Morphological and biochemical changes in the liver of various species in experimental phospholipidosis after diethylaminoethoxyhexestrol treatment

T~XICOL~GY

AND

APPLIED

PHARMACOLoGY

3,284

(1975)

Morphological and Biochemical Changes in the Liver of Various Species in Experimental Phospholipidosis After Diethylaminoethoxyhexestrol Treatment FELIX A.

DE LA

IGLESIA, GEORGE FEUER,EDWARDJ. MCGUIRE, AND AKIRA TAKADA~

Warner- Lambert Research Institute, Sheridan Park, Ontario, and Department of Clinical Biochemistry, University of Toronto, Ontario, Canada Received September 16,1974; accepted March 26,197s

Morphological and BiochemicalChangesin the Liver of Various Species in Experimental PhospholipidosisAfter Diethylaminoethoxyhexestrol Treatment. DE LA IGLESIA, F. A., FEVER, G., MCGUIRE, E. J. AND TAKADA, A. (1975). Toxicol. Appl. Pharmacol. 34, 28-4. The mechanismof druginduced experimentalphospholipidosiswas studied in severalspeciesby the administration of diethylaminoethoxyhexestrol. Rabbits, rats, mice, dogs, and guinea pigs developedmicroscopicand biochemicalabnormalities, while hamsterswere lessaffected. In the liver of affected species characteristicsubcellularchangeswere found, accompaniedby phospholipid accumulation.Hepatic lesionsconsistedof concentric lamellarbodies with varying degreesof osmic afhnity, representingsecondarylysosomes characterizedby cytochemicalmethods.Accumulation of thesebodieswas alsoseenin Kupffer, endothelial, and biliary epithelial cells.The intensity of the changeswas related to speciessusceptibility. Biochemicalstudies revealedan overall increaseof total phospholipidsin the affected species, togetherwith changesin the relative distribution of individual phospholipids and the appearanceof unidentifiedcomponents.The activity of microsomal drug metabolizing enzymesand microsomalphospholipid synthesiswere diminished. The lesionsclosely resembledthose observed in man after treatment with diethylaminoethoxyhexestrol and are related to altered phospholipid metabolismwith subsequentchangesin microsomaldrug metabolizing enzymeactivity. The long-term administration of the coronary vasodilator diethylaminoethoxyhexestro12to man has resulted in adverse liver reactions (Adachi et al., 1971; Matsuda et al., 1971; Oda et al., 1970; Onozawa et al., 1970)and subcellular changeswhich were characterized recently (de la Iglesia et al., 1974; Oda et al., 1970; Seki et al., 1971). These reports indicated a relatively wide spectrum of tissueresponseand differences in the changes between man and animals. This might be related to speciesdisposition affecting drug biotransformation or the structure or organelles involved. Adachi et al. (1972) and Yamamoto et al. (1971a) reported the experimental reproduction of phospholipidosis in the rat. However, the development of these changesin other speciesof i Presentaddress:Departmentof Internal Medicine,KanazawaMedical University, Uchinada, Japan. 2Coralgil,Maggioni,Milan, Italy, andTorii Pharmaceutical Co., Tokyo, Japan. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

28

EXPERIMENTAL

PHOSPHOLIPIDOSIS

29

laboratory animals is not known in detail. A comprehensive analysis of the drug-induced reaction in various species would, therefore, facilitate the interpretation of findings in man and would establish the mechanism by which lesions evolve. This report examines this possibility and reports for the first time the drug effects on several species. METHODS

