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Positive evidence of acceleration of lipoperoxidation in rat liver by carbon tetrachloride: in vitro experiments
Lüe Sciences Vol. 4, pp. 1521-1590, 1985. Great Britain .
Pergamon Press Ltd. Printed in
P08ITIVE EVIDErAC$ OF ACCSIißATION OF LIPOPBADaIDATION BY CARBON
TETßACHLOßIDB :
IN
VITßO
IN
ßAT LIVSß
SRPSRIl1EN7ô,*
Amiya â . Ghoshal and ßichard O . Recknagel** Department of Physiology, Nestern ßeserve IInivereity School of Medicine Cleveland, Ohio
(Received 14 May 1985)
Seventeen years ago Hove (1) observed that administration of
(X-tocopherol
prior to carbon tetrachloride protected rats against the toxic effects o! the latter .
Anti-oxidant protection against the hepatotoaic effects of carbon
tetrachloride was rediscovered by Gallagher (2, 8) .
Vitasin B, Biphenyl-para-
phenylenediamine (DPPD) and aeleniua all gave acme measure of protection . protective effect of DPPD was conü rmed by DiLuzio and Coatalee (4) .
The
It seems
highly likely that the protection afforded is due to the anti-oxidant property co®on to these agents .
If the latter assumption is correct, then by inference,
carbon tetrachloride can be assumed to exert its toxic eiiecta by initiation of destructive lipoperoxidation .
Priest et el . (S) sought for evidence of increased
lipoperoxidation 1n rat liver three hours after carbon tetrachloride poisoning . These workers measured hepatic levels of malonic dialdehyde with the thiobarbituric acid reaction and sound no increase is malonic dialdehyde content in carbon tetrachloride catty livers in camparisoa with non-toxic controls . * Supported by a research grant, No . AY-01489-08, from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health . U .S . Public Health Service . **Supported by Research Career Development Award 5-K3-0d-900-07 of the Department of Health, Education, and lleliare, United States Public Health Service .
1521
152 2
ACCELERATION OF LIPOPERORIDATION
Dahle et al .
(6) may be consulted ßor evidence that maloaic dialdehyde arisen
ßrom perosidative breakdava oß polyenoic Batty acids .
Vol. 4, No . 15
IIsing the thiobarbituric
acid method, we also could find no evidence oß increased maloaic dialdehyde production in rat liver ßrom 15 minutes to 25 hours aßter carbon tetrachloride poisoning (7) .
However, we have ahova that maloaic dialdehyde is readily
metabolized by rat liver mitochondria .
hashed mitochondria are inactive, but
activity oß the mitochondrial ßraction returns to the level oß whole homogenate when ATP, Yg~ ions, and inorganic phosphate are added (7) . The ßact that maloaic dialdehyde ie metabolized lessens greatly the signißicance oß the failure to ßiad this product oß lipoperoxidation in the toxic liver, i .e ., the ßailure to ßiad maloaic dialdehyde cannot be taken as a demon titration that lipoperoxidation has not occurred .
In this communication we
present positive evidence ßrom in vitro studies that carbon tetrachloride can accelerate lipoperoxidation . Experimental Procedures The procedure ßor measurement oß maloaic dialdehyde wsa based on the method oß Sohn and Liversedge (8) .
The TBA reagent wen 0 .12 M with respect
to TBA, and 0 .026 M with respect to Tris . to 7 .0 with riCl .
Final pH oß the reagent was adjusted
The reaction was carried out on protein-Brae aliquote oß
tissue extracts containing 5~ TCA .
The ßinal reaction mixture contained 2 .5 ml
oß the 5`X, TCA tissue extract, 0 .5 ml of 0 .6 N HCl, and 2 .0 ml oß the TBA reagent . The red color was developed by heating for 10 min at 100 ° , and was read at b35 m~ in a Beckman B spectrophotometer . was used as a standard (9) .
1,1,3,3-tetraethoxypropaae (TßP)
The color reaction was linear over the range ßrom
2 .2 to 22 4+g oß TSP per 5 ml, which 1a equivalent to 0 .72 to 7,2 hg oß maloaic dialdehyde per 5 ml . Yale rata (Holtzman Co ., Madison, wisc .) 350 to 400 grams body weight, were used . Rats were sacrißiced by decapitation .
A portion oß the liver was homogenized
in 0 .112 Y NaCl containing 0 .05 Y sodium phosphate at pH 5 .6 .
The homogenate
was centrißuged in a 3erva1l Reßrigerated Centrifuge at 0 ° ßor 12 min at 9000 x
Vol. 4, No . 15
ACCELERATION OF LIPOPEROJCIDATION RAT LIVER
1523
" RAT LIVER
40 G W V
S
6
a
!0
A
B
!0
0
lo
100
!00
b00
400
!00
!i
p mq AAICROSONES PLUS SUPERNATANT
60
7!
