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Albendazole induces apoptosis in adults and microfilariae of Setaria cervi
Experimental Parasitology 128 (2011) 236–242
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Albendazole induces apoptosis in adults and microfilariae of Setaria cervi A. Nayak a, P. Gayen a, P. Saini a, S. Maitra b, S.P. Sinha Babu a,⇑ a b
Parasitology Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal 731 235, India Physiology and Biochemistry Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal 731 235, India
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Article history: Received 6 June 2010 Received in revised form 29 December 2010 Accepted 16 March 2011 Available online 23 March 2011 Keywords: Lymphatic filariasis Setaria cervi Apoptosis Microfilaria (Mf) Albendazole (ABZ) Macrofilaricide
a b s t r a c t Setaria cervi, a filarial nematode of cattle, inhabits in the peritoneal cavity and has been used as a suitable model for screening antifilarial agents. Albendazole (ABZ), a tubulin-disrupting benzimidazole (BZ) and a potent microfilaricide binds to b-tubulin, is causing structural impairment of cytoskeleton and worm death. Our present study has revealed that exposure of microfilaria (Mf) and adult to gradually increasing concentration of ABZ leads to a dose-dependent gradual impairment of their motility followed by early death in vitro. We found extreme cellular disturbances in ABZ-treated worms characterized by nucleosomal DNA laddering and chromatin condensation. However, in the treated Mf no nucleosomal DNA laddering was found although presence of TUNEL reactive DNA was evident, thus indicating an apoptotic pathway independent of DNA fragmentation. We present data from molecular studies to provide evidence for ABZ-induced apoptosis in Mf and adult worms of S. cervi. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Setaria cervi is a filarial nematode parasite inhabiting in the peritoneal cavity of cattle and shows cosmopolitan distribution. Different antifilarial agents have been tested on S. cervi in rats and reported as a suitable model for screening antifilarial agents (Singhal et al., 1972). This is a suitable model because the rate of proper establishment of transplanted parasites in rats is 50% and microfilaria appears in the peripheral blood within 10–15 days after transplantation and persists for 120 days (Singhal et al., 1972). The community-based control of lymphatic filariasis is mainly aimed at interrupting parasite transmission (WHO, 2007). The antifilarial drugs presently used, diethylcarbamazine, ABZ and ivermectin, are selectively active against Mf in the blood, in reducing the numbers and limiting transmission. Recent antimicrofilarial or macrofilaricidal treatment regimens do have certain well documented limitations. Diethylcarbamazine, and ivermectin are effective at killing Mf but are associated with systemic and inflammatory adverse reactions (Cross et al., 2001; Molyneux et al., 2003). ABZ increases the efficacy of diethylcarbamazine and ivermectin and is used in combination with either of drugs as the basis of long term intervention programme, aimed at breaking disease transmission (Molyneux et al., 2003). ABZ is a safe and effective antifilarial drug belonging to the group BZ. Since its discovery in 1972, it has been used as a broad spectrum anthelmintic drug against round worms, tape worms and flukes of ⇑ Corresponding author. Fax: +91 3463 262672. E-mail address: [email protected] (S.P. Sinha Babu). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.03.005
domestic animals and humans (Theodorides et al., 1976). It plays a significant role in impairment of glucose uptake in the helminth parasites and also inhibits the helminth specific fumarate reductase (Marr et al., 2003). However, its primary role is to inhibit microtubule polymerization in susceptible nematodes by binding to the b-tubulin, a cytoskeletal element and thus reduce the glucose uptake (Lacey, 1990; Barrowman et al., 1984; Lacey and Watson, 1985; Martin, 1997). Cytoskeleton containing microtubules plays an important role in cell functioning. Microtubules exhibit highly dynamicity due to assembly and disassembly at their ends. The involvement of microtubules in critical process like mitosis helps to design suitable chemotherapeutic agents that show antitubulin activity and thus promoting programmed cell death (PCD) through different apoptotic pathways. Apoptosis is programmed cell death, characterized by nuclear condensation, membrane convolution and nucleosomal fragmentation (Kerr et al., 1972). Previous studies have shown an association between apoptosis and disintegrity of cytoskeletal components (Moss et al., 2006). The antitumor activity of ABZ has already been proved (Pourgholami et al., 2005) against human colorectal cell line. Recently, the elucidation of the cellular effects of ABZ and other BZ compounds, and especially their binding to tubulin in parasites, has prompted us to investigate whether ABZ could induce apoptosis in adults and Mf of S. cervi, a bovine filarial parasite. The discovery of programmed cell death in Caenorhabditis elegans, a free living nematode during post embryonic development and subsequent elucidation of its genetic control initiated a new field for apoptosis research (Sulston and Horvitz, 1977; Sulston et al., 1983; Ellis and
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Horvitz, 1986). Here we are reporting for the first time that ABZ, a safe and effective antihelminthic drug, shows strong antifilarial effect on adults and Mf of S. cervi by inducing apoptosis.
