JEZ 0543 492 S.C.JOURNAL LEARY ET OF AL. EXPERIMENTAL ZOOLOGY 284:492–499 (1999) Evaluation of Thyroid Hormone Economy in Elasmobranch Fishes, With Measurements of Hepatic 5¢-Monodeiodinase Activity in Wild Dogfish SCOT C. LEARY,1 JAMES S. BALLANTYNE,1 AND JOHN F. LEATHERLAND2* 1 Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 2 Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1 ABSTRACT This paper reviews the current understanding of thyroid hormone economy and homeostasis in elasmobranch fishes and considers those measures of the activity of the hypothalamic-pituitary gland-thyroid gland-peripheral tissue axis that are necessary for adequate assessment of thyroid hormone physiology. In particular, we focus on the value of measuring hepatic 5′-monodeiodinase (5′-MDA) activity as an indicator of the animal’s cellular production rate of the active thyroid hormone, triiodo-L-thyronine (T3). We also examine the characteristics of hepatic 5′MDA activity, in vitro, in adult female dogfish (Squalus acanthias) collected from Passamaquoddy Bay, New Brunswick, Canada, and in the embryos that they were carrying. T3 production from T4 by hepatic homogenates in vitro was time- and temperature-dependent, and was enhanced by the presence of a thiol donor. Michaelis constant (Km) and maximum reaction velocity (Vmax) values were 3.8 × 10–7 M and 0.29 nM T3/mg protein/hr, respectively. The inclusion of trimethylamine-Noxide (TMAO) or a mixture of urea, TMAO, betaine and sarcosine significantly enhanced T3 production. Hepatic 5′-MDA activity was depressed in fish fasted for 7 days. J. Exp. Zool. 284:492–499, 1999. © 1999 Wiley-Liss, Inc. THYROID HORMONE HOMEOSTASIS IN VERTEBRATES: AN OVERVIEW The thyroid gland or its progenitor, the endostyle, is a characteristic of all vertebrates, and its essential structure has been highly conserved throughout the vertebrate lineage (Leatherland, ’94). The gland is composed of follicles, formed by a single layer of thyrocytes, surrounding a colloidfilled lumen. The principal constituent of the colloid is the protein thyroglobulin, which contains within its structure the iodinated tyrosine units, monoiodotyrosine (MIT) and diiodotyrosine (DIT). When combined, these form the two hormones that are released from the thyroid gland, tetraiodothyronine, or L-thyroxine (T4) and triiodo-Lthyronine (T3). Our understanding of the processes associated with thyroid hormone synthesis and release is based largely on studies of mammals (Lissitzky et al., ’90), and few studies have been made in other classes of vertebrates; however, the information that is available suggests that the processes are very similar among vertebrates. © 1999 WILEY-LISS, INC. Hormone synthesis and release The protein that ultimately forms the iodinated thyroglobulin is synthesized by the thyrocytes and secreted, by exocytosis, into the lumen of the follicle. As the protein is secreted, it is iodinated at specific tyrosine sites, resulting in the formation of MIT and DIT units (Xiao et al., ’96). Further processing of the protein results in specific coupling of MIT and DIT units, to form T4 (two DIT units) or T3 (DIT + MIT). However, at this stage T4 and T3 are still part of the thyroglobulin matrix, and must be released from thyroglobulin in order to become hormonally viable. Both the initial iodination of the tyrosines and the coupling of the DIT and MIT residues are catalyzed by thyroid peroxidase and NADPH oxidase. These enzymes are integral proteins of the apical membrane of the thyrocytes. They have their ac- Grant sponsor: NSERC. *Correspondence to: Dr. J.F. Leatherland, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: email@example.com THYROID HORMONE ECONOMY OF DOGFISH tive site projecting into the lumen, and consequently, the iodination process per se occurs within the lumen (Golstein et al., ’95). The iodide required for iodination is provided by the thyrocytes, which actively sequester iodide from the blood by means of electrolytic pumps located on the basal cell membrane (Golstein et al., ’92). The iodide is then converted oxidatively to iodine and attached to the tyrosine units. The thyrocytes synthesize the peroxidase and NADPH oxidase, and secrete the hydrogen peroxide that is needed for the oxidative process. The thyroid hormones, T4 and T3, are released from the thyroglobulin by proteolytic digestion of the protein (Lissitzky et al., ’90). Colloid is taken up, by endocytosis, at the apical pole of the thyrocytes and membrane-defined vesicles appear within the cytoplasm. Lysosomes, also synthesized by the thyrocytes, fuse with the colloid-filled vesicles, the thyroglobulin is proteolytically