Testing of cigarette smoke inhalation for teratogenicity in rats

Toxicology, 4 (1975) 355--362 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

DIELDRIN TOXICITY AND IN VIVO INCORPORATION OF DL-[ 1-t 4C ] LEUCINE

K.K. KOHLI and T.A. VENKITASUBRAMANIAN Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007 (India)

(Received November 15th, 1974) (Revision received April 4th, 1975) (Accepted April llth, 1975)

SUMMARY The in vivo effect of a single oral dose (30 mg/kg b o d y weight) of dieldrin on proteolipid and phosphatidopeptide content of liver and brain and on total protein of liver, brain, plasma, muscle and kidney of rat was studied. Incorporation of [14C]leucin e into total protein of liver was increased whereas labelling of total protein of muscle was decreased. Labelling of total protein of other tissues was unchanged. Incorporation into liver phosphatidopeptides was increased and this was consistent with an involvement of these compounds in increasing protein synthesis in the dieldrin-treated group. Proteolipid protein content of brain was increased and that of liver unchanged. There was, however, no change in the labelling of brain or liver proteolipids.

INTRODUCTION

An increased rate of incorporation of [ 1 4C] leucine into liver proteins has been demonstrated using a cell-free system derived from DDT-treated rats [1]. Dieldrin has also been shown to increase protein synthesis in rat liver [2]. Phosphatidopeptides [3] are present in cell membranes and are metabolically very active. Since these compounds are important in the active transport of amino acids across the liver cell membranes [4], the present study was undertaken to investigate their role, if any, in dieldrin-treated rat liver and to determine whether proteolipid protein synthesis by liver is affected by dieldrin treatment. Proteolipids are also structural components of cell membranes [5] and have the unusual property of being soluble in chloroform and methanol (2 : 1, v/v} and insoluble in water [6]. Studies on brain were also included since chlorinated insecticides like DDT [7] have been shown to cause alterations in the metabolism of neurohormones and

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since brain proteolipid protein has been suggested to be the storage d e p o t of neurohormones in brain [8,9]. It is quite likely that proteolipid protein plays a role in the regulation of the metabolism of neurohormones. Thus in the present study the effect of dieldrin on proteolipid and phosphatidopeptide protein of liver and brain and on total protein of various tissues was investigated. The incorporation of DL-[ 1-14 C] leucine into these cell components was also studied. MATERIALS AND METHODS

Chemicals

DL-[1-1 4C] Leucine (specific activity 27.1 mCi/mmole) was obtained from Bhabha Atomic Research Centre, T r o m b a y (India). Dieldrin (1,2,3,4,10,10hexachloro-6,7-epoxy-l,4,4a,5, 6,7,8,8a-octahydro-exo-l,4-endo-5,8-dimethanonaphthalene) was a gift from Shell Chemical Company (U.S.A.). FolinCiocalteau reagent for protein estimation was purchased from Biochemicals Unit, V.P. Chest Institute, Delhi-7 (India). 2,5-Diphenyloxazole (PPO) and 1,4-bis[2-(5-phenyloxazole)]-benzene (POPOP) were obtained from Sigma Chemical Company (U.S.A.). All other chemicals and solvents were of "anal R' I grade and were purchased from British Drug Houses (India). Treatment of animals Male albino rats of Wistar Strain weighing about 100 g were used in this study. Animals in the experimental groups received a single oral dose (30 mg/kg b o d y weight) of dieldrin dissolved in groundnut oil (0.2--0.3 ml). Control animals received only groundnut oil. All animals were then fasted for 22--24 h and given water ad libitum. Incorporation studies Rats from experimental and control groups which had been fasted for 19--21 h were injected intraperitoneally with DL-[1-14C] leucine (specific activity 27.1 mCi/mmole) at a dose of 0.15 pCi/g b o d y weight. Rats were sacrificed 3 h after injection of the labelled leucine. Before sacrifice blood was collected from the jugular vein into heparinized tubes. Other organs like the brain, liver, kidney and a portion of hamstring muscle were quickly removed. Processing o f tissues Plasma was prepared by centrifuging blood at 2000 rev./min for 10 min. One portion of liver was used for analysis of proteolipids and another for protein. Brain was divided into two, one half was used for protein analysis and the other for proteolipid analysis. For proteolipid analysis tissues were weighed and immediately immersed in chloroform--methanol (2 : 1, v/v). For protein analysis, tissues were homogenised in ice-cold double distilled water and protein was precipitated with trichloroacetic acid. Lipids were removed from the precipitate by washing once with ethanol, twice with a

