Z. Ern,~hrungswiss. 17, 153-158 (1978) (~ 1978 Dr. Dietrich Steinkopff Verlag, Darmstadt ISSN 0044-264 X
National Research Centre, Dokki, Cairo (Egypt)
Microsomal enzymes inducers and serum minerals i n earbon-tetraehloride h e p a t o t o ~ c i t y R. A w a d a l l a h , E.A. E I - D e s s o u k e y , a n d H. M i k h a i l Tahani With 1 table (Received February 27, 1978)
Carbon tetrachloride in single or repeated doses produces characteristic hepatic lesions, including in t u r n in the liver f u r t h e r biochemical and pathological disturbance (38). E a r l y changes in calcium and potassium content of mitochondria after oral CC14 intoxication have been found b y ReynoZd et al. (30). Tahani et al. (35) reported an increase in s e r u m zinc, copper, iron, calcium, potassium and sodium, while for magnesium no significant change was observed in acute liver damage, due to a single intraperitoneal injection of carbon tetrachloride. Considerable evidence is now available suggesting that the production of liver d a m a g e is linked with an increased activity of the liver microsoreal enzymes (22). The effect of mircosomal enzymes inducers on hepatic function in mice and rats were studied w h e n phenobarbital-Na was given before or after poisoning with carbon tetrachloride (15). Phenobarbital p r e t r e a t m e n t enhanced the rise in s e r u m transaminase activity, hepatotoxicity and lethality induced b y carbon tetrachloride poisorting (20, 24, 14). However, w e found that phenobarbital had a protective effect w h e n given in repeated doses together with small doses of CCh. Protection was evident from reduced activity of plasma glutamic-pyruvic transaminase and reduced liver necrosis as demonstrated b y histologic evaluations (11). The purpose of the present w o r k is to study the effect of phenobarbital which is k n o w n to be an inducer of microsomal enzymes (6) and propionyl promazine which is an inhibitor (41) on serum zinc, copper, iron, calcium, magnesium, potassium and sodium in carbon-tetrachloride intoxication.
Material and methods Male Sprague-Dawley rats, weighing 150--200g, were used in all studies. The animals were maintained on an ad-libitum diet and water. Ten animals were included in each tretment group. The animals were divided into six groups: Control group, Group treated with CCh (0.1 mFkg s.c.), dilut528
154
Zeitschri]t ]i2r Ernghrungswissenschaft, Band 17, He~t 3 (1978)
ed in the ratio of 1:1 with paraffin oil, was administered daily for ten days. Phenobarbitone group (60 mg/kg i.p.), propionyl-promazine group (2 mg/kg i.m.), and two other groups were administered either phenobarbitone plus CC14 or propionyl promazine plus CC14. The animals were administered daily for a period of 10 days. The animals were then sacrificed by light ether anesthesia and blood samples were taken from the orbital plescus and blood was collected for serum minerals determination. The method of Sinaha and Gabrietti (34) was used for determination of serum zinc and copper. Serum iron, potassium and sodium were estimated by the method published in B e c k m a n Analytical method by Atomic Absorption Spectrophotometer. Serum Calcium and magnesium were determined using the method of Willis (39). Results and discussion In the present work, carbon tetrachloride in ten repeated doses of (0.1 ml/kg s.c.) resulted in a significant increase in serum iron, copper, zinc, calcium, potassium and sodium. Also in a previous w o r k we found that a single intraperitoneal injection of 2.5 ml/kg body weight has the same effect (35). This confirms the previous finding of VilleIa (38) that repeated small doses of CC14 have an additive effect, also the onset of the pathological changes depends on the route of administration. The results of the present w o r k also show that propionyl promazine when administered alone or togehter with CC14 has no effect on serum minerals. Phenobarbitone when administered alone increased serum minerals except sodium and magnesium, but to a lesser degree than CC14, while phenobarbitone when given repeatedly together with small doses of CC14 led to normalization of serum iron, potassium and calcium. Also serum zinc and copper were significantly lower than in case of CC14. The initial lesion produced by CC14 seems to be cytoplasmic, mitochondrial damage comes later (36, 1). In early stages of intoxication, cytological lesions were more pronounced in the ergastoplasm as confirmed by electron micrographs (2), leakage of cytoplasmic enzymes results from this i n j u r y (37). P r a s a d (25) showed that zinc is present in several metaloenzymes such as alkaline phosphatase and is important in their activities. Therefore, the increased serum zinc in CC14 phenobarbitone and phenobarbitone plus CC14 treated rats m a y be attributed to increased serum enzymes (11). K a z i m i e r z (17) found that in liver damage by CC14, the accumulation of radioactive zinc was highest in the mitochondria accompanied by a decrease in nuclear fraction radioactivity. This suggested displacement of zinc from nuclei to the cytoplasm. The level of blood zinc was also raised b y the administration of adrenaline (9). C a l v e r t and B r o d y (4) suggested that a predominant factor in the hepatoxicity of CC14 is an anoxia produced through the mediation of the sympathetic nervous system. Release of adrenaline from the adrenal medulla under sympathetic stimulation is also suggested.