Animals and treatment. Only male animals were used throughout the study: Wistar albino rats weighing 114-136 g; New Zealand albino rabbits, 2.0-2.2 kg; Syrian golden hamsters, 78-81 g; albino guinea pigs, 240-298 g; CF albino mice, 22-24 g; and Beagle dogs, 7.3-7.9 kg. Animals were housed in air-conditioned rooms at 22 + 2°C and 55-60 % relative humidity. Four to six animals were allocated to each treatment or control group, except in the dog experiments, where only two per group were used. Fresh tap water and laboratory ration was offered ad libitum. The treatment of rats, mice, hamsters, and guinea pigs consisted of po doses of 0.25 mmol(l17 mg) aqueous diethylaminoethoxyhexestrol/kg/day for 1 wk. An additional group of rats was given ip 0.08 mmol/kg/day for 1 wk. Due to lack of tolerance to high dose levels, dogs received seven daily po doses of 0.12 mmol/kg each. Rabbits were subjected to 0.25 mmol/kg po doses daily for 5 days. The last dose was given to all species 24 hr before sacrifice with an 8-hr fasting period. Under ether or barbiturate anesthesia, the livers were excised, blotted, weighed, and quickly processed according to the procedures described below. Microscopy and histochemistry. For light microscopy, liver tissue portions were fixed either in 1 “/o glutaraldehyde in phosphate buffer or by immersion in chromiumosmic solution (Dalton, 1955) and subsequently embedded in Epon-Araldite. Bright field and differential interference contrast microscopy were carried out in 1.5~,um sections stained with toluidine blue. For phospholipid histochemistry, tissue slices were fixed in form01 calcium solution, embedded in gelatin, and processed according to Baker (1947). Cytochemical studies by electron microscopy were performed in 20-pm sections fixed in glutaraldehyde buffer solution and assayed for acid phosphatase (using /?-glycerophosphate and cytidine 5-monophosphate substrates) (Novikof, 1963), inosine diphosphatase (Novikoff et al., 1962), adenosine triphosphatase (Wachstein and Meisel, 1957), and thiamine pyrophosphatase activities (Novikoff and Goldfischer, 1961). Ultrathin sections were obtained in Reichert ultramicrotomes with diamond knives and stained with lead. Cell organelle fractionation. Portions of liver were rinsed, weighed, homogenized, and cell organelles isolated. The final pellets were rinsed, reconstituted to the original volume (Cooper and Feuer, 1972) and purity was analyzed by electron microscopy. Phospholipid analysis. Liver homogenates and cell organelle fractions were analyzed for total content and individual phospholipid distribution. Phospholipids were extracted, purified (Folch et al., 1957) and quantitated (Bartlett, 1959; Marinetti, 1962). Individual phospholipids were separated by one and two dimensional thin-layer chromatography (Rouser et al., 1970; Skipsky et al., 1962). The spots were scraped and phospholipid phosphorus was determined (Cooper and Feuer, 1972). Enzyme assays. These were carried out on liver homogenates and subcellular fractions in treated and control rats, including acid phosphatase (Golberg et al., 1962), glucose

30

DE LA IGLESIA

ET AL.

6-phosphatase (Fetter et al., 1965), inosine diphosphatase (Novikoff and Heus, 1963), S-adenosyl-L-methionine:microsomal-phospholipid methyl transferase (Fetter et al., 1972), and coumarin 3-hydroxylase (Feuer, 1970). Protein content of homogenates and cell organelle fractions was measured (Lowry et al., 1951; Miller, 1959) and enzyme activities were expressed in units representing micromoles or nanomoles of substrate metabolized/hour/gram of liver or milligram of protein. Statistics. Analysis of numerical data was undertaken by means of Student’s t test and differences were considered significant when p < 0.05. RESULTS Light microscopy. No pathologic changes were detected using conventional light microscopic methods in paraffin-embedded material. The liver morphology of mice, rats, rabbits, guinea pigs, dogs, and hamsters by means of differential interference contrast microscopy is illustrated in Figs. l-7. Following drug administration, numerous dark, dense bodies were found in the vascular endothelium, Kupffer cells, and biliary epithelial cells. In hepatocytes, these dense bodies accumulated in the vicinity of bile canaliculi. Fewer dense bodies were seen in rats treated by ip administration (Fig. 3) or in hamsters (Fig. 7). In most species, these lamellated bodies were of uniform appearance, ranging 1 to 2.5 pm, evenly distributed throughout the liver lobule. In dogs, a preferential periportal disposition was observed. Neutral fat droplets of light contrast amplitude were associated with the dense bodies in rats and rabbits. No changes were seen in control livers. Phospholipid histochemistry. The phospholipid accumulation was characterized histochemically in liver and other organs. The reaction was faintly blue with finely granular cytoplasmic deposits. In the liver the deposits were observed in the cytoplasm of the hepatocytes and Kupffer cells and their distribution was similar to the localization of dense bodies seen by interference contrast microscopy. The reaction was also positive in other organs as indicated in Table 1. Histochemistry and cytochemistry of phosphatases. By light microscopy, the histochemical phosphatase reactions showed normal distribution in control rat liver (Biempica et al., 1967). Inosine diphosphatase was revealed by faint diffuse staining of the hepatocyte cytoplasm. By electron microscopy, this reaction was found in the endoplasmic reticulum cisternae. In treated animals, no reaction was seen in the dense bodies and adjacent cistemae (Fig. 8). Thiamine pyrophosphatase was not detectable in control livers by light microscopy. Small areas of reaction were seen in treated animals and by electron microscopy staining was observed in the Golgi apparatus, while dense bodies were negative (Fig. 9). Lysosomal phosphatases were manifest in cytoplasmic granules within the hepatocytes and Kupffer cells throughout the liver lobule. This reaction was more intense in treated animals and by electron microscopy it was found in lysosomes and in the outer membrane of the dense bodies (Figs. 10 and 11). Adenosine triphosphatase was present in bile canaliculi, intercellular membranes, and sinusoidal surfaces. A paradoxical, occasional deposition was seen in the outer membrane of some dense myelin bodies neighbouring the bile canaliculi (Fig. 12). This phenomenon was not noted in controls.