2. Materials and methods 2.1. Collection of parasites Adult worms of S. cervi were collected from the peritoneal cavity of freshly slaughtered cattle at local abattoirs, washed several times with Kreb’s Ringer bicarbonate buffer (Sigma), and kept in Ringer’s solution at 37 °C. Mf were obtained by dissecting gravid females, and kept in Ringer’s solution at 37 °C for 1 h. 2.2. Drug preparation Albendazole, purchased from Sigma Chemical Company (USA), was dissolved in DMSO to make a stock solution and diluted in RPMI-1640 to obtain the desired concentrations. 2.3. In vitro culture and treatment of adult worms and microfilaria Adult worms of S. cervi (1 male and 1 female) were incubated in 5 ml of complete media, CM (RPMI-1640 supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U/ml streptomycin, 100 lg/ml penicillin, 0.25 lg/ml of amphotericin B and 10% fetal calf serum), alone and in combination with ABZ at varying concentrations in a 24-well flat-bottomed culture plate (Tarson, India). Mf obtained from gravid adult females, were incubated in 200 ll of CM alone and in CM with ABZ, at selected concentrations in a 96-well flat-bottomed microtiter plate (Tarson, India). Both the cultures were maintained for 48 h at 37 °C in a humidified atmosphere of 5% CO2 (Rao and Weil, 2002). The cultures for adult worms were carried out in duplicate and for Mf in quadruplicate and repeated at least three times. 2.4. Assessment of parasite viability by MTT assay Viability of parasites was assessed quantitatively by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) reduction assay (Comley et al., 1989). After a brief microscopic assessment of parasite viability, Mf (n = 0.15 105) were suspended in 0.1 ml of PBS containing 0.5 mg of MTT ml 1 and then incubated for 2 h at 37 °C in dark. The dark blue crystals of formazan thus formed were then solubilized using 100 ll of DMSO in a 96-well microtiter plate. Adult worms were incubated in 0.5 ml of PBS containing 0.5 mg of MTT and formazan crystals were solubilized in 200 ll of DMSO. The absorbance intensity of each sample was read with an automatic microtiter plate reader (Beckman, USA) at a 595 nm wavelength by using DMSO as a blank. 2.5. Histology After 48 h, ABZ-treated adult worms were fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin and cut into 3-lm thick sections.
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2.7. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) There are many ways to detect apoptotic nuclei at different stages on histological sections; we used one of the most commonly used methods, TUNEL. According to manufacturer’s protocol (DeadEnd™ Colorimetric TUNEL System, Promega, USA), transverse sections were first deparaffinized, rehydrated in graded series of alcohol, digested by proteinase K (20 lg/ml) for 15 min at room temperature. Sections were then incubated in equilibration buffer for 8 min and the fragmented DNA were labeled by biotinylated nucleotide mix in the presence of recombinant deoxynucleotidyl transferase (rTdT) for 60 min in a humidified chamber. Finally, the labeled fragments were incubated with streptavidin horseradish peroxidase and visualized after diaminobenzidine color development in which a dark brown staining indicated apoptosis. A light microscope (Dewinter, Victory, Italy) was used for observations and photographs. 2.8. Hoechst staining For the assessment of chromatin condensation, the sections were stained as described previously (Su et al., 2001) using 8 lg/ ml of Hoechst 33258 in double distilled water at room temperature. The stained sections were examined with an Olympus fluorescent microscope (BX 41, Japan), using an excitation wavelength of 490 nm. 2.9. DNA ladder assay Total genomic DNA was extracted from ABZ-treated (48 h) Mf and adults as well as control worms by the phenol/chloroform method as described earlier (Datta et al., 2007) with slight modifications. In brief, the Mf and adult worms were digested in 500 ll of lysis buffer (Tris–HCl 20 mM, pH 8.0, EDTA 50 mM, SDS 0.5%, NaCl 100 mM, b-mercaptoethanol 1%, v/v), proteinase K (0.1 mg/ml) and incubated in a water bath at 37 °C for 2 h. This was followed by RNase (5 lg/ml) treatment for 2 h at 56 °C. After phenol–chloroform–isoamyl alcohol extraction and ethanol precipitation, the final DNA pellet was resuspended in 20 ll of sterile nuclease-free water and run on 2% agarose gel, stained with ethidium bromide and analyzed under ultraviolet illumination (BIO-RAD, USA). 2.10. Statistical analysis Data were analyzed by Student’s t-test using MS Excel software. Differences were considered significant with P values <0.05. 3. Results 3.1. Effect of ABZ on Mf viability The effects of ABZ on Mf viability (MTT assay) were represented graphically (Fig. 1B). It appears from the results that the gradual reduction in Mf motility (results not shown) was consistent with the reduction of Mf viability. After 48 h of incubation with ABZ at 2 and 4 lg/ml, the viability of Mf was significantly decreased to 50% (P < 0.05). At higher doses ABZ reduced Mf viability by more than 70%. Maximum reduction (about 90%) was recorded after 48 h of incubation with ABZ at 50 lg/ml.
2.6. Hematoxylin–eosin staining 3.2. Effect of ABZ on adult worm viability The sections were stained with hematoxylin and eosin to detect the extent of ABZ-induced tissue damage. The stained sections were examined with a light microscope (Dewinter, Victory, Italy).
