Some roles of free radicals in malaria

Free Radical Biology & Medicine, Vol. 6, pp. 315-321, 1989 Printed in the USA. All rights reserved.

0891-5849/89 $3.00 + .00 © 1989 Pergamon Press plc

Review Article SOME

ROLES

I. A.

OF FREE

CLARK,*tG.

RADICALS

CHAUDHRI,

IN MALARIA

and W. B. COWDEN

*Zoology Department and John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia (Received 4 December 1987; Revised and accepted 3 May 1988)

Abstract--Malaria parasites are very vulnerable to oxidant stress during the part of their life cycle when they inhabit erythrocytes. As the infection progresses they also activate macrophages, one consequence of which is extracellular release of reactive oxygen species. For these reasons free radicals are frequently discussed in the literature on antimalarial drugs, malarial immunity, and disease pathogenesis. They are also central to arguments explaining how the genetic mutations that lead to sickle cell disease, thalassemia and glucose-6-phosphate dehydrogenase have become so common in tropical regions. This review summarizes how these links between free radicals and this disease came to be understood, and the present state of the field. Keywords--Free radicals, Malaria, Gene selection, Tumor necrosis factor

quences for the parasite, and for the host, as discussed in this review. A quick synopsis of malaria is warranted here. The disease is caused by protozoan parasites belonging to the genus Plasmodium, four species of which infect human beings. These are P. falciparum (which is the chief concern, and often fatal), P. vivax, P. malariae, and P. ovale. Other mammals, and also reptiles and birds, have their own distinct malarial parasites, and all are transmitted by mosquitoes, again with distinct host specificity patterns. Malaria parasites have complex life cycles in both the mosquito and vertebrate hosts. In brief, both male and female gametocytes are ingested along with the blood meal of the female mosquito, and sexual reproduction occurs in its stomach. The zygote migrates through the stomach wall, and while attached to its outer surface forms a cyst, inside which it undergoes asexual division to form large numbers of sporozoites. These migrate to the salivary glands, and are injected when a blood meal is taken. Asexual multiplication occurs twice in the vertebrate host, once in the liver and once in circulating red cells. Most workers, including those concerned with the relationships between free radicals and the malaria parasite, have concentrated on the red cell stage of the life cycle, since the forms in this stage are most easily accessible and are the only forms associated with host illness.

INTRODUCTION

Of all the infectious agents, malaria parasites have the most intensely examined relationship with free radicals. This close association is inevitable, given this parasite's direct involvement with red cells and its capacity to activate the host's leukocytes, inducing them to release various mediators, including reactive forms of oxygen. These processes have important conse-

Ian Clark graduated in veterinary science from the University of Queensland, and received his PhD from the Institute of Basic Medical Sciences, University of London, under Professor J. Turk. For five years from 1972 he held a research position at the MRC Clinical Research Centre, Harrow. Since returning to Australia he has continued his research, mainly on mechanisms of cell-mediated immunity and pathology of infectious disease, as a Research Fellow at the Australian National University. Geeta Chaudhri obtained her BSc (Honours in Biochemistry) from the Australian National University in 1984 and her PhD from the John Curtin School for Medical Research, Australian National University in 1987. She is currently working as a Postdoctoral Fellow with lan Clark on cell-mediated immunity and pathology of infectious disease. William Cowden obtained his BSc in the USA and his PhD from the University of Queensland in 1979. Since then he has been a Research Fellow, first in the Medical Chemistry Group and later in the Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University. His current research interest is free radical chemistry, particularly in relation to pathological processes. tAuthor to whom correspondence should be addressed.

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I.A. CLARK,G. CHAUDHR1,and W. B. COWDEN

Susceptibility of intraerythrocytic malarial parasites to oxidant stress It took decades to develop the technique of continuous culture of cell forms of malarial parasites, largely because it was not realized how little oxygen they would tolerate in vitro. ~Closer study showed that 2 1% oxygen was deleterious, that best growth occurred at 3% oxygen, and that cultures survived well in oxygen concentrations as low as 0.5% provided that carbon dioxide did not exceed 2%. 2 Older in vivo studies had found that phenylhydrazine 3'4 and alloxan, 4 (agents not then known, but subsequently shown to generate reactive oxygen species in vivo) would inhibit P. knowlesi 3 and P. berghei 4 infections. Within the past five years it has been shown that injection of alloxan, 5 tert-butyl hydroperoxide6 or divicine, 7 all known radical generators, causes P. vinckei to disintegrate inside the circulating red cells of infected mice. Since pretreatment with either free radical scavengers or iron chelators protected the parasites, it was proposed that some ironcatalyzed reduction product of hydrogen peroxide, perhaps hydroxyl radical, was involved. Others have studied the phenomenon in vitro, using such radical generating systems as hydrogen peroxide, 8 xanthine plus xanthine oxidase, 9 and glucose plus oxidase 1~ against either P. yoelii or P. falciparum. More recent studies have suggested that aldehydes from oxidized lipids could be what actually kills the parasites, since a series of these agents that are toxic to malaria parasites 11 are generated under these conditions.12 Widespread resistance to chloroquine has generated a pressing need for new approaches to antimalarial drugs, and efforts are being made to harness the susceptibility of malarial parasites to oxidant stress for this purpose. Accordingly, we have synthesized and are testing a range of 5-hydroxypyrimidines,13 and have reasoned that qinghaosu (artemisinine), a sesquiterpine lactone endoperoxide, could act through generating oxidant stress.~3 Recent studies 14 are consistent with this possibility. There is also appreciable evidence that implicates radical-induced oxidant stress in the mechanism of dapsone 15 and primaquine, 16,17two well-established antimalarial drugs.