digested, and T4 and T3 are released. The hormones diffuse into the cytoplasm of the thyrocytes and then out of the cell. MIT and DIT residues are also released from the thyroglobulin during proteolysis, but are largely degraded and the iodide and amino acids recycled intracellularly. Hormone transport in the blood, peripheral metabolism of thyroid hormone and site of T3 action in target cells In all vertebrates studied to date, a significant amount (up to 99% in some mammals) of the thyroid hormones in the blood are attached to specific transport proteins (Hayashi and Refetoff, ’95). This binding of the hormones to transport proteins allows a greater concentration of hormone to be carried in the blood than could be accomplished if the hormone were in simple solution. In addition, loss of hormone via the kidney (and gills in aquatic vertebrates) is prevented, and there is a readily available supply of hormone present in the blood to meet the sometimes rapidly changing needs of the animal. An additional advantage for transporting thyroid hormones in a protein-bound form is that as blood perfuses an organ system, as free hormone leaves the blood to enter target cells, bound hormone dissociates from the transport protein. Thus, there is a source of available hormone in the blood in all regions of an organ system; this is particularly important for large organ systems, such as the liver. However, only the unbound (free) thyroid hormone in the blood is available for interaction with 493 receptors in target cells. Consequently, a dynamic equilibrium is established between free and protein-bound thyroid hormones in the blood. Factors that change the levels of the transport proteins, or interfere with the binding of hormone to these proteins will bring about changes in the dynamic equilibrium of free and bound hormones, and thus alter total thyroid hormone levels in the blood. Although both T3 and T4 are present in the blood of vertebrates, thyroid hormone-sensitive tissues have only T3 receptors. Thus, only T3 has biological activity and T4 acts as a pro-hormone, available for enzymatic conversion (monodeiodination) to form T3. In those taxa studied to date, most of the T3 present in the general circulation is generated by the monodeiodination of T4 in nonthyroidal tissues. The enzyme involved, 5′-monodeiodinase (5′-MDA), catalyzes the removal of one iodide from the outer tyrosine ring of T4, and has been characterized in representatives of all classes of vertebrates, except elasmobranch fishes (mammals: Visser, ’78; Toyoda et al., ’97; birds: McNabb et al., ’86; amphibians: Galton and Hiebert, ’87; teleost fishes: Leatherland et al., ’90b; lampreys: Leatherland et al.,’90a). T4 can also be subjected to monodeiodination by removal of an iodide from the inner tyrosine ring, a reaction that is catalyzed by 5-monodeiodinase (5-MDA) (Fig. 1). The product, called reverse T3 (rT3), has no proven biological activity to date. However, the degradation of T4 via a metabolite that has no biological activity allows for the elimination of excess T4 without generating the potent hormone, T3. The selective expression of 5′- and 5-MDA thus offers a cell the means by which the intracellular needs of T3 can be finely regulated. The cells of some organ systems manufacture sufficient T3 to meet only their own immediate needs. However, other peripheral tissues, such as the liver and kidney, not only supply their own intracellular needs, but also release T3 into the general circulation. T3 interacts with its target cell at receptor (T3R) sites that are located at specific positions on the nuclear DNA (Brent, ’94; Oppenheimer et al., ’95; Wagner et al., ’95). Additional nonnuclear sites of interaction have also been proposed, although the question as to whether these are receptor-mediated is still controversial. The nuclear T3R forms a heterodimer with the retinol-X receptor, and in the presence of T3, the receptor is activated and, depending on the specific target cell, will either stimulate or inhibit the expression of specific genes. 