V'uvhowsArch/vB

Virchows Arch. B Cell Path. 27, 279-306 (1978)

9 by Springer-Verlag 1978

Studies on the Cellular Toxicity of Polychlorinated Biphenyls (PCBs) I. Effect of PCBs on Microsomal Enzymes and an Synthesis and Turnover of Microsomal and Cytoplasmic Lipids of Rat LiverA Morphological and Biochemical Study David E. Hinton 1, Hans Glaumann 2 and Benjamin F. Trump 1 1 Comparative and Environmental Pathobiology Program, Department of Pathology, University of Maryland School of Medicine, 31 South Greene Street, Baltimore, Maryland, USA 21201 2 Department of Pathology, Huddinge Hospital, Karolinska Institutet, Stockholm, Sweden 11384

Summary. The acute effects of the PCB (polychlorinated biphenyls) mixture (Aroclor 1254) on microsomal enzymes and on synthesis and turnover of microsomal and cytoplasmic lipids of rat liver were investigated. Six daily i.p. injections of 25 and 50 mg PCB/kg body weight resulted in increased liver weight and liver to body weight ratios. When compared to controls PCB treatment resulted in a six-fold increase in amount of cytochrome P-450. Activities of NADPH-cytochrome c reductase, ethylmorphine demethylase and inosine diphosphatase were increased whereas glucose-6-phosphatase values were decreased by PCB exposure. Analysis of liver homogenate and microsomal fraction revealed an increase in lipid in PCB-exposed animals. Phospholipids, cholesterol and triglyceride were significantly increased after PCB exposure; however, the greatest percentage increase was seen in the triglyceride pool. The finding of an increase in microsomal triglyceride to phospholipid ratios with exposure to PCB is suggestive of an increase in membrane-enclosed lipid (liposomes). Studies with labelled glycerol indicated that the PCB-induced fatty liver resulted from increased half life but not increased synthesis of liver lipid moieties. The rate of incorporation of leucine into microsomal membrane and albumin was somewhat enhanced in rats exposed to PCB indicative of increased protein synthesis. Morphological studies showed increased occurrence of lipid material, both in cytoplasmic droplets and within rough and smooth-surfaced endoplasmic reticulum. Proliferation of smooth endoplasmic reticulum and flattened Golgi cisternae with no secretion granules containing lipoprotein particles characterized the liver from animals exposed for 6 days. The increase in lipid within membranes of the endoplasmic reticulum together with the This is contribution 390 from the Cellular Pathobiology Laboratory. Send offprint requests to B.F. Trump, M.D.

0340-6075/78/0027/0279/$05.60

280

D.E. Hinton et al. flattened Golgi lacking typical secretory vesicles indicates a defect in transport of lipoproteins from the endoplasmic reticulum to the Golgi apparatus and may be the cause of the PCB-induced fatty liver. Key words: Polychlorinated biphenyl (PCB) - Liver - Toxicity - Lipid Synthesis - Turnover - Microsomal fraction.

Introduction

Due to their chemical inertness and superior dielectric properties polychlorinated biphenyls (PCBs) have received wide industrial application (Kay, 1973). PCBs have been recognized as persistent environmental pollutants of potential toxicologic concern to animals and man (Fishbein, 1974). When rats were exposed to the commercial PCB mixture (Aroclor 1254) via the diet (Litterst et al., 1972; Litterst and Van Loon, 1972; Litterst and Van Loon, 1974; Turner and Green, 1974) gastric intubation (Redman et al., 1975), intraperitoneal injection (Alvares et al., 1973; Bickers et al., 1972; Bickers et al., 1974; Bruckner et al., 1973) or via cutaneous application (Bickers et al., 1975) a marked increase was seen in one microsomal hemoprotein, cytochrome P-450, with little or no change in cytochrome b5 (Bruckner et al., 1974; Reynolds et al., 1975; Turner and Green, 1974). Induction of enzymes coupled to the microsomal mixedfunction oxidative system was evident by increased activity of several drug metabolizing enzymes (Alvares et al., 1973; Bickers et al., 1974; Bruckner et al., 1974; Litterst et al., 1972; Litterst and Van Loon, 1974; Reynolds et al., 1975; Turner and Green, 1974). The above enzymatic changes accompany an increase in microsomal protein (Bickers et al., 1974; Goldstein et al., 1974; Litterst and Van Loon, 1972) and liver weight (Bickers et al., 1974; Litterst and Van Loon, 1972). The potency of PCBs has been established after comparison to other known inducing agents such as 1,1,1-trichloro-2,2-bis (P-chlorophenyl) ethane (DDT) and phenobarbital (PB). On a per weight basis, after intraperitoneal injection, PCB had twice the effect of DDT on liver weight, on microsomal protein and on cytochrome P-450 (Bickers et al., 1974). On an equimolar basis, after dietary exposure, PB and DDT were only 15 and 50%, respectively, as effective as PCB in increasing the amount of cytochrome P-450 (Litterst and Van Loon, 1972). Further, PCB had a greater effect than PB and an equal or greater effect than DDT on some specific microsomal enzymatic pathways such as demethylation and hydroxylation. Upon cessation of exposure, enzyme activity in PB treated animals decreased to control levels within a few days (Litterst and Van Loon, 1972; Orrenius et al., 1965). In contrast to PB, continuing rise in enzyme activity occurred after the cessation of a 7 day exposure to PCB (Litterst and Van Loon, 1974). Previous morphological studies of PCB in rat, mouse and rabbit have shown proliferation of smooth endoplasmic reticulum, disorientation of rough endoplasmic reticulum, and fatty livers (Kasza et al., 1976; Kimbrough et al., 1972; Koller and Zinkl, 1973; Nishizumi, 1970). The mechanisms for the PCB-induced fatty liver have not previously been investigated. Since the endoplasmic reticulum is the essential, if not the only locus for esterified lipid synthesis including

PCB and Liver Lipid

281

b o t h p h o s p h o l i p i d s ( P L P ) a n d t r i g l y c e r i d e s ( T G ) , we f o l l o w e d t h e c o n t e n t a n d the s y n t h e s i s a n d t u r n o v e r rates o f these t w o lipid m o i e t i e s in the h o m o g e n a t e a n d the m i c r o s o m a l f r a c t i o n after P C B e x p o s u r e . In o r d e r to f u r t h e r e l u c i d a t e the p a t h o g e n e s i s o f the P C B - i n d u c e d fatty liver, i n c o r p o r a t i o n rates o f i s o t o p i cally l a b e l l e d l e u c i n e i n t o m e m b r a n e ( " s t r u c t u r a l " ) m i c r o s o m a l p r o t e i n s a n d i n t o a " m e t a b o l i c " p r o t e i n ( a l b u m i n ) d e s t i n e d for the c i r c u l a t i o n w e r e also d e t e r m i n e d . In p a r t i c u l a r the l a b e l l i n g studies w e r e p e r f o r m e d to i n v e s t i g a t e w h e t h e r o r n o t t h e i n c r e a s e in lipids a n d the p o s t u l a t e d p r o l i f e r a t i o n o f E R m e m b r a n e s seen a f t e r P C B e x p o s u r e w e r e a t t r i b u t e d to i n c r e a s e d lipid a n d p r o t e i n s y n t h e s i s a n d / o r d e c r e a s e d b r e a k d o w n . F i n a l l y , the p u r p o s e o f the present w o r k was to c o r r e l a t e the b i o c h e m i c a l f i n d i n g s w i t h s u b c e l l u l a r c h a n g e s by m e a n s o f u l t r a s t r u c t u r a l analysis o f P C B - t r e a t e d rat livers.

