Indi an Journal of Experimental Biology Vol. 40, October 2002, pp. 1198-120 I

An appraisal of post-irradiation metabolic changes in toad liver (Bufo melanostictus)

J Mishra & A Mittra*

School of Life Sciences, Jyot i Vihar, Sambalpur University, Burla 768019, Indi a

Received I February 2002; revised 9 July 2002

Whole body 6OCO gamma irradiation of B. lIIelallosliclliS with two sublethal doses of 3.5 and 7 Gy resulted in a signifi­cant increase (P < 0.001) in total lipid (hyperlipidemi a), cholesterol content (hypercholesterolmia) and total free amino acid content, and significant decrease (P < 0.001) in total protein content in the li ver tissues o n post-irradiation day (PID) 1,5 and 10 as compared to controls. The ratio of total lipid to cholesterol showed a gradual declining trend by PID-IO in the treated groups as compared to cont rols. However, it was more pronounced in the 7 Gy treated group. An observation on the ratio of total protein to total free amino ac id content also showed a similar declining trend by PID-IO in both the treated groups as compared to controls.

The vertebrate liver is an important gland engaged in an array of synthetic and degradative processes. It also serves as a major storage site for carbohydrates, proteins, lipids etc. In addition, the amphibian liver specifically anurans has been reported to be haemato­poitic l.2 . Its varying sensitivity to ionizing radiation has been reported3

.4. Variable response of cholesterol and phospholipid concentrations has been reported. Whole body gamma irradiation of albino rats with sublethal doses of 1.75 and 3.5 Gy has been reported to augment the cholesterol level in liver and to decline in serum and aorta5

. Enhanced lipogenesis in liver of continuously y - irradiated rats6 ·and an elevated serum concentration of triglycerides, phospholipids and non­esterified fatty acids - but not total cholesterol­in comparison with unirradiated controls, has been reported7

. An increased level of serum lipids includ­ing cholesterol and phospholipids during continuous y-irradiation of male Wistar rats to a total dose of 30 Gy has also been reportedR

• A decrease in phosphat i­dyl choline and phosphatidyl ethanolamine, and in­crease in cholesterol in liver of rats exposed to 12 Gy y-irradiation has been reported9

. In rats , both X and y -irradiation induced increase in plasma cholesterol concentration and hepatic cholesterol synthesis have been also reported 10-12.

The structural and functional changes induced by d·· · . h b d13- 16 A d ra latlOn In proteIns ave een reporte . e-

*Correspondent author : Phone: (0663) 431879 (0) Fax: 0663-430158 E-mail: mjibanI970@yahoo.co.i n

crease in protein synthesis due to depression in the production of nuclear RNA i.e. specific types of mRNA has also been reported 17. Damage to mem­brane proteins, lipids, etc has been reported 18. Within living cells biomembranes serve multiple functions. Disturbance of membrane structure and function by physical or chemical agents may be of decisive importance with regard to the vitality of the cells 19.

Responses of amphibian tissues to whole body y -irradiation have not been pursued as extensively as in mammali an systems. Reports on radiation response of tropical anurans in particular are still scanty. These vertebrates are imperfect regulators having limited homeostatic abilities. They survive within a relati vely low range of physico-chemical variations in their en­vironment.

The present investigation focuses on the effects of two sublethal doses (3 .5 and 7 Gy) of whole body 60Co gamma irradiation on the changes in the total protein, total free amino acid, total lipid and choles­terol concentration in the liver ti ssues of the toad (Bufo melanostictus).

Experimental animals-The common Indian toad, Bufo melanostictus (Bloch and Schneider) were caught directly from their natural habitat and were immediately transported to the Animal Physiology Laboratory, School of Life Sciences, Sambalpur Uni­versity. The site of sample collection was Sambalpur University campus located in Sambalpur District of Orissa, India at 21 0

, 28' N latitude and 840, 04' E lon­

gitude and at an altitude of about 154 meter above sea level. The specimens were mai ntained together in

NOTES 1199

specially designed large cages containing moist sandy soil at 25° ±2°C and 12hr photoperiod. Freshly col­lected earthworms and termites were supplied twice daily. Water was provided ad libitum. All the animals were kept in the laboratory under these conditions for one-day (24 hr). A total of 162 randomly selected animals with body weight within one standard devia­tion of the population mean (50 ± 5 g) were equally distributed in three cages, i.e. 54 per cage and main­tained till the completion of each set of the experi­ment. The two groups were treated and the third served as control. The treatments and experiments were replicated thrice.