EXPERIMENTAL

PHOSPHOLIPlDOSlS

FIGS. l-7. Differential interference contrast microscopy of the liver diethylaminoethoxyhexestrol. Dense bodies (DB) can be observed in administration (2 and 3, respectively), rabbit (4), guinea pig (5), dog cell; BEC,biliary epithelial cell; BD, bile duct, LZ, lipid droplet. Small within vascular endothelial cells. Epoxy-embedded material, x400.

31

from various species treated with the mouse (l), rat after oral or ip (6), and hamster (7). KC Kupffer arrowheads indicate dense bodies

Electron microscopy. In all species hepatic subcellular changes appeared as concentric lamehated dense bodies in all types of cells. Their distribution was cytoplasmic and numerous morphological variations were observed. The bodies were close to bile canaliculi in hepatocytes and were prominent in the cytoplasm of Kupffer cells (Figs. 13-19). In mouse and rat liver, the bodies were associated with fat droplets and varied in size and shape. Small membrane whorls were closely associated with the Golgi apparatus 2

32

DE LA IGLESIA ET AL. TABLE

1

LIGHT MICROSCOPY HIST~CHJZMISTRY OF PHOSPHOLIPIDS IN VARIOUS ORGANS FROM EWERIMENTAL ANIMALS” Species Organ

Cell type

Liver Kidney Adrenal Pancreas Spleen Lymph nodes Thymus Lung Brain Gonad

Hepatocyte, Kupffer Tubular Cortex Acinar Macrophage Macrophage Reticular Alveolar II, macrophage Glia Seminiferous

Mouse +++ I!I + f ++ + zk Ii + +

a Graded - negative, + light, +/++ moderate, +++ means of subjective evaluation.

Rat

Rabbit

Guinea pig

Dog

++ z!I + 5 +t + -II!I + +

-I-+

-I+If:

+ zk +

++ + rlz rt + +

++ 5 + + 5 +

+ + + + + *

Hamster

marked, in terms of intensity of reaction by

FIGS. 8-12. Cytochemistry of phosphatases in treated rat liver. The reaction product is characterized by black deposits in the illustrations. Dense bodies (DB) display a hollow-image attributed to fixation or extraction artifacts. Inosine diphosphatase activity (8) localized in endoplasmic reticulum cisternae; arrows point at unreacting lamellae. Thiamine pyrophosphatase activity (9) localized in the Golgi apparatus (GO). Demonstration of acid phosphatase activity employing cytidine monophosphatase (10) and b-glycerophosphate (11) as substrate. Adenosine triphosphatase activity (12) visualized in the bile canaliculus (BC). Some paradoxic reaction, although of possible artifactual origin, occurred in the periphery of neighbouring dense bodies (arrows). Unstained sections, ~22,500.

