in vitro effect of free radicals on fasciola

7/30/2019 IN VITRO EFFECT OF FREE RADICALS ON FASCIOLA

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Juvenile Fasciola hepatica are resistant to killing in vitroby free

radicals compared with larvae ofSchistosoma mansoni

DAVID PIEDRAFITA1, 2 TERRY W.S P ITHILL1, 2, JOHN P .DALTON4, P AUL J.BRINDLEY3, MARK R.S ANDEMAN5,PAUL R.WOOD6 & JIM C.P ARS ONS1

1Victorian Institute of Animal Science, Attwood, Victoria, Australia,2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia,3

Molecular Parasitology Unit and Tropical Health Program, Queensland Institute of Medical Research, Herston, Queensland, Australia,4School of Biological Sciences, Dublin City University, Glasnevin, Dublin, Ireland,5School of Agriculture, La Trobe University, Bundoora, Victoria, Australia and6CSL, Veterinary Division, Parkville, Victoria, Australia

SUMMARY

Free radicals have previously been shown to kill the

immature stages of the trematode, Schistosoma mansoni

but their effect on newly excysted juvenile (NEJ) ukes of

Fasciola hepatica has not been established. Using acetalde-

hyde and xanthine oxidase to chemically generate reactive

oxygen intermediates (ROI), up to 61% of NEJ were killed

but only when exposed to high levels of ROI. At low

concentrations of acetaldehyde and xanthine oxidase as

sources of reactive oxygen intermediates, only 629% of

NEJ were killed compared with 70 92% of schistosomula.

Incubation with lipopolysaccharide (LPS)-stimulated rat

peritoneal lavage cells (PLCs) killed only 7 15% of NEJ

whereas 7887% of schistosomula were killed under thesame conditions by a mechanism dependent on the produc-

tion of reactive nitrogen intermediates. Relative to immature

and adult parasites, NEJ expressed 2520-fold lower levels

of superoxide dismutase and glutathione S-transferase but

no catalase activity was detected. Incubation of NEJ with

inhibitors of peroxidases and glutathione metabolism

increased the mean killing of NEJ by LPS-stimulated rat

PLCs to 4075%. These results demonstrate that, in com-

parison to schistosomula of S. mansoni, NEJ of F. hepatica

are relatively resistant to killing by free radicals and this

resistance could, in part, be due to the activity of oxidant

scavenger enzymes of NEJ.

Keywords rodent, monocyte/macrophages, Fasciola,

helminth parasites, free radicals

INTRODUCTION

Most work on the host immune effector mechanisms direc-ted against trematode parasites has involved studies on the

immature stages of Schistosoma mansoni. Several effector

mechanisms involved in the killing ofS. mansoni have been

identied in vitro, including cytotoxicity by eosinophils and

granulocytes (Capron & Dessaint 1987). The potential role

of these and other putative resistance mechanisms in vivo

are currently being investigated in various hosts (James et al.

1990, Gazzinelli et al. 1992, Oswald et al. 1994, Wynn et al.

1994, Coulson et al. 1998). However, the immune effector

mechanisms which mediate the killing of newly excysted

juveniles (NEJ) of Fasciola hepatica, an equivalent trema-

tode developmental stage to schistosomula of S. mansoni,are uncertain (reviewed in Hughes 1987). Ultrastructural

studies with NEJ of F. hepatica recovered from the perito-

neal cavity of rats, has shown eosinophils are in close

association with the surface of damaged NEJ, suggesting a

role for eosinophils in mediating resistance to F. hepatica

infection in rats (Davies & Goose 1981, Burden et al. 1983).

Hughes (1987), however, concluded that there is only

circumstantial evidence to implicate the eosinophil in kill-

ing of F. hepatica NEJ. Furthermore, in-vitro studies using

NEJ of F. hepatica have been unable to demonstrate

irreversible damage to this parasite following incubation

with eosinophils and granulocytes (Doy et al. 1980, Duffus

& Franks 1980, Doy & Hughes 1982, Glauert et al. 1985).

