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Development of resistance to anthelmintic drugs is an increasing problem that decreases the productivity of livestock and threatens the success of treatment in humans. It is essential to understand the mechanisms in the development of resistance so that alternative treatment strategies can be developed. Changes in genes or in gene expression in response to drugs enable the organism to survive treatment and might reflect evolution in a toxic environment in which drug resistance leads to 'survival of the fittest'. Here, we review knowledge of resistance mechanisms, focusing on changes in drugs (identified by single-nucleotide polymorphisms), the involvement of transport proteins and drug efflux that prevent the drug from reaching the target, and the role of detoxification mechanisms that modify the drug.

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a K(m) 2.19+/-1.5 microM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.

Widespread resistance to chemotherapeutic agents is one of the biggest challenges facing human health and the agricultural industry, with resistance to all current anthelmintics now recorded and few new agents or vaccines available. Understanding the development of drug resistance in parasitic nematodes is critical to prolonging the efficacy of current anthelmintics, developing markers for monitoring drug resistance and is beneficial in the design of new chemotherapeutic agents or targets. This study describes the development of ivermectin-resistant strains of the model nematode Caenorhabditis elegans through step-wise exposure to increasing doses of ivermectin commencing with a non-toxic dose of 1 ng/ml. Resistant strains were developed that displayed a multidrug resistance phenotype with cross-resistance to the related drug moxidectin and to other anthelmintics, levamisole and pyrantel, but not albendazole. Resistance was associated with increased expression of the multidrug resistance proteins (MRPs) and P-glycoproteins. Resistance to ivermectin was reversible by the co-administration of MRP, P-glycoprotein and glutathione biosynthesis inhibitors, confirming the involvement of these proteins in resistance. In our model, resistance to low levels of ivermectin (

Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.

Multidrug resistance mediated by the multidrug resistance-associated protein MRP1 is associated with decreased drug accumulation, which is in turn dependent on cellular glutathione. We have reported that verapamil, an inhibitor of drug transport, caused a decrease in cellular glutathione in CCRF-CEM/E1000 MRP1-overexpressing leukaemia cells (Biochem Pharmacol 55;1283--9, 1998). We now demonstrate that other inhibitors of MRP1-mediated drug transport (e.g. MK571, indomethacin, genistein, and nifedipine) deplete cellular glutathione in these leukaemia cells (>30% decrease; P < 0.01) while having no effect on the parental CCRF-CEM cells. However, treatment with etoposide or vincristine (at similar molar concentrations) caused a 20% decrease in glutathione. Verapamil-stimulated glutathione transport correlated with MRP1 expression in a series of drug-resistant cells, and glutathione was quantitatively recovered in the extracellular media. Further, verapamil-stimulated glutathione transport was rapid (50% decrease in 10 min), dose-dependent, and inhibited by vanadate, an inhibitor of ATPase activity, but not by sulphobromophthalein (BSP) or methionine, inhibitors of hepatic glutathione transporters. Incubation of CCRF-CEM/E1000 cells in 25 mM glutathione not only showed that verapamil-mediated efflux occurred against the concentration gradient, but also demonstrated the MRP1-mediated uptake of glutathione (P < 0.01 compared to the parental CCRF-CEM cells), which was not inhibited by vanadate. These results demonstrate that while MRP1 transports glutathione in the presence of inhibitors of drug transport, there is no convincing evidence for co-transport of glutathione with drug. They further demonstrate that MRP1 mediates the facilitated transport of glutathione into the MRP1-overexpressing CEM/E1000 cells, suggesting that MRP1 may play a major role in cellular glutathione homeostasis.

Multidrug resistance-associated protein (MRP) causes multidrug resistance (MDR) involving the anthracyclines and epipodophyllotoxins. Many studies show modulation of anthracycline levels and cytotoxicity in MRP-overexpressing cells, but there is limited data on the modulation of etoposide levels and cytotoxicity in MRP-overexpressing or in P-glycoprotein-expressing cells. Etoposide accumulation was 50% reduced in both the CEM/E1000 MRP-overexpressing subline and the CEM/VLB100 P-glycoprotein-expressing subline compared to the parental CEM cells, correlating with similar resistance to etoposide (200-fold) of the two sublines. For the CEM/VLB100 subline, the P-glycoprotein inhibitor SDZ PSC 833, but not verapamil, was able to increase etoposide accumulation and cytotoxicity. For the CEM/E1000 subline, neither SDZ PSC 833 nor verapamil had any effect on etoposide accumulation. However, verapamil caused a 4-fold sensitization to etoposide in this subline, along with an 80% decrease in cellular glutathione (P < 0.05). Buthionine sulfoximine (BSO), which depletes glutathione, also caused a 2.5-fold sensitization to etoposide with no effect on accumulation in the CEM/E1000 subline. In contrast, SDZ PSC 833 was able to increase daunorubicin accumulation in the CEM/E1000 subline (P < 0.05), but had no effect on daunorubicin cytotoxicity, or cellular glutathione. These results show that modulation of etoposide cytotoxicity in MRP-overexpressing cells may be through changes in glutathione metabolism rather than changes in accumulation and confirm that changes in drug accumulation are not related to drug resistance in MRP-overexpressing cells.

With the increasing use of inducers of cellular differentiation in the treatment of leukaemia, it is essential to understand the relationship between differentiation and the expression of the multidrug resistance. Using the K562 human leukaemia cell line and its multidrug resistant subline K562/E15B, differentiation was examined along two different pathways, megakaryocyte in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and erythroid in response to treatment with sodium butyrate, in the same cell line. P-glycoprotein expression was increased in the multidrug resistant K562/E15B subline, but not induced in the parental K562 cell line. However, both treatments conferred a different phenotype on the drug resistant subline. TPA treatment caused an increase in P-glycoprotein, increased drug resistance and decreased rhodamine-123 accumulation which was verapamil sensitive, demonstrating that TPA induced a fully functional P-glycoprotein. However, sodium butyrate treatment caused an increase in P-glycoprotein without increased drug resistance or without decreased rhodamine-123 accumulation suggesting that the P-glycoprotein induced by sodium butyrate was nonfunctional. These results stress the importance of examining not only the expression of P-glycoprotein in cells, but also the function of the P-glycoprotein induced.