(Figs. 23,25,26) and were interpreted as immature forms. These whorls coalesced into layered masses of intermediate appearance (Figs. 24 and 25). Well-developed dense bodies were usually larger and associated with fat droplets (Figs. 21 and 27) resembling autophagic vacuoles and engulfed smooth endoplasmic reticulum and glycogen (Figs. 20, 24, 27). In apparent final stages of development a distinct single outer membrane was seen (Fig. 22). Rabbits, guinea pigs, dogs, and hamsters revealed somewhat less dense bodies in the hepatocyte cytoplasm. In guinea pigs, they were also seen in the

EXPERIMENTAL

PHOSPHOLIPIDOSIS

33

FIG. 13. Dense bodies (DB) associated with small fat droplets in the vicinity of bile canaliculi of hepatocytes and closely packed in Kupffer cells (KC) in mouse liver. Lead stain, x3000. FIGS. 14 and 15. Rat liver fouowing oral treatment (14) or intraperitoneal drug administration (15). Dense bodies (DB) in both hepatocytes and Kupffer cells (KC’). Lead stain, x3000.

lumen of bile canafcuii (Fig. 17). Autophagic vacuoles were numerous in guinea pigs, hamsters, and mice. Observations on remaining cell organelles revealed no specific changes related to drug treatment. Total liver phospholipids. Significant increases were found in total phospholipid content in rats, rabbits, dogs, guinea pigs, and mice with no apparent change in hamsters. The percentage increase over control values varied (rat, 56 %; rabbit, 57 “/; ; dog,

34

DE LA IGLESIA

ET AL..

FIG. 16. Rabbit liver with multicentric dense bodies (DB) in hepatocytes and Kupffer cells (KC), together with neutral fat deposition (F). Arrows indicate dense bodies in the cell lining, the sinusoid and in the white blood cell. Lead stain, x3000. FIG. 17. Few dense bodies (DB) in guinea pig liver, including large units in Kupffer cells (KC). Circled areas indicate the presence of dense material in the lumina of bile canaliculi. Lead stain, x3000. FIG. 18. Dense bodies (BB) in the cytoplasm of canine hepatocytes. Lead stain, x3000. FIG. 19. Relatively small, infrequent dense bodies (MI) in Kupffer cells (KC) and hepatocytes of hamster liver. Lead stain, x3000.

EXPERIMENTAL

PHOSPHOLIPIDOSIS

35

FIGS. 20-27. A composite arrangement of high magnification electromicrographs representing relevant features of the lesions found. Rat liver, (20, 23, 25, 27); mouse liver, (21, 24, 26); hamster liver (22). Coalescence of small myeloid bodies with a lysosome (L Y) (20) or with fat droplets (21, F). Dense body (DB) surrounded by a distinct single membrane (22, arrows). In (23) immature dense bodies (ZOB) are seen next to the Golgi apparatus (GO). Typical aspect of a multicentric body (DB) close to an autophagic vacuole (A V, 24), or in apposition to a fat droplet (F, 25). Higher magnification of small myeloid bodies (arrows) in close relationship with Golgi apparatus (GO). Appearance of a fully developed myeloid body (DB), engulting glycogen particles (GL Y, 27). Lead stain, x41,500 (20); x24,500 (21,24-27); x37,500 (22), and x10,000 (23).

36

RELATIVE

DE LA IGLESIA

Rat Rabbit Dog Guinea pig Hamster Mouse

TABLE 2 PROTEIN CONTENT IN

WEIGHT,

Species

ET AL.

PHOSPHOLIPID, AND THE LIVER TREATED WITH DIETH~AMINOE~ZI~XYHEXESTROL~

No.

Treatment

6 6 4 4 2 2 4 4

None Drug None Drug None Drug None Drug None Drug None Drug

4 4 8 8

Relative liver weightb 4.58 f 0.16

5.01 It: 0.11e 4.57 kO.18 4.97 f 0.20 3.61 Itr 0.14 3.55 a 0.26 3.72 + 0.19 4.14 * 0.12 4.67 + 0.18 5.76_+0.21e 5.38 -t 0.14 5.61 + 0.06

Phospholipid’ 29.88 f 2.84 46&l+ 3.85' 33.54 f 2.91 52.56 f 4.16' 24.07 k 0.39 36.17 f 1.42' 33.48 f 6.15 57.47 f 1.42e 40.30f 2.65 41.18 f 3.02 27.74It: 1.14 43.62f2.O!Y