Thus, in comparison to schistosomula ofS. mansoni, NEJ of

F. hepatica may be resistant to several immune effector

mechanisms.

Currentevidence suggeststhat nonspecic defensemechan-

isms may be important in the resistance of various hosts to

infection by Schistosoma spp. and F. hepatica (Doy et al.

1981, Hayes & Mitrovic 1977, Oldham & Hughes 1982,

Parasite Immunology, 2000: 22: 287295

q 2000 Blackwell Science Ltd 287

Correspondence: David Piedrata, Department of Biochemistry and

Molecular Biology,Monash University, Clayton,Victoria 3168, Australia

Received: 25 June 1999

Accepted for publication: 27 January 2000


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Oldham 1983, Ford et al. 1987, Smith et al. 1992, Baeza

etal. 1994a,b). Cell-generated reactive nitrogen intermediates

(RNI) and chemically generated reactive oxygen intermedi-

ates (ROI) can kill > 80% of schistosomula of S. mansoni in

vitro (Mkoji et al. 1988a,b, James & Glaven 1989). Killing of

schistosomula by murine macrophage-generated RNI occurs

in the absence of parasite specic antibody and requires onlytransient contact between macrophages and schistosomula for

cytotoxicity to be mediated(McLaren & James1985, James &

Glaven 1989). In addition, low levels of acetaldehyde with

xanthine oxidase, as a source of ROI, kill 95100% of

schistosomula of S. mansoni in vitro (Mkoji et al. 1988a,b).

In order to determine whether RNI and ROI play a role in the

killing ofNEJ ofF. hepatica, we directly comparedthe relative

susceptibility of juvenile F. hepatica and S. mansoni schisto-

somula to killing by chemically and cell-generated RNI/ROI.

In addition, the role of certain antioxidant defense enzymes

expressed by NEJ in determining the susceptibility of NEJ to

killing by free radicals was also investigated.

MATERIALS AND METHODS

Animals, parasites and reagents

Cercariae of S. mansoni (NMRI strain) were obtained from

infected Biomphalaria globrata snails maintained at the

Queensland Institute of Medical Research, Brisbane,

Queensland. Mechanically transformed schistosomula

were prepared by vortex shearing of tails from cercariae,

followed by fractionation over a Percoll gradient and incu-

bation in DMEM with antibiotics for 3 h at 378C. Metacer-

cariae of Fasciola hepatica were purchased from ComptonPaddock Laboratories (Surrey, UK) and stored on cellophane

at 48C prior to excystment of NEJ (Smith & Clegg 1981).

Naive white male Wistar rats, 7 8 weeks old, used as donors

of peritoneal lavage cells were supplied by the Queensland

Institute of Medical Research, Queensland. Rats were

euthanized by asphyxiation with CO2 gas in a semiclosed

container and resident lavage cells from rats were collected

by simple lavage using sterile PBS containing 6 mM EDTA.

The total number of viable leucocytes was determined using

a Neubauer haemocytometer. DMEM was purchased from

Life Technologies, Inc., USA. L-arginine, azide, L-buthionine-

S,R-sulfoximine, 1-chloro-2,4-dinitrobenzene, cytochrome c,

gentamycin, naphthylenediamine dihydrochloride, nitrate,

nitrite, sulphanilamide and the tetrazolium, MTT, were

purchased from Sigma Chemical Co., St Louis, MO, USA.

Amphotericin B was purchased from Life Technologies

(Rockville, MD, USA). Glutathione, glutathione reductase,

superoxide dismutase and xanthine oxidase were purchased

from Boerhinger-Mannheim (IN, USA). Acetaldehyde was

purchased from Aldrich Chemical Co., USA. NG-monomethyl

arginine was purchased from CalbiochemBehring Corp. (La

Jolla, CA,USA). DMSO, hydrogen peroxide andtoluidine blue

were purchased from BDH Chemicals (Kilsyth, Australia) and

Ajax Chemicals (Australia), respectively. ELISA plates and

24-well tissue culture plates were purchased from Flow

Laboratories Inc. (CA, USA) and Greiner labortechnik

(Kremsmuenster, Austria), respectively. LPS of Eschericiacoli was kindly provided by Dr Paul Wood (CSIRO Division

of Animal Health, Melbourne, Australia).

