1 Equipe Multi-résistances et Antiparasitaires; 2 Atelier scientifique commun de Cytométrie en flux, INRA-Tours: UR086-BioAgresseurs, Santé et Environnement, 37380 Nouzilly, France
Received 24 October 2002; returned 12 March 2003; revised 28 March 2003; accepted 18 May 2003
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Abstract |
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Materials and methods: We used methyl-ß-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thiabendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites.
Results: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123.
Conclusion: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.
Keywords: P-glycoproteins, Haemonchus contortus, cholesterol, methyl-ß-cyclodextrin, thiabendazole
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Introduction |
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The specific mechanisms have been attributed to mutations in the intracellular targets for benzimidazoles, for example ß-tubulin.57 Different isotypes of ß-tubulin genes are produced.810 Structural changes in ß-tubulin have been shown to be related to gene deletions and have been observed in resistant isolates of H. contortus.6
The non-specific mechanisms have been less extensively studied. They are similar to multiple resistance phenomena conferred by the multidrug resistance (MDR) system in other eukaryotic systems.8,11 In these eukaryotes, the MDR genes and the activity of the proteins resulting from these genes are responsible for the development of chemotherapy-resistant tumour cells. The MDR system includes P-glycoprotein membrane pumps (Pgp) and multidrug resistance-associated proteins (MRP). These two transmembrane proteins are members of the ATP-binding cassette (ABC) superfamily of transporters and play key roles in the transport of xenobiotics.12,13 The pumps are implicated in cellular detoxification processes in various eukaryotic systems.14,15 In nematodes, genes associated with the MDR system are expressed11 and the Pgp membrane pumps may also play an important role in resistance phenomena.10,16 Nevertheless, our knowledge of the transmembrane transport mechanisms due to MDR in nematodes is still limited and only a small number of studies have looked at the functional activity of the pumps. We previously showed that drug transport is altered in several nematode species following treatment with both anthelmintics and verapamil, a Pgp-specific inhibitor.17 Efflux mechanisms due to pumps have also been studied in H. contortus using Pgp-specific substrates as described for tumour cells.18,19 Changes in the efflux of the fluorescent probe rhodamine 123 (R123), a substrate of Pgp, have been observed in H. contortus eggs submitted to treatment with the Pgp inhibitor verapamil.16 These results strongly suggest that Pgp activity plays an important role in the resistance of nematodes to anthelmintics. The use of inhibitors of pumps such as verapamil or lectins allowed up to 50% reversion of resistance.16,17,20 These preliminary results can probably be improved with other more potent inhibitors of the MDR system. They indicated a major contribution of non-specific mechanisms in the resistance of nematodes to anthelmintics.
In eukaryotic cells, the activity of Pgp seems to be conditioned by their membrane environment, in particular by the lipids, among which cholesterol, a major constituent of the eukaryotic cell membrane, plays an important role.21,22 Thus, we studied the effect of cholesterol concentration on the transport of drugs by membrane pumps in H. contortus eggs. The cholesterol concentration was modified by use of methyl-ß-cyclodextrin (MßCD) as described in other systems.2325 MßCD can deplete or load cell membranes with cholesterol.2325 The effects of these treatments were estimated by measuring the toxicity of anthelmintics using in vitro egg hatch assays in the presence of thiabendazole (TBZ). In parallel, biochemical assays were used to measure the cholesterol concentration directly in eggs.
The lipid environment of cell membranes also allows the passive transport of lipophilic compounds. Anthelmintics, which are lipophilic molecules, can thus pass passively through the membranes. To determine the role of passive transport, we used rhodamine 123, a cationic and lipophilic fluorescent probe, which crosses membranes mainly via Pgp pumps.19
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Materials and methods |
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Four Haemonchus contortus (Hc) isolates were studied: two susceptible (HcS) ones (HcS-WB for Weybridge, UK and HcS-C for Canada) and two resistant (HcR) ones, HcR-G for Guadeloupe and HcR-WR for White River. HcR-G is resistant to benzimidazoles and ivermectin and tolerant to moxidectin. HcR-WR (South African isolate) is resistant to benzimidazoles and ivermectin. Three-month-old sheep were infected with 3000 H. contortus infective larvae (L3) from each isolate 5 weeks before the assays. The experiments comply with the current French laws on animal experimentation. The life cycle of H. contortus includes several developmental stages. Eggs are the first free-living stage that can be obtained without slaughtering the donor animal. The studies were thus carried out on eggs which were isolated from faeces as described in Beaumont-Schwartz et al.26 The eggs were then stored in deionized water at 4°C for one night before being used.
