Lipidosis-like ultrastructural alterations in rat lymph nodes after treatment with tricyclic antidepressants or neuroleptics

Naunyn-Schmiedeberg's Arch. Pharmaeol. 286, 165--179 (1974) .�9 by Springer-Verlag 1974 Lipidosis-Like Uhrastruetural Alterations in Rat Lymph Nodes after Treatment with Tricyclic Antidepressants or Neuroleptics Renate L i i l lmann-Raueh Anatomisches Institut der Universitat Kiel Received August 9/Accepted October 7, 1974 Summary. Lymphocytes of rat lymph nodes have previously been revealed as reliable and sensitive indicators of a particular drug side effect, notably a generalized phospholipidosis, inducible by a compound of amphiphilie character (ehlorphenter- mine). The purpose of the present study was to examine the effects of other amphi- philie compounds upon rat lymphocytes. After oral administration of various trieyclie antidepressants (iprindole, imi- pramine, clomipramine, 1-ehloro-amitriptyline, 1-chloro-10,11-dehydro-amitripty- line, noxiptiline, amitriptyline), or of two neuroleptic drugs (ehlorpromazine, thio- ridazine) to rats, the popliteal lymph nodes were examined with the electron micro- scope. After a single application of either iprindole (50mg/kg), imipramine (100 mg/kg), or elomipramine (150 mg/kg) small proportions of lymphocytes were found to contain abnormal lamellated cytoplasmic inclusions. The size and number of inclusions and the number of affected lymphocytes increased with further treat- ment. Similar observations were made after treatment with the chlorinated ami- triptyline derivatives. On the other hand, only small numbers of lymphocytes were affected by noxiptiline, amitriptyline, chlorpromazine and thioridazine, even after prolonged treatments (up to 13 weeks) with high doses (up to 125--175 mg/kg). The present ultrastruetural findings are interpreted as representing lipidosis- like cellular alterations, yet of highly varying degrees. The large quantitative dif- ferences are tentatively suggested to be due to differences in the rate and mode of metabolism of the drugs, and due to differing degrees of amphiphilia of the com- pounds applied. Key words: Lipidosis -- Antidepressants -- Neuroleptics Lymphocytes -- Ultrastructure. Exper imenta l data on a part icular side effect of the anorectic drug ehlorphentermine have provided the basis for a recent ly proposed con- cept according to which compounds of pronounced amphiphi l ie character can induce a generalized phosphol ipidosis (L/i l lmann et al., 1973b). Pharmaeokinet ie, physieochemical, and biochemical findings have led to the suggestion that such drug molecules, due to their amphiphi l ie Send offprint requests to: Renate Liillmann-l~auch, Anatomisches Institut der Universitat, D-2300 Kiel, OlshausenstraBe, Federal Republic of Germany. 166 R. Lfillmann-Rauch character, become tightly, though not irreversibly associated to polar lipids (Liillmann et al., 1973 c; Scydel and Wassermann, 1973). This may result in a modification of the substrate properties of the polar lipids such that their metabolic degradation is impaired and that they will gradually accumulate within the affected cells (Schmien et al., 1974; Sciler and Wassermann, 1975). On the fine structural level, such an intracellular phospholipid accumulation is reflected by the formation of membrane- bound cytoplasmic inclusions of ]ysosoma] origin, with concentrically laminated or crystalloid internal patterns. Chlorphentermine-indueed ~ellular alterations of this type have been described in various tissues of several animal species (Liillmann-l~auch et al., 1972; Lfillmann et aL, 1973 a ; Liillmann- Rauch and Reil, 1973, 1974 b). Ultrastruetural observa- tions on cytological effects of other amphiphilic compounds, such as the hypocholesterolemic drug triparanol (Lfillmann et al., 1973a) and the anorectic drug fenfluramine (Liillmann-Rauch and Reil, 1974a) have supported the proposed concept. Recently, some tricyclic antidepressants (iprindole, imipramine, clomipramine, ]-chloro-amitriptyline) were also found to induce lipidosis- like alterations in various organs including neural tissues (Lfillmann- Rauch et al., 1973; Theiss et al., 1973; Karabelnik