The action of carbon tetrachloride on mitochondria in vitro

BIOCHIMICA ET BIOPHYSICA ACTA

I

BBA 4 I o I

THE ACTION OF CARBON TETRACHLORIDE ON M I T O C H O N D R I A

IN

VITRO

M. ARTIZZU, F. M. BACCINO AND M. U. DIANZANI Institute of General Pathology of the University o/Cagliari (Italy) (Received January 4th, 1963)

SUMMARY Carbon tetrachloride produces pronounced morphological and biochemical changes in mitochondria in vitro. At high concentrations, it increases the rate of spontaneous swelling of the particles; at low concentrations it delays the rate of spontaneous swelling. Addition of CC14 (saturated aqueous solution) to mitochondria in vitro results in extensive uncoupling of oxidative phosphorylation, as well as in abolition of respiratory control. ATPase activity is increased. The effect of CC14 can be observed also in homogenates. If these are prepared with sucrose solutions previously saturated with CC14, the mitochondria show evidence of biochemical changes, which are, however, less marked than those produced b y CC14 in the reaction medium. It is conceivable that part of the mitochondrial damage induced by CC14 is reversed by washing. Since the liver of CC14-treated rats contains CC14 in concentrations close to those shown to cause mitochondrial damage in vitro, the action of CC14 on mitochondria m a y account for the damage in vivo.

INTRODUCTION It is well-known t h a t f a t t y liver produced by treatment with CC14 is characterized b y the presence of intensive mitochondrial damage. The mitochondria are swollen 1-s and show uncoupling of oxidative phosphorylation and increased ATPase activity 5,9-i4. Respiratory control is abolished n-13 and the activity of DPN-dependent oxidases is decreased n-la, 15, 16 Octanoate oxidation is impaired n-13. A large proportion of intra-mitochondrial coenzymes or coiactors, e.g. DPNI", is, TPP1", ~°, cytochrome c21, adenylnucleotides 22, leaks out of the particles into the surrounding medium. The mechanism whereby such mitochondrial damage is produced is not well understood. According to CALVERT AND BRODY 23, adrenalectomy, adrenenergicblocking agents, pretreatment with reserpine and section of the spinal cord, all are active in preventing f a t t y infiltration and mitochondrial changes. CCl~ provokes a release from the adrenal medulla 24 of catecholamines which m a y influence the development of mitochondrial changes. In our experience ~5 adrenalectomy m a y delay the onset of lipid accumulation Abbreviation: TPP, thiamine pyrophosphate. Biochim. Biophys. Acta, 78 (1963) i - i i

2

M. ARTIZZU, F. M. BACCINO, M. U. D I A N Z A N I

within the liver, but is unable to prevent mitochondrial damage. J U D A H et al. ~6-a° showed that antihistamine drugs could protect mitochondria from swelling in vitro, and prevent liver damage in vivo. Evidence for a direct effect of CC14 on mitochondrial morphology was adduced by RECKNAGEL AND MALAMED31, who found that treatment of mitochondria by aqueous solutions of CC14 resulted in a marked increase in the rate of spontaneous swelling of the particles. This was confirmed by LEHNINGER32. CC14-treated particles show high Mg e+- and low dinitrophenol-stimulated ATPase. The activity of DPNdependent oxidases is strongly inhibitedaL12. As RECKNAGEL AND LITTERIA33 point out, ingested CC14 rapidly accumulates within the liver. The amount of this substance present within the liver I -2 h after stomach intubation is about 1-2 mg/g wet organ, a concentration which is approximately equal to or higher than that producing mitochondrial changes in vitro. Nevertheless mitochondrial activities were found to be normal at this stage by RECKNAGEL AND A N T H O N Y 11,12. In our experience 2s the change in mitochondrial morphology is evident I h after CC14-feeding. Uncoupling of oxidative phosphorylation occurs at 2 h. It seemed desirable therefore to test again the action of CCI 4 in vitro. The results obtained are reported in this paper. MATERIALS AND METHODS

