The effect of turpentine-induced inflammation on rat liver glycosyltransferases and golgi complex ultrastructure

Biochimica et Biophysica Acta, 629 (1980) 1--12

© Elsevier/North-HollandBiomedicalPress

BBA 29211

THE EFFECT OF TURPENTINE-INDUCED INFLAMMATION ON RAT LIVER GLYCOSYLTRANSFERASES AND GOLGI COMPLEX ULTRASTRUCTURE

CHRISTIAN LOMBART a,,, J E N N I F E R STURGESS a and H A RRY SCHACHTER b

Departments of a pathology and b Biochemistry, The Hospital for Sick Children, Toronto (Canada) (Received June 28th, 1979) (Revised manuscript received October 12th, 1979)

Key words: Turpentine; Inflammation; Glycosyltransferase; Golgi complex ultrastructure; (Rat liver)

Summary Turpentine-induced inflammation in the rat caused a 1.6--2.3-fold increase in liver homogenate sialyl-, galactosyl-and N-acetylglucosaminyltransferase total and specific enzyme activities. Peak transferase activities were achieved at about 40 h after turpentine injection; the rise and fall of these activities corresponded to a similar rise and fall in serum haptoglobin levels. Sialyl- and N-acetylglucosaminyltransferase activities were measured in both liver homogenates and Golgi-enriched membranes at 24 h after turpentine injection; both total and specific enzyme activities doubled in the homogenates following turpentine treatment but in the Golgi-enriched membranes only the total enzyme activities doubled while the specific enzyme activities increased only by about 20%. These findings suggest that turpentine injection results in an increase of Golgi complex protein relative to total cellular protein. This conclusion was supported by electron microscopic studies of rat liver at various times after turpentine injection. The increased glycosylation potential of the liver and the proliferation of liver Golgi complex may play an important role in the turpentine-induced secretion of acute-phase glycoproteins.

Introduction The injection of turpentine and other chemical inflammatory agents, the growth of turnouts and various other pathological conditions have long been * Present address: U . E . R . B i o m ~ d i c a l e des Saints-P6res, Paris, France. Abbreviations: Pipes, piperazine-N, Nt-bis(2-ethancsulphonic acid); Mes, 2-(N-morpholino)ethanesulphonic acid.

known to cause an increase in plasma total protein-bound carbohydrate in mammals [ 1,2]. Specific serum glycoproteins, known as acute-phase reactants [1], have been shown to contribute to this increase in plasma protein-bound carbohydrate, i.e., haptoglobin, fibrinogen, a:-globulins, al-acid glycoprotein and others. Numerous studies have indicated that the increase in plasma acutephase reactants was due to an increased synthesis of these glycoproteins by the liver [1--8]. Many of these studies involved the administration to animals of radioactive leucine or glucosamine followed by the isolation of glycoprotein fractions [2,6], a 1-acid glycoprotein [3--5], a2-macroglobulin [3] and haptoglobin [8] from serum and liver. Such studies have also been used to establish that the liver is the major site of synthesis of these plasma glycoproteins [8--10]. Albumin, a serum protein which does not contain carbohydrate, is assembled within the liver on membrane-bound ribosomes and passes through the liver's endomembrane system to the Golgi complex and then to the extracellular space [11]. There is a great deal of evidence that all glycoproteins secreted by the liver also pass through this endomembrane system [5,12,13] on their way out of the liver cell. The Golgi complex is an essential component of this process since it is the subcellular site at which the more peripheral sugars (L-fucose, sialic acid, D-galactose and some N-acetyl-D-glucosamine} are incorporated into protein-bound oligosaccharides [12--14]. It is therefore of great interest to determine the effect of turpentine injection on the structure and function of the Golgi complex. The effect of various drugs and disease processes on the Golgi complex has been reviewed recently [15]. For example, injection of the aminonucleoside portion of puromycin into rats caused an increase in liver glycoprotein synthesis, an increase in plasma glycoproteins and a 2--3-fold increase in the volume of the liver Golgi complex. An increase in Golgi complex volume may reflect either an increase in the Golgi membrane system with an accompanying increase in glycosylation potential, as appears to be the situation following aminonucleoside treatment [16--18], or a dilatation of the Golgi complex due to a block in the release of secretory material, as occurs in certain types of hepatocellular damage [ 15] or after colchicine treatment [ 19]. Golgi complex atrophy has been observed after prolonged protein or choline deficiency or following drug-induced inhibition of protein synthesis [15]. The effects of aminonucleoside and of the inhibition of protein synthesis on Golgi-localized glycosyltransferase activities have been studied but no major changes were observed [ 15]. Turchen et al. [20] have recently reported that turpentine-induced inflammation in the rat caused dilatation of liver endoplasmic reticulum, increased vesiculation of endoplasmic reticulum and increased amounts of smooth endoplasmic reticulum and Golgi complex relative to rough endoplasmic reticulum. These changes were most marked at 12--24 h after turpentine injection and were accompanied at 24 h after injection by a 2-fold increase in microsomal sialyl- and galactosyltransferase specific activity. The present study confirms the report of Turchen et al. [20] and, in addition, presents a quantitative morphometric analysis of Golgi complex hypertrophy, a kinetic comparison of serum haptoglobin levels and liver sialyl-, galactosyl- and N-acetylglucosaminyltransferase activities following turpentine injection, and, finally, a comparison of total and specific enzyme activity changes in homoge-