100
I!!
p mq YICROSOMES PLUS SUPERNATANT
Figure 1
Haloaic Disldehyde Production by Aat Liver Microsomes Plus Supernatant . A . The ordinate give s . malonic dialdehyde produced, in Fig per gram equivalent of microsomea plus supernatant, in 25 min at 38° . B . The ordinate gives malonic dialdehyde produced in 25 min at 38° by the amounts oP tissue shown on the abscissa, not reduced to a per gram basis .
gravity .
The sediment (nuclei plus mitochondria) was discarded .
The desired
volume of the microsome plus supernatant fraction was added to a 50 ml conical flask and adjusted to 4 ml final volume with the saline-phosphate buffer . tissue extract was incubated at 38 ° in a water bath with constant shaking . gas phase was air .
The The
The reaction was stopped by transferring the flasks to an
ice bath followed by addition of 1 ml of 25~ ice-cold TCA . the isolated microsome fraction was used .
For some experiments
To obtain the miarosome fraction, the
homogenate, free of nuclei and mitochondria, was centrifuged in the Spinco Preparative Centrifuge è~odel L ßor 60 minutes at 37,500 rpm .
Further details per-
tinent to the various experiments are provided with the figures and tables . Results Pro-oxidant effect of carbon tetrachloride on lipoperoxidation in rat liver microsome-supernatant fraction .
Production of malonic dialdehyde by the microsome-
152 4
ACCELERATION OF LIPOPEROXiDATION
Vol. 4, No. 15
supernatant traction oß rat liver falls sharply at high concentrations of the Barber (10) observed a similar effect for addition
tissue eatract (Fig. lA) .
of rat brain supernatant fraction to brain microsa®es . Figure 1B shave that production of malonic dialdehyde is proportional to the concentration of the microso®e-..supernatant fraction of rat liver up to 125 eq mg of tissue eatract per 4 ml ., One eq mg of the microsome-supernatant ßraction is that amount oß the microsame and supernatant fractions derived from 1 mg wet weight oß liver . 1Rien carbon tetrachloride was present during incubation cf the rat liver nicrosome-supernatant fraction at 38°, malonic dialdehyde production was enhanced . This occurred at low concentrations oß tissue eatract as well as at higher concentrations (Table I) .
No malonic dialdehyde ie produced at 0° with or with-
out carbon tetrachloride, and carbon tetrachloride hoe no eßfect on the final TBA reaction . TABLE I Acceleration oß Lipoperoxidation in Rat Liver Yicrosaaie-Supernatant Fraction by Carbon Tetrachloride . Yicroeomes plus supernatant eq mg per 4 ml
CC14 added «1
Yalonic dialdehyde production No. ~g per eq gm oß microsomea oß plue supernatant ea~erimente (mean and range)
125
none
8
23 .7 (22 .5-24 .8)
125
5
6
41 .5 (39 .2-43 .8)
300
none
5
13 .5 (12 .4-14 .8)
300
5
5
21 .9 (21 .2-23 .1)
Incubation was at 38° under air . Time oß incubation was 20 min ßor 125 eq mg oß micrbaomes plue supernatant, and 25 min for 300 eq mg of microsomes plus supernatant . Pro-oxidant effect of carbon tetrachloride in the presence of ~ -tocopheryl acetate .
The eßfect oß addition in vitro oP d-(y -tocopheryl acetate (Nutritional
Biochemicals Corp .) on malonic dialdehyde production was studied .
In experiments
Yol. 4, No. 15
ACCELERATION OF LIPOPEROXIDATION
1525
pith conditions exactly as in Table I, at a tissue concentration of 125 eq mg oß microsome-supernatant fraction per 4 ml, 5 x 10 -4 Y d-06-tocopheryl acetate completely inhibited ~alonic dialdehyde production in the presence or absence of added CClq .
Further experiments revealed that a concentration of 5 x 10 -5 H
d- CL-tocopheryl acetate wss critical, i .e ., this concentration of d-dC-tocopheryl acetate was just able to inhibit YDß production in the absence oß added CC1 4 , but not if CC1 4 were added to the system .
This infornatioa provided as oppor-
tunity to dramatize the pro-oxidant effect of CC1 4 (Fig . 2) .
The figure shove
80
O U
60 40
O
a a
20
0
0 10
20
30
40
M I N UTES AT 38°
50
60
Figure 2 Pro-oxidant gfßect of CC1 4 in Presence of M 5 x 10 d- CY-Tocopheryl Acetate Conditions : 100 eq mg microsomes plus supernatant ; saline-phosphate buffer, pH5 .6 " final volume 4 ml ; gas phase, air ; temperature, 38° ; M. d-CY-tocopheryl acetate, 5 x 10
1526
ACCELERATION OF LIPOPEROI®ATION
Vol. 4, No. 15
that in the presence oß 5 x 10'5 11 d-CY-tocopheryl acetate, there is a long induction period before malonic dialdehyde production finally begins .
