Drug-treated Worms Research Articles

Proteomic Changes during Disturbance of Cholesterol Metabolism by Azacoprostane Treatment in Caenorhabditis elegans

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro

The effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 microg ml(-1) PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation--an early event of the larval somatic differentiation to the adult worm-- was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro

AbstractThe effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 μg ml-1 PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation — an early event of the larval somatic differentiation to the adult worm — was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.

Temporal differences in praziquantel- and oxamniquine-induced tegumental damage to adult Schistosoma mansoni: implications for drug-antibody synergy

A temporal study of the effects on the tegument of Schistosoma mansoni adult worm following in vivo praziquantel and oxamniquine treatment was performed. Drug-induced damage to the tegument, exposure of surface antigens and attachment of host antibody occurred rapidly, within 1 h, following praziquantel treatment. Oxamniquine-treated worms required 4-8 days for these effects to be apparent. The 2 drugs differed in the degree and sites of damage on the worm surface. The administration of 2 different polyspecific rabbit sera with drug significantly increased the efficacy of praziquantel when administered with the drug, but not when given 6-9 days after drug treatment. In contrast, only 1 serum was synergistic with oxamniquine when administered with drug and both sera were synergistic when given 6-9 days after drug treatment. The effect of immune killing of drug-treated worms is discussed.

Efficacy of treatment of murine Schistosoma mansoni infections with praziquantel and oxamniquine correlates with infection intensity: role of host antibody.

The reduction in worm burden obtained by treatment of Schistosoma mansoni with praziquantel and oxamniquine was greater in mice with heavy infections than in relatively lightly infected animals. The reduction in worm burden achieved by each drug correlated with the size of the pre-treatment worm burden (r2 = 0.82 and 0.81 for praziquantel and oxamniquine, respectively). Intensity of infection did not affect the degree of tegumental damage and drug-induced antigen exposure on worms recovered soon after treatment with praziquantel. However, praziquantel-treated worms from mice with heavy infections had significantly more murine antibody attached to the treated-worm surface than worms from praziquantel-treated lightly infected mice. Heavily infected mice had greater levels of circulating anti-worm antibodies than lighter infected mice. The correlation between infection intensity and cure rates achieved by praziquantel and oxamniquine may thus be a reflection of the higher titres of relevant antibody in heavily infected mice mediating death of drug-treated worms.

Molecular modeling: a tool for predicting anthelmintic activity in vivo.

The structural and electronic features of the broad-spectrum benzimidazole anthelmintic mebendazole [MBZ, methyl 5-(benzoyl)-benzimidazole-2-carbamate] have been determined using a combination of quantum mechanics, molecular graphics, and molecular modeling techniques. Using conformational analyses and quantum mechanics, we found that the three-dimensional structure and electronic features of MBZ were consistent with those previously reported for highly active broad-spectrum benzimidazole anthelmintics and that in vivo drug efficacy against Hymenolepis diminuta depends upon the orientation of the benzoyl group at position 5 on the heterocyclic ring system, the magnitude of the molecular dipole moment, and the percentage of polar surface area. The chemotherapeutic actions of MBZ on H. diminuta in vivo were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of MBZ 24 h earlier were significantly smaller and contained much less glycogen (as a percentage of the wet weight) than worms from untreated controls. In MBZ-treated worms, protein concentrations rose at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in MBZ-treated worms were considerably lower than the corresponding control values. Differences in the absolute amounts of glycogen between control and drug-treated worms were even more profound. Administration of a curative dose of MBZ to the rat host produced in H. diminuta another change, the onset of which coincided with the gross alterations in worm weight and chemical composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of anthelmintics on the antioxidant system of Nippostrongylus Brasiliensis

To understand the mode of anthelmintic action of thiabendazole and methyl-[5-[[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1 H-benzimidazole-2-yl] carbamate (C.D.R.I. compound 81/470) against Nippostrongylus brasiliensis, their effect on the metabolism of reactive oxygen species in the parasite as well as in rat intestine was examined. Both drugs produced a significant depression in the levels of Superoxide dismutase (SOD) and reduced glutathione (GSH) of the parasite. Release of antioxidant enzymes by the drug-treated worms was also found to be appreciably lowered. Both thiabendazole and compound 81/470 induced a depression in the levels of all five constituents of the antioxidant system of rat intestine but significant alterations were detected only in the GSH content of infected and the SOD activity of normal intestine. The production of O − 2 by treated intestine was, on the other hand, markedly enhanced. Increased formation of O − 2 by the host intestine accompanied with the reduced level of SOD and GSH in N. brasiliensis appear to have a deleterious effect on the parasite. Consequently, the drug-treated worms are unable to retain themselves in situ and are ultimately expelled. The greater effect produced on these parameters by thiabendazole compared to compound 81/470 is consistent with the relative efficacy of these anthelmintics.