nates and Golgi-enriched membranes from rat liver to determine the nature of the increased microsomal glycosyltransferase levels observed by Turchen et al. [20]. The data indicate that turpentine injection causes a more than 2-fold increase in Golgi complex volume, that serum haptoglobin and liver glycosyltransferase levels follow similar kinetics after turpentine injection, and that the increased liver glycosyltransferase activities are due to a proliferation of Golgi membranes rather than the specific induction by turpentine of glycosyltransferases. Materials and Methods

Animals Male Wistar rats weighing 200--250 g were purchased from Canadian Farms and Breeding Laboratories Ltd., St. Constant, Quebec. Inflammation was induced by subcutaneous injection of 0.5 ml oil of turpentine/100 g body weight into the thigh. Control rats received a similar volume of saline. The animals were maintained on a diet of laboratory chow and water ad libitum and were fasted for 16 h prior to killing. Preparation o f rat serum samples and liver homogenates Rats were anesthesized with 0.2 ml 2.5% thiopental sodium by intramuscular injection. Blood was collected with a catheter from the aorta and was allowed to clot at room temperature. Serum was prepared by centrifuging at 2500 X g for 10 min and was stored at --20°C until used. The livers were excised rapidly, washed in ice-cold saline, blotted with filter paper and weighed. About 4 g liver were minced with scissors and homogenized in 2 vols. of ice-cold buffer A (0.05 M Tris-HC1, pH 7.0, 0.01 M MgC12, 0.001 M dithiothreitol and 0.25 M sucrose) by use of a motor-driven Potter-Elvehjem homogenizer with Teflon pestle. Three up-and-down strokes were carried out over a 2 min period. The homogenates were filtered through four layers of surgical gauze, their volumes were measured and they were kept frozen at --20°C until assayed. Preparation o f Golgi-rich membrane fractions After anesthesia, the rats were exsanguinated and serum prepared as above. The liver Golgi-rich fractions were prepared as described previously [21]. They were kept frozen at --20°C until assayed. G ly cosy l transferase assays The preparation of acceptors for the assays and the detailed assay procedures have been described previously [22,23]. Sialyltransferase (EC 2.4.99.1) assay. Each incubation contained 1 mg sialidase-treated a 1-acid glycoprotein, 2.5 pmol piperazine-N,N'-bis(2-ethanesulphonic acid) (Pipes), pH 7.2, 0.25 pl Triton X-100, 0.05 pmol CMP-N-[X4C]acetylneuraminic acid (0.74. l 0 s cpm/pmol) and enzyme in a final volume of 0.040 ml. The CMP-N-[ ~4C]acetylneuraminic acid was prepared as previously described [24,25]. Incubations were carried out at 37°C for 1 h, the reaction was stopped by addition of 0.010 ml 2% sodium tetraborate and 0.040 ml of this solution was then subjected to high-voltage paper electrophoresis in 1%