et al., 1974; Lfillmann and Lfillmann-Rauch, 1974; Liillmann-Raueh, 1974a, b, e). In contrast, two other antidepressants (amitriptyline and noxiptiline) and some neurolepties (chlorpromazine, thioridazine) could not be clearly stated to cause lipidosis-like alterations, at least not in rat lungs and liver (Liill- mann-lgaueh and Scheid, in preparation), nor in spinal cord and cerebel- lum of rats (Liillmann-Rauch, 1974c). According to general physico- chemical knowledge, all aforementioned psychotropic drugs display the characteristics of amphiphilia, since they all consist of an apolar aromatic ring system and a hydrophilic side chain with a positively charged nitro- gen atom (see Table 1 for molecular structures). All the more puzzling were the negative results from ultrastruetural examinations concerning some of the drugs mentioned above. I f these negative results are not taken as disproving the proposed concept, one might explain them partly by differences concerning the pharmaeokinetics, and partly also by differences in the degrees of am- phiphilia. Under this assumption, perhaps other tissues more suscep- tible than those examined so far, might be expected to show alterations typical of the drug side effect in question. As to the adverse action of chlorphentermine in rats, lymphatic tissue has been found so far to possess the most sensible cell types, notably lymphocytes and plasma cells, some of which contained abnormal lamellar inclusions already after a single oral application of the drug (i0 mg/kg) (Liillmann-Rauch ~nd Pietschmann, 1974). Drug-Induced Lipidosis 167 The present investigation was therefore designed to serve two pur- poses: 1. To establish lymphocytes and plasma cells as sensitive indi- eators of the lipidotic side effects also of some psychotropic drugs; for this purpose those trieyclic antidepressants were used, which had already clearly been shown to exert this adverse action within other tissues. 2. To examine the cytological side effects of those psychotropie drugs which have so far yielded negative results in other tissues. For this purpose attent ion was focused on the lymphoeytes, since in controls they were virtual ly never found to contain lamellated eytosomes of that kind to be described below, thus being the most reliable indicators. Materials und Methods ~u rats 1 of either sex, weighing 200--240 g (males) or 180--190 g (females), were kept on a dry pellet diet 2 and had free access to drinking water. Short term experiments (1--6 drug applications) were carried out on male rats only. The following tricyclic antidepressants were used: iprindole hydrochloridea; imipramine hydrochloridea; elomipramine hydrochlorideS; 1-chloro-amitriptyline hydrochloride s; 1-chloro-10,1 i-dehydro-amitriptyline s; noxiptiline hydroehloride ~ ; amitriptyline-hydrochloride s. The following neuroleptic compounds were used: chlorpromazine hydrochloride% thioridazine base 1~ The structural formulas of all compounds are summarized in Table 1. Thioridazine was dissolved in 0.1 N HC1, all other compounds were dissolved in distilled water. Drug concentrations were made up such that the intended doses could be administered in a volume of 0.5 ml/100 g body wt. Five times a week rats were force-fed with the drugs by means of a rigid stomach tube. The number of animals in each group, the single dosages, and the total num- ber of doses applied, are summarized in Table 2. In addition, 8 rats kept under identical conditions but receiving only water via the stomach tube, were used as controls. At 24 hrs after the last drug administration, rats were anaesthetized with pentobarbital, injected intraperitoneally. The lymph nodes of the popliteal region were removed, bisected, and fixed in cold 3O/o glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) for 2 hrs. Tissue samples were post-fixed in unbuffered 2~ OsO~ for 2 hrs, dehydrated with ethanol and embedded in Araldite. Ultrathin sections, cut on a Reichert microtome, were stained with uranyl acetate (saturated solution in 70O/o methanol) and lead citrate, for 5 rain each. Electron micrographs were taken on a Zeiss EM 9 or on a Philips EM 300 electron microscope. 