S.E. 4-
S.E. 4-
S.E. :h
S.E. _q-
S.E. ~
P : C v s P r o p . q- CC14 CCl~ v s P r o p . q- CCI~
P r o p . q- CC14
P: C vs prop.
Propionyl-Prom
P : C v s P h . q- CC14 CCI~ v s P h . + CC14
P h e n o q- CC14
P: C vs Phen.
Phenobarbitone
P : C vs CC14
CC14
232.8 11.57 0.005 n.s.
120.4 9.45 n.s.
164.1 9.22 0.005 0.005
174.0 11.29 0.05
213.3 7.27 0.005
133.4 4.27
Control
S.E. 4-
Zinc /zg%
Item
200.5 14.53 0.005 n.s.
134.5 8.74 n.s.
154.7 4,88 0.005 0.005
169.0 9.49 0.025
199.5 9.69 0.005
118.9 5.12
Copper /~g%
213.5 8.13 0.005 n.s.
153.8 8.47 n.s.
149.3 6.54 n.s. 0.005
187.2 12.21 0.05
218.5 10.0 0.005
135.2 6.13
Iron /tg%
Table 1.
9.81 0.21 0.005 n.s.
8.37 0.17 n.s.
8.49 0,59 n.s. 0.005
9.76 0.39 0.025
9.95 0.22 0.025
8.57 0.37
Calcium rag%
3.19 0.42 n.s. n.s,
3.88 0.11 n.s.
3.57 0.38 n.s. n.s.
3.72 0.30 n.s.
3.96 0.34 n.s.
4.00 0.13
Magnesium mg%
25.2 0.82 0.05 n.s.
22.2 0.47 n.s.
22.6 0.52 n.s. 0.05
25.1 1.12 0.05
26.6 0.78 0.05
22.8 0.36
Potassium rag%
283.5 12.17 0.005 n.s.
226.5 3.57 n.s.
291.0 4.91 0.005 0.05
248.5 11.57 n.s.
271.0 7.49 0.005
228.7 3.19
Sodium rag%
r
r f~
.q
0~
3"
b~
0~
o
-q
4
o,
156
Zeitschrift liar Ernt~hrungswissenschaft, Band 17, Heft 3 (1978)
On the other hand, liver disease was found to affect zinc in blood (13). The high level of s e r u m zinc was related to hepatic damage. A portion of this increase was due to hemolysis of e r y t h r o c y t e s (40). I n carbon tetrachloride t r e a t e d rats, the s e r u m copper was higher t h a n t h a t of the control. Phenobarbitone, p h e n o b a r b i t o n e plus CC14 h a v e the s a m e effect (table 1). Recknage~ and Litteria (27) found t h a t carbon tetrachloride diffuses r a pidly and is found in the liver to a m a x i m u m level 1'/2 hr a f t e r its administration. Christie and Judah (5) suggested that the change in the liver indicate a direct physical attack of the drug on liver mitochondria, degeneration and d e a t h of the cells results f r o m the disorganization of mitochondrial structure. Clearly t h e n carbon tetrachloride increase the p e r m e a b i l i t y of the m i tochondrial m e m b r a n e and high level of s e r u m copper is attributed to this hepatic d a m a g e (28). David et al. (8) showed that the hepatic and b r a i n copper concentration increased significantly as a result of p h e n o b a r b i t a l t r e a t m e n t , while blood s e r u m copper increased and muscle copper decreased. In the present work, the m e a n s e r u m iron in CC14 treated rats w h e n c o m p a r e d w i t h control rats, there is a statistically significant increase. Our finding agreed with t h a t obtained b y m a n y investigators (23, 21, 16). The increase of s e r u m iron w a s explained due to a significant decrease of utilization of iron for the hemoglobin synthesis in intoxication with carb o n tetrachloride (18). O k a w a r a (23) found t h a t incorporation of iron into the hemopoietic organs and iron storage organs w e r e r e t a r d e d b y CCh poisoning. Acute liver d a m a g e resulted in an increase in p l a s m a iron (29), while in various degrees of chronic liver d a m a g e the p l a s m a iron was not elevated in rats (10). Therefore, the increase in s e r u m iron results f r o m the disintegration of hepatic cells. O u r data indicated t h a t the s e r u m iron was still high u n d e r the effect of p h e n o b a r b i t o n e and p h e n o b a r b i t o n e plus CC14. The m e a n s e r u m calcium was significantly high in CC14 and p h e n o b a r bitone treated rats. In animals treated w i t h p h e n o b a r b i t o n e plus CC14 no significant change in s e r u m calcium was found. Ruichi (31) showed t h a t two hours a f t e r oral CC14 intoxication, calcium content of the mitochondria increased concomitantly w i t h a decrease in potassium ion. The t i m e course of mitochondrial degeneration in CC14 poisoning appears to follow the increase in concentration of calcium in liver mitochondria (32). Ernesto et al. (12) showed t h a t in CCh intoxication the balance between ADP phosphorylation and calcium u p t a k e is shifted in f a v o u r of the later, thus contributing to the accumulation of calcium in mitochondria. On the o t h e r hand, Kovacs (19) found t h a t CC14 did not change blood calcium. David et al. (8) showed t h a t p h e n o b a r b i t a l injection did not significantly change the concentration of m a g n e s i u m and calcium in liver.