sodium tetraborate and product was assayed as previously described [22,23]. N-A cety lglucosaminyltransferase (EC 2.4.1.51) assay. Each incubation contained 0.5 mg a l-acid glycoprotein pretreated with sialidase, fl-galactosidase and fl-N-acetylglucosaminidase [23], 5.0 pmol 2-(N-morpholino)ethanesulphonic acid (Mes), pH 5.7, 0.25 pl Triton X-100, 0.25 pmol MnC12, 0.028 pmol UDPN-acetyl-D-[1-14C]glucosamine ( 3 . 1 . 1 0 6 cpm/pmol) and enzyme in a total volume of 0.040 ml. UDP-N-acetyl-D-[1-14C]glucosamine (56.5 Ci/mol) was purchased from New England Nuclear and diluted with non-radioactive UDPN-acetylglucosamine from Sigma. Incubations were carried out at 37°C for 1 h. The reaction was stopped by addition of 0.040 ml 10% trichloroacetic acid/2% phosphotungstic acid. The resulting suspension was filtered with suction on a Whatman glass-fibre filter (type GF/C). The filter was washed with 30 ml ice-cold 5% trichloroacetic acid/l% phosphotungstic acid followed by 25 ml CHC13/CH3OH/H20 (1 : 1 : 0.3). The filters were dried under an infrared lamp and subjected to liquid scintillation counting in a toluene-based medium [22]. Galactosyltransferase (EC 2.4.1.38) assay. Each incubation contained 0.25 mg sialidase, fl-galactosidase-treated a 1-acid glucoprotein, 5.0 pmol Mes, pH 5.7, 0.3 pmol dithiothreitol, 3.0 pmol MnC12, 0.25 #l Triton X-100, 0.050 pmol UDP-[U-14C]galactose (3.0 • 106 cpm/pmol) and enzyme in a final volume of 0.050 ml. UDP-[U-~4C]galactose (281 Ci/mol) was purchased from New England Nuclear and diluted with non-radioactive UDP-galactose from Calbiochem. Incubations were carried out at 37°C for 1 h and the reactions were stopped and product formation determined as described above for N-acetylglucosaminyltransferase.

Serum haptoglobin assays The concentration of haptoglobin in the sera of normal and turpentinetreated rats was determined either by measurement of the peroxidase activity of the haptoglobin-hemoglobin complex [26] or by i m m u n o n e p h e l o m e t r y [27]. Protein assays Protein was determined by the m e t h o d of Lowry et al. [28] using bovine serum albumin as standard. Electron microscopy Electron microscopic studies were carried out on the livers of six groups of rats, namely, pair-matched controls and turpentine-injected rats at 24, 48 and 96 h following injection. Three rats were examined in each group. After exsanguination, samples of liver were resected from each rat, cut into blocks approximately 1 m m 3 in size and fixed with cold 2.7% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 3 h at 4°C. Tissues were post-fixed with 1% OsO4 in 0.1 M veronal acetate buffer, pH 7.4, block-stained with 1% uranyl acetate in 25% ethanol, dehydrated in graded ethanol solutions and embedded in Spurr low-viscosity epoxy resin. Ultrathin sections were stained with lead citrate and uranyl acetate and examined in a Philips EM 201 electron microscope at 60 kV. At least four blocks of liver from each rat were sectioned and examined by electron microscopy.