The effects of ABZ on viability (MTT assay) of adult worms, cultured in vitro were also studied (Fig. 1A). The gradual reduction in
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Fig. 1. Effect of ABZ on Setaria cervi viability. (A) Adult worms and (B) Mf. Parasites were incubated with ABZ for 24 and 48 h and viability was measured by the MTT reduction test. Results were analyzed as percent reduction in formazan production compared with control worms and expressed as percent reduction in viability.
worm viability with increasing drug concentrations was very compatible with gradual decrease in worm motility (results not shown). Viability of the treated worms was significantly decreased (P < 0.05) to 40% after incubation with ABZ at 100 and 500 lg/ml, and at higher dose, i.e. 1000 lg/ml the reduction reached up to 70%. Thus, ABZ produced strong microfilaricidal as well as macrofilaricidal effects on S. cervi in a dose-and time-dependent manner. However, normal untreated Mf and adult worms also show some degree of MTT reduction but the rate was negligible in comparison to the freshly collected worms (data not shown). 3.3. Hematoxylin–eosin staining ABZ treatment had caused damage to the outer cuticle and the underlying muscle layer (Fig. 2D). Cells from intestinal epithelium (Fig. 2E) and uterus lining (Fig. 2F) of treated worms showed
distorted appearance that seems to be apoptotic in nature. Such types of damage in cellular appearance were not observed in control worms (Fig. 2A–C). Besides there was huge depletion in the number of developing embryos in the uterus (figure not shown). 3.4. TUNEL The results of TUNEL assay clearly reveal presence of in situ DNA fragmentation in the nuclei of ABZ-treated tissue (Fig. 3B and D). Clusters of numerous dark brown apoptotic nuclei were found in the cells of outer epithelium and developing embryos within uterus as observed in the TUNEL stained transverse sections of 48 h ABZ-treated adult worms. Cells from the control sections (Fig. 3A and C) showed less or few number of apoptotic nuclei. Similar apoptotic nuclei were observed in a more dispersed fashion in the TUNEL stained body of 48 h ABZ-treated Mf
Fig. 2. H and E stained light micrographs of Setaria cervi female worms. Sections of untreated worms (A–C) showed no significant damage in worm morphology. Several abnormal morphological features were observed (indicated by arrows) in the sections of ABZ-treated (D–F) (100 lg/ml) worms: damaged hypodermal muscle layer (D); extensive cellular and nuclear disintegrity in intestinal epithelial cells (E); and distorted cellular and nuclear appearance in uterine epithelium (F). (A–F 400 magnification; cu, cuticle; hpd, hypodermis; int, intestine; ov, ovary; ut, uterus; lml, longitudinal muscle layer). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
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Fig. 3. TUNEL stained light micrographs of Setaria cervi adult worms. The TUNEL assay showed lack of apoptotic death in cells of the control sections (A and C), compared with the 48 h ABZ-treated (100 lg/ml) sections (B and D) of adult parasites. The cuticle, developing embryos and uterine epithelium showed numerous dark brown stained apoptotic nuclei (arrows) (A–D 400 magnification; cu, cuticle; hpd, hypodermis; ut, uterus; em, developing embryo). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
(Fig. 4B) compared to that of control Mf, which showed a very few number of such nuclei (Fig. 4A). 3.5. Hoechst staining For confirmation of apoptosis in ABZ-treated adult females, Hoechst 33258 staining was performed. Fig. 5B clearly shows condensed chromatin and in some cases fragmentation in ABZ-treated sections of parasites, both of which did not occur in cell nuclei of untreated worms (Fig. 5A).
3.6. DNA fragmentation assay The profile of genomic DNA in 2% agarose gel electrophoresis from 72 h ABZ-treated adult worms showed a typical dose-dependent nucleosomal DNA laddering (Fig. 6A). However, ABZ did not produce any DNA laddering in the genomic DNA isolated from ABZ-treated Mf even 120 h post-treatment (Fig. 6B). This suggests that ABZ even at higher doses could not activate endonuclease in the treated Mf.
Fig. 4. TUNEL stained light micrographs of Setaria cervi Mf treated with ABZ at 100 lg/ml in vitro. Several apoptotic nuclei (arrows) were found all over the Mf body of the treated parasite (B) which were absent in control worm (A). (A–B 400 magnification). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
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Fig. 5. Hoechst staining. For confirmation of apoptosis in ABZ-treated adult females, Hoechst 33258 staining was performed. Panel B shows clearly the chromatin condensation and in some cases fragmentation (arrows) in developing embryos and oocytes of ABZ-treated section of parasites, both of which were not found in cell nuclei of untreated parasites (Panel A).(A–B 400 magnification; em, developing embryos; oo, oocytes). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
Fig. 6. Detection of genomic DNA fragmentation. Agarose gel (2%) electrophoresis of genomic DNA extracts from untreated (control) and treated adult worms (A) and treated Mf (B). In both cases drug treatment was given for 48 h. A DNA fragmentation pattern was clearly observed from the genomic DNA of treated adult parasites which was not found in Mf [Lanes: Panel (A) M, molecular weight marker; C, control; ABZ 25, 50, 100, 500 and 1000 lg/ml Panel (B) M, molecular weight marker; C, control; ABZ 2, 4, 10, 20 and 50 lg/ml].
