Effect of larval trematode infection on inosine monophosphate catabolism in the freshwater pulmonate snail Lymnaea luteola

JOURNAL

OF INVERTEBRATE

PATHOLOGY

56,

25-30 (19%)

Effect of Larval Trematode Infection on lnosine Monophosphate Catabolism in the Freshwater Pulmonate Snail Lymnaea luteola B. RAMALAKSHMIREDDYAND Division of Pathobiology,

P. VENKATESWARARAO

Department of Zoology, Sri Venkateswara Tirupati, A.P. 517 502, India

University,

Received May 5, 1989; accepted October 16, 1989 Enzymes involved in purine nucleotide catabolism are studied in the major tissues of the uninfected and infected snails. All the catalytic enzymes lead to the formation of hypoxanthine and xanthine, but not uric acid. Their activity ratios reveal that inosine conservation takes place in digestive gland on infection. This contrasting behavior of digestive gland probably suggests the need for this nucleoside felt by the developing parasites. o 1990Academic press, IOC. KEY WORDS: Lymnaea luteola; enzymes; catabolism.

INTRODUCTION

tion of nucleic acid content of host tissues was reported for some helminthic infecA number of enzymes are involved in tions such as those with Schistosoma inosine monophosphate (IMP) catabolism. japonicum, Clonorchis sinensis, ParagoniA survey of the literature (Campbell and mus westermani, and Ascaris lumbricoides Bishop, 1970; Florkin and Bricteux(Von Brand, 1973). Practically no mention Gre’goire, 1972; Barankiewicz, 1973; was even made regarding impact of parasitBarankiewicz and Jezewska, 1972; Jezew- ism on the activity of any of the enzymes ska and Barankiewicz, 1977) reveals that involved in purine metabolism, in molmscs, none of these enzymes have been studied in although parasitism itself is being defined molluscs with special reference to experion the basis of metabolic dependence and mental or environmental conditions. Howmolecular exchanges (Lincicome, 1963). ever, their occurrence in different molluscs The only report relating to purine metabohas been often reported. Molluscs often lism is that of Gilbertson and Michelson play host to a variety of trematodes. (1969) who studied the incorporation of preWhether any parasitic infection will cause a cursors of RNA into the digestive gland of drastic change in nucleotide metabolism of uninfected and Schistosoma mansonithe host is unknown. infected Biomphalaria glabrata. The Sharma et al., (1978) reported higher xan- present investigation is therefore designed thine oxidase activity in the rat liver on in- to elucidate the alterations in IMP catabofection with Plasmodium berghei. In ani- lism caused by the developing intramollusmals infected with some neurotrophic vi- can larval trematodes . ruses hepatic xanthine oxidase activity MATERIALS AND METHODS increases (Bergmeyer, 1974). However, in Lymnaea luteola snails of medium size most of the other pathological conditions affecting liver, the activity of xanthine oxi- ranging from 300 to 400 mg (with shell) were dase decreases (Bauer and Bradley, 1956; collected from nearby paddy fields and susReid and Lewin, 1957). Bungener (1965) re- pended in isolation in 7.5 x 2.5-cm glass tubes containing 25 ml of dechlorinated tap ported an elevated nucleic acid catabolism during the P. berghei infection of rats. It water. Those shedding cercariae were conwas followed by increased levels of nu- sidered as infected. Those that did not shed cercariae after repeated suspensions were cleases, deaminases, nucleoside phosphotreated as uninfected tentatively and their hydrolases, and xanthine oxidase. Deple25 0022-201 mo $1.50 Copyright 0 1990 by Academic F’ress, Inc. All lights of reproduction in any form reserved.

