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Distribution of CCl4 among liver cell fractions
606
BIOCIKIMICA ET BIOPIiYSICA ACTA
Short Communications s¢ 23 o33
D i s t r i b u t i o n of CCI 4 a m o n g liver cell fractions CC14 is able to cause in vitro changes to occur in mitochondria z-~ and microsomes * isolated from rat liver, and to release hydrolytic activities from lysosomes in a ratliver homogenate 7. Such an effect occurs at concentrations of CC14 similar to those reached in vivo in the liver of orally or parenterally poisoned rats s-l°. These facts support the view that CC14 acts through a direct action on cell lipoprotein membranes. This may be true, even if CC14 is metabolized to some extent by tissues 11-14. In the present experiments, the distribution of CC14 among five subcellular fractions at different times after oral treatment is studied. Male albino rats, weighing about 15o g, fasted for 8 h before the treatment and for the time before killing, received 0.5 ml pure CC14 per IOO g body weight by a stomach tube. 14CC1, (The Radiochemical Centre, Amersham) was diluted with carrier CC14 to a specific activity of 1176000 counts/rain per ml. Each rat was killed by decapitation and bleeding; its liver was weighed and transferred to a cold room at 0-2 °. A tissue aliquot was homogenized with toluene (I5 ml per I g liver) for 60 sec in a P o t t e r - E l v e h j e m apparatus, allowed to stand for Io rain, then centrifuged. A sample of the toluene phase, dried over sodium sulphate, was counted for radioactivity in an ECKO Scintillation Counter. Another sample of tissue, IO % (w/v) in 0.25 M sucrose, was homogenized and submitted to differential centrifuging to separate the following fractions: I, nuclear (2000 x g for IO rain)" 2, mitochondrial (3300 ~'~ g for lO rain) ; 3, lysosomal (IOOOO ~ g for 15 rain) ; 4, microsomal (16000 x g for 60 rain) ; 5, final supernatant. Each fraction was resuspended and/or diluted to IO ml with distilled water, shaken with 15 ml toluene for 60 sec, allowed to stand for IO rain, and centrifuged. Samples of the toluene phase were counted for radioactivity. Total counts of the five fractions gave a 75-80 % recovery with respect to the toluene homogenates. CC14 (on the assumption that all the detected radioactivity belonged to CC14) rapidly accumulated within the liver during the first hour after treatment, reaching a level that remained steady until the fourth hour (Fig. I). The CCI~ level then slowly decreased. Top levels were higher than those observed by I~ECKNAGELAND LITTERIA °, who, however, used CC14 diluted I : I with mineral oil which is known to delay the intestinal absorption of CC147. In contrast with other reports9,~°, 15, we found that top levels of CCI~ were maintained in the liver for some hours. This means that, following our standard treatment, CC1a contacts liver cell structures at high concentrations for a relatively long time. Moreover RoQu~ A~,~DFEDORKO11 found by autoradiography that CC14 is unevenly distributed in hepatic lobules. Therefore, many liver cells presumably contained even greater amounts of CC1, than the averages found in our experiments. Distribution of CC14 among cell fractions is depicted in Fig. 2 (B, C, D). Distribution was not grossly different in our three experiments. The largest amount of CC1, Biochim. Biophys. Actor, 90 (I964) 606-608
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SHORT COMMUNICATIONS
(about 4 ° %) was found in the supernatant (Fraction 5); a substantial part (about 25 %) was recovered in the nuclear Fraction 2 (this also contained cell d6bris and red blood cells); microsomal, mitochondrial, and lysosomal fractions (4, 2, and 3) contained, respectively, 15, 13, and 9 % of the radioactivity. L
3
~2 E
t
L-
2
4 hours after
6 8 CC[4 Ieeding
Fig. i. CC14 c o n c e n t r a t i o n w i t h i n r a t liver a t different t i m e s a f t e r oral poisoning. E a c h p o i n t r e p r e s e n t s t h e m e a n of d u p l i c a t e s a m p l e s f r o m a single a n i m a l . Areas m a r k e d b y x r e p r e s e n t t h r e e s u p e r i m p o s i n g points. T h e line joins t h e p o i n t s c o r r e s p o n d i n g to t h e m e a n of e a c h group.
