrate 5'D occurred in liver, gill, and muscle, but not in kidney or RBC. The pH optimum was 7.0 and the optimum dithiothreitol (DTT) level ranged from 7 to 10 mM. The Km values (nM) were liver, 0.098; muscle, 0.198; and gill, 0.168. The Vmax values (pmol.hr-1.mg protein-1) were liver, 3.74; muscle, 0.79; and gill, 0.62. DTT affected both the Vmax and the Km, and propylthiouracil (PTU) inhibited the Vmax. These data suggest a ping-pong type mechanism. In contrast, the high substrate 5'D occurred only in liver (pH 7 optimum, DTT optimum 15 mM) and in kidney (pH optima 6 and 8, DTT optimum 15 mM). The Km values (nM) were liver, 10.0; and kidney, 14.7; the Vmax values (pmol.hr-1.mg protein-1) were liver, 8.21; and kidney, 5.76. DTT affected the Vmax but not the Km and PTU did not inhibit, indicating a sequential type mechanism. In conclusion, in rainbow trout there are at least two types of 5'D which differ in their tissue distribution, T4 substrate affinity, and enzyme mechanism, and which do not resemble in their combined properties the 5'D forms established in higher vertebrate taxa.

Intra- and extra-cellular sources of T3 binding to putative thyroid hormone receptors in liver, kidney, and gill nuclei of immature rainbow trout, Oncorhynchus mykiss.

The sources of extracellular and intracellular 3,5,3'-triiodo-L-thyronine (T3) binding to putative thyroid hormone receptors in liver, kidney, and gill nuclei were determined in vivo for immature rainbow trout at 12 degrees C. Both [131I]T3 and [125I]T4 were injected intraperitoneally, the plasma and tissues were examined at isotopic equilibrium at 20 h, and the proportions of intracellular [125I]T3 and extracellular [131I]T3 saturably bound in the nucleus were determined. Comparable total amounts of T3 were saturably bound in the nuclei of liver (7.2), kidney (8.0), and gill (9.7 moles x 10(-13) .mg DNA-1), but the percentage of nuclear T3 generated within the target cell was greater for gill (76%) than for liver (50%) and kidney (28%). Both gill and liver possess a low Km T4 5'monodeiodinase which could be responsible for the high proportion of the nuclear T3 generated within those tissues.

Stimulation of hepatic thyroxine 5'-deiodinase activity in rainbow trout (Oncorhynchus mykiss) by Pacific salmon growth hormone.

1. Growth hormone extracted from Pacific salmon pituitaries (sGH) was injected intra-peritoneally into rainbow trout to determine sGH effects on plasma levels of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and properties of the hepatic 5'-deiodinase enzyme (5'-D) responsible for T4 to T3 conversion. 2. After 24 hr, sGH (0.1 or 0.5 microgram/g) did not alter the plasma T4 level or 5'-D, Km, but elevated plasma T3 and especially 5'-D Vmax. 3. Thus sGH, like the previously tested human GH, acutely enhances the potential for extra-thyroidal T3 production in trout by increasing the functional level of hepatic 5'-D, but without changing the plasma T4 level.

Effects of cortisol on aspects of 3,5,3'-triiodo-L-thyronine metabolism in rainbow trout (Oncorhynchus mykiss).

Aspects of 3,5,3'-triiodo-L-thyronine (T3) metabolism were studied in fed rainbow trout (Oncorhynchus mykiss) held at 11.5-14 degrees and intraperitoneally implanted with hydrogenated corn oil (controls) or oil containing cortisol. Cortisol implants caused dose-related plasma cortisol elevations within the physiological range for 2-3 weeks, loss in body weight, and depression in plasma T3 and free T3 index with no consistent change in plasma thyroxine (T4) or free T4 index. Plasma T3 clearance rate and plasma T3 appearance rate were both increased by cortisol, with no change in hepatic microsomal T4 5'-monodeiodinase activity (Km or Vmax), but with a significant decrease in muscle T3 concentration. It is concluded that chronic physiologic cortisol treatment enhances plasma T3 clearance without change in hepatic T4 to T3 conversion, resulting in a decline in T3 concentration in both plasma and tissue (muscle) compartments.

Influence of dietary lipid and carbohydrate levels and chronic 3,5,3'-triiodo-L-thyronine treatment on thyroid function in immature rainbow trout, Oncorhynchus mykiss.

The influence of varying dietary levels of nonprotein energy sources (lipid, L; carbohydrate, C) and 3,5,3'-triiodo-L-thyronine (T3) on thyroid function in immature rainbow trout was studied. Three diets of equivalent available energy content and identical nutrient composition, except for dissimilar concentrations of L and C (diet 1, L = 7%, C = 28.3%; diet 2, L = 13%, C = 14.9%; diet 3, L = 19%, C = 1.5%), were each supplemented with 0, 4, 8, or 12 ppm T3 and fed to satiation to trout at 6.5 +/- 0.5 degrees on a 12-hr photoperiod for 12 weeks. Dietary L and C concentrations did not influence plasma total L-thyroxine (T4) or T3 levels, indices of free T4 or free T3 levels, hepatic T4 5'-monodeiodinase (5'D) activity, capacity or affinity of hepatic nuclear T3 receptors, or thyroid follicle epithelial cell height. T3 treatment elevated total and free T3 levels and decreased 5'D activity (Vmax) in approximate proportion to T3 dose, and without effect on plasma total or free T4 levels or T3 receptor properties. However, thyroid follicle epithelial cell height was depressed at 8 or 12 ppm dietary T3. In trout reverted for 20 days to a T3-free diet from a T3 (12 ppm) diet, plasma total T3 levels fell to 30% of those of control trout (0 ppm T3 throughout). It was concluded that, under our experimental conditions, (i) trout thyroid function was refractory to dietary concentrations of L and C, (ii) the primary response to T3 supplementation was suppressed hepatic 5'D level and T3 production, which was sustained for at least 20 days after T3 treatment ceased, and (iii) despite causing a probable indirect decrease in thyroidal secretion, T3 did not modify the set point of the hypothalamo-hypophyseal-thyroid axis based on plasma total or free T4 levels.

