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Glutathione S-transferases as markers of salicylanilide resistance in isolates of Fasciola hepatica
Pergamon
GL~TATHI~N~ SALICYLANTLTDE
C. M. *Division tElizabeth
of Biochemistry
Macarthur
S-TRANSFERASES AS MARKERS OF RESISTANCE IN ISOLATES OF I;ASCIULA HEPATICA
D.
MILLER,*
M.
J. HOWELL*
and
J. C.
BORAY~$
& Molecular
Agricultural
Biology. Faculty of Science, Australian National University, GPO Box 4, Canberra, A.C.T. 2601, Australia Institute, NSW Department of Agriculture, Private Mail Bag 8, Camden, NSW 2570, Australia
(Rrwiwd
5 July 1993: uccrp~ed 22 Augusr 1993)
A~tract-MlLL~R C. M. D., HOWELL M. J. and BOKAV J. C. 1994. Glutathione S-transferases as markers of salicyianilide resistance in isolates of Fffsciofa hrpulicu. illrernfff~~Jnf~1Journu/jbr Parasifoiogy 24: 533-542. A possible link between the level of glutathione S-transferase (CST, E.C. 2.5.1.18) activity and the development of salicylanilide resistance in Fasciolu hepaticu was investigated. Various isolates of F. heputiccl with varying susceptibilities to salicylanilides were isolated and maintained in the laboratory. Individual flukes of these isolates were surveyed for their level of GST activity and a correlation between the level of GST activity and drug efficacy was found. In contrast to most other studies, a decrease in GST activity was associated with an increase in drug resistance. Evidence was collected to show that this may be a selective process since flukes which had survived exposure to rafoxanide and ciosantel itl siao (in sheep) had lower activity levels of GST than flukes from untreated sheep. Treatment with other flukicides (oxyclozanide, luxabendazole and triclabendazole) did not have this effect. Furthermore, in viva treatment with closantel induced selection of particular isoenzymes in different isolates of F. heppuricuhaving different degrees of susceptibility to closantel. However, no single isoenzyme or isoenzyme profile was associated with resistance and. in total, up to 8 different isoenzymes could be present in a single isolate. Thus, GST has some potential as a marker enzyme for salicylanitide resistance in E: Irepaiica. However. the precise role of GST in resistance is unclear and the extensive inter- and intra-isolate variation in activity levels and isoenzyme characteristics of this enzyme indicate the need for considerably more study before application in field situations. INDEX KEY WORDS:
Fosciolu hepatica: glutathione
INTROJECTION important parasite to the sheep and cattle industries worldwide, causing major losses due to a variety of clinical and subclinical manifestations of fascioliasis (Dargie, 1986). The major form of control of the disease has been through the strategic administration of anthelmintics and among the most successful groups of flukicides in use are salicylanilides, such as rafoxanide and closantel. Resistance to these anthelmintics has been reported in the field, but tests such as the simple egg hatch assay or egg count reduction test to detect anthelmintic resistance in nematodes are not applicable because resistance is often manifest before patency. At present, resistance in F. heparica can be diagnosed only in special laboratories with established
all correspondence
should
salicylanilide
resistance;
sheep
snail colonies. Laboratory-bred intermediate host snails have to be infected with miracidia of the different isolates, metacercariae collected, groups of fluke-free sheep inoculated and the efficacy of titration treatments with anthelmintics evaluated in slaughter trials. The minimum duration of an experiment is approximately 6 months (Boray, 1990). For effective management of drug resistance a suitable test or marker is required to allow rapid identification of resistance to salicylanilides in the field. Glutathione S-transferases (GSTs) (E.C. 2.5.1.18) are a family of multifunctional proteins which appear to be concerned with detoxification of xenobiotics and endogenously derived toxic compounds using catalytic and binding mechanisms (Mannervik & Danielson, 1988; Brophy, Papadopoulos, Touraki, Coles, K6rting & Barrett, 1989). In the apparent absence of the cytochrome P-450 system in helminths (Precious & Barrett, 1989). GSTs are likely to be the principal
THE liver fluke, Fuscinla heputicu, is an economically
:To whom
S-transferases;
be addressed. 533
534
C. M. D.
detoxification enzymes in these parasites. Moreover, numerous studies have linked elevated GST activity levels with the development of drug resistance in a variety of situations including resistance of tumours to chemotherapy (Pickett & Lu, 1989), DDT resistance in the housefly (Clark, 1989) and resistance of Haemonthus contortus to cambendazole (Kawalek, Rew & Heavner, 1984). Previous work has shown that F. heputica contains high levels of GST (Howell, Board & Boray, 1988; Brophy, Crowley & Barrett, 1990), indicating that these enzymes may have an important physiological role. Therefore, the GSTs of F. hepatica were evaluated for use as biochemical markers of salicylanilide resistance. MATERIALS
AND METHODS
Parasites. Various isolates of F. hepatica with differing susceptibilities to various anthelmintics, including rafoxanide and closantel, were recovered from different properties in NSW and subsequently maintained in the laboratory in sheep using lymnaeid snails as intermediate hosts (Boray, & DeBono, 1989; Boray, 1990). Flukes susceptible to salicylanilides were also obtained from Compton Paddock Laboratories, Surrey, U.K. and were also maintained in the laboratory. Adult flukes were recovered 16 weeks pi. (IO weeks after treatment, where applicable) from experimentally infected sheep and the percentage efficacy of the anthelmintic used 6 weeks after inoculation (i.e. before the flukes were fully mature) was determined by comparing fluke counts in the treated sheep and in untreated controls at necropsy. The dose rates of the anthelmintics used in the study were: closantel and rafoxanide, 7.5 mg/kg; oxyclozanide, 26.7 mg/kg; triclabendazole, 4.5 mg/kg; luxabendazole, 7.5 or 11.25 mg/ kg. After removal from sheep, the flukes were placed in Hidon-Fleig saline (120 mMNaC1, 4mMKC1, 0.8 mMCa C1,.2H,O, 2 mMMgSO,.