AwadalIah et al., Microsomal enzymes inducers and serum minerals
157
No s i g n i f i c a n t c h a n g e i n s e r u m m a g n e s i u m i n o u r t r e a t e d g r o u p s w a s observed. A slight decrease in serum m a g n e s i u m was found in patients w i t h l i v e r disease (31). Carbon tetrachloride administration induced a significant increase in t h e l e v e l of s e r u m p o t a s s i u m a n d s o d i u m . T h e s e r u m p o t a s s i u m is still h i g h u n d e r t h e i n f l u e n c e of p h e n o b a r b i t o n e , w h i l e s e r u m s o d i u m is n o t s i g n i f i cantly higher than normal. Phenobarbitone administered together with CC14 n o r m a l i s e d s e r u m p o t a s s i u m . O u r f i n d i n g a g r e e d w i t h t h a t o b t a i n e d b y Boda et al. (3) w h o f o u n d t h a t s e r u m s o d i u m i n c r e a s e d w h i l e t i s s u e s o d i u m d e c r e a s e d d u e to c a r b o n tetrachloride. C a r b o n t e t r a c h l o r i d e i n t o x i c a t i o n c a u s e d a k i d n e y i n j u r y (33). W h e n s i g n s of i n t r a c e l l u l a r d e h y d r a t i o n occur, t h e p l a s m a p o t a s s i u m m a y b e s o m e w h a t h i g h e r t h a n n o r m a l (7). T h e i n c r e a s e i n s e r u m p o t a s s i u m m a y also b e f r o m t h e r e d cell h e m o l y s i s . That severe mitochondrial damage occurs in the liver after carbon tet r a c h l o r i d e p o i s o n i n g has b e e n a b u n d a n t l y c o n f i r m e d . L i v e r m i t o c h o n d r i a of r a t s p o i s o n e d w i t h c a r b o n t e t r a c h l o r i d e a r e s w o l l e n . Recknage~ a n d Mal a m e d (26) s h o w e d t h a t t h e m i t o c h o n d r i a l s w e l l i n g is d u e to a n i n c r e a s e i n m i t o c h o n d r i a l m e m b r a n e p e r m e a b i l i t y . A s a result, t h e m i t o c h o n d r i a r e lease i n t o t h e s u r r o u n d i n g m e d i u m c y t o c h r o m e C a n d p o t a s s i u m (28). I t is c o n c l u d e d t h a t a p o r t i o n of t h e rise i n s e r u m m i n e r a l s w a s d u e to i n c r e a s e d p e r m e a b i l i t y i n d a m a g e d m i t o c h o n d r i a l m e m b r a n e as a r e s u l t of t h e a c t i o n of c a r b o n t e t r a c h l o r i d e . Also t h e d e c r e a s e i n s e r u m m i n e r a l s w h e n CC14 w a s g i v e n t o g e t h e r w i t h p h e n o b a r b i t o n e m a y b e d u e to some p r o t e c t i v e effect of p h e n o b a r b i t o n e on l i v e r m i t o c h o n d r i a .
Summary The effect of ten repeated doses of carbon tetrachloride, phenobarbitone and propionyl promazine when administered alone or simultaneously with CC14 on serum minerals was investigated. Carbon tetraehloride resulted in a significant increase in serum iron, copper, zinc, calcium, potassium and sodium. A portion of this rise was due to increased permeability i n damaged mitochondrial m e m b r a n e as a result of the action of CCI~. Propionyl promazine when administered alone or together with CC14 has no effect on serum minerals. Phenobarbitone when administered alone increased serum minerals except sodium, but to a lesser degree t h a n CC14, while phenobarbitone when given repeatedly together with small doses of CC14 led to a normalization of serum iron, calcium and potassium. Also serum zinc a n d copper were lower t h a n in case of CC14. This may be due to some protective effect of phenobarbitone on liver mitochondria. Serum m a g n e s i u m was not affected in all the experimental groups. Relerences 1. Alfonso, O. R., E. Mitidieri, L. P. Ribeiro, G. G. V~llela, Prec. Soc. Exp. Biol., N.Y. 90, 527 (1955). - 2. Bassi, M., Exp. Ceil. Res. 20, 313 (1960). - 3. Boda, D., M. Galambos, Magyar Tudomanyos Akad-Biol. esorvosi: T u d o m a n y o k Osztaly a n a k Kozlemenyei 8, 172 (1957). - 4. Calvert, D. N., T. M. Brody, Amer. J. Physiol.