A
B
C
O
Fig. 2. P e r c e n t d i s t r i b u t i o n of 14C a m o n g cell f r a c t i o n s f r o m io % liver h o m o g e n a t e s p r e p a r e d in 0.25 M sucrose s a t u r a t e d w i t h CC14, c o n t a i n i n g s u i t a b l e a m o u n t s of 14CC1~ (A), a n d f r o m t h e liver of r a t s 30 m i n (B), I h (C), a n d 6 h (D) after t h e oral a d m i n i s t r a t i o n of 0. 5 ml p u r e CC14 ( c o n t a i n i n g p r o p e r a m o u n t s of 14CC14) per i o o g b o d y weight. T h e f r a c t i o n s are t h e following: i, n u c l e a r ; 2, m i t o c h o n d r i a l ; 3, l y s o s o m a l ; 4, m i c r o s o m a l ; 5, final s u p e r n a t a n t . Vertical b a r s r e p r e s e n t s t a n d a r d deviations. N u m b e r of e x p e r i m e n t s : 2 (A), 3 (B), 6 (C), 3 (D).
The distribution pattern we have obtained may not exactly correspond to that of liver in situ. Preparative manipulations (homogenization, centrifuging) might have caused CC14 to redistribute, at least in part. We therefore studied the distribution of radioactivity among fractions from IO % liver homogenates prepared in 0.25 M sucrose saturated with CCI~9, containing suitable amounts of 14CC1~, and allowed to stand for IO min in the cold room before differential centrifuging. The distribution pattern of radioactivity (Fig. 2, A) showed slight differences when compared with those previously described. These differences may have been due to small changes in the lipid composition of cells. It seems, therefore, that there were no active mechanisms controlling the diffusion of CCI~ throughout the structures of the liver cells. Fig. 3 shows the percentage distribution of total nitrogen among the liver cell Biochim. Biophys. Acta, 9o (1964) 6 o 6 - 6 o 8
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SHORT COMMUNICATIONS
fractions of control and CC14-poisoned rats. These histograms cannot be superimposed on those of the Fig. 2, the height of Column I being especially different. The results reported above show that, after rats were given o.5 ml pure CC14 per I00 g body weight per os, liver cell structures were exposed very early and for some hours to relatively high concentrations of CC14. There was no selective fixation of CC14 to a given cell particle, but the substance was widely distributed among all
A
B
C
O
Fig. 3. P e r c e n t d i s t r i b u t i o n of t o t a l n i t r o g e n a m o n g l i v e r cell f r a c t i o n s from n o r m a l (A) a n d CC14-poisoned rats, 3 ° m i n (B), 1 h (C), a n d 6 h (D) a f t e r t he t r e a t m e n t . F r a c t i o n s as in Fig. 2. Two e x p e r i m e n t s for e a c h group.
cell fractions. The fact that CCla, at similar concentrations, was able to damage liver particles in vitro is strongly suggestive of a direct damaging action of this substance in the liver in situ. Therefore, CC14 would act by affecting the physico-chemical properties of the lipoprotein membranes owing to its solubility in their lipid components. This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Rome.
Institutes of General Pathology of fhe Universities of Siena and Cagliari (Italy)
F . M. BACClNO G. SATTA L. I~{AMELI
O. RECKNAGEL AND S. MALAMED, J. Biol. Chem., 232 (1958) 705 . L. LEHNINGER, J. Biol. Chem., 234 (1959) 2465. O. RECKNAGEL AND D. D. ANTHONY, J. Biol. Chem., 234 (1959) lO52. O. RECKNAGEL AND B. LOM13ARDI, J. Biol. Chem., 236 (196I) 564. S. REYNOLDS, R. E. TI-IIERS AND 13. L. VALLEE, J. Biol. Chem., 237 (1962) 3546. ARTIZZU, 12. M. BACCINO AND M. U. DIANZANI, Biochim. Biophys. Aeta, 78 (I963) I. ARTIZZU, P. PANI, G. SATTA AND ,'V[. U. DIANZANI, Biochim. Biophys. Acla, 82 (1964) 454ARTIZZU AND M. U. D1ANZANI, Biochim. Biophys. ,4cla, 63 (1962) 453. O. RECKNAGEL AND M. LITTERIA, Am. J. Pathol., 36 (196o) 52i . J. R. DAWKINS, J. Palhol. Bacteriol., 85 (1963) 189. L. ROQU£ AND M. E. FEDORKO, J. Histochem. Cytochem., 9 (196t) 613. D. MCCOLLISTER, %V. H. BEAMER, G. J. ATCHISON AND H. C. SPENCER, J. Pharmacol. Exptl. Therap., lO2 {i9 5i ) 112. la 1~. S. REYNOLDS, Federalion Proc., 22 (1963) 37 o. 14 T. C. BUTLER, J. Pharmacol. Exptl. Therap., 134 (1961) 311. 15 A. J. MAXIMCHUK AND D. RUBINSTEIN, Ann. Occup. Hvg., 4 (1961) 49. 1 R. 2 A. 3 R. 4 R. 5 E. 6 M. 7 M. 8 M. 9 R. 10 M. 11 A. 1~ D.
Received April i5th , i964 Biochim. Biophys. Aeta, 90 (I964) 606-608.