Iß
carbon tetrachloride is added to the system, the induction period is much shorter .
Once initiated, the rate oY malonic dialdehyde production is nearly
the same in both cases, as is consistent with the autocatalytic nature of the process (see Discussion) . No pro-oxidant effect oß carbon t etrachloride in isolated rat liver microsa~aes is absence of the supernatant fraction .
Barber (10) showed that rat
brain microsonea did not peroxidize at 37° in the absence of the supernatant fraction, but peroxidation could be initiated by addition of ascorbic acid . Be have confirmed these findings for rat liver microsomes .
Liver microsomes,
when incubated at 38° in saline-phosphate buffer at pH 5 .8 exhibited no evidence of lipoperoaidation .
Ascorbic acid is a pro-oxidant for this system .
In an
ezperiment employing 100 eq mg of rat liver microsomea per 4 ml of salinephosphate buffer at pH 5 .8, it was found that optimum production of malonic dialdehyde was attained at final ascorbic acid concentrations ranging from 8 .25 x 10-5 lI to 2 .5 a 10 -4 ü .
1[alonic dialdehyde production falls oßß rapidly
at ascorbic acid concentrations below 6 .25 x 10-5 B and is negligible in absence of ascorbic acid . ße11 oßf slightly .
Above 2 .5 x 10 -4 ë ascorbic acid, malonic dialdehyde production The effect of graded amounts of carbon tetrachloride on
the ascorbic acid initiated lipoperoxidation of isolated liver microso~mes was studied .
Over a range from 0 .5 to 50 ~1 of added carbon tetrachloride no
pro-oxidant effect was observed, and at 20 to 50 ~1 of added carbon tetrachloride, malonic dialdehyde production was depressed slightly, about 10~ (Table II) . Carbon tetrachloride added to isolated microsomes without, added ascorbic acid did not initiate malonic dialdehyde production (Table II) .
Since neither
stimulation nor significant inhibition of lipoperoxidation occurred over a wide concentration range for carbon tetrachloride, it is evident that this halogenated hydrocarbon is a rather inert substance for lipoperoxidation processes in isolated microsomes is the absence oß the supernatant fraction,
This inertnesa
Vol. 4, No . 15
ACCELERATION OF LIPOPEROXIDATION
152 7
of carbon tetrachloride pith respect to isolated aicrosomea is in sharp contrast to the dramatic pro-oxidant effect of shall amounts of carbon tetrachloride on the microso~ fraction when the supernatant traction is present (Fig . 2) .
TABLE II No Pro-oxidant Effect of CCld oâ llicrosamea Alone
CC14 ~,1
Ascorbic Acid ~ moles
1~DA Production ~g per eq . P . oß microsomes
None
None
Zero
None
0 .5
51 .8
0.2
O .S
50 .0
1
0 .5
50 .8
3
O .S
51 .1
5
0 .5
48 .0
20
0 .5
44 .3
50
0 .5
42 .8
10
None
Zero
Conditions :
4 ml final volume ; 100 eq ag microsoates in saline-phosphate buffer at pH 5 .8 ; gas phase, air ; incubation at 38 ° for 30 min.
Discussion The fact that anti-oxidants can protect rata against carbon tetrachloride hepatotoxicity (1, 2, 3,
4) implies that the toxin exerts its eßfects via
accelerated lipoperoxidation .
The in vivo protection experineats do not show
directly that the toxic eßfecta of carbon tetrachloride do indeed involve destructive peroxidative attacks oa the unsaturated fatty acids of hepatic lipids .
To our lmowledge our preliainary report (11) and the more coaplete
date presented here constitute the first positive demonstration that carbon tetrachloride can initiate destructive lipoperoxidation in liver tissue .
152 8
ACCELERATION OF LIPOPEROIODATION
Vol . 4, No. 15
The in vitro data presented here compliment unpublished in vivo experimental work ßrom this laboratory .
It has been shown (R . O . Recknagel and A . H . Ghoshal,
unpublished) that as early as 90 minutes after carbon tetrachloride poisoning hepatic microsamal lipids exhibit an absorption maximum at 233 mp .
An intense
absorption maximum in the region Prom 230 to 235 mu is characteristic of the diene conjugation (12) .
Buch diene conjugation appears in peroaidized, unsat-
urated Batty acids such ae linoleic, linolenic, and arachidonic acids due to a displacement oß double bonds during resonance ßollowing ßree radical attack on the methylene bridges in these Batty acids (13, 14} .
Diene conjugation
absorption is not present in hepatic microsomal lipids oß intact rata .
These
Bindings have led one oß us (R,O .R,) to develop a new hypothesis ßor the mechanism oß action oß carbon tetrachloride at the organic chemical level oß organization .
The hypothesis rests in part on the work oß Rubinstein and
Hanks (15) who showed that the enzyme catalyzed conversion oß carbon tetrachloride to C0 2 , demonstrable ßor whole rat liver homogenates, took place largely in the microsome-supernatant fraction .
