Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from Thorax (1976), 31, 558.

Effect of chlorphentermine on the lipids of rat lungs

JOSEPHINE GLOSTER, DONALD HEATH, PHILIP HASLETON, and PETER HARRIS Cardiothoracic Institute, 2 Beaumont Street, London WIN 2DX and Department of Pathology, University of Liverpool, Liverpool

Gloster, Josephine, Heath, D., HaEleton, P., and Harris, P. (1976). Thorax, 31, 558-564. Effect of chlorphentermine on the lipids of rat lungs. Chronic administration of chlor- phentermine to rats resulted in a reduction of body weight compared to a normal control group. The weight of the heart, liver, kidney, and spleen was less in the treated group while the weight of the lungs was increased significantly. There was no change in the ratio of right ventricular to left ventricular weight in the rats treated with chlorphenter- mine, supporting the views that this drug does not cause pulmonary hypertension. Biochemical analysis showed that the increase in the weight of the lungs was due to the accumulation of phospholipid. All classes of phospholipid were affected, but particu- larly phosphatidyl choline, the tissue concentration of which increased nine times.

Chlorphentermine also increased the proportion of palmitate present in pulmonary copyright. phosphatidyl choline. Histological examination of the lung after treatment with chlor- phentermine showed evidence of this drug-induced lipidosis. No conclusion can as yet be reached as to the maechanism involved in the accumulation of phospholipid in the lung after chlorphentermine. http://thorax.bmj.com/ Chronic administration to rats of certain drugs lung and especially these cells are particularly including chlorphentermine (p-chloro-a, a-dimethyl susceptible to the effects of chlorphentermine. A phenethylamine hydrochloride) brings about a dis- pulmonary phospholipidosis is rapidly induced order of phospholipid metabolism (Schmien, with the appearance of myelin figures in not only Seiler, and Wassermann, 1974; Karabelnik and the granular pneumocytes and Clara cells but Zbinden, 1975; Seiler and Wassermann, 1975). also membranous pneumocytes and the en- This is characterized by the accumulation in dothelial cells of pulmonary capillaries (Smith several organs of lipids which appear ultra- et al., 1973). Many of the lamellar inclusions are structurally as intracellular myelin figures. Such shed into alveolar spaces where they lie enmeshed on September 26, 2021 by guest. Protected inclusions are especially common in the lung and in phospholipid lattices or become engulfed by have been reported in association with the ad- large numbers of pulmonary histiocytes which ministration to rats of chlorphentermine (Smith, pass into the alveolar spaces (Vijeyaratnam and Heath, and Hasleton, 1973; Smith, Heath, and Corrin, 1972; Smith et al., 1973). The purpose of Hasleton, 1974a) and of the antidepressant the investigation described in this paper was to iprindole which is an iminodibenzyl derivative determine the magnitude and nature of the pul- (5- (3-dimethyl-aminopropyl) -6,7,8,9,10,1 1-hexa- monary phospholipidosis induced by chlorphenter- hydrocyclo-oct(b) indole). mine. The reason for the predilection of phospholipi- dosis for the lung is the rapid turnover of METHODS phospholipid in that organ associated with the ANIMALS Seventeen young male Sprague Dawley formation of pulmonary surfactant by granular rats weighing 175-200 g were used in these studies. (type II) pneumocytes and possibly by Clara cells The animals were allowed free access to both (Smith, Heath, and Moosavi, 1974b). Hence the food and water. They were weighed at the outset 558 Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from Eflect of chlorphentermine on the lipids of rat lungs 559 of the experiment and twice weekly thereafter. The lower lipid-containing chloroform phase was Twelve of them were given intraperitoneal in- taken to dryness using a rotary evaporator. The jections of chlorphentermine hydrochloride in a total lipids from the lungs of the control rats dosage of 50 mg per kg body weight daily for six were immediately redissolved in 3-4 ml chloro- days in each week until 50 injections had been form, and the lipids from the lungs of the test given. The remaining five rats were kept as con- rats were redissolved in 20 ml chloroform. trols and received intraperitoneal injections of similar volumes of physiological saline. LIPID SEPARATION Preliminary separation of the total lipid extract from the lungs of both groups REMOVAL OF ORGANS At the end of the experi- of rats was made on small columns (1-5 g) of 100- ment the animals were weighed and then killed 200 mesh silicic acid (Mallincrodt) activated at by cervical dislocation. The heart, lungs, liver, 1100C overnight before use. The neutral lipids spleen, and kidneys were quickly removed and were eluted from the column by the use of 30 ml rinsed in ice-cold saline to remove blood. All chloroform and the phospholipids by the addition visible fat and connective tissue were removed, of 30 ml methanol. All the lipid extract from the and the organs were blotted dry with fllter paper control lungs and one-tenth of the total extract and weighed. The heart was weighed after the (2-0 ml) from the chlorphentermine lungs was great vessels had been dissected off. The atria separated in this way. were removed, and the free wall of the right The eluates from the columns were taken to ventricle was dissected, blotted, and weighed dryness with the use of a rotary evaporator and separately. The left ventricle was weighed to- immediately made up to volume with chloroform. gether with the interventricular septum. The neutral lipids were made up to a volume of Two portions of lung, weighing between 0-2 10 ml for the test lungs, or to a volume of and 0 5 g, were taken and dried by heating in an 2-0 ml in the case of the control lungs. The phos- oven at 500C, and by desiccation until a constant a total of pholipids were dissolved in volume copyright. weight had been obtained. The dried portions of 5*0 ml chloroform in both groups. lung were then reweighed to determine the amount of water during the drying procedure. THIN-LAYER CHROMATOGRAPHY Separation of the lipid classes was carried out on 0-25 mm thick HISTOLOGICAL EXAMINATION Part of the right layers of silica gel H (Merck). The plates were upper lobe was removed and fixed in 10% buffered prewashed in methanol: diethyl ether (1: 1 v/v) http://thorax.bmj.com/ formalin for histological examination, as described for several hours, allowed to dry, and then acti- previously (Heath, Smith, and Hastleton, 1973). vated for one hour at 1100C immediately before The remainder of the lungs was used for bio- use. chemical examination. The neutral lipid classes were separated using the solvent mixture petroleum ether (BP 40- EXTRACTION OF LIPID The remaining lung tissue 600C): diethyl ether: acetic acid (85:15 : 2 v/v). was weighed, minced finely with scissors, and Duplicate 100 ,ul aliquots were applied to the