494 S.C. LEARY ET AL. that code for 5′- and 5-MDA, and by the selective activation of these enzymes, as required. “Nonthyroidal” hormones, such as growth hormone (GH), can induce changes in 5′- and possibly 5MDA activity. As a result, the overall control of production of the biologically active thyroid hormone T3 imparts a considerable stability (homeostasis) to the regulation of the amount of hormone that is available in the blood and in specific target tissues. Evaluation of thyroid hormone homeostasis and economy Fig. 1. Schematic diagram showing the “typical” vertebrate model of the regulation of blood T4 levels by TSH regulation of thyroid gland synthesis and release, and the production of T3 and rT3 by outer- and inner ring monodeiodination of T4, respectively. Regulation of thyroid hormone secretion Thyroid gland activity in mammals is largely under the direct control of the pituitary hormone, thyrotropin (TSH). TSH stimulates many of the mechanisms associated with the synthesis of thyroglobulin, as well as the processes associated with the release of the hormones from the thyroglobulin. The synthesis and release of TSH are, in turn, stimulated by the hypothalamic hormone, thyrotropin-releasing hormone (TRH). In addition, several neurotransmitters influence the synthesis and/or release of TRH. This is the so-called hypothalamus-pituitary gland-thyroid gland (HPT) axis. There are marked species differences with regard to the dominant factors in the HPT axis of different vertebrates. For example, in some teleost fishes, TRH may play only a minor role, with dopamine, a potent inhibitor of TSH release, playing the major regulatory function (Leatherland, ’94). However, the fundamental HPT axis has been well conserved throughout vertebrate classes. In vitro studies have shown that there is a negative feedback regulation of TRH secretion by TSH. Moreover, in these experimental situations, TSH itself may be autoregulatory. In addition, T4, and to a lesser extent T3, exert a potent negative feedback control over the synthesis and secretion (release) of TSH, probably acting both at the level of the hypothalamus and the thyrotrophs. Thus, there is considerable stability in the maintenance of blood T4 levels. Furthermore, the production of T3 from T4 by peripheral tissues is itself highly regulated, by the selective expression of the genes The various negative feedback control systems that link the hypothalamus, pituitary gland and thyroid gland provide a significant stabilization of blood thyroid hormone levels. Furthermore, although the total blood hormone concentrations may be affected by several factors, including changes in the concentration of transport proteins, the free T3 concentrations in the blood tend to be adjusted dynamically to satisfy the needs of the animal. Added to this is the control of T3 and rT3 production by target tissues themselves. These regulatory processes, acting in concert, impart considerable stability to the overall thyroid hormone economy of the animal, and T3 homeostasis is maintained within fine limits. In order to monitor changes in the animal’s thyroidal status it is necessary to measure several parameters of thyroid physiology (Table 1). Measurement of only one parameter may not provide sufficient information to facilitate interpretation. Thyroid hormone economy and homeostasis in elasmobranchs Although it is assumed that many of the processes governing thyroid hormone homeostasis in elasmobranchs are similar to those found in mammals, there is very little direct evidence available. The morphology of the elasmobranch thyroid gland has received some attention (Olivereau, ’49; Woodhead, ’66; Honma et al., ’87), and the sensitivity of the elasmobranch gland to environmental goitrogens is well known (Leatherland et al., ’99). In addition, blood total thyroid hormone levels have been measured in a few elasmobranch species, and attempts have been made to relate changes in thyroid hormone concentrations to season and the reproductive state of the animals (e.g., Crow et al., ’99; Volkoff et al., ’99). However, compared with other vertebrate classes, the physiology of thyroid function in elasmobranch fishes has received very little attention. Consequently, there THYROID HORMONE ECONOMY OF DOGFISH TABLE 1. Variables to be measured for the assessment of thyroid function* Hypothalamus Expression of mRNA for TRH and other hypothalamic factors Thyroid gland Epithelial cell height and colloid content1 Rate of iodide incorporation2 Thyroid peroxidase activity Blood plasma or serum Total thyroid hormone concentration3 Free thyroid hormone concentration Transport protein concentration Thyrotrophin concentration TRH (and other hypothalamic hormone) concentration Thyroid hormone clearance Peripheral (non-thyroidal) tissues Tissue 5′-monodeiodinase (5′-MDA) activity4 Tissue 5-monodeiodinase (5-MDA) activity Expression of mRNA for 5′- and 5-MDA Expression of mRNA for T3 receptor (T3R) T3R content of target tissues *Superscripts indicate that some work has been documented on this aspect of thyroid hormone physiology for elasmobranch fishes: 1 Olivereau (’49), Woodhead (’66), Honma et al. (’87); 2Dimond (’63); 3 Crow et al. (’99), Volkoff et al. (’99); 4Leary et al. (this paper). are major gaps in