4. Discussion Programmed cell death (PCD) is a kind of cell fate involved in animal development and homeostasis (Hengartner and Horvitz, 1994). It helps in the precise regulation of cell number in developing or mature organism and has shown conservancy through evolution (Conradt and Horvitz, 1998). Drug induced apoptosis as reported here may be considered a type of homeostatic mechanism for the cells of a tissue to save its surroundings from destruction. The drug ABZ, a member of BZ family, is a well known anthelmintic for a long time (Pene et al., 1982). We used ABZ in this experiment at a much lower dose than the dose recommended for the human(s) (Belizario et al., 2003). Molecular mechanism of BZs as an anthelmintic relies on the impairment of microtubular organization by disrupting its polymerization when bind to the b-tubulin (Lacey, 1990; Barrowman et al., 1984; Lacey and Watson, 1985; Martin, 1997). These compounds also show great selectivity which is due to their differences in the binding affinity between helminth and mammalian b-tubulin. Its antitumor activity mediated through apoptosis in a human cancer line has also been reported earlier (Pourgholami et al., 2005). Disruption of normal cytoskeletal texture through inhibition in microtubule polymerization by ABZ is thought to be a key cause of apoptosis induction by this drug. BZs are mainly orally ingested by nematodes, which cause starvation to nematodes by intestinal disruption due to ABZ-induced apoptosis in these cells. Mitotic arrest and cytochrome C release by mebendazole, another potent BZ, have also
been reported previously (Mukhopadhyay et al., 2002; Sasaki et al., 2002). Here we are reporting for the first time, its antifilaremic activity mediated through apoptosis in S. cervi, a non-wolbachial filarial worm. A key role of microtubules during apoptotic changes especially in regulation of apoptotic chromatin dynamics and cellular fragmentation has been reported several times and is well established (Moss et al., 2006). The fact that antitubulin agents can induce the initiation of several apoptotic pathways through impairment of the cytoskeletal structure is well known (Kisurina-Evgen’eva et al., 2006). Analyzing all these informations with our results clearly indicate that ABZ induces apoptosis in bovine filarial worm S. cervi and thus acts as a very good antifilarial agent. ABZ produced a clear dose-dependent microfilaricidal activity over time as evident from viability assay. Similar results were observed against adult worms, and thus, we are reporting for the first time the macrofilaricidal effect of ABZ on S. cervi. In this paper we have also gathered several evidences that suggest the macrofilaricidal activity by ABZ is mediated through induction of apoptosis. The presence of numerous apoptotic nuclei in the tegument, developing embryos and uterine epithelium as seen in the TUNEL stained cross-sections of the ABZ-treated adult worms, establishes the fact that it does attack cells mainly from those areas by inducing apoptosis. The presence of clustered dark brown nuclei in a dispersed fashion all over the Mf body also implies the same phenomenon. The results from Hoechst staining are also consistent with the signs of apoptotic changes where condensed and fragmented chromatins are clearly visible. But the most interesting result from our experiment was the absence of oligonucleosomal DNA degradation in ABZ-treated Mf, even after a long exposure (5–6 days) to the drug at high dose. There may be several endonucleases, required for DNA fragmentation. This stage of apoptosis may give response to TUNEL well, which has been reported earlier in C. elegans (Wu et al., 2000) where no DNA degradation occurs due to mutation in the nuc 1 gene that encodes a homolog of mammalian DNase II, responsible for subsequent stages of DNA degradation. Thus there may be some abnormalities or inactivation of the genes required for the activation of nuc 1 gene or the nuc 1 itself (Horvitz, 2003). Besides, there are several reports at cellular level where apoptosis takes place in a way independent of DNA fragmentation (Yuste et al., 2001; Li et al., 2005). Further, there is possibility that the treated Mf may develop resistance to BZ. This phenomenon has been reported by several authors (Kwa et al., 1993a,b; Elard et al., 1996; Silvestre and Cabaret, 2002; Elard and Humbert, 1999). It occurs due to a single nucleotide change in the b-tubulin gene that leads to substitution of a single amino acid (phenyl alanine to tyrosine) at either position 167 or 200 of the nematode b-tubulin, the binding target of
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ABZ (Schwab et al., 2005). We propose that ABZ-induced apoptosis may be due to the activation of caspase-3 enzyme by the drug. Further works are in progress to establish the pathways involved in ABZ-induced apoptosis in S. cervi. In future, it would be interesting to investigate the apoptotic pathways in filarial parasites affecting human(s) that can serve as the targets for developing a new class of antifilarial drugs for the next generation of filarial control strategies.
Acknowledgments Financial assistance from Council of Scientific and Industrial Research, New Delhi, in the form of Senior and Junior Research Fellowships to PG and AN is acknowledged. We thank the Department of Biotechnology, New Delhi for a grant to support a part of the project. We are indebted to Dr. Goutam Ghosal, Professor, Department of English and Other Modern European Languages, Visva-Bharati for performing a critical review of the manuscript.
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Mr. Ananya Nayak, M.Sc., has been pursuing his doctoral work on the topic entitled ‘‘Improved efficacy of antiWolbachia antibiotics by albendazole: a new approach for the control of bancroftian filariasis,’’ in Department of Zoology, Visva-Bharati University, Santiniketan, India, since 2009.
Miss. Prajna Gayen, M.Sc., pursuing her doctoral work on the topic entitled ‘‘Epidemiology and control of bancroftian filariasis’’, in Department of Zoology, VisvaBharati, Santiniketan,India, since 2006.
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A. Nayak et al. / Experimental Parasitology 128 (2011) 236–242 Mr. Prasanta Saini, M.Sc., pursuing his doctoral work on the topic entitled ‘‘Acaciaside A: a novel compound for the control of bancroftian filariasis’’, in Department of Zoology, Visva-Bharati, Santiniketan, India, since 2009.
Dr. Sudipta Maitra M.Sc., Ph.D. did his Ph.D. from Visva Bharati University, Santiniketan, India on Endocrinology in the year 2000. Having more than 7 years of teaching and research experience, he is presently engaged in research on molecular endocrinology.
