Int Arch Occup Environ Hlth 36,229-234 (1976) © by Springer-Verlag 1976
Aortic Phospholipids Synthesis in Experimental Carbon Disulphide Intoxication in Rats* TERESA WRONSKA-NOFER Department of Biochemistry, Institute of Occupational Medicine, P.O Box 199, 90-950 Ld 2, Poland (Director: Prof J Nofer, M D )
Summary
The rate of incorporation of phosphate precursor into phospho-
lipids in vivo has been investigated in normal rats and in those intoxicated with C52 The specific activity of 32 p phospholipids was measured in plasma and the aorta wall at various times after i v
administration
of 0 2 mCi phosphate. In C52 intoxicated rats the specific activity of aorta phospholipids exceeded that in normal rats
The conclusion is drawn that the rise of
the aortic phospholipids specific activity does not derive from increased blood phospholipid concentration but is due to an increased rate of phospholipids synthesis in the aorta tissue. Key words: Carbon disulphide intoxication
Phospholipids synthesis.
INTRODUCTION In our previous
investigation
it was repeatedly
one of the lipid metabolism alterations
shown that
after intoxication
with C 52 is increased lipid level in blood l4,6-9 l It could be expected, that as a result of such alterations atheromatous changes
in vessel walls would develop Our tentative observations did not confirm these assumptions Eight months exposure
of rats to C 52
logical changes in the
does not
lead to significant histo-
aorta wall.
However, metabolic disorders
of lipids might be detectable
in this tissue regardless of whether morphological changes have occurred or not
In order to verify such a hypothesis
an
attempt was undertaken to make a thorough study of lipid metabolism in phospholipids
C
the aorta wall In this experiment in the aortic tissue was studied.
synthesis of
This investigation has been done under the Polish-American agreement No 05-008-2 with the Occupational Health Program, U S Public Health Service.
229
MATERIALS AND METHODS Used in this study female rats, of Wistar strain, and of mean body weight 180 ± 10g (at the beginning of experiment) were The exdivided into two groups: control and exposed to C 52 posure to C 52 was carried for 5 hrs daily, 6 days weekly, for The concentration of C 52 in the a total period of 6 months Both groups of animals were fed on a chamber was 1 5 mg/l The average daily food consumption was similar in stock diet In the course of the experiment the body weight both groups and the level of plasma phospholipids were determined. The lipid phosphorus in plasma was estimated according to In The blood was taken from the tail vein Dmochowski l2l the 6th month of exposure the rate of phospholipid synthesis was measured in vivo The method was based on the determination of the specific activity of 3 2 P-phospholipids following i v. 32 At administration of 0 2 m Ci of radioactive phosphate ( P) 32 p invarious time intervals (1,3,6,15, and 30 hrs) after jection, rats of control and intoxicated groups were decapitated in couples, and tissues (blood and aorta) were taken for determination of radioactivity and concentration of lipid The total lipids were extracted with 20 volumes phosphorus of methanol : chloroform (1 : 2) and for removing water soluble radioactive compounds the lipid extract was thoroughly washed according to the method of Folch et al slightly modified l3l. Two separate aliquots of purified extracts were used for estimating lipid phosphorus concentration l1l and radioactivity. The radioactivity was measured with a mica end window counter Specific activity of the tissue and with an efficiency of 45 % plasma phospholipid are expressed in counts/mg phospholipids/ min.
RESULTS AND DISCUSSION After 6 months, rats exposed to C 52 did not display any symptoms typical of long-term (10 months) intoxication with C 52 such as muscular weakness and paralysis of hind limbs E6,8J. Also the body weight of control and exposed to C 52 rats did not differ significantly. As a first step of the study the specific activity-time course of plasma and aortic phospholipid has been determined with the aim of estimating the most suitable time for the The data are evaluation of aortic phospholipids synthesis The slope of the curves of specific activity of in Fig 1 phospholipids in the aorta is similar for both control and Specific activity of phospholipids animals exposed to C 52
230
x 10 3 vn
'-
ov)*
?I
a ° W C
.6 _
00
d
8 U Cl)
Time lHoursl Fig 1 Specific radioactivity-time relation of plasma and aortic phospholipids in control
(
)
and C 52
rats were injected with
(
) exposed rats
32
Control and C52 exposed
Specific radioactivity of plasma
p-phosphate
and aorta phospholipids was estimated at different time-intervals after injection
Each point represents an average of 3-4 rats
given as mean ± S E
Table 1
Results are
For details see "Materials and Methods"
Specific radioactivity of phospholipids in aorta
Groups of
Number of
Specific radioactivity
animals
animals
cpm/mg of phospholipids
Control
8
6620 ± 273
C52
8
8800 ± 645 *
Specific radioactivity of 32 p phospholipids in the aorta was estimated 3 hrs after injection of 32 P-phosphate Methods". * Different from control
For details see "Materials and
(P < 0 002).