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m i x t u r e o f e t h a n o l e t h e r (3 : 1, v/v) and o n c e with ether. T h e p o w d e r t h u s o b t a i n e d was dissolved ix 85% f o r m i c acid and suitable aliquots were t a k e n f o r r a d i o a c t i v e c o u n t i n g and f o r p r o t e i n e s t i m a t i o n . F o r m i c acid was evaporated f r o m t h e aliquots t a k e n f o r p r o t e i n e s t i m a t i o n and residues were dissolved in a k n o w n a m o u n t o f 0.5 N s o d i u m h y d r o x i d e . A l i q u o t s o f this s o l u t i o n were a n a l y s e d f o r protein. T o t a l lipids and p h o s p h a t i d o p e p t i d e s were e x t r a c t e d f r o m liver and brain essentially as described b y G a i t o n d e [ 3 ] . Proteolipids were e x t r a c t e d [10] f r o m t o t a l lipid extracts and labelled as p r o t e o l i p i d extracts.

Estimation of protein P r o t e o l i p i d p r o t e i n and p h o s p h a t i d o p e p t i d e p r o t e i n were e s t i m a t e d in t h e p r o t e o l i p i d e x t r a c t s and p h o s p h a t i d o p e p t i d e e x t r a c t s b y t h e m e t h o d - C o f Hess and Lewin [ 1 1 ] . T h e same m e t h o d was e m p l o y e d for the e s t i m a t i o n o f t o t a l p r o t e i n in tissues. However, prior i n c u b a t i o n at 37 ° and e x t r a c t i o n o f lipids after d e v e l o p m e n t o f c o l o u r were n o t carried out. T h e m e t h o d is a slight m o d i f i c a t i o n o f t h e p r o c e d u r e described b y L o w r y et al. [ 1 2 ] .

Assay of radioactivity A l i q u o t s o f p r o t e o l i p i d extracts, p h o s p h a t i d o p e p t i d e e x t r a c t s and extracts o f t o t a l p r o t e i n in f o r m i c acid were a d d e d directly to c o u n t i n g vials. The f o r m i c acid and solvents f r o m all vials were e v a p o r a t e d a n d 5 ml of scintillation fluid (4 g o f PPO + 200 m g o f P O P O P in 1 0 0 0 ml o f t o l u e n e ) was a d d e d to each vial. The r a d i o a c t i v i t y was m e a s u r e d in a P a c k a r d liquid scintillation c o u n t e r (Model 3 0 0 3 ) with an e f f i c i e n c y o f 75% for 14C.

TABLE I EFFECT OF A SINGLE DOSE OF DIELDRIN (30 mg/kg BODY WEIGHT) ON BODY WEIGHT, LIVER WEIGHT, MUSCLE, PLASMA AND KIDNEY PROTEINS OF RAT Control Body weight Initial Fasting Liver weight, g, Total Per 100 g body weight Muscle proteins, mg/g tissue Ki.dney prot~eins, mg/g tissue Plasma proteins, mg/ml plasma

107 91

Dieldrin-treated -+ 3.4 + 3.6

3.29 -+ 0.13 3.6 +-0.13 128 150 68

-+ 1.4 + 6.8 + 2.3

104 89

+ 3.3 -+ 3.2

3.79 -+ 0.11 4.24-+ 0.11 127 153 79

+- 4.9 +3 + 2.9

p >0.05 >0.05 <0.02 <0.01 >0.05 >0.05 >0.05

Values are means -+SE from six animals in each group, p < 0.05 is considered statistically significant.

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T A B L E II E F F E C T O F A S I N G L E D O S E O F D I E L D R I N (30 mg/kg B O D Y W E I G H T ) ON T O T A L P R O T E I N , P R O T E O L I P I D P R O T E I N A N D P H O S P H A T I D O P E P T I D E P R O T E I N CONTENT OF RAT LIVER

Total protein Control Dieldrin-treated

Proteotipid protein Control Dieldrin-treated

P h o s p h a t i d o p e p t i d e protein Control Dieldrin-treated

mg/g fresh liver

m g / 1 0 0 g b o d y weight a

164 + 2.5 166 ± 4.7 p > 0.05

593 + 29 708 -+ 34 p > 0.05

0.77 + 0.02 0.72 ± 0.06 p > 0.05

2.74 + 3.05 + p > 0.05

5.07 + 0.20 3.49 ± 0.34 p < 0.01

18.5 + 0.66 14.7 -+ 1.41 p < 0.02

0.09 0.27

Values are m e a n +SE f r o m six animals in each group, p < 0.05 is considered statistically significant. a (mg/g fresh liver × whole liver w e i g h t ) / ( b o d y weight) × 100.