OF VARIOUS

SPECIES

Protein* 165.53 + 13.11 155.87 + 11.61

150.89 + 12.48 148.30 + 10.93

124.91 + 126.88 + 161.10 + 163.10 + 155.84 +

10.35 8.97 3.90 3.12 8.72

148.40 rt 10.50 232.61 f 10.09

233.61 f

9.42

a Mean f SE. b g/100 g body wt. c pmol. P/g liver. * mg/g liver. e Significantly different from control (p < 0.05). 50 %; mouse, 57 %; guinea pig, 72 %). The drug caused no effect on the protein content (Table 2). Tissue phospholipid distribution. Separation of individual phospholipids revealed drug-related changes in relative distribution (Table 3). Phosphatidylethanolamine was increased uniformly in all species; phosphatidylcholine was also enhanced, although not significantly in dogs. In rats, all other phospholipid fractions were elevated after treatment. In rabbits, phosphatidic acid, lysophosphatidylcholine and sphingomyelin were also significantly increased; in dogs, phosphatidylinositol and sphingomyelin; in guinea pigs, phosphatidic acid and phosphatidylinositol; and in mice, lysophosphatidylcholine and sphingomyelin were increased. By one-dimensional chromatography two unidentified phospholipids were found in significant amounts (unknown 1, Rf value of 0.25-0.28 and unknown 2, 0.78-0.81). The amounts of unidentified phospholipid detected in relation to total phospholipids are shown (Table 3). The chemical nature of these unknown phospholipids was not determined. Unknown 1 probably represents lysobisphosphatidic acid as described by Rouser et al. (1970). Although significantly elevated phospholipid content was found in the liver of treated animals, the relative distribution within individual phospholipids was similar to controls. Subcellular phospholipid distribution. Significantly increased phospholipids were localized in lysosomal and supernatant fractions (Table 4). The phospholipid content of mitochondrial and nuclear fractions was also increased, probably due to lysosomal contamination and phospholipid-laden debris. The supernatant fraction from control animals showed the lowest phospholipid content, while the largest drug-related increase was in this fraction. Microsomal phospholipid was reduced in treated rats.

None Drug

PA

1.49 0.15 1.83 +If: 0.08

PC

IN THE LIVER

6.22 +IL 0.45 7.46 0.47

++ + + + + + k 1.05d 0.73 2.59 2.71 0.73 0.49

0.49 0.61

12.68 ++ 0.45 0.62 16.97

21.38 14.38 13.82 11.89 16.08 26.41 19.50 15.26

TABLE

3

+ + + + +

0.03 0.13 0.42d 0.17 0.09d

+ 0.10

0.82 0.16 1.60 f+ 0.14

2.40 0.53 2.74 3.40 2.44 2.17

1.22 1.16 +f 0.09d 0.11

1.25 + + 0.06 0.07 1.81

SM

* * + f f

0.08 0.03 0.27 0.10 0.16d

1.54 + + 0.13 0.06 2.18

0.53 1.10 1.91 1.58 1.44

1.43 +- 0.11

3.77 0.50 f* 0.28 0.03

--

0.04f 0.01 0.01 f 0.01 1.02 + 0.23 -

1.19 + 0.19

1.24 + 0.27 0.04f 0.06

0.03 0.69 ff 0.01 0.13

Ul

u2

0.54 + 0.09 -

87 75

80

91

88 87 87 87

92 79

86 91

Recovery (%I

SM, sphingo-

0.03 1.06 ++ 0.01 0.08

0.02 + 0.00

-

--

0.07 + 0.04 0.99 &- 0.15

DIETHYLAMINOETHOXYHEXESTROL

1.30 *+ 0.12 2.22 0.29

WITH

b

0.61 +O.ll 0.64 f. 0.12 1.13 +_0.26d 1.36 + 0.14 1.26_+0.15d

1.11 rt0.24d

2.43 +& 0.31 0.93 0.12

0.82 + 0.11 1.92 + 0.35

LPC

Phospholipid”*

SPECIES TREATED

1.37 + 0.11 3.72 + 0.19

PI

OF VARIOUS

6.73 5 0.53 12.80 f 0.92 7.9 + 0.85“ 20.51 2~ 1.93

PE

CONTENT

13.56 7.99 +zk 0.91 0.50 1.19 + O.Ogd 8.22 f 0.61 1.16 + 0.11 4.96 2 0.54 1.21 f 0.10 8.78 2 1.69 2.61 310.09 13.65 f 1.61 0.88 + 0.10 9.34 f. 0.62 1.28 sf:0.14 8.86 + 0.67’