1 Wistar AF/HAN, Zentralinstitut fiir Versuchstierzfichtung, Hannover, Federal Republic of Germany. 2 Altromin | Altrogge, Lage (Lippe), Federal Republic of Germany. 3 Prondol | Wyeth, Mfinster, Federal Republic of Germany. 4 Tofranil | Thomae, Biberach a. R., Federal Republic of Germany. 5 Anafranil | Thomae, Biberach a. R., Federal Republic of Germany. Experimental compounds, kindly supplied by Hoffmann-La Roche and Co., Basle, Switzerland. 7 Agedal | Bayer, Leverkusen, Federal Republic of Germany. s Laroxyl | Hoffmann-La Roche and Co., Basle, Switzerland. 9 Megaphen | Bayer, Leverkusen, Federal Republic of Germany. 10 Melleril | Sandoz, Niirnberg, Federal Republic of Germany. 168 1~. Liillmann-Rauch Table 1. Molecular structures of compounds investigated ANTIDEPRESSANTS CH 3 Iprindole =ell - CH2-GH2- IN-~H C H 3 Amitriptyline Cl~ CH I~ 3 =CH - CH2-CH2- IN-H CH 3 1- Chloro-amitriptyline 1-Chloro_10,11CH3 dehydro-amit r iptyline " C H2- CH =.-o-o,2 I CH 3 Noxiptiline CH CH 3 Imipramine CI CH 3 -CH 2 - CH2- C H 2- i~-H CH 3 Clomipramine NEUROLEPTICS CJ ?.3 CH 3 Chlorpromazine S-CH 3 H ,,e .CH3 S~N - CH2-CH2-O Thioridazine For valuation of the present ultrastructural observations no strictly morpho- metrical and statistical methods were applied. In order to obtain a merely semi- quantitative estimate of the degree of alterations in lymph nodes os animals treated with low drug doses or with Compounds having little side effect, 4 6 grid squares (90 • 90 ~z) in the cortical lymph node regions, containing mainly lymphocytes (ap- proximately 160--190 profiles per grid square), were systematically scanned on the electron microscope screen, at a magnification of 10000, and the lymphocytes with lamellated eytosomes were counted. Cell profiles bearing abnormal inclusions were not taken into account, unless their nuclei were included in the plane of section, thus enabling the cells to be clearly identified as lymphocytes by the typical mor- phology of their nuclei (Mori and Lennert, 1969). Drug-Induced Lipidosis 169 Table 2. Details of drug treatment, and number of altered lymphocytes per grid square within cortical regions of the lymph nodes Compound No. of doses Dosage (mg/kg b.w. No. of 2kTo. of altered applied per day) animals lymphocytes per grid square, given for each animal Iprindole 1 50 2 8, 5 4, 5 50 2 9, 11 1 100 3 27, 19, 9 9, 16 100 2 numerous 25 (12 ~ 4 ~- 9) 100/125/150 1 a numerous 26 125 1 numerous 15 125 1 b few Imipramine 1 100 2 6, 5 2, 4 125 2 3, 13 10, 12 125 4 numerous 14 150 2 numerous 23 (16 ~- 7) 125/150 la numerous Clomipramine 1 100 2 none 1 150 1 2 6 150 2 18, 22 10, 11, 21 150 4 numerous 1-Chloro-ami- tr iptyl ine 1-Chloro-10, l l -dehydro- amitripty- line Noxiptil ine Amitriptyl ine Chlorpro- mazine Thioridazine 15, 50 120 3 numerous 10, 50 150 3 numerous i4, 39, 49 150 3 2, none, 0.5 63 (58 -4- 5) 150/175 1 �9 0.5 66 (56 ~ 10) 150/175 1 ~ 2 8, 10, 39 150 4 none 52 150 1 9 27 75 1 3 38 (28 -~ 10) 75/100 la 3 53 {27 -~ 18 -~ 8) 75/100/125 la 5 65 (2S + 16 + 21) 75/100/125 1 a 7 32 (20 ~- 12) 75/100 la 6 41 (19 ~- 11 6 + 5) 75/100/125/150 1 a none 53 (19 + 11 + 6 -- 14 + 3) 75/100/125/150/175 2 a 9,4 During the course of the experiment the drug doses were increased as listed in column 3. In column 2 the number of applied drug doses is stated separately for each dose level. b This animal was allowed to survive without drug treatment for a further la days before sacrification. 170 1~. Lfillmann-t~aueh Results Control Animals The ultrastructure of the popliteal lymph nodes of control animals was consistent with descriptions in the literature (Mori and Lennert, 1969). As noted previously (Lfillmann-l~aueh and Pietschmann, 1974), plasma cells very occasionally contained small lamellar cytoplasmic inclusions ; in lymphocytes lamellated eytosomes of that type to be de- scribed below were never encountered. Treated Animals A. Trieyelie Antidepressants a) lprindole. In lymph nodes of rats treated with iprindole for 2 to 5 weeks (9 to 25 • 100--150 mg/kg), numerous lymphoeytes (Fig. 1) and plasma cells (Fig.2) contained one or several abnormal lamellated in- clusions, measuring up to 2 ~ in diameter. The remaining cellular compo- nents were unchanged. Lymphocytes appeared equally affected in all regions of the popliteal lymph node. Occasionally, lymphocytes encount- ered within the walls or lumina of posteapillary venules or other blood vessels, also showed lamellated bodies, suggesting that ultrastruetural examination of the peripheral blood might be equally suitable to disclose the adverse drug action.