According to the hypothesis
developed in our laboratory, carbon tetrachloride is metabolized in liver endoplasmic reticulum . ßree radicals (16) .
The initial products are trichlormethyl and chlorine
Either one or both oß these attack the methylene bridges
separating double bonds oß the unsaturated Batty acids oß microsomal structural lipids .
The resulting organic ßree radical is either peroxidized immediately,
or after resonating .
The resonance shi8t oß the ßree radical electron leads
to displacement oß a double bond and appearance of diems conjugation (see in particular reßerence 14),
In either case organic peroxides are ßormed on
insertion oß oxygen at the ßree radical ßunction .
The resulting organic
peroxides are highly unstable and decompose to yield new ßree radicals which can initiate attacks on neighboring methylene bridges .
The process, once
Vol . 4, No . 15
ACCELERATION OF LIPOPEROXII)ATION
started, is autocatalytic .
152 9
This accounts for the tact that typical fatty liver
can be produced by exceedingly low doses o! the toxin .
The well known procliv-
ity o! carbon tetrachloride to attack the liver ie also consistent with the hypothesis, since carbon tetrachloride is metabolized in that organ .
Accord-
ing to the hypothesis, aetabolisa o! carbon tetrachloride ie a necessary p re lude to initiation o! eel!-propagating lipoperozidation .
The hypothesis would
also predlct that small concentrations o! carbon tetrachloride would have little effect on isolated micrneomes, since isolated microeosiea free oß supernatant traction s~etabolize the toxin poorly (15) .
Our findings to the effect
that carbon tetrachloride neither initiated, accelerated, nor aigaüicantly inhibited lipoperoxidation o! isolated microsomee conßirms the latter prediction .
In a recent report Smuckler (17) noted that carbon tetrachloride-
induced depression o! hepatic protein synthesis did not occur following exposure of isolated microsomee to media containing carbon tetrachloride .
Ii the carbon
tetrachloride-induced depression o! hepatic protein synthesis ie a consequence of carbon tetrachloride-induced perozidation o! micrneomal lipids, the report of Smuckler (17) would be rationalized by our observation that carbon tetrachloride acts as a pro-oxidant only under circumstances in which it can be metabolized . This hypothesis ßor the mechanism of the tonic action of carbon tetrachloride was made possible by application of principles of organic chemistry, especially tree radical chemistry (18), a highly useful
fte believe that it will prove to be
perhaps even a powerßul new theoretical frame of reference
for the study oß other hepatotoxina .
Re ße rence a 1.
B . L. HOVE, Arch . Biochem . 17, 467 (1948) .
2.
C . H . GALLAOHBR, Nature , 192, 881 (1961) .
3.
C . H, C~ALIAG~R, Austr . J . Sxp . Biol . an d Med . Sci . 40, 241 (1982) .
4.
N, R, DILDZIO and F . C08TAL83, Federation Proc . 23, 520 (1964) .
1530
ACCE LERATION OF LIPOPEROXiDATION
Vol . 4, No. 15
5.
R, S, PRI$ST, E. A, SYUCSIBR, O, A, Exp . Biol . Yed . 111, 50 (1982) .
IBSRI, and E, P, BENDITT,
8.
L, S, DAHIR, E, G, HILL, and R, T, HOLYAN, Arch . Hiochem. and Biophys . 98, 253 (1982) .
7.
R, 0 RECSNAGSL and AYIYA S, GHOSHAL, Exp. aad Molecular Pathol . (in press) .
8.
H. I. SOHN sad Y . LIVSRSEDGE, J. Pharmacol . Exptl . Therap . 82, 292 (1944) .
9.
R, O,
Proc . Soc .
SINNHUBER aad T, C. YU, Food Technology , 12, 9 (1958) .
10 .
A, A, BARBER, Radiation Rea . Supple . 3, 33 (1983) .
11 .
R, O RSCSNAGSL and AYIYA H, GHOSHAL, Federation Proc . 24, 299 (1965) .
12 .
R, B. "OODIYARD, J . Amer . Chem . Soc . 64, 72 (1942) .
13 .
J. L, HOLLAND and H, P, BOCH, J. Chem .Boc . p . 445 (1945) .
14 .
R, T, HOLYAN, Prog . in Chem . of Fats and Other Lipide , Vol . 2, p. 51, Ed . R. T, HOLYAN, A, O, LUNDBSRG, and T, YALSIN, Academic Preae Inc ., New York (1954) .
15 .
D, RUBINSTEIN and L. SANIC3, Caned . J . Bioch~a. 42, 1577 (1964) .
18 .
T. C . BU1'IER, J . Pharm. Exp er . The rap . 154, 311 (1981) .
17 .
8 . A, SYIICSLER,
18 .