Preparation of methyl-ß-cyclodextrin and cholesterol-methyl-ß-cyclodextrin inclusion complexes
MßCD (SigmaAldrich, Saint-Quentin, France) was dissolved in deionized water. The cholesterol-MßCD inclusion complexes (chol-MßCD) were prepared as described by Klein et al.24 Briefly, 30 mg of cholesterol (SigmaAldrich) dissolved in 1 mL of 99% isopropanol (SigmaAldrich) was added to 1 g of MßCD that had previously been dissolved in 19 mL of deionized water. The mixture was then stirred at 80°C until complete dissolution. The solution was then cooled to 20°C before being used to treat the eggs.
Preliminary studies: determination of the optimal experimental conditions for the treatment of eggs (MßCD and cholesterol concentrations, length of contact times)
The HcR-G isolate was chosen for these preliminary experiments.
The determination of egg cholesterol contents was carried out with 75 mM MßCD and two contact times (10 and 40 min) with washings, as described by Klein et al.24 For the loading assays, to keep the experimental conditions as close as possible to those used for the cholesterol depletion experiments, the concentrations in the complex were 7.8 mM for cholesterol and 75 mM for MßCD, but only one contact time was tested (10 min) with washings.
For the egg hatch assays and R123 assays, MßCD concentrations ranging from 1 mM to 75 mM were tested for different contact times: 1 x 1 h or 1 x 4 h without washings, 1 x 1 h, 1 x 4 h or 4 x 1 h with washings. Two cholesterol concentrations (3.8 and 7.8 mM), two MßCD concentrations (37.5 and 75 mM), but only one contact time (4 x 1 h) with washings were tested for the chol-MßCD experiments.
Treatments of H. contortus eggs with MßCD or chol-MßCD
To determine cholesterol contents, egg suspensions from each isolate (150 000 eggs/test tube) were incubated with either [75 mM MßCD] or [7.8 mM chol/75 mM MßCD] with shaking for 10 min. They were washed twice with deionized water. The eggs were then frozen in liquid nitrogen and stored at 20°C until cholesterol determinations.
For the egg hatch assays, 2500 eggs/test tube were incubated with either [2.25 mM MßCD] or [7.8 mM chol/75 mM MßCD] with shaking for 4 x 1 h with one washing with deionized water between each contact time.
For the R123 assays, 200 000 eggs/test tube were incubated with either [2.25 mM MßCD] or [7.8 mM chol/75 mM MßCD] with shaking for 4 x 1 h with one washing with deionized water between each contact time.
Determination of cholesterol content in eggs
The frozen eggs were crushed with a Dual tissue microgrinder (Teflon pestle, volume of 500 µL, Fisher Scientific, Elancourt, France). The cholesterol was extracted from the crushed eggs with 2 mL of chloroform/methanol (v/v; VWR international, Pessac, France) with manual shaking. The samples were centrifuged for 5 min at 500g and the lower phase collected and decanted into another test tube. The upper phase was then treated with 500 µL of chloroform with manual shaking. The samples were then centrifuged for 5 min at 500g and the lower phase collected and added to the first one. The samples were then evaporated in a double boiler at 65°C and the resulting dry extract was then dissolved in 50 µL of isopropanol. The samples were incubated in a double boiler at 45°C for 20 min. The cholesterol concentration was determined by the cholesterol oxidase method (RTU Kit, BioMérieux, Marcy lÉtoile, France). The pink colour was quantified by measurement of absorbance at 500 nm. The cholesterol concentration (ng/egg) was deduced from a calibration curve using a reference cholesterol solution.
