Acta Medica Okayamaousar.lib.okayama-u.ac.jp/files/public/3/32526/...Acta Merl, Okayama 23,...

Acta Medica OkayamaVolume 23, Issue 6 1969 Article 9

DECEMBER 1969

Biochemical pathology of fatty liver inducedby inhaled carbon tetrachloride, with specificreference to ATP and lipid metabolism in the

mouse liver

Shinsaku Watanabe∗

∗Okayama University,

Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.

Biochemical pathology of fatty liver inducedby inhaled carbon tetrachloride, with specificreference to ATP and lipid metabolism in the

mouse liver∗

Shinsaku Watanabe

Abstract

Cb strain female mice were exposed to 800 p.p.m. of carbon tetrachloride for 3 hours by theuse of newly devised gas chamber via constant current of gas. Contents of ATP, triglyceride andtotal lipid in the liver were measured at appropriate intervals after inhalation of carbon tetrachlorideand compared to non-treated controls. And P : 0 ratio of the liver mitochondria was measured byoxymeter and morphological changes of liver mitochondria were observed by electron microscopy.The following results were obtained. 1. ATP conten t in the liver decreased slightly immediatelyafter inhalation, rapidly decreased until 4 hours after inhalation and gradually decreased until 20hours after inhalation. 2. Contents of total lipids increased slightly immediately after the exposureand increased gradually until 20 hours later. Contents of triglyceride in the liver increased atalmost constant rate during and after the exposure. 3. P : 0 ratio of liver mitochondria did notchange immediately after the exposure and gradually increased after the exposure, keeping parallelrelation to decrease in ATP content in the liver. Decrease in ATP content in the liver after inhalationof carbon tetrachloride seems to be mainly due to uncoupling of oxidative phosphorylation of livermitochondria. 4. Morphological changes of liver mitochondria were observed at 4 hours after theexposure by electron microscopy. 5. Decrease in ATP levels of the liver suggested to have a closerelation to accumulation of lipid in the liver after the inhalation of carbon tetrachloride.

∗PMID: 4246443 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOL

Acta Merl, Okayama 23, 559-567 (1969)

BIOCHEMICAL PATHOLOGY OF FATTY LIVER INDUCEDBY INHALED CARBON TETRACHLORIDE, WITH

SPECIFIC REFERENCE TO ATP AND LIPIDMETABOLISM IN THE MOUSE LIVER

Shinsaku WATANABE

De/Jartment of Public Health, Okayama University Medical School,

Okayama, Japan (Director: Pro]. M. Ogata)

Receivedfor publication, August 20, 1969

Carbon tetrachloride is widely used in some industries as organicsolvent. The threshold limit of value of carbon tetrachloride gas in the airis decided to be lOp. p. m. (1).

Carbon tetrachloride is being used as experimental hepatotoxic agentsby many investigators. It is well known that carbon tetrachloride is toxicto the liver cells and causes fatty necrosis to the liver cells (2, 3). Butmechanisms of its hepatotoxity remain unsolved.

DIANZANII reported that carbon tetrachloride induced the uncouplingof oxidative phosphorylation of liver mitochondria accompanied by thedecreasing in the ATP content of the liver of rats 24 hours after injectionof carbon tetrachloride (4,5). RECKNAGEL reported that the activity ofMg-activated ATP-ase was enhanced after injection of carbon tetrachloride(6). However, very little information is available as to the toxic effect ofinhaled carbon tetrachloride to the liver.

A newly deviced gas chamber via constant current of the air was usedfor the experiments (7). Mice were exposed to 800 p. p. m. of carbontetrachloride gas for 3 hours. The contents of total lipid, triglyceride andATP in the liver were determined at appropriate intervals after exposureto carbon tetrachloride.

In order to clarify the cause of decrease in ATP content in the liver,P; 0 ratio of the liver mitochondria was studied by oxymeter and moraphology of liver mitochondria by electron microscopy.

MATERIALS AND METHODS

Cb strain female mice <weighing 16±2g) were exposed to 800 p. p. m. ofcarbon tetrachloride for 3 hours.

The mice were sacrificed by decapitation immediately after the inhalation,4,8,20 hours afterwards. By removing the liver, it was washed with ice cold

559

1

Watanabe: Biochemical pathology of fatty liver induced by inhaled carbon

Produced by The Berkeley Electronic Press, 1969

560 S. WATANABE

ATP conceritra tion

10' M log

ATP standard curveFig.

Jog

10'

10'

count

10'

saline and weighed.ATP content in the liver was determined by luciferine-Iuciferase reaction.The principles of luciferine-Iuciferase reactions.

