Defective DNA repair as a potential mechanism for the rapid development of drug resistance in Plasmodium falciparum

The development and spread of highly drug-resistant parasites pose a central problem in the control of malaria.Understanding mechanisms that regulate genomic stability, such as DNA repair, in drug-resistant parasites and during drug treatment may help determine whether this rapid onset of resistance is due to an increase in the rate at which resistance-causing mutations are generated. This is the first report to demonstrate DNA repair activities from the malaria-causing parasite Plasmodium falciparum that are specific for ultraviolet light-induced DNA damage. The efficiency of DNA repair differs dramatically among P. falciparum strains with varying drug sensitivities. Most notable is the markedly reduced level of repair in the highly drug-resistant W2 isolate, which has been shown to develop resistance to novel drugs at an increased rate when compared to drug-sensitive strains. Additionally, the antimalarial drug chloroquine and other quinoline-like compounds interfered with the DNA synthesis step of the repair process, most likely a result of direct binding to repair substrates. We propose that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may contribute to the accelerated development of drug resistance in the parasite.     

Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1

Cationic amphiphilic drugs like chlorpromazine, propranolol, and chloroquine inhibit lysosomal phospholipase A in vitro. Some workers have proposed that cationic amphiphilic drugs inhibit the activity of phospholipase A1 by forming substrate-drug complexes which cannot be degraded while others have reported competitive inhibition implying drug effects on the enzyme. To analyze the mechanism of inhibition, we examined the binding ability of these drugs to unilamellar vesicles of dioleoylphosphatidylcholine and correlated these results with a detailed kinetic analysis of phospholipase A. Chlorpromazine and propranolol bound to small unilamellar liposomes of dioleoylphosphatidylcholine substrate in a positive cooperative way consistent with two binding sites: a high-affinity site with low capacity and a low-affinity site with high capacity. The affinity of chlorpromazine for the high-affinity site was 2 times greater than that of propranolol (KA = 13 807 +/- 1722 vs. 8481 +/- 1078 M-1), and the saturation number for chlorpromazine was 3 times greater than for propranolol (N = 0.20 +/- 0.004 vs. 0.07 +/- 0.02 mol of drug/mol of phosphatidylcholine). Chloroquine did not bind to unilamellar liposomes of dioleoylphosphatidylcholine. We carried out detailed kinetic studies using purified lysosomal phospholipase A1 from rat liver. In the case of chloroquine inhibition, the Lineweaver-Burk double-reciprocal plots showed straight lines, but the slope replots were curved, indicating the formation of complexes having 2 mol of chloroquine/mol of enzyme (EI2 complexes). Thus, chloroquine is a competitive inhibitor which forms EI2 complexes with phospholipase A1. However, in the case of chlorpromazine and propranolol, the observed kinetic data do not fit to the same equilibrium used for the case of chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenicity examination of several non-steroidal anti-inflammatory drugs in bacterial systems

The mutagenicity of 6 marketed non-steroidal anti-inflammatory drugs (aspirin, flufenamic acid, diclofenac sodium, indomethacin, naproxen and chloroquine) as well as 2 new anti-inflammatory drugs (tenoxicam and carprofen) was examined by using in vitro bacterial systems (repair test and reversion test). None of them was mutagenic on Ames' reversion test. However, they differed in their responses to repair tests. Tenoxicam, carprofen, aspirin, flufenamic acid and naproxen were not mutagenic in either rec- or pol-assays, whereas chloroquine only showed positive results in the pol-assay system. Indomethacin and diclofenac sodium exhibited a slightly stronger inhibitory activity against B. subtilis rec- mutant than against its rec+ counterpart in rec-assay, which was much weaker than AF-2. Thus their mutagenicity was questionable. These results confirm the usefulness of DNA-repair assays as a complementary endpoint to gene mutation in assessing the genotoxic potential of environmental compounds.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

On the mechanism of chloroquine resistance in Plasmodium falciparum

Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.     

Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently considered models to explain the resistance phenotype include acquisition of a chloroquine efflux pump, changes in intracellular chloroquine partitioning, diminished binding affinity of chloroquine to its intracellular target, heme, and changes in heme crystallization. To challenge these different models, we have investigated chloroquine accumulation under trans-stimulation conditions and in the presence and absence of glucose. We show that, in chloroquine-sensitive strains, labeled chloroquine accumulation is steadily reduced as the pre-equilibrated chloroquine concentration is raised. In the resistant cells, the extent of accumulation is, strikingly, raised at the lower levels of preloading, in comparison with resistant controls in the absence of chloroquine. The trans-stimulation effect observed in chloroquine-resistant cells is strictly energy-dependent. The data are interpreted in terms of a model in which chloroquine is bound to intracellular binding sites, not different as between sensitive and resistant cells, but where, in resistant cells, there exists an energy-dependent carrier that moves chloroquine out of this intracellular compartment. A mathematical model describing the kinetics of these processes is presented.     

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.     

Repair between dose fractions: a simpler method of analyzing and reporting apparently biexponential repair

Increasing numbers of animal experiments in situ are reporting that repair of sublethal radiation damage in vivo slows down with time, usually described as two components of (monoexponential) repair. For repair of DNA strand breaks, plotting the reciprocal of proportion unrepaired as a function of time yielded straight lines. Two processes have been suggested as causing this: (1) a second-order process (bimolecular) instead of first-order (exponential) and (2) a skewed distribution of monoexponential rates. The present paper investigates whether such plots of hyperbolic or reciprocal repair are relevant for laboratory animal tissue results. Published repair data were reanalyzed from laboratory animal experiments that employed split doses or two fractions per day. Graphs are presented of the reciprocal proportion of damage remaining as a function of the interval between the two doses. If the reciprocal model applies, the graphs would be straight lines. Different animal data showed no inconsistency with straight reciprocal plots. These reciprocal plots describe well with one parameter tau, the first half-time, repair curves previously thought to be "biexponential", and to require three parameters. Straight reciprocal plots mean that in a constant time interval tau the unrepaired damage falls from 1 to (1/2), then from (1/2) to (1/3), then (1/3) to (1/4), etc. A much larger proportion of damage would therefore remain unrepaired at several half-times than is estimated by current mono- or biexponential models. The practical implications for clinical radiotherapy are important.     

Evidence for a pfcrt-associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.     

Chloroquine-induced phospholipid fatty liver. Measurement of drug and lipid concentrations in rat liver lysosomes

A method has been developed to measure the concentration of chloroquine in lysosomes isolated from the liver of rats. It employs 3H2O and [U-14C]sucrose to determine the intralysosomal water volume of purified lysosomes obtained by free flow electrophoresis. Twelve h after a single dose, the concentration of chloroquine in lysosomes was 6.3 mM and at 24 h it rose to 16.5 mM. With continued treatment, lysosomal chloroquine concentrations were 61 and 74 mM at 48 and 72 h. The lysosomal concentrations of chloroquine attained were sufficient to block intralysosomal phospholipase A1 activity. The lysosomal content of phospholipid rises 1.7-fold and 2.6-fold over that of control at 12 and 24 h, respectively. At 72 h, lysosomal phospholipid was 3.7-fold greater than that of control. Lysosomes show an increased negative surface charge with chloroquine administration which is due in part to an increased ratio of acidic to neutral phospholipids in the lysosomal membrane. The phosphatidylinositol content of lysosomes rose rapidly with chloroquine treatment and accounted for the early increase in the ratio. Bis(monoacylglycero)phosphate, an acidic phospholipid synthesized only in lysosomes, increased later in the course of chloroquine treatment and accounted for the continued increase in acidic phospholipids.     