C, IIALLING, Free Radicals in Solu~on , John lliley and Sona, Inc ., New York, (1957) .
Federation Proc . 24, 556 (1985) .
Pergamon Press Ltd. Printed in
P08ITIVE EVIDErAC$ OF ACCSIißATION OF LIPOPBADaIDATION BY CARBON
TETßACHLOßIDB :
IN
VITßO
IN
ßAT LIVSß
SRPSRIl1EN7ô,*
Amiya â . Ghoshal and ßichard O . Recknagel** Department of Physiology, Nestern ßeserve IInivereity School of Medicine Cleveland, Ohio
(Received 14 May 1985)
Seventeen years ago Hove (1) observed that administration of
(X-tocopherol
prior to carbon tetrachloride protected rats against the toxic effects o! the latter .
Anti-oxidant protection against the hepatotoaic effects of carbon
tetrachloride was rediscovered by Gallagher (2, 8) .
Vitasin B, Biphenyl-para-
phenylenediamine (DPPD) and aeleniua all gave acme measure of protection . protective effect of DPPD was conü rmed by DiLuzio and Coatalee (4) .
The
It seems
highly likely that the protection afforded is due to the anti-oxidant property co®on to these agents .
If the latter assumption is correct, then by inference,
carbon tetrachloride can be assumed to exert its toxic eiiecta by initiation of destructive lipoperoxidation .
Priest et el . (S) sought for evidence of increased
lipoperoxidation 1n rat liver three hours after carbon tetrachloride poisoning . These workers measured hepatic levels of malonic dialdehyde with the thiobarbituric acid reaction and sound no increase is malonic dialdehyde content in carbon tetrachloride catty livers in camparisoa with non-toxic controls . * Supported by a research grant, No . AY-01489-08, from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health . U .S . Public Health Service . **Supported by Research Career Development Award 5-K3-0d-900-07 of the Department of Health, Education, and lleliare, United States Public Health Service .
1521
152 2
ACCELERATION OF LIPOPERORIDATION
Dahle et al .
(6) may be consulted ßor evidence that maloaic dialdehyde arisen
ßrom perosidative breakdava oß polyenoic Batty acids .
Vol. 4, No . 15
IIsing the thiobarbituric
acid method, we also could find no evidence oß increased maloaic dialdehyde production in rat liver ßrom 15 minutes to 25 hours aßter carbon tetrachloride poisoning (7) .
However, we have ahova that maloaic dialdehyde is readily
metabolized by rat liver mitochondria .
hashed mitochondria are inactive, but
activity oß the mitochondrial ßraction returns to the level oß whole homogenate when ATP, Yg~ ions, and inorganic phosphate are added (7) . The ßact that maloaic dialdehyde ie metabolized lessens greatly the signißicance oß the failure to ßiad this product oß lipoperoxidation in the toxic liver, i .e ., the ßailure to ßiad maloaic dialdehyde cannot be taken as a demon titration that lipoperoxidation has not occurred .
In this communication we
present positive evidence ßrom in vitro studies that carbon tetrachloride can accelerate lipoperoxidation . Experimental Procedures The procedure ßor measurement oß maloaic dialdehyde wsa based on the method oß Sohn and Liversedge (8) .
The TBA reagent wen 0 .12 M with respect
to TBA, and 0 .026 M with respect to Tris . to 7 .0 with riCl .
Final pH oß the reagent was adjusted
The reaction was carried out on protein-Brae aliquote oß
tissue extracts containing 5~ TCA .
The ßinal reaction mixture contained 2 .5 ml
oß the 5`X, TCA tissue extract, 0 .5 ml of 0 .6 N HCl, and 2 .0 ml oß the TBA reagent . The red color was developed by heating for 10 min at 100 ° , and was read at b35 m~ in a Beckman B spectrophotometer . was used as a standard (9) .
1,1,3,3-tetraethoxypropaae (TßP)
The color reaction was linear over the range ßrom
2 .2 to 22 4+g oß TSP per 5 ml, which 1a equivalent to 0 .72 to 7,2 hg oß maloaic dialdehyde per 5 ml . Yale rata (Holtzman Co ., Madison, wisc .) 350 to 400 grams body weight, were used . Rats were sacrißiced by decapitation .
A portion oß the liver was homogenized
in 0 .112 Y NaCl containing 0 .05 Y sodium phosphate at pH 5 .6 .
The homogenate
was centrißuged in a 3erva1l Reßrigerated Centrifuge at 0 ° ßor 12 min at 9000 x
Vol. 4, No . 15
ACCELERATION OF LIPOPEROJCIDATION RAT LIVER
1523
" RAT LIVER
40 G W V
S
6
a
!0
A
B
!0
0
lo
100
!00
b00
400
!00
!i
p mq AAICROSONES PLUS SUPERNATANT
60
7!