Growth hormone stimulates hepatic thyroxine 5'-monodeiodinase activity and 3,5,3'-triiodothyronine levels in rainbow trout (Salmo gairdneri).

Intraperitoneal injection of rainbow trout (Salmo gairdneri) with 0.4 microgram/g of human growth hormone (hGH) increased plasma levels of 3,5,3'-triiodo-L-thyronine (T3) and Vmax of the hepatic microsomal 5'-monodeiodinase enzyme (5'D) that converts thyroxine (T4) to T3, with no effect on Km or plasma T4 levels. A dose of 0.4 microgram hGH/g increased both plasma T3 and Vmax as early as 8 hr postinjection (pi). Maximal levels for both parameters occurred at 24 hr pi and significant stimulation was sustained to 48 hr pi. In trout injected with hGH over a dose range of 0.1-1.0 microgram/g and sampled at 24 hr pi, both Vmax and plasma T3 increased in a dose-dependent manner. It is concluded that the increase in the level of functional hepatic 5'D may contribute to the rapid hGH-induced elevation in plasma T3.

The relationship between T3 production and energy balance in salmonids and other teleosts.

Extrathyroidal T4 5'-monodeiodination, demonstrated in several teleost species, generates T3 which binds more effectively than T4 to putative nuclear receptors and is probably the active thyroid hormone. T4 to T3 conversion is sensitive to the physiological state and provides a pivotal regulatory link between the environment and thyroid hormone action. T3 generation is enhanced in anabolic states (positive energy balance or conditions favoring somatic growth; food intake or treatment with androgens or growth hormone) and is suppressed in catabolic states (negative energy balance or conditions not favoring somatic growth; starvation, stress, or high estradiol levels associated with vitellogenesis). In fish, as in mammals, thyroidal status may be finely tuned to energy balance and through T3 production regulate energy-demanding processes, which in fish include somatic growth, development and early gonadal maturation.

Short-term treatment with testosterone increases plasma 3,5,3'-triiodo-L-thyronine and hepatic L-thyroxine 5'-monodeiodinase levels in arctic charr, Salvelinus alpinus.

Testosterone (T), methyl testosterone (MT), and testosterone propionate (TP) (0.5 mg/100 g body wt in 40 microliter peanut oil) or peanut oil alone were injected (ip) on Days 0 and 3 into immature arctic charr (Salvelinus alpinus). Charr were sampled on Days 7 and 12 and plasma testosterone, L-thyroxine (T4), 3,5,3'-triiodo-L-thyronine (T3), and hepatic microsomal T4 5'-monodeiodinase (T4 5'D) measured. Plasma androgen levels were elevated by all androgen treatments to levels similar to those observed in spawning salmonids. Plasma T3 was significantly increased by all forms of testosterone on Days 7 and 12. T4 levels remained unchanged or significantly decreased on Day 7, with no significant differences on Day 12. T4 5'D activity was increased on both Days 7 and 12 in the experimental groups due to increases in the Vmax (1.3 to 5.9 X control groups) with negligible changes in the Km. In conclusion, T, MT, and TP all stimulate thyroidal status by increasing plasma T3 levels in part as a result of increased hepatic T4 5'D activity.

The influence of short-term 17 beta-estradiol treatment on plasma T3 levels and in vitro hepatic T4 5'-monodeiodinase activity in immature rainbow trout, Salmo gairdneri.

To determine the effects of 17 beta-estradiol (E2) on aspects of thyroid function, immature rainbow trout were intraperitoneally injected with estradiol benzoate (0.5 mg/100 g) on Days 0 and 3 and sampled on Days 7 and 12. This protocol created plasma E2 concentrations during the first 7 days comparable to those during naturally induced vitellogenesis. Control trout received peanut oil alone. Plasma levels of 3,5,3'-triiodothyronine (T3) were significantly depressed on Day 7 but returned to levels by Day 12. Plasma thyroxine (T4) levels were not altered consistently by E2 treatment. Hepatic microsomal T4 5'-monodeiodinase (5'D) activity responsible for conversion of T4 to T3 was significantly depressed on Day 7 but returned to control levels by Day 12. Lineweaver-Burke plots showed that the lower hepatic 5'D resulted from a 10-fold decrease in Vmax, indicating a lower enzyme concentration. A slight reduction in Km was also observed. These results confirm that high E2 levels, comparable to those in vitellogenesis, depress plasma T3 levels in trout and show that, at least in part, this effect is the result of a decrease in the amount of functional hepatic 5'D.