in the aorta significantly exceeds
that in plasma at the first 32
intervals of time after administration of imental groups of rats
(Fig 1)
3 hrs after
specific activity of aortic phospholipids high as
the specific
activity in plasma
p
32
In both experp injection
the
is about twice as Later that of plasma
rises very rapidly, while that of aorta increases more slowly. On the ground of the above results
3 hrs time interval
after
231
Table 2 Comparative results of the effect of carbon disulphide on concentration of serum phospholipids in rats Concentration
Duration of
Serum
of C52 (mg/l)
exposure
phospholipids
(months)
(mg/%)
C52 (1 5-1 8) Control
5
84 ± 16 1 78 ± 9 5
Present study
C52
4
95 ± 18 2
8
References
a
(1 7)
Control C52
79 ±
(O 55)
6
98 ± 14 6 83 ± 6 3
6
114 ± 18 5 76 ± 20 0
Control C52 (1 5-1 8) Control
7 8
6
Each value represents an average of 18 rats. a The level of phospholipids had been estimated in the 5th month of exposure before the radioactive
32
p was injected
Details are given in
"Materials and Methods".
32
p
administration was
phospholipids Zilversmit
synthesis
10,11 l
taken for the evaluation of aortic The
6 hrs
time interval chosen by
for studying this process
in rabbits does
not seem to be appropriate in the case of rats The results of the study of phospholipids synthesis are shown in Table 1. It is evident from this table that 3 hrs after 3 2 p injection the specific activity of phospholipids exposed to C 52 the
exceeds
same time the specific activity of
lipids
in the aorta of rats
that in control by about 35 %
As at
the aortic phospho-
is much higher than it had been in plasma allows us
to conclude that the increase of the phospholipids in the aorta wall in C 52 intoxicated rats is due mainly to a higher rate of synthesis The possibility that a significant amount of radioactivity
is derived from the blood can be excluded. Comparing the specific activity time curves of the plasma phospholipids between control and C 52 intoxicated rats, some slight differences in the efficiency of 32 p incorporation between both groups are noticable
Specific activity of the plasma phospholipids is higher in rats exposed to C 52 in all time intervals (Fig 1) Since the total amount of phospholipids contained in the plasma is derived from the liver 11l it may be suggested that C 52 is inducing some changes in the turnover-rate of liver phospholipids, which are then reflected
232
by changes in the specific activity of the phospholipids in the blood This hypothesis could not be proved statistically, because of the small number of animals used in the experiment. Intoxication with C52 brings about elevation of the level of phospholipids in the blood, but as it is shown in Table 2 this increase is not high, and depending on experimental conditions it ranges from 15 to 50% This magnitude of change does not seem to be sufficient to account for the changes in the synthesis of phospholipids in the aorta wall According to Zilversmit 5,10,11l even if the level of phospholipids in blood exceeds many times the normal level, 90% of newly appearing phospholipids in the aorta are due to de novo synthesis and the rate of 32 p incorporation into aortic phospholipids does not depend on the level of plasma lipids Thus, it may be suggested that also in the case of C52 intoxication, the level of the plasma lipids does not influence the rate of 32 p incorporation into aortic phospholipids. Comparing the results obtained in this experiment and the results of Zilversmit 5,10,11 l it is concluded that: 1) In C52 intoxicated rats the rise of the specific activity of aortic phospholipids is due to an increased rate of phospholipids synthesis. 2) The possibility that a significant fraction of aortic phospholipids derives from plasma is excluded by the fact that in the time interval selected for the analysis the specific activity of plasma phospholipids was much lower than the specific activity in aorta. 3) In rats chronically exposed to C52 some changes in lipid metabolism in the aorta wall are detectable before histological ones.