158
Zeitschrift fiir Ern~hrungswissenschaft, Band 17, Heft 3 (1978)
198, 668 (1960). - 5. Christie, G. S., J. D. Judah, P r o c . Soc. B 142, 241 (1954). - 6. Conney, A. H., C. Davidson, R. Gastel, B. B. Burns, J. P h a r m a c . E x p . T h e s . 130, 1 (1960). - 7. Darrow, D. S., P h y s i o l . R e v . 38, 114 (1958). - 8. David, H., M. S. Fahim, D. G. Hall, E. Pickett, T r a c e S u b s t . E n v i r o n m . H e a l t h 5, P r o c . U n i v . M o . A n n u . C o n f . , 5 t h , 235, 1971 ( P u b . 1972). - 9. Dennis, E., R. Tupper, A. Warmall, B i o l . J. 78, 578 (1961). - 10. E1-Dessoukey, E. A., R. AwadaIlah, S. El-Attar, Biochemical changes under the effect of carbon-tetrachloride i n t o x i c a t i o n . Z. Ern~hrungswiss. ( u n d e r p u b l i c a t i o n ) . - 11. El-Dessoukey, E. A., Tahani H. Mikhail, R. Awadallah, Zinat H. Aly, Nadia A. Kotb, E f f e c t o f p h e n o b a r b i t o n e and propionyl-promazine on serum enzymes in carbon tetrachloride hepatoxicity ( u n d e r p u b l i c a t i o n ) . - 12. Ernesto, C., T. Roberta, E x p . M o l . P a t h . 9 (1), 131 (1968). - 13. Fayez, M., S. Aziz, A. El-Sheikh, M. T. El-Gengehy, F. Fouad, Y. A. Habib, J. E g y p t . M e d . A s s o c . 56, 2 (1973). - 14. Gans, J. H., K. Roy, H. Joab, B i o c h e m . P h a r m a c o l . 25 (5), 577 (1976). - 15. Hurwitz, A., T o x i c o l . a p p l . P h a r m . 22 (3), 339 (1972). - 16. Kaszewska, J. I. G. Marian, PoI. A r c h . M e d . W e w n 45 (4), 531 (1970). - I7. Kazimierz, W., J. Hanfy, L. Owezarek, W. Fenrych, S. Gorsk, C. M e j e w s k , A c t a M e d . P o l . 7 (3), 253 (1966). - 18. Keiderling, W., I. Reissmer, W. Kaboth, N u c l . M e d . 2, 173 (1961). - 19. Kovacs, F., G. Tolgyes, M a g y a r A l l a t o r v o s k L o p j a 15, 277 (1960). - 20. Litterst, C. L., T. M. Father, E. J. Vanloon, T o x i c o l . a p p l . P h a r m a c o l . 25 (3), 354 (1973). - 21. Lob, T. T., J. S. Juggi, A u s t . J. E x p . B i o l . M e d . Sc. 49 (5), 493 (1971). - 22. Mclean, A. E. M., E. K. Mclean, B i o c h e m . J. 100, 564 (1966). - 23. Okawara, Y., N i p p o n N a i k G a k k a i Z a s s h i 49, 1484 (1961). 24. Pani, P., L. Gabriel, M. V. Torrielli, E. Gravela, B i o c h e m . Soc. T r a n s . 1 (4), 976 (1973). - 25. Prasad, A. S., A m . J . C l i n . N u t r . 22, 1215 (1969). - 26. Recknagel, R. 0., S. Malamed, J. B i o l . C h e m . 232, 705 (1958). - 27. Recknagel, R. 0., M. Litteria, A m e r . J . P a t h . 36, 521 (1960). - 28. Recknagel, R. 0., P h a r m a c o l . R e v . 19, 145 (1967). - 29. Reissmann, K. R., M. R. Dietrich, J. C l i n . I n v e s t . 35, 580 (1956). 30. Reynolds, E. S., R. E. Thiers, B. L. Vallee, J. B i o l . C h e m . 237, 3546 (1962). 31. Ruichi, I., q u o t e d f r o m C h e m . A b s t . 70, 27099 w (1969). - 32. Share, L., R. O. Recknagel, A m e r . J. P h y s i o l . 197, 121 (1959). - 33. Sherlock, S., i n : D r u g i n c l u d e d D i s e a s e s , L. Mayer a n d H. M. Peck, E d s 157 ( A s s e r t 1962). - 34. Sinaha, S. N., E. R. Gabrielli, A m e r . J. C l i n . P a t h . 64, 570 (1970). - 35. Tahani H. Mikhail, R. Awadallah, E. A. El-Dessoukey, E f f e c t o f A M P o n s e r u m m i n e r a l s i n c a r b o n t e t r a c h l o r i d e h e p a t o x i c i t y . Z. E r n a h r u n g s w i s s . ( u n d e r p u b l i c a t i o n ) . - 36. Villela, G. G., E. Mitidieri, N a t u r e , L o n d . 176, 208 (1955). - 37. Villela, G. G., N a t u r e , L o n d . 190, 807 (1961). - 38. Villela, G. G., B i o c h e m . P h a r m . 13, 665 (1964). - 39. Willis, J. B., C l i n . C h e m . 77, 251 (1965). - 49. Wol]f, H., H. Maske, B. Stavnpfl, F. Bau?ngarten, N a u n y n . S c h m i e d e b e r g ' s A r c h . E x p t l . P a t h . P h a r m a k o l . 216, 440 (1952). - 41. Zinat H. Aly, Bahria A. Fahim, M. A. Sherif, V e t . M e d . A s s . J. (in press). Author's address: D r . Raa]at Awadallah, B i o c h e m i s t r y D e p a r t m e n t , Dokki, Cairo (Egypt)
National Research Centre,
National Research Centre, Dokki, Cairo (Egypt)
Microsomal enzymes inducers and serum minerals i n earbon-tetraehloride h e p a t o t o ~ c i t y R. A w a d a l l a h , E.A. E I - D e s s o u k e y , a n d H. M i k h a i l Tahani With 1 table (Received February 27, 1978)