Incubation of schistosomula and/or NEJ with cell-and

chemically generated free radicals

Incubations were carried out at 378C in 24-well plates or 96-

well at-bottom ELISA plates, in a humidied incubator in the

presence of 95% air and 5% CO2. Aliquots of 50100 3 h

transformed schistosomula or NEJ were incubated using a 24

well plate for 48 h to assess the effects of reactive oxygen

intermediates on parasite killing: each well contained 500ml of

DMEM and 0 22 mM acetaldehyde and/or 1040 mU/mlxanthine oxidase. Lavage cells were incubated with NEJ and/

or schistosomula (13 larvae/well) using a 96-well plate in

200ml DMEM, containing 10% heat-inactivated FCS, 2mg/ml

amphotericin B, 10mg/ml gentamycin and with or without

05mg/ml LPS, 05 mM NG-monomethyl arginine or combina-

tionsof these reagentsat anE:T ratioof 052 105 :1forupto

5 days. Larval killing wasdetermined microscopically.Briey,

killing of schistosomula was accompanied by progressive

internal disorganization of the parasite, development of

lucent areas in the tegumental cytoplasm and loss of motility.

Dead schistosomula had an intact tegument and membrane, but

had an opaque appearance with lucent areas in the tegumentalcytoplasm and a characteristic swelling (McLaren & James

1985). Larval killing of NEJ was determined microscopically

as loss of motility, an opaque appearance and an inability to

reduce the tetrazolium, MTT (Comley et al. 1989).

Nitrite levels

The levels of nitrite in culture supernatants of lavage cells were

determined at the end of an incubation period and used as an

indicator of nitric oxide production by lavage cells (Hibbs etal.

1988, Ignarro et al. 1993). Nitrite concentration in the culture

media was assayed by a standard Greiss reaction (Green et al.

1982). Briey, half (100 ml) of the culture supernatant was

added to 50ml of 1% sulphanilamide in 25% H3PO4 (085%)

and 50ml of 01% naphthylenediamine dihydrochloride. After

15 min, the absorbance was read at 540 nm using a Titerteck

Multiskan MCC spectrophotometer (Titerteck Instruments

Inc., AL, USA). Nitrite concentration was determined with

reference to a standard curve generated using concentrations

from 1mM to 280 mM sodium nitrite in culture media.

D.Piedrata et al. Parasite Immunology

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 287295288


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Determination of oxidant scavenger enzymes

On each of two occasions, approximately 5000 NEJ were

manually homogenized in 100 mM Tris-HCL buffer, pH 74,

containing 150m M KCl in an ice-cooled ground glass

homogeniser for 5 min and the homogenate centrifuged in

a microcentrifuge at 48C at 13000g for 20 s. Similarly, 20

50 juvenile or adult parasites from each sheep were brieyhomogenized, in a beaker on ice, using a blender at

8500 r.p.m. (IKA Ultra-turrax T25, NC, USA) for 20 s and

the crude extract further homogenized in a ground glass

homogenizer. Crude enzyme extracts were centrifuged in a

microcentrifuge at 48C at 13 000g for 20 s. The activity of

superoxide dismutase, glutathione peroxidase, glutathione

S-transferase and catalase was measured in triplicate in the

resultant supernatant. All reactions were carried out at 258C

in a Shimadzu UV-160 spectrophotometer (Shimadzu

Corporation, Kyoto, Japan) in a total reaction mixture of

1 ml, and the change in absorbance monitored continuously

for 2 min. Superoxide dismutase activity was determined bythe cytochrome c reduction method (Flohe & Otting 1984),

using bovine erythrocyte superoxide dismutase as a standard.