100
I!!
p mq YICROSOMES PLUS SUPERNATANT
Figure 1
Haloaic Disldehyde Production by Aat Liver Microsomes Plus Supernatant . A . The ordinate give s . malonic dialdehyde produced, in Fig per gram equivalent of microsomea plus supernatant, in 25 min at 38° . B . The ordinate gives malonic dialdehyde produced in 25 min at 38° by the amounts oP tissue shown on the abscissa, not reduced to a per gram basis .
gravity .
The sediment (nuclei plus mitochondria) was discarded .
The desired
volume of the microsome plus supernatant fraction was added to a 50 ml conical flask and adjusted to 4 ml final volume with the saline-phosphate buffer . tissue extract was incubated at 38 ° in a water bath with constant shaking . gas phase was air .
The The
The reaction was stopped by transferring the flasks to an
ice bath followed by addition of 1 ml of 25~ ice-cold TCA . the isolated microsome fraction was used .
For some experiments
To obtain the miarosome fraction, the
homogenate, free of nuclei and mitochondria, was centrifuged in the Spinco Preparative Centrifuge è~odel L ßor 60 minutes at 37,500 rpm .
Further details per-
tinent to the various experiments are provided with the figures and tables . Results Pro-oxidant effect of carbon tetrachloride on lipoperoxidation in rat liver microsome-supernatant fraction .
Production of malonic dialdehyde by the microsome-
152 4
ACCELERATION OF LIPOPEROXiDATION
Vol. 4, No. 15
supernatant traction oß rat liver falls sharply at high concentrations of the Barber (10) observed a similar effect for addition
tissue eatract (Fig. lA) .
of rat brain supernatant fraction to brain microsa®es . Figure 1B shave that production of malonic dialdehyde is proportional to the concentration of the microso®e-..supernatant fraction of rat liver up to 125 eq mg of tissue eatract per 4 ml ., One eq mg of the microsome-supernatant ßraction is that amount oß the microsame and supernatant fractions derived from 1 mg wet weight oß liver . 1Rien carbon tetrachloride was present during incubation cf the rat liver nicrosome-supernatant fraction at 38°, malonic dialdehyde production was enhanced . This occurred at low concentrations oß tissue eatract as well as at higher concentrations (Table I) .
No malonic dialdehyde ie produced at 0° with or with-
out carbon tetrachloride, and carbon tetrachloride hoe no eßfect on the final TBA reaction . TABLE I Acceleration oß Lipoperoxidation in Rat Liver Yicrosaaie-Supernatant Fraction by Carbon Tetrachloride . Yicroeomes plus supernatant eq mg per 4 ml
CC14 added «1
Yalonic dialdehyde production No. ~g per eq gm oß microsomea oß plue supernatant ea~erimente (mean and range)
125
none
8
23 .7 (22 .5-24 .8)
125
5
6
41 .5 (39 .2-43 .8)
300
none
5
13 .5 (12 .4-14 .8)
300
5
5
21 .9 (21 .2-23 .1)
Incubation was at 38° under air . Time oß incubation was 20 min ßor 125 eq mg oß micrbaomes plue supernatant, and 25 min for 300 eq mg of microsomes plus supernatant . Pro-oxidant effect of carbon tetrachloride in the presence of ~ -tocopheryl acetate .
The eßfect oß addition in vitro oP d-(y -tocopheryl acetate (Nutritional
Biochemicals Corp .) on malonic dialdehyde production was studied .
In experiments
Yol. 4, No. 15
ACCELERATION OF LIPOPEROXIDATION
1525
pith conditions exactly as in Table I, at a tissue concentration of 125 eq mg oß microsome-supernatant fraction per 4 ml, 5 x 10 -4 Y d-06-tocopheryl acetate completely inhibited ~alonic dialdehyde production in the presence or absence of added CClq .
Further experiments revealed that a concentration of 5 x 10 -5 H
d- CL-tocopheryl acetate wss critical, i .e ., this concentration of d-dC-tocopheryl acetate was just able to inhibit YDß production in the absence oß added CC1 4 , but not if CC1 4 were added to the system .
This infornatioa provided as oppor-
tunity to dramatize the pro-oxidant effect of CC1 4 (Fig . 2) .
The figure shove
80
O U
60 40
O
a a
20
0
0 10
20
30
40
M I N UTES AT 38°
50
60
Figure 2 Pro-oxidant gfßect of CC1 4 in Presence of M 5 x 10 d- CY-Tocopheryl Acetate Conditions : 100 eq mg microsomes plus supernatant ; saline-phosphate buffer, pH5 .6 " final volume 4 ml ; gas phase, air ; temperature, 38° ; M. d-CY-tocopheryl acetate, 5 x 10
1526
ACCELERATION OF LIPOPEROI®ATION
Vol. 4, No. 15
that in the presence oß 5 x 10'5 11 d-CY-tocopheryl acetate, there is a long induction period before malonic dialdehyde production finally begins .
Iß
carbon tetrachloride is added to the system, the induction period is much shorter .