BIOCIKIMICA ET BIOPIiYSICA ACTA
Short Communications s¢ 23 o33
D i s t r i b u t i o n of CCI 4 a m o n g liver cell fractions CC14 is able to cause in vitro changes to occur in mitochondria z-~ and microsomes * isolated from rat liver, and to release hydrolytic activities from lysosomes in a ratliver homogenate 7. Such an effect occurs at concentrations of CC14 similar to those reached in vivo in the liver of orally or parenterally poisoned rats s-l°. These facts support the view that CC14 acts through a direct action on cell lipoprotein membranes. This may be true, even if CC14 is metabolized to some extent by tissues 11-14. In the present experiments, the distribution of CC14 among five subcellular fractions at different times after oral treatment is studied. Male albino rats, weighing about 15o g, fasted for 8 h before the treatment and for the time before killing, received 0.5 ml pure CC14 per IOO g body weight by a stomach tube. 14CC1, (The Radiochemical Centre, Amersham) was diluted with carrier CC14 to a specific activity of 1176000 counts/rain per ml. Each rat was killed by decapitation and bleeding; its liver was weighed and transferred to a cold room at 0-2 °. A tissue aliquot was homogenized with toluene (I5 ml per I g liver) for 60 sec in a P o t t e r - E l v e h j e m apparatus, allowed to stand for Io rain, then centrifuged. A sample of the toluene phase, dried over sodium sulphate, was counted for radioactivity in an ECKO Scintillation Counter. Another sample of tissue, IO % (w/v) in 0.25 M sucrose, was homogenized and submitted to differential centrifuging to separate the following fractions: I, nuclear (2000 x g for IO rain)" 2, mitochondrial (3300 ~'~ g for lO rain) ; 3, lysosomal (IOOOO ~ g for 15 rain) ; 4, microsomal (16000 x g for 60 rain) ; 5, final supernatant. Each fraction was resuspended and/or diluted to IO ml with distilled water, shaken with 15 ml toluene for 60 sec, allowed to stand for IO rain, and centrifuged. Samples of the toluene phase were counted for radioactivity. Total counts of the five fractions gave a 75-80 % recovery with respect to the toluene homogenates. CC14 (on the assumption that all the detected radioactivity belonged to CC14) rapidly accumulated within the liver during the first hour after treatment, reaching a level that remained steady until the fourth hour (Fig. I). The CCI~ level then slowly decreased. Top levels were higher than those observed by I~ECKNAGELAND LITTERIA °, who, however, used CC14 diluted I : I with mineral oil which is known to delay the intestinal absorption of CC147. In contrast with other reports9,~°, 15, we found that top levels of CCI~ were maintained in the liver for some hours. This means that, following our standard treatment, CC1a contacts liver cell structures at high concentrations for a relatively long time. Moreover RoQu~ A~,~DFEDORKO11 found by autoradiography that CC14 is unevenly distributed in hepatic lobules. Therefore, many liver cells presumably contained even greater amounts of CC1, than the averages found in our experiments. Distribution of CC14 among cell fractions is depicted in Fig. 2 (B, C, D). Distribution was not grossly different in our three experiments. The largest amount of CC1, Biochim. Biophys. Actor, 90 (I964) 606-608
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(about 4 ° %) was found in the supernatant (Fraction 5); a substantial part (about 25 %) was recovered in the nuclear Fraction 2 (this also contained cell d6bris and red blood cells); microsomal, mitochondrial, and lysosomal fractions (4, 2, and 3) contained, respectively, 15, 13, and 9 % of the radioactivity. L
3
~2 E
t
L-
2
4 hours after
6 8 CC[4 Ieeding
Fig. i. CC14 c o n c e n t r a t i o n w i t h i n r a t liver a t different t i m e s a f t e r oral poisoning. E a c h p o i n t r e p r e s e n t s t h e m e a n of d u p l i c a t e s a m p l e s f r o m a single a n i m a l . Areas m a r k e d b y x r e p r e s e n t t h r e e s u p e r i m p o s i n g points. T h e line joins t h e p o i n t s c o r r e s p o n d i n g to t h e m e a n of e a c h group.
A
B
C
O
Fig. 2. P e r c e n t d i s t r i b u t i o n of 14C a m o n g cell f r a c t i o n s f r o m io % liver h o m o g e n a t e s p r e p a r e d in 0.25 M sucrose s a t u r a t e d w i t h CC14, c o n t a i n i n g s u i t a b l e a m o u n t s of 14CC1~ (A), a n d f r o m t h e liver of r a t s 30 m i n (B), I h (C), a n d 6 h (D) after t h e oral a d m i n i s t r a t i o n of 0. 5 ml p u r e CC14 ( c o n t a i n i n g p r o p e r a m o u n t s of 14CC14) per i o o g b o d y weight. T h e f r a c t i o n s are t h e following: i, n u c l e a r ; 2, m i t o c h o n d r i a l ; 3, l y s o s o m a l ; 4, m i c r o s o m a l ; 5, final s u p e r n a t a n t . Vertical b a r s r e p r e s e n t s t a n d a r d deviations. N u m b e r of e x p e r i m e n t s : 2 (A), 3 (B), 6 (C), 3 (D).