Chloroquine Is a Zinc Ionophore

Chloroquine is an established antimalarial agent that has been recently tested in clinical trials for its anticancer activity. The favorable effect of chloroquine appears to be due to its ability to sensitize cancerous cells to chemotherapy, radiation therapy, and induce apoptosis. The present study investigated the interaction of zinc ions with chloroquine in a human ovarian cancer cell line (A2780). Chloroquine enhanced zinc uptake by A2780 cells in a concentration-dependent manner, as assayed using a fluorescent zinc probe. This enhancement was attenuated by TPEN, a high affinity metal-binding compound, indicating the specificity of the zinc uptake. Furthermore, addition of copper or iron ions had no effect on chloroquine-induced zinc uptake. Fluorescent microscopic examination of intracellular zinc distribution demonstrated that free zinc ions are more concentrated in the lysosomes after addition of chloroquine, which is consistent with previous reports showing that chloroquine inhibits lysosome function. The combination of chloroquine with zinc enhanced chloroquine''s cytotoxicity and induced apoptosis in A2780 cells. Thus chloroquine is a zinc ionophore, a property that may contribute to chloroquine''s anticancer activity.     

Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.

The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.     

Short-term effect of chloroquine on the infectivity of Plasmodium chabaudi gametocytes.

The short-term enhancing effect of chloroquine on gametocyte infectivity was investigated with Plasmodium chabaudi chabaudi, a synchronous parasite which is highly sensitive to chloroquine. In comparison with control groups, oocyst numbers increased in mosquitoes fed on mice 12 hours after the injection of 5 mg/kg chloroquine (180% of controls) although it was not statistically significant. No effect was seen with 1 mg/kg chloroquine. The authors interpretation is that chloroquine impaired the schizogony, thus reducing also the release of toxic material of parasite origin which blocks gametocytes infectivity. Results of similar experiments with other rodent species of Plasmodium are compared and discussed in relation with the chronobiological characteristics of these parasites.     

Chloroquine induces activation of nuclear factor-kappaB and subsequent expression of pro-inflammatory cytokines by human astroglial cells

Chloroquine, an antimalarial lysosomotropic base, is known for its anti-inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti-inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS-induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS-induced expression of pro-inflammatory cytokines such as lymphotoxin (LT)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta and IL-6 in human astroglial cells, while the same treatment suppressed LPS-induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro-inflammatory cytokines in a dose- and time-dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor-kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti- or pro-inflammatory responses in the CNS depending on the cellular context.     

Plasmodium chabaudi: in vivo effects of Ca2+ antagonists on chloroquine-resistant and chloroquine-sensitive parasites

The effects of Ca2+ antagonists, verapamil, nicardipine, and diltiazem, on susceptibility to chloroquine were examined in mice infected with chloroquine-sensitive and chloroquine-resistant lines of Plasmodium chabaudi. In mice that received no chloroquine, daily injections of 50 mg/kg of verapamil, nicardipine, or diltiazem did not affect the growth of both sensitive and resistant parasites. When mice were injected daily with verapamil plus 2 to 3 mg/kg chloroquine, the chloroquine-sensitive parasite became more susceptible to chloroquine than the parasite in mice given chloroquine alone. On the other hand, in mice infected with chloroquine-resistant parasites, verapamil severely suppressed the growth of the parasite when accompanied by daily injections of 2 to 3 mg/kg of chloroquine, at which doses resistant parasites grew steadily in the absence of verapamil, indicating reversal of chloroquine resistance. This reversal was dose-dependent between 5 and 50 mg/kg of verapamil. Daily injections of nicardipine or diltiazem at 50 mg/kg also reversed resistance to chloroquine in resistant parasites. These results indicate that Ca2+ antagonists increase the susceptibility to chloroquine in a sensitive line of P. chabaudi and reverse chloroquine resistance in a resistant line.     