Prof. S.P. Sinha Babu M.Sc., Ph.D. did his Ph.D. from Visva-Bharati University, Santiniketan, India on parasitology in the year 1984. Having more than 20 years of teaching and research experience, he is presently engaged in research on the development of drugs against lymphatic filariasis.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Albendazole induces apoptosis in adults and microfilariae of Setaria cervi A. Nayak a, P. Gayen a, P. Saini a, S. Maitra b, S.P. Sinha Babu a,⇑ a b
Parasitology Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal 731 235, India Physiology and Biochemistry Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal 731 235, India
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Article history: Received 6 June 2010 Received in revised form 29 December 2010 Accepted 16 March 2011 Available online 23 March 2011 Keywords: Lymphatic filariasis Setaria cervi Apoptosis Microfilaria (Mf) Albendazole (ABZ) Macrofilaricide
a b s t r a c t Setaria cervi, a filarial nematode of cattle, inhabits in the peritoneal cavity and has been used as a suitable model for screening antifilarial agents. Albendazole (ABZ), a tubulin-disrupting benzimidazole (BZ) and a potent microfilaricide binds to b-tubulin, is causing structural impairment of cytoskeleton and worm death. Our present study has revealed that exposure of microfilaria (Mf) and adult to gradually increasing concentration of ABZ leads to a dose-dependent gradual impairment of their motility followed by early death in vitro. We found extreme cellular disturbances in ABZ-treated worms characterized by nucleosomal DNA laddering and chromatin condensation. However, in the treated Mf no nucleosomal DNA laddering was found although presence of TUNEL reactive DNA was evident, thus indicating an apoptotic pathway independent of DNA fragmentation. We present data from molecular studies to provide evidence for ABZ-induced apoptosis in Mf and adult worms of S. cervi. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Setaria cervi is a filarial nematode parasite inhabiting in the peritoneal cavity of cattle and shows cosmopolitan distribution. Different antifilarial agents have been tested on S. cervi in rats and reported as a suitable model for screening antifilarial agents (Singhal et al., 1972). This is a suitable model because the rate of proper establishment of transplanted parasites in rats is 50% and microfilaria appears in the peripheral blood within 10–15 days after transplantation and persists for 120 days (Singhal et al., 1972). The community-based control of lymphatic filariasis is mainly aimed at interrupting parasite transmission (WHO, 2007). The antifilarial drugs presently used, diethylcarbamazine, ABZ and ivermectin, are selectively active against Mf in the blood, in reducing the numbers and limiting transmission. Recent antimicrofilarial or macrofilaricidal treatment regimens do have certain well documented limitations. Diethylcarbamazine, and ivermectin are effective at killing Mf but are associated with systemic and inflammatory adverse reactions (Cross et al., 2001; Molyneux et al., 2003). ABZ increases the efficacy of diethylcarbamazine and ivermectin and is used in combination with either of drugs as the basis of long term intervention programme, aimed at breaking disease transmission (Molyneux et al., 2003). ABZ is a safe and effective antifilarial drug belonging to the group BZ. Since its discovery in 1972, it has been used as a broad spectrum anthelmintic drug against round worms, tape worms and flukes of ⇑ Corresponding author. Fax: +91 3463 262672. E-mail address: [email protected] (S.P. Sinha Babu). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.03.005
domestic animals and humans (Theodorides et al., 1976). It plays a significant role in impairment of glucose uptake in the helminth parasites and also inhibits the helminth specific fumarate reductase (Marr et al., 2003). However, its primary role is to inhibit microtubule polymerization in susceptible nematodes by binding to the b-tubulin, a cytoskeletal element and thus reduce the glucose uptake (Lacey, 1990; Barrowman et al., 1984; Lacey and Watson, 1985; Martin, 1997). Cytoskeleton containing microtubules plays an important role in cell functioning. Microtubules exhibit highly dynamicity due to assembly and disassembly at their ends. The involvement of microtubules in critical process like mitosis helps to design suitable chemotherapeutic agents that show antitubulin activity and thus promoting programmed cell death (PCD) through different apoptotic pathways. Apoptosis is programmed cell death, characterized by nuclear condensation, membrane convolution and nucleosomal fragmentation (Kerr et al., 1972). Previous studies have shown an association between apoptosis and disintegrity of cytoskeletal components (Moss et al., 2006). The antitumor activity of ABZ has already been proved (Pourgholami et al., 2005) against human colorectal cell line. Recently, the elucidation of the cellular effects of ABZ and other BZ compounds, and especially their binding to tubulin in parasites, has prompted us to investigate whether ABZ could induce apoptosis in adults and Mf of S. cervi, a bovine filarial parasite. The discovery of programmed cell death in Caenorhabditis elegans, a free living nematode during post embryonic development and subsequent elucidation of its genetic control initiated a new field for apoptosis research (Sulston and Horvitz, 1977; Sulston et al., 1983; Ellis and
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Horvitz, 1986). Here we are reporting for the first time that ABZ, a safe and effective antihelminthic drug, shows strong antifilarial effect on adults and Mf of S. cervi by inducing apoptosis.