‘TH,O, 1.5% NaHCOj, 4% glucose) at 37°C. Parasites were allowed to disgorge caecal contents before individual flukes were homogenised in 1 ml of 0.1% Triton X-100 (BDH), briefly centrifuged at 12,000 g and the supernatant stored at - 20°C until required. Glurathione S-transferase assays. GST activity was assayed spectrophotometrically at 37°C with 1-chloro-2,4-dinitrobenzene (CDNB) as the standard second substrate following the method of Jaffe & Lambert (1986). The amount of protein in the samples was estimated by the method of Bradford (1976) using purified bovine serum albumin as the standard. One unit of activity is defined as the amount of enzyme required to conjugate 1 ~01 ofCDNB to glutathione (GSH) in 1 min at 37°C. Polyacrylamide gel elecrrophoresis (PAGE) and Western blofiing. Profiles of GST isoenzymes (forms of GST exhibiting charge differences) of samples from individual flukes were analysed by native PAGE (non-reducing conditions without sodium dodecyl sulphate; SDS) followed by Western blotting and probing with antisera to fluke GSTs
MILLER et a/
using standard procedures. An equal volume of 2X sample buffer (125 mMTris_Cl pH 6.8, 20% glycerol, 0.4% bromophenol blue) was added to the samples which were loaded onto 12% polyacrylamide gels and run at 40 mA for 34 h in a Biorad Protean Cell. Purified F. hepatica GST (Howell et ul., 1988) provided by Dr P.G. Board, JCSMR. ANU, was run as an internal control. For Western blotting. the gels were set up in a Biorad transblot apparatus and the proteins transferred to nitrocellulose (Schleicher & Schiill) at 20 v and 150 mA overnight. The nitrocellulose was blocked with 5% skim milk powder (Diploma) in TBS (20 mMTris_Cl pH 7.4, 0.5 MNaCl) then incubated with sheep serum containing antibodies to GST at 1: 100. The anti-GST serum was obtained from sheep that had received 2 injections of purified F. hepaticu GST, 3 weeks apart. The serum was prepared 6 weeks after the 2nd injection. Rabbit anti-sheep Ig coupled to horseradish peroxidase (HRP) (Cappel Laboratories) was used as a second antibody (1 : 1000) and bands were visualised by incubation in Biorad HRP colour development reagent (4-chloro-1-naphthol) dissolved in methanol than added to TBS containing 0.015% H,Oz. For subunit analysis, polyacrylamide gels were run under reducing conditions (SDS--PAGE) following standard procedures. Samples were boiled for 90 s in an equal volume of sample buffer containing 10% SDS and 10% viv p mercaptoethanol prior to loading on to 12% SDS PAGE gels. Purified GST (Howell et al., 1988) was run as an internal control and Rainbow markers (Amersham) were run to allow mol. wt determinations to be made of the subunits. Inhibition assays. Inhibition of GST activity was measured as an I,,, which is defined as the concentration of anthelmintic at which 50% of enzyme activity is inhibited. This was determined by measuring GST activity using CDNB as the second substrate in the presence of 4 different concentrations of closantel (Janssen Pharmaceutics Belgium) dissolved in dimethylsulphoxide (DMSO, BDH), or rafoxanide (Merck, Sharp & Dohme) dissolved in PEG 8000 (Aldrich). The samples were incubated for 5 min at 37°C in the presence of anthelmintic before the reaction was started by the addition of CDNB. Activity was also measured in the presence of DMSO or PEG 8000 without anthelmintic and percentage inhibition in the presence of anthelmintic determined from this value. The percentage inhibition of activity was plotted against the concentration of anthelmintic al which that inhibition occurred and the I,, value estimated from the graph.
RESULTS of individualyukes The level of activity detected, using CDNB as second substrate, was found to vary widely from isolate to isolate and there was also variation in activity from fluke to fluke within an isolate. This ranged from a small difference in some isolates to a considerable difference in others. For example, GST activities of individual flukes in the Morven isolate ranged from 0.13 to 0.38 wol min-lmg protein-‘, whereas GST activities in individual flukes in GST activity in homogenates
GSTs and salicylanilide
in F. hepatica
resistance
535
Rafoxanide 6-
. 6-
Closantel 6-
. 60
f 4-
2-
OL I
I
I
I
I
0
25
50
75
100
Drug Efficacy
(%)
FIG. 1. GST activity in crude homogenates of individual flukes. Each measurement has been plotted against efficacy of closantel and rafoxanide for the isolate. Each point represents specific GST activity in an individual fluke. The regression of GST activity vs efficacy has been plotted 0, = 0.021 x + 0.539; r = 0.494; P < 0.0001 for closantel andy = 0.02x + 0.347; I = 0.417; P < 0.0001 for rafoxanide; Spearman’s rank correlation). Isolates are: Hampton II (V), Walcha (V), Paddock 16 ( n ), Jerangle W (O), Jerangle K (+). Morven ( l ), Lancashire (0). Compton ( V), Sofala I (A).
Compton ranged from 0.27 to 6.52 ~01 min.1 mg protein-‘. When individual activities were plotted against the percentage efficacy of closantel or rafoxanide, strong positive correlations emerged (Fig.
1). The correlations were highly significant with Ye = 0.494, P < 0.0001 for closantel and rs = 0.417, P < 0.0001 for rafoxanide (Spearman’s non-parametric test). However, there were two isolates which showed a
C. M. D.
536 TABLE I-GST FLLKCS
ACTIVIII~S
Ok DIbtLRLNl-
VARIOUS
Isolate
(MOAN
ISOLATES
*
s t M
EXPOSED
l&12)
=
IN IO
AN’IHELMINTICS
GST activitv (units mg protein
Anthelminttc
Unexposed Lancashire Walcha Hampton II Jerangle K Lancashire Comvton Jerangle W Paddock 16 Morven Sofala I
II
OK UNtXPOStD
Closantel Closantcl Closantel Closantcl Rafoxanide Rafoxanide Oxyclozanide Triclabendazole Luxabendazole Luxabendaaole
2.35 0.42 0.64 1.15 2.35 3.48 2.23 0.49 0.24 1.14
f f f f + f f + + f
I)
from
Jerangle
W had
the general relatively
trend: high
0.35 0.07 0.07 0.43 0.35 0.51 0.30 0.06 0.03 0.27
some
levels
which showed no significant change. Amongst isolates exposed to non-salicylanilide anthelmintics, Paddock I6 (triclabcndazolc) and Morvcn (luxabendazolc) showed a trend towards an increase in GST activity following cxposurc while Sofala I (luxabendazolc) showed no change in GST activity following exposure.