Quantitative studies were carried out on electron micrographs from control and turpentine-treated rat livers to determine changes in the Golgi apparatus. The livers were quantified using randomly sampled electron micrographs as described previously [29]. Golgi cisternal elements were identified on every micrograph and were joined together to outline an approximate shape for the Golgi complex; the largest diameter of this shape was then measured on every micrograph and an average diameter determined for every group of livers. Since the amount of Golgi complex visible in any one section varies, this method gives only a relative measure of Golgi complex hypertrophy. The number of cisternae per Golgi complex was also determined and averages were calculated for every group of livers. Results and Discussion

Figs. 1 and 2 show the changes in liver homogenate glycosyltransferase specific activities as a function of time after the administration of turpentine. The extent of the acute inflammatory response to turpentine in the rats used for the transferase studies is demonstrated by the increase in serum haptoglobin concentrations which are also plotted in Figs. 1 and 2. The maximum haptoglobin concentration is achieved at about 40 h after turpentine injection. The liver sialyl- and N-acetylglucosaminyltransferase specific activities also peak at about 40 h after turpentine administration while the galactosyltransferase levels

GAL, S.A

Hp

mglml

"a:

/

/

0

20

40

hrs

after

gO

8'0

turpent.ine

160

Fig, I . E f f e c t o f t u r p e n t i n e - i n d u c e d i n f l a m m a t i o n o n s l a l y l t r a n s f e r a s e (S.A., e ) a n d g a l a c t o s y l t r a n s f e r a s e (Gal, i ) specific activities ( e x p r e s s e d as n m o l of sugar t r a n s f e r r e d l m g p r o t e i n p e r h ) in r a t l i v e r h o m o g e h a t e s . S e r u m h a p t o g l o b i n c o n c e n t r a t i o n s ( H p , o) are also p l o t t e d (in m g / m l ) . E a c h t i m e P o i n t r e p r e s e n t s the m e a n v a l u e f r o m several a n i m a l s ; t h e n u m b e r o f a n i m a l s u s e d a t e a c h t i m e p o i n t is i n d i c a t e d in p a r e n theses. T r a n s f e r a s e assays o n e a c h liver h o m o g e n a t e w e r e c a r r i e d o u t a t t w o p r o t e i n c o n c e n t r a t i o n s u n d e r c o n d i t i o n s of p r o p o r t i o n a l i t y w i t h r e s p e c t t o e n z y m e p r o t e i n a n d t i m e of i n c u b a t i o n , T h e ve1"tical b a r a t e a c h p o i n t i n d i c a t e s t h e S.E.; this c a l c u l a t i o n w a s c a r r i e d o u t o n l y if m o r e t h a n t h r e e a n i m a l s w e r e u s e d . T h e r e p r o d u c i b i l i t y o f assays b e t w e e n d i f f e r e n t a n i m a l s a t the s a m e t i m e p o i n t w a s e x c e l l e n t .

Hp m g l rnl

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o

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8'0

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Fig. 2. E f f e c t o f i n f l a m m a t i o n on liver h o m o g e n a t e N - a c e t y l g l u c o s a m i n y l t r a n s f e r a s e (e) specific a c t i v i t y a n d o n s e r u m h a p t o g l o b i n c o n c e n t r a t i o n ( H p , Q). E a c h t i m e p o i n t r e p r e s e n t s t h e m e a n v a l u e f r o m six diff e r e n t animals. T r a n s f e r a s e assays w e r e c a r r i e d o u t as d e s c r i b e d in the legend to Fig. 1. T h e vertical b a r at each t i m e p o i n t represents the S.E.

