Int.Arch.Arbeitsmed.34,221-229 9 by S p r i n g e r - V e r l a g 1975

(1975)

Studies on in vivo Incorporation of 14C-Acetate and 14C-Mevalonate into Cholesterol in the Liver of Rat Intoxicated with Carbon Disulphide* Teresa Wr6nska-No~r Department Institute

of

Biochemistry,

of

(Director: Received

Occupational

Prof. April

J.

Medicine,

Nofer,

19,

1974

L6d~

M.D.)

/ Accepted

October

Summary. As an extension of former results, cation with CS 2 raises

the b i o s y n t h e s i s

aim of this experiment

was to investigate

of the 14C-acetate and to determine

into cholesterol

which

with CS 2. The studies into

intoxicated

rats

after a single

(concentration

injection

of the e x p e r i m e n t

1.5 mg/l,

indicated

that exposure radioactivity

14C-acetate

and 14C-mevalonate

injection,

biosynthesis

and 14C-mevalonate

both after

of choles-

than with mevalonate;

liver weight;

c) faster

and decline

in the serum c o r r e s p o n d i n g

times

The results

of liver cholesterol

concentration

cholesterol

at various

but incorporation

faster with acetate

d) increase

and CS 2

of rats to CS 2 causes:

in the cholesterol

in the liver;

to CS2,

is a c c e l e r a t e d

over 7 months)

b) no changes cholesterol

the

of incorporation

or 14C-mevalonate.

in the specific

increase

that intoxi-

in the liver,

the dynamics

of 14C-acetate

of 14C-acetate

proceeds

testified

out in vivo, in the control

a) increase

terol p r e c u r s o r s

1974

in the liver of rats exposed

of i n c o r p o r a t i o n was carried

which

of cholesterol

step of cholesterol

liver cholesterol

23,

in the liver and in the

of specific

in the specific to the changes

activity of

radioactivity observed

of

in the

liver. It has been c o n c l u d e d I. CS 2 increased turnover

the cholesterol

rate of cholesterol

2. stimulating is located between effect,

that:

effect of CS 2 in the process acetate

behind m e v a l o n a t e

and m e v a l o n a t e

investigation

and a c c e l e r a t e d

the

of cholesterol

although

another

synthesis

site of CS 2

cannot be excluded.

Key words: Carbon Disulphide

This

biosynthesis

in the liver;

Intoxication

has been carried

- Cholesterol

out p a r t l y

Synthesis.

under the Polish-

A m e r i c a n a g r e e m e n t No. 05-008-2 with the Occupational U.S. Public Health Service.

Health Program,

221

INTRODUCTION It was found in e a r l i e r s t u d i e s that i n t o x i c a t i o n w i t h CS 2 r e s u l t e d in e l e v a t e d levels of c h o l e s t e r o l in s e r u m E8,15-20~. It was also shown that CS 2 a c c e l e r a t e d s i g n i f i c a n t l y the inc o r p o r a t i o n of a c e t a t e into c h o l e s t e r o l in the liver b o t h w h e n the i n t o x i c a t i o n was c o n d u c t e d for a long time [14,16, 17,19~ and d u r i n g short e x p o s u r e s ~4]. Since the liver p l a y s an e s s e n t i a l p a r t in the m e t a b o l i s m of s e r u m c h o l e s t e r o l , the c o n c l u s i o n has b e e n d r a w n that inc r e a s e d b i o s y n t h e s i s of c h o l e s t e r o l in the liver m a y a c c o u n t for the i n c r e a s e d level of s e r u m c h o l e s t e r o l . In o r d e r to inv e s t i g a t e the rate of s y n t h e s i s and to d e t e r m i n e w h i c h stage of b i o s y n t h e s i s of c h o l e s t e r o l in the liver is a f f e c t e d by CS2, the study was c a r r i e d out on i n c o r p o r a t i o n of a c e t a t e and m e v a l o n a t e into the liver c h o l e s t e r o l of rats c h r o n i c l y e x p o s e d to CS 2.