knowledge (Table 1), and interpretation of the available data has to be made with care. Of particular note is the absence of any work related to the characterization of MDAs in peripheral tissues of elasmobranchs. The following section reports on such a study of hepatic 5′MDA in dogfish, Squalus acanthias, collected from the wild. Hepatic 5¢-monodeiodinase activity in Squalus acanthias To date, there have been no studies of peripheral monodeiodination of T4 in any elasmobranch species. As discussed above, this is a key aspect of thyroid hormone homeostasis since monodeiodination of T4 by peripheral tissues represents the major T3 generating process, and some of the earliest responses to changing physiological state of the animal are to be found in the altered MDA activity in peripheral tissues. For example, in teleostean fishes, 5′-MDA activity falls rapidly when animals are fasted (Farbridge and Leatherland, ’92; Leatherland, ’94). As a consequence of this lability, measurements of MDA activity in tissues of fish taken from the wild have proven difficult because most wild species will not readily adapt to captive conditions. The study of MDA activity in elasmobranchs is further complicated by the fact that appropriate enzyme assay conditions, vis-à-vis the potential ef- 495 fects of organic osmolytes on tissue enzyme activity, have not been determined. These osmolytes, such as urea and trimethylamine-N-oxide (TMAO), are known to be required for optimal enzyme activity for some metabolic enzymes (Yancey and Somero, ’80; Lin and Timasheff, ’94). Thus, we undertook to investigate the requirements for, and effects of these osmolytes on hepatic 5′-MDA activity in adult female dogfish collected from the north Atlantic Ocean, all of which showed evidence of recent feeding activity. The animals were sacrificed and the enzyme assays performed shortly after their capture to minimize any loss of enzyme activity associated with food deprivation. Additional animals were sampled after a fasting period to evaluate whether the response to food deprivation in this species is comparable to that of salmonid teleostean species. We also measured hepatic 5′-MDA activity in the embryos taken from the pregnant dogfish. These studies were of considerable interest since parallel studies in teleostean species (tilapia, Oreochromis mossambicus: Reddy et al., ’92; rainbow trout, Oncorhynchus mykiss and Arctic charr, Salvelinus alpinus: P.K. Reddy, M.N. Khan and J.F. Leatherland, unpublished data) have failed to detect 5′MDA activity in yolksac embryos; enzyme activity appeared only after full yolk absorption. MATERIALS AND METHODS Five adult female dogfish (2.68 ± 0.25 kg, range 2.1–3.4 kg) were caught by otter trawl from Passamaquoddy Bay, New Brunswick, and placed temporarily in a live well aboard the trawling vessel until transfer to 2 m3 aquaria supplied with continuous running sea water at 15°C at the Huntsman Marine Science Centre. Three animals were sampled following a 6 hr acclimation, and the two remaining animals were sampled after 7 days; the fish were not fed during this period. Five embryos, obtained from three of the pregnant females, were also used in the study. For comparative purposes, we examined the effects of organic osmolytes on 5′-MDA activity in both rainbow trout and dogfish liver homogenates. Trout were chosen because the kinetics of 5′-MDA are well studied in this species, and in vivo organic osmolyte levels are very low. Three immature rainbow trout (410 ± 9 g, range 394–424 g) were acclimated for at least 2 months to laboratory conditions in constantly running aerated well water at 10°C, and were fed daily, ad libitum, with a commercial trout chow (Martin’s Feed Mills, Elmira). 496 S.C. LEARY ET AL. Blood samples were taken by heparinized syringe from the caudal vein of the dogfish only, the plasma was separated by centrifugation and stored frozen at –80°C. The dogfish were pithed and the rainbow trout were anaesthetized in MS222 before dissection of the liver. We used the hepatic 5′-MDA assay described previously (Flett and Leatherland, ’89). Briefly, pieces of freshly dissected liver were homogenized in three volumes of ice-cold phosphate buffer (0.1 M Na2HPO4/ KH2PO4; pH 7.0) using a Polytron homogenizer (setting 6, three × 5 sec bursts). The homogenates were centrifuged at 150g for 15 min and the lipid layer was removed (dogfish). Unless otherwise stated, the reaction vessel contained 50 µl of homogenate, 150 µl of phosphate buffer and dithiothreitol (DTT) and T4 to final concentrations of 10 mM and 50 nM, respectively. After 20 min at 15°C, the reaction was stopped by the addition of four volumes of