Materials and Methods Animals. Male Sprague-Dawley rats 150-200 g in weight were used. Animals were housed in a temperature and humidity controlled room with a 12 h light and dark cycle. Food, standard laboratory chow (Antisimex, Stockholm), and water were available ad libitum. Animals were starved 16 hrs prior to sacrifice. Perfusion of Livers. In order to remove all blood from livers, a perfusion of 1-2 min duration with Ringer's solution at 37~ was performed. Animals were lightly anesthetized with ether and their peritoneal cavities were opened. The inferior vena cava of the lumbar region was exposed and cannulated. After flow (5 ml/min) was initiated by peristaltic pump, the hepatic artery and portal vein were severed and the inferior vena cava was clamped above the diaphragm (Glaumann and Trump, 1975). Blood for serum lipoprotein determination was withdrawn from the aorta. Homogenization. Following perfusion, livers were removed, blotted, weighed and 4 g were minced and homogenized in ice-cold 0.3 M sucrose. Homogenization was carried out by 4 complete passes of a glass-Teflon homogenizer at 200 rpm. A 25% homogenate (w/v) was sedimented at 10,000 • for 15 min. The supernatant was sedimented at 100,000 x g for 60 min. The resultant microsomal pellet was suspended in 0.3 M sucrose (0.5 g/ml). Injections of PCB. In order to prevent interference by solvent oil with lipid synthesis and turnover, a PCB-Ringer's emulsion was prepared. PCB (Aroclor 1254) was suspended in Ringer's solution, sonicated for 10 min and injected immediately. The emulsion contained 50 mg of PCB per ml and a volume of 0.05 or 0.1 ml/100 g rat (25 and 50 mg/kg) was injected intraperitoneally once daily for 3 or 6 days. Controls received injections of Ringer's solution daily as above. Labelling. 10 laCi of C14-glycerol-I from the Radiochemical Centre, Amersham, U.K. (20 mCi/ mMole) was injected into the femoral vein. For the studies on lipid synthesis, two intervals of 5 and 10min were used. In these experiments perfusion was performed for exactly 1 min. In turnover (decay) studies the same procedure was employed but the intervals studied were 90 min and 3,6, and 9 h after injection of the isotope. Radioactivity was measured as described previously (Glaumann and Trump, 1975). For protein labelling studies, DL-(I-C 14) leucine (10 laCi/100 g) in Ringer's solution was injected into the femoral vein and animals were sacrificed at indicated time intervals. Radioactivity was measured using TCA precipitated protein dissolved in 10 ml of Aquasol. The counting efficiency was approximately 89%. Chemical Assays. For lipid extraction an aliquot of the homogenate or microsomal fraction was precipitated in 5% cold TCA washed twice in 5% TCA and rinsed in distilled water. Extraction was performed in ethanol-chloroform (1:1). Extracts were washed twice in cold 0.1 N HC1 and

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the chloroform phase was used for silicic acid column chromatography (Borgstrom, 1952). The lipid extract was added onto the column and neutral lipids were collected in the chloroform eluate. PLP were eluted with methanol. PLP and TG were determined as described previously (Glaumann and Dallner, 1968) and protein and RNA were assayed by the techniques of Lowry et al. (1951) and Dallner et al. (1966) respectively.

Enzyme Assays. Cytochrome P-450 was determined according to Omura and Sato (1964). An extinction coefficient of 91 mM 1 • cm- 1 was used for the estimation of the amount of cytochrome P-450. Since the livers were thoroughly perfused, no hemoglobin remained to interfere with the cytochrome P-450 spectrum. Ethylmorphine N-demethylase was determined according to Gnosspelius et al. (1969) by measuring formaldehyde produced with Nash reagent. NADPH-cytochrome c-reductase (in NADPH-cyt. c red) was determined according to Dallner (1963). Glucose-6-phosphatase (G6Pase) and inosine diphosphatase (IDPase) activities were measured as previously described (Glaumann and Dallner, 1970).

Light and Electron Microscopic Techniques. In order to visualize lipid within hepatic lobules, frozen sections of formalin fixed livers were stained with oil red O. Livers for electron microscopic examination were perfusion fixed, as previously described (Glaumann et al., 1975a), with 2% glutaraldehyde in 0.067 M sodium cacodylate buffer (pH 7.4). Following perfusion, liver pieces were minced in pools of fixative and 1 mm pieces were washed in buffer and postfiexed in 1% osmium tetroxide, dehydrated in graded ethanol solutions and embedded in Epon (Luft, 1961). Thin sections (600 to 1000 A,) of Epon-embedded material were stained with uranyl magnesium acetate and lead citrate and viewed with a JEOL 100 B electron microscope.

Results Liver Weight T h e effect o f e x p o s u r e t o P C B f o r 3 a n d 6 d a y s o n t h e r a t i o s o f l i v e r s t o b o d y w e i g h t is s h o w n in T a b l e 1, N o s i g n i f i c a n t d i f f e r e n c e s w e r e s e e n a t e i t h e r 3 o r 6 d a y s b e t w e e n t h e t w o d o s a g e s (25 a n d 50 p p m ) . H o w e v e r , b o t h e x p e r i m e n t a l g r o u p s w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s a f t e r six d a y s o f e x p o s u r e (P<0.001), In comparison with the data in the literature, the liver to body

Table 1. Effect of PCB treatment on the ratio of liver to body weight Dosage per kg of body weight

Number of injections

Control 3 6

25 mg/kg

50 mg/kg

0.03904-0.02 0.0438___0.03

0.0367+_0.05 0.0449_+0.02

0.0345 4- 0.06

The animals received PCB in a sonicated emulsion in Ringer's solution (0.05 ml and 0.1 ml per 100 g of body weight respectively) as an intraperitoneal injection, at the same time each day. The animals were starved for 16 h before sacrifice. The peritoneum was opened and the liver was perfused via the inferior caval vein with Ringer's solution at 37~ C for approximately 2 rain, removed and weighed. The values are the means of at least 15 experiments + one standard error of the mean

PCB and Liver Lipid

283

weight ratio in our experiments was somewhat higher. This could well be accounted for by the fact that we perfused the livers, which is known to increase liver weight by approximately 25% (Glaumann, 1973). It has been shown that after DL-ethionine administration (Schlunk and Lombardi, 1967)and also following orotic acid-induced fatty livers (Rajalakshmi et al., 1969) the sedimentation properties of microsomes are altered, mainly because of accumulation of lipids within the ER. After homogenization, this lipid material is enclosed in microsomes resulting in a decreased equilibrium density of microsomes. In order to investigate if PCB also changes the density of liver microsomes, control and PCB 10,000 g • 15 min supernatant were centrifuged for 16 h through a sucrose-H20 gradient. In both cases, the distribution patterns covered the whole range of the gradient from 1.07 to 1.29 g/cm 3 (not shown in figure). For control microsomes, the median density was at about 1.168 g/cm 3, whereas it was at 1.150 g/cm 3 for PCB microsomes. Equilibrium density centrifugation of both control and PCB treated microsomes gave rise to a small pellet consisting mainly of ribosomes. It can therefore be concluded that short term treatment with PCB does not significantly alter the mean equilibrium density of liver microsomes. Consequently, PCB treated microsomes will sediment at 100,000 g • 60 rain in 0.30 M sucrose (density ~ 1.04).