~TERIAL

AND METHODS

The e x p e r i m e n t was p e r f o r m e d on f e m a l e rats, W i s t a r strain, of a m e a n b o d y w e i g h t of 165 f 5 g (at the b e g i n n i n g of the e x p e r i m e n t ) . The a n i m a l s w e r e d i v i d e d into two groups: I = rats e x p o s e d to CS2, and II = c o n t r o l rats kept u n d e r the same c o n d i t i o n s as g r o u p I, but not e x p o s e d to CS 2. The rats w e r e i n t o x i c a t e d w i t h CS 2 at the c o n c e n t r a t i o n 1.5 (1.4 - 1.7) mg/l, 5 hrs daily, 6 days a w e e k for 7 months. In the c o u r s e of the e x p e r i m e n t all a n i m a l s w e r e fed a s t a n d a r d d i e t I. The in ~ v o i n c o r p o r a t i o n of 2 - 1 4 C - a c e t a t e and 2 - 1 4 C - m e v a l o n a t e was i n v e s t i g a t e d at the end of 7 m o n t h s of e x p o s u r e to CS 2. A f t e r the last d a i l y e x p o s u r e to CS 2 and the f o l l o w i n g 18 hrs of ,fasting, the a n i m a l s of b o t h g r o u p s w e r e g i v e n i n t r a p e r i t o n e a l l y L 2 - 1 4 C ~ a c e t a t e 2 (15 ~ C / I O O g b o d y wt.) or ~ 2 - 1 4 C ] m e v a l o n a t e 3 (2 ~ C / I O O g b o d y wt.). The rats w e r e k i l l e d in couples: c o n t r o l and i n t o x i c a t e d rats 15, 30, 60, 180, and 360 m i n a f t e r i n j e c t i o n of E 2 - 1 4 ~ a c e t a t e and 120 min a f t e r i n j e c t i o n of E 2 - 1 4 C 3 m e v a l o n a t e . The b l o o d was c o l l e c t e d and the liver was taken out immed i a t e l y a f t e r d e c a p i t a t i o n and the c o n c e n t r a t i o n and r a d i o a c t i v i t y of total c h o l e s t e r o l w e r e m e a s u r e d . 1

Standard diet for rats produced by Wytwbrnia Pasz, ~owicz, Poland. E2-14C]sodium acetate, specific activity 10,3 mCi/mM produced by Institute of Nuclear Research, ~wierk, Poland. 3 E2-14C]mevalonate acid, DBED salt, specific activity 4,2 mCi/mM produced by Radiochemical Centre, Amersham, England. 2

222

S e r u m and liver c h o l e s t e r o l a n a l y s i s : s e r u m was e x t r a c t e d w i t h a l c o h o l : a c e t o n e (I : I) and a f t e r s a p o n i f i c a t i o n of two s e p a r a t e s s a m p l e s the total c h o l e s t e r o l was p r e c i p i t a t e d as a d i g i t o n i d e for e s t i m a t i o n of c h o l e s t e r o l by the m e t h o d of S p e r r y & V e b b [10] and for the m e a s u r e m e n t of r a d i o a c tivity. The r a d i o a c t i v i t y of d i g i t o n i d e was m e a s u r e d in a gas f l o w c o u n t e r at a c o n s t a n t t h i c k n e s s p l a t i n g of d i g i t o nide. The liver c h o l e s t e r o l was e x t r a c t e d f r o m the t i s s u e w i t h a l c o h o l : a c e t o n e : e t h e r (4 : 4 : I), and r e e x t r a c t e d w i t h p e t r o l e u m ether a c c o r d i n g to S w e l l E11]. The e x t r a c t was e v a p o r a t e d to d r y n e s s then r e d i s s o l v e d in a l c o h o l : acetone (I : I), s a p o n i f i c a t e d , and t o t a l c h o l e s t e r o l was prec i p i t a t e d w i t h d i g i t o n i n e . Two s e p a r a t e a l i q u o t s of the prec i p i t a t e w e r e u s e d to m e a s u r e c o n c e n t r a t i o n ~9] and radioa c t i v i t y of c h o l e s t e r o l as for serum. To c h e c k the p u r i t y of the r a d i o a c t i v e c h o l e s t e r o l i s o l a t e d from the liver of c o n t r o l and CS 2 i n t o x i c a t e d rats, some s a m p l e s of c h o l e s t e rol i s o l a t e d as a d i g i t o n i d e w e r e p u r i f i e d by m e a n s of b r o m i n a t i o n a c c o r d i n g to K a b a r a ~7]. C r y s t a l i n e 5,6 d i b r o m o C h o l e s t a n - 3 B - o l was d e b r o m i n a t e d (using zinc and a c e t i c acid) i s o l a t e d a g a i n as a d i g i t o n i d e and s p e c i f i c r a d i o a c t i v i t y of c h o l e s t e r o l d i g i t o n i d e was a g a i n m e a s u r e d . C a l c u l a t i o n : The results, e x p r e s s e d as the a r i t h m e t i c m e a n s • S.D./ and the s i g n i f i c a n c e of d i f f e r e n c e s w e r e e v a l u ated by the S t u d e n t ' s t - t e s t of s i g n i f i c a n c e . The r e g r e s s i o n lines for the s p e c i f i c a c t i v i t y of c h o l e s t e r o l w e r e c a l c u latedusing the m e t h o d of the l e a s t - m e a n - s q u a r e - f i t , and t h e i r slope c o e f f i c i e n t s w e r e c o m p a r e d by m e a n s the S t u d e n t ' s t-test.