Carbon tetrachloride in single or repeated doses produces characteristic hepatic lesions, including in t u r n in the liver f u r t h e r biochemical and pathological disturbance (38). E a r l y changes in calcium and potassium content of mitochondria after oral CC14 intoxication have been found b y ReynoZd et al. (30). Tahani et al. (35) reported an increase in s e r u m zinc, copper, iron, calcium, potassium and sodium, while for magnesium no significant change was observed in acute liver damage, due to a single intraperitoneal injection of carbon tetrachloride. Considerable evidence is now available suggesting that the production of liver d a m a g e is linked with an increased activity of the liver microsoreal enzymes (22). The effect of mircosomal enzymes inducers on hepatic function in mice and rats were studied w h e n phenobarbital-Na was given before or after poisoning with carbon tetrachloride (15). Phenobarbital p r e t r e a t m e n t enhanced the rise in s e r u m transaminase activity, hepatotoxicity and lethality induced b y carbon tetrachloride poisorting (20, 24, 14). However, w e found that phenobarbital had a protective effect w h e n given in repeated doses together with small doses of CCh. Protection was evident from reduced activity of plasma glutamic-pyruvic transaminase and reduced liver necrosis as demonstrated b y histologic evaluations (11). The purpose of the present w o r k is to study the effect of phenobarbital which is k n o w n to be an inducer of microsomal enzymes (6) and propionyl promazine which is an inhibitor (41) on serum zinc, copper, iron, calcium, magnesium, potassium and sodium in carbon-tetrachloride intoxication.
Material and methods Male Sprague-Dawley rats, weighing 150--200g, were used in all studies. The animals were maintained on an ad-libitum diet and water. Ten animals were included in each tretment group. The animals were divided into six groups: Control group, Group treated with CCh (0.1 mFkg s.c.), dilut528
154
Zeitschri]t ]i2r Ernghrungswissenschaft, Band 17, He~t 3 (1978)
ed in the ratio of 1:1 with paraffin oil, was administered daily for ten days. Phenobarbitone group (60 mg/kg i.p.), propionyl-promazine group (2 mg/kg i.m.), and two other groups were administered either phenobarbitone plus CC14 or propionyl promazine plus CC14. The animals were administered daily for a period of 10 days. The animals were then sacrificed by light ether anesthesia and blood samples were taken from the orbital plescus and blood was collected for serum minerals determination. The method of Sinaha and Gabrietti (34) was used for determination of serum zinc and copper. Serum iron, potassium and sodium were estimated by the method published in B e c k m a n Analytical method by Atomic Absorption Spectrophotometer. Serum Calcium and magnesium were determined using the method of Willis (39). Results and discussion In the present work, carbon tetrachloride in ten repeated doses of (0.1 ml/kg s.c.) resulted in a significant increase in serum iron, copper, zinc, calcium, potassium and sodium. Also in a previous w o r k we found that a single intraperitoneal injection of 2.5 ml/kg body weight has the same effect (35). This confirms the previous finding of VilleIa (38) that repeated small doses of CC14 have an additive effect, also the onset of the pathological changes depends on the route of administration. The results of the present w o r k also show that propionyl promazine when administered alone or togehter with CC14 has no effect on serum minerals. Phenobarbitone when administered alone increased serum minerals except sodium and magnesium, but to a lesser degree than CC14, while phenobarbitone when given repeatedly together with small doses of CC14 led to normalization of serum iron, potassium and calcium. Also serum zinc and copper were significantly lower than in case of CC14. The initial lesion produced by CC14 seems to be cytoplasmic, mitochondrial damage comes later (36, 1). In early stages of intoxication, cytological lesions were more pronounced in the ergastoplasm as confirmed by electron micrographs (2), leakage of cytoplasmic enzymes results from this i n j u r y (37). P r a s a d (25) showed that zinc is present in several metaloenzymes such as alkaline phosphatase and is important in their activities. Therefore, the increased serum zinc in CC14 phenobarbitone and phenobarbitone plus CC14 treated rats m a y be attributed to increased serum enzymes (11). K a z i m i e r z (17) found that in liver damage by CC14, the accumulation of radioactive zinc was highest in the mitochondria accompanied by a decrease in nuclear fraction radioactivity. This suggested displacement of zinc from nuclei to the cytoplasm. The level of blood zinc was also raised b y the administration of adrenaline (9). C a l v e r t and B r o d y (4) suggested that a predominant factor in the hepatoxicity of CC14 is an anoxia produced through the mediation of the sympathetic nervous system. Release of adrenaline from the adrenal medulla under sympathetic stimulation is also suggested.
S.E. 4-
S.E. 4-
S.E. :h
S.E. _q-
S.E. ~
P : C v s P r o p . q- CC14 CCl~ v s P r o p . q- CCI~
P r o p . q- CC14
P: C vs prop.