Enzymatic Composition As is seen in Figure 1, PCB was a potent inducer of microsomal cytochrome P-450. More specifically, there was a linear response with time during the 6 day interval. With the 25 and 50 mg/kg dose the cytochrome P-450 amounts after 6 days were 300% and 400%, respectively, over control values. Taking the amount of microsomal protein into consideration, the total amount of cytochrome P-450 per gram of liver was increased six-fold. As can be seen from Table 2, there was a moderate increase (80%) of both NADPH-cytochrome c-reductase and ethylmorphine demethylase. PCB caused some decrease in the activity of glucose 6 phosphatase (G6Pase) whereas inosine-diphosphatase (IDPase) activities were stimulated, similarly to other inducers of microsomal en-

50 mg eCB/kg

3

Fig. 1. Dose-response relationship of cytochrome P-450 after daily injections of 25 and 50 mg PCB per kg of rat weight. Each point represents the mean value of a minimum of 8 PCBtreated rats. Vertical bars represent the standard error of the mean (SE)

i

25 mg PCB/kg 2

E

DkYS OF EXPOSURE

284

D.E. Hinton et al.

Table 2. Effect of PCB treatment on some typical microsomal "marker" enzyme activities Enzyme

Control

PCB treatment

NADPH-cytochromee reductase a Ethylmorphine demethylase b Cytochrome P-450 ~ G6Pase d IDPase a

258 (43%)

478 (39%)

a

b c d

3.0 0.86 (46%) 5.7 (36%) 16.7

5.2 2.6 (41%) 4.9 (34%) 21.5

Nanomoles cyt.c reduced/min/mg protein Nanomoles malonaldehyde/min/mg protein lamoles P/20 min/mg protein Nanomoles/mg protein

PCB was injected once daily for 6 days (50 mg/kg). The values are the means of 6 experiments, Variation from one exp. to another was in the range of 20 percent. The numbers within parentheses denote total enzyme activity (amount) in the microsomal fraction as compared to the homogenate (= 100%)

zymes such as phenobarbital and methylcholanthrene (Glaumann, 1970) and pregnenolone-16-c~-carbonitrile (Lindeborg and Glaumann, 1974). Finally, this table demonstrates that the recovery of the total microsomal fraction from control and from PCB-treated rats was almost identical or in the range of 36 to 46% for control microsomes and 34 to 41% for PCB microsomes as judged from the total content or activity of cytochrome P-450, NADPH-cyt. c red and G6Pase. Consequently, there was no significantly enhanced loss of microsomes due to the PCB treatment.

Chemical Composition The effect of PCB on the content of liver protein, RNA, phospholipids (PLP), cholesterol and triglycerides (TG) is shown in Table 3. In the total liver homogenate (Table 3a) no significant change in protein or RNA was seen. After 6 days of exposure to 50 mg PCB/kg the PLP levels were increased by 24% over control values (P<0.001). This increase seemed to be fully counted for by the increase in microsomal PLP (see Table 3). Cholesterol values in animals treated for 6 days at both dosage levels were approximately 30% higher than those of controls (P< 0.01). The relatively moderate increase in microsomal cholesterol does not fully explain the homogenate enhancement. In contrast to protein, RNA, and the other two lipid moieties (PLP and cholesterol) the amount of TG was substantially increased from about 5 mg to 11 mg per gram of liver. Consequently, there was almost no change in the PLP/protein or in the cholesterol/PLP ratios, whereas the TG/PLP ratio was nearly doubled. The effect of PCB on the content of liver microsomal protein, RNA, phospholipids, cholesterol and TG is shown in Table 3 b. After 6 days of PCB treatment

PCB and Liver Lipid

285

Table 3. Amounts of liver protein, phospholipid, triglyceride, cholesterol and RNA for control and PCB-treated animals a Homogenate (nag/gram of liver) Triglyceride

RNA

PLP/ ChoPro- lestein terol/ Phospholipid

Triglyceride/ Phospholipid

21.86+0.45 2.22-+0.32

4,98+0.32

8.16_+0.83

0.116 0.102

0.228

25 mg/kg 3days 184.1+13.14 19.36_+0.62 2.09-+0.23 6days 192.9_+7.12 24.40_+0.74 2.86-+0.24

3.52_+0.35 9.58_+0.90

7.96+1.01

0.105 0.108 0.126 0.117

0.182 0.393

20.97_+0.86 2.53+0.21 4.00+0.45 27.03_+0.52 2.94-+0.19 11.22_+1.14

7.74_+0.91

0.112 0.121 0.136 0.109

0.191 0.415

Control

Protein

Phospholipid

188.8_+3.85

50 mg/kg 3days 187.6-+6.76 6days 197.9+5.30

Cholesterol

Treatment with PCB and measurements were performed as described in Materials and Methods b Microsomes (rag/gram of liver) Protein

Phospholipid

Cholesterol

Triglyceride

RNA

PLP/ ChoPro- lestein terol/ Phospholipid

Triglyceride/ Phospholipid

19.44_+0.66

4.20_+0.11

0.41_+0.02

0.41-+0.02

2.04+0.12

0.216 0.098

0.098

25 mg/kg 3days 18.50-+1.34 6days 22.19_+1.13

4.71_+0.14 5.35_+0.22

0.50_+0.04 0.48_+0.11

0.51-+0.03 0.66-+0.08

2.06_+0.18

0.255 0.106 0.241 0.090

0.108 0.123

50 mg/kg 3 days 20.28-+ 1.00 6 days 26.38-+ 1.34

5.18+0.27 6.60_+0.22

0.56-+0.02 0.58-+0.05

0.56-+0.04 0.83_+0.09

1.94_+0.16

0.255 0.108 0.250 0.088

0.108 0.126

Control

All values are the means of at least 16 experiments_+ standard error of the mean e Floating lipid layer (mg/gram of liver)

Control PCB, 50 mg/kg, 6 days

Protein

RNA

PLP

TG

0.22 0.42

0.016 0.017

0.069 0.095

1.12 1.86

Total homogenates were sedimented at 100,000gx60 min. The floating lipid layer was pipetted off, washed twice at 100,000 g x 60 rain and analyzed. Each value represents the mean of 4 experiments. The variation from one experiment to another was within_+ 13%

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D.E. Hintonet al.

microsomal protein values in the 50 mg/kg dose groups were 36% higher than in controls (P<0,001). Corresponding increases were 57% and 41% for PLP and cholesterol, respectively. A comparison of values for PLP and protein at 3 and 6 days in the 50 mg/kg group demonstrates a greater increase for PLP than for protein. Microsomal TG was more than doubled with the 50 mg/kg dose and was 41% higher in the 25 mg/kg groups. Microsomal RNA did not change. Consequently, the increase in microsomal protein decreased RNA/ protein ratios by 10% and 30% in the low and high dose group at 6 days, respectively. The PLP/protein ratio in the microsomal fraction remained at control levels or slightly above while the TG]PLP ratio showed an increase of about 30% after 6 days of treatment. It has been postulated that the liver cell contains at least two TG pools, one associated with the microsomal fraction (Glaumann and Dallner, 1968; Stein and Shapiro, 1959), and another localized in the cytoplasm. In order to determine if PCB also affects the cytoplasmic pool, in addition to the total TG compartment (=homogenate) and the microsomal TG pool as described above, the "floating lipid layer" derived from the 100,000 g supernatant of thoroughly perfused livers was also analyzed chemically and ultrastructurally after PCB treatment (Table 3c). In comparison with the TG, PLP and RNA content is very low. This would indicate that the floating material consists primarily of fat droplets. There was a 50% increase in the amount of TG content of the floating lipid layer from PCB treated rats as compared to control. Electron micrographs of "floating lipid layers" revealed few membrane limited spherical bodies and only one to two ribosome-studded membranes per low magnification field in PCB-treated animals. In this respect, PCB differs from the effect of orotic acid (Rajalskshmi et al., 1969) and ethionine (Schlunk and Lombardi, 1967) which gave rise to a significant increase of the floating lipid layer in the form of spherical bodies, all limited by membranes sometimes studded with ribosomes. The presence of ribosomes identified these liposomes as derived from the ER. Consequently and in contrast to PCB, some microsomes after treatment with orotic acid or ethionine did not sediment but floated.