Once initiated, the rate oY malonic dialdehyde production is nearly
the same in both cases, as is consistent with the autocatalytic nature of the process (see Discussion) . No pro-oxidant effect oß carbon t etrachloride in isolated rat liver microsa~aes is absence of the supernatant fraction .
Barber (10) showed that rat
brain microsonea did not peroxidize at 37° in the absence of the supernatant fraction, but peroxidation could be initiated by addition of ascorbic acid . Be have confirmed these findings for rat liver microsomes .
Liver microsomes,
when incubated at 38° in saline-phosphate buffer at pH 5 .8 exhibited no evidence of lipoperoaidation .
Ascorbic acid is a pro-oxidant for this system .
In an
ezperiment employing 100 eq mg of rat liver microsomea per 4 ml of salinephosphate buffer at pH 5 .8, it was found that optimum production of malonic dialdehyde was attained at final ascorbic acid concentrations ranging from 8 .25 x 10-5 lI to 2 .5 a 10 -4 ü .
1[alonic dialdehyde production falls oßß rapidly
at ascorbic acid concentrations below 6 .25 x 10-5 B and is negligible in absence of ascorbic acid . ße11 oßf slightly .
Above 2 .5 x 10 -4 ë ascorbic acid, malonic dialdehyde production The effect of graded amounts of carbon tetrachloride on
the ascorbic acid initiated lipoperoxidation of isolated liver microso~mes was studied .
Over a range from 0 .5 to 50 ~1 of added carbon tetrachloride no
pro-oxidant effect was observed, and at 20 to 50 ~1 of added carbon tetrachloride, malonic dialdehyde production was depressed slightly, about 10~ (Table II) . Carbon tetrachloride added to isolated microsomes without, added ascorbic acid did not initiate malonic dialdehyde production (Table II) .
Since neither
stimulation nor significant inhibition of lipoperoxidation occurred over a wide concentration range for carbon tetrachloride, it is evident that this halogenated hydrocarbon is a rather inert substance for lipoperoxidation processes in isolated microsomes is the absence oß the supernatant fraction,
This inertnesa
Vol. 4, No . 15
ACCELERATION OF LIPOPEROXIDATION
152 7
of carbon tetrachloride pith respect to isolated aicrosomea is in sharp contrast to the dramatic pro-oxidant effect of shall amounts of carbon tetrachloride on the microso~ fraction when the supernatant traction is present (Fig . 2) .
TABLE II No Pro-oxidant Effect of CCld oâ llicrosamea Alone
CC14 ~,1
Ascorbic Acid ~ moles
1~DA Production ~g per eq . P . oß microsomes
None
None
Zero
None
0 .5
51 .8
0.2
O .S
50 .0
1
0 .5
50 .8
3
O .S
51 .1
5
0 .5
48 .0
20
0 .5
44 .3
50
0 .5
42 .8
10
None
Zero
Conditions :
4 ml final volume ; 100 eq ag microsoates in saline-phosphate buffer at pH 5 .8 ; gas phase, air ; incubation at 38 ° for 30 min.
Discussion The fact that anti-oxidants can protect rata against carbon tetrachloride hepatotoxicity (1, 2, 3,
4) implies that the toxin exerts its eßfects via
accelerated lipoperoxidation .
The in vivo protection experineats do not show
directly that the toxic eßfecta of carbon tetrachloride do indeed involve destructive peroxidative attacks oa the unsaturated fatty acids of hepatic lipids .
To our lmowledge our preliainary report (11) and the more coaplete
date presented here constitute the first positive demonstration that carbon tetrachloride can initiate destructive lipoperoxidation in liver tissue .
152 8
ACCELERATION OF LIPOPEROIODATION
Vol . 4, No. 15
The in vitro data presented here compliment unpublished in vivo experimental work ßrom this laboratory .
It has been shown (R . O . Recknagel and A . H . Ghoshal,
unpublished) that as early as 90 minutes after carbon tetrachloride poisoning hepatic microsamal lipids exhibit an absorption maximum at 233 mp .
An intense
absorption maximum in the region Prom 230 to 235 mu is characteristic of the diene conjugation (12) .
Buch diene conjugation appears in peroaidized, unsat-
urated Batty acids such ae linoleic, linolenic, and arachidonic acids due to a displacement oß double bonds during resonance ßollowing ßree radical attack on the methylene bridges in these Batty acids (13, 14} .
Diene conjugation
absorption is not present in hepatic microsomal lipids oß intact rata .
These
Bindings have led one oß us (R,O .R,) to develop a new hypothesis ßor the mechanism oß action oß carbon tetrachloride at the organic chemical level oß organization .
The hypothesis rests in part on the work oß Rubinstein and
Hanks (15) who showed that the enzyme catalyzed conversion oß carbon tetrachloride to C0 2 , demonstrable ßor whole rat liver homogenates, took place largely in the microsome-supernatant fraction .