Exposed 1.00 0.18 1.25 1.03 1.20 1.51 4.57 1.05 0.78 0.95
f 0.10* f 0.02* f 0.32t jz 0.29 f 0.27* f 0.17* + 0.63t f 0.25 & 0.34 f 0.13
*Significant decrease in GST activity following exposure (P < 0.05 Mann-Whitney c’-test) iSignificant increase in GST activity following exposure (P < 0.05. Mann-Whitney u-test) Other comparisons not signihcant (P > 0.05. Mann Whitney /I-test)
departure
MILLER e/ cd.
specimens
of
of GST activity
Sofala I individuals had relatively low levels for the efficacy displayed. There was also a strong positive correlation between rafoxanide efficacy and closantel efficacy; rs = 0.723 P < 0.05. Inspection of Fig. I also shows that the range of activities among individuals of resistant isolates was less than for susceptible isolates. Activity was measured in individual flukes and values cannot bc assumed to bc normally distributed. Therefore, individual activities are plotted rather than mean S.E M. for the group of flukes. and
Table I shows the values for GST activity obtained from crude homogenates of individual flukes which survived in riro exposure (in sheep) to various anthelmintics along with values obtained from unexposed flukes from the same isolate. The isolates arc grouped according to which anthelmintic they were exposed to. After exposure to salicylanilides (Jcranglc K. Hampton II, Walcha, Lancashire, Compton and Jerangle W) most of the isolates showed a decrease in GST activity, i.e. there appeared to be a greater proportion of individuals with lower GST activity, resulting in a decrease in the mean GST activity for the population of flukes examined. The exceptions were Hampton II (closantel) and Jerangle W (oxyclozanidc). both of which showed a significant increase in GST activity following exposure P i 0.05, Mann-Whitney U-test) and Jerangle K (closantcl)
Isoenzyme profiles of individual flukes were examined using native PAGE and Western blotting in an attempt to link a particular isoenzyme or profile with resistance. However, a large amount of genetic variation was discovered with differences in profiles between and within the isolates. Examples of profiles of individual flukes from Hampton II, Jeranglc K. Compton and Lancashire are shown in Fig. 2. As isoenzymes were separated on the basis of net charge rather than size, it was not possible to make direct comparisons between isolates run on different gels. However. GST purified by Howell et u/. (I 988) was run as a reference marker and it is obvious that no isoenzymc profile, nor indeed any single isocnzymc was common to all isolates. In flukes of the Hampton II isolate a total of 7 different isocnzymcs wcrc detected yielding 5 different banding patterns. 6 isocnzymcs wcrc rccognised in Jeranglc K flukes yielding 4 banding patterns (discounting lanes 3 and 5 where no isoenzymes were detected), IO different isocnzymcs wcrc idcntitied in Compton yielding IO banding patterns and 7 isoenzymes giving 9 dilferent banding patterns wcrc idcntificd in Lancashire. This extensive variation precluded the discovery of a common isoenzyme linked with salicylanilidc rcsistance. However. when exposed and unexposed flukes from Hampton 11 and Common were processed togcthcr thcrc was some evidence of selection of a particular profile following exposure to anthelmintics. as the dominant protilc in a population appcarcd to change following treatment ofthe host. In Hampton II (Fig. 3, upper), in unexposed samples (lanes 7 12) thcrc was a total of 7 different isoenzymes yielding 3 different banding patterns. Lanes 8. IO and I I contained a pattern designated HI and lanes 7 and Y contained H2. Lane 12 was designated H3. In the exposed samples (lanes I--6) thcrc wcrc 8 different isoenzymcs with 4 different banding patterns. All except for one sample (lane 5. designated H4) had a banding pattern that w’as found in the unexposed samples. However. the relative proportions of the banding patterns in cxposcd as opposed to unexposed samples were different (Fig. 3. lower). Pattern HI was found in 50% of unexposed samples but only 17% of cxposcd samples. Likcwisc. H2 was found in 33% of unexposed samples and 5O’% of exposed samples while H3 remained constant
GSTs and salicylanilide
resistance
537
in F. h~~uticu
-‘-
lJ_-_---------_
m-w_----
I q,
-a=
===a5z!s-=---m_
CIC)
=
==3-$ia===-
m--w-
-= ==ptonn
l
,_____--------
m----------___--_
0
11-1-mm------
I=-
1-----11
-
-c3---
=a
a
-
c;I-
=
0
+
,--a=
-
mm
P
lAOC”hh
1
13
45
6
,
8
P
10111lll
1
2
s
4
66
‘I
8
9
1011,213
FIG. 2. Diagrams of Western blots of 12% native PAGE gels of crude homogenates of individual flukes. The blot was probed with serum from sheep injected with fluke GST (Howell er ul., 1988). Negative results with control serum from a colostrumdeprived uninfected sheep confirmed the specificity of the anti-GST serum (data not shown). Approximately 40 pg of protein was loaded for each sample: 2 fig of purified GST (Howell et ~1.. 1988) was loaded as a positive control. Bands of darker intensity are shaded while fainter bands are open. Blots are for: Hampton II: lanes 1 12 samples from individual flukes of this isolate; lane 13 purified GST. Jetangle K: lanes I 12 samples from individual flukes of this isolate; lane 13 purified GST. Compton: lane I purified GST; lanes 2-l 3 samples from individual flukes of this isolate. Lancashire: lane I purified GST; lanes 2-13 samples from individual flukes of this isolate.
between the two groups. Pattern H4 was found in one fluke that had been exposed to anthelmintic but the only difference between H3 and H4 was that a cathodal band was located closer to the origin in H4. None of the banding patterns resembled that of the purified GST of Howell et al. (1988) used as a positive control. In Compton (Fig. 4, upper), in unexposed samples (lanes 2-7) there wcrc 10 diffcrcnt isoenzymes yielding 3 different banding patterns. Lanes 3-6 had identical banding patterns (Cl) while lane 2 had C2 and lane 7 had C3. In exposed samples (lanes 8-13) there were 12 different isoenzymes producing 4 banding patterns, one of which was not found in unexposed samples (lane IO). As seen for Hampton II, the frequency of the banding patterns in exposed flukes differed from unexposed flukes (Fig. 4, lower). Cl occurred in 67% of unexposed samples and 50% of exposed samples while C2 was found in 17% of unexposed flukes and 50% of exposed flukes. The frequency of C3 was constant in the two groups while C4 appeared in one fluke that had been exposed to anthelmintic. Profiles C3 and C4 are similar with the only
only difference being that a cathodal band is located closer to the origin in C4. The GST purified by Howell et al. (1988) again shows its unique nature compared with the other samples. The origin of the flukes the purified GST was derived from is believed to be sheep but the only isoenzyme profiles showing any similarity come from GST purified from flukes isolated from a kangaroo (data not shown). However, it is clear that these GSTs are not related to the isolates studied. Subunit unalysis using SDS-PAGE Western blots of crude homogenates of individual flukes from all isolates (data not shown) separated on SDS-PAGE showed 2 bands (23/23.5 k) reported previously (Howell et d., 1988). Exposure to anthelmintics had no effect on the number of subunits observed (Fig. 5). Although a few Compton individuals also had a third band (24 kDa) slightly larger than the other two (data not shown), the prevalence of this did not change following exposure to anthelmintics. The high mol. wt bands observed (6&70 k and occasionally higher) appear to result from GST binding to other proteins rather than high mol. wt
C. M. D. MILLER efd
538
1
234567
8
9
10
11
12
1s
Isoenzyme
HI
Profile *_
I
2345678
10
9
11
12
13
ISOenZpC3
H2 -
H3 -
H4
Cl
Profile
..-
-
o-
c2
c3
-
-
c4 lYZ=i
+ %Unexposed
50
33
17
0
hUnexposed
67
17
17
0
%Exposed
17
50
17
17
%Exposed
17
50
17
17
FIG. 3. Diagram (upper) of Western blot of 10% native PAGE gel of crude homogenates of individual flukes from Hampton II. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample; 2 pg of purified GST was loaded as a control. Bands of darker intensity are shaded while fainter bands are open. The lower diagram identifies the various isoenzyme profiles found (Hl, H2, H3 and H4) and details the frequency of occurrence of each in exposed and unexposed flukes. Lanes l&6: samples from exposed flukes; lanes 7-12: sampies from unexposed flukes; lane 13: purified GST (Howell er al., 1988).