How sensitivity of malarial parasites to oxidant stress has shaped the human genome Normal red cells are by no means an ideal home for parasites susceptible to oxidant stress, since the free radical flux to which these cells are exposed is finely balanced by their antioxidant capacity. This flux is inevitably high, since oxygen radicals are formed continuously through hemoglobin autooxidation.18.J9 Lacking both mitochondria and a nucleus, these cells

can neither metabolize oxygen along the cytochrome pathway (thus avoiding toxic intermediate forms) nor synthesize proteins and thus replenish antioxidant enzymes. Furthermore, red cells are laden with iron, and regularly pass through the pulmonary alveoli, where oxygen tension is highest. This area has been well reviewed earlier by Carrel et al. 2° and, more recently, by Hebbell. 2~ Details of how oxygen radicals damage the various components of red cells have not yet been resolved. This is exemplified by the recent studies of Davies and Goldberg, 22 who showed that proteolysis is a much more sensitive indicator of exposure of red cells to oxidant stress than is lipid peroxidation. They also found that these two types of damage, although both caused by radicals, are separately controlled. Whether these relationships hold true in parasitized red cells, and in the parasites themselves, has yet to be examined. The problems this creates for malarial parasites is exacerbated in individuals whose red cells carry genetically-determined abnormalities that increase oxidant stress on the red cell and its contents. One would expect genetic abnormalities that produce aberrant hemoglobins and deficiencies in such key enzymes as glucose-6-phosphate dehydrogenase to be rare, but in tropical areas they can be very common indeed. P. falciparum evidently is at some disadvantage in these altered red cells, giving the genetically abnormal host a survival advantage. This has provided the opportunity for a profound degree of natural selection of human genes in malarial areas. The historic concept that infectious disease could be an important evolutionary agent was first proposed, in general terms, by J. B. S. Haldane, 23 and in the same year he suggested that P. falciparum had increased the incidence of thalassemia in this way. 24 He argued that in terms of gene survival the loss of homozygotes before breeding age might be more than compensated for by increased survival, in the face of falciparum malaria, of heterozygotes compared to normal individuals. Thus malaria could increase gene frequency in a population. This concept was first developed with sickle cell disease, 25'26 and, as reviewed by Livingstone 27 and Luzzatto, 28 now extends to a series of gene mutations that are common in the tropical world but rare, except in immigrants, elsewhere. Some examples, summarizing the evidence for free radical involvement, are given below.

(a) Sickle cell disease. The root cause of this condition is an abnormal form of hemoglobin (HbS), which, after inheritance in a simple Mendelian fashion, comprises all of the hemoglobin of individuals homozygous for the gene, and about two-fifths of the hemoglobin of heteroxygous carriers. 29 This abnormal form of hemo-

Malaria and free radicals

globin evidently places an increased oxidant stress on red cells, since there is an increased flux of radical formation 3° and an accelerated oxidation of membrane protein thiols 31 in red cells containing HbS. It has been argued that iron decompartmentalization, as evidenced by the membranes of affected cells containing abnormally high amounts of heme, 32 is the essential cause of these changes. 33 As noted above, malaria parasites are very susceptible to increases in oxidant stress, and the changes described here 33 could contribute to their poor growth in red cells containing HbS. 34 Orjih et al. have produced evidence consistent with the concept that parasites in such red cells are damaged by oxidatively-denatured hemoglobin. 35

