Lysosomal Storage Of Sulfated Glycosaminoglycans Research Articles

Morphological and functional changes due to drug-induced lysosomal storage of sulphated glycosaminoglycans in the rat retina.

A series of dicationic amphiphilic drugs, most of them immunomodulatory agents, are known to induce generalised lysosomal storage of sulphated glycosaminoglycans (GAGs) in rats and in cultured cells of several species including man. The present study deals with the cytological effects of two experimental immunomodulatory acridine derivatives upon the retina of rats. The animals were treated orally with compound CL-90.100 (3,6-bis[2-(diethylamino)ethoxy]acridine) or an analogue for periods up to 22 weeks at a dose range of 60-90 mg/kg body weight and the retinae examined by light and electron microcopy. ERG measurements were done initially and after 16 weeks of treatment. All types of retinal cells developed abnormal cytoplasmic vacuoles which represented the ultrastructural counterpart of lysosomal GAG storage as demonstrated by histochemical and cytochemical staining experiments. The retinal pigment epithelium and the Müller cells were most prominently affected, photoreceptor cells to a lesser degree, and retinal neurons to varying degrees. The topographical distribution of the drug as detected by fluorescence microscopy closely resembled the distribution of the GAG accumulation in the retinal layers. After treatment for 16 weeks, the a-and b-wave amplitudes in the ERG were significantly reduced compared with the controls. the glycosaminoglycan storage in pigment epithelium is reminiscent of that seen in some inherited mucopolysaccharidoses of humans. When a given cell type shows lysosomal accumulation of glycosaminoglycans as a consequence of impaired degradation, it can be assumed to be engaged in the turnover of glycosaminoglycans under normal conditions. Thus the present results suggest that not only the retinal pigment epithelium but also Müller cells, photoreceptor cells, and, to variable degree, retinal neurons are normally involved in the catabolism of sulphated glycosaminoglycans. We believe that the lysosomal storage of glycosaminoglycans caused secondary cellular disturbance responsible for the functional changes shown by electroretinography.

Lysosomal storage of sulphated glycosaminoglycans induced by dicationic amphiphilic drug molecules: significance of the central planar ring system.

The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and several congeners are known to disturb the lysosomal degradation of sulphated glycosaminoglycans and thereby induce lysosomal storage of glycosaminoglycans in cultured cells and intact organisms. The molecules of tilorone and congeners consist of a planar aromatic ring system symmetrically substituted with two aliphatic side chains each carrying a protonizable nitrogen. In a previous study it was proposed that non-degradable glycosaminoglycan-drug complexes are formed by electrostatic interactions and that additionally intermolecular interactions between the drug molecules due to electronic coupling of their central planar ring system are important for formation and stabilization of the glycosaminoglycan-drug complexes and thus for the drug side effect in question. The significance of the central planar ring system was tested in the present study by comparing tilorone and the compound bis(beta-diethylamino-ethylether)hexestrol (DH) with respect to their potencies to cause lysosomal glycosaminoglycan storage in cultured bovine corneal fibroblasts. DH has the same side chains as tilorone, but its central apolar moiety lacks planarity. At a concentration (1.75 muM) which did not cause enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52), DH was significantly less potent than tilorone in causing storage of [35S]glycosaminoglycans. This is taken as support of the hypothesis that the planar tricyclic ring system is essential for the high potency of tilorone and its congeners to exert this adverse action.

The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo

The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo. In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS). In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the M PS-inducing potency as compared to chloroquine carrying an isoquinoline ring system. Tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) known as a potent inducer of MPS served as reference compound. The compounds were compared at a concentration (3 μM) which did not enhance the secretion of the lysosomal enzyme β-hexosaminidase (E.C. 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell. Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms. Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine. The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system. On the other hand, quinacrine was significantly less potent than tilorone and the symmetrically substituted acridine derivative 3,6-bis[2-(diethylamino)ethoxy]acridine investigated previously. This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.

Tilorone-induced lysosomal lesions: the bisbasic character of the drug is essential for its high potency to cause storage of sulphated glycosaminoglycans.