Cytochrome c reduction was monitored at 550 nm. One unit

of superoxide dismutase activity was dened as the amount

of enzyme necessary to inhibit the rate of reduction of

cytochrome c by 50%. The catalase assay (Ganschow &

Schimke 1969) measured the disproportion of hydrogen

peroxide to water and oxygen as a decrease in absorbance

at a wavelength of 240 nm. One unit of catalase activity is

dened as that amount of enzyme required to decompose

50% of 20mM hydrogen peroxide in 1 min at 258C. The

glutathione peroxidase assay (Hopkins & Tudhope 1973)

used the NADPH-coupled reduction of oxidized glutathione,

catalysed by glutathione reductase. The specic activity was

expressed as nmoles of glutathione oxidized per minute

per mg of protein. The glutathione S-transferase assay

(Habig et al. 1974) measured the conversion of glutathione

from the oxidized form to the reducedform andwas monitored

spectrophotometrically as an increase in absorbance at

340 nm. The specic activity of glutathione S-transferase

was dened as the amount of 1-chloro-2,4-dinitrobenzene

conjugated per min per mg of protein. Protein concentration

was determined using the Bio-Rad DC colourimetric assay

for protein concentration following detergent solubilization

(Lowry et al. 1951).

Incubation of NEJ with azide, L-buthionine-S,R-

sulfoximine and 1-chloro-2,4-dinitrobenzene

Aliquots of 100200 NEJ/well were initially incubated in

2 ml of DMEM in 24-well plates with either 14 mM azide,

14mM 1-chloro-2,4-dinitrobenzene or 6 mM L-buthionine-

S,R-sulfoximine for 2 h. The DMEM was removed and

NEJ, in a nal volume of approximately 100 ml, within the

well were washed twice with 2 ml of DMEM. NEJ were

subsequently transferred to 96-well ELISA plates and incu-

bated with 2 105 rat peritoneal lavage cells for 5 days and

the percentage of dead NEJ scored as described above.

Azide, 1-chloro-2,4-dinitrobenzene and L-buthionine-S,R-

sulfoximine were added directly to some incubations of NEJand rat lavage cells.

RESULTS

NEJ are resistant to killing by chemically generated

free radicals

The effect of ROI on the viability of NEJ in vitro was

determined using free radicals generated by xanthine oxidase/

acetaldehyde. Only high levels of acetaldehyde and xanthine

oxidase were able to kill a substantial number of NEJ in vitro

with up to 61% killing observed at the highest concentra-tions of the free radical generating system (Table 1). These

high concentrations required to attain substantial killing of

NEJ appeared to contrast with the reported lower levels

required to kill schistosomula of S. mansoni (Mkoji et al.

1988a,b, Nare et al. 1990). We directly compared the

susceptibility of NEJ and schistosomula to ROI using

levels of acetaldehyde and xanthine oxidase which had

previously been shown to kill the majority of schistosomula

in vitro (Mkoji et al. 1988a,b, Nare et al. 1990). When

exposed to ROI chemically generated by low levels of

xanthine oxidase (10 mU/ml) and 0205 mM acetaldehyde,

only 621% of NEJ were killed compared with 7092% of

schistosomula (Table 1); although only a single experiment,

this level of killing of schistosomula is similar to that

reported in earlier studies (Mkoji et al. 1988a,b, Nare et al.

1990). These results suggest that NEJ are only susceptible to

killing by ROI at relatively high ROI concentrations.

NEJ are not killed by RNI released by rat peritoneal

lavage cells

The effect of RNI on the viability of NEJ in vitro was

determined using LPS-stimulated rat peritoneal lavage cells

(PLCs). Previously, we determined that maximal nitrite

levels were obtained in culture supernatants by increasing

the E:T ratio to 2 105 : 1. However, when NEJ were

incubated for 5 days with LPS-stimulated rat PLCs at an

E:T ratio of 2 105 : 1 the level of parasite mortality was

only 10% (Table 2) even though high levels of nitrite (75

97 mM) were detected in the culture supernatants (data not

shown). This nding suggested NEJ could be resistant to

cytotoxicity by RNI, as schistosomula have been previously

Volume 22, Number 6, June 2000 Free radical damage to juvenile F. hepatica and larvae of S. mansoni