--In addition to lymphocytes and plasma cells, other cell types of the lymph node such as macrophages and phagocytic reticulum cells also contained increased numbers of lamellated cyto- somes. The same was found in rats treated with imipramine, elomipram- ine, 1-chloro-amitriptyline and l-chloro-10,11-dehydrc-amitriptyline. Since these cell types often possessed a few lamellar bodies also in controls, they appeared not suitable as reliable indicators for the drug side effect in question, and they will not be mentioned further in this report. At 24 hrs after a single application of iprindole (50 mg/kg or 100 mg/kg) low proportions of lymphocytes (about 6 cells per grid square, or about 18 cells per grid square) were found to contain small abnormal inclusions, which displayed the early stages of lamellae formation. Such inclusions (Figs. 3 and 4) were delimited by a single unit membrane, and contained Fig. i. Low power electron micrograph from a cortical region of the popliteal lymph node of a rat treated with iprindole (25• 100--150 mg/kg). Several lymphocytes contain abnormal lamellar inclusions (arrows). RC reticuhm cell. • Fig. 2. Low power view of a medullary lymph node region of the same rat as in Fig. 1. Plasma cells contain large abnormal lamellar inclusions, which at this magnification may appear as electron dense bodies (arrows). • 5400. Inset: Lamellar body in a plasma cell of a rat treated with a single dose of iprindole (1 • 100 mg/kg), x 24600 Drug-Induced Lipidosis 171 Figs. 1 ~nd 2 172 g. Liillmann-Raueh lamellae emanating from an amorphous dense matrix. In favourable places, the lamellated material was seen to display a periodicity measuring approximately 4 nm (Fig. 5). At this stage of drug treatment already, a few lymphocytes with abnormal lamellar bodies were found also in the lumina of blood vessels. After 4 or 5 applications of iprindole (4--5 • 50 mg/kg) the number of affected lymphocytes had not markedly in- creased as compared to that found after a single administration of 50 mg/kg, but many inclusions had grown in size. In animals treated with 9 or more doses of iprindole (100--150 mg/kg) the numbers of affected lymphocytes were too large to be counted accurately in the described manner. The alterations of plasma cells paralleled those described for lympho- eytes. Lamellated inclusions, clearly larger and more numerous than in controls, were seen in plasma cells already after a single application of iprindole (50 mg/kg and 100 mg/kg) (Fig. 2, inset). To examine the reversibility of the iprindole-induced cellular alter- ations, one animal, which had been treated with the drug for 3 weeks (15 • 125 mg/kg), was allowed to survive without treatment for a further 13 days before saerifieation. Under this condition, only a few lymphoey- tes and plasma cells were found containing lamellar bodies. b) Imipramine and Clomipramine. The cytological alterations in- duced by imipramine and clomipramine were morphologically identical with those described for iprindole. The side effects of these two drugs were, however, slightly less pronounced. After a single application of imipramine (1 • 100 mg/kg) a low proportion of lymphocytes (approxi- mately 5 cells per grid square) was found to contain lamellated bodies (Fig. 6a). After a single administration of elomipramine (1 • 100 mg/kg) cellular alterations could not be stated with certainty, while a dose of 1 • 150 mg/kg induced clearly abnormal inclusions in a few lymphocytes (approximately 2 cells per grid square) (Fig.7a). During further treat- ments with imipramine or with e]omipramine the size of inclusions (Figs. 6 b and 7 b) and the number of affected lymphocytes increased. After 4 applications of imipramine (4 • 125 mg/kg) and after 6 applications of clomipramine (6 • 150 mg/kg) the numbers of altered lymphocytes were comparable to those found after a single dose ofiprindole (1 • 100 mg/kg). After still longer periods of treatment with either compound, the affected lymphoeytes were too numerous to be counted by the simple method presently applied.