Creation of chronic physiological elevations of plasma thyroxine in brook trout, Salvelinus fontinalis (mitchill) and other teleosts

GENERAL

AND

COMPARATIVE

22, 209-217 (1974)

ENDOCRINOLOGY

Creation of Chronic Physiological Thyroxine in Brook Trout, (Mitchill)

and

Elevations of Plasma Salvelinus fontinalis

Other

Teleosts

J. G. EALES’ Department

of Zoology,

University

of Manitoba,

Winnipeg,

Manitoba,

Canada

Received May 1, 1973 Immersion and intraperitoneal injection methods were examined for producing small chronic elevations of plasma thyroxine (T,) in brook trout and other teleosts. Continual immersion of starved yearling brook trout in a solution of 10 pg T1/lOO ml water at 1213°C raised plasma T, within hours from 1.0 pg/lOO ml (or less) to l-3 ~g/lOO ml. This increase was sustained during 27 days of treatment. An ambient T, level of 2 pg/lOO ml produced a smaller but distinct increase ; 50 pg of T4/100 ml water was considered pharmacological. The influence of ambient Th on plasma T, depended on body size and temperature, with possible effects due to feeding state and species. Trout immersed in radioactive T, (5 pg/lOO ml) by 3 hr reached a steady state, with plasma T, considerably less than ambient T,. Both deiodination and biliary excretion of T, contributed to the steady state by removing radioactive T, still entering the fish. A single intraperitoneal T, injection was found to be impractical owing to rapid T, turnover. Thus frequent injections would be required which would probably not eliminate surges in plasma T, due to intraperitoneal injection. water at 1243°C before use. One-year-old or twoyear-old brook trout, or rainbow trout, Salmo gairdneri, from a local hatchery were fed dried trout pellets supplied by Victor Fox Foods Ltd., Winnipeg (series A and C) or Ewos Trout Grower (Astra-Ewos AB, Sodertalje, Sweden) (series B). Black bullhead, Ictalurus melas, and channel catfish, I. punctatus, were seined or angled, respectively, from the Red River drainage. Black bullhead were fed Ewos Salmon Grower. Owing to their reluctance to feed, the channel catfish were starved for the period of 3-4 weeks intervening between capture and use. With few exceptions (stated in Results) experimental fish were held at 12-13°C and starved from the start of each experiment. The water temperature of fish held in closed containers was maintained by use of a controlled environment room. Series A. The objective was to determine the influence of ambient T, of different concentrations on plasma T, levels of brook trout and other species. Groups of fish were held in plastic tanks con-

While numerous data exist on the effects of thyroxine (T4) on teleosts (reviews by Pickford and Atz, 1957; Dodd and Matty, 1964; Gorbman, 1969), they are difficult to interpret, as physiological doses of T, are not defined for fish. The present objective was to investigate procedures for creating small, chronic elevations of plasma T, in brook trout, Salvelinus fontinalis, and other teleosts. Plasma T, levels were measured either after a single intraperitoneal injection of T, or during immersion of fish in a Tq solution, and comparisons made with control fish. From the data a protocol was derived for e’stablishing chronic physiological elevations of plasma T,. MATERIALS

AND

METHODS

Fish maintenance. All fish were held for at least 3 weeks in the laboratory in running fresh‘Supported the National

by grants-in-aid Research Council

of research of Canada.

by 209

Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

210

J.

G.