The distribution pattern we have obtained may not exactly correspond to that of liver in situ. Preparative manipulations (homogenization, centrifuging) might have caused CC14 to redistribute, at least in part. We therefore studied the distribution of radioactivity among fractions from IO % liver homogenates prepared in 0.25 M sucrose saturated with CCI~9, containing suitable amounts of 14CC1~, and allowed to stand for IO min in the cold room before differential centrifuging. The distribution pattern of radioactivity (Fig. 2, A) showed slight differences when compared with those previously described. These differences may have been due to small changes in the lipid composition of cells. It seems, therefore, that there were no active mechanisms controlling the diffusion of CCI~ throughout the structures of the liver cells. Fig. 3 shows the percentage distribution of total nitrogen among the liver cell Biochim. Biophys. Acta, 9o (1964) 6 o 6 - 6 o 8
60~
SHORT COMMUNICATIONS
fractions of control and CC14-poisoned rats. These histograms cannot be superimposed on those of the Fig. 2, the height of Column I being especially different. The results reported above show that, after rats were given o.5 ml pure CC14 per I00 g body weight per os, liver cell structures were exposed very early and for some hours to relatively high concentrations of CC14. There was no selective fixation of CC14 to a given cell particle, but the substance was widely distributed among all
A
B
C
O
Fig. 3. P e r c e n t d i s t r i b u t i o n of t o t a l n i t r o g e n a m o n g l i v e r cell f r a c t i o n s from n o r m a l (A) a n d CC14-poisoned rats, 3 ° m i n (B), 1 h (C), a n d 6 h (D) a f t e r t he t r e a t m e n t . F r a c t i o n s as in Fig. 2. Two e x p e r i m e n t s for e a c h group.
cell fractions. The fact that CCla, at similar concentrations, was able to damage liver particles in vitro is strongly suggestive of a direct damaging action of this substance in the liver in situ. Therefore, CC14 would act by affecting the physico-chemical properties of the lipoprotein membranes owing to its solubility in their lipid components. This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Rome.
Institutes of General Pathology of fhe Universities of Siena and Cagliari (Italy)
F . M. BACClNO G. SATTA L. I~{AMELI
O. RECKNAGEL AND S. MALAMED, J. Biol. Chem., 232 (1958) 705 . L. LEHNINGER, J. Biol. Chem., 234 (1959) 2465. O. RECKNAGEL AND D. D. ANTHONY, J. Biol. Chem., 234 (1959) lO52. O. RECKNAGEL AND B. LOM13ARDI, J. Biol. Chem., 236 (196I) 564. S. REYNOLDS, R. E. TI-IIERS AND 13. L. VALLEE, J. Biol. Chem., 237 (1962) 3546. ARTIZZU, 12. M. BACCINO AND M. U. DIANZANI, Biochim. Biophys. Aeta, 78 (I963) I. ARTIZZU, P. PANI, G. SATTA AND ,'V[. U. DIANZANI, Biochim. Biophys. Acla, 82 (1964) 454ARTIZZU AND M. U. D1ANZANI, Biochim. Biophys. ,4cla, 63 (1962) 453. O. RECKNAGEL AND M. LITTERIA, Am. J. Pathol., 36 (196o) 52i . J. R. DAWKINS, J. Palhol. Bacteriol., 85 (1963) 189. L. ROQU£ AND M. E. FEDORKO, J. Histochem. Cytochem., 9 (196t) 613. D. MCCOLLISTER, %V. H. BEAMER, G. J. ATCHISON AND H. C. SPENCER, J. Pharmacol. Exptl. Therap., lO2 {i9 5i ) 112. la 1~. S. REYNOLDS, Federalion Proc., 22 (1963) 37 o. 14 T. C. BUTLER, J. Pharmacol. Exptl. Therap., 134 (1961) 311. 15 A. J. MAXIMCHUK AND D. RUBINSTEIN, Ann. Occup. Hvg., 4 (1961) 49. 1 R. 2 A. 3 R. 4 R. 5 E. 6 M. 7 M. 8 M. 9 R. 10 M. 11 A. 1~ D.
Received April i5th , i964 Biochim. Biophys. Aeta, 90 (I964) 606-608.