REFERENCES 1
Bartlett, G R : Phosphorus assay in column chromatography 234, 466 (1959)
2
Dmochowski, A , Krajewski, T , Urbanek, H : Microdetermination of phosphorus after dry mineralization lin Polishl 683 (1960)
3
J biol Chem.
Chemia Analityczna 5,
Folch, J , Less, M , Sloane Stanley, G H : A simple method for the isolation and purification of total lipids from animal tissues Chem
4
Nofer,
226, 497-509
J biol.
(1957)
J , Chojnowski, J , Kawecka, M , Kie E, E , Wrohska-Szpakowa, T :
Effect of occupational exposure to carbon disulphide lin Polishl. Medycyna Pracy 12, 101-118 (1961) 5
Shore, M L , Zilversmit, D B , Ackerman, R F : Plasma phospholipide deposition and aortic phospholipide synthesis in experimental atherosclerosis
Amer J Physiol
181,
527-531 (1955)
233
6
Wrofska-Nofer, T : The influence of nicotinic acid upon the disturbances in lipid metabolism caused by carbon disulphide in rats Arch Arbeitsmed
7
Int.
27, 221-227 (1970)
Wrohska-Nofer, T : The influence of low doses of nicotinic acid upon the development of lipid disturbances in rats chronically exposed to
8
carbon disulphide Int Arch Arbeitsmed 29, 285-290 (1972) Wrohska-Nofer, T : Disturbances of lipids metabolism in rats in dependence upon carbon disulphide concentrations in the air Lavoro 64, 8-12
9
Med.
(1973)
Wrohska-Nofer, T , Knobloch, K : The influence of variation of the daily rhythm of exposure upon the magnitude of biological effects: a comparison of X-ray irradiation and carbon disulphide
Bull Acad.
10
Pol Sci 20, 813-819 (1972) Zilversmit, D B , McCandless, E L : Independence of arterial phospholipide synthesis from alterations in blood lipids J Lip Res 1,
11
Zilversmit, D B , Shore, M L , Ackerman, R F : The origin of aortic
118-124 (1959) phospholipide in rabbit atheromatosis
Circulation 9, 581-585
Received August 18, 1975 / Accepted November 3, 1975
234
(1954)
Aortic Phospholipids Synthesis in Experimental Carbon Disulphide Intoxication in Rats* TERESA WRONSKA-NOFER Department of Biochemistry, Institute of Occupational Medicine, P.O Box 199, 90-950 Ld 2, Poland (Director: Prof J Nofer, M D )
Summary
The rate of incorporation of phosphate precursor into phospho-
lipids in vivo has been investigated in normal rats and in those intoxicated with C52 The specific activity of 32 p phospholipids was measured in plasma and the aorta wall at various times after i v
administration
of 0 2 mCi phosphate. In C52 intoxicated rats the specific activity of aorta phospholipids exceeded that in normal rats
The conclusion is drawn that the rise of
the aortic phospholipids specific activity does not derive from increased blood phospholipid concentration but is due to an increased rate of phospholipids synthesis in the aorta tissue. Key words: Carbon disulphide intoxication
Phospholipids synthesis.
INTRODUCTION In our previous
investigation
it was repeatedly
one of the lipid metabolism alterations
shown that
after intoxication
with C 52 is increased lipid level in blood l4,6-9 l It could be expected, that as a result of such alterations atheromatous changes
in vessel walls would develop Our tentative observations did not confirm these assumptions Eight months exposure
of rats to C 52
logical changes in the
does not
lead to significant histo-
aorta wall.
However, metabolic disorders
of lipids might be detectable
in this tissue regardless of whether morphological changes have occurred or not
In order to verify such a hypothesis
an
attempt was undertaken to make a thorough study of lipid metabolism in phospholipids
C
the aorta wall In this experiment in the aortic tissue was studied.
synthesis of
This investigation has been done under the Polish-American agreement No 05-008-2 with the Occupational Health Program, U S Public Health Service.