RESULTS Liver weight was increased in dieldrin-treated rats. There was no change in total protein of muscle, plasma and kidney (Table I). There was no significant change in the incorporation of [1-1 4C] leucine into kidney and plasma proteins. Incorporation into muscle proteins was significantly decreased

T A B L E III E F F E C T O F A S I N G L E D O S E O F D I E L D R I N (30 m g / k g BODY W E I G H T ) ON T O T A L PROTEIN, PROTEOLIPID PROTEIN, PHOSPHATIDOPEPTIDE PROTEIN CONTENT OF RAT BRAIN mg/g of fresh tissue

Total protein Proteolipid protein P h o s p h a t i d o p e p t i d e protein

p

Control

Dieldrin-treated

87 + 2.4 1.13 + 0.03 2.45 + 0.21

85 + 3.7 1.31 + 0.03 2.23 + 0.05

>0.05 <0.01 >0.05

Values are m e a n ±S.E. f r o m six animals in each group, p < 0.05 is considered statistically significant.

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TABLEIV E F F E C T O F A S I N G L E DOSE O F D I E L D R I N (30 m g / k g BODY WEIGHT) ON T H E I N C O R P O R A T I O N OF D L - [ 1 - ~ 4 C ] L E U C I N E D U R I N G A P E R I O D O F T H R E E H O U R S INTO T O T A L P R O T E I N S , P R O T E O L I P I D S A N D P H O S P H A T I D O P E V P I D E S O F R A T LIVER Radioactivity incorporated cpm/g fresh liver Total p r o t e i n Control Dieldrin-treated

Proteolipid protein Control Dieldrin-treated

Phosphatidopeptideprotein Control Dieldrin-treated

95 977 -+ 5 059 142 098 +-- 6 647 p < 0.001

cpm/100 g body weighta

cpm/mg protein

347 053 + 25 609 601 325 +- 18 748 p < 0.001

585 + 34 860 + 60 p < 0.01

1 779 -+ 1 680 -+ p > 0.05

65 64

6 401 + 7 100 + p > 0.05

289 174

2 332 + 99 2 410 + 230 p > 0.05

2 125 -+ 2 836 -+ p < 0.01

108 116

7 664 + 11 983 -+ p < 0.001

505 293

405 + 19 852 + 180 p < 0.001

Values are m e a n +SE f r o m six animals in each group, p < 0.05 is c o n s i d e r e d statistically significant. a As in Table II. TABLE V E F F E C T O F A S I N G L E DOSE OF D I E L D R I N (30 m g / k g BODY WEIGHT) ON THE I N C O R P O R A T I O N OF D L [ 1 - ~ 4 C ] L E U C I N E D U R I N G A P E R I O D O F T H R E E H O U R S INTO T O T A L P R O T E I N , P R O T E O L I P I D S A N D P H O S P H A T I D O P E P T I D E S O F R A T BRAIN Radioactivity incorporated

Total p r o t e i n Control Dieldrin-treated

Proteolipids Control Dieldrin-treated

Phosphatidopeptides Control Dieldrin-treated

c p m / g fresh tissue

cpm/mg protein

36 126 -+ 4 212 32 169 + 1 565 p > 0.05

419 -+ 56 381 +- 18 p > 0.05

443 + 522 -+ p > 0.05

34 64

393 + 28 397 + 38 p > 0.05

789 -+ 869 + p > 0.05

66 47

324 + 20 391 + 22 p < 0.05

Values are m e a n -+SE f r o m six animals in each group, p < 0.05 is c o n s i d e r e d statistically significant.

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TABLE VI EFFECT OF A SINGLE DOSE OF DIELDRIN (30 mg/kg BODY WEIGHT) ON THE INCORPORATION OF DL[1-14C]LEUCINE DURING A PERIOD OF THREE HOURS INTO TOTAL PROTEINS OF MUSCLE AND PLASMA Radioactivity incorporated Control

Dieldrin-treated

Muscle protein cpm/gtissue cpm/mg protein

21 779-+1748 170 -+ 13

12 296-+ 618 98 + 6

<0.001 <0.001

Plasma protein cpm/ml plasma cpm/mg protein

51 342 + 4 092 771 -+ 78

63 998 -+ 5 801 836 -+ 64

>0.05 >0.05

Values are mean -+SE from six animals in each group, p < 0.05 is considered statistically significant.