1) ATP+ luciferine luciferase+ MgH- ...... adenyl-Iuciferine+ pyrrophosphate2) Adenyl-Iuciferine + to ...... adenyl-oxyluciferine + light

The light generated from this reaction was measured by liquid scintillationsrectrophotometer (Pachard Model 3324) (9).

The preparation of luciferine-Iuciferase solution (10).Fifty mg of desicated tails of firefly lanterns (made in Sigma Co) was homo

genized with Potter Evelyne type glass homogenizer in the ice cold bath for 5 min,and frozen at -20°C as the stock solution.

Standard ATP solution was prepared by100-fold dilution of stock solution. Liquidscintillation counter was set at gain 50 %,window red, width of the window 50-1000and preset count O. 1 min.

Distilled water, diluted standard solutionand luciferine-luciferase solution (1.0 ml: 1.0ml; 0.1 ml vIv) were mixed well. Immediatelyafter the start of enzyme reaction, thelight generated was measured by liquid scintillation counter.

The standard curve thus obtained waslinear at the ATP concentration of 10-6 M to10-8 M (as shown in Fig. 1).

st ate 4

st ate 3

s! ate 3

state 5--------1

st ate I

state 2

a. nlitoc/lOlldrla addedstate 4 b. Substrate added

c. ADP added

Time ....... I m111

490 ml'Oatom /ml(245mll O,/ml)

Fig. 2 Determination of ADP : 0 ratio ofmitochondria with oxymeter.Respiratory control ratio

_ 0 Consumption of ~tate 3/min- () Consu~ption of State 2/min

ADP : 0 ratio (P: 0 ratio)_ ADP addded (!~ moles)- -0- Contumptionof ~tate3(;moles (jato~f

Preparation of ATP solution fromthe mouse liver.

Mouse was killed by decapitationand it's liver excised, washed withice cold saline, weighed, put in theboiling water, homogenized andquickly chilled in the ice cold waterand centrifuged. The supernatantobtained at each time served as thesample solution. ATP concentrationin the sample solution was calculatedfrom the standard curve. The interval from sacrifice to boiling the liverwas strictly fixed at 2 minutes.

The total lipid was measured byFOLCH method (11) and triglyceridewas measured by VAN HANDELSmethod (12).

Liver mitochondria were pre-

2

Acta Medica Okayama, Vol. 23 [1969], Iss. 6, Art. 9

http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss6/9

Hepatotoxity of Inhaled CCl4 561

pared by a modified method of HOGEBOOM and SCHNEIDER (13).P : 0 ratio (ADP : 0 ratio) was measured by oxymeter (14). Reaction in ox

ymeter was performed at 25°C. The respiratory control ratio was obtained bythe O 2 consumption of state 2 per minute devided by the O 2 consumption of state3 per minute (as shown in Fig. 2). ATP contents of the liver was also determinedat the same time. After staining the liver mitochondria with 2 % permanganate,their morphology after the inhalation of carbon tetrachloride' was observed byelectron microscopy.

RESULTS

Change in the contents of ATP, triglyceride and total lipids In theliver after inhalation of carbon tetrachloride are shown in Table 1.

Table I Change in ATP, total lipids and triglycerides in the liverafter inhalation of 800 p. p. m. of CCl4 for 3 hours

Hours No. ATP in the Liver Total Lipides Triglycerides

after of in the Liver in the Liver

inhalation cases mg/g w.w % mg/g body % mg/g w.w. %weight

Control 6 0.811 ±0.215* 100 2.13±0.16* 100 7.13±O.82* 100

0 5 0.725±0.198 89 2.39±0.18 112 14.63±3.32 205

4 5 0.294±0.106 36 3.23±O.29 152 31.55±2.86 442

8 6 0.163±O.082 20 3.62±0.62 195 47.72±5.20 669

20 6 O.143±0.065 18 7.03± 1.48 330 71.07±7.81 1005

* M ± 6

ATP content (mgjg liver wet weight) was 89% of the control, immediately after the exposure, 36 % at 4 hours, 20 % at 8 hours and 18 % at 20

ATPmg/g liver

0.8

0.7 .

0.6

Tr iglycerldemg/g liver

·······lM.70.' .' 6

.... _.. ~.. . 60

0.5 .

0.4

0.3 .

0.2

'1'0.1

,"h~lat Ion of eel,

.... ']'--ATP•....••• Triglyceride

·50

·40

• 30

20hrs

Fig. 3 Change in ATP and triglyceride content in the liver after inhalationof 800 p. p. m. of CC14 for 3 hours

3



562 S. WATANABE

hours after the exposure.Triglyceride content increased to 205 % of control immediately after

the exposure. Relationship between ATP content and triglyceride contentin the liver is illustrated in Fig. 3.