26

RAMALAKSHMI

REDDY

actual status was determined at the time of tissue isolation by dissection under a stereobinocular microscope. Snails thus sorted out were acclimated to laboratory conditions and fed ad libitum with leaves of Amaranthus viridis. The snails infected with xiphidiocercariae of Prosthogonimus sp. alone were chosen for experimental purposes. The level of infection is approximately the same in all these field-infected snails of this size range, as already reported through planimetric studies, the ratio between parasite and host digestive gland tissues being 1.2-2.32 (Manohar and Venkateswara Rao , 1978). 5’ (IMP)-nucleotiduse. The method of Uhich Gerlach and Walter Hiby as given by Bergmeyer (1974) was followed for the assay of 5’ (IMP)-nucleotidase, and was carried out in the presence and absence of nickel ions. In their presence, the activity of nonspecific phosphatase was determined while in their absence the sum of the activities of nonspecific phosphatases and S-nucleotidase was measured. The difference in the amount of phosphate ion liberated per unit time in the two assays as determined by the method of Fiske and Subbarow (1925) was used a measure of 5’ IMP-nucleotidase activity. Tissue homogenates were prepared in Verona1 buffer (40 mm), pH 7.5, in a cold room and the supernatants were used as enzyme source. Reaction mixture contained 30 mM Verona1 buffer, pH 7.5, magnesium sulfate, 10 mM nickel chloride, 1 mM 5’-inosine monophosphate, and enzyme source. Incubation was for 30 min at 37°C in a metabolic shaking incubator. Znosine phosphorylase. Inosine phosphorylase activity was determined following the procedure of Barankiewicz and Jezewska (1972). Tissue homogenates were prepared in 0.1 M Tris-chloride buffer, pH 8, in a cold room and the supernatants were used as enzyme source. The reaction mixture contained 1.5 mM inosine, 0.1 M Trischloride, pH 8, and enzyme source in a final volume of 2 ml. Incubation was for 1 hr at

AND

VENKATESWARA

RAO

37°C in a metabolic shaking incubator. The product (ribose) formed during incubation period was determined by the method of Tracey ( 1950). Xanthine oxidase. Xanthine oxidase activity was determined following the procedure given by Grossman and Moldave (1967). Enzymatic activity is most conveniently determined spectrophotometrically by following the increase in absorbance at 293 nm upon aerobic oxidation of hypoxanthine and xanthine to uric acid, and the increase in absorbance at 280 nm due to the conversion of hypoxanthine to xanthine. Tissue homogenates were prepared in 0.5 M glycine buffer, pH 8.8, containing 700 pg of bovine serum albumin per milliliter of buffer in a cold room and the supernatants are used as enzyme source. The reaction mixture contained 1 pmol of hypoxanthine or xanthine, 490 pg of bovine serum albumin, 0.5 M glycine, pH 8.8, and enzyme source in a final volume of 3 ml. Incubation was for 30 min at 37°C in a metabolic shaking incubator. Protein was determined according to the method of Lowry et al. (1951). For all the experiments the samples were read in a Bausch and Lomb Spectronic-2000, UVVIS double beam recording spectrophotometer system. Analar grade reagents were used for all the experiments. For standard deviation and probability tests, i.e., Student’s t, test the method given by Garrett and Woodworth (1961) is adopted. If the calculated value exceeded the table value at the 5% level, it was treated as significant at that level. RESULTS 5’ (IMP)-Nucleotidase. The activity of this enzyme increased on infection in all the three tissues (Table 1). Its specific activity in infected foot was almost double that of uninfected foot and the difference was statistically significant. Activity of this enzyme though elevated by 63.83 and 20.72% in mantle and digestive gland, respectively, on infection was not statistically different

IMP

CONSOLIDATED

TABLE

SHOWING

CATABOLISM

THE ENZYME

IN Lymnaea

TABLE 1 ACTIVITIES IN THE TISSUES OF UNINFECTED Lymnaea luteola

Foot Enzyme 5’ (IMP)-nucleotidase (pm01 of Pi formed/ mg . proteink) PC Inosine phosphorylase (km01 of ribose formed/ mg . proteiti) PC Xanthine oxidase (hypoxanthinexanthine) (Fmol of xaothine formed/ mg . proteink) PC Xanthine oxidase (hypoxanthine-uric acid) (pmol of uric acid formed/ m;;protein/hr) Xanthine oxidase (xanthineuric acid) (pm01 of uric acid formed/ proteinhr) PC

Uninfected

Mantle Infected

0.130(8) +0.110

0.247*(6) kO.133

+90.00 0.438(6)

0.473(6) k-o.263

kO.401

Uninfected

0.01218) +0.011 - 100.00

0.033(8) ~0.032 +42.42

Digestive Uninfected

0.188(6) 20.152 +63.83

0.308(6) kO.157

kO.330

0.728(6) k0.327

0.675(6) 20.250

2.780(6)