Propionyl-Prom
P : C v s P h . q- CC14 CCI~ v s P h . + CC14
P h e n o q- CC14
P: C vs Phen.
Phenobarbitone
P : C vs CC14
CC14
232.8 11.57 0.005 n.s.
120.4 9.45 n.s.
164.1 9.22 0.005 0.005
174.0 11.29 0.05
213.3 7.27 0.005
133.4 4.27
Control
S.E. 4-
Zinc /zg%
Item
200.5 14.53 0.005 n.s.
134.5 8.74 n.s.
154.7 4,88 0.005 0.005
169.0 9.49 0.025
199.5 9.69 0.005
118.9 5.12
Copper /~g%
213.5 8.13 0.005 n.s.
153.8 8.47 n.s.
149.3 6.54 n.s. 0.005
187.2 12.21 0.05
218.5 10.0 0.005
135.2 6.13
Iron /tg%
Table 1.
9.81 0.21 0.005 n.s.
8.37 0.17 n.s.
8.49 0,59 n.s. 0.005
9.76 0.39 0.025
9.95 0.22 0.025
8.57 0.37
Calcium rag%
3.19 0.42 n.s. n.s,
3.88 0.11 n.s.
3.57 0.38 n.s. n.s.
3.72 0.30 n.s.
3.96 0.34 n.s.
4.00 0.13
Magnesium mg%
25.2 0.82 0.05 n.s.
22.2 0.47 n.s.
22.6 0.52 n.s. 0.05
25.1 1.12 0.05
26.6 0.78 0.05
22.8 0.36
Potassium rag%
283.5 12.17 0.005 n.s.
226.5 3.57 n.s.
291.0 4.91 0.005 0.05
248.5 11.57 n.s.
271.0 7.49 0.005
228.7 3.19
Sodium rag%
r
r f~
.q
0~
3"
b~
0~
o
-q
4
o,
156
Zeitschrift liar Ernt~hrungswissenschaft, Band 17, Heft 3 (1978)
On the other hand, liver disease was found to affect zinc in blood (13). The high level of s e r u m zinc was related to hepatic damage. A portion of this increase was due to hemolysis of e r y t h r o c y t e s (40). I n carbon tetrachloride t r e a t e d rats, the s e r u m copper was higher t h a n t h a t of the control. Phenobarbitone, p h e n o b a r b i t o n e plus CC14 h a v e the s a m e effect (table 1). Recknage~ and Litteria (27) found t h a t carbon tetrachloride diffuses r a pidly and is found in the liver to a m a x i m u m level 1'/2 hr a f t e r its administration. Christie and Judah (5) suggested that the change in the liver indicate a direct physical attack of the drug on liver mitochondria, degeneration and d e a t h of the cells results f r o m the disorganization of mitochondrial structure. Clearly t h e n carbon tetrachloride increase the p e r m e a b i l i t y of the m i tochondrial m e m b r a n e and high level of s e r u m copper is attributed to this hepatic d a m a g e (28). David et al. (8) showed that the hepatic and b r a i n copper concentration increased significantly as a result of p h e n o b a r b i t a l t r e a t m e n t , while blood s e r u m copper increased and muscle copper decreased. In the present work, the m e a n s e r u m iron in CC14 treated rats w h e n c o m p a r e d w i t h control rats, there is a statistically significant increase. Our finding agreed with t h a t obtained b y m a n y investigators (23, 21, 16). The increase of s e r u m iron w a s explained due to a significant decrease of utilization of iron for the hemoglobin synthesis in intoxication with carb o n tetrachloride (18). O k a w a r a (23) found t h a t incorporation of iron into the hemopoietic organs and iron storage organs w e r e r e t a r d e d b y CCh poisoning. Acute liver d a m a g e resulted in an increase in p l a s m a iron (29), while in various degrees of chronic liver d a m a g e the p l a s m a iron was not elevated in rats (10). Therefore, the increase in s e r u m iron results f r o m the disintegration of hepatic cells. O u r data indicated t h a t the s e r u m iron was still high u n d e r the effect of p h e n o b a r b i t o n e and p h e n o b a r b i t o n e plus CC14. The m e a n s e r u m calcium was significantly high in CC14 and p h e n o b a r bitone treated rats. In animals treated w i t h p h e n o b a r b i t o n e plus CC14 no significant change in s e r u m calcium was found. Ruichi (31) showed t h a t two hours a f t e r oral CC14 intoxication, calcium content of the mitochondria increased concomitantly w i t h a decrease in potassium ion. The t i m e course of mitochondrial degeneration in CC14 poisoning appears to follow the increase in concentration of calcium in liver mitochondria (32). Ernesto et al. (12) showed t h a t in CCh intoxication the balance between ADP phosphorylation and calcium u p t a k e is shifted in f a v o u r of the later, thus contributing to the accumulation of calcium in mitochondria. On the o t h e r hand, Kovacs (19) found t h a t CC14 did not change blood calcium. David et al. (8) showed t h a t p h e n o b a r b i t a l injection did not significantly change the concentration of m a g n e s i u m and calcium in liver.