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 287295 289


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play a role in protecting NEJ from free radical damage, as

shown previously for schistosomula (Mkoji et al. 1988a,b,

Nare et al. 1990). Azide or 1-chloro-2,4-dinitrobenzene

were used as inhibitors of peroxidases and glutathionemetabolism, respectively (Paul & Barrett 1980, Arrick

et al. 1982). Incubation of NEJ with LPS-stimulated rat

PLCs at an E:T ratio of 2 105 : 1 for 5 days resulted in only

15% killing (Table 4), similar to results shown above

(Table 2). However, when NEJ were cultured for 2 h with

each of these inhibitors, washed and incubated with LPS-

stimulated rat PLCs, 45 75% of NEJ were killed (Table 4);killing was strongly inhibited by the addition of NG-mono-

methyl arginine which inhibited nitrite production by PLCs

(Table 4). Thorough washing of NEJ, after initial incubation



Table 3 Specic activities of some oxidant scavenger enzymes of F. hepatica

Parasite stage2

Oxidant Scavenger Enzyme Activity

(nmol/min/mg protein) NEJ1 7 weeks 12 weeks 16 weeks

Superoxide dismutasea 27, 25 NA NA 676 14

Glutathione peroxidase 37, 66b

396 9b

416 11b

526 12b

Glutathione S-transferase 150, 190c 8606 320d 21806 770e 29306 880e

Catalase BD BD BD BD

1 Values represent the mean enzyme activity of three determinations from each of two separate preparations of 5000 NEJ; 2 Values represent the

mean 6 SD from separate preparations of 2050 parasites collected from four donor sheep infected with 300 metacercariae. a Superoxide

dismutase activity is expressed as U/mg protein. BD, Below detectable limit of assay; na, not assayed. b,c,d,e Values within each row with different

superscripts could be signicantly differentiated by a one-way ANOVA for a completely randomized design after transforming on a log scale;

P< 005.

Table 2 Comparative susceptibility of NEJ of F. hepatica and schistosomula ofS. mansoni to killing by LPS-stimulated peritoneal lavage cells of

rats

NEJ Schistosomula

Number dead/ % dead Number dead/ Number dead/ % dead

Incubation total number 6SD total number % dead total number 6SD

Effector: target ratio 2 105 : 1a 105 : 1b 105 : 1c

Schistosomula or NEJ LPS 1/10, 1/10, 1/10, (56 5) 2/15, 1/15, (86 3)

1/10, 0/10, 0/10, 0/15e (0) 1/15, 1/15e

0/10, 0/10

Schistosomula or NEJ LPS cells 1/1 0, 2/10, 1/1 0, ( 106 8) 12/15, 11/15, (786 3)

2/10, 1/10, 0/10, 1/15e (7) 12/15, 12/15e

0/10, 1/10

Schistosomula or NEJ LPS cells 1/10, 1/10, 1/10, (86 4) 1/15, 2/15, (106 8)

NG

-monomethyl arginine (05 mM) 0/10, 1/10, 1/10 1/15e

(7) 0/15, 3/15e

Effector: target ratio 05 105 : 1d

Schistosomula NEJ LPS 0/15 (0) 0/15 (0)

Schistosomula NEJ LPS cells 1/15 (7) 13/15 (87)

Schistosomula NEJ LPS cells 0/15 (0) 2/15 (13)

NG-monomethyl arginine (05 mM)

a Ten separate wells each containing one NEJ were incubated with or without cells and/or inhibitors for 5 days and the mean number of dead

parasites subsequently determined. b,c,dFive separate wells each containing three schistosomula and/or three NEJ were incubated with or without

cells and/or inhibitors for 3 days and the mean number of dead parasites subsequently determined. a,cData from 48 separate experiments. d Note

for incubations with schistosomula and NEJ together the E:T ratio was 05 105

: 1.e

Values with this superscript represent incubations with PLCs

from the same rat.


