Inhibitory influence of thiourea on brain of singi fish (Heteropneustes fossilis bloch) and subsequent recovery by l -triiodothyronine

Neurochem. Int. Vol. 6, No. 4, pp. 527-532, 1984

0197-0186/8453.00 +0.00 © 1984 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

INHIBITORY INFLUENCE OF THIOUREA ON BRAIN OF SINGI FISH (HETEROPNEUSTES FOSSILIS BLOCH) AND SUBSEQUENT RECOVERY BY L-TRIIODOTHYRONINE AJIT K. MEDDA and RANJIT K. GHOSH Department of Animal Physiology, Bose Institute, Kankurgachi, Calcutta-700054, India (Received 16 August 1983; Accepted 5 January 1984) Abstract--Immersion of Singi fish in thiourea-containing medium (1 mg/ml) for 45 days significantly decreased the cranio-somatic index, weight of different parts of brain, viz. cerebrum, cerebellum, midbrain and medulla oblongata, and also protein and RNA contents of these different regions. The DNA content of these substructures remained unchanged. In other sets of experiment, thiourea treatment for 33 days caused reduction in mitochondrial cytochrome-linked ~-glycerophosphate dehydrogenase (EC 1.1.99.5) activity, and total protein and RNA contents of whole brain, while the amount of mitochondrial protein and total DNA content of whole brain did not undergo significant variation. A single injection of L-triiodothyronine (0.5/~g/g) enhanced this enzyme activity, mitochondrial protein amount and total protein and RNA contents of whole brain of thiourea-treated fish to almost such levels as obtained by L-triiodothyronine injection in normal (control) Singi fish within 3 days. The CSI in normal fish increased by triiodothyronine injection. In thiourea-treated fish, the reduction of CSI was restored to just control level by triiodothyronine. The results are, therefore, discussed as additional supportive evidences of the responsiveness of fish brain to thyroid hormone.

A number of reports have been made on the dependency of mammalian brain on thyroid hormone for its biochemical and morphological development (Geel and Timiras, 1970; Sokoloff and Kennedy, 1973). Hypothyroidism in early life is responsible for various biochemical and structural changes in brain resulting in irreversible mental retardation (Eayrs, 1964; Eberhardt et al., 1976, 1980). These changes can be prevented by thyroid hormone replacement therapy initiated before development of a critical period (Hamburgh and Flexner, 1957; Naidoo et al., 1978; Dainat and Rebi6re, 1978, 1980). Although the adult mammalian brain has been shown unresponsive to exogenous thyroid hormone with respect to different metabolic activities (Barker and Klitgaard, 1952; Sokoloff, 1977; Eberhardt et al., 1980), it is known that alterations in the thyroidal state in adult cause various neurological and psychological abnormalities. Moreover, the presence of thyroid hormone receptors in mammalian brain at all postnatal ages from neonatal to adult (Eberhardt et al., 1975, 1976; Address for correspondence: Department of Animal Physiology, Bose Institute, P-l/12 CIT Scheme VII M, Kankurgachi, Calcutta 700054, India.

Naidoo et al., 1978; Schwartz and Oppenheimer, 1978) clearly indicate the brain as a target tissue for thyroid hormone. On this background and as a sequel of the investigations undertaken in this laboratory on the effects of thyroid hormone and antithyroid drug on various physiological processes in different freshwater fish (Ray et al., 1975; Ray and Medda, 1976, 1977; Medda and Ray, 1979; Dasgupta, 1979; Bhaumik, 1983), the brain of Singi fish (Heteropneustes fossilis Bloch) has been chosen as an organ for studying the role of thyroid hormone in different metabolic parameters. The thyroid hormone-induced changes in protein, RNA, DNA, glycogen, cholesterol and total lipid contents and in mitochondrial ~t-glycerophosphate dehydrogenase (~-GPD, E.C. 1.1.99.5) activity of Singi fish brain have been reported recently (Ghosh and Medda, 1982; Ghosh et al., 1983; Ghosh and Medda, 1984). The present investigations on the effect of thiourea on protein, RNA and DNA contents and mitochondrial ~-GPD activity and mitochondrial protein content have been undertaken with a view to provide more supportive evidence on the thyroid hormone actions in fish brain. The possibility of corrections of biochemical