taining 32 liters of static aerated water. For channel catfish (size experiment), 106 liters of water in Fiberglas tanks were used. Every 24 hr the fish were removed by net and transferred to an identical tank of clean water. Since the net was almost as wide as the tank, most fish were caught in the first sweep of the net. The total netting procedure lasted but a few seconds. Prior to addition of fish, 5 ml of 0.1 N NaOH containing T, (experimental group) or 5 ml of 0.1 N NaOH (control group) was thoroughly mixed wit.h the water. [The pentahydrate monosodium salt of L-thyroxine (Sigma) was used to make up solutions, but all T4 concentrations refer to the free acid.1 At intervals fish were removed, anesthetized (MS 222 ; concentration species-dependent) and bled from the caudal artery using a heparinized l-ml syringe with a needle appropriate for the size of the fish. In some cases (Figs. 3 and 4 in Results) fish were returned to the tank for later resampling. Plasma was separated from the corby centrifugation (Eales, 1970) and puscles either ana!yzed immediately or stored for up to 2 weeks at -20°C. Plasma stable T, levels were measured by a competitive-binding method previously tested for fish plasma (Higgs and Eales, 1973)) using plasma volumes of 0.0250.2 ml, depending on the T, level. Samples with very high T, levels were diluted with 0.1 N NaOH before application to the Sephadex columns. In one experiment, small water samples (IO-100 pl, depending on the ambient T, level) were removed with an automatic pipette sampler and added directly to the columns used for T, determinations. Measurements of T, levels in water were more variable than measurements of T1 levels in plasma. The reasons are not known. but one problem was undoubtedly the absorption of T, from the aqueous medium to the pipette walls. Series B. The objective was to examine the fate of ambient radiothyroxine after its uptake by the fish. Owing to the cost of radiothyroxine, observations were confined to a 24-hr period. Fifteen brook trout were placed in plastic tanks containing 10 liters of static aerated water. A volume of 0.5 ml of 50% aqueous glycol containing 100 pCi of radioactive T4 (AmershamSearle, sp. act. 35 pCi/pg, labeled with 12’1 in the phenol ring) and carrier T4 had been previously added to the water to raise the ambient T, level to 5 ag/lOO ml. Fish were killed at intervals up to 24 hr. Blood was obtained by cutting through the caudal peduncle and the plasma radioactivity

EALES analyzed separately as pr,otein-bound radioiodine (PB”“1; includes most of the plasma radioactive T,) and inorganic radioiodine (I”“1) (Eales, 1970, 1972). The radioactivity in either fraction was, for purposes of standardization, expressed relative to the total radioactivity in the water. This plasma/water (P/W) ratio was calculated as follows : p/w cpm in either PBiZ51 or ImI fraction/ml plasma = total cpm/ml water (measured at the same time) The gall bladder, liver, and intestine (each including contents) were removed whole; their radioactivity was measured (Eales, 1970) and expressed as cpm in whole organ X loo/body weight (g), thereby standardizing to a fish weight of 100 g. Series C. The objective was to determine the influence of injected T, of different doses on

FIG. 1. Plasma T4 concentrations (#g/100 ml) for starved control and treated brook trout up to 9 days after immersion in T, solutions of 0, 2, 10, and 50 pg/lOO ml at 12-13°C. Each point represents a mean of values from 3 fish. Mean body weights (g) and ranges were: controls (28.3; 17.0-46.4); 2 fig/100 ml (25.8; 15.8-35.4); 10 rg/lOO ml (28.6; 18.5-46.2); 50 fig/100 ml (27.1; 18.4-46.9).

THYROXINE

DOSES

plasma T, levels of brook trout. Anesthetized brook trout were injected in the midventral coelom with 0.05 ml of 50% propylene glycol (control) or 0.05 ml of 50% propylene glycol containing 8 or 0.8 pg T,. They were held in plastic tanks containing 32 liters of static aerated water. Every 23-26 hr, water was changed as in series A. At intervals, fish were killed and bled and their plasma was analyzed for stable T, as in series A. RESULTS

Series A Plasma T, levels of brook trout during 9 days immersion in T, solutions are shown in Fig. 1. Figure 2 shows the expected and measured water concentrations of T, in the 3 experimental tanks. %ontrol plasma T, levels were usually below 0.5 pg/lOO ml. An ambient Tq concentration of 2 pg/lOO ml caused a small but distinct

FOR

TROUT

211

elevation in plasma T,. At 10 pg/lOO ml water, plasma T, fell within the range 1.5 to 4 &lo0 ml (3-8 times control). An ambient T, level of 50 pg/lOO ml resulted in high or increasing plasma T,, attaining 18 ,ug/lOO ml plasma (36 times control) after 7 days. Plasma T, values following longer exposure to ambient Tq (Tables 1 and 2) demonstrate that small sustained elevations in plasma T, can be achieved in brook trout by this method. Table 3 shows that an ambient T, concentration of 10 pg/lOO ml raised plasma T, in fed trout, although the increase was not as great as for starved trout used in the previous experiments (Tables 1 and 2). Table 4 shows that the response to ambient T, can be modified by temperature acclimation. For T,-treated trout, plasma T, levels were significantly lower at 17.519.O”C than at 12-13°C.