--The alterations in plasma cells again paralleled those in lymphoeytes, after treatment with either drug. c) 1-Chloro-Amitriptyline and 1-Chloro-lO,11-dehydro-amitriptyline. Either compound caused conspicuous cytological alterations, morpho- logically identical with those described in the foregoing sections. The Drug-Induced Lipidosis 173 Figs.3--5. Abnormal lamellated inclusions in lymphocytes of rats treated with a single dose of iprindole Fig.3. Iprindole 1 x50 mg/kg. The abnormal inclusion is clearly delimited by a single membrane (arrows) and contains an amorphous matrix from which concentric lamellae appear to emanate. X 64000 Fig.4. Iprindolc 1 x 100 mg/kg. Two abnormal inclusions are seen, at the larger of which the delimiting membrane is conspicuous (arrows). From the amorphous matrix lamellac seem to emanate, which display a periodic structure. X64000 Fig. 5. Rectangular area indicated in Fig. 4, at higher magnification. A periodicity of approximately 4 nm is visible, x 199000 .degree of alterations found after t reatment with 1-chloro-10,11-dehydro- amitr iptyl ine ( lO• and with 1-chloro-amitriptyline (15 • 120 mg/kg) appeared comparable with that evoked by imipramine under similar conditions. However, since short term experiments were not per- 174 g. Liillmann-l~auch Fig. 6 a and b. Lymphocytes of rats treated with imipramine. (a) Imipramine t • 100 mg/kg, Three small abnormal inclusions are seen. N nucleus. • (b) Imi- pramine 10 • 125 mg/kg. Two adjacent lymphocytes contain abnormal inclusions (arrow@ • 10000 Fig. 7 a and b. Lymphocytes of rats treated with clomipramine. (a) Clomipramine 1 • 150 mg/kg. Four small abnormal inclusions are seen. M mitochondrium. • 30300 (b) Clomipramine 6 X 150 mg/kg. A lymphocyte, encountered in the lumen of a postcapillary venule, contains a lamellated inclusion (arrow). E vascular endo- thel ium; EC erythrocyte; L another lymphocyte, x 10000 Drug-Induced Lipidosis 175 Fig. 8 a and b. Lymphocyte of a rat treated with ehlorpromazine (65 • 75-- 125 mg/kg). One abnormal inclusion is present, which is seen in (b) at higher magnification. (a) • (b) • Fig. 9 a and b. Lymphocyte of a rat treated with thioridazine (53 • 75-- 175 mg/kg). The cytoplasm contains two abnormal inclusions, which are seen in (b) at higher magnification. (a) • 13500; (b) • 61500 formed, a quantitat ive comparison with the side effects of the other antidepressants can only be tentative. d) Noxiptiline and Amitriptyline. In contrast to the antidepressants mentioned so far, noxipti l ine and amitr iptyl ine showed a very low ac- t iv i ty with respect to the side effect in question. In 4 out of 5 animals, 176 R. Lfillmann-Rauch noxiptiline given for 3--13 weeks caused the formation of abnormal lamellated inclusions in a few lymphocytes, but the inclusions were always small and the number of affected lymphocytes never exceeded 2 cells per grid square. In one animal treated for 8 weeks, no abnormal inclusions were found in lymphocytes. In 3 animals, which had received amitriptyline for 2 and 8 weeks, alterations in lymphocytes could not be stated with certainty. Only in one rat treated for 10 weeks, clearly abnormal lamellar bodies were found in a few lymphocytes. ]3. Neuroleptic Drugs In lymph nodes of all 4 animals treated with chlorpromazine (75--125 mg/kg) for 5--13 weeks and in 3 out of 4 rats treated with thioridazine (75--175mg/kg) for 6--10weeks, a low proportion of lymphocytes (3--9 ceils per grid square) contained clearly abnormal lamellated inclusions (Figs. 8 and 9). The remaining cellular constituents of affected lymphocytes appeared normal. Discussion The cytological effects of iprindole, clomipramine, imipramine and of the chlorinated amitriptyline derivatives upon rat lymph nodes are iden- tical in morphology and comparable in extent with the alterations pre- viously found in this tissue and in peripheral