Egg hatch assays after treatment with MßCD or chol-MßCD
In the in vitro egg hatch assays, the eggs were treated with MßCD or chol-MßCD and then incubated for 48 h at 22°C with concentrations of thiabendazole ranging from 0.02 to 0.08 µg/mL for the susceptible isolates and from 0.24 to 1.26 µg/mL for the resistant ones. Thiabendazole was chosen as a model for benzimidazole anthelmintics. Hatching rates were compared to those of control eggs treated with deionized water and thiabendazole only. LD50 values were calculated as described previously by Beaumont-Schwartz et al.26
Functional assays for drug transport activity
These assays were carried out on the HcR-G isolate. Preliminary assays were carried out on the modulation of the transport of R123 (SigmaAldrich). We used 0.25 µg/mL of R123 as previously described by Kerboeuf et al.27 Green fluorescence was measured using a FACStarPlus cytometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA, USA), equipped with an argon ion laser with the excitation wavelength set at 488 nm. Instrument settings were chosen such that 2000 eggs were analysed per sample. The intensity of green fluorescence was measured through a 530/30 nm bandpass filter and expressed in relative arbitrary units, calculated as the difference between the fluorescence of eggs without R123 and that of eggs stained with R123 to eliminate the native green fluorescence that differs between two isolates.27 Three replicates were analysed for each treatment.
Statistical analysis
The results of the egg hatch assays were subjected to covariance analyses as described in Snedecor & Cochran.28 This analysis combined the features of variance and regression analyses. The test included comparison of regression lines and intercepts. The results of cholesterol assays were subjected to a two-way analysis of variance.
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Results |
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Cholesterol depletion: The changes in cholesterol concentration between control and eggs treated with 75 mM MßCD were greater for a contact time of 10 min, than for 40 min (control eggs mean ± S.D.: 0.091 ± 0.006 ng/egg; treated eggs 10 min mean ± S.D.: 0.058 ± 0.006 ng/egg; treated eggs 40 min mean ± S.D.: 0.068 ± 0.005 ng/egg). The contact time chosen for the treatment of eggs was thus 10 min.
For the egg hatch assays, no significant difference in LD50 was observed with contact times ranging from 10 to 60 min and with 75 mM MßCD to keep the experimental conditions as close as possible to those used for the determination of the cholesterol concentration. To obtain a biological effect, it was necessary to modulate the MßCD concentration, the contact time and the applications of washings (Table 1).
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Cholesterol loading: Treatment with [7.8 mM chol/75 mM MßCD] induced a significant change in cholesterol concentration between control and treated eggs.
These concentrations also induced a significant biological effect (P < 0.05) as shown by egg hatch assays. Another concentration was also tested [3.8 mM chol/37.5 mM MßCD] and the biological effects were compared with those obtained previously. Both cholesterol concentrations induced a significant decrease (P < 0.05) in hatching rates of eggs in the presence of thiabendazole. This decrease was proportional to the dose of cholesterol. The effect obtained with 7.8 mM cholesterol was more independent of TBZ concentration (Figure 1). Thus, this cholesterol concentration was chosen for the egg hatch assays and cytometric analyses.
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The mean ± S.D. cholesterol concentration in control eggs was similar for HcS-WB, HcR-WR and HcR-G isolates (0.049 ng/egg ± 0.007) but was significantly lower for the HcS-C isolate (0.031 ng/egg ± 0.009, P < 0.05, Figure 2).
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Changes in resistance to TBZ by modulation of cholesterol content
The MßCD treatment increased the LD50 of TBZ for the four isolates (Table 2). Nevertheless, the difference between control and treated eggs was only significant for the two resistant isolates (P < 0.05). Chol-MßCD induced a significant decrease in the LD50 of TBZ. The differences between control and treated eggs were significant for the four isolates (P < 0.05).
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The difference in LD50, expressed as the percentage variation between the control and MßCD-treated eggs, showed that the effect was two times greater for the resistant isolates than for the susceptible ones. In contrast, the chol-MßCD treatment induced a similar relative difference whatever the isolate.