ice-cold ethanol. The reaction tubes were refrigerated overnight at 4°C and then centrifuged at 2000g for 12 min. Appropriate volumes of the supernatant were transferred into glass tubes, dried at room temperature, and the T3 content was measured by radioimmunoassay (RIA). Blanks containing either no homogenate (blank 1) or with ethanol added prior to the introduction of the T4 substrate (blank 2) were prepared with each enzyme assay. The blank 2 values were subtracted from the test reaction vessels before calculating the reaction rate as pM of T3 formed per mg of homogenate protein per hour. Homogenate protein concentration was measured by the biuret method using human albumin as the standard. The T3 content of each tube and plasma T3 and T4 concentrations were measured using commercial RIAs (Amersham Amerlex Assay, Kodak Clinical Diagnostics, Toronto, ON) previously validated for use with fish tissues (Leatherland and Farbridge, ’92). Hepatic 5′-MDA kinetics were examined in pooled homogenates of the 6-hr-acclimated adult female dogfish. The following parameters were evaluated: incubation time from 0 to 60 min, substrate concentration from 0 to 125 nM, DTT concentration from 0 to 100 mM, pH from 6.5 to 8.0, temperature from 0 to 40°C, organic osmolytes [urea or TMAO alone (400 and 200 mM, respectively) or a mixture of osmolytes that simulate the concentrations found in elasmobranch liver and muscle cells (urea, TMAO, betaine and sarcosine at 400, 180, 13 and 7 mM, respectively)] (Yancey and Somero, ’80; Robertson et al., ’91). The same osmolyte concentrations were used for the measurement of hepatic 5′-MDA activity in trout, and in both the trout and dogfish preparations, the effect was examined over a 0–125 nM range of substrate concentrations. Regression lines were calculated for LineweaverBurk plots and the correlation coefficients for each line tested for significance (P < 0.05). Km and Vmax values were derived from the Lineweaver-Burk regression lines. RESULTS In the adult dogfish liver homogenates, T3 production from the T4 substrate was time-dependent, increasing rapidly over the first 20 min and reaching a plateau within 60 min (Fig. 2a). The Km and Vmax values were 3.8 × 10–7 M and 0.29 nM T3/mg protein/hr. The reaction rate was temperature-dependent over a range of 0–40°C (Fig. 2b), had a pH optimum of 7.5 at 15°C (Fig. 2c), and was linearly dependent on DTT concentrations over a range of 0–100 nM (Fig. 2d). The inclusion of organic osmolytes in the reaction vessel markedly reduced the Vmax and Km values for the hepatic 5′-MDA of rainbow trout (Table 2). However, in the dogfish, the Vmax and Km values were reduced (12 and 51%, respectively) in the presence of urea, and enhanced (418 and 190%, respectively) in the presence of TMAO (Table 2). In the presence of the organic osmolyte mixture, hepatic 5′-MDA Vmax and Km values increased (125%) and decreased (31%), respectively, when compared with the preparation incubated with buffer alone (Table 2). Plasma T3 and T4 concentrations and hepatic T3 production rates of the dogfish fasted for 7 days were markedly lower than those of the fish sampled on the day of capture (Table 3). Moreover, the hepatic 5′-MDA activity of the yolksac embryos was considerably lower than that of the 6 hr acclimated adults, and in the same range as that of the 7-day-fasted fish (Table 3). DISCUSSION This study shows that the kinetic characteristics of hepatic 5′-MDA activity of the dogfish resemble those of the high affinity hepatic 5′-MDA of trout (e.g., Flett and Leatherland, ’89; MacLatchy and Eales, ’92). The dogfish enzyme appeared to be relatively thermally stable, and, as in the trout, its activity was enhanced in the presence of the thiol, DTT. The apparent enzyme pH optimum of 7.5 at 15°C is similar to the reported intracellular pH of elasmobranch muscle cells (Heisler et al., ’76). The study also shows that in THYROID HORMONE ECONOMY OF DOGFISH 497 Fig. 2. Kinetics of T3 production from T4 substrate by liver homogenates from adult female dogfish: effects of temperature (a), temperature (b), pH (c), and dithiothreitol (DTT) concentration (d). Data represent mean ± SEM of 3 replicates. order to measure 5′-MDA activity in this species, an appropriate organic osmolyte or mixture of osmolytes needs to be added to the assay buffer. Whereas the organic osmolytes inhibited rainbow trout hepatic 5′-MDA activity, as evidenced by the depressed Vmax value, the Vmax value for the 5′-MDA in adult dogfish was enhanced in the presence of either TMAO alone or the 2:1 urea to methylamine organic osmolyte mixture (Table 2). Although the addition of these organic osmolytes to the