Female Cb mice weighing 20-23 g were exposed to 800 ppm (in average) of 1, I, 2, 2.tetrachloroethane for 3 hours. Both triglyceride and phospholipid in the liver and plasma were determined at varying times after the exposure. On the other hand, there were observed the ultraviolet
absorption spectra of microwmallipids in the liver at 90 minutes after the 1, 1,2, 2-tetrachloroethane or the carbon tetrachloride exposure. The results thus obtained are summarized as follows: 1. The increase of hepatic triglyceride contents attained the maximum level in the period between 20 and 25 hours after the exposure and declined to the initial levels at 90 hours later. 2. The plasma triglyceride levels decreased until 25 hours after the exposure, then tended to increase significantly and were much higher than the control levels in the period between 70 and 90 hours later. 3. Both liver and plasma phospholipid levels decreased gradually up to 25 hours after the exposure, then slowly recovered with almost the same rate of increase. 4. It was suggested that the inhalation of the above vapors induced a little change in microsomal lipids in the liver.</p

Tomokuni, Katsumaro

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Studies on hepatotoxicity induced by chlorinated hydrocarbons. II. Lipid metabolism and absorption spectrum of microsomal lipid in the mice exposed to 1, 1, 2, 2-tetrachloroethane

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Acta Medica OkayamaVolume 24, Issue 3 1970 Article 5JUNE 1970Studies on hepatotoxicity induced bychlorinated hydrocarbons. II. Lipidmetabolism and absorption spectrum ofmicrosomal lipid in the mice exposed to 1, 1,2, 2-tetrachloroethaneKatsumaro Tomokuni∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on hepatotoxicity induced bychlorinated hydrocarbons. II. Lipidmetabolism and absorption spectrum ofmicrosomal lipid in the mice exposed to 1, 1,2, 2-tetrachloroethane∗Katsumaro TomokuniAbstractFemale Cb mice weighing 20-23 g were exposed to 800 ppm (in average) of 1, I, 2, 2.tetra-chloroethane for 3 hours. Both triglyceride and phospholipid in the liver and plasma were de-termined at varying times after the exposure. On the other hand, there were observed the ul-traviolet absorption spectra of microwmallipids in the liver at 90 minutes after the 1, 1,2, 2-tetrachloroethane or the carbon tetrachloride exposure. The results thus obtained are summarizedas follows: 1. The increase of hepatic triglyceride contents attained the maximum level in theperiod between 20 and 25 hours after the exposure and declined to the initial levels at 90 hourslater. 2. The plasma triglyceride levels decreased until 25 hours after the exposure, then tendedto increase significantly and were much higher than the control levels in the period between 70and 90 hours later. 3. Both liver and plasma phospholipid levels decreased gradually up to 25hours after the exposure, then slowly recovered with almost the same rate of increase. 4. It wassuggested that the inhalation of the above vapors induced a little change in microsomal lipids inthe liver.∗PMID: 4249888 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 24, 315-322 (1970)STUDffiS ON HEPATOTOXICITY INDUCED BYCHLORINATED HYDROCARBONSII. LIPID METABOLISM AND ABSORPTION SPECTRUMOF MICROSOMAL LIPID IN THE MICE EXPOSEDTO 1, 1, 2, 2·TETRACHLOROETHANEKatsumaro TOMOKUNIDepartment of Public Health, Okayama University Medical School, Okayama,Japan (Directol: Prof M. Ogata)Received for publication, Feb. 27, 1970The chlorinated hydrocarbons are widely used as a detergent agentin industry, but it is said that a long term inhalation of these vapon willinduce liver disturbances. Especially, in the animal experiments, it hasbeen shown by some investigaton that chlorinated hydrocarbon vaporsinduce fatty liver 0-4). Up to date, however, there has not been anystudy on the restitution process of fatty liver, that is, the problem howthe fatty liver once induced by the chlorinated hydrocarbon vapors dimini.shes with lapse of time after the exposure. Therefore, the studies on therestitution process of fatty liver would be valuable in the field of industrialhygiene. On the other hand, RECKNAGEL et at. (5) have