FIG. 2. Ambient T, concentrations (pg/lOO ml) at various stages during the experiment shown in Fig. 1. The theoretical ambient TJ concentration is shown by a broken line. Individual measured values are represented. With the exception of the first point, samples were taken just prior to transfer of the fish to another tank.

212

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G.

EALES

TABLE

1

PLASMA T, FOR ST.~R~ED CONTROL BROOK TROUT~ AND STARVED TREATED TROU@ IMMERSED UP T O 27 11.4~s IN A SOLUTION OF 10 @G Td/lOO ML WATER AT 12-13°C Controls n

Immersion

Td-treated

f

(days1

Range

(fig/100ml)

(cLg/l@J ml)

0

3

0.41

0.23-0.60

1 7 14 21 27

3 3 3 5

0.43 0.24 0.28 0.58 -

0.37-0.54 0.23-0.29 0.20-0.31 0.35-0.80 -

-

a Average weight b Average weight c P/W represents

P/W=

3 3 3 3 8

36.7 g; range 24.5-5U.3 g. 29.8 g; range 19.8-47.7 g. the mean ratio of plasma T, for treated

fish relaGve

-

1.15 1.25 1.56 1.73 1.52

0.92-1.45 0.70-1.68 1.53-1.61 1.22-2.13 1.11-1.99

to the ambient

0.12 0.13 0.16 0.17 0.15

T,.

was little correlation (T = -0.14) between body weight and plasma T, for untreated fish, a much higher correlation (T = -0.67) was obtained for treated fish, suggesting an influence of body size on T, uptake from the water.

Figure 3 shows that plasma T, can be raised in other species by immersion in a T, solution of 10 pg/lOO ml, but suggests species differences, as channel catfish and rainbow trout had lower values than brook trout or black bullhead. It is possible that the serial blood sampling of the channel catfish and rainbow trout may have contributed to these differences. However, both the channel catfish and rainbow trout were much larger than the brook trout or black bullhead, and body weight could be the important variable. Figure 4 shows the relationship between body weight of channel catfish and plasma T, measured immediately before immersion in T, (10 pg/lOO ml water) and then after 6 days of immersion. While there

Series B The ambient level of radioactivity did not change significantly during the 24-hr exposure period. From the data in series A (Fig. 2) one can assume that this radioactivity represents mainly T,. Plasma PB1251 (expressed as ‘P/W) increased rapidly to 3 hr and showed no further increase up to 24 hr (Fig. 5). P/W values for PB’*“I were of the same order as those obtained with stable Tq in series

TABLE

2

PLASMA TI FOR STARVED CONTROL BROOK TROUT AND STARVED TREATED TROUT IMMERSED UP TO 19 DAYS IN SOLUTIONS OF Tp RANGING FROM 5 TO 25 p6/100 ML AT 12-13°C Fish Ambient T4

Immersion

(days) 15-19 8-13 .

15-18 ..Lk

a P/W

represents

n

(&loo ml) Controls 5 Controls 10 Controls 25 the mean

ratio

weight 1

Plasma SE

3

of plasma

29.1 29.7 38.5 36.3 27.8 36.27 Th for treated

SE

P/Wa

WI00 ml)

Cd 21 20 23 21 17 18

T,

0.98 2.02 1.19 1.94 1.62 2.29 fish relative

0.40 1.04 0.28 1.67 0.34 4.01’ to the ambient

0.08 0.14 0.02 0.20 0.05 0.31 T4.