homogenized in methanol using a Teflon in glass plates as bands about 2-5 cm wide. A standard on September 26, 2021 by guest. Protected homogenizer. The lipids were extracted accord- mixture was also run on each plate to aid ing to the procedure described by Folch, Lees, identification of the separated lipid classes. The and Sloane-Stanley (1957). Sufficient chloroform neutral lipids were located by spraying the plates was added to the methanol homogenate to give a with 10% phosphomolybdic acid in methanol. The 2: 1 mixture by volume of chloroform and lipid-containing zones and corresponding blank methanol. A total volume of 60 ml chloroform: areas were scraped off the plates. Each lipid methanol (2: 1 v/v) was used for the extraction fraction was eluted by being shaken with two of the control lungs, and 100 ml of the same 5 ml portions of diethyl ether, and centrifuging mixture was used for the extraction of lung tissue between each extraction. The combined extracts from the chlorphentermine treated rats. After were taken to dryness. Cholesterol and cholesterol standing overnight at room temperature the esters were determined by the method of extract was filtered, and the residue was re- Maclntyre and Ralston (1954). Triglyceride was extracted for a second time. After filtration the determined by the modification of Van Handel extracts were combined and 20 vol % of (1961) and free fatty acids by the colorimetric 01 M NaCl was added to form a two-phase system. method of Duncombe (1964). Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from 560 Josephine Gloster, Donald Heath, Philip Hasleton, and Peter Harris