RESULTS

AND DISCUSSION

In T a b l e I are l i s t e d the v a l u e s of the s p e c i f i c a c t i v i t y of liver c h o l e s t e r o l a f t e r b r o m i n a t i o n . This e x p e r i m e n t has b e e n p e r f o r m e d to c h e c k the p u r i t y of r a d i o a c t i v e c h o l e s t e r o l i s o l a t e d as a d i g i t o n i d e . As it has b e e n shown, p u r i f i c a t i o n of the c h o l e s t e r o l by d i b r o m i d e p r e c i p i t a t i o n does not cause any d e t e c t a b l e c h a n g e s in the s p e c i f i c r a d i o a c t i v i t y of the l i v e r c h o l e s t e r o l ; and the d i f f e r e n c e s f o u n d in the s p e c i f i c a c t i v i t y of c h o l e s t e r o l b e t w e e n the c o n t r o l and CS 2 i n t o x i c a t e d rats r e m a i n the same. T h e s e d a t a a g r e e w i t h the results o f S c h w e n k & W e r t h e s s e n t E9], w h o f o u n d that 4 hrs a f t e r i n j e c t i o n of rat w i t h 1 4 C - a c e t a t e f u r t h e r p u r i f i c a t i o n w i t h d i b r o m i d e did not a l t e r the s p e c i f i c a c t i v i t y of t i s s u e c h o l e s t e r o l and was not n e c e s s a r y . In all of those e x p e r i m e n t s d e s c r i b e d a b o v e and t h o s e f o l l o w i n g , c h o l e s t e r o l iso-

223

Table I Radiochemical purity of cholesterol isolated as a digitonide from the a liver of control rats and those exposed to CS 2 Cholesterol-14C

Group of animals

Control

CS

Digitonide

Dibromide

(cpm/min/mg)

(cpm/min/mg)

58 (4)

53,5 (4)

153

2

149

(5)

(5)

Specific radioactivity of 14C-cholesterol was estimated in the liver 3 hrs after the intraperitoneal injection of 14C-sodium acetate. Unlabeled cholesterol was added as a carrier to each sample estimated by digitonide and dibromide precipitation. The specific activity values were not corrected for dilution by added carrier. The numbers of animals are given in parentheses. For experimental details see "Methods".