reaction mixture caused small pH changes (up to pH 7.2), the changes in Vmax and Km of the preparations incubated with the osmolytes cannot be attributed to the small pH shift (see Fig. 2c). The fact that organic osmolytes influence hepatic enzyme activity in dogfish but not trout suggests that they play a physiological role in 5′-MDA function in elasmobranch fishes. Some studies have shown that urea and TMAO affect protein structure by altering the solubility of nonpolar residues and of the polypeptide backbone (Mashino and Fridovich, ’87; Lin and Timasheff, ’94). Furthermore, both osmolytes have been shown to exert enzyme-specific and species-specific stimulatory or inhibitory effects on enzyme activities (Yancey and Somero, ’80; Mashino and Fridovich, ’87; Yancey, ’93). As with fasting salmonid fishes (Farbridge and Leatherland, ’92), the hepatic 5′-MDA activity and plasma thyroid hormone concentrations of the adult female dogfish declined when the fish were held without food for 7 days (Table 3). The fall in plasma T3 levels, presumably in part resulting from a decreased peripheral T3 production rate, is likely a caloric sparing strategy, comparable to that seen in ration-restricted teleost fishes (Leatherland, ’94). The finding of hepatic 5′-MDA activity (albeit 498 S.C. LEARY ET AL. TABLE 2. Effect of organic osmolytes on the Vmax and Km values of hepatic 5¢-monodeiodinase of dogfish and rainbow trout* Assay conditions Buffer alone Buffer + urea (400 mM) Buffer + TMAO (200 mM) Buffer + mix3 Vmax1 Km 2 Dogfish Trout Dogfish Trout 0.29 0.26 0.35 0.18 382 188 59 27 1.21 0.13 726 11 0.36 0.17 225 7 *Vmax and Km values were derived from a Lineweaver-Burk plot of the effect of substrate concentration (0–125 nM range). The correlation coefficients of the regression lines for all dogfish trials and the buffer alone trout trial were >0.94, those for the trout trials with organic osmolytes were 0.85, 0.79 and 0.56 for the urea, TMAO, and mixed osmolyte trials, respectively; all regression lines were significantly different (P < 0.01) from 0. 1 pmol T3/mg protein/hr. 2 nM T4. 3 Urea (400 mM); TMA0 (200 mM); betaine (13 mM) and sarcosine (7 mM). at low levels) in dogfish yolksac embryos is of considerable interest. In several comparable studies made of embryonic teleost fish, 5′-MDA activity (whole body) was present only after the yolk had been absorbed (Reddy et al., ’92). The yolk of some teleost fish eggs contains significant levels of thyroid hormone. The hormones appear to be of maternal origin and may inhibit the development of a functional HPT axis, and may delay the onset of hepatic 5′-MDA activity (Tagawa et al., ’90; Leatherland and Barrett, ’93; Leatherland, ’94). The finding of 5′-MDA activity in the dogfish embryos suggests that a similar inhibition of the enzymes involved in T 4 monodeiodination is not present. TABLE 3. Plasma L-thyroxine (T4) and triiodo-L-thyronine (T3) concentrations and hepatic 5¢-monodeiodinase (5¢-MDA) activity in adult female dogfish and hepatic 5¢-MDA activity in dogfish embryos1 Adult females [6 hour retention in holding aquarium] Adult females [7 day retention in holding aquarium] Embryos 1 T4 T3 5′-MDA activity 3.3 2.3 2.3 nd nd 4.3 1.5 3.8 nd nd 0.27 0.06 0.07 0.01 0.01 nm nm 0.02 ± 0.003 Plasma thyroid hormone concentrations (nM) and hepatic 5′-MDA activity (pmol/mg protein/hr) of adult dogfish are shown as individual values; hepatic MDA activity of the embryos is shown as mean ± SEM (n = 5); nd, not detectable; nm, not measured. CONCLUSION There is clearly much that needs to be done to examine the various components of thyroid function in elasmobranch fishes. To date only a handful of studies have been made of blood hormone levels at different times of development and of season, and the present report is the first to describe the presence of monodeiodinase activity in any elasmobranch tissues. Any study of the effect of environmental factors on thyroid hormone physiology of elasmobranch fishes has to include measurements of several parameters in order for the results to be interpreted with confidence. This study shows that 5′-MDA is present in the liver of dogfish, and it is likely also present in other tissues, and that changes in enzyme activity reflect the altered physiological status of the animal. They are therefore useful indicators for any studies of thyroid hormone economy or thyroid hormone homeostasis in elasmobranch fishes. ACKNOWLEDGMENTS The work was supported by grants from NSERC to J.S.B. and J.F.L. Thanks to A.W. Bially for assisting with the data collection and to S. BrettWelsh for technical support. 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