FIG. 4. Diagram (upper) of Western blot of 10% native PAGE gel of crude homogenates of individual flukes from Compton. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample: 2 pg of purified GST was loaded as a control. Bands of darker intensity are shaded while fainter bands are open. The lower diagram identifies the various isoenzyme profiles found (C 1, C2, C3 and C4) and shows the frequency of occurrence of each in unexposed and exposed samples. Lane 1: purified GST (Howell et al., 1988); lanes 2-7: samples from unexposed flukes: lanes S-13: samples from exposed flukes.
subunits. Alternatively, they could result from nonspecific binding of antibody although a negative control using serum from a color&rum-deprived uninfected sheep confirmed the specificity of the antiGST serum used (data not shown).
rafoxanide was tested on exposed and unexposed flukes from Compton, Lancashire, Hampton II and Jerangte K isolates. The IW value for both anthelmintics were determined for individual fluke homogenates and the results presented as a scattergram (Fig. 6). There appeared to be a trend towards higher closantel I,, vlaues in homogenates of exposed flukes of the relatively susceptible Compton and Lancashire iso-
Inhibition assays The inhibition
of GST
in vitro by closantel
and
GSTs and salicylanilide
resistance
in F. hepatica
539
FIG. 5. Western blot of 10% SDS-PAGE gel of crude homogenates of individual flukes from Compton and Lancashire. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample; 2 pg of purified GST was loaded as a control. Lane 1: purified GST (Howell et al., 1988); lanes 24: samples of exposed Compton flukes; lanes 5-7: samples of unexposed Compton flukes; lanes 8-10: samples of exposed Lancashire flukes; lanes 1 l-13: samples of unexposed Lancashire flukes.
lates, i.e. the number of individuals present with high Iso values was greater following exposure but this was not significant P > 0.05, Mann-Whitney U-test). There appeared to be no differences between closantel Iso values among flukes of the relatively resistant Hampton II and Jerangle K isolates following exposure. Chemotherapy appeared to have the opposite effect on rafoxanide Is0 values among individual homogenates from Compton and Hampton II isolates. There was a trend towards lower Iso values following exposure but the changes were not statistically significant. There were no significant differences between rafoxanide I,, values among flukes of the Lancashire and Jerangle K isolates. DISCUSSION two striking features of GST in F. hepatica. First, there is a strong positive correlation between GST activity (using CDNB) as second substrate) and the efficacy of rafoxanide and closantel in eliminating flukes. In other words, isolates This study
has revealed
in which the efficacies of rafoxanide and closantel were low, generally had low levels of GST activity. This is surprising since GST has generally been found to be overexpressed in drug-resistant cells/organisms (Pickett & Lu, 1989), including H. contortus (Kawalek et al., 1984) and is therefore more usually associated with drug resistance rather than susceptibility. However, most examples of overexpression of GST in association with drug resistance appear to be due to induction of novel isoenzymes (Pickett & Lu, 1989). This phenomenon was not observed in this study. Analysis of the scattergram in Fig. 1 shows a pattern in which susceptible isolates exhibit a wide range of activities among individuals whereas resistant isolates have a narrow range of low activities. Hence, selection may act to eliminate individuals with high GST activity. Evidence that this hypothesis may be correct is provided in Table 1 and further supported by Figs. 3,4 and 6. Thus, comparison of GST activity in adult flukes of isolates exposed to rafoxanide and closantel as juveniles with that in unexposed flukes from the same population generally showed a significant
540
C. M. D. MILLERC~ al. r
,
lides, and two possibilities are apparent. First, Brophy, Southan & Barrett (1989) have shown that GSTs of Moniesiu c.upansa bind anthelmintics rather than conjugate them and it is possible that the same is true of the GSTs of F.~~~~~jc~ (Brophy et al., 1990). Binding of the drug may increase uptake into the parasite and, in the absence of any apparent selection l l v for decreased binding of drug by the enzyme, a 4 mechanism whereby drug uptake could be reduced is through lowered levels of GST. Therefore, lower levels of GST may limit the amount of drug entering the parasite, thereby increasing the chances of parasite survival. Another possibility is that the products of mctabolism of the anthelmintics are more toxic than the drug 0 itself. There is some evidence that not all metabolites V produced by GST conjugation are less toxic than the original compound, particularly for halogenated hydrocarbons (Pickett & Lu, 1989). Metabolism of V 0 halogenated hydrocarbons may also result in the T release of free radicals. For example, in the reaction of GST with CDNB, the initial step in the breakdown of V CDNB involves the replacement of Cl atom with glutathione; a byproduct is HCI. However. the possibility exists of generation of Cl free radicals (Cl.) or aromatic ring-centered free radicals which may be more toxic than the original compound. Most U E u E U E U E flukicides contain halogens and may undergo similar FIG.6. Inhibition of Iluke GST in virro with closantel and metabolism. For example, carbon tetrachloride (a tafoxanide. The I,, values (i.e. that concentration of anthelmintic at which 50% of GST activity is inhibited) were successful flukicide) is metabolised in the liver to determined for each Auke and plotted as a scattergram. produce toxic radical species (Cl.) which cause severe isolates are: Compton (V), Lancashire(C). I-Iampton II (V) membrane damage, probably via lipid peroxidation and Jerangle K (+). (Prichard, 1978). In these circumstances it would also be advantageous to have lower levels of GST and be less efficient at metabolizing the drug. Either possibidecrease in GST activity and a restriction in the range lity discussed could explain the low levels of GST in of activities following exposure (Table I) (the drug-resistant flukes observed in this study. exceptions Hampton II and Jerangle K will be Another interesting point arising from this study is discussed below). In addition, analysis of isoenzyme the apparent specificity of the effect seen. The decrease profiles of exposed and unexposed Compton and in GST activity was confined to isolates exposed to Hampton II flukes showed a change in the proportions rafoxanide and closantel; isolates exposed to other in the population of existing isoenzymes following anthelmintics do not appear to give rise to lower levels exposure rather than induction of new isoenzymes. of GST among surviving fluke populations. The effect Induction cannot be ruled