0.21 0.17 0.31

THYROXINE TABLE PLASMA T, (kc/100 ML) BROOK TROUT~ AND TROU&~ IMMERSED IN A SOLUTION OF ML WATER AT

3 FOR FED CONTROL FED TREATED UP TO 9 DAYS 10 PG T,/lOO 12-13°C Plasma WlOO

Immersion (days 1 3 6 9

DOSES

T, ml)

Treatment

n

%

SE

control T&-treated control T.-treated control T,-treated

6 6 6 6 6 8

0.18 0.75 0.45 0.75 0.29 0.97

0.013 0.080 0.062 0.066 0.048 0.043

0 Average weight 36.0 g; 0 Average weight 39.0 g; c Fed trout received an weight of Victor Fox trout

range 23.9-63.4 g. range 27.7-57.4 g. average of 2.Oyo body pellets per day.

A (Tables 1 and 2). In al1 instances P/W values were considerably below 1.0. Plasma Pz51 (Fig. 5) increased between 3 and 24 hr, as did radioactivity in the gall bladder and intestine (Fig. 6). Liver radioactivity did not increase appreciably beyond 7 hr.

FOR

213

TROUT

Series C Figure 7 shows plasma T, levels for control fish and for experimental fish killed at different times after intraperitoneal injection of T,. Control plasma T, levels were all below 1 rg/lOO ml. Injection of 0.8 pg T, (an average of 18 rig/g body weight) increased mean plasma T, levels 27-fold after 2 hr. By 20 hr (or earlier) control T, levels were reestablished, indicating rapid T, elimination from the circulation. Injection of 8 pg (an average of 210 rig/g body weight) increased plasma T, levels 170-fold after 2 hr, but by 48 hr plasma T, levels were comparable to control fish. The biological half-lives (tn) of the administered T, in the plasma were calculated as 6.4 and 4.5 (or less) hr for groups receiving 8 and 0.8 pg, respectively. DISCUSSION

Control plasma T, levels for the species studied were usually less than 0.5 pg/lOO ml, agreeing with previous values (Higgs and Eales, 1973). Several separate experiments showed that immersion of starved underyearling

12 t

-.. f RAINBOW TROUT

0

--...... ” CHANNEL CATFISH

DAYS FIG. brook mersed values bullhead

3. Plasma Td concentrations (fig/100 ml) in black bullhead (av. body wt. 81.3 g; range 50.1-171.5 g); trout (32.0; 20.0-42.9 g); rainbow trout (338; 279-390 g) and channel catfish (464; 352-581 g) imup to 9 days in a T, solution of 10 pg/lOO ml at 12-13°C. Except at 4 and 5 days, when individual are shown, each point represents a mean of determinations from 3-4 fish. Brook trout and black were killed at sampling; rainbow trout and channel catfish were serially sampled.

214

J. G. EALEH TABLE

4

PLASMA T4 (~~/100 ML) FOR STARV~;.D CONTROL BROOK TROUT AND TRK.~T~;D TROUT IMMERSED FOR 3 OR 7 Days IN A RoL~TIoN OF 10 PG T~/~OO ML WATER AT ll-13°C OR 17.5519”Ca Day

(6

Treatment

12-13

(rg/lOO

Control T,-treated Control T,treated

17.5-19

6 6 6 6

(pg/lOO

ml)

0.44 2.29” 0.26 1.50*

0.07 0.11 0.02 0.20

7 6 5 8

0.48 2.00 0.42 1.59

7

ml) 0.03 0.17 0.09 0.11

a Trout were acclimated for 1 week at the higher temperature before T, was given. Average fish weight at 12-13°C was 46.0 g (range 21.1-64.2 g) and at 17.5-19°C was 43.6 (range 21.1-59.5 g). b Plasma T4 differed significantly (p < 0.05) between temperatures for Td-treated fish at 3 days. Plasma T4 differed significantly (p < 0.01) between temperatures for TJ-treated fish when a-day and ‘I-day groups were combined.

brook trout in water at 12-13°C containing 10 lug TJlOO ml (1: 107) usually raised plasma T, by lOO-300%. The slight variation in values between experiments may be due to size or other factors considered below. This increase was sustained with little change during 27 days of immersion

(Table 1). An ambient T, level of 2 pg/ 100 ml caused a measurable small increase in plasma T,; a level of 50 ~g/lOO ml produced plasma T, levels up to 18 pg/lOO ml with no achievement of a steady state in the plasma by 9 days (Fig. 1). All the variables examined (feeding,

0 4

t 0 0

E 38 T %

0

0 00

0 I-b u ZE 2 i

0

2-

O 0

0

0

-

0

0 I-

.