The immunomodulatory agent tilorone -2,7-bis-[2-(diethyl-amino)ethoxy]fluoren-9-one- and congeners are potent inducers of lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and cultured fibroblasts of animals and man. All potent inducers of GAG storage hitherto described are bisbasic polycyclic aromatic compounds. They are accumulated in lysosomes and disturb the degradation of GAGs, mainly dermatan sulphate. It has been proposed that the drugs cross-link the polyanionic GAG chains giving rise to undergradable drug-GAG complexes. This hypothesis implies that the bisbasic character of the drug molecules is essential for the side effect in question. In the present study, this was tested by comparing tilorone and its monobasic derivative (MT) with respect to (i) induction of GAG storage in cultured bovine corneal fibroblasts and (ii) physicochemical interactions with GAGs in vitro. The intralysosomal concentration of MT achieved after 1-3 days was of the same order of magnitude as previously shown for tilorone. Nevertheless, under conditions that did not enhance the secretion of a lysosomal enzyme (beta-hexosaminidase, EC 3.2.1.52), the ability of MT to cause storage of [35S]GAGs was significantly lower than that of tilorone. Morphological observations showed that MT was much more potent in causing lysosomal storage of polar lipids than of GAGs. CD spectroscopy with tilorone revealed that the presence of GAGs caused the primarily achiral drug molecules to display CD. This suggested a helical orientation of the tilorone molecules within GAG-drug complexes, and short intermolecular distances which allowed electronic coupling of the aromatic ring systems of adjacent drug molecules. In contrast, MT failed to display any induced optical activity, indicating the absence of highly ordered GAG-drug complexes. In conclusion, the present results show that the substitution of the planar aromatic ring system with two basic side chains is essential for the high potency of tilorone in inducing lysosomal GAG storage. This is paralleled by, and presumably causally related to, strong physicochemical interactions with GAGs.

Lysosomal storage of sulfated glycosaminoglycans induced by two bis-aminomethyl anthrachinones.

Several immunomomodulatory drugs, all of them symmetrically substituted dicationic amphiphilic compounds, are known to cause lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact animals and cultured fibroblasts. The storage is due to impaired GAG degradation. The standard compound is tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one). In the present study two bis-aminomethyl anthrachinones were examined for their ability to induce lysosomal GAG storage in cultured bovine corneal fibroblasts. For reference, a bis-aminoethoxy-anthrachinone compound (RMI-10.024) was included, which is known to be a potent inducer of lysosomal GAG storage. The present morphological, radiochemical, and biochemical results show that the bis-aminomethyl anthrachinone compounds investigated cause lysosomal storage of GAGs, although with significantly lower potencies than the bis-aminoethoxy anthrachinone. Dermatan sulfate contributed approximately 90% to the drug-induced increment of intracellular GAGs. The present results suggest that the length of the side chains, i.e., the distance between the aromatic ring system and the protonizable nitrogen of the side chains, and the position of the side chains relative to the aromatic ring system are important molecular features influencing the potency of inducing lysosomal GAG storage.

Tilorone-induced lysosomal storage of sulphated glycosaminoglycans can be separated from tilorone-induced enhancement of lysosomal enzyme secretion

This investigation deals with a drug side-effect. The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and congeners induce lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and in cultured cells. At high tilorone concentrations, GAG storage in cultured fibroblasts was previously reported to be accompanied, and presumably caused by, disturbance of intracellular targeting of lysosomal enzyme precursors, which leads to enhanced secretion and thus loss of lysosomal enzymes. The purpose of the present study was to examine whether the GAG storage induced in cultured bovine fibroblasts by low tilorone concentrations is also accompanied by enhanced lysosomal enzyme release. Enhanced secretion of β-hexosaminidase (EC 3.2.1.52) was taken as indicating the intracellular mistargeting of lysosomal enzyme precursors. Dose-response curves were established for (a) the intracellular accumulation of 35S-GAGs and (b) the release of β-hexosaminidase after exposure (72 hr) to tilorone (1–35 μM). For positive controls, the classical lysosomotropic agents NH 4C1 (1–30 mM) and chloroquine (1–60 μM) were used. With NH 4C1, 35S-GAG storage was accompanied by enhanced enzyme release throughout the concentration range ( ec 50 at 3.3 mM for either effect). With chloroquine, low concentrations (⩽ 5 μM) caused a small increase in 35S-GAG accumulation without abnormal enzyme secretion; at higher concentrations both drug effects were produced ( ec 50 around 15 μM for either effect). With tilorone, low concentrations (⩽5 μM) caused marked 35S-GAG accumulation without enhancement of enzyme release. The ec 50 for tilorone-induced 35S-GAG storage was 3 μM, as opposed to 15 μM for enzyme release. The results indicate that GAG storage induced by low concentrations of tilorone is due to mechanisms other than mistargeting and loss of lysosomal enzymes. On the basis of previous results it may be hypothesized that tilorone and other symmetrically substituted dicationic compounds form complexes with the polyanionic GAG chains and thereby impair their enzymic degradation.