Malaria chemoprophylaxis in travellers to east Africa: a comparative prospective study of chloroquine plus proguanil with chloroquine plus sulfadoxine-pyrimethamine

As malaria caused by Plasmodium falciparum has become resistant to chloroquine alternative drug regimens need to be developed. The prophylactic efficacy against malaria and the side effects of chloroquine phosphate 500 mg weekly with proguanil hydrochloride 200 mg daily was compared with the efficacy of chloroquine 500 mg weekly with sulfadoxine 500 mg-pyrimethamine 25 mg weekly in a randomised study of Scandinavian travellers to Kenya and Tanzania during 1984-5. A total of 767 subjects (416 male and 351 female; 384 taking chloroquine phosphate with proguanil hydrochloride and 383 taking chloroquine with sulfadoxine-pyrimethamine) completed a diary on the breakthrough of malaria and the side effects of treatment while taking the drugs. They were also asked to make thick blood films when symptoms like those of malaria occurred, which were sent to and analysed in Denmark. Four subjects taking chloroquine with proguanil hydrochloride and three taking chloroquine with sulfadoxine-pyrimethamine developed falciparum malaria, which was verified microscopically. Side effects were reported by 36 subjects taking chloroquine phosphate with proguanil hydrochloride and 55 taking the other regimen (p=0·043). The side effects of both regimens were generally mild, but the combination of chloroquine phosphate with proguanil hydrochloride is recommended because it results in fewer side effects.As breakthroughs of malaria occurred at the earliest after seven weeks self treatment should not be recommended for travellers staying only a short time. Thick blood films are useful for diagnosis of suspected cases of malaria, can be prepared by non-specialists in Africa, and can be analysed successfully after long delays.     

Transplasma membrane electron and proton transport is inhibited by chloroquine

Chloroquine is a weak base which has been shown to inhibit lysosomal acidification. Chloroquine inhibits iron uptake in reticulocytes at a concentration of 0.5 mM. It is also effective in the control of malaria and other parasitic diseases. We now report that chloroquine inhibits NADH diferric transferrin reductase as well as the proton release stimulated by diferric transferrin from liver and HeLa cells. Ammonium chloride which also inhibits endosome acidification does not significantly inhibit the NADH diferric transferrin reduction. NADH diferric transferrin reductase of isolated rat liver plasma membrane is inhibited by chloroquine at concentrations similar to those required for inhibition of diferric transferrin reduction by whole cells. Ferricyanide reduction by whole cells is also inhibited by chloroquine. These observations provide an alternative mechanism for chloroquine control of acidification of endosomes and suggests a new approach to control of protozoal parasites through inhibition of a transmembrane oxidoreductase which controls transmembrane proton movement.     

Methylene blue as an antimalarial agent

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

Chloroquine accumulation by erythrocytes: a latent capability

Treatment of normal mouse erythrocytes with a nonspecific protease from streptomyces griseus activates a saturable process for chloroquine accumulation. The apparent dissociation constant for the interaction of chloroquine with the saturable component of this latent process is 50 nM. This value is similar to that of a high-affinity process of chloroquine accumulation which hitherto has been observed only in erythrocytes infected with malaria parasites. In addition, the capacity of the latent process, approximately 30 μmoles per Kg of erythrocytes, is sufficient to account for the accumulation of chloroquine by erythrocytes infected with malaria parasites. These findings demonstrate the plausibility of attributing the ability to accumulate chloroquine with high affinity to the erythrocyte host rather than to the parasite.     

Evolutionary paradigm of chloroquine-resistant malaria in India

Drug pressure in the field is believed to be responsible for the emergence of drug-resistant Plasmodium falciparum, the parasite that causes malaria. Variants of the P. falciparum chloroquine resistance transporter (pfcrt) gene have been shown to be responsible for conferring resistance to the commonly used drug chloroquine. In particular, an amino acid mutation, K76T, was shown to have a strong positive correlation with the chloroquine-resistant varieties of malaria parasites. Global studies have reported highly reduced genetic diversity surrounding K76T in the pfcrt gene, which indicates that the mutation has been a target of positive Darwinian natural selection. However, two recent studies of P. falciparum in India found high genetic diversity in the pfcrt gene, which, at first sight, do not support the role of natural selection in the evolution of chloroquine resistance in India.