527 NCI 6/4~H

528

AJIT K. MEDDA and RANJIT K. GHOSH

changes in hypothyroid condition by thyroid horm o n e administration has also been explored. EXPERIMENTAL

PROCEDURES

Singi fishes (Heteropneusteslbssilis Bloch), body weight 7_+lg, length 10-11cm, were purchased from the local supplier and acclimated in laboratory conditions at 25~C for about a week before experiments. They were fed ad libitum with Tubifex tubifex during acclimatized period. The animals were distributed randomly in different groups and kept in polythene tray containing tap water (1 l/fish) at 25'C. Thiourea (E. Merck, A. G. Darmstadt) was dissolved in the medium (1 mg/ml) in which the fishes were immersed for 45 days. The control fishes were kept in the same volume of tap water. The medium was changed at every alternate day and fresh thiourea treatment was made. All fishes were fed as usual. These experimental animals were sacrificed just after 45 days of treatment. The brain from each animal was excised and the different parts of brain, viz. cerebrum, cerebellum, midbrain and medulla oblongata, were quickly dissected out, weighed and taken for the estimation of protein, RNA and DNA contents as followed previously (Ghosh and Medda, 19821. The cranio-somatic index (CSI) was also determined as (total brain weight/body weight) x 100. In other sets of experiments, some of the thiourea-treated fishes were divided into two major groups on day 30, one group was maintained in thiourea containing medium (1 mg/ml) and the other in tap water. The latter group of animals were given on day 30 a single injection of L-triiodothyronine (T~) at the dose of 0.5 #g/g. For injection, T 3 obtained from Sigma Chemical Company, U.S.A., was dissolved with minimum volume of 0.1 M NaOH and the required volume was made by 0.65°; saline. Similarly, the control animals were divided into two groups, one for T~ injection as above and the other to run as usual control. The injected volume, either of T 3 solution or alkaline 0.65'~i saline for the control, did not exceed 35/d/animal. The fishes were fed. All these experimental animals were killed on days 31, 32 and 33 list, 2nd and 3rd day after T~ injection). The brain was quickly dissected out, weighed and homogenized in a glass Teflon homogenizer in cold 0.25 M sucrose solution. The procedure for the preparation of the mitochondria for the assay of :c-GPD activity was the same as followed in our previous studies (Ghosh et al., 19831. The

brains l¥om 6 or 7 animals were pooled in each set. Mitochondrial :~-GPD activity was measured by the method of Lee and Lardy (19651. The mean specific activity of :~-GPD from each of six such sets was expressed as mean_+SE of change in optical density at 500nm per min per mg of protein and DNA. Protein content was estimated by the method of Lowry et al. (1951). RNA was determined by the method of Mejbaum (1939) as modified by Munro and Fleck (1966), and DNA by Burton (1956) as modified by Croft and Lubran (19651. All data were statistically analyzed by using Student's t-test. Values of P < 0.05 were taken as significant. RESULTS

Effect o[ thiourea on CSI, weight and protein, RNA and DNA contents of d(fferent parts of brain C o m p a r e d to the control, the cranio-somatic index (CSI) decreased (P < 0.05) after 45 days o f thiourea treatment by immersion. The CSI values o f the control and thiourea-treated groups were 0.63 _+ 0.02 and 0.57_+0.01 respectively. Thiourea treatment caused a significant decrease in the weight o f different substructures o f brain, viz. cerebrum, cerebellum, midbrain and medulla oblongata (Table 1). There was no significant change in body weight after thiourea treatment. The protein and R N A contents of cerebrum, cerebellum, midbrain and medulla oblongata also decreased after thiourea treatment for 45 days. But the D N A content of these regions of brain did not alter significantly after thiourea treatment.