30 E c_ E

2B

8

~

2.2

ZC

I

60

Fig.1 Incorporation

I

120

of

I

180 Time [mini

I

I

240

~2-14C]acetate

300

into

the

I

360

liver

cholesterol

of rats intoxicated with CS 2 and control rats. The groups of control rats and those exposed to CS 2 were injected intraperitoneally with E2-14C]acetate. Specific radioactivity of liver cholesterol was estimated at different intervals after injection. Each point represents an average of 3-5 rats. Results are given as mean • S.D. For details see "Methods". x = Different from the corresponding control (P < 0 . 0 5 )

224

lated f r o m t i s s u e s was p u r i f i e d by p r e c i p i t a t i o n of digitonide, and the r a d i o a c t i v i t y of the d i g i t o n i d e of c h o l e s terol was m e a s u r e d . Fig. 1 d e p i c t s the s p e c i f i c a c t i v i t y of c h o l e s t e r o l in the liver of c o n t r o l and e x p e r i m e n t a l rats. I n c o r p o r a t i o n of a c e t a t e into c h o l e s t e r o l in rats is v e r y r a p i d and the peak of r a d i o a c t i v i t y o c c u r s w i t h i n the f i r s t 15 min after injection of a c e t a t e in b o t h c o n t r o l rats and t h o s e e x p o s e d to CS 2 . T h e r e a f t e r , the s p e c i f i c r a d i o a c t i v i t y of c h o l e s t e r o l declines. The rate of d e c l i n e b e t w e e n 15 m i n and 3 hrs a f t e r i n j e c t i o n of 1 4 C - a c e t a t e is g r e a t e r in rats e x p o s e d to CS 2 (compared r e g r e s s i o n c o e f f i c i e n t s are s i g n i f i c a n t l y d i f f e r e n t ; P < 0.05). The s p e c i f i c r a d i o a c t i v i t y of c h o l e s t e r o l in the liver of rats i n t o x i c a t e d w i t h CS 2 at all time i n t e r v a l s s t u d i e d is a b o u t twice as g r e a t as in c o n t r o l a n i m a l s (Fig.l). T h e s e two facts s u g g e s t that e x p o s u r e to CS 2 s t i m u l a t e s the i n c o r p o r a t i o n of a c e t a t e into c h o l e s t e r o l and a c c e l e r a t e s the t u r n o v e r r a t e of c h o l e s t e r o l in the liver. The r i s e in the rate of s y n t h e s i s of h e p a t i c c h o l e s t e r o l is not r e f l e c t e d by the c h a n g e s in the level of liver chole s t e r o l and the w e i g h t of the liver. The c o n c e n t r a t i o n of total c h o l e s t e r o l in the liver and w e i g h t of the liver in CS 2 i n t o x i c a t e d rats, r e m a i n e s e n t i r e l y u n c h a n g e d as c o m p a r e d w i t h the c o n t r o l ones (Table 2). T h e s e r e s u l t s are s i m i l a r to t h o s e of G o u l d et el. E5~, B e r n d t & G a u m e r t [I~ and W r o ~ s k a N o f e r & K n o b l o c h E I ~ , w h o found an i n c r e a s e d i n c o r p o r a t i o n of a c e t a t e also in X - i r r a d i a t e d a n i m a l s w i t h o u t any c h a n g e s in the c o n c e n t r a t i o n of t o t a l c h o l e s t e r o l . The s p e c i f i c a c t i v i t y of s e r u m c h o l e s t e r o l (Fig.2) in both e x p e r i m e n t a l g r o u p s was s o m e w h a t lower than in the liver at c o r r e s p o n d i n g time i n t e r v a l s . The p e a k of r a d i o a c t i v i t y of s e r u m c h o l e s t e r o l o c c u r s at a p p r o x i m a t e l y 60 min a f t e r the i n j e c t i o n of 1 4 C - a c e t a t e in both c o n t r o l and CS 2 i n t o x i c a t e d rats. The i n c r e a s e of the s p e c i f i c a c t i v i t y in s e r u m o b s e r v e d in the rats e x p o s e d to CS 2 r e f l e c t s the c h a n g e s found in the liver, the m a g n i t u d e of w h i c h b e g i n n i n g f r o m 3 hrs a f t e r a c e t a t e i n j e c t i o n are similar. T a b l e 3 i l l u s t r a t e s the r e s u l t s of the e x p e r i m e n t in w h i c h E 2 - ! 4 C ~ m e v a l o n a t e was u s e d as a l a b e l e d p r e c u r s o r in the synt h e s i s of c h o l e s t e r o l in the liver. The i n c r e a s e of i n c o r p o r a t i o n of m e v a l o n a t e into c h o l e s t e r o l in the rats e x p o s e d to CS 2 a m o u n t e d to a b o u t 30%. T h e s e r e s u l t s i n d i c a t e that the b i o s y n t h e s i s of c h o l e s t e r o l in rats i n t o x i c a t e d w i t h CS 2 is i n c r e a s e d b o t h f r o m 1 4 C - a c e t a t e and 1 4 C - m e v a l o n a t e , but the e x t e n t of that rise is m u c h h i g h e r w h e n 1 4 C - a c e t a t e is u s e d as a p r e c u r s o r . C o n s i d e r i n g these data, one m i g h t c o n c l u d e