(Table VI). Total protein and proteolipid protein contents of liver were not affected. However, the c o n t e n t of p h o s p h a t i d o p e p t i d e protein was significantly decreased (Table II). I n c o r p o r a t i o n of [1-1 4C] leucine into liver total protein and p h o s p h a t i d o p e p t i d e protein was significantly increased while incorporation into proteolipids remained unaffect ed (Table IV). Total protein and p h o s p h a t i d o p e p t i d e protein c o n t e n t of dieldrin-treated brain remained unchanged while proteolipid protein c o n t e n t increased significantly (Table III). There was no change in the incorporation of [1-14C] leucine into brain protein (Table V). DISCUSSION Liver weight and c ons equent l y the liver : b o d y weight ratio was significantly increased in dieldrin-treated rats, This confirms previous reports from this l a b o r a t o r y [2,13] as well as those of o t h e r investigators [ 1 4 - - 1 6 ] . This effect could be due to an increase in cell mass. In this c o n t e x t dieldrin has been shown to cause an increase in the sm oot h endoplasmic reticulum [ 1 5 , 1 7 , 1 8 ] . The increased liver weight m ay be an adaptive mechanism, n o t necessarily reflecting the toxic effect of insecticide [ 1 9 - - 2 2 ] . A highly significant increase in the incorporation of [1-14C] leucine into liver total protein on all parameters expressed was observed. This clearly shows th at protein synthesis is increased in dieldrin-treated rat liver, confirming a previous r e p o r t [ 2 ] . DDT has been shown to have a similar effect [ 1 ] . This increase in the rate of protein synthesis is also compatible with the earlier r e p o r t of increased e n z y m e activity in dieldrin-treated animals [2,23] and o f dieldrin-induced h y p e r t r o p h y of the s m o o t h endoplasmic reticulum [ 1 5 , 1 7 , 1 8 ] . Since total protein was n o t depleted in muscle, the reduced

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incorporation of 1 4C into muscle protein would be consistent with decreased tu r n over or to a decrease in availability of [ 14C] leucine to muscle because o f increased i nc or por a t i on elsewhere, e.g. liver. The specific activity o f phosphatidopeptides was observed to be significantly increased (p < 0.001) in dieldrin-treated rat liver. Specific activities of total protein and phosphatidopeptides observed in this study were quite comparable. Phosphatidopeptides [3] have been suggested as precursors o f protein synthesis. However, the current study provides no i nform at i on on this aspect. This would explain the increased i ncor por at i on of [ 1 aC] leucine into phosphatidopeptides observed in t he experimental group. Phosphatidopeptides have been shown to be functioning in the active transport of amino acids across the liver cell membranes [ 4 ] . T he possibility t hat these c o m p o u n d s increase the transport of amino acids into t he cells in the livers of experimental animals, however, c a n n o t be ruled out. Since no effect has been observed on liver proteolipids which are mostly synthesised by mitochondrial protein synthesising system [ 2 4 , 2 5 ] , it may be postulated t hat dieldrin does n o t affect the mitochondrial protein synthesising system in liver. In contrast to liver, t he proteolipid c o n t e n t of brain was increased in dieldrin-treated rats. There was, however, no significant effect on the e x t e n t of in co r p o r a t i on of 14C into brain proteolipids. It should be emphasised that the i n c o r p o r a t i o n studies were p e r f o r m e d 19--21 h after dieldrin administration so th at any earlier effect would n o t have been detected. The increase in brain proteolipid may be associated with changes in the metabolism of neur o h o r mo n es . Thus certain biogenic amines have been shown to bind strongly to brain proteolipids [26,27] and it has been suggested t hat these latter c o m p o n e n t s may act as a storage d e p o t for n e u r o h o r m o n e s [8, 9]. A study of effects o f dieldrin on the metabolism of serotonin is currently in progress. ACKNOWLEDGEMENTS One o f us (K.K.K.) is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of a Senior Research Fellowship. We are grateful to Shell Chemical C om pany, New York, U.S.A. for a gift of recrystalline dieldrin (HEOD). Thanks are due to Mr. A.K. Tyagi for technical assistance. REFERENCES

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