Labelling Studies The next step in this study was to investigate the effect of PCB on lipid synthesis and turnover in order to evaluate the mechanism resulting in PCB-induced fatty liver. The rate of incorporation of C14-1abelled glycerol into the PLP fraction of total liver homogenate is shown in Figure 2 at 5 and 10 rain intervals. At both intervals the specific rate of incorporation was somewhat lower for the PCB-treated (50 mg/kg) animals than for controls. However, this difference was no longer apparent when the radioactivity was expressed as DPM per liver (PLP-fraction) due to the fact that the PCB-treated liver increased in amount of PLP with respect to control. The rate of incorporation of C14-glycerol into PLP of liver microsomes is shown in Figure 3. When specific activity was studied, microsomes isolated from PCB-treated animals revealed lower rates of incorporation. However, when

PCB and Liver Lipid

287

HOMOUENATE

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"-~o100

l '

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1'0 MINUTESOF C14-GLYCEROLINCORPORATION

Fig. 2. Incorporation of C14-glycerol into PLP of total liver homogenate. C~%glycerol in distilled water was injected into the femoral vein (10 p,Ci/100 gram). PCB indicates experiments in which rats were injected intraperitoneally with PCB (50 mg/kg) once daily for 6 days. At each time point, 6 rats were analyzed. "Per liver" denotes liver from 100 grams of rat. Vertical bars represent SE

MICROSOMES

I

~'

Fig. 3. Incorporation of C14-glycerol into microsomal PLP. For experimental conditions see Figure 2

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1|'

S i MINUTES AFTER CN-OLYCEIISLADMINISTRATION

II

activity was expressed as DPM per liver, instead of on a PLP basis, PCB-treated animals revealed slightly higher rates of incorporation at both intervals than did controls. The rate of incorporation of C14-glycerol into TG of liver homogenate is shown in Figure 4. With PCB-treated animals, a decrease in the specific rate of incorporation was seen in comparison to that of controls. However, the marked increase in TG content after PCB exposure compensated for the decrease in specific rate of labelling when the activity was expressed as DPM per liver (TG fraction). Figure 5 shows the specific rate of incorporation of Ct4-glycerol into TG of microsomes from control and PCB-treated liver. Exposure to PCB resulted in a decreased rate of incorporation of label into the microsomal TG. However,

288

D.E. Hinton et al. HOMOOEllATE

'51y /y,c, +~

io

s

lo

MINUTES OF CI+-OLYCEIIOLINCORPORATION

Fig. 4. Incorporation of C14-glycerol into TG of total liver homogenate. C14-glycerol in distilled water was injected into the femoral vein (t0 ~tCi/100 gram). "Per liver" denotes liver from 100 grams of rat. Means of 6 experiments for each time point

MICUOSOMES

20.

flOL

Y 0

i20 '0

i

io

1'o

Fig. 5. Incorporation of C14-glycerol into microsomal TG. For experimental conditions see Figure 4

MINUTESOF CN-'-OLYCESOLINCOllPOllATION

as with the homogenate, when the activity was expressed as DPM per liver, no significant difference in labelling between control and PCB-treated livers was observed. The decrease in radioactivity of PLP and TG after a single injection of C14-glycerol was also followed in order to determine if PCB caused any change in the turnover of liver lipids. The decay curve for the total PLP pool of the homogenate at 90 rain and 3, 6 and 9 h after injection is shown in Figure 6. When expressed either as specific activity or as DPM per liver, the total PLP pool from PCB-treated livers demonstrated a prolonged half-life from about 10 h for the controls to about 20h after exposure to PCB. Decay for the microsomal PLP (Fig. 7) pool similarly revealed differences in turnover between control and PCB-treated livers; the slope for the microsomal PLP from PCB-treated animals had a calculated half-life of 11 h (per liver) to 13 h (specific activity) which is about double in comparison with the controls: 6 h (per liver) and 8 h (specific activity) respectively. These findings indicate that PCB slows the utilization of PLP and consequently increases the apparent half-life of this lipid.

PCB and Liver Lipid

289

HOMOQENATE

l

t

~ll

,.IN

m{ i

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NOUNSOF CN-OtYCEIIOLINCORPORATION

Fig. 6. Decay of radioactivity of C14-glycerol in PLP of total liver homogenate. C14-glycerol (10 laCi/100 gram) was injected into the femoral vein at zero-time. 6 rats were used for each time point. PCB (50 mg/kg) was injected once daily for 6 days. "Per liver" denotes liver from 100 grams of rat MIClIOSOMU 4O CONTROL PGB

~ Fig. 7. Decay of radioactivity of C ~4-glycerol in microsomal PLP. For experimental conditions, see Figure 6

1 5 84

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HUllS OF CN-OLYIEROLINCOilPOIIAnON

The decay of radioactivity of C14-glycerol labelled T G in liver homogenates of control and PCB-exposed rats was also followed and the results are shown in Figure 8. The most striking finding was a slower disappearance of radioactivity in the PCB-treated rats in comparison with non-treated animals. Thus, the estimated T 1/2 increased from about 3 h in the controls to about 6 h for the PCB-treated rats. With respect to decay of radioactivity of microsomal T G a different pattern was seen in comparison with that of the homogenate (Fig. 9). For control animals the microsomal decay curve was triphasic with the first slope having a faster decay (1 h), than the second (3.5 h) and the third (12 h). The half-life of T G in PCB-treated rats is also shown in Figure 9. The "three-slope-appearance" still characterizes this microsomal lipid after exposure to PCB. However, all three slopes show approximately a doubling of the half-lives after PCB administration, indicative of an overall decreased turnover rate. The calculated

290

D.E. Hinton et al. HOMOGENATE

10,0

200

OONTROL

.•5,0

-- 100. PCB

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It ,~ 5o

2~

26,

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HOURS OF Clk GLYCEROL INCORPORATION

Fig. 8. Decay of radioactivity of C14-g]ycerol in TG of total liver homogenate. For experimental conditions, see Figure 6. Means of 8 experiments. Vertical bars represent SE

MICROSOMES

CROTROL

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HOURSOF Gm'-GLYCEROLINCORPORATIOR

Fig. 9. Decay of radioactivity of C14-glycerol in microsomal TG. For experimental conditions, see Figure 6

apparent half-lives for PLP and T G in control and in PCB-treated rat liver are tabulated in Table 4. Another set of experiments was undertaken to determine the influence of PCB on the in vivo rate of incorporation of amino acids into microsomal "membrane" proteins and into a "metabolic" protein destined for the circulation, namely albumin. Exposure to PCB resulted in an increased rate of uptake of labelled leucine when measured as total radioactivity in the homogenate of thoroughly perfused livers. More specifically rate of C 14- leucine incorporation into both membrane proteins and albumin (Fig. 10a) was increased. This increase is even more apparent when related to weight of liver since some elevation of the microsomal proteins occurred as a result of the PCB treatment (Table 3b).