225

Table 2 Weight of the liver and liver cholesterol concentration in control and exposed to CS 2 rats a Group of animals

Cholesterol concentration (rag/g)

Liver weight (g)

Control

2.1 + 0.22 (35)

6.7 • I.o (35)

CS

2.0 • 0.27 (32)

6.8 -+ 0.8 (32)

2

The data given in Table 2 are derived from the study of the rats used in all experiments presented in this paper. Number of rats in group is given in parenthesis. Each value represents an average • S.D.

~

2.6

.c_

1,1

== 24

2~

22

"r.

~

1.8

I 60

I 120

~

k

I

I

180

2/-,0

300

360

Time [mini

Fig.2 Specific radioactivity 14C-cholesterol in s e r u m of c o n t r o l and intoxicated w i t h CS 2 rats. T h e g r o u p s of c o n t r o l r a t s a n d t h o s e e x p o s e d to CS 2 w e r e i n j e c t e d i n t r a p e r i t o n e a l l y with E2-14C~acetate. Specific radioactivity of serum cholesterol was estimated at d i f f e r e n t intervals after injection. Each point represents an a v e r a g e of 3-5 rats. R e s u l t s a r e g i v e n as m e a n • S.D. F o r d e t a i l s s e e " M e t h o d s " . • = D i f f e r e n t from the corresponding control (P < 0.05)

that the stimulating e f f e c t of CS 2 in t h e p r o c e s s of c h o l e s t e r o l s y n t h e s i s is l o c a t e d b e t w e e n a c e t a t e a n d m e v a l o n a t e , a l t h o u g h a n o t h e r s i t e of CS 2 e f f e c t , b e h i n d m e v a l o n a t e cannot be excluded. Formation of m e v a l o n a t e from 6-hydroxy-B-methylglutaryl-Co A according to G o u l d & P o p j a k E ~ a n d B u c h e r e t al. [ ~ is t h e r a t e - l i m i t i n g s t e p in c h o l e s t e r o l biosyn-

226

Table 3 Incorporation of E2-14C]mevalonate into liver cholesterol of CS 2 intoxicated and control rats a Group of animals

Specific radioactivity of liver cholesterol

(cpm/mg) Control

2025 • 170

CS 2

2647 • 169

b

The groups of control rats and those exposed to CS 2 were injected intraperitoneally with 14C-mevalonate. Specific radioactivity of 14C-cholesterol in liver was estimated 2 hrs after injection. Each value represents an average of 5 rats • S.D. For details see "Methods". Different from control (P < 0.002).

thesis. esterol

The above synthesis

stage on the biosynthetic is c h a n g e d b y s t a r v a t i o n

pathway D2,1~,

of c h o l X-ray

irradiation D,2,41 and after treatment of the animals with actinomycin D E3~. C S 2 a n d X - r a y s h a v e a s i m i l a r e f f e c t o n the process of cholesterol synthesis. In t h e c a s e o f X - r a y irradiation, it is s u g g e s t e d t h a t t h e s t i m u l a t i o n o f t h e synthesis of cholesterol is p l a c e d b e t w e e n a c e t a t e a n d m e v a l o n a t e a n d is c o n n e c t e d w i t h t h e a c t i v i t y o f t h e e n z y m e limiting cholesterol synthesis, i.e. B - h y d r o x y - 8 - m e t h y l glutaryl-Co A reductase. Similar disturbance i n v o l v e d in t h e p r o c e s s o f CS 2 i n t o x i c a t i o n and X-ray irradiation E19~ suggest the same mechanism of the effect of CS 2 on cholest e r o l s y n t h e s i s in t h e r a t s . T h e h y p o t h e s i s should be directly v e r i f i e d i n t h e f u t u r e r e s e a r c h .