Duplicate 50 ,lI aliquots of the phospholipid there was a linking strand of cytoplasm extending containing extract were taken for the direct deter- through a Cohn's pore. The foamy appearance mination of total phospholipid phosphorus by the of the cells was due to numerous clear intra- method of Bartlett (1959). Duplicate 100 ,ul cytoplasmic vesicles which had aggregated in aliquots were then separated on thin layers of some cells to produce larger clear areas. In silica gel H using a solvent system of chloro- many alveolar spaces the foam cells had fused form : methanol: acetic acid: water (65: 45: 1: 4 together to produce a granular pale-staining mass. v/v) and solvent tanks lined with filter paper. At higher magnification this apparently amor- The position of the lipids was visualized by iodine phous material still showed the vesicular pattern vapour. The lipid zones were scraped off the characteristic of the cytoplasm of the viable cells. plates, and the phospholipids eluted with two Some of the intra-alveolar cells in tissue fixed successive 5 ml portions of a mixture of chloro- in formalin gave a vivid magenta colour on stain- form : methanol : water: acetic acid (50 : 50: 15: ing with the periodic acid-Schiff reaction. Other I v/v). The volume of the extract was reduced foam cells, usually smaller, in the same section by evaporation using a current of air and the gave a negative reaction with this stain. The phosphorus content of each phospholipid class granular debris within the alveoli which had was determined by the method of Bartlett (1959). resulted from breakdown of groups of the intra- Appropriate blank areas from a plate were pro- alveolar cells gave a cherry-pink reaction with cessed in the same way. the PAS stain. None of the intra-alveolar cells gave a positive reaction for haemosiderin with GAS LIQUID CHROMATOGRAPHY The fatty acid the Prussian blue reaction. There was no evidence composition of the lecithin fraction was deter- of fibrosing alveolitis. Around many of the mined by gas liquid chromatography. Two bronchi was a cellular exudate of plasma cells, hundred microlitres of phospholipid extract was lymphocytes, and mononuclears. applied to a thin-layer plate and the phos- copyright. pholipids were separated as described above. The EFFECT OF CHLORPHENTERMINE ON THE BODY AND were with 10% phosphomolybdic plates sprayed ORGAN WEIGHTS OF THE RAT The body weights of the The lecithin was acid to locate phospholipids. the rats at the start of the experiment and at the scraped off the plate and methylated directly by of the for 6 h at end, together with the wet weights lung, refluxing with 5% H,SO4-methanol and spleen, are given in Table heart, liver, kidneys http://thorax.bmj.com/ 720C. The methylated fatty acids were extracted the two groups of ether (BP 40-600C). A Perkin I. The starting body weights of with petroleum rats were similar. After 50 days of chlorphenter- gas chromatograph with a Elmer Model 881 treatment the mean body weight of the test was used for the mine flame ionization detector was 83 g less than the mean weight of the were carried out on group analysis. The separations rats. This difference in body weight was m and +-inch control dual stainless steel columns ( 2 long reflected in the smaller size of the heart, liver, OD) packed with 10% polyethylene glycol adipate in the Chromosorb W. The initial tem- kidneys, and spleen chlorphentermine on 80-100 mesh group. The notable exception to this was in the perature of the column was 1650C programmed on September 26, 2021 by guest. Protected for a temperature rise of 1OC/min to a maximum of 2000C. The area under each peak was deter- mined by means of an Infotronics electronic TABLE I integrator (Model CRS-100). BODY WEIGHTS AND ORGAN WEIGHTS IN THE RAT AFTER TREATMENT WITH CHLORPHENTERMINE RESULTS Control Test n=5 n=12 HISTOPATHOLOGY OF THE LUNG AFTER CHLORPHEN- (wt in g ± SD) (wt in g ± SD) TERMINE In all the rats the alveolar spaces were Body weight-start 184 ± 6 180 ± 8 with large ovoid cells, 30X15 ,um, which end 405±36 322+29* packed Lung 2-366±0-4 3 701±0 5* had foamy cytoplasm. Many of the cells were Whole heart 1 204i0-2 0.833±0.1* Left ventricle 0 802±0 1 0.576±0.1* single and had an eccentric nucleus. Some con- Right ventricle 0.179 ±0-02 0-136±0-01 * or more and it seems likely Liver 14 539 ±2-7 11*792±0 9t tained two nuclei, Kidneys 3t198±04 2-974±+01 that in these the cytoplasm of two or more cells Spleen 0720±0-2 0512+0-1t had fused to form a syncytium-like mass. In some *P < 0001 instances, the cells were dumb-bell-shaped where t Pc0.01 Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from

Effect of chlorphentermine on the lipids of rat lungs 561

case of the lungs which weighed significantly finding, however, was the sevenfold increase of more than in the control rats (P

mean of 80-5-+-17% water, while the figure was http://thorax.bmj.com/ reduced to 76 6+1-6% water in the test rats given chlorphentermine (P

TrABLE III Sp LIPID CONTENT OF RAT LUNGS AFTER TREATMENT WITH CHLORPHENTERMINE 0 10 20 30 40 50 60 70 Control Test % n=5 n=12 distribution (mg lipid ± SD) (mg lipid ± SD) F IGURE Percentage distribution of phospholipid Cholesterol ester 13 ±0i3 2-4±0.5* classes in rat lung after treatment with chlorphenter- Cholesterol 7 1±P10 15.8±3.5* mine. SF=material at Free fatty acid 0 4±01 1.1±0.3* migrating the solvent front. Triglyceride 19*7±9*3 6-3±3.2* Tentatively suggested to be a mixture of phosphatidic Phospholipid 411 ± 8-4 2837 ±62-5* Total lipid present acid and phosphatidyl glycerol; PE=phosphatidyl in lung 69-6±18-1 309 2 ± 66-9 ethanolamine; Pl=phosphatidyl inositol; PS=phos- phatidyl serine; PC=phosphatidyl choline; Sp= *p < 0 001 The content has been expressed as mg lipid present in both lungs. sphingomyelin. Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from