blood after administration of chlorphentermine (Lfillmann-Rauch and Pietschmann, 1974; Lfill- mann-l~auch, 1975). They furthermore correspond to cellular changes induced by these antidepressants in other tissues, such as liver, lung, retina and brain (Lfillmann-t~auch et al., 1973 ; Lfillmann-I~auch, 1974a, b, e). The present fine structural observations are consistent with the concept of a drug-induced phospholipidosis. The results of short term experiments on iprindole, imipramine and clomipramine indicate that rat lymphocytes and plasma cells are rather susceptible for the drug side effect, more than other tissues (lungs and liver) so far studied under this aspect (Ltillmann-t~aueh and Scheid, in preparation). In support of the previous findings on chlorphentermine, the present observations on antidepressants thus establish lymphoeytes and plasma cells of rats as particularly sensitive indicators of the potency of an amphiphilie compound to induce a generalized lipidosis. The findings on lymphocytes of animals chronically treated with noxiptiline or amitriptyline, or with the neuroleptics ehlorpromazine or thioridazine may, on morphological grounds, likewise be interpreted as lipidosis-like cellular alterations, yet of very modest degrees. Thus, by using rat lymphoeytes as sensitive and reliable indicators, some psychotropic drugs of amphiphilie character, which had so far yielded negative ultra- Drug-Induced Lipidosis 177 structural results, could be disclosed to fit into the proposed concept of a drug-induced lipidosis. I t seems, however, obvious that the present results do not permit any conclusions as to the biological significance of the rather weak side effects exerted by noxiptiline, amitriptyline, chlor- promazine and thioridazine. It should be mentioned that in the case of the more effective amphiphilic compounds the functional significance of the induced lipidosis is not yet understood either. On the other hand, the phenomenon should not be neglected as such, since there are reports on severe human cases of drug-induced phospholipidosis, after treatment with the coronary vasodilator 4,4'-diethylaminoethoxy-hexcstrol, being also an amphiphilic compound (Yamamoto et al., 1971; Shikata et al., 1972; de la Iglesia et al., 1974). The most intricating outcome of the present investigation is the finding that the applied compounds, regardless of their close relation- ships concerning chemistry and main pharmacological actions, show large differences in their potencies to cause lipidosis-like cellular alterations. At present, no experimental evidence is available to explain these differen- ces conclusively. On the basis of the proposed concept, which implicates the pronounced amphiphilic character of a drug molecule as one of the main prerequisites, some possible explanations may be offered. One might be based on differences in the rate and mode of drug metabolism. Metabolic alterations at the aromatic ring system, as for example hy- droxylation, would decrease the apolarity of this moiety and thus reduce the amphiphilia of the resulting metabolites. This explanation might account at least partly for the present findings on chlorpromazine and thioridazine. In a simplified biological system, i.e., in cultured ganglion cells, which are presumably incapable of a quantitatively significant metabolic modification of the drug, chlorpromazine has, in fact, been demonstrated to induce lamellated cytoplasmic inclusions (Brosnan et al., 1970). Another explanation, applicable to the observations on anti- depressants, might be based upon structural differences in the aromatic ring systems and upon the presence or absence of a double bond con- necting the side chain to the ring system. Such differences, :whieh are evident when noxiptiline and amitriptyline are compared for example with imipramine, might result in a lower degree of amphiphilia in the case of the former. The introduction of a chlorine atom in 1-position, however, enhances the apolarity of the aromatic ring system, thus producing a compound of higher amphiphilia, which is more potent in in- ducing a lipidosis. 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