advanced thelipoperoxidation hypothesis to explain the causative factor of carbontetrachloride-fatty liver.Previously (3), the author demonstrated that the inhalation of I, I, 2,2-tetrachloroethane induced fatty liver. The present study was conductedin order to investigate the restitution process of fatty liver, especiallycentering around lipid metabolism in the liver and plasma of mice exposedto 1, I, 2, 2-tetrachloroethane. Spectrophotometric observations were alsocarried out on the microsomal lipid in the liver of mice at the onset offatty liver after the 1, 1, 2, 2·tetrachloroethane inhalation, and the resultwas compared to that of the carbon tetrachloride inhalation.MATERIALS AND METHODSAnimals: Female Cb mice weighing 20-23 g were used. They were fed onsolid feed of Oriental Yeast Company and water was given ad libitum until theywere sacrificed.Exposure: The mice were placed in the exposure chamber (3) supplied withthe constant current of air via a vaporizing unit, and were exposed to 800 ppm3151Tomokuni: Studies on hepatotoxicity induced by chlorinated hydrocarbons.Produced by The Berkeley Electronic Press, 1970316 K. TOMOKUNI(in average) of 1, 1,2, 2-tetrachloroethane for 3 hours. The gas concentration wasmeasured by gas chromatography once every 30 minutes during the exposure.During exposure the mice received neither food nor water.Preparation of mouse liver and plarma: After the mice were lightly anesthetizedwith ethyl ether at varying times after the exposure, the blood was withdrawninto heparinized capillary tube by orbital bleeding technic and transferred intoglass centrifuge tube. Then the mice were sacrificed by decapitation and theliver was excised and weighed for lipid analyses.Determination of liver and plasma triglycerides: Liver triglyceride was measuredby the modification of BUTLER, et al. (6) and plasma triglyceride by the method ofVAN HANDEL (7).Determination of liver and plasma phospholipids: Lipids were extracted from liverand plasma by the procedure of FOLCH et al. (8). An aliquot of chloroform-methanol extract containing phospholipids was placed into the 10 ml-glass stop-pered tube and the solvent was evaporated in the water bath. The phospholipidswere digested in the glass tube with perchloric acid by means of broiling over asmall open fire for about 3 minutes, and phosphorus was determined by themethod of FISKE and SUBBAROW (9). The weight of phospholipids was calculatedby multiplying the weight of phosphorus by 25.Isolation of microsome in the liver: Fundamentally, this experiment was per-formed by a modification of the RECKNAGEL et al. procedure (10). Twelve micewere divided into two groups. One group was employed for the exposure of 1,1,2, 2-tetrachloroethane and the other group for that of carbon tetrachloride. Themice were exposed to 800 ppm (in average) of the agents for 3 hours. At 90minutes after exposure, the mice were killed by decapitation, the liver wasimmediately excised and chilled in the ice-cold sucrose-EDTA medium. About2.8 g of liver gathered from 3 mice was homogenized in 18 ml of ice-cold 0.3 Msucrose, 0.003 M EDTA medium. The homogenate was centrifuged for 15 minutesat 8,000 g in the RP 40 rotor of the Hitachi 55PA ultracentrifuge. The superna-tant fraction was then centrifuged for 60 minutes at 94,000 g in the same rotor.Microsomal lipid extraction and spectrophotometric analysis: The microsome fractionthus obtained was transferred with 5 ml of methanol into a 50 ml-glass stopperedcylinder, 10 ml of chloroform and 15 ml of chloroform-methanol (2: 1) were added.The mixture was shaken gently for 5 minutes and allowed to stand overnight ina cold room. The mixture was filtered and 10 ml of water was added to the filt-rate. The two-phase system thus obtained was shaken gently and centrifuged.The upper phase was removed. The lower chloroform phase was evaporatedunder a stream of nitrogen at 35-40°C and dried. The total lipid thus extractedwas dissolved in cyclohexane to a concentration of 0.5 mg per m!. Optical den-sities were determined against a cyclohexane blank over the range from 220 to230 mit, in Hitachi 139 ultraviolet spectrophotometer in cells with l-cm light path.RESULTSChanges in hepatic lipids: The amounts of triglyceride and pho3pholipid2Acta Medica Okayama, Vol. 24 [1970], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss3/5Hepatotoxicity Induced by Chlorinated Hydrocarbons 317in the liver of mice at appropriate intervals after 1, 1,2, 2-tetrachloroethaneinhalation are shown in Table 1. Triglyceride contents remarkably in-creased with lapse of time after the exposure, the concentration reachingthe maximum level in the period between 20 and 25 hours later (p<O.OonThen, the triglyceride contents gradually decreased and almost recoveredto the initial level at 90 hour.; later. On the contrary, phospholipid con-tents gradually decreased with lapse of time after the exposure and reachedthe minimum level at 25 hours later (P<O. 001). Then, they increasedslowly and almost recovered to the control level at 90 hours later.TABLE I. TRIGLYCERIDE AND PHOSPHOLIPID CONTENTS IN THE LIVER OF MICE AT VARIOUSTIMES AFTER ExPOSURE TO 800 PPM OF 1,1,2, 2-TETRACHOROETHANE FOR 3 HOURS.EACH VALUE REPRESENTS THE MEAN ± STANDARD DEVIATION.Time after Number of mice Liver lipids mg/g (w. w.)exposure (hour) Triglycerides PhospholipidsControl 8 8.4 ± 1.0 23.2 ± 2.3.5 5 21.0 ± 3.4 22.5 ± 1.820 5 50.4 ± 8.7 18.3 ± 2.725 5 50.0 ± 9.5 16.7 ± 2.230 5 40.6 ± 6.9 18.3 ± 1.845 5 26.4 ± 5.6 18.0 ± 1.970 5 15.0 ± 3.2 21.6 ± 2.990 5 9.1 ± 1.5 22.1 ± 1.9Changes in plasma lipids: The amounts of triglyceride and phospholipidin the plasma of mice at varying times after 1, 1, 2, 2.tetrachloroethaneexposure are shown in Table 2. Both triglyceride and phospholipid con-centrations in the plasma decreased after the exposure, and continued todecrease until 25 hours later (p<o.oon With lapse of time after that,the plasma phospholipid levels gradually rose and recovered to the controlTABLE 2. TRIGLYCERIDE AND PHOSPHOLIPID CONTENTS IN THE PLASMA OF MICE AT VARIOUSTIMES AFTER EXPOSURE TO 800 PPM OF 1,1,2, 2-TETRACHLOROETHANE FOR 3 HOURS.EACH VALUE REPRESENTS THE MEAN ~I~ STANDARD DEVIATION.Time aftere:q:osure (hour)Control5202530457090Number of mice85555555PlasmaT rig1ycerides0.98 ± 0.130.62 ± 0.120.43 ± 0.100.39 ± 0.060.52 :i= 0.110.67 ± 0.141.63 ± 0.241.37 ± 0.31lipids mg/mlPhospholipids0.84 ± 0.090.64 ± 0.060.58 ± 0.090.54 ± 0.080.69 ± 0.100.71 ± 0.090.87 ± 0.080.82 ± 0.103Tomokuni: Studies on hepatotoxicity induced by chlorinated hydrocarbons.Produced by The Berkeley Electronic Press, 1970318 K. TOMOKUNIlevel at 90 hours after the exposure, while the plasma triglyceride concent-rations subsequently recovered and were much higher than the controllevel in the period between 70 and 90 hours after the exposure (P<O. 00l).Changes in hepatic and plasma triglycerides: As can be seen in Fig. 1,liver triglyceride levels rapidly rose during 20 hours after the exposure.The average rate of increase up to 20 hours later was approximately 1.8mg/g liver w. w. per hour. The concentration of hepatic triglyceridesattained the maximum level in the period between 20 and 25 hours afterthe exposure, and then they decreased slowly and almost recovered to thecontrol level at 90 hours later. The half life of hepatic triglycerides con-comitant with restitution was about 25 hours. On the cotrary, the plasmatriglyceride levels decreased significantly after the exposure, reaching theminimum level at 25 hours later and then showing a tendency of recovery.However, the concentration of plasma triglyceride was more than that ofthe controls following a decrease in hepatic triglyceride levels, especiallyin the period between 70 and 90 houn after the exposure.60~ Liver T. G, (!?i 50 . 2.0 Eb/)----b/)----8b/) 40 1.68 V)Q)V)-0Q) .;:-0 30 1.2 Q)';: uQ) ;>.u bobo 20 0.8 °5-5 til.. ~Q).::. 