Albino rats (Wistar strain), weighing 12o-15o g, fed on a semi-synthetic diet (M. I. L. Morini, S. Polo d'Enza, Reggio Emilia (Italy)), were used for this investigation. They were killed b y decapitation. The livers were immediately removed, weighed and transferred to the cold room (4°). Homogenates (lO%) were prepared in a P o t t e r - E l v e h j e m type apparatus, using 0.3 M sucrose containing 0.02 M Tris-HC1 buffer (pH 7.4). In most cases the sucrose solution contained also o.ooi M EDTA. Mitochondria were isolated by differential centrifugation of the homogenate in a Lourdes type AB centrifuge (rotor 9RA). Nuclei, red cells and debris were sedimented at 800 ~i g for 5 rain. Mitochondria were then sedimented at 3300 ~" g for IO min. They were washed once in most experiments. The rate of mitochondrial swelling was studied by measuring the decrease in absorbancy at 52o m/, in a Beckman model DU spectrophotometer. Each cuvette contained 0.3 M sucrose buffered with Tris-HC1 buffer (pH 7.4) and mitochondria equivalent to 50 mg wet liver. The nitrogen content was about 0.3 rag. The final volume was 3.2 ml. The absorbancy was recorded every minute for 20 rain, and then at io-min intervals up to 70 min. The percent decrease in absorbancy after 70 min (A %) was taken as a measure of the extent of swelling. The rate of swelling was calculated from the time necessary to attain 50% decrease in absorbancy (50% T). In some experiments, the influence of the following substances on mitochondrial swelling was tested: ATP, 2,4-dinitrophenol, succinate, glutamate, pyruvate, pyrur a t e + malate, all as potassium salts. The final concentration in the medium was in each case I" IO-4 M. Oxidative phosphorylation was measured in a Warburg apparatus, b y the method described previonsly2S.The reaction medium contained: 0.oo67 M MgSO4, 0.025 M KC1, O.Ol3 M KF, 0.02 M Na2HPO4-KH2PO~ buffer (pH 7.4), o.ooi M AMP (potassium salt), yeast hexokinase (Sigma, Type III) 15 ° K.M. units, 0.026 M glucose, 30 t, moles Biochim. Biopky~..4eta, 78 (I9(J3) i t f

MITOCHONDRIAL DAMAGE BY CC14 in vitro

3

potassium glutamate, mitochondria corresponding to 17o mg wet liver. These were suspended in o.25 M sucrose containing o.o2 M Tris-HC1 buffer (pH 7.4). 0.2 ml 3o % KOH were placed into the central well of each flask. The reaction was stopped by adding I ml 3o% trichloroacetic acid. Oxygen uptake was measured for IO min. The time allowed for temperature equilibration was 7 min. Inorganic orthophosphate was determined on the deproteinized filtrate according to BERENBLUMAND CHAIN34. Respiratory control of mitochondria was studied by the method described elsewhere 25, by determining the oxygen uptake: (a) in the system previously described for the measurement of oxidative phosphorylation; (b) in the same medium, from which hexokinase 4-glucose were omitted; (c) in the same medium, from which hexokinase + glucose + AMP were omitted. ATPase activity was studied according to the method of RECKNAGEL AND ANTHONYTM. In each case, the enzymic activity was studied: (a) in a basal medium from which activators were omitted; (b) in a medium containing 3"1o-5 M DNP; (c) in a medium containing 0.002 M MgSO~. The basal medium contained: 6/zmoles ATP (disodium salt) (pH 7.4), 30/,moles Tris-HC1 buffer (pH 7.4), 325/,moles sucrose, mitochondria corresponding to 12.5 mg wet liver. The final volume was 1.5 ml. The incubation was made at 25 ° for 20 min. After this time the reaction was stopped by addition of 0.5 ml 40 % trichloroacetic acid. Inorganic orthophosphate was determined on the deproteinized filtrate according to BERENBLUM AND CHAIN34. Each experiment was made in duplicate. The nitrogen content of mitochondria or homogenates was determined by the usual micro-Kjeldahl method. CC14 solutions were prepared by the diffusion equilibrium method described by RECKNAGEL AND LITTERIA :~3. The CC14 concentration was determined by a colorimetric method 33. The statistical significance of the data was evaluated by means of Fisher's " t " test. Most of the chemicals used were Merck or Sigma products. Tris was a product of British Drug Houses. EDTA was obtained from Siegfried, Zofingen (Switzerland). CC14 was a redistilled Merck product. RESULTS Action of CCl 4 on mitochondrial swelling