Total lipid content in the liver increased to 112 % of control immedi.ately after the exposure, 152 % at 4 hours, 193 % at 8 hours, and 330 %at 20 hours after the exposure. Relationship between ATP content andtotal lipid content in the liver is illustrated in Fig. 4.

0.5

ATPmgig Liver

0.8 ~0.7 •

0.6 .

\0.4 .

0.3

0.2

.' - - _.' -' r__ T

Total Lipidmg /g body weight

8.0

5.0

--ATP 4.0...... Total Lipid

. 3.0

2.0

0.1Inhalation of eel,

"-,-------~M

6 1.0

20hrs

Fig. 4 Change in ATP and total lipid content in the liver after inhalationof 800 p. p. m. of CCl4 for 3 hours

Triglyceride: total lipid, ratio III the liver was 16.4 96 in controlimmediately after the exposure and 36.3 % at 4 hours after inhalation.

These results suggest that the increase in total lipid contents of theliver was mainly due to the increase in triglyceride content of the liver.

In order to clarify the mechanism of decrease in ATP levels of the

Table 2 Change in ADP : 0 ratio of liver mitochondria afterinhalation of 800 p. p. m. of CCl4 for 3 hours.

Hours Succinate Glutamate ATP in the Liverafter

Inhalation respiratory control ADP :0 respiratory control ADP:O mg/g liver w. w.ratio ratio ratio ratio

Control 4.8 1.53 5.4 2.60 0.700*

0 4.5 1.53 5.6 2.77 0.722

4 2.8 1.40 3.6 1.94 0.492

8 1.3 1.18 1.3 1.17 0.193

* means of 3 casesMitochondria were obtained from the homogenate of the livers of the 3 mice in each.

4



Hepatotoxity of Inhaled CC14 563

2.0

p:o ratio3.0

liver, the P : aratio of the liver mito·chondria was determined by meansof oxymeter, using succinate andglutamate as substrate. ATP contentof the liver was determined by luciferine.luciferase method at the sametime. The results are shown in Fig. 5and Table 3.

Respiratory control ratio, P: aratio and ATP content in the liverdid not change immediately afterinhalation, but moderately decreasedat 4 hours and markedly decreased at8 hours after the exposure.

P : aratio and respiratory controlratio changed almost parallel to the

ATP/g lover w.w.

·1.O mg

n

1.0

8 hrs..after inhala,tion

mDI eel. inhalation for 3hrs

.I P: 0 ratIo : glutamate as substrate

. n P:O ratio :' succinate as substrate

Fig. 5 Change in ATP content andP : 0 ratio after inhalation of 800 p. p. m.of CCl4

ATP content in the liver.These results suggests that the decrease in ATP content of the liver

after inhalation of carbon tetrachloride was mainly due to the uncouplingof oxidative phosphorylation of liver mitochondria. Electron micrographof the liver mitochondria 4 hours after inhalation of carbon tetrachlorideshowed appearance of giant mitochondria and morphological alteration ofmitochondria which suggests the fusion of mitochondria (as shown in Fig. 6)

Decrease in number of mitochondria was observed in treated casescompared to non-treated cases. Increase in number of mitochondrialchristae and elongation of mitochondrial cristae were also observed intreated cases.

These morphological changes may give some explanation to the functional changes which were obtained in the experiment of oxymeter.

DISCUSSION

Hepatotoxity of carbon tetrachloride was studied by many investigaters, but most of experiments were performed by intraperitoneal injectionor intragastric administration of carbon tetrachloride. However, in thefield of industrial hygiene, carbon tetrachloride-gas intoxication is veryimportant.

K YLINE stated that carbon tetrachloride inhaled caused fatty liver (15).In the present experiment, a newly devised gas chamber via constant

current of gas was employed. The ATP and lipid metabolisms after inha-

5



564 s. WATANABE

lation of carbon tetrachloride were studied.DIANZANII reported that ATP content decreased 24 hours after injec.

tion of carbon tetrachloride, and he demonstrated that uncoupling ofoxidative phosphorylation of liver mitochondria (4) and suggested that thedecrease in ATP content of the liver induced inhibition of fatty aciddecomposition.

We have found that the decrease in ATP content of the liver has aclose relation to the accumulation of lipid in the liver after inhalation oftetrachloroethylene, trichloroethylene and carbon tetrachloride (16).

FARBER reported that the fall in the ATP levels of liver by ethionineinduced accumulation of triglyceride in the liver (17) and he also demonstrated that the decrease in hepatic ATP levels induced inhibition of proteinsynthesis of the liver (18), and explained that inhibition of the serumlipoprotein synthesis induced accumulation of triglyceride in the liver (19).