REFERENCES I. Berndt, J., Gaumert, R.: The in vitro biosynthesis of cholesterol and the cholesterol content in the liver of x-irradiated mice. Radiat.Res. 4_~2, 292-304 (1970) 2. Bucher, N.L.R., McGarrahan, K., Gould, E., Loud, A.V.: Cholesterol biosynthesis in preparation of liver from normal, fasting, X-irradiated cholesterol-fed, triton or ~4-cholesten-3-one-treated rats. J.Biol. Chem. 234, 262-267 (1959) 3. De Matteis, F.: Stimulation of liver cholesterol synthesis by actinomycin D. Biochem.J. 109, 775-785 (1968)

227

4. Gould, R.G., Bell, V.L., Lilly, E.M.: Effects of x-irradiation on cholesterol, fatty acid and protein synthesis in rat tissues. Radiat. Res. 5, 609 (1956) 5. Gould, R.G., Bell, V.L., Lilly, E.H.: Stimulation of cholesterol biosynthesis from acetate in rat liver and adrenals by whole body irradiation. Amer.J.Physiol.

196, 1231-1237

(1959)

6. Gould, R.G., Popjak, G.: Biosynthesis of cholesterol in vivo and in vitro from

DL-~-hydroxy-B-methyl-~-(2-14C)-valerolactone.

Biochem.J.

66, 51P (1957) 7. Kabara, J.J., McLaughlin, J.T.: A micro dibromide procedure for purifying radioactive cholesterol. J.Lip.Res. 2, 283-285

(1961)

8. Nofer, J., Chojnowski, J., Kawecka, M., Kie~, E., Wroflska-Szpakowa,T., Wyszomirska, Z.: Effect of occupational hazard to carbon disulphide on the pathogenesis of atherosclerosis (Polish). Med.Pracy 12,

101-118 (1961) 9. Schwenk, E., Werthessen, N.T.: Studies on the biosynthesis of cholesterol. IV. Higher counting substances accompanying Cl4-cholesterol in the intact rat. Arch.Biochem. Biophys. 42, 91-93 (1953) 10. Sperry, W.M., Vebb, M.: A revision of the Schoenheimer-Sperry method for cholesterol determination. J.Biol.Chem. 187, 97-105 (1959) Ii. Swell, L., Trout, E.C., Field, H., Treadwell, C.R.: The role of lymph cholesterol in the regulation of endogenous cholesterol and cholesterol ester synthesis. J.Biol.Chem. 230, 631-641 (1958) 12. Tomkins, G.M., Chaikoff, I.L.: Cholesterol synthesis by liver. I. Influence of fasting and of diet. J.Biol.Chem. 196, 569-573

(1952)

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19. W r o h s k a - N o f e r ,

T., Knobloch,

K.: The i n f l u e n c e of v a r i a t i o n of the

d a i l y r h y t h m of e x p o s u r e upon the m a g n i t u d e of b i o l o g i c a l a c o m p a r i s o n of X - r a y i r r a d i a t i o n Bull.Acad. Pol. Sci. 20. W r o h s k a - N o f e r ,

20, 8 1 3 - 8 1 9

T., Nofer,

t r o u b l e s du m @ t a b o l i s m e carbone. pp.

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effects:

intoxication.

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J.: R e c h e r c h e s

exp~rimentales

des lipides sous l ' i n f l u e n c e

Excerpta Medica Monograph

161-164,

and c a r b o n d i s u l p h i d e

sur les

du sulfure de

" T o x i c o l o g y of C a r b o n Disulphide",

(1966)

Dr. T e r e s a W r o h s k a - N o f e r Department

of B i o c h e m i s t r y

I n s t i t u t e of O c c u p a t i o n a l M e d i c i n e P.O.

Box 199

90-950 ~Sd~,

Poland

229