[Effect of amyxine, loramyxine and their composites with yeast RNA on spontaneous and induced mutagenesis in Salmonella typhymurium].

It was found out that amyxine, loramyxine and their composites with yeast DNA do not increase reliably the frequency of spontaneous mutations in test-strain S. typhymurium TA 98. The studied substances do not possess the antimutagen activity in respect of mutations, induced by potassium bichromate in S. typhymurium TA 98 and do not increase their quantity.

[Tilorone-induced functional changes in the rat retina]. / Tiloron-induzierte funktionelle Veränderungen der Rattenretina.

BACKGROUND: The amphiphilic interferon-inducing drug tilorone belongs to the group of synthetic fluorenone with antitumorus and antiviral effects and is known to cause lipidosis in the human and rat retina. METHODS: We treated female albino Wistar-Kyoto-Rats and Spontaneously-Hypertensive-Rats orally with tilorone for 8 weeks. The animals were submitted to electroretinography, and the retinae were prepared for histological investigations. RESULTS: Tilorone caused moderate lipidosis in the ganglion cells and slight lipidosis in the pigment epithelium after 8 weeks of treatment with a stronger effect in Spontaneously-Hypertensive-Rats. The b-wave amplitude was reduced by 30% compared to controls in Spontaneously-Hypertensive-Rats and by 10% in the Wistar-Kyoto-group. The a-wave amplitude was reduced by 25% in Spontaneously-Hypertensive-Rats. CONCLUSION: Tilorone induced lipidosis shows a clear affinity to ganglion cells. The electroretinographic changes in Spontaneously-Hypertensive-Rats are most likely caused by the severe lipidosis in the neuroretina compared to the Wistar-Kyoto-group.

Lysosomal glycosaminoglycan storage as induced by dicationic amphiphilic drugs: investigation into the mechanisms underlying the slow reversibility.

Several dicationic amphiphilic compounds, such as the immunomodulator tilorone and analogues, impair the lysosomal catabolism of sulphated glycosaminoglycans (GAGs). Thereby they cause lysosomal GAG storage in rats and in cultured fibroblasts of several species including man. The GAG storage is rather slowly reversible in vivo; it persists for months after discontinuance of drug treatment. In the present study, we investigated the mechanisms underlying the slow reversibility. Cultured bovine corneal fibroblasts were pretreated for 4 days with tilorone (5 and 20 microM) or with compound CL-90.100 (3 and 10 microM) and further cultured in drug-free medium for periods up to 11 days. The intracellular GAG storage was analysed biochemically and demonstrated histochemically. The subcellular drug distribution (CL-90.100) was demonstrated by fluorescence microscopy. Dermatan sulphate (DS) provided the predominant contribution towards the GAG storage. After pretreatments with the low, as well as the high concentrations of either drug, the storage of DS was irreversible during the period of observation, whereas the minor storage of heparan sulphate was resolved. The enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52) caused by pretreatment with the high concentration of tilorone was also readily reversible. Thus, enzyme deprivation could not be the explanation for the sustained DS storage. The localization of the drug-related fluorescence within perinuclear cell organelles, presumably lysosomes, resembled that of the stored GAGs as visualized by histochemical staining. Both, the fluorescence and the positive GAG staining persisted with unchanged intracellular distribution throughout the recovery period. The present results suggest that the persistence of the DS storage is due to the formation of long-lived, non-degradable DS-drug complexes within the lysosomes.

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 MPS-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 microM) which did not enhance the secretion of the lysosomal enzyme beta-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.

[The interferonogenic properties of yeast RNA-tilorone complexes in a cell culture]. / Izuchenie interferonogennykh svoistv kompleksov drozhzhevaia DNK--tiloron v kul'ture kletok.

The use of molecular complexes of yeast RNA with thyloron as inductors of type I interferons (alpha/beta-interferons) in the culture of cells L929 is described. It was shown that the complexes induced the interferon synthesis in the cells at the level comparable to that of the standard inductors of the polyribonucleotide nature i.e. larifan and poly(I)-poly(C). In the experimental doses the complexes proved to be nontoxic. It was concluded that the use of the yeast RNA and thyloron complexes as inductors in large-scale production of type I interferons was promising.

Prevention of human recombinant interleukin-1 beta (rhIL-1 beta) embryolethality with progesterone or indomethacin.