2. Materials and methods 2.1. Collection of parasites Adult worms of S. cervi were collected from the peritoneal cavity of freshly slaughtered cattle at local abattoirs, washed several times with Kreb’s Ringer bicarbonate buffer (Sigma), and kept in Ringer’s solution at 37 °C. Mf were obtained by dissecting gravid females, and kept in Ringer’s solution at 37 °C for 1 h. 2.2. Drug preparation Albendazole, purchased from Sigma Chemical Company (USA), was dissolved in DMSO to make a stock solution and diluted in RPMI-1640 to obtain the desired concentrations. 2.3. In vitro culture and treatment of adult worms and microfilaria Adult worms of S. cervi (1 male and 1 female) were incubated in 5 ml of complete media, CM (RPMI-1640 supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U/ml streptomycin, 100 lg/ml penicillin, 0.25 lg/ml of amphotericin B and 10% fetal calf serum), alone and in combination with ABZ at varying concentrations in a 24-well flat-bottomed culture plate (Tarson, India). Mf obtained from gravid adult females, were incubated in 200 ll of CM alone and in CM with ABZ, at selected concentrations in a 96-well flat-bottomed microtiter plate (Tarson, India). Both the cultures were maintained for 48 h at 37 °C in a humidified atmosphere of 5% CO2 (Rao and Weil, 2002). The cultures for adult worms were carried out in duplicate and for Mf in quadruplicate and repeated at least three times. 2.4. Assessment of parasite viability by MTT assay Viability of parasites was assessed quantitatively by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) reduction assay (Comley et al., 1989). After a brief microscopic assessment of parasite viability, Mf (n = 0.15 105) were suspended in 0.1 ml of PBS containing 0.5 mg of MTT ml 1 and then incubated for 2 h at 37 °C in dark. The dark blue crystals of formazan thus formed were then solubilized using 100 ll of DMSO in a 96-well microtiter plate. Adult worms were incubated in 0.5 ml of PBS containing 0.5 mg of MTT and formazan crystals were solubilized in 200 ll of DMSO. The absorbance intensity of each sample was read with an automatic microtiter plate reader (Beckman, USA) at a 595 nm wavelength by using DMSO as a blank. 2.5. Histology After 48 h, ABZ-treated adult worms were fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin and cut into 3-lm thick sections.
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2.7. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) There are many ways to detect apoptotic nuclei at different stages on histological sections; we used one of the most commonly used methods, TUNEL. According to manufacturer’s protocol (DeadEnd™ Colorimetric TUNEL System, Promega, USA), transverse sections were first deparaffinized, rehydrated in graded series of alcohol, digested by proteinase K (20 lg/ml) for 15 min at room temperature. Sections were then incubated in equilibration buffer for 8 min and the fragmented DNA were labeled by biotinylated nucleotide mix in the presence of recombinant deoxynucleotidyl transferase (rTdT) for 60 min in a humidified chamber. Finally, the labeled fragments were incubated with streptavidin horseradish peroxidase and visualized after diaminobenzidine color development in which a dark brown staining indicated apoptosis. A light microscope (Dewinter, Victory, Italy) was used for observations and photographs. 2.8. Hoechst staining For the assessment of chromatin condensation, the sections were stained as described previously (Su et al., 2001) using 8 lg/ ml of Hoechst 33258 in double distilled water at room temperature. The stained sections were examined with an Olympus fluorescent microscope (BX 41, Japan), using an excitation wavelength of 490 nm. 2.9. DNA ladder assay Total genomic DNA was extracted from ABZ-treated (48 h) Mf and adults as well as control worms by the phenol/chloroform method as described earlier (Datta et al., 2007) with slight modifications. In brief, the Mf and adult worms were digested in 500 ll of lysis buffer (Tris–HCl 20 mM, pH 8.0, EDTA 50 mM, SDS 0.5%, NaCl 100 mM, b-mercaptoethanol 1%, v/v), proteinase K (0.1 mg/ml) and incubated in a water bath at 37 °C for 2 h. This was followed by RNase (5 lg/ml) treatment for 2 h at 56 °C. After phenol–chloroform–isoamyl alcohol extraction and ethanol precipitation, the final DNA pellet was resuspended in 20 ll of sterile nuclease-free water and run on 2% agarose gel, stained with ethidium bromide and analyzed under ultraviolet illumination (BIO-RAD, USA). 2.10. Statistical analysis Data were analyzed by Student’s t-test using MS Excel software. Differences were considered significant with P values <0.05. 3. Results 3.1. Effect of ABZ on Mf viability The effects of ABZ on Mf viability (MTT assay) were represented graphically (Fig. 1B). It appears from the results that the gradual reduction in Mf motility (results not shown) was consistent with the reduction of Mf viability. After 48 h of incubation with ABZ at 2 and 4 lg/ml, the viability of Mf was significantly decreased to 50% (P < 0.05). At higher doses ABZ reduced Mf viability by more than 70%. Maximum reduction (about 90%) was recorded after 48 h of incubation with ABZ at 50 lg/ml.
2.6. Hematoxylin–eosin staining 3.2. Effect of ABZ on adult worm viability The sections were stained with hematoxylin and eosin to detect the extent of ABZ-induced tissue damage. The stained sections were examined with a light microscope (Dewinter, Victory, Italy).
The effects of ABZ on viability (MTT assay) of adult worms, cultured in vitro were also studied (Fig. 1A). The gradual reduction in
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Fig. 1. Effect of ABZ on Setaria cervi viability. (A) Adult worms and (B) Mf. Parasites were incubated with ABZ for 24 and 48 h and viability was measured by the MTT reduction test. Results were analyzed as percent reduction in formazan production compared with control worms and expressed as percent reduction in viability.