E ~ w t o/thiourea on CSI, mitochondrial ~-GPD actirit.;', mitochondrial protein, total protein, RNA and DNA contents o[ whole brain atut subsequent recorerv by 7~, (Table 2) Thiourea treatment by immersion for 31 33 days caused a reduction in CSI, mitochondrial 7 - G P D activity ( A O D / m i n / m g protein or D N A ) and total protein and R N A contents o f whole brain, while the

Table 1. Effect of thiourea on organ weight, protein, R N A and D N A contents of different parts o f brain (cerebrum. cerebellum, midbrain and medulla oblongata) of Singi fish (Heteropneustes/ossilis Bloch) O r g a n weight (mg/g body wt) Mean _+ SE

Protein (rag/100 mg tissue) Mean + SE

Control Thiourea

1.46 _+ 0.06 1.22 ± ().04~

9.41 + 0.20 6.7(I + 0.63,}

173.6 + 6.7 143.2 _+ 12. I*

162.9 + 5.8 155.7 + 9.5

Cerebellum

Control Thiourea

1.58 + 0.08 1.36 + 0.06*

10.26 + [).64 8.26 + 0.28§

187. I + 6.5 143.5 + I q.6*

628.4 ± 26.0 633.2 + 18.8

Midbrain

Control Thiourea

2.27 + 0.04 2.01 + 0.(19"

9.35 + 0.18 8.22 + 0.29§

130.4 + 6.4 104.8 + 9.7*

[ 6~.3 ± 8.5 156.7 ± 8.8

Medulla oblongata

Control Thiourea

1.54 + 0.08 I. 12 + 0.03§

9.88 + 0.23 8.94 + 0.32"

137.5 ± 7N 1(17.4 ± 9.6*

2 1 7 4 + 13.6 217.2 + 8.1

Respective organ

Treatment

Cerebrum

RNA (itg:100 m g tissue) Mean + SE

DNA t # g ' 100 m g tissue) Mean - SE

SE = Standard error, t-test probability differences between the control and treated group: P-value: * = P < 0.05. ~ - t' < 0.01. The fishes were immersed in thiourea-containing medium (1 mg/ml) for 45 days at 25 C and fed. Each group (treated or control) consisted of 20 animals. Change in body weight after treatment in each group was not significant.

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amount of mitochondrial protein as well as DNA content of whole brain were found not to undergo a significant alteration under such treatment. A single injection of T~ at the dose of 0.5 #g/g body weight increased the ~-GPD activity (AOD/min/mg protein or DNA) of whole brain of normal (control) fish on the 1st day after T~ injection (day 31 of the experimental period) and more so on the 3rd day (day 33). This dose of T~ when given to the thiourea-treated hypothyroid fish in a single injection on day 30 restored the ~-GPD activity to the normal or control level on the 1st day after injection and then exceeded the activity over the control value to reach the level obtained by T) injection in normal (control) fish on the 3rd day (day 33). The mitochondrial protein and total RNA content of brain of normal fish significantly increased after T, injection (single, 0.5/~g/g) on the 2nd and 3rd day (days 32 and 33). Although mitochondrial protein content remained unchanged in thiourea-treated fish, it was enhanced by T~ injection in both thioureatreated and normal fish on the 2nd and 3rd day. The T~-induced recovery of RNA content in hypothyroid fish took place on the 2nd day, and on the 3rd day the RNA amount surpassed the normal control value with T> The total protein content of normal fish increased only on the 3rd day after T~ injection (day 33). The restoration of total protein content to the control level by T~ injection in thiourea-treated fish was found on the 2nd day, and next day (day 33) the protein value reached to a higher level as observed on the 3rd day in normal fish given T 3 injection. The CSI also increased by T 3 injection on the 3rd day in normal fish. The reduction of CSI in hypothyroid fish was also recovered to just control level by a single injection of T~ (0.5/~g/g) on the 3rd day. The DNA content of whole brain did not show any variation after T~ injection in normal (control) or thioureatreated hypothyroid fish. DISCUSSION

In continuation of our previous studies on the responsiveness of Singi fish brain to thyroid hormone with regard to various metabolic alterations (Ghosh and Medda, 1982; Ghosh et al., 1983; Ghosh and Medda, 1984), the present experiments have been designed with these animals to further explore the changes in weight and protein and nucleic acid contents of different substructures of brain, mitochondrial ~-GPD activity, mitochondrial proteim total protein, RNA and DNA contents of whole brain and CS| of thiourea-treated hypothyroid fish.