The influence of CC14 on mitochondrial swelling in vitro is shown in Table I, and in Figs. I and 2. It is interesting to note that the rate of spontaneous swelling of untreated mitochondria depends upon the washing of the particles. In fact, in some experiments in which unwashed mitochondfia were used, swelling occurred at a rate which was significantly higher than that observed with washed mitochondria. A " t " value of 5.533 was found for the difference between the A % observed with 13 unwashed (4o.6 ± 5.2) and 22 twice-washed mitochondrial preparations (25.3 ± 8.9). The value of " t " was 2.552, corresponding to P < o.o2, in the case of the difference between the 5o % transmittance values observed in the first group of experiments (946 ~= 247) and those seen in the second group (12o6 ± 3o2). It is possible that the cause of this difference is the presence in unwashed mitochondria of substances capable of increasing the rate of spontaneous swelling. In Biochim. Biophys. Acta, 78 (I963) i - I I

4

M. ARTIZZU, F. M. BACCIN0, M. U. DIANZANI

fact, m a n y substances present in the homogenate, e.g. oxidative substrates and fatty acids, are known to exert this effect s5-a°. The action of CC14 on the spontaneous swelling was diphasic depending upon its concentration. In fact, concentrations of CC14 equal to or higher than 0.2 mg per cuvette produced in each case an evident acceleration of the rate of spontaneous swelling. This can be deduced both from the A% and from the 50 % transmittance values. In each case the differences were of high statistical significance. The addition of ATP, which is known to protect against the spontaneous swelling, does not influence the acceleration produced by CC14. These results agree with those reported by other authors al-33.

0.500

0.40C

o 1

0.450

0.3

0

2

0.40O

.

2

5

0 ~ 30 Time (min)

60

Fig. I. Effects of CC14 on s p o n t a n e o u s swelling of mitochondria. Ordinate, a b s o r b a n c y at 52o m # ; abscissa, t i m e (min). Additions: I, o.I m g CC14; 2, o.i m g CC14 + ATP; 3, o.I m g CCI 4 + dinitrophenol; 4, no additions; 5, o. 4 m g CC14; 6, 0. 4 m g CC14 + dinitrophenol; 7, o-4 m g CC14 + ATP.

Time (rain)

Fig. 2. Effects of CC1¢ on s p o n t a n e o u s swelling of mitochondria. The d a t a are presented as in Fig. i. Additions: I, o.I m g CC14; 2, no additions; 3, 0.4 m g CC14 + g l u t a m a t e ; 4, o.4 mg CC14 + succinate; 5, g l u t a m a t e ; 6, succinate.

In some experiments, mitochondrial swelling was studied by suspending the particles in an aqueous solution saturated with CC14, containing about I mg CC14/ml. The amount of CC14 present per cuvette was about 3 mg. Immediate swelling of the particles occurred in this medium; it was followed in each case by flocculation of the proteins. Low concentrations of CC14, ranging from o.o8 to o.12 mg per cuvette, exert, on the contrary, a delaying action on the spontaneous swelling. This is evident both from the A % and from the 50 % transmittance values, but the differences were particularly significant in the latter case. CC14 concentrations lower than 0.04 mg per cuvette did not exert any effect on the A % values. The 50% transmittance values showed some difference, but this was not statistically significant. Biochim. Biophys. Acta, 78 (i963) i - t i

MITOCHONDRIAL DAMAGE BY CC14 in vitro TABLE ACTION

OF

CCI~

ON

THE

SPONTANEOUS

5

I SWELLING

OF

MITOCHONDRIA

in vitro

The values for the percent decrease in absorbancy recorded after 7 ° rain are given under the heading Zl %, those for t h e t i m e (sec) necessary to a t t a i n a 5° % decrease in a b s o r b a n c y are given u n d e r 5 ° % T. Means ± s t a n d a r d deviation. The " t " value refers to the d a t a obtained in the absence and in the presence of CC14. Amount of Number of CC1, added experi(rag) ments

0.02 0.04 0.08 o.io o.12 o.15 0.20 0.30 0.40

3* 3* 13" 22 12" 2* 2 6 5

A% No CCI,

44 .8 44 .8 40.6 25.3 38.1 39.2 30.5 24.8 23.3

± ± 4± ± ± ± ± •

3.3 3.3 5.2 8.9 7.1 I.I 2.8 4.6 4 .2

50% T

+ CCl4

43.7 42.6 33.4 17. 7 27.6 31.6 42.7 42.6 43.5

No CC14

"t"

