Reactive oxygen intermediates metabolizing enzymes in ancylostoma ceylanicum and nippostrongylus brasiliensis

FreeRadicalBiology& Medicine,Vol. 8, pp. 271-274, 1990 Printed in the USA.All rightsreserved.

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Brief Communication REACTIVE OXYGEN INTERMEDIATES METABOLIZING ENZYMES IN ANCYLOSTOMA

CEYLANICUM

AND

NIPPOSTRONGYLUS

BRASILIENSIS

SANJAY BATRA, S. P. S1NGH, S. GUPTA,* J. C. KATIYAR,* and V. M. L. SRIVASTAVA~ Divisions of Biochemistry and Parasitology,* Central Drug Research Institute, Lucknow226001, India

(Received 20 March 1989;Accepted 22 November 1989) Abstract--Adult worms of Ancylostoma ceylanicum and Nippostrongylus brasiliensis were found to possess an

active system for the detoxification of reactive oxygen intermediates. Xanthine oxidase, which is known to produce superoxide anion, was detected in both the nematode parasites in significant activities. Superoxide anion, thus produced, may quickly be eliminated by superoxide dismutase. Both parasites also exhibited the presence of catalase, peroxidase, and glutathione peroxidase for efficient removal of hydrogen peroxide. Glutathione reductase and glucose-6-phosphate dehydrogenase were, however, detected in low levels of activities. Endowment of A. ceylanicum and N. brasiliensis with these antioxidant enzymes, therefore, enables them to evade the host's effector mechanism for their survival. Superoxide dismutase of both these nematodes showed marked inhibition by KCN and, hence, the enzyme appears to be of copper-zinc type. Keywords--A. ceylanicum, N. brasiliensis, Xanthine oxidase, Superoxide dismutase, Catalase, Glutathione per-

oxidase, Free radicals

tinal tract as well as during aerobic incubation in vitro for a considerable period. Hence, they must be endowed with a mechanism to detoxify reactive oxygen intermediates (ROI). With this view in mind, a study of ROI metabolism in A. ceylanicum and N. brasiliensis was undertaken. This communication deals with the presence of antioxidant defense enzymes in the two nematode parasites named above.

INTRODUCTION

The ubiquity and high reactivity of oxygen reduction products have forced a choice on all living organisms that they must either evolve a system to deal with these toxic products or find some anaerobic abode. Intestinal tract, which is considered one of the safest niches for helminth parasites, is not completely devoid of oxygen. The intestinal tissue contains xanthine oxidase and superoxide dismutase, which are known to generate superoxide anion (O2-) and hydrogen peroxide (H200, respectively, the two highly toxic intermediates of oxygen metabolism. ~- 3Furthermore, the mitochondria of a few helminths, namely, Hymenolepis diminuta 4 and Setaria cervi, 5 have also been reported to produce H202. Ancylostoma ceylanicum and Nippostrongylus brasiliensis use significant amounts of oxygen and possess a modifiedrespiratory chain, e According to Kohler, 7 a respiratory component in Ascaris suum which is insensitive to antimycin-A and cyanide, terminates in H202 production. By virtue of this property, A. ceylanicum and N. brasiliensis should also produce H202. These parasites, however, survive in the gastrointes-

MATERIALS AND METHODS

Parasites Male hamsters (Mesocricetus auratus, 6 0 - 8 0 g) were infected by administering orally 70 -+ 5 infective larvae of A. ceylanicum, while rats (Druckrey, 4 0 - 6 0 g) were subcutaneously inoculated with 2000 +- 100 infective larvae of N. brasiliensis. Adult worms were obtained from the intestines of infected animals after patency, that is, on days 18 and 9, respectively.

Preparation of homogenate The worms were washed in normal saline and homogenized (5% w/v) in isotonic KC1. The homogenate was centrifuged at 900 g for 10 min. The supernate was further centrifuged at 9000 g for 30 min followed

tAuthor to whom correspondence should be addressed. 271

S. BATRA et al.

272

by recentrifugation at 105,000 g for 60 min. The mitochondrial and microsomal pallets were suspended in KCI. The microsomal fraction was sonicated for 4 x 30 s with 60-s successive cooling intervals, employing a Heat Systems-cell disruptor, Model W220F. The sonicated microsomes were centrifuged at 9000 g for 30 min to remove the membranous fraction.