229
MATERIALS AND METHODS Used in this study female rats, of Wistar strain, and of mean body weight 180 ± 10g (at the beginning of experiment) were The exdivided into two groups: control and exposed to C 52 posure to C 52 was carried for 5 hrs daily, 6 days weekly, for The concentration of C 52 in the a total period of 6 months Both groups of animals were fed on a chamber was 1 5 mg/l The average daily food consumption was similar in stock diet In the course of the experiment the body weight both groups and the level of plasma phospholipids were determined. The lipid phosphorus in plasma was estimated according to In The blood was taken from the tail vein Dmochowski l2l the 6th month of exposure the rate of phospholipid synthesis was measured in vivo The method was based on the determination of the specific activity of 3 2 P-phospholipids following i v. 32 At administration of 0 2 m Ci of radioactive phosphate ( P) 32 p invarious time intervals (1,3,6,15, and 30 hrs) after jection, rats of control and intoxicated groups were decapitated in couples, and tissues (blood and aorta) were taken for determination of radioactivity and concentration of lipid The total lipids were extracted with 20 volumes phosphorus of methanol : chloroform (1 : 2) and for removing water soluble radioactive compounds the lipid extract was thoroughly washed according to the method of Folch et al slightly modified l3l. Two separate aliquots of purified extracts were used for estimating lipid phosphorus concentration l1l and radioactivity. The radioactivity was measured with a mica end window counter Specific activity of the tissue and with an efficiency of 45 % plasma phospholipid are expressed in counts/mg phospholipids/ min.
RESULTS AND DISCUSSION After 6 months, rats exposed to C 52 did not display any symptoms typical of long-term (10 months) intoxication with C 52 such as muscular weakness and paralysis of hind limbs E6,8J. Also the body weight of control and exposed to C 52 rats did not differ significantly. As a first step of the study the specific activity-time course of plasma and aortic phospholipid has been determined with the aim of estimating the most suitable time for the The data are evaluation of aortic phospholipids synthesis The slope of the curves of specific activity of in Fig 1 phospholipids in the aorta is similar for both control and Specific activity of phospholipids animals exposed to C 52
230
x 10 3 vn
'-
ov)*
?I
a ° W C
.6 _
00
d
8 U Cl)
Time lHoursl Fig 1 Specific radioactivity-time relation of plasma and aortic phospholipids in control
(
)
and C 52
rats were injected with
(
) exposed rats
32
Control and C52 exposed
Specific radioactivity of plasma
p-phosphate
and aorta phospholipids was estimated at different time-intervals after injection
Each point represents an average of 3-4 rats
given as mean ± S E
Table 1
Results are
For details see "Materials and Methods"
Specific radioactivity of phospholipids in aorta
Groups of
Number of
Specific radioactivity
animals
animals
cpm/mg of phospholipids
Control
8
6620 ± 273
C52
8
8800 ± 645 *
Specific radioactivity of 32 p phospholipids in the aorta was estimated 3 hrs after injection of 32 P-phosphate Methods". * Different from control
For details see "Materials and
(P < 0 002).
in the aorta significantly exceeds
that in plasma at the first 32
intervals of time after administration of imental groups of rats
(Fig 1)
3 hrs after
specific activity of aortic phospholipids high as
the specific
activity in plasma
p
32
In both experp injection
the
is about twice as Later that of plasma
rises very rapidly, while that of aorta increases more slowly. On the ground of the above results
3 hrs time interval
after
231
Table 2 Comparative results of the effect of carbon disulphide on concentration of serum phospholipids in rats Concentration
Duration of
Serum
of C52 (mg/l)
exposure
phospholipids
(months)
(mg/%)
C52 (1 5-1 8) Control
5
84 ± 16 1 78 ± 9 5
Present study
C52
4
95 ± 18 2
8
References
a
(1 7)
Control C52
79 ±
(O 55)
6
98 ± 14 6 83 ± 6 3
6
114 ± 18 5 76 ± 20 0
Control C52 (1 5-1 8) Control
7 8
6
Each value represents an average of 18 rats. a The level of phospholipids had been estimated in the 5th month of exposure before the radioactive
32
p was injected
Details are given in
"Materials and Methods".