peak somewhat earlier. The liver sialyl- and galactosyltransferase specific activities decrease slightly in the first 5--10 h after turpentine injection (Fig. 1); a similar p h e n o m e n o n is observed with serum haptoglobin levels (Fig. 1) and with liver haptoglobin levels (data not shown). Since early time points were not taken for N-acetylglucosaminyltransferase levels (Fig. 2), it is not known if the early decrease also occurs with this enzyme. The significance of this early decline is n o t known but it appears that turpentine injection at first inhibits biosynthesis of acute-phase glycoproteins such as haptoglobin. The maximum stimulations for the three transferases are 2.3-fold, 2.0-fold and 1.6-fold for the sialyl-, galactosyl- and N-acetylglucosaminyltransferases, respectively. In an a t t e m p t to determine the nature of the transferase stimulation, sialyltransferase activity was measured in liver homogenates and in Golgi-rich membranes prepared from the livers of control rats and rats 24 h after turpentine injection (Table I). The liver homogenates showed over 2-fold stimulation in both the specific activity and total activity of sialyltransferase. These observations with liver homogenates indicate either an increased synthesis or an activation of the transferases relative to the total protein of the liver. The Golgi apparatus in rat liver is known to be the major subcellular site of the transferases studied in the present report [12,13,22]. It was therefore important to study Golgi transferase levels after turpentine injection. The total sialyltransferase activity in Golgi-rich membranes was doubled at 24 h after turpentine injection, as was the case for the liver homogenates, but the Golgi specific activity increased very little. The recovery of sialyltransferase activity in Golgi-rich membranes was 22--23% for both control and treated animals. The drop in

Controls 24 h a f t e r t u r p e n t i n e

Treatment

0.0192 ± 0.0013 0.0398 ± 0.0029

2 3 . 3 + 1.5 4 9 . 7 ± 3.7

1.06 + 0.08 1.29 ± 0 . 1 6

Specific a c t i v i t y (~mol/mg protein per h)

Specific a c t i v i t y (/~mol/mg protein per h)

Total activity Gttmol/liver p e r h )

Golgi-rich m e m b r a n e s

Liver homogenates

5.31 ± 0 . 3 1 11.1 ± 1.11

Total activity (#tool/liver per h)

55.2 32.4

Purification factor

E n z y m e a c t i v i t y d e t e r m i n a t i o n s w e r e c a r r i e d o u t o n h o m o g e n a t e s a n d G o l g i - r l c h m e m b r a n e s f r o m five s e p a r a t e r a t s i n e a c h g r o u p a n d t h e r e s u l t s are r e p o r t e d as t h e m e a n ± S.E.

S I A L Y L T R A N S F E R A S E L E V E L S OF R A T L I V E R H O M O G E N A T E S A N D G O L G I - R I C H M E M B R A N E S

TABLE I

purification factor on turpentine treatment (from 55.2 to 32.4) indicates that there is an approximately 1.7-fold increase in Golgi membranes relative to total cell protein, provided other factors remain unchanged. The slightly higher specific activity in the Golgi-rich membranes from turpentine-treated rats may, however, indicate a higher degree of purity in these Golgi preparations; an analogous observation was reported for Golgi-rich membranes isolated from the livers of rats after ethanol administration [ 30]. Golgi-rich membranes were also assayed for N-acetylglucosaminyltransferase activities and data very similar to that shown in Table I were obtained (not shown). These findings suggest that the increased liver transferase levels are due to an increase in Golgi apparatus membranes relative to other components of the liver. Control experiments were carried o u t to determine if turpentine injection produced factors in the liver which might be either stimulatory or inhibitory towards glycosyltransferase activities. Table II shows that the addition of liver homogenate from control and treated rats to a Golgi-rich preparation from a control liver caused a 27% and 17% stimulation of sialyltransferase activity, respectively. Thus the enzyme increase in liver homogenates from treated rats cannot be due to a turpentine-induced factor which stimulates sialyltransferase activity; the existence of such a factor would have been detected by an appreciably higher enzyme activity in the mixture containing homogenate from turpentine-treated rats and Golgi from control rats than in the mixture containing control homogenate and control Golgi m e m b r a n e s . Similar results were obtained for liver galactosyltransferase (data n o t shown). The conclusion was therefore drawn that turpentine injection caused a proliferation of Golgi complex membranes with an accompanying increased synthesis of Golgi-located glycosyltransferases relative to total liver protein. To obtain support for this hypothesis and to determine whether other liver membranes also proliferate after turpentine injection, extensive electron micro-