Enzyme activities The cytosol fraction was assayed spectrophotometrically for xanthine oxidase, 8 superoxide dismutase, 9 catalase, ~° glutathione peroxidase, 1~ glutathione reductase, ~2 and glucose-6-phosphate dehydrogenase. t3 Lipid peroxidation in the mitochondrial fraction was determined according to Henry et al.,~4 while peroxidase activity in the supernate fraction of the disrupted microsomes was measured according to Bergmeyer and Bernt.~5 Protein content was determined colorimetrically. J6

Chemicals and reagents Xanthine, NADP, hydrogen peroxide, glucose-6phosphate, and glutathiones (oxidized and reduced) were the products of SISCO Research Laboratories, Bombay. Bovine serum albumin, NADPH, cytochrome-c (oxidized), glutathione reductase, and catalase were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Epinephrine and o-dianisidine were purchased from Romali and V. P. Chest Institute, Delhi, respectively, while thiobarbituric acid was procured from Loba Chemie, Bombay. RESULTS

Data summarized in Table 1 indicate that both A. ceylanicum and N. brasiliensis possess all the enzymes

Table 1. Enzymes Mediating the Metabolism of Reactive Oxygen Intermediates in A. ceylanicum and N. brasiliensis Enzymes

A. ceylanicum

N. brasiliensis

Xanthine oxidase* Superoxide dismutaset Catalase* Peroxidase* Lipid peroxidation Glutathione peroxidase* Glucose-6-phosphate dehydrogenase* Glutathione reductase*

0.703 -+ 0.039 1.064 -+ 0.123 0.794 + 0.055 0.842 ± 0.052 Traces 0.249 -+ 0.027 0.011 ± 0.003

0.257 - 0.025 0.393 -+ 0.017 0.879 --- 0.039 53.232 - 5.441 Traces 1.453 - 0.208 0.016 ± 0.002

0.027 ± 0.005

0.025 ± 0.004

*lt mol/min/mg protein. tOne enzyme unit is that amount of protein that inhibits the reduction of Nitroblue tetrazolium by 50%. Data are mean -+ SE of three experiments.

required for the formation and elimination of O2- and described in Figure 1. Xanthine oxidase, which is one of the several enzymes leading to the production of O2-, was present in greater amounts in A. ceylanicum than in N. brasiliensis. Superoxide dismutase, the enzyme responsible for the conversion of O2- into H 2 0 2 , exhibited a similar pattern. Catalase, glutathione peroxidase, and peroxidase, involved in the detoxification of H 2 0 2 , w e r e also detected in significant amounts. The very high activity of peroxidase in N. brasiliensis is very surprising. In contrast to the above enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase showed low levels of activities. Lipid peroxidation, which is represented by the total of enzymatic and nonenzymatic formation of malondialdehyde, proceeded at extremely low rate. Superoxide dismutase of both the nematodes showed marked sensitivity to cyanide (Table 2). At 0.67 mM concentration, KCN inhibited the enzyme by 50%, while almost complete inhibition was recorded at 2 mM concentration. These data suggest for the presence of a Cu-Zn type of dismutase in these parasites. H202

DISCUSSION

Processes induced by reactive oxygen intermediates, 02- and H202 in particular, are now appreciated to contribute significantly to the tissue damage in a variety of pathological conditions.~7'18 These intermediates are also known to kill a diverse array of infectious agents ~9 and thereby to represent a potential effector mechanism of host against invaders. Recent studies with hemoflagellates suggest that the parasites which are able to detoxify these intermediates can survive in the host, whereas those which lack this capacity are unable to survive. 2°'2' This rule applies to helminth parasites, also. For instance, adult worms and muscle stage larvae of Trichinella spiralis compared to newly hatched larvae contain five times higher activities of superoxide dismutase and glutathione peroxidase, and, hence, the former stages are more tolerant to H202. 22 On the other hand, Moniezia expansa and H. diminuta, which lack both catalase and glutathione peroxidase, appear to eliminate H202 by cytochrome-c dependent peroxidase. 23 Data of the present study clearly indicate that both A. ceylanicum and N. brasiliensis can effectively produce and also detoxify 02- and H202. Catalase and peroxide in these parasites appear to be the major defense enzyme against H202 while glutathione peroxidase seems to be of lesser importance. The capacity of the latter enzyme to detoxify H202 may be restricted further due to the limited regeneration of reduced glu-

02 metabolism in intestinal helminths

I XANTHINE-~-. "URATE ,~ ~ 2 OR 02 HYPOXANTHINE

0 2 ~

>U202

273

3

•OH

2

H20 + ~_02

D~AGETO LIPIDSAND PROTEINS GSH/

N ¢kgP >

GSS

NADP

CELL TOXICITY H20 Fig. 1. Antioxidant defense mechanism coupled with the metabolism of toxicants. 1, Xanthine oxidase; 2, superoxide dismutase; 3, peroxidase; 4, catalase; 5, glutathione peroxidase; 6, glutathione reductase; 7, glucose-6-phosphate dehydrogenase; GSH, glutathione reduced; GSSG, glutathione oxidized; A, acceptor.