-- 4 .8 ± 5.8 ± 12.o ± 8. 7 ~2 11.8 4- 0.9 4- 0.2 i 2.7 -- 4 .8

--1.923 2.815"* 2.573*** --7.435** 6.412"*

828 828 946 12o6 iOlO lO65 855 1237 118o

=c 42 ~ 42 ± 247 ± 302 ± 307 =E 195 =ix 21 ± 294 ~: 141

+ CCI4

11o 4 122o 1583 1944 18o9 lO5O 385 358 408

"t"

± 169 ± 585 ± 33 ° ± 629 =c 502 ~- 12o ~ 78 -- 15o ~ 200

--4.967** 4.871.* 4.591"* --6-047** 6.316"*

* E x p e r i m e n t s m a d e w i t h u n w a s h e d mitochondria. ** P

(

0.0I.

*** P

<

0.02.

TABLE II EFFECT

OF CC14

in vitro

ON O X I D A T I V E P H O S P H O R Y L A T I O N

E x p t . I : m i t o c h o n d r i a from i g liver, suspended in 3 ml o. 25 M s u c r o s e - T r i s - E D T A , s a t u r a t e d w i t h CCI~; p r e i n c u b a t i o n for io rain at 25 °. E x p t . 2 : h o m o g e n a t e p r e p a r e d as follows: I g liver + 0.25 M s u c r o s e - T r i s - E D T A to 3 ml, s a t u r a t e d w i t h CC14; p r e i n c u b a t i o n for io rain at 25 °. E x p t . 3: m i t o c h o n d r i a isolated f r o m I g liver homogenized in io ml 0. 3 M s u c r o s e - E D T A , s a t u r a t e d w i t h CCla, after i o m i n p r e i n c u b a t i o n at 25 °. Mitochondria were washed once w i t h o. 3 M s u c r o s e - E D T A a n d resuspended in 0.25 M s u c r o s e - T r i s - E D T A . Expt.

I

2

3

Number of experiments

Mitochondria : (a) No CC14 (b) +CC14 Homogenates : (a) No CCla (b) +CC14 Mitochondria from preincubated homogenates : (a) No CC14 (b) +CCI~

I~atomsP/mg N

t,atoms O/mg N

P/O

3.93 i 0.46 2.26±0.73

2.1o ~- 0.20 2.61 ± 0 . 2 4 *

1.9o ± o.31 0.84±0.36*

2.79 ± 0.70 o.26±o.11"

1.33 ± 0.20 1.31 ~ o . I 7

2.18 I 0-75 o.2o ~- o. Io*

6.92 ± o.71 2.03 ± 0.92*

5.17 -L 0.67 5.33 ± 0-36

1.38 ± o.18 0.39 ± o. I9"

6

4

3

" Difference b e t w e e n (a) and (b) significant, P < o.oi.

The addition of dinitrophenol or ATP, known to decrease the rate of mitochondrial swelling, does not increase the delaying action of CC14. The addition of oxidative substrates, e.g. succinate, glutamate, pyruvate, pyruvate + malate, increased the rate of swelling of untreated mitochondria, in agreement with the data reported b y others s5-4°. In the presence of 0.12 nag CC14, the addition of these substrates produced an increase in the rate of swelling which was slightly less than that observed in the absence of CC14 (Fig. 2). Biochim. Biophys. Acta, 78 (1963) i - i i

()