(b) Thalassemia. This term is used to describe a complex family of hemoglobinopathies in which formation of either the o~ or 13 globin chains (hence a or 13 thalassemia) of the hemoglobin molecule is defective. The range of possible defects is large,36.37 and the sequelae for the affected individual can be complex, but the net result, in the context of disease-induced gene selection, is that affected red cells do not support the growth of P. falciparum as well as do normal erythrocytes. 38,39 Large quantities of ferritin-like iron, probably from hemoglobin denaturation, have been reported in red cells with various forms of et and 13 thalassemia, 4° and superoxide is generated from autoxidation of isolated and 13 chains of human h e m o g l o b i n f These parts of the hemoglobin molecule are in relative excess in 13 and a thalassemia respectively. Thus, given the evidence that oxidatively-stressed ferritin can release iron that can catalyze formation of very reactive radical species,42 the stage is evidently set for oxidative damage to malaria parasites. Rice-Evans has recently reviewed the iron-driven oxidative changes that occur in thalassemic red cells. 43 Homozygous 13 thalassaemia is such a severely hemolytic condition (Cooley's anemia) that regular blood transfusions are necessary to sustain life. As in any similar circumstance this eventually leads to transfusion siderosis, since the iron released during continuous haemolysis inevitably accumulates, despite treatment with desferrioxamine. Systemic radical-induced tissue damage occurs, its distribution being determined by the pattern of deposition of the transition metal and local antioxidant defenses. (c) Glucose-6-phosphate dehydrogenase deficiency. Human G-6-PD deficiency encompasses a complex series of genetic changes, and some hundreds of distinct mutations that have led to this condition have been described. 44 In each case the functional defect is the s a m e - - a poor capacity to keep glutathione in its re-

317

duced form, with all the consequences this implies for oxidant balance, particularly in red cells. Thus, after treatment with the thiol-oxidizing agent diamide, G-6PD deficient red cells are much slower than are normal erythrocytes to regain normal membrane thiol status, and the growth of any resident P. falciparum parasites is correspondingly inhibited. 45 The first link between G-6-PD deficiency and malaria was indirect, and intriguing, having to do with the introduction in the 1920s of the 8-aminoquinoline antimalarial drug, pamaquine and later its less toxic relative, primaquine. Both were hemolytic in a proportion of Asians, but not in North Europeans, and red cells from susceptible individuals proved to be deficient in G-6-PD. Hence primaquine sensitivity became G-6-PD deficiency. 46'47 The studies of Eaton and Eckman 48 demonstrated that malaria parasites exert oxidant stress upon the host red cell, implying that G-6-PD deficient red cells are liable to lyse before the parasite could attain maturity. Nevertheless direct evidence that malaria has been a strong enough pressure to select these otherwise disadvantageous mutations, such that they are carried by tens of millions of people in the warmer, but not temperate zones, has been slow in coming. The data on geographical correlation with malaria is conflicting, 49'5° and G-6-PD deficient individuals have not always proved more resistant to P. falciparum than those with normal G-6-PD activity.5~ The correlation appears to be much better if dietary oxidant stress is taken into account. This story is most clearcut in the Mediterranean and Middle East countries, where G-6-PD deficiency is typically more severe and common than in tropical Africa, and where Vicia faba (the fava bean) is a staple foodstuff. These beans contain two 13-glucosides, vicine and convicine, which are hydrolyzed in the gut to form their unstable pyrimidine aglycones, divicine, and isouramil. It has been known for some decades that the oxidized forms of these compounds readily deplete red cells of reduced glutathione, 52 and that G-6-PD-deficient erythrocytes, with their poor capacity to recycle glutathione, are easily lysed in their presence? 3 More recently it was shown that these aglycones generate hydrogen peroxide, 54 and that divicine autoxidation can proceed along a variety of pathways, some involving radical species. 55 On the face of it, fatal hemolysis after consumption of fava beans (favism) should have lowered the frequency of G-6-PD deficiency genes in the very area where they are most common. Huheey and Martin 5° rationalized this paradox by proposing that fava beans have antimalarial properties that operated best in G-6PD deficient individuals. Thus, they reasoned, fava bean ingestion plus malaria, not malaria alone, has selected for the higher incidences of G-6-PD deficiency

318

I.A. CLARK,G. CHAUDHRI,and W. B. COWDEN

in people from Mediterranean regions. Experimental evidence consistent with these ideas has now emerged: amounts of isouramil that inhibit P. falciparum growth in G-6-PD-deficient erythrocytes in vitro will not affect these parasites when they are in normal red cells, 57 and divicine indeed has in vivo antimalarial activity. 7 Both desferrioxamine and butylated hydroxyanisole prevented hemolysis and parasite death in these in vivo studies, 7 so it appears that a radical species formed on transition metal-induced reduction of hydrogen peroxide may be involved. Thus dietary oxidant stress, plus the historic prevalence of P. falciparum, may, as suggested by Huheey and Martin, account for the high severity and frequency of G-6-PD in Mediterranean countries. Other agents with properties similar to fava beans may have operated, in conjunction with malaria, elsewhere, and helped determine the uneven distribution of human G-6-PD deficiency genes across the wet tropics.