T A B L E II SIALYLTRANSFERASE RICH MEMBRANES

A C T I V I T I E S O F M I X T U R E S OF L I V E R H O M O G E N A T E S

AND GOLGI-

Golgi-rich m e m b r a n e s w e r e p r e p a r e d f r o m n o r m a l r a t liver a n d w e r e u s e d t o detect the presence of a stimulatory or i n h i b i t o r y f a c t o r in liver homogenates prepared from both c o n t r o l a n d turpentine-injected rats. The latter were killed 24 h a f t e r i n j e c t i o n . S i a l y l t r a n s f e r a s e activities are averages of d u p l i c a t e d e t e r minations. E n z y m e source

Sialyltransferase activity (cpm in the incubation/h)

5 ~zl Golgi-rich m e m b r a n e s 5 jul control homogenate 5 ~ul Golgi + 5 ~l control homogenate Expected activity, if a d d i t i v e Stimulation by control homogenate (%)

690 1270 2490

5/~I Golgi-rich m e m b r a n e s 5/~I 'turpentine homogenate' 5/~I Golgi + 5 ~I 'turpentine homogenate' Expected activity, if additive Stimulation by 'turpentine h o m o g e n a t e ' (%)

690 1250 2270

1960 27.0

1940 17.0

9

scopic examination of livers from control and turpentine-treated rats was carried out. All livers from control rats showed a characteristic normal hepatic ultrastructure. At 24 h after turpentine injection, the rough endoplasmic reticulum appeared normal and was arranged in well-ordered arrays suggesting that the hepatocytes were actively engaged in protein synthesis; mitochondria and smooth endoplasmic reticulum also appeared normal in these livers. However, the livers at 24 h after injection showed a marked proliferation of the Golgi complex (Fig. 3a). Large numbers of transition vesicles were observed budding from the rough endoplasmic reticulum towards the forming face of the Golgi apparatus (Fig. 3b). The Golgi cisternae at the mature face of the Golgi complex were dilated and contained flocculent electron-dense material (Fig. 3c). In addition, there was an increase in lipid droplets and numerous small vesicles were observed at the plasma membrane adjoining the space of Disse. These changes are more consistent with an increased export of secretory materials from the hepatocytes than with a Golgi dilatation secondary to a block in secretion. At 48 h after turpentine injection a further increase in Golgi complex hypertrophy was observed and Golgi elements were often seen to be arranged in large rings (Fig. 3d). Microtubules were prominent and were arranged in arrays parallel to the surfaces of the Golgi cisternae. Extensive secretory vesicles were evident (Fig. 3e). At 96 h after injection, changes were evident in organelles other than the Golgi complex. The rough endoplasmic reticulum showed degranulation and vesiculation, mitochondria showed swelling with prominent dense granules in the matrix, and numerous fatty droplets were observed in the cytoplasm. Although the Golgi complex was still larger than normal, regressive changes were now evident; the cisternae appeared fragmented and the cisternal contents showed a marked increase in electron-dense material (Fig. 3f). Table III shows a quantitative assessment of Golgi complex changes following turpentine injection. It is seen that the average number of cisternae per Golgi stack increases. The average diameter of the Golgi complex visible in the electron micrograph sections increases over 2-fold. These changes indicate proliferation of Golgi complex membranes, primarily in the cisternae, following turpentine injection.