10 0.4 .!..J .....10 20 30 .40 50 60 70 80 90FI Exposure Time after exposure (hour)Fig. 1 Changes in triglyceride levels in the liver and plasma of mice atappropriate intervals after exr;osure to 800 ppm of 1,1,2, 2-tetrachloroethane for3 hours. Each point represents the mean ::1= SEM.Changes in hepatic and plasma phospholipids: As shown in Fig. 2, bothliver and plasma phospholipid levels were significantly reduced until 25hours after the exposure, then recovered gradually with lapse of time.There was almost a parallel relationship between the change in the hepa-tic phospholipid levels and that in the plasma phospholipid levels,4Acta Medica Okayama, Vol. 24 [1970], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss3/5Hepatotoxicity Induced by Chlorinatedr Hydrocarbons 319a.........bOS0.60.41.00.20.8908070605040Liver P-lipids C~)Plasma P-lipidsC$)302010403020, 0iii Exposure Time after exposure (hour)Fig. 2 Changes in phospholipid levels in the liver and plasma of mice atappropriate intervals after exposure to 800 ppm of 1,1,2, 2-tetrachloroethane for3 hours. Each point represents the mean ± SEM.The absorption spectra of microsomal lipids: Figs. 3 and 4 show theultraviolet abwrptic.m spectra of microsomal lipids in the mouse liver at 90minutes after the 1, 1, 2, 2-tetrachloroethane and the carbon. tetrachlorideexposures, respectively. As shown in Figs. 3 and 4, the maximum absorp-tion on difference spectrum was detected at 225 m,1J. in the case of 1, 1,2,.~ DifferenceE 0.3 spectrum1.4 I)-0C;0.21.2 .:: ~a.~ 01.0 0.1E CHC12·CHClzI)-0 exposed (0)c; 0.8 0 0 0 0 0.:: N ...., ~ OJ'>'"a N N N N N0 0.6 Wave length mJl0.4Control (e)0.2220 230 240 250 260 270 280 290 300Wave length m,llFig. 3 Absorption spectrum of liver microsomal lipids in mice 90 minutesafter exposure to 800 ppm of 1,1,2, 2-tetrachloroethane for 3 hours. Each pointshows the mean obtained from two exreriments.5Tomokuni: Studies on hepatotoxicity induced by chlorinated hydrocarbons.Produced by The Berkeley Electronic Press, 1970320T. £1.2.~ 1.0::~"0 0.8-;.!::::0.. 0.600.£0.2K. TOMOKUNI.f' 0.3:: Difference~ 0 2 spectrum-; ..!::::0..o 0.1, !000'" U) .....'" '" '"Wave length m.~!..220 230 2£0 250 260 270 280 29.0 300Wave length m.uFig. 4 Absorption sFectrum of liver microsomal lipids in mice 90 minutesalter exposure to 800 ppm of carbon tetrachloride for 3 hours. Each Foint showsthe mean obtai~1ed from two experiments.2-tetrachloroethane exposure, while it was detected at 230 m/" in the caseof carbon tetrachloride exposure.DISCUSSIONIn 1965, LOMBARDI et ai. (11) observed the changes in hepatic andserum triglycerides during onset time of intoxication after carbon tetra-chloride administration to the rats, and they found that the formerremained constant during the first hour of intoxication, then rose rapidly,while the latter decreased very sharply during the fir.:.t hour of intoxica-tion, then adjusted to a slower rate of decline. Moreover, they (11)demonstrated a marked inhibition of serum lipoprotein synthesis, especiallythat of very low density (VLD-) lipoprotein. In the same year, STERN etai. (12) have stated that there are marked changes in triglyceride levels,determining the lipid components in rat liver and plasma with lapse oftime after carbon tetrachloride administration.In this study on 1, 1,2, 2-tetrachloroethane inhalation by mice, it wasdemonstrated that: i) judging from the increase in hepatic triglyceridelevels, the development of fatty liver reached the maximum peak in theperiod between 20 and 25 hours after the exposure, and declined at 90hours lq,ter (Table 1, Fig. 1), ii) the plasma triglyceride levels signifi-6Acta Medica Okayama, Vol. 24 [1970], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss3/5Hepatotoxicity Induced by Chlorinated Hydrocarbons 321candy decreased until 25 hours after the exposure, then increasedgradually with recovery of hepatic triglyceride levels and were muchhigher than the control levels in the period between 70 and 90 hours later(Table 2, Fig. 1), and iii) both liver and plasma phospholipid levels de·creased gradually until 25 hours after the exposure, then recovered slowly(Fig. 2).In 1968, RAO et ai. (0) found a marked diene conjugation absorptionin liver microsomal lipids 5 minutes after carbon tetrachloride administra-tion to the rats, and proposed the lipoperoxidation hypothesis for carbontetrachloride-fatty liver.In the present study, the author also observed the absorption spectra ofmicrosomal lipids 90 minutes after 1, 1, 2, 2-tetrachloroethane and carbontetrachloride inhalations, and found that the former had the maximumabsorption at 225 m,fl., while the latter at 230 m/I. on the difference spectrum(Figs. 3 and 4). These findings suggest that the inhalation of 1, 1, 2, 