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Table 4. Calculated half-lives of liver lipid after PCB treatment Half-lives in h Triglycerides

Homogenate Microsomes Interval 0-3 h Interval 3-6h Interval 6-9 h

PLP

Control

PCB

Control

PCB

3.2+0.3

5.6+_0.4

9.5+ 1

19+- 1

1.0+-0.3 3.5+_1 12+-2

1.5+-0.2 6.0+_0.5 20+3

7+-1 7+1 7+ 1

12+_2 12+2 12+2

MIURQSOMES lOB,

aN-

PUB

i| .,~ H I , E

I

l:

E ~IINE

1

y/y

E

MINUTESAFTERCN-LEUUINEAOMINISTATION

Fig. 10. a Incorporation of C14-1eucine into microsomal membrane protein and albumin of liver microsomes from PCB-treated rat. PCB (50 mg/kg) was given once daily for five days. The label (10 l.tCi/100 gram) was given in 0.5 ml Ringer's solution into the femoral vein. Membrane protein denotes microsomes washed in alkaline Tris buffer, sonicated in distilled water and subsequently rewashed in KCI-EDTA. Albumin was isolated from sonicated microsomes as described earlier (Glaumann and Dallner, 1970). Means of 4 experiments for each time point. h Logarithmic decay of Cl'Lleucine in microsomal membrane proteins. For experimental conditions see Figure 10a

20-

| 10

.\ 2 L o

7

i h

i

i

i

DAYSAFTEReN-LEUOINEINJECTION

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Figs. 11-13 are toluidine blue stained semithin sections of Epon-embedded liver from control and PCB-treated rats Fig. 11. Section of liver from control rat. Sinusoidal borders are uniform and do not protrude toward lumens. Note the even distribution of darkly stained cytoplasmic components, x 1800 Fig. 12. Section of liver from rat exposed to 50 mg PCB/kg body weight for 3 days. Note enlargement of hepatocytes and presence of lipid vacuoles (arrows). Hepatocyte borders are rounded and often protrude into space of Disse. Cytoplasm shows increased areas occupied by lightly stained components, x 1800 Fig. 13. Section of liver from rat exposed to 50 mg PCB/kg body weight for 6 days. Hepatocytomegaly accompanies increases in lightly stained "ground-glass" areas of cytoplasm. Lipid droplets (arrows) are seen in cells at right of field, x 1800 Fig. 14. Perinuclear area of hepatocyte from control rat showing parallel arrays of rough endoplasmic reticulum (RER). N-Nucleus, x 26,000 Fig. 15. Junctional zone of rough (RER) and smooth (SER) endoplasmic reticulum in control hepatocyte. GLY-glycogen x 26,000 Fig. 16. Golgi apparatus in control hepatocyte. Secretory vesicles (arrows) associated with the Golgi apparatus contain numerous lipoprotein particles (LP). GLY-glycogen. x 26,000 Fig. 17. Perinuclear region of hepatocyte from rat exposed to 50 mg PCB/kg body weight for 3 days shows increased amount of smooth endoplasmic reticulum (SER). Rough surfaced endoplasmic reticulum shows disorientation and appears (arrow) to have been "pushed aside" by SER. x 26,000

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Fig. 18. Ultrastructure of hepatocyte from liver of rat exposed to 50 mg PCB/kg body weight for 3 days. Several Golgi-associated secretory vesicles (arrows) contain lipoprotein. N-nucleus. x 26,000 Fig. 19. Ultrastructure of hepatocytes after 3 days exposure to 50 mg PCB/kg body weight. Smooth endoplasmic reticulum (SER) extends to periphery of cells. Rough endoplasmic reticulum (RER) is limited to small area between mitochondria, Membrane-bounded lipoprotein particles (arrows) are seen near bile canaliculus and at cell margin in lower left of field, x 26,000

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Figure 10b demonstrates the effect of PCB on the turnover rate of total microsomal membrane proteins. As is shown, PCB treatment caused a slight increase in the half-life from around 2.4 days in the controls to 2.8 days for the PCB-treated animals.

Morphologic Studies. Toluidine blue-stained sections of Epon-embedded livers are shown in Figures 11, 12 and 13. Control hepatocytes (Fig. 11) were relatively uniform in size and contained dark staining areas of cytoplasm in perinuclear and peripheral sites. Subsequent electron microscopic examination showed mitochondria, lysosomes and peroxisomes in these sites. Small and lightly stained areas were regularly interspersed between darkly stained zones of cytoplasm. After 3 days of exposure to PCB, an increase in size of hepatocytes was seen (Fig. 12). Furthermore, and in contrast to controls, lipid droplets and large areas of lightly stained homogeneous cytoplasm with a ground-glass appearance were seen. These areas were more conspicuous in 6 day exposure animals (Fig. 13), and in some cells appeared to occupy all but a zone of cytoplasm at the periphery of the cell. Subsequent electron microscopic study showed these areas to be endoplasmic reticulum. Hepatocytomegaly (Figs. 12 and 13) was a constant finding after exposure to PCB. Electron microscopic examination confirmed and extended the light microscopic observations. Near nuclei, cytoplasm of control hepatocytes (Fig. 14) contained parallel arrays of rough endoplasmic reticulum (RER), occasional mitochondria and glycogen in typical rosette pattern. At the periphery of RER arrays (Fig. 15), a transition from RER to SER was seen. The peripheral part of Golgi apparatus of control hepatocytes (Fig. 16) showed secretory vesicles filled with particles of medium electron density (400-800 A in diameter). These were interpreted as precursors of serum very low density lipoproteins (Glaumann et al., 1975b; Jones et al., 1967). Following 3 and especially 6 days of exposure to PCB, areas of hepatocyte cytoplasm were filled with SER. This proliferation was encountered both near the nucleus (Fig. 17) and at the periphery of the cell (Fig. 19). Some disarray of RER was noted (Fig. 17). Cytoplasmic vesicles containing electron-dense material (Figs. 18 and 19) resembling the lipoproteins in Golgi secretory vesicles of controls, were seen after 3 days of exposure to PCB.

Fig. 20. Ultrastructure of hepatocyte following 6 days exposure to 50 mg PCB/kg body weight. Numerous profiles of smooth endoplasmic reticulum fill cytoplasm between mitochondria. Only occasional profiles of rough endoplasmic reticulum (RER) are seen in this field. • 21,000 Fig. 21. Proliferation of smooth endoplasmic reticulum in hepatocyte following 6 days exposure to 50 mg PCB/kg body weight. Normal appearing parallel arrays of rough endoplasmic reticulum remain (upper left hand corner) despite marked change in smooth endoplasmic reticulum. • 21,000 Fig. 22. Golgi apparatus (GA) in hepatocyte following 6 days exposure to 50 mg PCB/kg body weight. All saccules are flattened and no secretory vesicles are seen. Proliferated smooth endoplasmic reticulum fills remainder of field. • 26,000