O

. . . . I l

.

0’

’ 30

.

.

’ I”“’ 60

I

100

,

200

BODY

Fro. 4. Relationships between body weight prior to (control) and 6 days after immersion 12-13°C.

l .08 .

. .

.

.

s,

.

G

I I11111 500

WEIGHT

000 9

o* le

‘e 1000

l

f

t 3000

(9)

and T, concentrations in plasma of channel catfish sampled in a solution of 10 g T&99 ml. Water temperature was

THYROXINE

IC

DOSES

FOR

215

TROUT

I-

WATER

RADIOACTIVITY

0.E I-

0.E

P/W

0.4

0.2

HOURS

01

1 0

I 5

I IO

1

I 20

I 25

FIG. 6. Levels of radioactivity physical decay and body weight) (including bile), liver, and intestine tents) for the fish shown in Fig. 5.

(corrected

for

in gall bladder (in&ding

con-

HOUR:

FIG. 5. Radioactivity-(cpm/mlplasmafraction)/ (cpm/ml HzO), (P/W)-for PBlz61(x) and Pa61(0) plasma fractions of brook t,rout immersed up to 24 hr in 10 liters of water (12-13°C) containing 100 PCi of radiothyroxine and 5 fig/l00 ml of carrier Tb. Ambient radioactivity is represented by a P/W value of 1.0. Values are shown for individual fish. The mean 18.0-38.9 g.

body

weight

was

30.0

g;

range

temperature, species and body size) influenced to some extent the response to an ambient T, level of 10 ~g/lOO ml. The low plasma T, levels in fed fish (Table 3) could reflect lowered ambient T, due to T, absorption to food or fecal particles not present during starvation; they may be caused also by more effective T, degradation or biliary excretion in fed fish. The lower plasma T, levels at the higher acclimation temperature (Table 4) could arise from a more rapid T, turnover at the higher temperature, as suggested in brook trout by Drury and Eales (1968)

and in the eel by Leloup (1965). The differences between species could in part be due to size differences, as suggested by the inverse correlation between body size of channel catfish and their plasma T, levels following exposure to T,. However, this relationship may not be as evident for all species. Unlike most teleosts, catfish lack scales. Significant T, may enter via the skin and the inverse relationship between body weight and plasma T, could be due to the larger skin surface relative to body mass for smaller fish. The channel catfish were also starved for 34 weeks, and this may have bearing on the data. Biliary excretion of T, has already been shown to be a prominent excretory route for T, in brook trout (Eales, 1970) and may be important in stabilizing plasma T, levels. The contribution of deiodination to T, clearance from the blood is less well understood. T, deiodination has been demonstrated in who for a number of

216

J.

0.1 I 0

I

I I

I

G.

I 2

EALES

I

I 3

I

k 4

I

I 5

DAYS FIG. 7. Plasma T, concentrations &g/100 ml) for control and treated brook trout at 1%13°C up to 5 days after a single intraperitoneal inject.ion of various doses of Tq. Each point represents a mean of values from 3 fish. Mean body weights (g) and ranges were: controls (38.2; 27.3-56.9); 18 rig/g (45.1; 27.4-67.6); 210 rig/g (38.3; 25.7-56.5).