M. A R T I Z Z U , F. M. B A C C I N O , M. U. D I A N Z A N I

Action of CCl 4 on oxidative phosphorvlation In other experiments, the action of CC14 on oxidative phosphorylation was studied. Mitochondria from I g liver were suspended in 3 ml 0.25 M sucrose, containing Tris and EDTA, previously saturated with CCI4. The total amount of CC14 present was about 3 mg. The mixture was preincubated at 25 ° for IO rain, and then used for the measurement of oxidative phosphorylation. In each case, a blank was done in which mitochondria were preincubated in the same mixture without CCI4. As Table I I shows, P/O ratios obtained with mitochondria in the absence of CC14 were lower that the theoretical ones. That this may be due to the preincubation at 25 ° is indicated by the observation that mitochondria, without preincubation, yielded normal P/O ratios ranging from 2.3 to 2. 9. Such an effect of preincubation on the P/O ratio has been described 41. In the case of mitochondria preincubated in the presence of CC14, very low P/O ratios were obtained. The difference between these and normal values is statistically highly significant. The decrease in P/O ratios is related both to the depressed esterification of inorganic orthophosphate and to the more rapid rate of oxidation, both being significantly different from normal. In the experiments of RECKNAGELAND ANTHONY11,12, mitochondria isolated from the liver of CC14-treated rats were found to behave normally during the first hours; the CC14 concentration in the liver was of the same order of magnitude as that which produced uncoupling of oxidative phosphorylation in the present experiments. It seemed possible that the failure by RECKNAGELAND ANTHONY11, 12 to find mitochondrial damage might be due to the presence in the cell of substances capable of inhibiting the action of CC14. For this reason it seemed interesting to study the action of CC14 on the whole homogenate. Homogenates were prepared in 0.25 M sucrose solution containing Tris and EDTA, previously saturated with CC14 by the diffusion equilibrium method. They were preincubated at 25 ° for I0 rain and then used for the measurement of oxidative phosphorylation. As Table II shows, preincubation with CC14-containing sucrose produced a high degree of uncoupling of oxidative phosphorylation, due to decreased esterification of inorganic orthophosphate. Oxygen uptake remained unchanged. It is interesting to note that homogenization results in some loss of CC14from the solution 33, in our experiments to 15-20 %. This means that the effect described above must be attributed to CC14 concentrations below saturation. It seemed interesting to test if the presence of CC14 in the incubation medium was necessary to produce uncoupling of oxidative phosphorylation. In fact, uncoupling by CC14 was thought possible to be reversible. To test this possibility, I0 % homogenates were prepared in 0.3 M sucrose, containing Tris and EDTA and saturated with CC14. After a I0 min pre-incubation at 25 °, the mitochondria were isolated from these homogenates and washed once with 0.3 M sucrose, not containing CC14. Oxidative phosphorytation was found to be uncoupled with these mitochondria also. However, low P/O ratios and high oxidation values were obtained also with mitochondria treated similarly, but in the absence of CC14. It is possible that this phenomenon is partly due to removal of foreign material in the washing. The difference between the two experiments was, however, of high statistical significance.

Action of CC14 on mitochondrial respiratory control It seemed interesting to assess also the e-ctent of respiratory control, which is Biochim. /~iophys..qcla, 7 8 ( I 9 6 3 ) I I

7

g

~D

-g

g 57.88 ± 7-47 59.55 :k 4-04

No CC14

d-CCI4

preincubated homogenates :

3

14.8o ~- 1.38

+ CC14

Mitochondria from

14.86 d: 2.29

4

No CC14

Homogenates :

3[.99 ± 3-81"

+CC14

20.63 qz 6.75

15.79 ~ 1.64

14.52 X- 2.26

13-57 :L 2.41

24.76 + 3.79*

13.8o ± 2.44

b

* Difference between e x p e r i m e n t s in presence and in absence of CC14 significant, P < o.oi.

3

2

23.77 ± 2.90

5

a

No CC1a

1

Mitochondria :

Number of experiments

Expt.

8.87 :L 1.14

7.21 q 1.31

4 .68 i o.51

5.03 ~_ 1.38

7-59 d- 2.41

7.07 ± 1.31

c

3.15 -- 1.12

3.69 ~ o.51

1.22 ~ o.13"

1.75 ± 0.22

a/b

6.74 ± 0.20

9.56 ± 1.9o

4.33 ~ 1.47

3.48 ± 0.87

a/c

The values are given as ttl 0 2 t a k e n u p / m g N. Mitochondria, h o m o g e n a t e s and m i t o c h o n d r i a isolated f r o m CCla h o m o g e n a t e s were p r e p a r e d as described in Table II, under E x p t s . 1-3: a, complete s y s t e m ; b, s y s t e m w i t h o u t a d d i t i o n of glucose + hexokinase; c, s y s t e m n o t containing glucose + h e x o k i n a s e + AMP.