out, however, as native on GST activity may be related to the mode of action PAGE/Western blotting simply shows the presence/ of the drug and its pharmacokinetics in the host. absence of isoenzymes. Therefore, an increase in Rafoxanide and closantel bind very strongly to plasma production of an isoenzyme, particularly one with protein and persist at subtherapeutic concentrations in perhaps low activity using CDNB as second substrate, the host for up to 90 days following treatment, will not be detected. There were also no significant whereas oxyclozanide and luxabendazole reach peak changes in I,, values with closantel and rafoxanide, in the blood very quickly and are which possibly indicates that the ability of F.hepatica concentration excreted equally rapidly (Lacey, 1988; Boray, 1990). GST to bind salicylanilides is unaffected by exposure Triclabendazole is metabolized in the liver into sulfone of isolates to these anthelmintics. and sulphoxide metabolites which bind to serum These results may be indicative of the kind of interaction that occurs between GST and salicylanialbumin and have an extended circulation in this form, I
1
I
GSTs and salic~laniljde although the actual mode of action is unknown (Boray, 1990). Thus, the different modes of action of, and metabolites produced by, the different anthelmintics may require different responses on the part of the parasite. It seems that F. ~e~uf~cff GST is diffcrcnti~lly responsive to different anthelmintics, as would be expected in an enzyme that is multifunctional, and could be expected to be the major line of defence in F. hepuficu against toxins. This plasticity and responsiveness on the part of GST underscores the second striking feature of GST in F. l7epffti~ff - the large amount of variation found in activities and isoenzyme profiles both within and between isolates. Variation appears to be an important feature and may help to explain the anomalies in the data, i.e. Jerdngle K flukes show no difference in activity and Hampton II flukes show a significant increase following exposure to closantel. Different isolates may respond differently to the same stimulus. Individual GST subunits possess a unique substrate specificity and relative concentrations will determine a fluke’s detoxification capacity. Therefore, the repertoire of GST isoenzymes present in the isolates may play a large part in deterlnining the overall response of the parasite population to anthelmintic pressure. In conclusion, there appears to be potential for GST activity to bc used as a marker of closantcl and rafoxanide resistance in F. hepatica. However, the large amount of variation found and the apparent plasticity of the response to various stimuli suggests a multiplicity of functions. There appears to be greater selective pressure to maintain genetic variation and, therefore, the capacity to respond to a variety of situations than to maintain a low level ofGST activity. Evidence that selection for increased variation overrides anthelmintic selection for lower levels of GST activity comes from samples of Hampton If taken during the course of the study. Early in the study exposed flukes had significantly lower levels of GST activity compared with unexposed flukes. Midway through the study exposed flukes showed no significant difference in GST activity compared with unexposed flukes while in the final samples taken exposed flukes had significantly higher levels of GST activity than unexposed flukes (Table 1, C. M. D. Miller, M. J. Howell & J. C. Boray, unpublished observations). Maintaining a high level of variation within a population may help to promote survival of enough parasites to produce a generation able to respond to a variety of circumstances. GST activity is clearly influenced by a multitude of factors indicating that the role of GST in detoxification in F. hepatica is almost certainly a complex phenomenon requiring further studies before GST can be established as a field
resistance
in F. iz~p~r~~u
marker for salicylanilide long
resistance
541 in F. izeputicain the
term.
Acknol~icrlRrme,lr,s-Thanks to Doreen DeBono for technical assistance and Dr Nick Smith for many helpful comments. This work was funded, including the award of an Australian Wool Corporation Postgraduate Scholarship to C. M. D. Miller, by the Australian Wool Corporation. REFERENCES BORAY J. C. & DE BONO D. 1989. Drug resistance in &sciokc heputic~. In: Au.~truliun Adwut7c~.s in ~~~~~jfiur~ Scknc~ (Edited by OUTERIIX P. M. & RICHARDS R. B.), pp. 166169. Australian Veterinary Association, Sydney. BORAY J. C. 1990. Drug resistance in Fusciolu hepaticu In: Reristanw of Parasites to Antiparositic Drugs. Round table conference at the VIIth international Congress of Parasitology Paris August 1990 (Edited by BOKAV J. C., MARTIN P. J. & ROUSH R. T.), pp. 51 60. MSD AGVET Rahway, New Jersey. BRADFORD M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana~vticul Biochemistry 72: 248-254. BROFHY P. M., PAPAUOPOULOS A., TOUKAKI M., COLKS B., KORTING W. &BARRETT J. 1989. Purification ofcytosolic glutathione transferases from Schistowphalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation. Molecular and Biochemicul Parasitology 36: 187-196. BROPHY P. M.. SOUTHAN C.&BARRETT J. 1989. Giutathione transferases in the tapeworm Monkin espnnsa. Biochemical Journal 262: 939-946. BROFHY P. M., CROWLEY P. & BARRETT J. 1990. Detoxification reactions of Fusciolu heputicu cytosolic glutathione transferases. Molecular and Biochemical Para.sitol0g.y 39: 155-l 62. CLARK A. G. 1989. The comparative enzymology of the glu~dthion~ S-Iran&erases from non-vertebrate organisms. ~~~~parati~,e ~iff~hernistr~ urtd Physiology 92B: 419446. DARGIE J. D. 1986. The impact on production and mechanisms of pathogenesis of trematode infections in cattle and sheep. In: Parasi/olog,y-- Quo Vadit?Proceedings of the Sixth International Congress of Parasitology, Brisbane, 1986 (Edited by HOWELL M. .I.), pp. 453-463. Australian Academy of Science, Canberra. HOWELL M. J., BOARD P. G. & BORAV J. C. 1988. Glutathione S-transferases in Fusciola hepatica. Jotrmul of Parasitology 74: 7 15-718. JAFFE J. J. & LAMBERT R. A. 1986. Glutathione S-transferase in adult Dirqjilariu immitis and Brugia pahangi. Molecular and Biochemical Parasitoiog.)’ 20: 199 206. KAWALEK J. C., REW R. S. & HEAVNER J. 1984. Glutathion~ S-transferase, a possible drug-metabolizing enzyme, in Harmonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. lnternationul Journaljbr Purusitology 14: 173-175. LAC~Y E. 1988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to
C. M. D. MILLER rr al.
542
benzimidazoles. International Journcrl,fi,r Parasitology 18: 885-936. MANNERVIK B. & DANIELSON U. H. 1988. Glutathione transferases structure and catalytic activity. CRC Critical
Reviews in Biochemistry
PICKETTCB. & Lu A.Y.H. gene structure,
regulation
Review of Biochemisrry
23: 283 337.