OGATA demonstrated that inhibition of the serum albumin and lipoprotein in the liver after injection of carbon tetrachloride by studying theincorporation of C14 glycine into protein part of serum lipoprotein andserum albumin (20, 21).

RECKNAGEL stated that change in microsomal enzyme activitypreceded the change in the mitochondrial enzyme activity (23). SAKAI

reported that activity of cytochrome b 5 or p 450 of microsome decreasedearlier than the decrease in P : 0 ratio of liver mitochondria (24).

SLATER suggested that free radical metabolite (CPCO) injured mitochondria and microsome in the liver by lipid peroxidation (25).

This was comfirmed by RECKNAGEL (26).Morphological changes of liver mitochondria and alteration of micro

somes in carbon tetrachloride liver injury were confirmed by BASSEI andHABA by electron microscopy (27, 28).

In the present experiment, it was suggested that the decrease in theATP content of the liver and accumulation of triglyceride occurred afterinhalation of carbon tetrachloride and decrease in the ATP content of theliver and accumulation of triglyceride occured after inhalation of carbontetrachloride and decrease in the ATP content of the liver accompaniedby decreased P : 0 ratio of the liver mitochondria was mainly due to theuncoupling of oxidative phosphorylation. Activation of Mg-activated ATPase reported by RECKNAGEL seems to be another cause of decrease in ATPcontent in the liver. (6).

It was suggested that the decrease in hepatic ATP levels and decreasedtemplate activity of liver microsomes caused inhibition of protein part ofthe very low density lipoprotein synthesis in the liver disturbed the secre-

6



Hepatotoxity of Inhaled CCl4 565

tion of triglyceride from the liver (29). Disturbance of secretion of triglyceride from the liver induces accumulation of triglyceride in the liver. Thedecrease in ATP contents of the liver and impaired function of livermitochondria seem to take another important role for accumulation oftriglyceride in the liver by inhibition of fatty acid oxidation in the livermitochondria.

CONCLUSION

Cb strain female mice were exposed to 800 p. p. m. of carbon tetrachloride for 3 hours by the use of newly devised gas chamber via constantcurrent of gas.

Contents of ATP, triglyceride and total lipid in the liver were measured at appropriate intervals after inhalation of carbon tetrachloride andcompared to non-treated controls. And P : 0 ratio of the liver mitochondriawas measured by oxymeter and morphological changes of liver mitochondria were observed by electron microscopy.

The following results were obtained.1. ATP conten t in the liver decreased slightly immediately after

inhalation, rapidly decreased until 4 hours after inhalation and graduallydecreased until 20 hours after inhalation.

2. Contents of total lipids increased slightly immediately after theexposure and increased gradually until 20 hours later. Contents of triglyceride in the liver increased at almost constant rate during and after theexposure.

3. P : 0 ratio of liver mitochondria did not change immediately afterthe exposure and gradually increased after the exposure, keeping parallelrelation to decrease in ATP content in the liver. Decrease in ATP contentin the liver after inhalation of carbon tetrachloride seems to be mainly dueto uncoupling of oxidative phosphorylation of liver mitochondria.

4. Morphological changes of liver mitochondria were observed at 4hours after the exposure by electron microscopy.

5. Decrease in ATP levels of the liver suggested to have a closerelation to accumulation of lipid in the liver after the inhalation of carbontetrachloride.

ACKNOWLEDGEMENT

The author is indebted much to Professor M. OGATA for invaluable advices and to Mr.K. TOMOKUNI and Miss S. UEKI for technical assistances.

7



566 S. WATANABE

REFERENCES

1. American Conference of Governmental Industrial Hygienists: Threshold limit values for1964: Arch Environ. Health 2, 545, 1964

2. GABRIEL, L. et al: CC14 induced liver damage. Archives of Environ. Health 9, 536, 19643. RECKNAGEL, R. Carbon tetrachloride Hepatotoxity. Pharmacol. Reviws. 19, 145, 19674. DIANZANII, M. U.: Uncoupling of oxidative phosphorylation in mitochondria from

fatty livers. Biochemical et Biophisica Acta 14, 514, 19545. DIANZANII, M. U.: The content of adenosine polyphosphates in fatty liver. Biochemical

j. 65, 116, 19676. RECKNAGRL, R.: Biochemical changes in carbon tetrachloride fatty liver. Separation of

fatty changes from mitochondrial degeneration. j. Bio. Chem. 234, 1052, 19677. OGATA, M., TOMOKUNI, K. and WATANABE, S.: Content of adenosine triphosphate

and total lipids in the liver of mice exposed to tetrachloroethylene. Medicine and biology74, 22, 1967