1.65 +5~0.16 0.63 0.10

1.42 5 0.17 2.06 _+0.20

~-

PHOSPHOLIPID

a PA, phosphatidic acid; PE, phosphatidylethanolamine: PC, phosphatidylcholine; PI, phosphatidylinositol; LPC,lysophosphatidylcholine; myelin; Ul and U2, unidentified phospholipids. b pmol phospholipid: P/g liver. c Number of animals. d Not significantly different from controls (p > 0.05); all other drug treatment values are significantly different from controls (p < 0.05).

Mouse

Dog Guinea Pig Hamster

88

Drug None

44 2 2 4 44 4

Drug None None Dwz None Drug

None Drug

6 6

Rat

Rabbit

NC Treatment

Species

INDIVIDUAL

Y

gv, E

6

8

8 %

2 !2

2

170.71 93.85 5.29 7.08 21.16 101.40

from

control

+ 2.94 _+ 2.70 + 1.09’ f 0.75 f 1.37’ f 1.44

4

PHOSPHATASE

TABLE ACTIVITY

+ 0.42 _+ 1.35 + 0.26 + 0.09 _+ 0.45 + 0.06

f f f t f f

1.47” 0.48’ 0.23e 0.31” 0.69e 1.07’

Treated 61.11 25.52 5.63 3.80 7.36 5.52

Phospholipid’

0.046 0.156 2.797 2.146 0.435

f f f k + -

IN

0.005 0.021 0.877 0.041 0.089

Control

DIETHYLAMINOETHOX~HEXESTROL~

AND ACID

Control 38.99 18.70 1.71 2.85 9.55 0.93

PHOSPHOLIPID,

(p < 0.05).

175.71 100.46 10.80 6.49 14.53 94.44

Treated

OF PROTEIN,

l?roteinb

k 15.08 f 2.95 k 1.02 f 0.55 f 0.94 + 2.95

Control

’ Mean + SE. b mg/g liver. C pm01 P/g liver. d wol P/hr/mg protein. l Significantly different

Total liver Nuclei Mitochondria Lysosomes Microsomes Supernate

Fraction

DISTRIBUTION

Acid

0.108 0.189 2.420 1.086 2.084

+_ 0.005’ + 0.028 _+ 0.064 + 0.181’ f 0.334” -

Control

With

0.220 + 0.027 0.327 + 0.035 3.818 f 0.313 6.302 k 0.708 0.570 + 0.121 -

phosphatase”

OF RATS TREATED WITH

Treated

THE LIVER

0.347 0.276 1.954 2.118 1.311

Triton

+ 0.008= f 0.030 * 0.064’ f 0.439’ &- 0.138 -

Treated

“n E $ g

E -

EXPERIMENTAL

39

PHOSPHOLIPIWSIS

Lysosomal enzyme activity. The drug treatment induced a significant increase in whole liver acid phosphatase activity (Table 4). However, this enzyme activity was significantly reduced in lysosomal fractions. Following Triton X-100 preincubation, increased acid phosphatase activity was noted in almost all fractions. TABLE PHOSPHOLIPID

DISTRIBUTION

5

AND ENZYME ACTIVITY IN MICROSOME~ WITH DIETHYLAMINOETHO~YHEXESTROL

FROM RAT LIVER

TREATED

Treated Parameter” Enzymeb Inosine diphosphatase Glucose 6-phosphatase Coumarin f-hydroxylase MT-aseC Phospholipidd Total Phosphatidic acid Phosphatidylethanolamine Phosphatidylcholine Phosphatidylinositol + sphingomyelin Lysophosphatidylcholine Unidentified

Control

oral

ip

257.10 f 227.79 + 4.11 f 6.19 f.