1989. GlutathioneS-transferases: and biological function. Annual 58: 743 -764.
PRKIOUS W.Y.
& BARRETI J. 1989. Xenobiotic metabolism in helminths. Parasitology Today 5: 1% 160. PRICHARDR.K. 1978. Sheep anthelmintics In: T/w Epidemiology and Control
o~Gastro-inrrstinai
Parasites
of’Sheep
iti
(Edited by DONALD A.D., SOUTH~OTTW.H. & DINEEN J.K.), pp. 75-107. Division of Animal Health, CSIRO, Melbourne.
Australia
GL~TATHI~N~ SALICYLANTLTDE
C. M. *Division tElizabeth
of Biochemistry
Macarthur
S-TRANSFERASES AS MARKERS OF RESISTANCE IN ISOLATES OF I;ASCIULA HEPATICA
D.
MILLER,*
M.
J. HOWELL*
and
J. C.
BORAY~$
& Molecular
Agricultural
Biology. Faculty of Science, Australian National University, GPO Box 4, Canberra, A.C.T. 2601, Australia Institute, NSW Department of Agriculture, Private Mail Bag 8, Camden, NSW 2570, Australia
(Rrwiwd
5 July 1993: uccrp~ed 22 Augusr 1993)
A~tract-MlLL~R C. M. D., HOWELL M. J. and BOKAV J. C. 1994. Glutathione S-transferases as markers of salicyianilide resistance in isolates of Fffsciofa hrpulicu. illrernfff~~Jnf~1Journu/jbr Parasifoiogy 24: 533-542. A possible link between the level of glutathione S-transferase (CST, E.C. 2.5.1.18) activity and the development of salicylanilide resistance in Fasciolu hepaticu was investigated. Various isolates of F. heputiccl with varying susceptibilities to salicylanilides were isolated and maintained in the laboratory. Individual flukes of these isolates were surveyed for their level of GST activity and a correlation between the level of GST activity and drug efficacy was found. In contrast to most other studies, a decrease in GST activity was associated with an increase in drug resistance. Evidence was collected to show that this may be a selective process since flukes which had survived exposure to rafoxanide and ciosantel itl siao (in sheep) had lower activity levels of GST than flukes from untreated sheep. Treatment with other flukicides (oxyclozanide, luxabendazole and triclabendazole) did not have this effect. Furthermore, in viva treatment with closantel induced selection of particular isoenzymes in different isolates of F. heppuricuhaving different degrees of susceptibility to closantel. However, no single isoenzyme or isoenzyme profile was associated with resistance and. in total, up to 8 different isoenzymes could be present in a single isolate. Thus, GST has some potential as a marker enzyme for salicylanitide resistance in E: Irepaiica. However. the precise role of GST in resistance is unclear and the extensive inter- and intra-isolate variation in activity levels and isoenzyme characteristics of this enzyme indicate the need for considerably more study before application in field situations. INDEX KEY WORDS:
Fosciolu hepatica: glutathione
INTROJECTION important parasite to the sheep and cattle industries worldwide, causing major losses due to a variety of clinical and subclinical manifestations of fascioliasis (Dargie, 1986). The major form of control of the disease has been through the strategic administration of anthelmintics and among the most successful groups of flukicides in use are salicylanilides, such as rafoxanide and closantel. Resistance to these anthelmintics has been reported in the field, but tests such as the simple egg hatch assay or egg count reduction test to detect anthelmintic resistance in nematodes are not applicable because resistance is often manifest before patency. At present, resistance in F. heparica can be diagnosed only in special laboratories with established
all correspondence
should
salicylanilide
resistance;
sheep
snail colonies. Laboratory-bred intermediate host snails have to be infected with miracidia of the different isolates, metacercariae collected, groups of fluke-free sheep inoculated and the efficacy of titration treatments with anthelmintics evaluated in slaughter trials. The minimum duration of an experiment is approximately 6 months (Boray, 1990). For effective management of drug resistance a suitable test or marker is required to allow rapid identification of resistance to salicylanilides in the field. Glutathione S-transferases (GSTs) (E.C. 2.5.1.18) are a family of multifunctional proteins which appear to be concerned with detoxification of xenobiotics and endogenously derived toxic compounds using catalytic and binding mechanisms (Mannervik & Danielson, 1988; Brophy, Papadopoulos, Touraki, Coles, K6rting & Barrett, 1989). In the apparent absence of the cytochrome P-450 system in helminths (Precious & Barrett, 1989). GSTs are likely to be the principal
THE liver fluke, Fuscinla heputicu, is an economically
:To whom
S-transferases;