Structure-Activity Relationships of Benzothiazole and Benzimidazole Anthelmintics: A Molecular Modeling Approach to In vivo Drug Efficacy

An investigation of the biochemical effects of an anthelmintic, tioxidazole (TIOX, methyl 6-[n-propoxy]benzothiazole-2-carbamate), on Hymenolepis diminuta in experimentally infected rats is reported. The chemotherapeutic actions of TIOX on H. diminuta in vivo were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TIOX 24 hr earlier were significantly smaller and contained much less glycogen (as a percentage of the wet weight) than worms from untreated controls. In TIOX-treated worms, protein concentrations rose at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TIOX-treated worms were considerably lower than the corresponding control-values. Differences in the absolute amounts of glycogen and protein between control and drug-treated worms were even more pronounced. Administration of a subcurative dose of TIOX to the rat produced in H. diminuta another change, the onset of which preceded the gross alterations in worm weight and chemical composition. In vitro studies, carried out 18 hr after treatment, revealed that TIOX-treated worms absorbed and metabolized much smaller quantities of exogenous glucose than did the controls and that the ability of the worm to accumulate glucose against a concentration difference was significantly depressed. A mode of action common to the structurally related benzothiazole and benzimidazole anthelmintics is indicated by the similarity of their biochemical and physiological effects on the tapeworms and their time course of action when administered to rats infected with H. diminuta. Molecular modeling revealed that the benzothiazole and benzimidazole anthelminitics are congruent electronically and structurally. In vivo drug efficacy depends upon the magnitude of the molecular dipole moment and the percentage of polar surface area. Within the benzimidazole series, structural and electronic congruence is found between the 2-thiazolyl and 2-methyl carbamate groups, suggesting that these groups behave similarly in transport to, and binding at, the active site. Finally, anthelmintics that have the 5' substituents twisted out-of-plane were more active than those anthelminitics with 5' substituents in-plane. All of these factors implicate a highly polar, L-shaped cleft to which the anthelmintics bind at the active site.

Schistosoma mansoni:morphology and ultrastructure of adult worms recovered from cyclosporin A-treated mice

Cyclosporin A administered to Schistosoma mansoni-infected mice at around day 20 of infection reduces the worm burden by greater than 60%, as assessed by portal perfusion on days 28 and 86. Those worms recovered at perfusion have been examined by light and electron microscopy for drug-induced changes in morphology. Gross parasite damage was characterized by massive bolus formation and subsequent herniation of the gut. This event was attributed to the abnormal accumulation of crystalline structures in the lumen; the crystals were closely associated with lipid droplets, and were shown by X-ray micro-analysis to contain iron. Such crystals were seen only rarely in the intestines of control worms, but they too gave small iron peaks when examined by X-ray micro-analysis. In some drug-treated worms the caecal epithelium had ruptured, thereby releasing luminal contents throughout the worm body. In addition, herniations of the gut were seen protruding through the tegument causing surface deformation and disruption of tegumental and parenchymal tissues. The structural integrity of the worm was ultimately compromised allowing access to cytotoxic effector cells of the host. The combined effects of drug action and cellular cytotoxicity presumably account for the very significant levels of worm killing achieved by CsA treatment of the host.

Glutathione redox state, lipid peroxide levels, and activities of glutathione enzymes in oltipraz-treated adult Schistosoma mansoni

A decrease in reduced glutathione (GSH) levels in adult Schistosoma mansoni exposed in vitro to the antischistosomal drug oltipraz (OPZ) (20–60 nM) was accompanied by a significant increase in oxidized glutathione (GSSG) levels. The total glutathione (GSH + GSSG) levels also diminished in drug-treated parasites. The activities of the parasite glutathione peroxidase (GPO), utilizing cumene hydroperoxide as a substrate, and glutathione 5-transferase (GST), measured 18 hr after in vitro incubation with the drug, were elevated significantly, but there were no significant alterations in the activities of the GPO, utilizing H 2O 2, or glutathione reductase (GR). Drug-treated worms showed increased lipid peroxidation. In vivo, the proportion of the worms recovered from infected mice given OPZ (100 mg/kg body wt) gradually declined with time, to about 30% of that recovered from infected untreated control mice by day 14 after drug administration, and consisted predominantly of male worms. Accompanying this significant decline in the proportion of worms recovered were significant decreases in the activities of the enzymes GR and GST in drug-exposed worms. On the other hand, a slight initial increase in the GPO activity with cumene hydroperoxide was followed by a return to control values, and the GPO activity with H 2O 2 was decreased only slightly with time. Interestingly, the 4-hydroxyalk-2-enal aldehydes, known products of lipid peroxidation, inhibited the GST reaction with 1-chloro-2,4-dinitrobenzene (CDNB). The OPZ-induced changes in S. mansoni could increase parasite susceptibility to oxidative attack by host phagocytes, and are probably linked with the antischistosomal action of the drug in vivo.

Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens

We have demonstrated previously in a mouse model that effective chemotherapy against Schistosoma mansoni with praziquantel (PZQ) is dependent upon an intact host antibody response. In the same study, it was found that worms recovered from PZQ-treated animals display surface-bound antibodies. In order to identify the target antigens of the antibodies involved in the synergy between PZQ and the immune response, monoclonal antibodies (mAbs) and polyclonal antisera recognizing different tegumental components were tested by indirect immunofluorescence (IF) assay for their ability to bind in vitro to the surface of 6-week-old schistosomes perfused from nude (athymic) mice 1 h after PZQ treatment. Nude mice were used as hosts because worms from these animals were found to lack bound anti-schistosome antibodies. Only 5 of the 21 antibodies tested reacted with drug-treated worms. This indicated that the damage caused by PZQ to the schistosome tegument is restricted to specific tegumental components. Of the positive reactions, one group of antibodies gave IF patterns different from, whereas the other group gave IF reactions similar to those seen with worms perfused from immunologically intact mice. Antibodies against a schistosome esterase and alkaline phosphatase produced reaction patterns in the former category. In contrast, two out of three monoclonal antibodies recognizing different epitopes on a 200-kDa glycoprotein abundant in worm tubercles gave IF patterns very similar to those observed on schistosomes from drug-treated, intact mice. The biological significance of these reactions was confirmed by demonstrating that transfer of one of the positive monoclonal antibodies to 6-week-infected, B cell-depleted (μ-suppressed) mice reconstitutes the efficacy of PZQ treatment to normal levels. The above results suggest that the antibodies involved in the mechanism of action of PZQ react with a limited set of antigens. Furthermore, they implicate the 200-kDa tubercle protein as a major target of this response in naturally infected hosts.

The effects of amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on wild-type and resistant mutants of Caenorhabditis elegans

The anthelmintic drugs amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) inhibited the growth of wild-type (N2) Caenorhabditis elegans but had little effect on development or reproductive capacity. Inhibition of growth correlated well with drug-induced paralysis, both becoming maximal at around 1.0 mM concentration of either drug. Egg laying was delayed by about 24 h and the rate of laying was only about 60–70% of the controls. However, the period during which eggs were laid was extended by a similar amount and the total number of eggs laid was the same for controls and drug-treated worms. Five drug-resistant mutants (T114, T22, T26, T216, and T226) were isolated following ethylmethanesulphonate mutagenesis. All were shorter than N2 at 96 h on drug-free medium; their growth was not further impaired by either of the anthelmintic drugs. All except T114 exhibited a normal pattern of sexual maturation. Cultures of T114 at 96 h contained many immature worms. This mutant also exhibited the most impaired motility, being severely uncoordinated in liquid suspension. The other mutants could swim normally but were noticeably slower than N2. Genetic analysis indicated that each mutant was the result of a single genetic lesion, that the mutants were recessive, and that there were two genes for amidantel resistance (adt1 and adt2). In vitro studies on representatives of each class (T114 and T22) indicated a defect in the acetylcholine receptor. T22 mutants showed a moderate decrease in sensitivity towards typical cholinergic agonists as well as the anthelmintic drugs, while T114 mutants were apparently devoid of functional pharmacological acetylcholine receptors.

Hymenolepis diminuta: Effects of Amoscanate on energy metabolism and ultrastructure

Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16–61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25–30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanate-treated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure.

Biochemical effects of fenbensazole on Hymenolepis diminuta in vivo

An investigation of the chemotherapeutic and biochemical effects of a new broad-spectrum anthelmintic, fenbendazole, (FBZ, methyl 5-[phenylthio]-benzimadazole-2-carbamate), on Hymenolepis diminuta in experimentally infected rats is reported. FBZ proved to be highly active against H. diminuta; a single oral dose of 12.5, 25 and 50 mg/kg body wt. on day 15 of infection eliminated 77, 100 and 88% of the tapeworms respectively as determined at necropsy 24 h after treatment. The chemotherapeutic actions of FBZ on H. diminuta in vivo were accompanied by marked changes in worm wt. and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of FBZ 18 h earlier were significantly smaller and contained much less glycogen (as a percent of the fresh wt.) than worms from unmedicated controls. Protein concentrations rose in FBZ-treated worms, but more slowly than the rate of decline in glycogen concentration. Glycogen/protein ratios in FBZ-treated worms were considerably lower than the corresponding control values. Differences in the absolute amounts of glycogen and protein between control and drug-treated worms were even more profound. Administration of a single oral dose of FBZ (14 mg/kg) to the rat produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm wt. and chemical composition. In vitro studies, carried out 16 h after treatment, revealed that FBZ-treated worms absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability to the worm to accumulate glucose against a concentration difference was significantly depressed.