2·tetrachloroethane or carbon tetrachloride by the mice will lead to a littledecomposition of microsomal lipids in the liver.On the other hand, in vivo, WAGLE et ai. (3) reported the inhibitionof protein biosynthesis in vitamin Bl2-deficient rat liver in 1958. In 1966,VIVIANI et ai. (14) stated that a fatty liver produced in the lysine. andthreonine-deficient rats. These reports seem to suggest that the vitaminBl2.deficiency in vivo could lead to a fatty liver. However, the author hasnot yet obtained any evidence to support this hypothesis, in the case ofthe 1, 1,2, 2-tetrachloroethane-fatty liver.CONCLUSIONFemale Cb mice weighing 20-23 g were exposed to 800 ppm (inaverage) of 1, I, 2, 2.tetrachloroethane for 3 hours. Both triglyceride andphospholipid in the liver and plasma were determined at varying timesafter the exposure. On the other hand, there were observed the ultravioletabsorption spectra of microwmallipids in the liver at 90 minutes after the1, 1,2, 2-tetrachloroethane or the carbon tetrachloride exposure. Theresults thus obtained are summarized as follows: 1. The increase ofhepatic triglyceride contents attained the maximum level in the periodbetween 20 and 25 hours after the exposure and declined to the initiallevels at 90 hours later. 2. The plasma triglyceride levels decreased until25 hours after the exposure, then tended to increase significantly and weremuch higher than the control levels in the period between 70 and 90 hourslater. 3. Both liver and plasma phospholipid levels decreased gradually up7Tomokuni: Studies on hepatotoxicity induced by chlorinated hydrocarbons.Produced by The Berkeley Electronic Press, 1970322 K. TOMOKUNIto 25 hours after the exposure, then slowly recovered with almost the samerate of increase. 4. It was suggested that the inhalation of the abovevapors induced a little change in microsomal lipids in the liver.ACKNOWLEDGEMENTThe author wishes to acknowledge the support and encouragement given by FrofessorMASANA OGATA.REFERENCES1. KYLIN, B., REICHARD, H., SUMEGI, I. and VLLNER, S.: Hepatotoxicity of inhaledtrichloroethylene, tetrachloroethylene and chloroform. Single exposure. Acta pharmacal.et toxicol. 20, 16, 19632. OGATA, M., TOMOKUNI, K. and WATANABE, S.: ATP and lipid contents in the liverof mice after inhalation of chlorinated hydrocarbons. Industrial Health 6, 116, 19683. TOMOKUNI, K.: Studies on hepatotoxicity induced by chlorinated hydrocarbons. Lipidand ATP metabolisms in the liver of mice exposed to 1, 1, 2, 2-tetrachloroethane. ActaAled. Okayama 23, 273, 19694. WATANABE, S.: Biochemical pathology of fatty liver induced by inhaled carbon tetrachlo-ride, with specific reference to ATP and lipid metabolism in the mouse liver. ActaAled. Okayama 23, 559, 19695. RECKNAGEL, R. O. and GHOSHAL, A. K.: Lipoperoxidation as a vector in carbontetrachloride he?atotoxicity. Lab. Invest. 15, 132, 19666. BUTLER, W. M., MALING, H. M., HORNING, M. G. and BRODIE, B. B.: The directdetermination of liver triglycerides. j. Lipid Res. 2, 95, 19617. VAN HANDEL, E.: Suggested modifications of the micro-determination of triglycerides.Clin. Chem. 7, 249, 19618. FOLCH, J., LEES, M. and SLOANESTANLEY, G. H.: A simple method for the isolationand purification of total lipides from animal tissues. j. Biol. Chem. 226, 497, 19579. FISKE, C. H. and SUBBAROW, Y.: The colorimetric determination of phosphorus. j.Biol. Chem. 66, 37.5, 192510. RAO, K. S. and RECKNAGEL, R. 0.: Early onset of lipoperoxidation in rat liver aftercarbon tetrachloride administration. Exp. Alol. Pathol. 9, 271, 196811. LOMBARDI, B. and UGAZIO, G.: ~erum li;:oiJroteins in rats with carbon tetrachloride-induced fatty liver. j. Lipid Res. 6, 498, 196512. STERN, P. H., FURUKAWA, T. and BRODY, T. M.: Eat liver and plasma lipids aftercarbon tetrachloride administration. j. Lipid Res. 6, 278, 196513. WAGLE, S. R., MEHTA, R. and JOHNSON, B. C.: Vitamin B12 and protein biosynthesisIV. In vivo and in vitro studies. j. Biol. Chem. 230, 137, 195814. VIVIANI, R., SECHI, A. M. and LENAZ, G.: Lipid metabolism in fatty liver of lysine-and threonine-deficient rats. j. Lipid Res. 7, 473, 19668Acta Medica Okayama, Vol. 24 [1970], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss3/5