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with azide or 1-chloro-2,4-dinitrobenzene, was necessary

before incubation with PLCs as the presence of these com-

pounds inhibited nitrite production by LPS-stimulated PLCs

(data not shown). Incubation of NEJ and LPS-stimulated ratPLCs with L-buthionine-S,R-sulfoximine, anotherglutathione

depleting agent (Arricket al. 1981, Grifth & Meister 1979),

resulted in killing of 40% of NEJ and this killing was also

signicantly inhibited by the addition of NG-monomethyl

arginine (Table 4). L-buthionine-S,R-sulfoximine, a rever-

sible inhibitor of glutathione synthesis (Grifth & Meister

1979, Arrick et al. 1981), was required throughout the

period of culture as no killing resulted when NEJ were

initially cultured for 2 h in L-buthionine-S,R-sulfoximine,

washed with DMEM, and subsequently incubated with LPS-

stimulated rat PLCs (data not shown). These results suggest

that peroxidases and glutathione play a role in protecting

NEJ against damage by free radicals.

DISCUSSION

In this study, we have demonstrated that NEJ of F. hepatica,

when compared to schistosomula ofS. mansoni, are resistant

to killing by ROI/RNI. When NEJ were exposed to low

concentrations of xanthine oxidase and acetaldehyde, only

629% of NEJ were killed and high levels of xanthine

oxidase and acetaldehyde were necessary to achieve a

signicant (4461%) degree of killing. Up to 7092% of

schistosomula were killed when exposed to low levels ofacetaldehyde and xanthine oxidase, a degree of killing

similar to that reported by others using this oxidant-generating

system (Mkoji et al. 1988a,b). This suggests that NEJ are less

susceptible to killing by ROI. Similarly, incubation of NEJ

or schistosomula with LPS-stimulated rat PLCs resulted in

only 710% mortality of NEJ but greater than 78% killing

of schistosomula and this killing was mediated by RNI.

These studies as a whole conrm that, in comparison to

schistosomula, NEJ are highly resistant to killing mediated

by chemically generated ROI and cell-generated RNI in vitro.

The survival of NEJ in the presence of ROI and RNI

suggests that NEJ have effective defenses against these toxic

molecules. Oxidant scavenger enzymes are one of the major

ways in which parasites detoxify free radicals and their

intermediates (Callahan et al. 1988). Here, we have demon-

strated for the rst time that NEJ of F. hepatica contain a

range of oxidant scavenger enzymes. The levels of super-

oxide dismutase and glutathione S-transferase were 220-

fold lower than the levels detected in immature or adult

parasites whereas glutathione peroxidase activity was similar.



Table 4 Effect of inhibitors of peroxidases and glutathione metabolism on the killing of NEJ of F. hepatica by LPS-stimulated rat peritoneal

lavage cells

NEJ

Number dead/total number Nitrite (mM)

Incubation Experiment 1 Experiment 2 (mean % dead) Experiment 1 Experiment 2

NEJ LPS 1/10, 0/10 5


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The levels of glutathione S-transferase and superoxide

dismutase in the immature or adult parasites are similar in

range to those reported previously (Howell et al. 1988,

Brophy et al. 1990, Piacenza et al. 1998). In general,

detoxication of oxidants generated by chemicals or cells

external to the parasite suggests the requirement for the

oxidant scavenger enzymes to be cytosolic, on the surface ofthe parasiteor secreted/excreted by the parasite(Callahan etal.

1988). The distribution of defense enzymes in the NEJ is

largely unknown. Glutathione S-transferase is distributed

throughout the tissues of NEJ, including the subtegumental

region but not at the parasite surface (Creaney et al. 1995),

whereas glutathione S-transferase has been localized to the

surface tegument and gut lamellae of adult ukes (Howell

et al. 1988, Wijffels et al. 1992). Oxidant scavenger

enzymes have been found in the excretory/secretory (ES)

materials of several parasites (Callahan et al. 1988, Henkle

et al. 1991, Cookson et al. 1992, Britton et al. 1994, James

et al. 1994, Tang et al. 1994) and the ES material of NEJ

may contain enzymes capable of scavenging oxidants.Indirect evidence exists for the release of low levels of

glutathione S-transferase in the ES material of adult

F. hepatica (Howell et al. 1988, Sexton et al. 1990, Hillyer

et al. 1992). A cDNA encoding a peroxiredoxin homologue

has been cloned from F. hepatica using sera raised against

adult ES products suggesting that adult ukes release a

peroxiredoxin-like enzyme which may protect ukes against

hydrogen peroxide and other ROI (McGonigle et al. 1997).