0.888*(s) +0.090

0.940(8) 20.140

O&%7(8) ~0.040

0.037(8) lrO.046 +10.81

mantle and diges-

Znosine phosphorylase. The activity of this enzyme decreases in mantle and digestive gland on infection but the change was statistically significant only in the latter (Table 1). The 7.99% increase in its activity in infected foot was not significant. Xanthine oxidase. Xanthine oxidase activity, with hypoxanthine as substrate was found to be elevated significantly in foot and digestive gland and declined signiticantly in mantle when the measured product was xanthine. With hypoxanthine as substrate and activity measured as uric acid formed, the activity could be detected in uninfected foot but not in infected foot. The activity similarly measured in mantle and digestive gland of infected snails showed a 55 and 27% drop, respectively. The changes are significant only in foot and mantle but not in digestive gland. With xanthine as substrate and activity measured as

3.356(6)

1.110(6)

0.755*(6) ~0.126

-31.98

0.710*(8) ~0.056

0.058(S) *0.030 -55.17

Infected

20.818

kO.359

0.590(8)

0.750*(g)

~0.100

-24.47

BLD (8)

gland

+20.72

-7.28

Note. Figures in the parentheses indicate number of individual samples. *Significant PC, percentage change in infected snail. BLD, below level of detection.

from that of uninfected tive gland.

AND INFECTED

Infected

+7.99

0.760(8) +0.059 +16.84

27

luteola

kO.120 +27.12

0.026*(8)

~0.010

0.029(8) kO.024

0.021(8) 20.019 -27.58

0.041(8) +0.045

BLD (8)

BLD (8)

at 5% level according to Student’s t test.

uric acid formed, the activity could not be detected in the digestive gland of uninfected and infected snails. Changes observed in infected foot and mantle were not statistically significant. It is evident from Table 1 that xanthine oxidase activity decreased substantially when the measured product was uric acid with both hypoxanthine and xanthine as substrates. DISCUSSION

The presence of an active enzyme of any metabolic pathway only raises the possibility that the pathway is operative. The activities of the enzymes were, of course, assayed under optimal conditions in vitro, and may or may not be applicable to the in vivo situation. The absolute values by themselves do not tell anything about the actual metabolic flux along that pathway. However, the metabolic program can more easily be understood by computing enzyme activity ratios which may be or may not be

28

RAMALAKSHMI

REDDY AND VENKATESWARA

through the adenosine deaminase pathway than the 5’ (IMP)-nucleotidase pathway since the adenosine deaminasel5’ (IMP)nucleotidase ratio is more than one. The enzyme activity ratios (Table 2) suggest there was a shift toward hypoxanthine formation from adenosine and adenine on infection. It is interesting to see from xanthine oxidase activity that xanthine formation was high in all tissues compared to uric acid formation as revealed by the activity ratios (Table 2). It is noteworthy that on infection, xanthine formation increased in foot and digestive gland while uric acid formation dropped in all tissues. The adenine deaminase to xanthine oxidase activity ratio shows that on infection, the rate of hypoxanthine formation was higher than the rate of xanthine formation or rate of uric acid formation. This is further supported by the reported low levels of uric acid in mantle and digestive gland and traces in foot (Kondaiah, 1977). The decrease in xanthine oxidase activity toward uric acid formation on infection might be to provide hypoxanthine and xanthine to the developing larval trematodes. It appears, on the whole, that hypoxanthine or xanthine accumulation takes place in the tissues of Lymnaea luteala as well as inosine conservation in diges-

discriminating (Pette and Bucher, 1963; Bass et al., 1969). Barankiewicz et al. (1979) suggested that besides the adenine cycle, there is another pathway starting from IMP leading to the formation of the major nitrogenous end products, namely uric acid, and this they considered as the main pathway of protein catabolism in Helix pomatiu (See Fig, 1 for the pathways. Adopted from Prosser, 1973). In the present investigation, 5’ (IMP)-nucleotidase and inosine phosphorylase activities were highest in digestive gland followed by mantle and foot. Their activity ratios (Table 2) reveal that only in digestive gland, where it is more than one, was there a tendency for inosine formation from IMP to exceed inosine conversion to hypoxanthine. In infected snail tissues, there was a general increase in 5’ (IMP)nucleotidase where there was no change or drop in inosine phosphorylase activity-the drop being statistically significant only in digestive gland. The ratio of these enzyme activities increased on infection (Table 2) in all the three tissues, the increase being nearly 100%. This may be construed to mean that there was an effort on the part of the infected snail to accumulate inosine. Inosine formation appears to be higher