ACTION OF CC14 ON MITOCHONDRIAL RESPIRATORY CONTROL i n v i t r o

TABLE III

"-.I

~0

> N >

o

o

M. ARTIZZU, F. M. BACCINO, M. U. DIANZANI

thought to be an index of mitochondrial adequacyn,12, ~2. As Table III shows, preincubation of mitochondria with CCla caused release of respiratory control. In fact, the ratio a/b is significantly lower than that in the control experiments. The ratio a/c was, however, not significantly different. The oxidation values obtained with the complete system (a) or with the system devoid of hexokinase + glucose (system b), were significantly higher with treated than with untreated mitochondria. When AMP -hexokinase + glucose were omitted, however, the oxidation values were very similar in both cases. It is possible that this high degree of AMP dependence of treated mitochondria is due to adenylnucleotide depletion. Swollen mitochondria release into the suspension medium a large amount of their internal AMP 22. Respiratory control was assessed also in mitochondria isolated from homogenates prepared with o.3 M sucrose saturated with CC14. The oxygen uptake in the complete system was higher with these mitochondria than with those in the preceding experiment. It is possible that this difference m a y result from removal of a certain amount of foreign nitrogen in the washing. There was no difference between the respiratory control of normal and of treated mitochondria. The oxidation values of homogenates were not modified by the presence of CC14 in the incubation medium. Also in this case, the oxygen uptake was strongly stimulated by AMP, but no difference was found between CCI~ homogenates and the controls. TABLE EFFECTS

/~

IV

Vi[~'O OF C O l 4 ON ATPAsE A C T I V I T Y

A T P a s e a c t i v i t y is g i v e n as /tg i n o r g a n i c p h o s p h a t e liberated from A T P per m g N in 20 min. T h e e x p e r i m e n t s w e r e carried o u t : a, w i t h o u t a d d i t i o n of a n y a c t i v a t o r ; b, in t h e p r e s e n c e of 3' IO-5 M d i n i t r o p h e n o l a n d 2 / ~ m o l e s E D T A ; c, in t h e p r e s e n c e of 0.002 M Mg z+. E x p t . i : no p r e i n c u b a t i o n ; a b o u t 2 m g CC14 w e r e a d d e d to e a c h tube. E x p t . 2: m i t o c h o n d r i a from i g l i v e r s u s p e n d e d in 0.3 M s u c r o s e - E D T A (to i o ml) and diluted i :4 w i t h 0.3 M s u c r o s e - E D T A s a t u r a t e d w i t h CC14; IO m i n p r e i n c u b a t i o n at 25 °. E x p t . 3: i g liver h o m o g e n i z e d in 0.3 M s u c r o s e - E D T A to i o ml, diluted 1:4 w i t h 0. 3 M s u c r o s e - E D T A s a t u r a t e d w i t h CC14 and p r e i n c u b a t e d for i o rain a t 25 °. E x p t . 4 : m i t o c h o n d r i a i s o l a t e d from a i o % h o m o g e n a t e in 0. 3 M s u c r o s e - E D T A s a t u r a t e d w i t h CC14; IO rain p r e i n c u b a t i o n at 25 °. M i t o c h o n d r i a were t h e n w a s h e d once a n d r e s u s p e n d e d in i o m l 0. 3 M s u c r o s e - E D T A . T h e y w e r e t h e n diluted 1:4 w i t h this solution. Number of experiments

Expt.

I

2

3

4

Mitochondria NO CCI 4 "+CC14 Mitochondria : No CCI 4 +CCI 4 Homogenates : No CCI4 +CC14 Mitochondria from preincubated homogenates : No CC14 + CC14 No CC14 + CC14 No CC14 + CC14

[ Dinitrophenol

= M g a+

N o activat*.r~

3 423 .8 ~ 51.7 449-0 ± 53.3

234.7 ± 43.1 257.2 ~ 22.9

185"5 ~ 44,7 246"0 ~: I°4"5

272. 5 ± 46,3 856.9 @~217.6"*

242. 4 ~ 56.3 637.8 ~ 371.7 *

126.6 :" 56.0 425.5 - 136.6"*

i66.6 ~ 17.o** 289.7 ~ 33-4

174.3 ± 47.2 351.4 ~ 89.5

71.8 ± 5 1 . 3 . . 223.8 ~: 21.3

6

4

231. 9 135. 4 181.o 142.6 339.6 191.2

21o.2 234. i 2oo. 9 242.6 279.7 376.2

12o. 4 136.7 89.8 93.9 158-6 176.5

* p ~ o . o 5. ** P ~ O.Ol.