60C . d" d I o gamma lrra/atlOn an treatment protoco -The experimental groups I and II of 54 animals each were treated with doses of 3.5 and 7 Gy of whole body gamma irradiation from a 60Co gamma source. The animals were irradiated at Department of Radio­therapy, Y. S. S. Medical College, Burla. To avoid crowding and to ensure proper irradiation animals were taken in small groups of 9 each. Each group was individually irradiated in a 25 x 25 cm rotating field, in a thin , transparent and well perforated polyethylene bag so as to restrict their mobility and without inter­rupting oxygen supply during irradiation .

The source of irradiation was a Janus, 60Co source, Elpro International. The samples for whole body irra­diation were kept at a distance of 60 cm from the source. The dose calibrations were done using a vic­toreen dosimeter. The dose rate was 1.22 Gy/min. After irradi ation the animals were transferred to sepa­rate cages as described earlier.

Biochemical analyses-The animals were anaes­thetised using chloroform vapour and sacri ficed on the stipulated days after irradi ation [post-irradi ation day (PID) I, 5 and 10]. Both the lobes of liver were removed carefully. The sacrificed animals showing any internal abnormalities or parasitic infections were rejected. Total ti ssue mass was gently squeezed / pressed and blotted with blotting paper to absorb the excess water, body fluids and blood from it. The ex­cised liver tissues were transferred to ice bucket then taken for biochemical estimations.

The estimations of different biochemical parame­ters were carried out spectrophometrically using JASCO UYIVIS spectrophotometer (Mode l 7800), following specific establi shed procedures as per vari­ous authors.

Total lipid-Total lipid from known fresh weight liver ti ssues of the control and the treated groups fol­lowing the method described by Zollner and Kirsch20

using vanillin orthophosphoric reagent. The optical

density (O.D.) was measured at 525 nm. Standard curve was prepared using cholesterol (l mg of choles­terol in 5 ml of chloroform). The results were ex­pressed in mg g" fresh weight liver tissue.

Cholesterol-Cholesterol estimation was done ac­cording to Libermann Burchard and the procedure was adopted from Plumer2'. The standard curve was prepared taking 2 mg of cholesterol in 1 ml of chloro­form. The extinction was measured at 680 nm and the results were expressed in mg g" fresh weight liver ti ssue.

Total protein-The estimation of total protein was n

done as per Lowry et at. --. The O.D. was measured at 750 nm. The standard curve was prepared by taking bovine serum albumin (l mg/l ml O.IN NaOH). The results were expressed in terms of mg g" fresh weight liver tissue.

Total free amino acids-Estimation of total free amino acids was done following the method of Yemm and Cocking23. The O.D. was measured at 550 nm. A standard curve was prepared using glycine (10 mg/1 00 ml dry wt) Results were expressed as )lg g" fresh weight liver tissue.

Statistical analyses -Statistical analyses made by comparison of individual values of each group on three days (PID-I, 5 and 10) as well as among the individual values of three groups (control, 3.5 and 7 Gy treated groups). For thi s purpose one-way analys is of variance (ANOY A) as per Sokal and Rohl f24 was employed.

The control values for all the parameters measured remained constant throughout the period of observa­tion. Irradiation affected the parameters concerned proportional to dose and in progression with time after exposure.

Total lipid and cholesterol content-The tota l lipid and cholesterol content in the fresh liver ti ssues of the control groups remained constant on PID-I , 5 and 10, whereas s ignificant differences were observed in the total lipid and cholesterol content of the fresh li ver tissues in the 3.5 and 7 Gy treated groups on PID- I, 5 and 10. The total lipid content and choles­terol levels were raised with dose and time after expo­sure (Table I).

Total protein and free amino acid content-A comparison between the total protein and free amino ac id content in the liver ti ssues of the control group remained constant on PID-l , 5 and 10, whereas same for the treated groups showed significant increase which was again dose and time dependent (Table 1).

In the present study a dose dependent increase in the total lipid and cholesterol content of liver ti ssues

1200 INDIAN J EXP BIOL, OCTOBER 2002

Table I -Total lipid, cholesterol, total protein , total free amino acid content in liver tissue of B. lIIelanostictus on post-irradiation

day (PID) I, 5 and 10

[Values are mean ± SO from 18 animals in each group] PID Control 3.5 Gy 7 Gy

I

5

10

10

1

5

10

I

5 10

Total lipid (mg g.1 tissue)

30.59 ± 0.69 3 1.09 ± 0.86

30.21 ± 1.2 33.56 ± 0.31

30.46 ± 0.98 34.96 ± 0.42

Cholesterol (mg g.1 tissue)

9.65 ± 0.76 9.98 ± 0.42

9.59 ± 0.52 11 .52 ± 0.39

9.62 ± 0.91 12.9 ± 1.05

Total protein (mg g-I ti ssue)