That well-marked goitre characterised by packed mass of thyroidal epithelial cells with little or no colloid in the gland develops in thiourea-treated fish has been shown by Ray and Medda (1972). Moreover, thiourea as a well known antithyroid drug causing inhibition of thyroid hormone synthesis (Eales, 1979), has also been shown to inhibit the iodination of tyrosine as well as the uptake of radioiodine by the perfused lamprey thyroid (Pickering, 1976). In our thiourea-treated Singi fish, the reductions in CSI, weight and protein and RNA contents of different substructures of brain as well as of whole brain, and mitochondrial :~-GPD activity are believed to be due to thyroid hormone deficiency that possibly resulted in decreased synthesis of protein and RNA and also specific enzyme protein in brain tissue. This can be supported by a vast number of reports which amply focus the indispensable role o1' thyroid hormone in the induction of protein and nucleic acid synthesis in different organs of different animals (Frieden and Just, 1970: Kohl, 1972: Sokoloff and Kennedy, 1973: Tata, 1974: Hoch, 1974: Bernal and Refetofl\ 1977: Medda and Ray, 1979; Dainat and Rebidre, 1980). In our present experiments also, T~ injection in normal Singi fish enhanced the CSI, ~-GPD activity, mitochondrial protein, total protein and RNA contents of brain. These changes are naturally supposed to be the results of induction of syntheses of protein, nucleic acid and also specific enzyme protein. In Lata fish (Ophicephalus punctatus), thiourea treatment has been found to cause significant reduction in protein and RNA contents of liver and muscle (Ray and Medda, 1977). That the reduction in protein and RNA contents and ~-GPD activity of thiourea-treated Singi fish brain is due to the inadequacy of thyroid hormone in the body has been experimentally verified by the administration of T~ which not only restored the levels of these cellular constituents to normal state, but also exceeded the amounts/enzyme activity to reach the levels as found by T~ injection in normal fish within 3 days. The T3-induced recovery to the normal level occurs within 24 h in case of :~-GPD activity, but in case of total protein and RNA content the recovery was found on the 2nd day after T~ injection and for CSI the minimum time for recovery was 3 days. These recovery changes reflect the time required for physiological manifestations such as net resultant changes in enzyme activity, amount of protein and RNA and total organ weight. Although we do not know the circulating thyroid hormone level in normal or thiourea-treated Singi fish. careful scrutiny of the experimental results re-

Thiourea-induced changes in fish brain veals that the magnitude of T3-induced increase in protein and R N A contents and ~ - G P D activity was more marked in thiourea-treated hypothyroid Singi fish than that in normal (control) fish given T3 injection. It is likely that the thyroid hormoneinduced changes in Singi fish brain are due to receptor-hormone binding resulting in specific transcriptional as well as translational processes, since the thyroid hormone receptors are present not only in mammalian brain (Naidoo et al., 1978; Schwartz and Oppenheimer, 1978; Eberhardt et al., 1980) but also in fish brain (Darling et al., 1982). The higher extent of increase in the cellular constituents in thioureatreated fish by T3 administration is possibly due to more uptake of the exogenous hormone by the nuclear receptors in case of less availability of the endogenous hormone. It is to be pointed out in this connection that neonatal thyroidectomy or hypothyroidism increases the thyroid hormone receptor concentration in mammalian brain tissue (Valcana and Timiras, 1978; Ishiguro et al., 1980) and antithyroid drug treatment decreases the plasma thyroid hormone concentration (Ishiguro et al., 1980). The results of thiourea-induced changes in Singi fish brain in the present experiments are thus additional supportive evidences of the responsiveness of fish brain to thyroid hormone. REFERENCES

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