be addressed. 533
534
C. M. D.
detoxification enzymes in these parasites. Moreover, numerous studies have linked elevated GST activity levels with the development of drug resistance in a variety of situations including resistance of tumours to chemotherapy (Pickett & Lu, 1989), DDT resistance in the housefly (Clark, 1989) and resistance of Haemonthus contortus to cambendazole (Kawalek, Rew & Heavner, 1984). Previous work has shown that F. heputica contains high levels of GST (Howell, Board & Boray, 1988; Brophy, Crowley & Barrett, 1990), indicating that these enzymes may have an important physiological role. Therefore, the GSTs of F. hepatica were evaluated for use as biochemical markers of salicylanilide resistance. MATERIALS
AND METHODS
Parasites. Various isolates of F. hepatica with differing susceptibilities to various anthelmintics, including rafoxanide and closantel, were recovered from different properties in NSW and subsequently maintained in the laboratory in sheep using lymnaeid snails as intermediate hosts (Boray, & DeBono, 1989; Boray, 1990). Flukes susceptible to salicylanilides were also obtained from Compton Paddock Laboratories, Surrey, U.K. and were also maintained in the laboratory. Adult flukes were recovered 16 weeks pi. (IO weeks after treatment, where applicable) from experimentally infected sheep and the percentage efficacy of the anthelmintic used 6 weeks after inoculation (i.e. before the flukes were fully mature) was determined by comparing fluke counts in the treated sheep and in untreated controls at necropsy. The dose rates of the anthelmintics used in the study were: closantel and rafoxanide, 7.5 mg/kg; oxyclozanide, 26.7 mg/kg; triclabendazole, 4.5 mg/kg; luxabendazole, 7.5 or 11.25 mg/ kg. After removal from sheep, the flukes were placed in Hidon-Fleig saline (120 mMNaC1, 4mMKC1, 0.8 mMCa C1,.2H,O, 2 mMMgSO,.‘TH,O, 1.5% NaHCOj, 4% glucose) at 37°C. Parasites were allowed to disgorge caecal contents before individual flukes were homogenised in 1 ml of 0.1% Triton X-100 (BDH), briefly centrifuged at 12,000 g and the supernatant stored at - 20°C until required. Glurathione S-transferase assays. GST activity was assayed spectrophotometrically at 37°C with 1-chloro-2,4-dinitrobenzene (CDNB) as the standard second substrate following the method of Jaffe & Lambert (1986). The amount of protein in the samples was estimated by the method of Bradford (1976) using purified bovine serum albumin as the standard. One unit of activity is defined as the amount of enzyme required to conjugate 1 ~01 ofCDNB to glutathione (GSH) in 1 min at 37°C. Polyacrylamide gel elecrrophoresis (PAGE) and Western blofiing. Profiles of GST isoenzymes (forms of GST exhibiting charge differences) of samples from individual flukes were analysed by native PAGE (non-reducing conditions without sodium dodecyl sulphate; SDS) followed by Western blotting and probing with antisera to fluke GSTs
MILLER et a/
using standard procedures. An equal volume of 2X sample buffer (125 mMTris_Cl pH 6.8, 20% glycerol, 0.4% bromophenol blue) was added to the samples which were loaded onto 12% polyacrylamide gels and run at 40 mA for 34 h in a Biorad Protean Cell. Purified F. hepatica GST (Howell et ul., 1988) provided by Dr P.G. Board, JCSMR. ANU, was run as an internal control. For Western blotting. the gels were set up in a Biorad transblot apparatus and the proteins transferred to nitrocellulose (Schleicher & Schiill) at 20 v and 150 mA overnight. The nitrocellulose was blocked with 5% skim milk powder (Diploma) in TBS (20 mMTris_Cl pH 7.4, 0.5 MNaCl) then incubated with sheep serum containing antibodies to GST at 1: 100. The anti-GST serum was obtained from sheep that had received 2 injections of purified F. hepaticu GST, 3 weeks apart. The serum was prepared 6 weeks after the 2nd injection. Rabbit anti-sheep Ig coupled to horseradish peroxidase (HRP) (Cappel Laboratories) was used as a second antibody (1 : 1000) and bands were visualised by incubation in Biorad HRP colour development reagent (4-chloro-1-naphthol) dissolved in methanol than added to TBS containing 0.015% H,Oz. For subunit analysis, polyacrylamide gels were run under reducing conditions (SDS--PAGE) following standard procedures. Samples were boiled for 90 s in an equal volume of sample buffer containing 10% SDS and 10% viv p mercaptoethanol prior to loading on to 12% SDS PAGE gels. Purified GST (Howell et al., 1988) was run as an internal control and Rainbow markers (Amersham) were run to allow mol. wt determinations to be made of the subunits. Inhibition assays. Inhibition of GST activity was measured as an I,,, which is defined as the concentration of anthelmintic at which 50% of enzyme activity is inhibited. This was determined by measuring GST activity using CDNB as the second substrate in the presence of 4 different concentrations of closantel (Janssen Pharmaceutics Belgium) dissolved in dimethylsulphoxide (DMSO, BDH), or rafoxanide (Merck, Sharp & Dohme) dissolved in PEG 8000 (Aldrich). The samples were incubated for 5 min at 37°C in the presence of anthelmintic before the reaction was started by the addition of CDNB. Activity was also measured in the presence of DMSO or PEG 8000 without anthelmintic and percentage inhibition in the presence of anthelmintic determined from this value. The percentage inhibition of activity was plotted against the concentration of anthelmintic al which that inhibition occurred and the I,, value estimated from the graph.
RESULTS of individualyukes The level of activity detected, using CDNB as second substrate, was found to vary widely from isolate to isolate and there was also variation in activity from fluke to fluke within an isolate. This ranged from a small difference in some isolates to a considerable difference in others. For example, GST activities of individual flukes in the Morven isolate ranged from 0.13 to 0.38 wol min-lmg protein-‘, whereas GST activities in individual flukes in GST activity in homogenates
GSTs and salicylanilide
in F. hepatica
resistance
535
Rafoxanide 6-
. 6-
Closantel 6-
. 60
f 4-
2-
OL I
I
I
I
I
0
25
50
75
100
Drug Efficacy
(%)
FIG. 1. GST activity in crude homogenates of individual flukes. Each measurement has been plotted against efficacy of closantel and rafoxanide for the isolate. Each point represents specific GST activity in an individual fluke. The regression of GST activity vs efficacy has been plotted 0, = 0.021 x + 0.539; r = 0.494; P < 0.0001 for closantel andy = 0.02x + 0.347; I = 0.417; P < 0.0001 for rafoxanide; Spearman’s rank correlation). Isolates are: Hampton II (V), Walcha (V), Paddock 16 ( n ), Jerangle W (O), Jerangle K (+). Morven ( l ), Lancashire (0). Compton ( V), Sofala I (A).
Compton ranged from 0.27 to 6.52 ~01 min.1 mg protein-‘. When individual activities were plotted against the percentage efficacy of closantel or rafoxanide, strong positive correlations emerged (Fig.
1). The correlations were highly significant with Ye = 0.494, P < 0.0001 for closantel and rs = 0.417, P < 0.0001 for rafoxanide (Spearman’s non-parametric test). However, there were two isolates which showed a
C. M. D.
536 TABLE I-GST FLLKCS
ACTIVIII~S
Ok DIbtLRLNl-
VARIOUS
Isolate
(MOAN
ISOLATES
*
s t M
EXPOSED
l&12)
=
IN IO
AN’IHELMINTICS
GST activitv (units mg protein
Anthelminttc
Unexposed Lancashire Walcha Hampton II Jerangle K Lancashire Comvton Jerangle W Paddock 16 Morven Sofala I
II
OK UNtXPOStD
Closantel Closantcl Closantel Closantcl Rafoxanide Rafoxanide Oxyclozanide Triclabendazole Luxabendazole Luxabendaaole
2.35 0.42 0.64 1.15 2.35 3.48 2.23 0.49 0.24 1.14
f f f f + f f + + f
I)
from
Jerangle
W had
the general relatively
trend: high
0.35 0.07 0.07 0.43 0.35 0.51 0.30 0.06 0.03 0.27
some
levels
which showed no significant change. Amongst isolates exposed to non-salicylanilide anthelmintics, Paddock I6 (triclabcndazolc) and Morvcn (luxabendazolc) showed a trend towards an increase in GST activity following cxposurc while Sofala I (luxabendazolc) showed no change in GST activity following exposure.