Recently, superoxide dismutase activity has been detected

in ES products of immature (35-week-old ukes) parasites

(Piacenza et al. 1998). Thus, oxidant scavenger enzymes

could be one way in which NEJ render chemically or cell-generated oxidants ineffective.

Previous studies have suggested a relationship between

the level of oxidant scavenger enzymes in parasites and the

degree of resistance of parasites to killing by reactive

oxygen intermediates (Kazura & Meshnick 1984, Callahan

et al. 1988, Nare et al. 1990). The specic enzyme activities

of superoxide dismutase and glutathione peroxidase of NEJ

found in this study were up to 10-fold higher than those

reported for schistosomula using the same enzyme assay

system (Mkoji et al. 1988a, Nare et al. 1990) and may

therefore explain the greater resistance of NEJ to killing by

chemically generated reactive oxygen intermediates. The

addition of inhibitors of peroxidases and glutathione

metabolism, such as azide, 1-chloro-2,4-dinitrobenzene

and L-buthionine-S,R-sulfoximine, rendered 40 75% of NEJ

susceptible to killing by LPS-stimulated rat PLCs. These

inhibitors have previously been shown to render schistoso-

mula, trypanosomes, newborn larvae of Trichinella, and

tumour cells susceptible to damage by reactive oxygen

intermediates (Bass & Szejda 1979, Grifth & Meister

1979, Nathan et al. 1980, Paul & Barrett 1980, Arrick et al.

1981, Arrick et al. 1982, Mkoji et al. 1988b, Nare et al.

1990). However, the killing of NEJ, following the addition

of inhibitors, was apparently mediated by RNI, not primarily

by ROI, as the inclusion of NG-monomethyl arginine

reversed the cytotoxicity of LPS-stimulated rat PLCs

against NEJ. Thus, the increase in killing of NEJ followingexposure to these inhibitors may have resulted from inhibi-

tion of NEJ oxidant scavenger enzymes and suggests that

these enzymes play a role in the protection of the parasite

against cytotoxicity by RNI intermediates. In agreement

with these nding, evidence is now emerging suggesting an

important role of parasite oxidant scavenger enzymes

against RNI toxicity (Piedrata & Liew 1998).

The relative resistance of NEJto free radical killing in vitro,

when compared to schistosomula, may also indirectly

depend on the differences in energy metabolism between

the two parasites. Newly transformed schistosomula are

dependent on mitochondrial respiration and exposure to

RNI results in the inactivation of cell mitochondria anddeath of the parasite over a 48-h period (McLaren & James

1985, James & Glaven 1989; James 1991). Conversely, NEJ

are not dependent on aerobic metabolism (Tielens 1994).

Exposure of NEJ to levels of RNI, which kill schistosomula,

may also lead to mitochondrial damage, but NEJ may obtain

their energy requirements through alternate pathways (Tielens

1994) and, hence, survive. RNI-mediated killing of NEJ

may thus result from the inhibition of other cellular targets

such as aconitase or other iron-sulphur enzymes (Piedrata

& Liew 1998).

In conclusion, these studies demonstrate that NEJ of

F. hepatica are resistant to killing by ROI/RNI and thisresistance could, in part, be mediated by oxidant scaven-

ger enzymes of NEJ. Further studies on the biochemical

basis for the resistance of NEJ of F. hepatica to killing by

ROI/RNI may lead to the identication of new potential

targets for rational chemotherapeutic or immunological

control of F. hepatica infection in production animals

and humans.

ACKNOWLEDGEMENTS

These studies were supported by DARATECH Pty. Ltd,

Ciba Animal Health Australia, Agriculture Victoria and

Monash University. David Piedrata was supported by a

PhD scholarship from the Victorian Education Foundation.

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