203.1 ± 6.145 189.46 ± 4.35

197.39 ± 5.29 170.6 ± 2.96

20 1.087±5.18 162.5±2.52

33.2 ± 0.36

35.11± 0.52

36.23 ± 0.73

10.965 ± 0.98

13 .8 ± 0.62

14.29 ± 0.75

172.9 ± 2.78

161.52 ± 3.42

149. 11 ± 2.22

Total free amino acid (Jlg mg· 1 tissue)

1.27 ± 0.057

1.308 ± 0.012 1.296 ± 0.048

2.341 ± 0.036

5.931 ± 0.055 3.45 ± 0.038

1.516 ± 0.021

3.79 ± 0.075 2.2 1 ± 0.03

Values in vertical column except control and values in each hori ­zontallines are significantly different at P < 0.001

was observed in the irradiated groups as compared to controls. Whole body irradiation has been reported to cause many and quite different changes depending on a number of factors notably radiation dose, type of animal and their physical conditions25

. Hyperlipide­mia and inhibition of lipoprotein lipase activity have been reported to occur in rabbits after exposure to lethal levels of whole body ionizing radiation2

6-28 . In the present study, the increased total lipid content may possibly be due to either increased lipogenesis or sup­pressed translocation/transportation of lipid to plasma. Inactivation and/or denaturation of lipoprotein lipase may also be a cause of radiation-induced augmenta­tion of the lipid store of this tissue.

Exposure of animals to ionizing radiation of vary­ing f1uences increased the level of total cholesterol in liver, when mostly lethal doses of radiation were em­ployed9

. An elevated level of cholesterol also has been re.E0rted in liver and other tissues of X-irradiated mice5

.2

.30. The effect of the doses of whole body irra­diation seems to have acted in some way to depress lipogenolysis possibly by denaturing, or by inactivat­ing some of the lipolytic enzymes as suggested earlier26

.27 or by hampering the transp0rtation of these

molecules to other steroidogenic tissues via the

Table 2-Total lipid/cholesterol (a) and total protein/free amino

ac id (b) ratio in the li ver tissues of B. lIIelanosticlUS on post-

irradiation day (PID) I , 5 and 10

PID-I PID-5 PID- 10

Con to I a 3. 169 3. 15 3.166

b 6.639 6.533 6.601

3.5 Gy a 3. 11 2.9 13 2.7 1

b 6.093 5.083 4.648

7 Gy a 3.027 2.544 2.535

b 5.2 4.6 4.115

plasma pool Jue to alteration in membrane functions. Therefore the enhanced level of cholesterol concen­tration may have contributed to an over-all increase in the total lipid pool of the liver ti ssues of the irradi ated animals. The ratio of total lipid to cholesterol showed an interesting decreased trend in the treated groups as compared to controls by PIO-IO (Table 2), indicating at these doses a fraction of hepatocytes population is possibly being killed leading to a disturbance in the rates of mobilization of both cholesterol and other lipid fractions.

A dose-dependent significant decrease in the total protein content and a concomitant increase in the total free amino acid content in the liver tissues of irradi­ated animals were observed. It may either be due to degradation/denaturation of the selective i.e . more radiosensiti ve proteins leading to an increased ami no acid pool or due to the alteration in the rates of amino acid incorporation produced by gamma radiation in­duced depression in protein synthesis. This is possibly due to decrease in the production of specific RNA i.e. mRNA as reported earlier17

• Gamma radiation inter­ference with the biosynthesi s of new proteins could result from damage to the genome or from partial de­rangement of the cytoplasmic machinery involved in protein synthesis. The decrease in the total protein content in the liver ti ssues of treated animals by PIO-10 may be due to lesser incorporation of the amino acids in the translational process. A slight fluctuation in the concentration of total free amino acid concen­tration between PID-l and 10 indicates a possible variation in the rate process involved.

In both doses the size of total free amino acid pool showed a significant increase. However, in the low dose group the increase was higher reaching a peak on PIO-5 and then showed a decline on PIO-lO. Whereas in the higher dose treated group the increase though significantly higher than the controls, is relatively less

NOTES 1201

as compared to the lower dose group. Possibly at this higher dose a fraction of the free amino acid pool have been mobilized to the plasma pool. The ratio of total protein to total free amino acid showed a de­creasing trend in the irradiated animals as compared to controls by PID-lO (Table 2), indicating a distinct decrease in the total protein pool size and an increase in the total free amino acid pool size. The present re­sults indicate that in anuran liver both protein and lipid metabolism are significantly influenced at both the doses via whole body irradiation.

At both the doses employed, it is likely that a frac­tion of the hepatocyte population has been killed fol­lowed by some protein degradation . Though it is known that the mammalian liver has regeneration po­tentialit/J1

, the amphibian liver in this experiment does not show any sign of recovery in terms of pro­tein concentration till PID-lO.

Thanks are also due to the Head and the Health Physicist, Cancer and Radiotherapy Division, V.S.S. Medical College, Burla for generous help.

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