PROBLEM: Bropirimine and tilorone were found in earlier studies to be embryolethal when administered to Crl:TUC(SD)spf (TUC) rats on gestation day 10. Progesterone or indomethacin could, at least partially, prevent this effect. The immunomodulators appeared to mimic the luteolytic effects of PGF2 alpha, resulting in a shutdown in progesterone release by the corpora lutea, followed by a disruption in maternal support to the pregnant uterus and embryolethality. Since bropirimine has been shown to induce interleukin-1, and since this cytokine has been found to increase PGF2 alpha levels in human decidual cells, the decision was made to investigate whether human interleukin-1 beta might act in an analogous manner to bropirimine and tilorone. METHOD: Bropirimine (400 mg/kg, p.o.) or rhIL-1 beta (20, 30, or 40 micrograms/kg, s.c.) was administered on gestation day 10 to Crl:CD[BR] (CD) or TUC rats, alone and in combination with progesterone (2 mg/kg/day, s.c.) or indomethacin (0.6 mg/day, s.c., days 9-11). On gestation day 14 the dams were killed and their uterine contents examined. RESULTS: rhIL-1 beta (30-40 micrograms/kg) was embryolethal when administered to CD or TUC rats on gestation day 10. Progesterone or indomethacin coadministration prevented, at least partially, the embryolethality seen when rhIL-1 beta was administered (30 micrograms/kg) to TUC rats. CONCLUSION: Evidence was obtained in support of the hypothesis that interleukin-1 is involved in the embryolethal actions of the immunomodulators bropirimine and tilorone.

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.

Prevention of tilorone developmental toxicity with progesterone.

The immunomodulator tilorone hydrochloride was administered (gastric intubation) once to time-pregnant Upj:TUC(SD)spf (Sprague-Dawley) rats in four experiments. In experiment 1, tilorone (250 or 500 mg/kg) was administered on day 10 of gestation. The dams were killed 4 or 72 hr after dosing. Interferon-like activity and drug levels were determined in maternal blood, spleen, and thymus, as well as in the embryos. In experiment 2, the test groups received progesterone (2 mg/kg), or tilorone (200 or 400 mg/kg), or progesterone and tilorone. The dams from each group were killed 24 or 48 hr after receiving tilorone. Experiment 3 was similar to experiment 2, except that the dams were killed on gestation day 20. In experiment 4, tilorone (400 mg/kg) was administered on gestation day 17, 18, or 19, and the dams were killed 24 hr after dosing or on gestation day 20. In all four experiments, tilorone-related maternal toxicity (regardless of whether progesterone also was administered) was observed, as characterized by marked decreases in weight gain, the occurrence of clinical signs, and in experiment 1 by decreased thymus weights, 72 hr post-dosing. Dose-related increases in the mean number of dead embryos and in serum interferon titers occurred 72 hr postdosing. In experiment 2, there was an increase in the number of dams in the 400-mg/kg (tilorone only) group with dead embryos only, 24 hr postdosing; similar results occurred in both the 200- and 400-mg/kg groups, 48 hr postdosing. However, in the groups that also received progesterone, a partial prevention of such embryolethality was evident. In experiment 3, embryotoxicity again was observed in both tilorone-treated groups, whereas several of the dams that were also given progesterone through day 19 of gestation experienced at least a partial prevention of the embryolethal effects of tilorone. In experiment 4, no fetotoxicity was observed despite the severe maternal toxicity evident.

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.

Mucopolysaccharidosis (MPS) in ocular tissues as induced by amphiphilic di-cationic drugs.

The immunostimulatory drug tilorone (2,7-bis(2- (diethylamino)ethoxy)-9H-fluorene-9-one) has previously been described to induce keratopathy in patients and in rats; in the latter, lysosomal storage of sulfated glycosaminoglycans (mucopolysaccharidosis, MPS) was found to be the reason for corneal clouding. In the present histochemical and ultrastructural study, several ocular tissues of rats were examined after chronic treatment with tilorone and two other immunostimulatory agents (3,6-bis(2-(dipiperidyl)ethoxy)-acridine and the diethylamino analogue). The acridine derivatives hardly affected the cornea and sclera. Tilorone as well as the acridine derivatives induced significant MPS in ciliary body, iris, and choroid. While the functional consequences of drug-induced MPS in ocular tissues are not known yet, except for the cornea, this adverse drug action should be taken into account when new compounds are designed that share certain physicochemical features with tilorone. One molecular feature essential for the MPS-inducing potency of a drug appears to be the dicationic amphiphilic character.

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.

Mucopolysaccharidosis-like cellular alterations in chondrocytes of rats treated with tilorone.