an array of endogenous antioxidants, all red cells, parasitized and unparasitized alike, appear to be under oxidant stress during this infection. 67 Since Friedman had reported that malaria parasites inside thalassemic or G-6-PD deficient red cells suffered more than those in normal erythrocytes,68 we have argued 5 that oxidant stress of leukocytic origin could accentuate the antimalarial effects of these genetic defects, to the survival advantage of the genes that control them. One of the agents that can prime leukocytes for the release of reactive oxygen species is tumor necrosis factor (TNF), 69,7° a monokine that we proposed, some years ago, 58.59 could be central to how macrophages can kill malaria parasites inside circulating red cells. TNF will not kill malaria parasites in vitro,71'72 but will in vivo, 71,65 so some host-mediated step evidently occurs. Whether this involves free radicals has not yet formally been shown.

Free radicals and disease pathogenesis in malaria Influence of free radicals of leukocyte origin on malaria parasites Reports that malaria parasites die inside circulating erythrocytes during the immune response had no conventional explanation, so we proposed that release of soluble factors from macrophages were involved. 58,59 This raised the possibility that damage to these parasites, as with intramacrophage protozoa, might be contributed to by reactive forms of oxygen. In the hope of simulating this effect we injected alloxan 5 or tertbutyl hydroperoxide,6 both of which induced rapid disintegration of P. vinckei inside circulating erythrocytes. As noted earlier, other sources of reactive oxygen species have been studied in vitro in this context. 8-1° Ockenhouse and Shear 6° studied this possibility directly, using macrophages and malaria parasites in vitro. They found that parasites separated from macrophages by a filter were susceptible to H202 released by lymphokine-activated macrophages incubated with phorbol myristate acetate, opsonized zymosan or P. yoelii antigen. Catalase prevented parasite inhibition. Various groups have now tied in vitro suppression of malaria parasites to secretion of reactive forms of oxygen by both macrophages 6~'62and neutrophils, 63,64and recently we have reported enhancement of P. chabaudi (a parasite that dies inside red cells during the immune response) by feeding infected mice a diet rich in butylated hydroxyanisole. 6s The antimalarial properties of desferrioxamine and other iron chelators preclude their use in this type of experiment. Additionally, luminol-reactive chemiluminescence from blood leukocytes increases dramatically during P. vinckei infection, 66 and, as evidenced by changes in

Malaria is not a simple disease to understand; somehow a parasite, often so rare (in human malaria) at onset of illness that it can be difficult to find, causes systemic disease that affects many tissues other than the red cells in which the parasites dwell. We have proposed 58,59 that the syndrome is best explained by malarial antigens (which are released in regular showers into the plasma as an inevitable part of the parasite's life cycle) triggering macrophages to release a range of soluble mediators. TNF has been central to these arguments, 73-75 but involvement of free oxygen radicals warrants serious consideration, since macrophages 68 and neutrophils 69 are sensitized by TNF to agents that induce these cells to generate and release superoxide. We have recently reported that iron chelators and phenolic radical scavengers will inhibit, and low concentrations of hydrogen peroxide or sodium periodate increase, TNF release from macrophages. 76 Thus oxidant stress may amplify TNF-induced pathology, part of which, in turn, appears to be mediated by reactive oxygen species. These concepts make it possible to explain why TNF and free radicals offer parallel explanations for important aspects of malarial pathology. It is possible, for example, to prevent the cerebral pathology ofP. berghei malaria with ethoxyquin, 77butylated hydroxyanisole77or antibody to TNF, 78and both TNF 79 and vitamin E deficiency8°-82 will cause dyserythropoiesis and erythrophagocytosis, two important contributors to human malarial anemia. 83 In addition, pulmonary accumulation of intravascular neutrophils, which occurs as part of the pulmonary oedema of falciparum malaria 84 and has a well-established niche in the free radical literature on endothelial damage, 85 can

Malaria and free radicals

be induced by injecting TNF into experimental animals. 86,73 There is no reason to believe that this cooperative activity between oxidant stress and monokines is restricted to TNF; interleukin-1, which shares many functions with TNF (reviewed in ref. 87) is a likely candidate for such cooperation. Understanding these interactions should provide useful extensions of our knowledge of free radical-induced pathology.

16. 17. 18. 19. 20.

Acknowledgements--This study was supported by the malaria component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Disease and the National Health and Medical Research Council of Australia. We wish to thank Elizabeth Jackson for kindly typing the manuscript.

21. 22.

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