The regression curves were parallel for the different treatments (Figure 3) with significant differences between treated and control eggs (P < 0.05) for the MßCD and chol-MßCD treatments, except for the HcS-C isolate treated with MßCD.
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The mean ± S.D. calculated fluorescence intensity was 162 ± 7.8 in the untreated eggs, 126 ± 14.0 in the eggs treated with MßCD and 12 ± 1.0 in the eggs treated with chol-MßCD. The MßCD or chol-MßCD treatments induced a significant decrease in green fluorescence intensity (P < 0.05). This decrease was much more marked with the chol-MßCD treatment (P < 0.001).
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Discussion |
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We hypothesized that changes in the lipid composition of H. contortus eggshells, in particular the cholesterol concentration, would modulate the Pgp activity and thus their resistance to anthelmintics. As in other eukaryotes, cholesterol is involved in the organization of lipid bilayers in nematodes and thus, the development of nematode eggs requires cholesterol. The mechanisms by which cholesterol is distributed and transported in nematodes have been described.22 Nevertheless, nematodes cannot synthesize sterols de novo and therefore display a nutritional requirement for sterols from their animal or plant hosts.21,38
The first step of our experiment was to obtain changes in the cholesterol contents of H. contortus eggs. These eggs have more complex structures than cells. The eggshell which protects the embryo includes a vitelline layer, a medial chitinous layer and a basal lipid/protein layer.39 In other models, treatments with cyclodextrins, such as the ß-cyclodextrins, which have a high affinity for lipids, in particular sterols, have been described. Moreover, the methyl form (MßCD) preferentially extracts cholesterol from membrane cells24 and can be used as a rather specific cholesterol acceptor. Methyl-ß-cyclodextrin complex inclusions make it possible to load cells with cholesterol. This method for changing the sterol concentration in cells is precise and reproducible.40 In our study, treatment with MßCD or chol-MßCD also changed the cholesterol concentration in nematode eggs, as demonstrated by biochemical assays that showed changes in total cholesterol contents for the four isolates and both treatments. The cholesterol content of eggs was decreased by MßCD and increased by chol-MßCD. Nevertheless, it was easier to increase the cholesterol content in eggs than to decrease it. It can be hypothesized that the MßCD treatment, which extracts the cholesterol, highly disorganizes the eggshell, a complex and biochemically stable structure in natural conditions. The method seems to be adequate for the modulation of the cholesterol concentration in eggs but it has several limitations. MßCD has an affinity for all sterols, therefore the exact nature of the components extracted from the eggs is not known. Moreover, the biochemical assay for the determination of cholesterol does not differentiate cholesterol from cholesterol esters. The treatments applied to eggs could have caused cholesterol to migrate from the blastomeres towards the shell. This type of migration is known to occur in cellular systems.22,41 It is thus possible that the cholesterol movements in eggs at least partially hid the effect of the treatment.
Before studying the biological consequences of these cholesterol changes on Pgp activity, it was necessary to adapt the experimental conditions used in the biological tests on eggs. High concentrations of MßCD combined with long contact times induced toxic effects. These effects were strong, as shown by the sharp decrease in hatching rates from 100 per 100 to 0 per 100 between two relatively close MßCD concentrations. Conversely, high concentrations of MßCD combined with short contact times made it possible to modulate the cholesterol concentration, without inducing biological changes. With the lowest concentrations, MßCD tended towards a state of saturation after a long contact time and few biological effects were induced. To limit the toxicity and to obtain a significant biological effect, the eggs were washed between and after the treatments. This increased the tolerance of eggs to the highest concentrations of MßCD. It induced a biological effect even for the lowest concentration of MßCD, but caused the degradation of eggs with the highest concentrations of MßCD. If the complexes formed are regularly eliminated and new MßCD added, the capacity to create inclusion complexes is increased. The relationship between cholesterol and MßCD followed a chemical equilibrium.23 Several short contact times plus a low MßCD concentration resulted in a biological effect without too high a mortality and made it possible to estimate the pump activity by measuring the transport of R123.