0.: Early onset of lipoperoxidation in rat liver after carbon tetrachloride administration.

A simple method for the isolation and purification of total lipides from animal tissues.

ATP and lipid contents in the liver of mice after inhalation of chlorinated hydrocarbons.

Biochemical pathology of fatty liver induced by inhaled carbon tetrachloride, with specific reference to ATP and lipid metabolism in the mouse liver. Acta Aled.

E.: Suggested modifications of the micro-determination of triglycerides.

Eat liver and plasma lipids after carbon tetrachloride administration.

erum li;:oiJroteins in rats with carbon tetrachlorideinduced fatty liver.

Hepatotoxicity of inhaled trichloroethylene, tetrachloroethylene and chloroform. Single exposure. Acta pharmacal. et toxicol.

Lipid metabolism in fatty liver of lysineand threonine-deficient rats.

Lipoperoxidation as a vector in carbon tetrachloride he?atotoxicity.

Studies on hepatotoxicity induced by chlorinated hydrocarbons. Lipid and ATP metabolisms in the liver of mice exposed to 1, 1, 2, 2-tetrachloroethane. Acta Aled.

The colorimetric determination of phosphorus.

The direct determination of liver triglycerides.

Vitamin B12 and protein biosynthesis IV. In vivo and in vitro studies.

Studies on hepatotoxicity induced by chlorinated hydrocarbons. II. Lipid metabolism and absorption spectrum of microsomal lipid in the mice exposed to 1, 1, 2, 2-tetrachloroethane

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