AwadalIah et al., Microsomal enzymes inducers and serum minerals
157
No s i g n i f i c a n t c h a n g e i n s e r u m m a g n e s i u m i n o u r t r e a t e d g r o u p s w a s observed. A slight decrease in serum m a g n e s i u m was found in patients w i t h l i v e r disease (31). Carbon tetrachloride administration induced a significant increase in t h e l e v e l of s e r u m p o t a s s i u m a n d s o d i u m . T h e s e r u m p o t a s s i u m is still h i g h u n d e r t h e i n f l u e n c e of p h e n o b a r b i t o n e , w h i l e s e r u m s o d i u m is n o t s i g n i f i cantly higher than normal. Phenobarbitone administered together with CC14 n o r m a l i s e d s e r u m p o t a s s i u m . O u r f i n d i n g a g r e e d w i t h t h a t o b t a i n e d b y Boda et al. (3) w h o f o u n d t h a t s e r u m s o d i u m i n c r e a s e d w h i l e t i s s u e s o d i u m d e c r e a s e d d u e to c a r b o n tetrachloride. C a r b o n t e t r a c h l o r i d e i n t o x i c a t i o n c a u s e d a k i d n e y i n j u r y (33). W h e n s i g n s of i n t r a c e l l u l a r d e h y d r a t i o n occur, t h e p l a s m a p o t a s s i u m m a y b e s o m e w h a t h i g h e r t h a n n o r m a l (7). T h e i n c r e a s e i n s e r u m p o t a s s i u m m a y also b e f r o m t h e r e d cell h e m o l y s i s . That severe mitochondrial damage occurs in the liver after carbon tet r a c h l o r i d e p o i s o n i n g has b e e n a b u n d a n t l y c o n f i r m e d . L i v e r m i t o c h o n d r i a of r a t s p o i s o n e d w i t h c a r b o n t e t r a c h l o r i d e a r e s w o l l e n . Recknage~ a n d Mal a m e d (26) s h o w e d t h a t t h e m i t o c h o n d r i a l s w e l l i n g is d u e to a n i n c r e a s e i n m i t o c h o n d r i a l m e m b r a n e p e r m e a b i l i t y . A s a result, t h e m i t o c h o n d r i a r e lease i n t o t h e s u r r o u n d i n g m e d i u m c y t o c h r o m e C a n d p o t a s s i u m (28). I t is c o n c l u d e d t h a t a p o r t i o n of t h e rise i n s e r u m m i n e r a l s w a s d u e to i n c r e a s e d p e r m e a b i l i t y i n d a m a g e d m i t o c h o n d r i a l m e m b r a n e as a r e s u l t of t h e a c t i o n of c a r b o n t e t r a c h l o r i d e . Also t h e d e c r e a s e i n s e r u m m i n e r a l s w h e n CC14 w a s g i v e n t o g e t h e r w i t h p h e n o b a r b i t o n e m a y b e d u e to some p r o t e c t i v e effect of p h e n o b a r b i t o n e on l i v e r m i t o c h o n d r i a .
Summary The effect of ten repeated doses of carbon tetrachloride, phenobarbitone and propionyl promazine when administered alone or simultaneously with CC14 on serum minerals was investigated. Carbon tetraehloride resulted in a significant increase in serum iron, copper, zinc, calcium, potassium and sodium. A portion of this rise was due to increased permeability i n damaged mitochondrial m e m b r a n e as a result of the action of CCI~. Propionyl promazine when administered alone or together with CC14 has no effect on serum minerals. Phenobarbitone when administered alone increased serum minerals except sodium, but to a lesser degree t h a n CC14, while phenobarbitone when given repeatedly together with small doses of CC14 led to a normalization of serum iron, calcium and potassium. Also serum zinc a n d copper were lower t h a n in case of CC14. This may be due to some protective effect of phenobarbitone on liver mitochondria. Serum m a g n e s i u m was not affected in all the experimental groups. Relerences 1. Alfonso, O. R., E. Mitidieri, L. P. Ribeiro, G. G. V~llela, Prec. Soc. Exp. Biol., N.Y. 90, 527 (1955). - 2. Bassi, M., Exp. Ceil. Res. 20, 313 (1960). - 3. Boda, D., M. Galambos, Magyar Tudomanyos Akad-Biol. esorvosi: T u d o m a n y o k Osztaly a n a k Kozlemenyei 8, 172 (1957). - 4. Calvert, D. N., T. M. Brody, Amer. J. Physiol.