worm viability with increasing drug concentrations was very compatible with gradual decrease in worm motility (results not shown). Viability of the treated worms was significantly decreased (P < 0.05) to 40% after incubation with ABZ at 100 and 500 lg/ml, and at higher dose, i.e. 1000 lg/ml the reduction reached up to 70%. Thus, ABZ produced strong microfilaricidal as well as macrofilaricidal effects on S. cervi in a dose-and time-dependent manner. However, normal untreated Mf and adult worms also show some degree of MTT reduction but the rate was negligible in comparison to the freshly collected worms (data not shown). 3.3. Hematoxylin–eosin staining ABZ treatment had caused damage to the outer cuticle and the underlying muscle layer (Fig. 2D). Cells from intestinal epithelium (Fig. 2E) and uterus lining (Fig. 2F) of treated worms showed
distorted appearance that seems to be apoptotic in nature. Such types of damage in cellular appearance were not observed in control worms (Fig. 2A–C). Besides there was huge depletion in the number of developing embryos in the uterus (figure not shown). 3.4. TUNEL The results of TUNEL assay clearly reveal presence of in situ DNA fragmentation in the nuclei of ABZ-treated tissue (Fig. 3B and D). Clusters of numerous dark brown apoptotic nuclei were found in the cells of outer epithelium and developing embryos within uterus as observed in the TUNEL stained transverse sections of 48 h ABZ-treated adult worms. Cells from the control sections (Fig. 3A and C) showed less or few number of apoptotic nuclei. Similar apoptotic nuclei were observed in a more dispersed fashion in the TUNEL stained body of 48 h ABZ-treated Mf
Fig. 2. H and E stained light micrographs of Setaria cervi female worms. Sections of untreated worms (A–C) showed no significant damage in worm morphology. Several abnormal morphological features were observed (indicated by arrows) in the sections of ABZ-treated (D–F) (100 lg/ml) worms: damaged hypodermal muscle layer (D); extensive cellular and nuclear disintegrity in intestinal epithelial cells (E); and distorted cellular and nuclear appearance in uterine epithelium (F). (A–F 400 magnification; cu, cuticle; hpd, hypodermis; int, intestine; ov, ovary; ut, uterus; lml, longitudinal muscle layer). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
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Fig. 3. TUNEL stained light micrographs of Setaria cervi adult worms. The TUNEL assay showed lack of apoptotic death in cells of the control sections (A and C), compared with the 48 h ABZ-treated (100 lg/ml) sections (B and D) of adult parasites. The cuticle, developing embryos and uterine epithelium showed numerous dark brown stained apoptotic nuclei (arrows) (A–D 400 magnification; cu, cuticle; hpd, hypodermis; ut, uterus; em, developing embryo). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
(Fig. 4B) compared to that of control Mf, which showed a very few number of such nuclei (Fig. 4A). 3.5. Hoechst staining For confirmation of apoptosis in ABZ-treated adult females, Hoechst 33258 staining was performed. Fig. 5B clearly shows condensed chromatin and in some cases fragmentation in ABZ-treated sections of parasites, both of which did not occur in cell nuclei of untreated worms (Fig. 5A).
3.6. DNA fragmentation assay The profile of genomic DNA in 2% agarose gel electrophoresis from 72 h ABZ-treated adult worms showed a typical dose-dependent nucleosomal DNA laddering (Fig. 6A). However, ABZ did not produce any DNA laddering in the genomic DNA isolated from ABZ-treated Mf even 120 h post-treatment (Fig. 6B). This suggests that ABZ even at higher doses could not activate endonuclease in the treated Mf.
Fig. 4. TUNEL stained light micrographs of Setaria cervi Mf treated with ABZ at 100 lg/ml in vitro. Several apoptotic nuclei (arrows) were found all over the Mf body of the treated parasite (B) which were absent in control worm (A). (A–B 400 magnification). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
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Fig. 5. Hoechst staining. For confirmation of apoptosis in ABZ-treated adult females, Hoechst 33258 staining was performed. Panel B shows clearly the chromatin condensation and in some cases fragmentation (arrows) in developing embryos and oocytes of ABZ-treated section of parasites, both of which were not found in cell nuclei of untreated parasites (Panel A).(A–B 400 magnification; em, developing embryos; oo, oocytes). Scale bars have been incorporated using Dewinter Biowizard 4.2 (Scale bar = 190 lm).
Fig. 6. Detection of genomic DNA fragmentation. Agarose gel (2%) electrophoresis of genomic DNA extracts from untreated (control) and treated adult worms (A) and treated Mf (B). In both cases drug treatment was given for 48 h. A DNA fragmentation pattern was clearly observed from the genomic DNA of treated adult parasites which was not found in Mf [Lanes: Panel (A) M, molecular weight marker; C, control; ABZ 25, 50, 100, 500 and 1000 lg/ml Panel (B) M, molecular weight marker; C, control; ABZ 2, 4, 10, 20 and 50 lg/ml].