Drug-induced intralysosomal storage of sulfated glycosaminoglycans (GAGs): A methodical pitfall occurring with acridine derivatives

The present communication deals with an adverse drug action which is exerted by a series of dicationic amphiphilic compounds such as the immunomodulatory drug tilorone and congeners. The drugs induce lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact organisms and in cultured cells by impairing the lysosomal GAG degradation. This impairment was proposed to be due to the formation of non-degradable GAG-drug complexes. GAGs are highly water-soluble and not preservable by aldehyde fixatives. Therefore, usually the lysosomes appear optically empty in histological preparations, unless the fixative is supplemented with a GAG-precipitating agent. When acridine derivatives were used for the induction of GAG-storage, the lysosomal storage material displayed unexpected and unsystematic variability with regard to its preservability and ultrastructure. In the present study, evidence is presented that the acridine derivatives (a) remain bound to the stored GAGs for some time after glutaraldehyde fixation; and (b) they precipitate GAGs in vitro. Thus, apart from their unwanted action in the living cell, i.e., disturbing lysosomal GAG-degradation, the drugs function as precipitants and "fixatives" for the intralysosomal GAGs. The uncontrolled persistence of the drugs after tissue fixation leads to variable degree of GAG-preservation and thus to unpredictable variability of the ultrastructure of the storage lysosomes. If this pitfall is not realized, the resulting inconsistencies may rise confusion among toxicologic pathologists who deal with drug-induced lysosomal storage disorders.

Drug-induced lysosomal storage of sulfated glycosaminoglycans. Studies on the underlying structure-activity relationships

Some immunomodulatory drugs have previously been shown to induce lysosomal storage of sulfated glycosaminoglycans (sGAG) in intact organisms and cultured cells. These compounds consist of a planary aromatic ring system and two symmetric side chains each carrying a protonizable nitrogen. The purpose of this study was to test a larger collection of such compounds for their potencies to induce lysosomal storage of sGAG in cultured fibroblasts of rat cornea. The cells were exposed (72 h) to various compounds differing with respect to the aromatic ring system or the side chains. Lysosomal sGAG-storage was demonstrated by selective cytochemical staining with cuprolinic blue. The threshold concentration, i.e., the concentration necessary to induce cuprolinic blue-positive cytoplasmic inclusions in at least 1% of the cells, was determined for each compound. The threshold concentrations were distributed over a range of 0.3–30 μM. It should be emphasized that the threshold concentration of a given compound is not a constant, but depends on the volume of cell culture medium per surface area of cell monolayer, since the lysosomal accumulation lowers the initial drug concentration in the medium. If the ratio of medium volume : cell monolayer surface is increased as compared with standard cell culture conditions, the threshold concentration will be lowered. The compounds were ranked according to their threshold concentrations as determined under standard conditions. The following conclusions can be drawn from the ranking: the type of the central aromatic ring system and the distance between the ring system and the protonizable nitrogen atoms of the side chains influence the potency to induce lysosomal sGAG-storage. Regarding the ring system, the potency decreases as follows: acridine ∼ anthrachinone > fenfluorenone ∼ fenfluorene > xanthenone; xanthene > dibenzofuran ∼ dibenzothiophene. In intact organisms, these structure-activity relationships may be superimposed by drug metabolism and pharmacokinetic factors.

Lysosomal storage of sulfated glycosaminoglycans in cultured fibroblasts exposed to immunostimulatory acridine derivatives

The purpose of the present cytological and radiochemical study was to investigate whether the immunomodulatory agent 3,6-bis[2-(diethylamino)ethoxy]acridine (CL-90.100) and three congeners induce lysosomal storage of sulfated glycosaminoglycans (sGAG) in cultured rat corneal fibroblasts. The reason for asking this question was as follows: The four acridine derivatives have molecular similarities with the dicationic amphiphilic compound tilorone, which has previously been shown to cause sGAG storage in cultured cells and in intact rats. The cells were exposed to the drugs for 72 hr. Tilorone served as reference. All acridine derivatives caused cytological alterations which, on the basis of the cytochemical results, were indicative of lysosomal sGAG storage. The threshold concentrations ranged from 0.3 to 0.7 μ m. Radiochemical experiments showed that Cl-90.100 up to 10 μ m induced [ 35S]GAG storage in a dose-dependent manner, with an EC 50 of 2 μ m. Concentrations above 10 μ m were cytotoxic. Experiments with equimolar concentrations (3 μ m) demonstrated that three of the acridine derivatives were more potent and one was less potent than tilorone. Additionally, CL-90.100 was tested on bovine corneal fibroblasts, with cytochemical and radiochemical results similar to those in rat cells. The present findings show that (a) the four acridine derivatives induce lysosomal sGAG storage; (b) the acridine ring, compared with the fenfluorenone ring (tilorone), enhances this potency; and (c) the substituents at the nitrogens can have some influence on the potency to induce sGAG storage.