562 Josephine Gloster, Donald Heath, Philip Hasleton, and Peter Harris percentage of phosphatidyl choline was increased peritoneal injection in rats caused a reduction in to 61% of the total. Proportionally this increase body weight compared to a normal control group. occurred at the expense of phosphatidyl ethano- Lullmann et al. (1972) found a similar reduction lamine, phosphatidyl serine, and sphingomyelin whether the chlorphentermine was given by whose percentage distribution was, therefore, mouth or intraperitoneally. Young adult male lower. The percentage of phosphatidyl inositol rats were chosen for these studies so it would be present in the lungs after chlorphentermine treat- expected that they would be in a phase of active ment was increased slightly. In all the thin-layer growth at this period. Anorectic drugs lead to a separations there was material migrating above suppression of appetite, so it is important to phosphatidyl ethanolamine at the solvent front. distinguish those changes in organ weights due to This material was believed to be a mixture of the action of the drug from those occurring due phosphatidic acid and phosphatidyl glycerol al- to the effect of the reduced food intake. A pre- though no definite identification could be made. vious study on the effect of restricted food intake After chlorphentermine treatment the percentage on the rat (Gloster, Heath, and Harris, 1972a) of this material was also increased. The above showed a decrease in weight of all organs studied figures refer to the percentage composition; the compared with free-fed animals. After chlor- absolute quantity of each class of phospholipid phentermine, however, the weight of the rat lung per gram dry weight of lung was increased after was found to be significantly higher than in the treatment with chlorphentermine. controls. This increase in weight could not be Gas liquid chromatography was used to accounted for by the presence of oedema as the examine the fatty acid composition of the phos- percentage of water present in the lungs from phatidyl choline fraction of the lung. The per- the test animals was in fact decreased. The lung/ centage fatty acid composition determined in body weight ratio found in the previous study this way is given in Table IV. A characteristic (Gloster et al., 1972a) was the same in both free- of tissue is the high proportion of feature lung fed and food-restricted rats. The doubling of copyright. the saturated fatty acids occurring in the phos- lung/body weight ratio observed after chlorphen- pholipids. Thus in the lungs from the control termine can thus be accounted for by the action rats palmitic acid formed 68% of the total fatty of the drug and not simply by diet. acids in phosphatidyl choline. After chlorphenter- Similarly, a restricted diet had no effect on the mine treatment the amount of palmitic acid pre- lipid content of the lungs of the rat (Gloster, sent in phosphatidyl choline was increased to Heath, and Harris, 1972b). After chlorphenter-http://thorax.bmj.com/ 80 0% of the total (P<0 001). The total percentage mine, however, the total lipid content was of saturated acids was increased from a figure of increased, the biggest rise occurring in the phos- 74 8% in the lungs of the control rats to a figure pholipids. The histological changes occurring of 85-1% in the lungs of the test rats after after treatment with chlorphentermine show chlorphentermine treatment. evidence of this drug-induced lipidosis. The histological appearances of the lung were identical seen in a study in which TABLE IV with those previous

chlorphentermine was administered to rats on September 26, 2021 by guest. Protected PERCENTAGE FATTY ACID COMPOSITION OF LECITHIN FROM RAT LUNG AFTER TREATMENT WITH (Heath et al., 1973). CHLORPHIENTERMINE The occurrence of the large foam cells with Control Test lamellated inclusions in the alveoli of chronically n=5 n=12 (%±SD) (%+SD) treated rats was first reported by Franken, Lull- and has since been Myristic acid [14:01 1-5 +02 3.3 l05 mann, and Siegfriedt (1970) Palmitic acid [16:01 68-0±3t3 80-0±3-0 confirmed by other workers (Lullmann-Rauch Palmitoleic acid [16:11 6-4+0-9 59+0 9 Stearic acid [18:01 5-3+0 9 1-8±0-3 et al., 1972: Smith et al., 1973). Electron micro- Oleic acid [18:11 10.1+1.0 3-9±1:0 Linoleic acid [18:21 5*4i1-6 3-4+10 scopy was not carried out on lung tissue from the Arachidonic acid [20:41 3-1±+05 1-8+0-7 present animals but a previous study (Smith et al., Total saturated acids 74-8 85S1 1973) demonstrated that the alveolar 'foam cells' contained lamellar inclusions. They proved to be pulmonary histiocytes which had ingested DISCUSSION lamellar material extruded from granular pneumo- As would be expected, chronic administration of cytes and bronchiolar Clara cells. The intra- the anorectic drug chlorphentermine by intra- alveolar debris was found to contain fragments of Thorax: first published as 10.1136/thx.31.5.558 on 1 October 1976. Downloaded from