Biochim. Biophvs. Acta, 78 (1963) 1-i i

MITOCHONDRIAL DAMAGE BY CCI 4 in vitro

9

Action of CC14 on A TPase activity The influence of CC14 treatment on ATPase activity was studied in three reaction systems : (I) in the presence of dinitrophenol and EDTA (2/,moles) ; (2) in the presence of Mg 2+; (3) in the absence of activators. Table IV shows that the addition of CC14 to the reaction media, without previous incubation, did not provoke significant changes in ATPase activity. When mitochondria were preincubated at 25 ° for io min, a significant increase of ATPase was seen in all three systems. A study of activity in homogenates revealed a significant increase in the absence of activators and in the presence of dinitroplienol. Also the Mg2+-activated ATPase was found to be increased, but less significantly. ATPase activity of mitochondria isolated from CC14-sucrose homogenates showed a slight increase in the presence of Mg 2+, as well as a strong decrease in the presence of dinitrophenol. DISCUSSION

The experiments described in this paper show that CC14 damages mitochondria in vitro in concentrations similar to or lower than those met with in the liver during the first hours after CCI 4 feeding. The effect of CC14 on mitochondria includes a decrease of the P/O ratio, slackening of respiratory control and an increase in ATPase activity. The effect on mitochondrial absorbancy is diphasic, depending upon the concentration of CCI 4 in the ieaction system. High amounts of CC14, similar to or lower than those capable of producing biochemical damage, produce rapid swelling of mitochondria. Smaller amounts (from 0.08 to 0.12 mg per cuvette), on the contrary, exert a delaying action on spontaneous swelling. Swelling is generally believed to be related to the flow of electrons through the respiratory channel 43, 44, or to an oxidized state of the chain 45,48. On the other hand, protection from swelling is related to a decreased electron flow, or to a reduced state of the chain. The protective action of small amounts of CC14 m a y be related to an initial damage to the enzymes. In each case, respiratory enzymes are not greatly affected, as addition of oxidative substrates, e.g. succinate, pyruvate, pyruvate + malate, or glutamate, stimulates mitochondrial swelling even in the presence of CC14. Several authors al-a3 have established that swelling induced b y CC14 is not reversible. The present paper shows that mitochondria isolated from homogenates pre-incubated with sucrose containing CC14 and washed, are severely damaged. These experiments show that the damage provoked by CC14 is not entirely reversed b y the removal of CC14 from the medium. It is, however, possible that some reversal of biochemical changes takes place. In fact, these changes were more marked when CC14 was present in the incubation medium than when it was removed b y washing. Homogenates do not contain substances capable of affording complete protection of mitoehondria against the action of CC14, as indicated by the action of CCI 4 on oxidative phosphorylation and ATPase in such systems, The present experiments clearly show that CC] 4 can produce direct mitochondrial damage. Thus it seems very probable that the damage observed by several authors in mitochondria isolated from CC14 f a t t y livers is due to a direct action of this substance. The close similarity of the CC14 concentration in the liver during the first few hours after CC14 feeding and the concentration resulting in mitochondrial damage in vitro, suggests that damage in vivo occurs b y a similar mechanism. Changes in liver-mitochondrial morphology are evident even 30-60 min after Biochim. Biophys. dcta, 78 (1963) I - I i

I0

M. ARTIZZU, F. M. BACCINO, M. U. DIANZAN1

CC14 feeding. The fact that biochemical changes are not found at this time might be due to a partial reversibility of the damage. The demonstration of a direct action of CC14 on mitochondria clearly does not exclude the possibility of an action on other cell structures and functions. It is interesting to note that marked changes in glucose-6-phosphatase, an enzyme occurring within the endoplasmic reticulum, were observed before the onset of mitochondrial changesla, 47, and that substantial damage to the endoplasmic reticulum was evident, by electron microscopy4, 5, at a very early stage in CC14 poisoning. The accumulation of fat in the liver may not be due to mitochondrial damage. In fact, several authors have shown that the secretion, by the liver, of triglycerides into the blood stream is greatly reduced after CC14 poisoning 48-5°. The hepatic synthesis of fat is increased 5°. ACKNOWLEDGEMENT

This paper was supported b y a grant from the Consiglio Nazionale delle Ricerche, Rome. It is included in the Festschrift dedicated to H. YON EULER.

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