T A B L E III QUANTITATION

OF HEPATOCYTE GOLGI COMPLEX Controls

T i m e after t u r p e n t i n e injection 24 h

48 h

96 h

N u m b e r o f cisternae per Golgi s t a c k Mean Range

2.6 2--4

3.2 2--4

3.1 2--6

3.6 2--7

D i a m e t e r o f G o l g i c o m p l e x ~um) Mean S.E.

1.6 0.08

2.6 0.11

3.1 0.13

3.7 0.32

10

Fig. 3. E l e c t r o n m i c r o g r a p h s o f h e p a t o c y t e s f r o m r a t liver s h o w i n g u l t r a s t r u c t u r a l c h a n g e s in t h e Golgi a p p a r a t u s at v a r i o u s t i m e s a f t e r a d m i n i s t r a t i o n of t u r p e n t i n e . (a) 24 h: the Golgi a p p a r a t u s is e x t e n s i v e a n d establishes c o n t i n u i t y in t h e c y t o p l a s m ( a r r o w s ) . T h e d i a m e t e r of this Golgi c o m p l e x f o r the p u r p o s e s o f q u a n t i t a t i o n ( T a b l e I I I ) w o u l d b e t h e d i s t a n c e b e t w e e n t h e a r r o w s n e a r e s t e a c h edge of t h e m i c r o g r a p h . M a g n i f i c a t i o n : 13 0 0 0 . (b) 2 4 h: a t r a n s i t i o n vesicle ( a r r o w ) is seen arising f r o m the r o u g h e n d o p l a s m i c r e t i c u l u m (rer) a n d p o i n t i n g t o w a r d s t h e Golgi c o m p l e x (c). M a g n i f i c a t i o n : 45 000. (c) 2 4 h: a c i s t e r n a (c)

11

a t t h e m a t u r e face of t h e Golgi c o m p l e x is d i s t e n d e d w i t h e l e c t r o n - d e n s e m a t e r i a l ( a r r o w ) . M i c r o t u b u l e s ( m ) are p r o m i n e n t in t h e c y t o p l a s m s u r r o u n d i n g t h e Golgi c o m p l e x . M a g n i f i c a t i o n : 34 000. (d) 48 h: a h y p e r t r o p h i e d Golgi c o m p l e x f o r m i n g a ring-like s t r u c t u r e ( a r r o w s ) . N u m e r o u s s e c r e t o r y vesicles are visible. T h e d i a m e t e r o f this Golgi c o m p l e x f o r t h e p u r p o s e s o f q u a n t i t a t i o n ( T a b l e I I I ) w o u l d be t h e largest d i a m e t e r o f t h e ring-like s t r u c t u r e , i.e., r o u g h l y t h e d i s t a n c e b e t w e e n the a r r o w s in the l e f t u p p e r a n d r i g h t l o w e r c o r n e r s o f t h e m i c r o g r a p h . M a g n i f i c a t i o n : 27 0 0 0 . (e) 4 8 h: s e c r e t o r y vesicles ( a r r o w s ) are seen b u d d i n g f r o m t h e c i s t e r n a e a t t h e m a t u r e face of a Golgi c o m p l e x . M a g n i f i c a t i o n : 34 0 0 0 . (f) 9 6 h: t h e c i s t e r n a e o f t h e Golgi c o m p l e x ( a r r o w s ) are f l a t t e n e d , f r a g m e n t e d a n d c o n t a i n v e r y e l e c t r o n - d e n s e material. Magnification: 66 000.

12 The electron microscopic studies therefore support the glycosyltxansferase data (Table I) that turpentine does not induce a limited number of transferases in a specific manner but rather causes a generalized proliferation of Golgi membranes. This proliferation and the accompanying increased glycosylation potential of the liver probably play important roles in the increased synthesis of acute-phase glycoproteins by the livers of turpentine-injected rats.

Acknowledgements This research was supported by grants from the Medical Research Council of Canada to J.S. and H.S. and by grant 76-10693 from the 'Institut National de la Sant~ et de la Recherche M~dicale' to C.L.

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