Effect of chlorphentermine on the lipids of rat lungs 563 lamellar material and phospholipid lattices. The fact that triglyceride is reduced after Similar laminated inclusions are found in granular chlorphentermine could be an indication that the pneumocytes, membranous pneumocytes, bron- synthetic pathway involving 1,2 diglycerides is chiolar Clara cells, pulmonary macrophages, and directed towards phospholipid synthesis rather endothelial cells of pulmonary capillaries. Lamel- than for neutral glyceride formation. The parti- lar inclusions of similar appearance have been cular effect of chlorphentermine on pulmonary found in other organs of rats and guinea-pigs phospholipid may be due not only to the high (Lullmann, Lullmann-Rauch, and Reil, 1973a). rate of phospholipid turnover in the lung but also These lamellations have a periodicity of between to the high concentration of the drug in that 4 and 5 nm, which is the same as described by organ. Lullmann, Rossen, and Seiler (1973b) Stoeckenius (1962) for phospholipid micelles in an found that the lung displays a very marked bind- aqueous phase. ing affinity for chlorphentermine with a low rate Biochemically, chlorphentermine treatment was of exchange of the bound material. Nuclear found to result in a large increase in the phos- magnetic resonance studies by Seydel and Wasser- pholipids of rat lungs while the total neutral lipid mann (1973) showed that chlorphentermine was content was almost unchanged. The neutral and bound specifically to lecithin. phospholipid contents in the control rats were There appear to be considerable species dif- similar to the values reported by Rooney, Cana- ferences regarding the histological and bio- van, and Motoyama (1974). Neutral lipids made chemical changes occurring after treatment with up 41% of the total lipids, while phosphatidyl chlorphentermine. The lungs of rats are greatly choline was the major component of the phos- affected, those of guinea-pigs less so, while the pholipids. After chlorphentermine the proportion lungs of mice showed no significant increase in of neutral lipids was reduced to only 8% of the lipid (Seiler and Wassermann, 1975). It is of in- total lipids. The percentage changes in the distri- terest that the closely related drug, phentermine, bution of phospholipid classes observed in this is not accumulated by the lung and does not cause copyright. study after giving chlorphentermine were similar the same histological changes as are shown by to those reported by Karabelnik and Zbinden chlorphentermine (Lullmann et al., 1973b). (1975). The major increase was in the amount of The lack of change in the ratio of right ventri- phosphatidyl choline present. cular to left ventricular weight in the rats treated with chlorphentermine speaks against the occur- Whole lung was used in the present study. No http://thorax.bmj.com/ differentiation can thus be made between lung rence of pulmonary hypertension. The muscular tissue and alveolar fluid. It has been well docu- pulmonary arteries of the test rats had appear- mented that the pulmonary surfactant making up ances identical with those described by Smith the active lining layer of the alveolar walls is rich et al. (1974a) in normal rats. Lullmann et al. (1972) in dipalmitoyl phosphatidyl choline. Chevalier also reported that the pulmonary vasculature was and Collett (1972) suggested that phosphatidyl unaffected although they observed an increased choline synthesized in the endoplasmic reticulum pulmonary arterial pressure in rats treated with of the type II alveolar cell was transferred chlorphentermine. into the on September 26, 2021 by guest. Protected through the Golgi complex, incorporated This work was supported by a grant from the Clinical lamellar bodies, and eventually released to the Research Fund of the National Heart and Chest surface. The lamellar body is thus probably Hospitals. composed largely of surfactant. Hallman and the Gluck (1975) have provided evidence that REFERENCES lamellar inclusion bodies have the same phos- Bartlett, G. R. (1959). Phosphorus assay in column pholipid content as surfactant. Another possible chromatography. Journal of Biological Chemistry, site of pulmonary surfactant production could be 234, 466. the bronchiolar Clara cells which show a marked Chevalier, G. and Collett, A. J. (1972). 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564 Josephine Gloster, Donald Heath, Philip Hasleton, and Peter Harris

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