De novo n Nucleotides

{

RAO

I

synthesis Adenytosoccinate

,P@ I’

Nucleosides Free

bases

{

C

19 \ \ \

1’

0

“Gus

Adc /’

I

m

URIC

ACID

FIG. 1. Interconversions of the nucleotides, nucleosides, and free bases in animals. The “shunt pathway” is indicated by heavy arrows and the “salvage pathway” by dashed arrows. The enzymes mediating the interconversions are a, 5 nucleotidases; b, guanine deaminase (guanase); c, adenosine deaminase; d, AMP deaminase; e, nucleoside phosphorylase; f, adenine phosphoribosyltransferase; g, hypoxanthine-guanine phosphoribosyltransferase; h, adenosine kinase; i, adenylosuccinate synthetase; j, adenylosuccinate lyase; k, IMP dehydrogenase; 1, XMP aminase; m, xanthine oxidase; n, GMP reductase; and o, adenine deaminase (adenase).

IMP CATABOLISM

ENZYME

ACTIVITY

RATIOS

29

luteola

TABLE 2 OF UNINFECTED AND INFECTED Lymnaea

IN THE TISSUES

Foot Ratios S’(IMP)-nucleotidase/ inosine phosphorylase Adenine deaminase/xanthine oxidase (hypoxanthinexanthine) Adenine deaminasel xanthine oxidase (xanthineuric acid) Inosine phosphorylase/ xanthine oxidase (hypoxanthinc-uric acid) Adenosine deaminase/ S’(IMP) nucleotidase Adenine deaminasel xanthine oxidase (hypoxanthine-uric acid) Adenine deamtiase/ inosine phosphorylase

IN Lymnaea

Uninfected 0.297 22.97 529.10

27.37

Infected 0.522 21.15 399.15

0

7.04

4.28

1091.25

0

39.88

39.70

tive gland. These findings are explained in the light of the following evidence. Although all the reactions catalyzing AMP and IMP led to hypoxanthine and xanthine formation, low levels of these purines are detected in all the tissues on infection (Ramalakshmi Reddy, 1985). The increased activity of the enzymes concerned with formation of inosine in digestive gland and hypoxanthine and xanthine in all the tissues might serve to meet the parasites’ biochemical requirement. Parasitic withdrawal of the IMP catabolic products seems to be more or less certain in view of the following reports. Senft et al. (1972, 1973) reported that during incubation of live S. mansoni in the presence of labeled adenosine or inosine, large amounts of hypoxanthine are accumulated in the medium, both hypoxanthine and a considerable amount of IMP being detected later in the worm homogenate supernatant. Wong and Ko (1979) reported that analysis of purine content in the medium after the worm Angiostrongylus cantonensis was incubated with inosine for 2 hr revealed that only 33% of the added inosine remained and 68% of the total radioactivity was found to be associated with hypoxanthine. Basch (1981) successfully obtained adult S. mansoni through culture using the medium which necessarily contained hypo-

Mantle Uninfected Infected 0.258

0.456

luteola

Digestive gland Uninfected Infected 2.50

4.44

17.69

20.32

0

0

13.72

20.79

344.59

360.73

12.55

25.96

28.83

26.%

2.87

6.72

2.19

2.13

222.40

567.69

17.72

21.86

271.17 9.04

544.29 20.18

xanthine. These findings suggest that inosine and hypoxanthine are essential for the parasites. Moreover the parasites are not able to synthesize their purines de novo (Senft et al., 1972; Coles, 1970; Dovey et al., 1984). This is further supported by the lipophylic nature of hypoxanthine and its known ability to freely penetrate red cell membranes (Benke et al., 1973). After taking inosine, hypoxanthine, and xanthine from the host, the parasites might be able to synthesize their nucleotides through salvage pathways (Dovey et al., 1984). Therefore, it is suggested that utilization of preformed purines produced by the host is a highly specialized parasitic adaptation resulting in the establishment of a hostdependent system. ACKNOWLEDGMENTS The authors thank the head of the Department of Zoology, Sri Venkateswara University College, Tirupati, for facilities.

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BARANKIEWICZ, Acid in Helix

pomatia

30

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REDDY

AND

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