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Fig. 23. Perinuclear region of hepatocyte following 6 days exposure to 50 mg PCB/kg body weight. Numerous membrane-bound particles of relatively low electron density (arrows) are seen in this field. These liposomes are associated with smooth surfaced membranes. • 26,000 Fig. 24. Peripheral area of hepatocyte following 6 days exposure to 50 mg PCB/kg body weight. Liposomes are associated with rough (vertical arrows) and smooth (horizontal arrows) surfaced endoplasmic reticulum, x 26,000 Fig. 25. Ultrastructure of hepatocyte following 6 days expsoure to 50 mg PCB/kg body weight. Two large lipid vacuoles are shown. Arrows point to liposomes, x 26,000

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Fig. 26. Electron micrograph of microsomal pellet from control rat liver. • 50,000 Fig. 27. Electron micrograph of microsomal pellet from PCB-treatedrat liver. • 50,000

Sections of livers from rats exposed to PCB for 6 days revealed even greater areas of SER proliferation (Figs. 20 and 21). Interestingly, parallel stacks of RER remained intact (Fig. 21). In contrast to control rats, the Golgi apparatus, after 6 days of exposure, appeared flattened and no secretory vesicles with lipoprotein particles were seen (Fig. 22). Membrane-bounded lipid (liposomes) (Figs. 23 and 24) were occasionally encountered after 3 days of exposure but were much more frequent after 6 days. Membrane elements of both the smooth (Fig. 23) and rough (Fig. 24) endoplasmic reticulum were associated with these liposomes although the former were much more common. The finding of a flattened Golgi apparatus lacking the secretory vesicles together with numerous liposomes in ER suggested an intracellular block in lipoprotein transport. Electron density of lipid within liposomes was similar to that of the large cytoplasmic lipid droplets (Fig. 25). The latter, however, were devoid of membranes or only partially surrounded by parallel arrays or cisternae of the endoplasmic reticulum (Fig. 25). The partial association of these lipid droplets with endoplasmic reticulum was interpreted as impingement of free cytoplasmic lipid against neighboring membranes. No necrosis of hepatocytes was observed at either the light or the electron microscopic level. On electron micrographs of microsomal pellets from control and PCB-treated livers, membranes with ribosomes attached were encountered together with smooth surfaced profiles (Figs. 26 and 27). This finding, in addition to the above described micrographs from hepatocytes, illustrates that the microsomal membrane structure and the attachment of ribosomes to microsomal membranes was not significantly altered by acute exposure to PCB.

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Discussion

Increases in the following parameters: liver to body weight ratio, cytochrome P-450, and microsomal hydroxylating enzymes conclusively demonstrate that livers of exposed rats responded to PCB under the selected method of administration.

Method of Delivery of PCB Since the primary objective of this study was to determine the effect of PCB on lipid synthesis, transport, and turnover in rat liver, a lipid-free suspension of PCB in Ringer's solution was considered necessary. Previous work had shown that corn and mineral oils affect lipid metabolism in the rat hepatocyte by increasing the number of lipoprotein particles in SER, Golgi and space of Disse (Ashworth et al., 1960; Ashworth etal., 1965; Ashworth etal., 1966). The use of the above system together with effective perfusion to remove serum lipids permitted us to detect the PCB effects upon lipid metabolism in the hepatocyte.

Recovery of Microsomes after PCB Treatment Treatment with orotic acid (Rajalakshmi et al., 1969) and ethionine (Schlunk and Lombardi, 1967) gave rise to massive fatty liver resulting from accumulation of triglycerides in the ER. This results in a shift of the density of microsomes, some of which did not sediment at 100,000g but floated. If this were the case also for microsomes derived from PCB-treated livers, any comparison between control microsomal and PCB microsomal fractions may not necessarily be justified. In order to investigate this possibility, the recovery of the microsomal fraction was calculated on the basis of three typical marker enzymes. These experiments (Table 2) demonstrated that there was no increased loss of microsomes after PCB treatment as compared to control. This is consistent with the chemical and morphological analysis of the floating lipid layer, which consisted of large lipid droplets and only trace amounts of microsomes. Further support for the notion that PCB did not significantly alter the sedimentation properties of microsomes was gained from the experiments on equilibrium density, which demonstrated that PCB-treated microsomes will in fact sediment at 100,000gx 60 min in 0.30 M sucrose. Apparently, the relatively moderate increase of TG and other lipids in the microsomal fraction only slightly decreased the density of microsomes. Taken together, these results indicate that, at least in the case of the short term treatment with PCB as performed in this study, comparison between control and PCB microsomal fractions seems justified.

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Microsomal Enzyme Effects. The increases in the microsomal cytochrome P-450 and in the activities of the other drug metabolizing enzymes, NADPH-cyt. c-red and ethylmorphine demethylase, confirm previous studies with various Aroclors (Litterst et al., 1972; Litterst and Van Loon, 1974), and chemically pure isomers of PCB, particularly those with chlorine substitution at the 4 and 4' positions (Ecobichon and Comeau, 1975). Our findings with respect to the activities of certain microsomal "marker" enzymes demonstrate a qualitative similarity between PCB and other inducers including: the organochlorine pesticides, hexachlorobenzene (HCB) and DDT; the polycyclic hydrocarbon carcinogen, 3-methylcholanthrene (3-MC); and phenobarbital (PB). All the above compounds increased cytochrome P-450 amounts (Bickers et al., 1972; Bickers et al., 1974; Litterst et al., 1972; Litterst and Van Loon, 1974; Reynolds et al., 1975). PCB, the organochlorines and PB, but not 3-MC caused increased activity of N-demethylase (Bickers et al., 1974; Litterst et al., 1972; Litterst and Van Loon, 1972; Stonard, 1975; Turner and Green, 1974) and aniline hydroxylase (Bickers et al., 1972; Bickers et al., 1974; Litterst and Van Loon, 1972; Stonard, 1975; Turner and Green, 1974) activities. PCB, 3-MC and PB caused increased activity of NADPH-cyt. c-red (Reynolds et al., 1975). Our finding of decreased activity of G-6-Pase after PCB is similar to that seen after PB (Glaumann, 1970; Menard et al., 1974). Furthermore, Alvares et al. (1973) demonstrated a strong similarity between PCB and 3-MC with respect to shifts in carbon monoxide difference spectra of microsomes from exposed rats, indicating that these compounds give rise to an increase of a specific type of cytochrome P-450. With respect to carcinogenicity, PCB has been shown to produce premalignant alteration of gastric mucosa of Rhesus monkey (Allen and Norbach, 1973), and hepatocellular carcinoma in mice (Ito et al., 1973; Kimbrough and Linder, 1974) and in female rats of the Sherman strain (Kimbrough et al., 1975).

Correlation of Biochemical and Morphological Studies Correlation between the biochemical and morphological findings of the present study was particularly true with respect to the increase in protein, enzyme activity, and PLP in the microsomal fraction and the marked proliferation of SER. The electron micrographs of liver from PCB-exposed animals showed a preferential increase in SER with little to no change in RER. Chemically, this finding was substantiated by a higher degree of increase in microsomal PLP than in protein. Since the SER is enriched in PLP in comparison with RER, mainly due to the presence of attached ribosomes in RER (Glaumann and Dallner, 1968), this difference reflects a disproportionate increase in one segment, namely, the SER over the other (RER). Microsomal RNA values did not change with exposure to PCB. This finding confirmed the morphological

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299

data of intact arrays of RER with ribosomes attached to the membranes after 6 days of PCB exposure. The RNA]protein ratio of the total microsomal fraction decreased as a result of a preferential increase of smooth surfaced profiles upon exposure to PCB. It should be noted that the decrease in RNA/protein ratio was small compared to that seen after CC14, which causes stripping of ribosomes from RER membranes (Smuckler and Arcasoy, 1969).