Our results showed that changes in the cholesterol content of eggs modulated their hatching rate in the presence of thiabendazole (TBZ) and induced a change in their resistance to TBZ. Cholesterol depletion increased resistance and cholesterol loading decreased resistance. The biological consequences of these changes were qualitatively similar for the susceptible isolates and the resistant ones. Nevertheless, the resistant isolates were much more affected by MßCD and by chol-MßCD than were the susceptible ones. The differences observed between the susceptible isolates and the resistant ones could be explained by the following hypotheses: (1) presence of a larger number of Pgp pumps in the resistant isolates compared to the susceptible ones, (2) resistant isolates possess as many Pgp pumps as the susceptible ones but the pumps are more active and (3) changes in the membrane environment (cholesterol) could induce structural modifications of the Pgp that are greater in the resistant isolates than in the susceptible ones. These three hypotheses are not mutually exclusive. In resistant isolates, the decrease in cholesterol content might have stimulated the activity of Pgp, possibly by causing structural changes.35 This effect could have reinforced an already more active efflux due to the higher number of pumps in the resistant isolates than in the susceptible ones. Complementary studies are necessary to quantify the Pgp molecules in nematode eggs.
The mechanisms by which changes in cholesterol content modified the functionality of Pgp remain to be explain. This can result directly from an increased drug transport activity, but also from an increased affinity of Pgp for the specific substrates. Changes in the cholesterol content of the other cellular systems have been shown to affect: (a) their affinity for the substrate of transmembrane proteins such as hormonal receptors24,42 or (b) the transduction of the intracellular signal.43,44 Nevertheless in our study, the parallelism between the regression curves obtained from the egg hatch assays is not compatible with the hypothesis of a change in Pgp affinity. In our experimental conditions, it seems that the mechanism is more likely due to a modulation of transport. Two transport mechanisms have been described: passive diffusion and active transport. Active transport requires membrane pumps. We hypothesize that TBZ, a hydrophobic compound, could have been absorbed passively through lipid-rich membranes. The enrichment of eggs with cholesterol could have increased this passive transport. The flow cytometric assays on the fluorescence of HcR-G eggs resulting from the contact with R123 allowed us to observe this mechanism more directly. R123, another hydrophobic molecule, enters the cells mainly via Pgp pumps. It can also enter the membranes passively. Nevertheless, only a small amount of R123 is taken up passively and this process is very slow. Therefore, the fluorescence of eggs after contact with R123 was mainly representative of the activity of Pgp.31 The intensity of the green fluorescence decreased significantly after both MßCD and chol-MßCD treatment. The cholesterol concentration in eggs may act differently according to the concentration.29 Thus, a decrease in fluorescence after MßCD treatment might be attributed to a stimulated Pgp activity resulting from the decrease in the cholesterol content. Conversely, the chol-MßCD treatment might have blocked the Pgp activity by increasing the cholesterol content.
Our results confirmed that cholesterol can modulate Pgp activity in nematode eggs and change the level of resistance to anthelmintics. The mechanisms by which the lipids modulate the activity of the pumps and the possible role of other membrane lipids remain to be investigated.29 An hypothesis could be an alteration of the membrane fluidity as described by Rothnie et al. in cancer cells.12
Our results confirmed that Pgps are essential for the transport of drugs in nematodes, as shown in other models such as fluconazole-resistant Candida albicans isolates45,46 or chemoresistant tumour cells12 or other parasites.3 The role of the MDR system is thus significant in ruminant nematodes as in human nematodes. Studies on Onchocerca volvulus showed that expression of MDR genes coding for different Pgp also exists although the function of these genes has not yet been established.47,48 Resistance is apparently multifactorial and results from both specific and non-specific mechanisms. The use of Pgp inhibitors has already allowed up to 50% reversion in resistance.16,17,20 Changes in membrane lipid contents could provide another way to improve the reversion of resistance and to increase the efficacy of anthelmintics.
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Acknowledgements |
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Footnotes |
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