brook trout tissue homogenates (Law and Eales, 1973) including gills. Active deiodination by gill tissue might prevent extensive entry of T, into the plasma in the first place. Intraperitoneal T, injection is an impractical method for creating any physiological chronic elevation of plasma T,. Small T, doses are rapidly cleared from the plasma, probably owing to loss in the bile. Thus frequent injections would be required involving considerable stress on the fish. However, even the best-planned protocol would probably not eliminate major surges in plasma T, due to intraperitoneal injection. Injection at other sites (e.g., intramuscular) or pellet implantation (Peter, 1972) might treat more sustained plasma T, changes with less stress on the fish. Treatment of fish with ambient T, has advantages over other methods of T, administration in (i) creating small predictable and sustained elevations in plasma T,, (ii) producing this plasma steady state of T, within hours, (iii) causing less stress

than with injection procedures, (iv) causing no high local T, level as will arise from injection or implantation, and (v) obviating problems of leakage loss of injected hormone or effect of injection solvent. Nevertheless, studies involving gills, skin, and other sites of exchange between the water and the fish require careful interpretation. Since the ambient T, level is always considerably greater than that for the blood, the exchange surfaces will be exposed to much higher levels than the internal tissues. The upper limit of a physiological T, dose for fish will probably always be debatable. I adopt the view that it should not raise plasma T, beyond the level which the fish itself could maintain by endogenous production. While plasma T4 for brook trout is usually less than 0.5 pg/lOO ml, it can exceed 1 ag/lOO ml and sometimes attain 3 pg/lOO ml (Higgs and Eales, 1973). In addition, brook trout at 12-13°C can be stimulated by acute or chronic bovine TSH treatment to raise plasma T, over 3 ,ug/lOO ml and occasionally up to

THYROXINE

DOSES

5 p&100 ml (Chan and Eales, unpublished). Using these data and assumptions, an ambient T, level of 10 ,ug/lOO ml, which usually raises plasma T, to l-2 plLg/lOO ml, may be considered physiological. ACKNOWLEDGMENTS The author thanks Miss Ruth Baruch, Miss Janet Collicutt, and Mrs. Tara Narayansingh for technical help. The brook trout and rainbow trout were kindly provided by the Province of Manitoba Trout Hatchery; black bullhead were donated by Dr. J. H. Gee. Drs. J. C. Rauch, R. E. Peter, D. Beatty, T. Hara, and P. E. Johansen are thanked for their criticisms of the manuscript. REFERENCES DODD, J. M., AKD MATTY, A. J. (1964). Comparative aspects of thyroid function. In “The Thyroid Gland” (R. Pitt-Rivers and W. R. Trotter, eds.), Vol. I, pp. 30%356. Butterworths, London. DRURY, D. E., AND EALES, J. G. (1968). The influence of temperature on histological and radiochemical measurements of thyroid activity in the eastern brook trout, Salvelinus fontin& (Mitchill). Can. J. Zool. 46, l-9. EALES, J. G. (1970). Biliary excretion of radio-

FOR

TROUT

217

thyroxine by the brook trout, Salvelinus fontinulis (Mitchill). Gen. Comp. Endocrinol. 14, 385-395. EALEB, J. G. (1972). Radiothyroxine metabolism in several freshwater teleost fishes. Can. J. 2001. 50, 623-631. GORBMAN, A. (1969). Thyroid function and its control in fishes. In “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. II, pp. 241-274. Academic Press, New York. HIGGS, D. A., AND EALES, J. G. (1973). Measurement of circulating thyroxine in several freshwater teleosts by competitive binding analysis. Can. .I. Zool. 51, 49-53. LAW, Y. M. C., AND EALES, J. G. (1973). Deiodination of radiothyroxine by tissue homogenates of brook trout, Salvelinus fontinalti (Mitchill). Comp. Biochem. Physiol. 44B, 11751183. LELOUP, J. (1965). Mktabolisme de la thyroxine chez I’Anguille normale et hypophysectomiske en fonction de la tempkrature. Gen. Comp. Endocrinol. 5, 66. PETER, R. E. (1972). Feedback effects of thyroxine in goldfish Carassius auratus with an autotransplanted pituitary. Neuroendocrinology 17, 273-281. PICKFORD, G. E., AND ATZ, J. W. (1957). “The physiology of the Pituitary Gland of Fishes,” p. 613, N. Y. Zool. Sot., New York.