According to the hypothesis
developed in our laboratory, carbon tetrachloride is metabolized in liver endoplasmic reticulum . ßree radicals (16) .
The initial products are trichlormethyl and chlorine
Either one or both oß these attack the methylene bridges
separating double bonds oß the unsaturated Batty acids oß microsomal structural lipids .
The resulting organic ßree radical is either peroxidized immediately,
or after resonating .
The resonance shi8t oß the ßree radical electron leads
to displacement oß a double bond and appearance of diems conjugation (see in particular reßerence 14),
In either case organic peroxides are ßormed on
insertion oß oxygen at the ßree radical ßunction .
The resulting organic
peroxides are highly unstable and decompose to yield new ßree radicals which can initiate attacks on neighboring methylene bridges .
The process, once
Vol . 4, No . 15
ACCELERATION OF LIPOPEROXII)ATION
started, is autocatalytic .
152 9
This accounts for the tact that typical fatty liver
can be produced by exceedingly low doses o! the toxin .
The well known procliv-
ity o! carbon tetrachloride to attack the liver ie also consistent with the hypothesis, since carbon tetrachloride is metabolized in that organ .
Accord-
ing to the hypothesis, aetabolisa o! carbon tetrachloride ie a necessary p re lude to initiation o! eel!-propagating lipoperozidation .
The hypothesis would
also predlct that small concentrations o! carbon tetrachloride would have little effect on isolated micrneomes, since isolated microeosiea free oß supernatant traction s~etabolize the toxin poorly (15) .
Our findings to the effect
that carbon tetrachloride neither initiated, accelerated, nor aigaüicantly inhibited lipoperoxidation o! isolated microsomee conßirms the latter prediction .
In a recent report Smuckler (17) noted that carbon tetrachloride-
induced depression o! hepatic protein synthesis did not occur following exposure of isolated microsomee to media containing carbon tetrachloride .
Ii the carbon
tetrachloride-induced depression o! hepatic protein synthesis ie a consequence of carbon tetrachloride-induced perozidation o! micrneomal lipids, the report of Smuckler (17) would be rationalized by our observation that carbon tetrachloride acts as a pro-oxidant only under circumstances in which it can be metabolized . This hypothesis ßor the mechanism of the tonic action of carbon tetrachloride was made possible by application of principles of organic chemistry, especially tree radical chemistry (18), a highly useful
fte believe that it will prove to be
perhaps even a powerßul new theoretical frame of reference
for the study oß other hepatotoxina .
Re ße rence a 1.
B . L. HOVE, Arch . Biochem . 17, 467 (1948) .
2.
C . H . GALLAOHBR, Nature , 192, 881 (1961) .
3.
C . H, C~ALIAG~R, Austr . J . Sxp . Biol . an d Med . Sci . 40, 241 (1982) .
4.
N, R, DILDZIO and F . C08TAL83, Federation Proc . 23, 520 (1964) .
1530
ACCE LERATION OF LIPOPEROXiDATION
Vol . 4, No. 15
5.
R, S, PRI$ST, E. A, SYUCSIBR, O, A, Exp . Biol . Yed . 111, 50 (1982) .
IBSRI, and E, P, BENDITT,
8.
L, S, DAHIR, E, G, HILL, and R, T, HOLYAN, Arch . Hiochem. and Biophys . 98, 253 (1982) .
7.
R, 0 RECSNAGSL and AYIYA S, GHOSHAL, Exp. aad Molecular Pathol . (in press) .
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H. I. SOHN sad Y . LIVSRSEDGE, J. Pharmacol . Exptl . Therap . 82, 292 (1944) .
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R, O,
Proc . Soc .
SINNHUBER aad T, C. YU, Food Technology , 12, 9 (1958) .
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A, A, BARBER, Radiation Rea . Supple . 3, 33 (1983) .
11 .
R, O RSCSNAGSL and AYIYA H, GHOSHAL, Federation Proc . 24, 299 (1965) .
12 .
R, B. "OODIYARD, J . Amer . Chem . Soc . 64, 72 (1942) .
13 .
J. L, HOLLAND and H, P, BOCH, J. Chem .Boc . p . 445 (1945) .
14 .
R, T, HOLYAN, Prog . in Chem . of Fats and Other Lipide , Vol . 2, p. 51, Ed . R. T, HOLYAN, A, O, LUNDBSRG, and T, YALSIN, Academic Preae Inc ., New York (1954) .
15 .
D, RUBINSTEIN and L. SANIC3, Caned . J . Bioch~a. 42, 1577 (1964) .
18 .
T. C . BU1'IER, J . Pharm. Exp er . The rap . 154, 311 (1981) .
17 .
8 . A, SYIICSLER,
18 .
C, IIALLING, Free Radicals in Solu~on , John lliley and Sona, Inc ., New York, (1957) .
Federation Proc . 24, 556 (1985) .