tathione by poorly active glutathione reductase and glucose-6-phosphate dehydrogenase. Comprehensive investigations on N. brasiliensis in rats suggest that a combination of direct antibody-mediated damage and a subsequent cell-mediated event is responsible for the expulsion of adult worm, with a nonimmunoglobin-bearing lymphocyte involved in the final stages. 24-26According to Mackenzie, 27neutrophils adhere through small areas of membrane flattening, at which nitroblue tetrazolium reaction can be observed. This indicates for the presence of some reactive oxygen intermediate, possibly of O2-, at the surface. However, no severe damage to the parasite'may be expected because of the quick elimination of the superoxide anion by active scavenging system (Table 1). Likewise, A. ceylanicum may avoid the external and internal risks by its antioxidant defense machinery. N. brasiliensis, compared to A. ceylanicum, contains only one-third activity of xanthine oxidase, but at the same time, it has about 63-fold higher activity of peroxidase. Since there is no appreciable difference in the rate of oxygen consumption (8.48 --- 1.35 and 11.89 + 1.37 n atom/mg wet weight/min forN. braTable 2. Effect of Potassium Cyanide on Superoxide Dismutase Activity Inhibition (%) Concentration (mM)

N. brasiliensis

A. ceylanicum

0.33 0.67 1.00 2.00

23.4 53.1 84.4 96.3

nd 49.8 81.7 92.5

nd = not done.

siliensis and A. ceylanicum, respectively),6 no definite explanation can be offered to the significantly high activity of peroxidase in N. brasiliensis. However, it certainly indicates for the greater resistance ofN. brasiliensis to peroxide (H202). Most interesting, during aerobic incubation in vitro, N. brasiliensis can thrive well for more than 5 days, while about 50% A. ceylanicum become sluggish and lose motility on the third day (Singh, S. P.; Katiyar, J. C.; Srivastava, V. M. L.: Unpublished data). This indicates that N. brasiliensis can tolerate more oxygen than A. ceylanicum. It is pertinent to add that the relative activity of xanthine oxidase in the two parasites may not be linked directly with their rates of 02- production since, in addition, this radical may be generated by several other means. It may therefore be inferred that both A. ceylanicum and N. brasiliensis possess an active antioxidant enzyme system, which confers upon them the capacity to tolerate the host's effector mechanism for survival in the host. The results of the present study are also in good accord with the general conclusion made by Butterworth 28 that nematode parasites differ from mammalian cells, bacteria, and protozoa in respect of their marked resistance against conventional oxidative killing mechanisms, especially those dependent on the production of H202.

Note: As this manuscript was completed, A. ceylanicum and N. brasili~nsis were found to release superoxide dismutase, catalase, and glutathione peroxidase into the ambient medium at a rate of 4.2, 1.3, 0.05, and 1.2, 0.63 & 0.03 units/h/g, respectively at 37°C. This i,, important because, the released enzymes may protect the parasite,, against reactive oxygen intermediates present in their immediate surroundings.

274

S. BATRAet al.

Acknowledgement--Two of the authors, Sanjay Batra, and S. P. Singh are grateful to Indian Council of Medical Research, New Delhi, and Council of Scientific and Industrial Research, New Delhi, respectively, for the award of Junior Research Fellowship.