The dicationic amphiphilic compound tilorone was previously shown to interfere with the lysosomal degradation of sulfated glycosaminoglycans (GAGs) of cultured cells and to cause mucopolysaccharidosis-like alterations in several organs of rats. Since chondrocytes belong to those cells that are severely affected in cases of inherited mucopolysaccharidoses, we wished to know whether this also holds true of the experimentally induced mucopolysaccharidosis. Young rats were treated with tilorone (50-80 mg/kg of body weight) for 2-17 weeks; chondrocytes in tracheal and costal cartilage and in the epiphyseal growth plates of the tibia and ribs were examined by electron microscopy and cytochemistry (acid trimetaphosphatase and staining with Cuprolinic Blue). The tracheal and costal chondrocytes showed numerous abnormal clear vacuoles. Some of them could be identified cytochemically as lysosomes filled with polyanionic storage material, probably sulfated GAGs. In the epiphyseal plate, only the resting chondrocytes were markedly affected, whereas those of the remaining zones were hardly altered; this may be due to the short life span of these chondrocytes in rat growth plates. The present results show that chondrocytes participate in the experimental mucopolysaccharidosis and suggest that, under normal conditions, the lysosomes of chondrocytes have to cope with a considerable load of GAGs.

Genotoxicity assessment of low-molecular weight interferon inducers by the SOS Chromotest.

Tilorone and its aza-analogs, as well as CMA and its butyric analog (CNPA) were investigated as potential genotoxic agents by the SOS Chromotest. The SOS-inducing potency values (SOSIP) were 0.0033 and 0.0009 for SAF and vivakorfen, respectively, after activation with S9 fraction of mouse liver only. In contrast, an SOSIP value for tilorone of 0.0011 was observed in a non-activated assay. The SOSIPs of investigated compounds were low and comparable to the lowest values determined for other genotoxins. CMA and CNPA were not SOS inducers in any test system.

Mucopolysaccharidosis-like alterations in cardiac valves of rats treated with tilorone.

The purpose of this study was to examine whether the aortic and mitral valves of rats are involved in the mucopolysaccharidosis-like disorder induced by tilorone. Rats were treated with large doses of the drug for periods of 1-21 weeks. After chronic drug treatment the leaflets of both heart valves were thickened and opaque. In all treated animals the spongiosa layer of the stroma was crowded with vacuolated cells; the fibrosa layer was altered only after prolonged treatment. Ultrastructurally, the vacuolated cells of the spongiosa could be identified as histiocytes and fibroblasts, the former being the most susceptible cell type. The fibroblasts of the fibrosa represented the least sensitive cell type. The histochemical results showed that the clear cytoplasmic vacuoles in the spongiosa cells were due to lysosomal storage of polyanionic material with staining characteristics similar to cartilage matrix. After discontinuation of drug treatment the alterations persisted for several weeks. The present study shows that heart valves are involved in the mucopolysaccharidosis-like disorder induced by tilorone. The molecular pathomechanism of the disorder and the exact identification of the storage material must await further analysis.

Tilorone-induced lysosomal storage mimicking the features of mucopolysaccharidosis and of lipidosis in rat liver.

This ultrastructural and histochemical study deals with the lysosomal storage phenomena occurring in the rat liver after repeated oral administration of tilorone, an agent with anti-tumor and anti-viral activities. In the sinusoidal endothelium and in Kupffer cells, the lysosomes were changed into large vacuoles which contained material with the histochemical characteristics of acid glycosaminoglycans. The alterations closely resembled those previously observed in the splenic red pulp of tilorone-treated rats. In hepatocytes, the lysosomes were converted into large multilamellated inclusions indicating storage of polar lipids. The results show that, in the rat liver, tilorone induces cellular alterations mimicking those of inherited mucopolysaccharidoses and lipidoses. After discontinuing drug treatment the two storage phenomena gradually faded at different rates: The lipidosis disappeared within 2 to 4 weeks, whilst mucopolysaccharidosis-like changes were still found 15 weeks after drug withdrawal. The occurrence of lipidosis is not surprising, since by its molecular structure tilorone can be regarded as belonging to the group of amphiphilic cationic drugs which often have this side effect. Much more surprising is the occurrence of mucopolysaccharidosis-like alterations. The exact biochemical identification of the polyanionic storage material and the molecular mechanisms responsible for this drug side effect remain to be established.

Fine structure of contrasting choroid plexus lesions caused by tertiary amines or cyclophosphamide.

Hydropic alterations of the fine structure of the choroid plexus epithelium following administration of three different tertiary amines to rats were compared with alterations produced by cyclophosphamide and with normal choroid plexus. Single doses of 2,7-bis(2-diethylamino)-ethoxy)-4-methyl-1,8-naphthyridine HCl or 2,6-di-(omega-dimethyl aminoethoxy)-pyridine produced dramatic accumulations of membrane-limited vacuoles. Multiple doses of 2,7-bis-(diethylaminoethoxy)fluoren-9-one HCl (tilorone) produced accumulations of dense cytoplasmic bodies in choroid epithelial cells in addition to the hydropic vacuoles. Differential responses to these agents suggested functional specialization or temporal variation in function among adjacent choroidal cells. Despite the occurrence of pronounced plasma exudation in the plexitis following cyclophosphamide treatment, there was no hydropic vacuolization.