Mechanism of SER Membrane Increase As to the mechanism leading to proliferation of the SER, this study has demonstrated that PCB causes moderate increase in the rate of incorporation of C 14leucine into both membrane proteins as well as into albumin (Fig. 10a). Some precaution is, however, called for in the interpretation of these incorporation rate studies, because PCB may have changed the free amino acid pool, which in turn may influence the results. Future measurements of the leucine pool in control and in PCB-treated rat livers may answer this question. It is likely, although not conclusively shown in this study, that increased rate of membrane biogenesis is one plausible explanation for the hypertrophy of SER. However, the turnover studies of the total microsomal membrane protein fraction indicated that not only increased synthesis but also decreased breakdown occurred after exposure to PCB. Thus, in addition to stimulating protein synthesis, PCB seems to reduce the catabolism (or exchange) of microsomal proteins. This effect of PCB resembles the effect of phenobarbital (Kuriyama et al., 1969; Shuster and Jick, 1966). It should be noted in this context that the turnover studies of membrane proteins were performed during an interval of 1 to 4 days after the injection of the isotope together with extensive washing procedures in order to minimize interference with proteins destined for the circulation, such as albumin (Glaumann and Ericsson, 1970). The rate of synthesis and turnover of the other main component of microsomal membranes, namely PLP, was followed after injection of C14-glycerol, because glycerol constitutes the " b a c k b o n e " of the PLP molecule, easily penetrates biological membranes and is not subjected to exchange reactions and reutilization (Eriksson, 1973) which is the case for several other PLP precursors, such as p32 and ethanolamine. At first sight the results may seem to indicate that PCB causes an impaired rate of PLP synthesis, since the specific rate of labelling (DPM/mg PLP) decreased after exposure to PCB; however, when the increased amount of PLP resulting from the PCB administration is taken into account, it becomes obvious that no dramatic change occurred as to the total PLP synthesis after PCB treatment. The main effect of PCB on PLP metabolism was instead an increase of the half-life (Fig. 7). This prolongation in half-life for PLP may well explain the finding that, in spite of any increase in specific labelling of PLP, PCB gave rise to increased amounts of microsomal PLP. Ultrastructurally, the increase of microsomal protein and PLP was noted as a hypertrophy of SER.

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Pathogenesis of the PCB-lnduced Fatty Liver Increases in the number and size of cytoplasmic lipid droplets and membranebound profiles of lipid (liposomes) constitute morphologic evidence in addition to the biochemical findings that the exposed rats responded to PCB with an accumulation of liver lipid. Fatty liver has been reported following exposure to commercial (Kasza et al., 1976; Kimbrough et al., 1972; Koller and Zinkl, 1973 ; Nishizumi, 1970; Vos and Notenboom-Ram, 1972) and chemically defined PCB isomers with chlorine substitution at the 4 and 4'positions (Hansell and Ecobichon, 1974). The chemical characterization of the various lipid moieties in the PCB-induced fatty liver of this study demonstrates conclusively that the highest percentage increase (approximately 200%) of lipid was in the TGpool. Similarly, Litterst et al. (1972) reported up to a 300% increase in TG from rat liver homogenate after a one month exposure to four different Aroclors. In this regard, the PCB-induced fatty liver is similar to the response brought about by a variety of agents (Lombardi, 1966). Two lines of evidence seem to indicate that the increase in hepatic TG is related to drug metabolizing enzymes. First, in order to establish a minimum inductive dose for Aroclors with 42, 48, 54 and 60% chlorine by weight, Litterst et al. (1972) fed rats diets containing 500, 50, 5 and 0.5 ppm of each compound. TG in homogenate and activities of microsomal enzymes were determined and correlated. Both increased TG and induction of microsomal enzymes were seen at all dose levels. If the two effects were unrelated, one would expect to observe an exposure level at which one, but not the other, of the two effects is seen. Secondly, studies designed to determine the effects of chemically pure isomers of PCB upon microsomal enzyme activities (Ecobichon and Comeau, 1975) and liver morphology (Hansell and Ecobichon, 1974) demonstrate that the isomers which cause increased enzyme activities also give rise to lipid accumulation as determined morphologically. With respect to the pathogenesis of the PCB-induced fatty liver, our data demonstrate that the net rate of synthesis of hepatic TG was not increased by exposure to PCB. In fact, there was a decrease in the specific rate of labelling. Based on recent studies on lipid synthetic enzymes in liver microsomal fraction of rats exposed to PCB, Holub et al. (1975) showed no elevation of activity in PCB exposed rats and thus arrived at a similar conclusion to ours. Furthermore, our data on the rate of turnover of labelled TG demonstrates an increased half-life and/or disturbed removal of labelled TG from the liver. Thus, it would appear that the basic mechanism of the PCB-induced fatty liver is one in which the rate of synthesis of hepatic TG is normal, but there is a block in their utilization. This observation fits "Category 1" of the Lombardi classification (Lombardi, 1966) of pathogenetic mechanisms leading to fatty liver. More precisely, the complexity of the decay curve for microsomal TG indicates that this lipid is localized in two (or possibly three) compartments; one with short half-life ranging from 1 to 3 h and one with a longer half-life of around 12 h. It is plausible to assume that the compartment with the most rapid turnover corresponds to TG-enriched lipoprotein particles (very low density lipoproteins) destined for the circulation (Glaumann et al., 1975b). The second TG compart-

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ment with a half-life of about 12 h most likely corresponds to the cytoplasmic pool as judged from earlier studies by Stein and co-workers (Stein and Stein, 1967; Stein and Shapiro, 1959) and Glaumann et al. (1975b). In the interpretation of our results, we have assumed that there is no extensive reutilization of labelled glycerol made available by degradation during the interval studied. For this reason, glycerol was preferred as precursor (cf. Eriksson, 1973, for a discussion). In addition it should be noted in this context that the observed differences in turnover rate between the TG pools may be masked to some extent by exchange of TG between the cytoplasm and ER membranes (including its intracisternal content). Such exchange reactions appear to occur between various lipoprotein species in the circulation (Levy et al., 1971). It is known that upon incubation of the 105,000 x g supernatant with p32-1abelled microsomes, the phospholipids in the supernatant become labelled as a result of phospholipid exchange. Several proteins have also been isolated with high phospholipid exchange activities (Wirtz, 1974). In order to investigate possible differences in the exchanges of TG from control and PCB-treated rats between microsomes and cytoplasm (supernatant), a set of experiments was performed in analogy with earlier studies on exchange reactions of PLP (Wirtz, 1974). As can be seen from Table 5, exchange reactions for TG apparently take place in vitro between prelabelled microsomes and supernatant although at a limited extent (slow reaction?). The relatively slow exchange of TG compared to PLP might be related to the fact that TG is mainly associated with intracisternal VLDL particles whereas PLP is a true Table 5. In vitro exchange of TG from microsomal fraction to supernatant and from supernatant

to microsomal fraction Microsomes

Labelled microsomes

Supernatant

Control

PCB

4.547 (100%)

DPM 3.400 (100%)

Labelled supernatant Mixture labelled microsomes unlabelled supernatant Mixture unlabelled microsomes labelled supernatant

3.174 (70%) 514 (1%)

2.440 (72%) 200 (1%)

Control

PCB

18.971 (100%)

34.130 (100%)

980 (22%)

924 (27%)

17.035 (90%)

22.235 (92%)