REFERENCES 1. Connock, M.; Pover, W. E. R. Catalase particles in the epithelial cells of the guinea pig small intestine. Histochem. J. 2 (5): 371380; 1970. 2. Riario-sforza, G.; Carcassi, A.; Bayeli, P. F.; Marcolonga, R.; Marinello, E.; Montagnani, M. Xanthine oxidase in the mucosa of fasted subjects with gout. Bull. Soc. Ital. Biol. Sper. 45 (12): 785-786; 1969. 3. Singh, S. P.; Batra, S.; Gupta, S.; Katiyar, J. C.; Srivastava, V. M. L. Effect of Ancylostoma ceylanicum infection in hamsters on enzymes that metabolize reactive oxygen intermediates. Med. Sci. Res. 17 (11): 493-495; 1989. 4. Fioravanti, C. F. Mitochondrial NADH oxidase activity of adult Hymenolepis diminuta (Cestoda). Comp. Biochem. Physiol. 72B: 591--596; 1982. 5. Agarwal, N.; Srivastava, V. M. L. Mitochondrial NADH oxidase activity of Setaria cervi. Vet. Parasitol. (Communicated). 6. Agarwal, N.; Srivastava, V. M. L.; Gupta, S.; Katiyar, J. C. Respiratory electron transport in Ancylostoma ceylanicum and Nippostrongylus brasiliensis. Ind. J. Exptl. Biol. 26: 724--727; 1988. 7. Kohler, P. The strategies of energy conservation in Helminths. Mol. Biochem. Parasitol. 17: 1-18; 1985. 8. Horecker, B. L.; Heppel, A. L. The reduction of cytochromec by xanthine oxidase. J. Biol. Chem. 178: 683-690; 1949. 9. Beauchamp, C.; Fridovich, I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analyt. Biochem. 44: 276-287; 1971. 10. Aebi, H. Catalase in vitro. In: Packer, L., ed. Methods in enzymology. Vol. 105. New York: Academic Press; 1984: 121126. 11. Leopold, F.; Wolfgang, A. G. Assays of Glutathione peroxidase. In: Packer, L., ed. Methods in enzymology. Vol. 105. New York: Academic Press; 1984:114-121. 12. Racker, E. Glutathione reductase from Baker's yeast beef liver. J. Biol. Chem. 217: 855-865; 1955. 13. Shonk, C. E.; Boxer, G. E. Enzyme patterns in human tissues. I. Methods for the determination of glycolytic enzymes. Cancer Res. 24: 709-721; 1964. 14. Utley, G. G.; Bernheim, F.; Hochstein, P. Effect of sulphydryl

reagents on peroxidation in microsomes. Arch. Biochem. Biophys. 118: 29-32; 1967. 15. Bergmeyer, H. U.; Bernt, E. D-Glucose: determination with glucose oxidase and peroxidase. In: Bergmeyer, H. U., ed. Methods in enzymatic analysis. Weinheim: Buchdruckerei 1963: 123-126. 16. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-276; 1951. 17. Clark, I. A.; Mackie, E. J.; Cowden, W. B. Injection of free radical generators causes premature onset of tissue damage in malaria infected mice. J. Pathol. 148: 301-305; 1986. 18. Clark, I. A.; Chowdhri, G.; Cowden, W. B. Interplay of RO species and TNF in tissue injury. UCLA. Sym. Mol. Cell. Biol. New Ser. (oxy-Radicals Mol. Biol. Path.). 82:53-60 (Eng); 1988. 19. Klebanoff, S. J. Oxygen intermediates and the microbicidal event. In: Van Furth, R., ed. Mononuclear phagocytes, functional aspects. Martinus Nijhoff: The Hague; 1980:1105-1137. 20. Nathan, C. F. Mechanisms of macrophage antimicrobial activity. Trans. Royal Soc. Trop. Med. Hyg. 77: 620-630; 1983. 21. Docampo, R.; Moreno, S. N. J. Free radical intermediates in the antiparasitic action of drugs and phagocytic cells. In: Pryor, W. A., ed. Free radicals in biology. Vol. VI. New York: Academic Press; 1984: 243-288. 22. Kazura, J. W.; Meshnick, S. R. Scavenger enzymes and resistance to oxygen-mediated damage to Trichinella spiralis. Mol. Biochem. Parasitol. 10: 1-10; 1984. 23. Paul, J. M.; Barrett, J. Peroxide metabolism in the cestodes Hymenolepis diminuta and Moniezia Expansa. Int. J. Parasitol. 10: 121-124; 1980. 24. Ogilvie, B. M.; Mackenzie, C. D.; Love, R. J. Lymphocyte and eosinophils in the immune response of rats to initial and subsequent infections with Nippostrongylus brasiliensis. Am. J. Trop. Med. Hyg. 26: 61-67; 1977. 25. Ogilvie, B. M.; Love, R. J.; Jarra, W.; Brown, K. N. Nippostrongylus brasiliensis infected rats. The cellular requirement for worm expulsion. Immunol. 521-528; 1977. 26. Ogilvie, B. M.; Askenase, P. W.; Rose, M. E. Basophils and eosinophils in three strains of rats and in athymic (nude) rats following infection with the nematodes Nippostrongylus brasiliensis and Trichinella spiralis, lmmunol. 39: 385-389; 1980. 27. Mackenzie, C. D.; Jungery, M.; Taylor, P. M.; Ogilvie, B. M. The in vitro interaction of eosinophils, neutrophils, macrophages and mast cells with nematode surfaces in the presence of complement or antibodies. J. Pathol. 133: 161-175; 1981. 28. Butterworth, A. E. Cell-mediated damage to Helminths. Adv. Parasitol. 23: 143-235; 1984.