32
p
administration was
phospholipids Zilversmit
synthesis
10,11 l
taken for the evaluation of aortic The
6 hrs
time interval chosen by
for studying this process
in rabbits does
not seem to be appropriate in the case of rats The results of the study of phospholipids synthesis are shown in Table 1. It is evident from this table that 3 hrs after 3 2 p injection the specific activity of phospholipids exposed to C 52 the
exceeds
same time the specific activity of
lipids
in the aorta of rats
that in control by about 35 %
As at
the aortic phospho-
is much higher than it had been in plasma allows us
to conclude that the increase of the phospholipids in the aorta wall in C 52 intoxicated rats is due mainly to a higher rate of synthesis The possibility that a significant amount of radioactivity
is derived from the blood can be excluded. Comparing the specific activity time curves of the plasma phospholipids between control and C 52 intoxicated rats, some slight differences in the efficiency of 32 p incorporation between both groups are noticable
Specific activity of the plasma phospholipids is higher in rats exposed to C 52 in all time intervals (Fig 1) Since the total amount of phospholipids contained in the plasma is derived from the liver 11l it may be suggested that C 52 is inducing some changes in the turnover-rate of liver phospholipids, which are then reflected
232
by changes in the specific activity of the phospholipids in the blood This hypothesis could not be proved statistically, because of the small number of animals used in the experiment. Intoxication with C52 brings about elevation of the level of phospholipids in the blood, but as it is shown in Table 2 this increase is not high, and depending on experimental conditions it ranges from 15 to 50% This magnitude of change does not seem to be sufficient to account for the changes in the synthesis of phospholipids in the aorta wall According to Zilversmit 5,10,11l even if the level of phospholipids in blood exceeds many times the normal level, 90% of newly appearing phospholipids in the aorta are due to de novo synthesis and the rate of 32 p incorporation into aortic phospholipids does not depend on the level of plasma lipids Thus, it may be suggested that also in the case of C52 intoxication, the level of the plasma lipids does not influence the rate of 32 p incorporation into aortic phospholipids. Comparing the results obtained in this experiment and the results of Zilversmit 5,10,11 l it is concluded that: 1) In C52 intoxicated rats the rise of the specific activity of aortic phospholipids is due to an increased rate of phospholipids synthesis. 2) The possibility that a significant fraction of aortic phospholipids derives from plasma is excluded by the fact that in the time interval selected for the analysis the specific activity of plasma phospholipids was much lower than the specific activity in aorta. 3) In rats chronically exposed to C52 some changes in lipid metabolism in the aorta wall are detectable before histological ones.
REFERENCES 1
Bartlett, G R : Phosphorus assay in column chromatography 234, 466 (1959)
2
Dmochowski, A , Krajewski, T , Urbanek, H : Microdetermination of phosphorus after dry mineralization lin Polishl 683 (1960)
3
J biol Chem.
Chemia Analityczna 5,
Folch, J , Less, M , Sloane Stanley, G H : A simple method for the isolation and purification of total lipids from animal tissues Chem
4
Nofer,
226, 497-509
J biol.
(1957)
J , Chojnowski, J , Kawecka, M , Kie E, E , Wrohska-Szpakowa, T :
Effect of occupational exposure to carbon disulphide lin Polishl. Medycyna Pracy 12, 101-118 (1961) 5
Shore, M L , Zilversmit, D B , Ackerman, R F : Plasma phospholipide deposition and aortic phospholipide synthesis in experimental atherosclerosis
Amer J Physiol
181,
527-531 (1955)
233
6
Wrofska-Nofer, T : The influence of nicotinic acid upon the disturbances in lipid metabolism caused by carbon disulphide in rats Arch Arbeitsmed
7
Int.
27, 221-227 (1970)
Wrohska-Nofer, T : The influence of low doses of nicotinic acid upon the development of lipid disturbances in rats chronically exposed to
8
carbon disulphide Int Arch Arbeitsmed 29, 285-290 (1972) Wrohska-Nofer, T : Disturbances of lipids metabolism in rats in dependence upon carbon disulphide concentrations in the air Lavoro 64, 8-12
9
Med.
(1973)
Wrohska-Nofer, T , Knobloch, K : The influence of variation of the daily rhythm of exposure upon the magnitude of biological effects: a comparison of X-ray irradiation and carbon disulphide
Bull Acad.
10
Pol Sci 20, 813-819 (1972) Zilversmit, D B , McCandless, E L : Independence of arterial phospholipide synthesis from alterations in blood lipids J Lip Res 1,
11
Zilversmit, D B , Shore, M L , Ackerman, R F : The origin of aortic
118-124 (1959) phospholipide in rabbit atheromatosis
Circulation 9, 581-585
Received August 18, 1975 / Accepted November 3, 1975
234
(1954)