8. OGATA, M. and WATANABE, S.: Rapid determination of micro-quantity of ATP withluciferine-luciferase reaction and liquid scintillation counter. Medicine and biology 74, 36,1967

9. STREHLER, B. L.: Adenocine-5-triphosphate and creatine phosphate determination withluciferase, Method in enzymatic Analysis, Edited by Bergmeyer, H. Academie Press,New York and London, 1965

10. ADDANKI, S., JUAN, F. S. and PHILLIP, D. R.: Rapid determination of picomolequantities of ATP with a liquid scintillation counter. Anal. Biochem. 14, 261, 1966

11. FOLCH, et al: A simple method for the isolation and purification of total lipids fromanimal tissues. j. Biol. Chem. 226, 497, 1957

12. VAN, HANDEL, E.: Suggested modifications of micro-determination of triglycerides. Clin.

Chem. 7, 249, 196113. HOGEBOOM, G. H. and SCHNEIDER, W. C.: Intracellular distribution of enzymes. j.

Biol, Chem. 204, 233, 195314. HAGlHARA, B.: Tecknique for application of polarography to mitochondrial respiration.

Biochem. Biophys. Acta 46, 134, 196115. KYLINE, B. et al: Hepatotoxity of inhaled trichloroethylene, tetrachloroethylene and

chloroform, single exposure. Acta Pharmacol. 20, 16, 196316. OGATA, M. WATANABE, S. and TOMOKUNI, K.: ATP and lipid content in the liver

of mice after inhalation of chlorinated hydrocarbons. Ind. Health 6, 116, 196817. FARBER, E & CASTILLO, A. E.: Formation of S-adenocylethionine during the develop

ment of ethionine induced fatty liver. Fed. Proc. 22, 370, 196318. TREVINO, S. V., SCHULL, K. and FARBER, E.: The role of adenosine triphosphate

deficiency in ethionine-induced inhibition of protein synthesis. j. Bio. Chem. 238, 175,1963

19. FARBER, E.: Biochemical pathology of acute hepatic ATP deficiency. Nature 203, 34,1964

20. OGATA, M.: Study on the protein synthesis in Poisonings I: Acta med. Okayama 16, 1,1962

21. OGATA, M.: Study on the protein synthsis in Poisonings II. Acta med. Okayama 16, I,

196222. RECKNAGEL, R.: A new insight into pathogenesis of carbon tetrachloride accumulation.

Pro. Soc. Exp. Biol. Med. 104, 608, 195823. RECKNAGEL, B.: Studies of biochemical changes in subcellular particle of. rat liver and

their relationship of carbon tetrachloride fat accumulation. j. Bio. Chem. 336, 564, 1960

8



Hepatotoxity of Inhaled CC14 567

24. SAKAI, S.: Fatty liver and abnormal fatty acid metabolism. Records of Nippon RinshoTaisha Gakkai 3, 100, 1966

25. SLATER, T. F.: Necrogenic action of carbon tetrachloride. Nature 209, 36, 196626. RECKNAGEL, R.: Lipoperoxidation as a vector in carbon tetrachloride hepatotoxity. Lab.

Investi. 15, 132, 196627. HABA, K.: Morphology of oxidative phosphorylation in mitochondria. Morphologic

study of the ret liver and it's mitochondria in carbon-tetrachloride poisonings. Acta med.Okayama 14, 227, 1960

28. BASSEI, M.: Electron microscopy of rat liver after carbon tetrachloride poisonings. Exp.Cell Res. 20, 313, 1960

29. LOMBARDI, B. and UGAZIA, G. Serum lipoproteins in rats with carbon tetrachlorideinduced fatty liver. Journal of Lipid Research 6, 498, 1965

9



568

Fig. (j llectnl"l miC:'og;aph of the liver n~it()chondria of the D'OUSC. :)O~)() X

l. '1 he live ': itocho::dria of U"Jtre31ed co.1trol

:2, c), '1 he livt"l' n:itocho:~drja 0" the rrou~c 4 hours after i:1halation of ((:CI4)

M. mitochondria ,\. accurr,ulation of lipid in the liver Cf·lls P. Swelling and fusionof mitochondria

10



Acta Medica Okayamaousar.lib.okayama-u.ac.jp/files/public/3/32526/...Acta Merl, Okayama 23,...

Documents

Transcript of Acta Medica Okayamaousar.lib.okayama-u.ac.jp/files/public/3/32526/...Acta Merl, Okayama 23,...