Induction of mucopolysaccharidosis in rats by treatment with immunostimulatory acridine derivatives

In the accompanying paper, four dibasic acridine derivatives were reported to induce lysosomal storage of sulfated glycosaminoglycans (sGAG), i.e., mucopolysaccharidosis, in cultured fibroblast (Handrock et al. Toxicol. Appl. Pharmacol. 114, 1992). The purpose of the present morphological and biochemical investigation was to examine whether two representatives of the acridine derivatives, namely 3,6-bis[2-(diethylamino)ethoxy]-acridine and the piperidino analogue, induce mucopolysaccharidosis in intact organisms. Rats were orally treated with 60–80 mg/kg up to 22 weeks. Morphological examination of liver, spleen, and blood lymphocytes yielded cytochemical evidence of mucopolysaccharidosis. Biochemically, up to a 48-fold increase of the urinary excretion of sGAG was found. In the liver and spleen of chronically treated rats, the sGAG contents were elevated by factors up to 56 and 23, respectively. Heparan sulfate and dermatan sulfate contributed most to the total increase of sGAG; chondroitin sulfate was stored to a minor degree. For one compound, the tissue concentrations were determined. It was found that the drug was accumulated in the tissues. Due to their fluorescent properties, the drugs could be detected by fluorescence microscopy to be present in high concentrations within the sGAG-storing lysosomes. On the basis of these observations and of the biochemical data it appears justified to assume a ratio of at least one drug molecule per disaccharide unit of the sGAG to be present in the lysosomes. It is proposed that this leads to the formation of sGAG-drug complexes in the lysosomes. Such complexes may be indigestible substrates for the lysosomal enzymes, thus leading to mucopolysaccharidosis. For toxicologic practice, the cytochemical examination of lymphocytes is recommended as a simple measure for early detection and monitoring of this adverse drug effect.

Drug-induced lysosomal storage of sulfated glycosaminoglycans in cultured bovine and human fibroblasts

Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 microM and 5 microM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action.

Cultured corneal fibroblasts as a model system for the demonstration of drug-induced mucopolysaccharidosis

The purpose of the present investigation was to establish a cell culture system suitable for demonstrating the drug-induced lysosomal storage of sulfated glycosaminoglycans (GAGs). This is a drug side-effect which was previously studied in animals treated with the di-cationic amphiphilic compound tilorone and congeners, and which is likely to occur in humans, too. Cultured corneal fibroblasts of rats were exposed to tilorone for 72 h. They developed histochemical and cytochemical alterations indicative of mucopolysaccharidosis and resembling those occurring in vivo. The threshold drug concentration was found to be below 0.7 microM. The reversibility of the lysosomal GAG storage was low. An increase in the drug concentration to 10 microM produced additional unspecific lysosomal alterations, while the mucopolysaccharidosis-like lesions became less prominent. Concentrations of 40 microM and 80 microM caused unspecific cytoplasmic vacuolation and cell death, respectively. The present model system appears suitable for screening investigations of newly developed drugs with respect to their mucopolysaccharidosis-inducing potential and for investigating the structure-activity relationships underlying this adverse drug effect. Care should be taken not to use too high drug concentrations which cause unspecific lysosomal lesions.

Mucopolysaccharidosis and lipidosis in rats treated with tilorone analogues

The experimental immunomodulatory agent tilorone was previously reported to induce generalized mucopolysaccharidosis (lysosomal storage of sulfated glycosaminoglycans) and lipidosis in rats. While lipidosis is a side effect common to many cationic amphiphilic compounds, none of them except tilorone has been known to cause mucopolysaccharidosis in intact animals. The purpose of the present histochemical and ultrastructural study was to examine whether or not mucopolysaccharidosis can be induced by tilorone analogues. Three analogues were selected and administered to rats in short-term and subchronic experiments, and liver, spleen, kidney, and cornea were examined. The analogues caused generalized cellular lesions which had the same histochemical and cytological characteristics and the same distribution as the lesions produced by tilorone. The results show that the ability to induce mucopolysaccharidosis is not a unique property of tilorone; this drug side effect should be taken into account when developing new drugs with molecular structures resembling that of tilorone.

Lysosomal storage of sulfated glycosaminoglycans in renal interstitial cells of rats treated with tilorone.

This investigation provides histochemical evidence for lysosomal storage of sulfated glycosaminoglycans (GAGs) in the interstitial cells of the renal cortex and in macrophage-like cells of the renal medullary zones of rats chronically treated with the drug tilorone. This compound is known to interfere with lysosomal degradation of sulfated GAGs; therefore cells that develop GAG-storage can be assumed to be involved in the turnover of GAGs. In view of this consideration, the most remarkable and still unexplained finding was that the intrinsic interstitial cells in the papilla, which is known to be particularly rich in sulfated GAGs, did not show the cytological symptoms of lysosomal GAG-storage. The present findings may stimulate further studies focused on the cellular sites of turnover of the sulfated GAGs present in the renal medullary interstitium.