4. Discussion Programmed cell death (PCD) is a kind of cell fate involved in animal development and homeostasis (Hengartner and Horvitz, 1994). It helps in the precise regulation of cell number in developing or mature organism and has shown conservancy through evolution (Conradt and Horvitz, 1998). Drug induced apoptosis as reported here may be considered a type of homeostatic mechanism for the cells of a tissue to save its surroundings from destruction. The drug ABZ, a member of BZ family, is a well known anthelmintic for a long time (Pene et al., 1982). We used ABZ in this experiment at a much lower dose than the dose recommended for the human(s) (Belizario et al., 2003). Molecular mechanism of BZs as an anthelmintic relies on the impairment of microtubular organization by disrupting its polymerization when bind to the b-tubulin (Lacey, 1990; Barrowman et al., 1984; Lacey and Watson, 1985; Martin, 1997). These compounds also show great selectivity which is due to their differences in the binding affinity between helminth and mammalian b-tubulin. Its antitumor activity mediated through apoptosis in a human cancer line has also been reported earlier (Pourgholami et al., 2005). Disruption of normal cytoskeletal texture through inhibition in microtubule polymerization by ABZ is thought to be a key cause of apoptosis induction by this drug. BZs are mainly orally ingested by nematodes, which cause starvation to nematodes by intestinal disruption due to ABZ-induced apoptosis in these cells. Mitotic arrest and cytochrome C release by mebendazole, another potent BZ, have also
been reported previously (Mukhopadhyay et al., 2002; Sasaki et al., 2002). Here we are reporting for the first time, its antifilaremic activity mediated through apoptosis in S. cervi, a non-wolbachial filarial worm. A key role of microtubules during apoptotic changes especially in regulation of apoptotic chromatin dynamics and cellular fragmentation has been reported several times and is well established (Moss et al., 2006). The fact that antitubulin agents can induce the initiation of several apoptotic pathways through impairment of the cytoskeletal structure is well known (Kisurina-Evgen’eva et al., 2006). Analyzing all these informations with our results clearly indicate that ABZ induces apoptosis in bovine filarial worm S. cervi and thus acts as a very good antifilarial agent. ABZ produced a clear dose-dependent microfilaricidal activity over time as evident from viability assay. Similar results were observed against adult worms, and thus, we are reporting for the first time the macrofilaricidal effect of ABZ on S. cervi. In this paper we have also gathered several evidences that suggest the macrofilaricidal activity by ABZ is mediated through induction of apoptosis. The presence of numerous apoptotic nuclei in the tegument, developing embryos and uterine epithelium as seen in the TUNEL stained cross-sections of the ABZ-treated adult worms, establishes the fact that it does attack cells mainly from those areas by inducing apoptosis. The presence of clustered dark brown nuclei in a dispersed fashion all over the Mf body also implies the same phenomenon. The results from Hoechst staining are also consistent with the signs of apoptotic changes where condensed and fragmented chromatins are clearly visible. But the most interesting result from our experiment was the absence of oligonucleosomal DNA degradation in ABZ-treated Mf, even after a long exposure (5–6 days) to the drug at high dose. There may be several endonucleases, required for DNA fragmentation. This stage of apoptosis may give response to TUNEL well, which has been reported earlier in C. elegans (Wu et al., 2000) where no DNA degradation occurs due to mutation in the nuc 1 gene that encodes a homolog of mammalian DNase II, responsible for subsequent stages of DNA degradation. Thus there may be some abnormalities or inactivation of the genes required for the activation of nuc 1 gene or the nuc 1 itself (Horvitz, 2003). Besides, there are several reports at cellular level where apoptosis takes place in a way independent of DNA fragmentation (Yuste et al., 2001; Li et al., 2005). Further, there is possibility that the treated Mf may develop resistance to BZ. This phenomenon has been reported by several authors (Kwa et al., 1993a,b; Elard et al., 1996; Silvestre and Cabaret, 2002; Elard and Humbert, 1999). It occurs due to a single nucleotide change in the b-tubulin gene that leads to substitution of a single amino acid (phenyl alanine to tyrosine) at either position 167 or 200 of the nematode b-tubulin, the binding target of
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ABZ (Schwab et al., 2005). We propose that ABZ-induced apoptosis may be due to the activation of caspase-3 enzyme by the drug. Further works are in progress to establish the pathways involved in ABZ-induced apoptosis in S. cervi. In future, it would be interesting to investigate the apoptotic pathways in filarial parasites affecting human(s) that can serve as the targets for developing a new class of antifilarial drugs for the next generation of filarial control strategies.
Acknowledgments Financial assistance from Council of Scientific and Industrial Research, New Delhi, in the form of Senior and Junior Research Fellowships to PG and AN is acknowledged. We thank the Department of Biotechnology, New Delhi for a grant to support a part of the project. We are indebted to Dr. Goutam Ghosal, Professor, Department of English and Other Modern European Languages, Visva-Bharati for performing a critical review of the manuscript.
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Mr. Ananya Nayak, M.Sc., has been pursuing his doctoral work on the topic entitled ‘‘Improved efficacy of antiWolbachia antibiotics by albendazole: a new approach for the control of bancroftian filariasis,’’ in Department of Zoology, Visva-Bharati University, Santiniketan, India, since 2009.
Miss. Prajna Gayen, M.Sc., pursuing her doctoral work on the topic entitled ‘‘Epidemiology and control of bancroftian filariasis’’, in Department of Zoology, VisvaBharati, Santiniketan,India, since 2006.
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A. Nayak et al. / Experimental Parasitology 128 (2011) 236–242 Mr. Prasanta Saini, M.Sc., pursuing his doctoral work on the topic entitled ‘‘Acaciaside A: a novel compound for the control of bancroftian filariasis’’, in Department of Zoology, Visva-Bharati, Santiniketan, India, since 2009.
Dr. Sudipta Maitra M.Sc., Ph.D. did his Ph.D. from Visva Bharati University, Santiniketan, India on Endocrinology in the year 2000. Having more than 7 years of teaching and research experience, he is presently engaged in research on molecular endocrinology.
Prof. S.P. Sinha Babu M.Sc., Ph.D. did his Ph.D. from Visva-Bharati University, Santiniketan, India on parasitology in the year 1984. Having more than 20 years of teaching and research experience, he is presently engaged in research on the development of drugs against lymphatic filariasis.