Rats were injected 2 h before sacrifice with 20 laCi C14-glycerolper 100 g. Microsomes and 100,000 g supernatant were isolated as described in Materials and Methods. For washing, the microsomes were resuspended in Tris buffer, pH 8.0 (Glaumann and Dallner, 1968) and sedimented at 150,000 g • 60 min. The resuspended microsomal fraction (from labelled and unlabelled rats) was incubated with labelled and unlabelled supernatant for 1 h at 37~ C. Following incubation, the suspension was sedimented at 100,000 g • 60 min and TG was extracted from the microsomal pellet and supernatant

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microsomal membrane component (Glaumann and Dallner, 1968; Glaumann et al., 1975b). For an exchange reaction to take place, the TG molecule would have to penetrate the microsomal membrane, which thus may serve as a barrier. However, as we see it, the main conclusion is not the degree of exchange per se because it might in an in vitro system be quite different from the in vivo situation. Instead, we would stress the finding that there was no difference in the extent of exchange between control and PCB-treated rats. If this would not have been the case, an additional possibility, namely decreased rate of exchange of TG would have to be taken into account as an explanation for the prolonged half-life of TG after PCB treatment. Our data do not support this alternative. In summary, we admit however, that it cannot be excluded that limited reutilization and exchange reactions may interfere with our calculation. Our results strongly support a delayed utilization and not increased net synthesis as the main mechanism for the PCB-induced accumulation of TG, for which our data give maximal but not necessarily actual values. In the rat, the primary pathway of utilization of hepatic TG is secretion into the circulation in the form of lipoproteins (Glaumann et al., 1975b; Jones et al., 1967; Stein and Stein, 1973). This process appears to involve the following steps: synthesis of protein and of TG and other lipid moieties; conjugation of lipid and protein; intracellular transport via the ER and the Golgi apparatus; and secretion into the extracellular space. Our data indicate that PCB does not interfere with protein synthesis, rather, enhanced incorporation rate of labelled amino acid was seen. This is in contrast to other agents such as CC14 (Smuckler et al., 1962), ethionine (Farber et al., 1964) and puromycin (Jones et al., 1967) which cause fatty liver at least partially due to interference with protein synthesis. Our data indicate that impairment of intracellular lipid transport as well as a decreased degree of utilization of the cytoplasmic (storage) pool occur upon exposure to PCB. This was evidenced by prolongation of the half-lives of both the compartment with a rapid turnover (VLDL) and that with a longer half-life (cytoplasmic lipids). Morphologically, the interference of PCB with intracellular transport of VLDL precursors was most conspicuous regarding the Golgi apparatus in animals exposed for 6 days. In these rats, the Golgi appeared flattened and lacked typical secretory vesicles with lipoprotein particles. This finding suggests a block in the route of transport between the endoplasmic reticulum and the Golgi apparatus. Similar alterations were reported after ethionine exposure (Arstila and Trump, 1972) and were accompanied by decreased cellular levels of ATP (Farber et al., 1964). The intracellular migration of export proteins is energy dependent (cf. Palade, 1975) which may help to explain the "piling up" of lipoprotein particles proximal to the Golgi apparatus after ethionine treatment. In this regard, it should be mentioned, however, that no reduction in liver ATP levels was seen after 12 days ingestion of a diet containing 75 and 400 ppm PCB (Mehlman et al., 1974). Colchicine and vinblastine cause disruption of the microtubular apparatus and lead to a fatty liver by the accumulation of Golgi-derived secretory vesicles containing lipoprotein particles (Orci et al., 1973; Stein and Stein, 1973; Stein et al., 1974). However, not all investigators agree that the colchicine-induced accumulation of lipoprotein particles in the Golgi apparatus can be ascribed to microtubule

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depolymerization (Redman et al., 1975). In contrast to colchicine, PCB caused a flattening of the Golgi cisternae with a disappearance of Golgi-derived, lipoprotein-containing secretory vesicles. Furthermore, in the PCB induced fatty liver, lipoprotein accumulated within the endoplasmic reticulum and/or "shuttling vesicles" (Palade, 1975) between the endoplasmic reticulum and the Golgi. As described above we saw no evidence of necrosis during acute exposure to PCB. Furthermore, chronic studies with PCB at even higher dose levels than those used in this study failed to demonstrate liver necrosis (Kasza et al., 1976). Thus, on the basis of increased numbers of liposomes and total lack of necrosis, the PCB-induced fatty liver, in some respects at least, appears more closely akin to that caused by ethionine than carbon tetrachloride, which causes hepatic necrosis (Smuckler and Arcasoy, 1969). Although additional work is needed to precisely define the specific molecular alterations in the associated organelles involved in the pathogenesis of the PCBinduced fatty liver, it is clear that the lipid accumulation occurs simultaneously with a hypertrophic, hyperactive SER during an adaptive rather than a degenerative (Hutterer et al., 1969) phase of response. The main mechanism by which PCB produces fatty accumulation in the liver seems to be interference with intracellular transport or utilization of lipoprotein particles. Acknowledgements. This work was initiated during the term of a post-doctoral visit of Dr. David Hinton to the Department of Pathology, Karolinska Institute, Stockholm and completed with the aid of a grant from the Swedish Cancer Society and by a grant from the United States Public Health Service for graduate training in Pathology, GM-00431-14. The authors wish to thank Mrs. Helena Jansson and Mrs. Lena Rodin for skillful technical assistance.

References Allen, J.R., Norback, D.H. : Polychlorinated biphenyl and triphenyl-induced gastric mucosal hyperplasia in primates. Science 179, 498 (1973) Alvares, A.P., Bickers, D.R., Kappas, A.: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc. Nat. Acad. Sci, 70, 1321 (1973) Arstila, A.U., Trump, B.F. : Ethionine induced alterations in the Golgi apparatus and in the endoplasmic reticulum. Virchows Arch. Abt. B Zellpath. 10, 344 (1972) Ashworth, C.T., Stembridge, V.A., Sanders, E. : Lipid absorption, transport and hepatic assimilation with electron microscopy. Am. J. Physiol. 198, 1326 (1960) Ashwort, C.T., Wrightsman, F., Cooper, B., DiLuzio, N.R.: Cellular aspects of ethanol-induced fatty liver: A correlated ultrastructural and chemical study. J. Lipid Res. 6, 258 (1965) Ashworth, C.T., Leonard, J.S., Eigenbrodt, E.H., Wrightsman, F.J.: Hepatic intracellular osmiophilic droplets. Effect of lipid solvents during tissue preparation. J. Cell Biol. 31, 301 (1966) Bickers, D.R., Harber, L.C., Kappas, A., Atvares, A.P.: Polychlorinated biphenyls: Comparative effects of high and low chlorine containing Aroclors on hepatic mixed function oxidases. Res. Commun. Chem. Path. Pharmacol. 3, 505 (1972) Bickers, D.R., Kappas, A., Alvares, A.P.: Differences in inducibility of cutaneous and hepatic drug metabolizing enzymes and cytochrome P-450 by polychlorinated biphenyls and 1,1,l-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT). J. Pharmacol. Exp. Ther. 188, 300 (1974) Borgstrom, B. : Investigation on lipid separation methods. Separation of phospholipids from neutral lipids and fatty acids. Acta Physiol. Scand. 25, 101 (1952) Bruckner, J.V., Khanna, K.L., Cornish, H.H.: Biological responses of the rat to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 24, 434 (1973)

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Received January 7, 1978