14.38 31.81 0.52 0.21

220.30 f 36.02 233.69 + 20.46 4.54 + 0.56 2.75 + 0.35d

227.85 f 176.72 f 2.28 + 3.68 +

15.18 26.59 0.31de 0.75d

6.31 + 0.08 + 1.18 + 3.1Ort 0.97 f

0.40 0.02 0.08 0.16 0.15

7.13 + 0.19 0.16 + 0.02” 1.44 * 0.02” 3.56 + 0.13’ 1.22 +_ 0.13

6.04 & 0.16 + 1.15 k 2.84 + 1.45 rt

0.17 + 0.15 0.03 + 0.02

0.27 + 0.05 0.13 + 0.04’

0.30 + 0.07 0.06 + 0.02

0.261 0.01’ 0.06’ 0.22* 0.24

0 Mean + SE, four animals per group. b pmol/lir/g liver. c 4adenosyl-L-methionine microsomal-phospholipid methyl transferase activity in pmol/hr/g liver. d pm01 phospholipid: P/g liver. e Significantly different from control (p < 0.05). ’ Significantly different from oral treatment group (p < 0.05).

Following treatment inosine diphosphatase and glucose 6-phosphatase activities were reduced; coumarin 3-hydroxylase was decreased in the group which was treated ip only. S-adenosyl+methionine microsomal-phospholipid : methyl transferase was diminished, while no appreciable changes developed in the concentration and distribution of phospholipids (Table 5). Microsomal

enzyme activities

and phospholipids.

DISCUSSION Nature of concentric, lamellated, or myeloid bodies. In various studies, numerous concentric lamellar bodies were found in human liver related to somewhat prolonged diethylaminoethoxyhexestrol therapy (Adachi et al., 1971; Matsuda et al., 1971; Oda et al., 1970; Shikata et al., 1970, 1972). The causal relationship was established by Yamamoto et al. (1971b) and confirmed later by others (Itoh and Tsukada, 1973:

40

DE LA IGLESIA

ET AL.

Iwamura, 1973). The reproducibility of these changes was tested in rats (Adachi et al., 1972; Yamamoto et al., 1971a) and in other species with different degrees of response by the data presented here. Morphological changes similar to those in our study have been described in several organs and tissues. Lamellated bodies appeared in response to the administration of various chemically heterogeneous compounds with a wide range of therapeutic applications, including antimalarials, anti-inflammatory, and cholesterol-lowering agents (Abraham, 1968; Dietert and Scallen, 1969; Dodson, 1973; Flodh and Magnusson, 1973; Gray et al., 1971; Hendy et al., 1969; Hildebrand et al., 1973; Hruban et al., 1972, 1973; Liillman et al., 1973; Vijeyaratnam and Corrin, 1973). Some resemblance existed with myeloid bodies normally occurring in tissues (Balis and Conen, 1964). The drug-related nature of these changes, however, would suggest different mechanisms of formation. The lesions have been shown to be reversible after discontinuation of treatment (Kitani 1972). Based on morphological and histochemical studies, these lesions were interpreted as modified or secondary lysosomes (Abraham et al., 1968; Hruban et al., 1972). Cytidine monophosphatase and nonspecific acid phosphatase reactions were observed in the limiting membrane of these bodies, rather than in the matrix. The morphological variation of these lesions would suggest a series of changes leading to the formation of lamellated bodies with peripheral acid hydrolase activity. Similar findings were attributed to metabolic changes affecting the lysosomal membrane (Abraham et al., 1968). This seems possible since the membranes underwent significant structural modifications indicated by changes in phospholipid content and distribution. Abundant amounts of normal as well as abnormal phospholipids were produced, but this was not paralleled by structural protein changes. The altered phospholipid: protein ratio might have resulted in some aberrant binding and changes in the spatial configuration of the molecular arrangement within the membrane. Subsequently, a circular distribution took place and the small membrane whorls evolved into larger concentric bodies. The Golgi apparatus and endoplasmic reticulum participated in the development of these lesions, indicated by their increased thiamine pyrophosphatase and acid phosphatase activities respectively. Although the importance of acid phosphatase in the endoplasmic reticulum has not been clearly established (Buvat, 1971), the rise in this fraction at least would indicate a rearrangement of enzyme synthesis or an activation of autophagic processes. Congruent with this idea, multicentric druginduced bodies were interpreted (Hruban et al., 1972) as intermediate forms of cytoplasmic degradation. These accounted for the increased activity of acid hydrolases reflecting an accelerated autophagy and elimination of abnormal constituents. Phospholipid metabolism. The morphological and biochemical data revealed a significant impairment of hepatic phospholipid metabolism as a result of the drug treatment. The simultaneous appearance of the lamellar bodies and the changes in phospholipid content and composition by diethylaminohexestrol suggested an association. The greatest increase in phospholipids was evident in lysosomes and in lysosomederived lamellar bodies with an overflow appearing in the supernatant fraction (Table 5), indicating that the drug action probably originated in these cell organelles. Furthermore, the reduced phospholipid levels in the microsomal fraction suggested a shift in the normal balance of enzyme activities which govern the synthesis and utilization of phospholipids among various cellular particles.