158
Zeitschrift fiir Ern~hrungswissenschaft, Band 17, Heft 3 (1978)
198, 668 (1960). - 5. Christie, G. S., J. D. Judah, P r o c . Soc. B 142, 241 (1954). - 6. Conney, A. H., C. Davidson, R. Gastel, B. B. Burns, J. P h a r m a c . E x p . T h e s . 130, 1 (1960). - 7. Darrow, D. S., P h y s i o l . R e v . 38, 114 (1958). - 8. David, H., M. S. Fahim, D. G. Hall, E. Pickett, T r a c e S u b s t . E n v i r o n m . H e a l t h 5, P r o c . U n i v . M o . A n n u . C o n f . , 5 t h , 235, 1971 ( P u b . 1972). - 9. Dennis, E., R. Tupper, A. Warmall, B i o l . J. 78, 578 (1961). - 10. E1-Dessoukey, E. A., R. AwadaIlah, S. El-Attar, Biochemical changes under the effect of carbon-tetrachloride i n t o x i c a t i o n . Z. Ern~hrungswiss. ( u n d e r p u b l i c a t i o n ) . - 11. El-Dessoukey, E. A., Tahani H. Mikhail, R. Awadallah, Zinat H. Aly, Nadia A. Kotb, E f f e c t o f p h e n o b a r b i t o n e and propionyl-promazine on serum enzymes in carbon tetrachloride hepatoxicity ( u n d e r p u b l i c a t i o n ) . - 12. Ernesto, C., T. Roberta, E x p . M o l . P a t h . 9 (1), 131 (1968). - 13. Fayez, M., S. Aziz, A. El-Sheikh, M. T. El-Gengehy, F. Fouad, Y. A. Habib, J. E g y p t . M e d . A s s o c . 56, 2 (1973). - 14. Gans, J. H., K. Roy, H. Joab, B i o c h e m . P h a r m a c o l . 25 (5), 577 (1976). - 15. Hurwitz, A., T o x i c o l . a p p l . P h a r m . 22 (3), 339 (1972). - 16. Kaszewska, J. I. G. Marian, PoI. A r c h . M e d . W e w n 45 (4), 531 (1970). - I7. Kazimierz, W., J. Hanfy, L. Owezarek, W. Fenrych, S. Gorsk, C. M e j e w s k , A c t a M e d . P o l . 7 (3), 253 (1966). - 18. Keiderling, W., I. Reissmer, W. Kaboth, N u c l . M e d . 2, 173 (1961). - 19. Kovacs, F., G. Tolgyes, M a g y a r A l l a t o r v o s k L o p j a 15, 277 (1960). - 20. Litterst, C. L., T. M. Father, E. J. Vanloon, T o x i c o l . a p p l . P h a r m a c o l . 25 (3), 354 (1973). - 21. Lob, T. T., J. S. Juggi, A u s t . J. E x p . B i o l . M e d . Sc. 49 (5), 493 (1971). - 22. Mclean, A. E. M., E. K. Mclean, B i o c h e m . J. 100, 564 (1966). - 23. Okawara, Y., N i p p o n N a i k G a k k a i Z a s s h i 49, 1484 (1961). 24. Pani, P., L. Gabriel, M. V. Torrielli, E. Gravela, B i o c h e m . Soc. T r a n s . 1 (4), 976 (1973). - 25. Prasad, A. S., A m . J . C l i n . N u t r . 22, 1215 (1969). - 26. Recknagel, R. 0., S. Malamed, J. B i o l . C h e m . 232, 705 (1958). - 27. Recknagel, R. 0., M. Litteria, A m e r . J . P a t h . 36, 521 (1960). - 28. Recknagel, R. 0., P h a r m a c o l . R e v . 19, 145 (1967). - 29. Reissmann, K. R., M. R. Dietrich, J. C l i n . I n v e s t . 35, 580 (1956). 30. Reynolds, E. S., R. E. Thiers, B. L. Vallee, J. B i o l . C h e m . 237, 3546 (1962). 31. Ruichi, I., q u o t e d f r o m C h e m . A b s t . 70, 27099 w (1969). - 32. Share, L., R. O. Recknagel, A m e r . J. P h y s i o l . 197, 121 (1959). - 33. Sherlock, S., i n : D r u g i n c l u d e d D i s e a s e s , L. Mayer a n d H. M. Peck, E d s 157 ( A s s e r t 1962). - 34. Sinaha, S. N., E. R. Gabrielli, A m e r . J. C l i n . P a t h . 64, 570 (1970). - 35. Tahani H. Mikhail, R. Awadallah, E. A. El-Dessoukey, E f f e c t o f A M P o n s e r u m m i n e r a l s i n c a r b o n t e t r a c h l o r i d e h e p a t o x i c i t y . Z. E r n a h r u n g s w i s s . ( u n d e r p u b l i c a t i o n ) . - 36. Villela, G. G., E. Mitidieri, N a t u r e , L o n d . 176, 208 (1955). - 37. Villela, G. G., N a t u r e , L o n d . 190, 807 (1961). - 38. Villela, G. G., B i o c h e m . P h a r m . 13, 665 (1964). - 39. Willis, J. B., C l i n . C h e m . 77, 251 (1965). - 49. Wol]f, H., H. Maske, B. Stavnpfl, F. Bau?ngarten, N a u n y n . S c h m i e d e b e r g ' s A r c h . E x p t l . P a t h . P h a r m a k o l . 216, 440 (1952). - 41. Zinat H. Aly, Bahria A. Fahim, M. A. Sherif, V e t . M e d . A s s . J. (in press). Author's address: D r . Raa]at Awadallah, B i o c h e m i s t r y D e p a r t m e n t , Dokki, Cairo (Egypt)
National Research Centre,