EXPERIMENTAL

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41

The alteration of phospholipid metabolism was most prominent in rats and not significant in guinea pigs correlating with ultrastructural findings. Some morphological effects were seen in hamsters and biochemical changes were minimal. This might be associated with a difference in the ability to metabolize the compound, while in rabbits the increased susceptibility would be due to a more generalized toxic reaction (e.g., the accumulation of neutral fat). Different metabolic pathways for diethylaminoethoxyhexestrol might exist in the species studied here, as indicated by differences observed in our histochemical studies and in other studies (Abraham et al., 1968; Hruban et al., 1972, 1973). In spite of the account of subtle species differences, the overall elevation of the hepatic phospholipid level, the appearance of abnormal phospholipids and the development of concentric lamellated bodies resembled the action of diethylaminoethoxyhexestrol in man (de la Iglesia et al., 1974; Oda et al., 1970; Seki et al., 1971; Yamamoto et al., 1971b). Enzyme changes.The reduced lysosomal enzyme activity, measured by the representative acid phosphatase activity was unrelated to changes in binding. Although adequate treatment with Triton X-100 released more enzymes in liver preparations isolated from both control and drug-treated animals, acid phosphatase activity still remained lower in the treated group. The decreased phospholipid content in the microsomal fractions was probably associated with diminished methyl transferase activity (Table 6). Microsomal phospholipids are required for the function of the endoplasmic reticulum (Feuer et al., 1972). Consequently, their reduction was probably associated with these decreased enzyme activities. Since several microsomal and lysosomal enzymes showed an impairment, alternate series of steps could evolve leading to the formation of secondary lysosomes, via compensatory mechanisms. Implications to human therapy. Various drugs have been shown to cause an accumulation of phospholipid-laden bodies and biochemical changes in a wide range of species, including man (Hildebrand et al., 1973; Hruban et al., 1972, 1973). This response seemed prompt but reversible upon cessation of treatment. Changes in microsomal phospholipids and the manifestation of drug effects bear close association (Feuer et al., 1972). In this relationship the endoplasmic reticulum constitutes a primary role, since this is the target organ evoking the adverse response to foreign compounds. The alteration of microsomal phospholipid metabolism and the distribution of individual phospholipids, somewhat preceded overt functional changes, allowing a possible differentiation between the action of drugs and hepatotoxins (Feuer et al., 1974). This study provided observations on the mechanism of action of drug-induced phospholipidosis. Further development of tests for phospholipid metabolism may result in practical applications. Careful monitoring of these parameters could reveal adverse drug reactions, thus permitting advantageous use of potential therapeutic agents. REFERENCES R., HENDY, R. AND GRASSO,P. (1968). Formation of myeloid bodiesin rat liver lysosomesafter chloroquine administration. Exp. Mol. Pathol. 9,212-229. ADACHI,S.,MATSUZAWA,Y., YOKOMURA, T., ISHIKAWA, K., UHARA,S.,YAMAMOTO, A. AND NISHIKAWA, M. (1972).Studieson drug-inducedlipidosis(V). Changesin the lipid composition of rat liver and spleenfollowing the administrationof 4,4’-diethylaminoethoxyhexestrol.

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