Exposed 1.00 0.18 1.25 1.03 1.20 1.51 4.57 1.05 0.78 0.95
f 0.10* f 0.02* f 0.32t jz 0.29 f 0.27* f 0.17* + 0.63t f 0.25 & 0.34 f 0.13
*Significant decrease in GST activity following exposure (P < 0.05 Mann-Whitney c’-test) iSignificant increase in GST activity following exposure (P < 0.05. Mann-Whitney u-test) Other comparisons not signihcant (P > 0.05. Mann Whitney /I-test)
departure
MILLER e/ cd.
specimens
of
of GST activity
Sofala I individuals had relatively low levels for the efficacy displayed. There was also a strong positive correlation between rafoxanide efficacy and closantel efficacy; rs = 0.723 P < 0.05. Inspection of Fig. I also shows that the range of activities among individuals of resistant isolates was less than for susceptible isolates. Activity was measured in individual flukes and values cannot bc assumed to bc normally distributed. Therefore, individual activities are plotted rather than mean S.E M. for the group of flukes. and
Table I shows the values for GST activity obtained from crude homogenates of individual flukes which survived in riro exposure (in sheep) to various anthelmintics along with values obtained from unexposed flukes from the same isolate. The isolates arc grouped according to which anthelmintic they were exposed to. After exposure to salicylanilides (Jcranglc K. Hampton II, Walcha, Lancashire, Compton and Jerangle W) most of the isolates showed a decrease in GST activity, i.e. there appeared to be a greater proportion of individuals with lower GST activity, resulting in a decrease in the mean GST activity for the population of flukes examined. The exceptions were Hampton II (closantel) and Jerangle W (oxyclozanidc). both of which showed a significant increase in GST activity following exposure P i 0.05, Mann-Whitney U-test) and Jerangle K (closantcl)
Isoenzyme profiles of individual flukes were examined using native PAGE and Western blotting in an attempt to link a particular isoenzyme or profile with resistance. However, a large amount of genetic variation was discovered with differences in profiles between and within the isolates. Examples of profiles of individual flukes from Hampton II, Jeranglc K. Compton and Lancashire are shown in Fig. 2. As isoenzymes were separated on the basis of net charge rather than size, it was not possible to make direct comparisons between isolates run on different gels. However. GST purified by Howell et u/. (I 988) was run as a reference marker and it is obvious that no isoenzymc profile, nor indeed any single isocnzymc was common to all isolates. In flukes of the Hampton II isolate a total of 7 different isocnzymcs wcrc detected yielding 5 different banding patterns. 6 isocnzymcs wcrc rccognised in Jeranglc K flukes yielding 4 banding patterns (discounting lanes 3 and 5 where no isoenzymes were detected), IO different isocnzymcs wcrc idcntitied in Compton yielding IO banding patterns and 7 isoenzymes giving 9 dilferent banding patterns wcrc idcntificd in Lancashire. This extensive variation precluded the discovery of a common isoenzyme linked with salicylanilidc rcsistance. However. when exposed and unexposed flukes from Hampton 11 and Common were processed togcthcr thcrc was some evidence of selection of a particular profile following exposure to anthelmintics. as the dominant protilc in a population appcarcd to change following treatment ofthe host. In Hampton II (Fig. 3, upper), in unexposed samples (lanes 7 12) thcrc was a total of 7 different isoenzymes yielding 3 different banding patterns. Lanes 8. IO and I I contained a pattern designated HI and lanes 7 and Y contained H2. Lane 12 was designated H3. In the exposed samples (lanes I--6) thcrc wcrc 8 different isoenzymcs with 4 different banding patterns. All except for one sample (lane 5. designated H4) had a banding pattern that w’as found in the unexposed samples. However. the relative proportions of the banding patterns in cxposcd as opposed to unexposed samples were different (Fig. 3. lower). Pattern HI was found in 50% of unexposed samples but only 17% of cxposcd samples. Likcwisc. H2 was found in 33% of unexposed samples and 5O’% of exposed samples while H3 remained constant
GSTs and salicylanilide
resistance
537
in F. h~~uticu
-‘-
lJ_-_---------_
m-w_----
I q,
-a=
===a5z!s-=---m_
CIC)
=
==3-$ia===-
m--w-
-= ==ptonn
l
,_____--------
m----------___--_
0
11-1-mm------
I=-
1-----11
-
-c3---
=a
a
-
c;I-
=
0
+
,--a=
-
mm
P
lAOC”hh
1
13
45
6
,
8
P
10111lll
1
2
s
4
66
‘I
8
9
1011,213
FIG. 2. Diagrams of Western blots of 12% native PAGE gels of crude homogenates of individual flukes. The blot was probed with serum from sheep injected with fluke GST (Howell er ul., 1988). Negative results with control serum from a colostrumdeprived uninfected sheep confirmed the specificity of the anti-GST serum (data not shown). Approximately 40 pg of protein was loaded for each sample: 2 fig of purified GST (Howell et ~1.. 1988) was loaded as a positive control. Bands of darker intensity are shaded while fainter bands are open. Blots are for: Hampton II: lanes 1 12 samples from individual flukes of this isolate; lane 13 purified GST. Jetangle K: lanes I 12 samples from individual flukes of this isolate; lane 13 purified GST. Compton: lane I purified GST; lanes 2-l 3 samples from individual flukes of this isolate. Lancashire: lane I purified GST; lanes 2-13 samples from individual flukes of this isolate.
between the two groups. Pattern H4 was found in one fluke that had been exposed to anthelmintic but the only difference between H3 and H4 was that a cathodal band was located closer to the origin in H4. None of the banding patterns resembled that of the purified GST of Howell et al. (1988) used as a positive control. In Compton (Fig. 4, upper), in unexposed samples (lanes 2-7) there wcrc 10 diffcrcnt isoenzymes yielding 3 different banding patterns. Lanes 3-6 had identical banding patterns (Cl) while lane 2 had C2 and lane 7 had C3. In exposed samples (lanes 8-13) there were 12 different isoenzymes producing 4 banding patterns, one of which was not found in unexposed samples (lane IO). As seen for Hampton II, the frequency of the banding patterns in exposed flukes differed from unexposed flukes (Fig. 4, lower). Cl occurred in 67% of unexposed samples and 50% of exposed samples while C2 was found in 17% of unexposed flukes and 50% of exposed flukes. The frequency of C3 was constant in the two groups while C4 appeared in one fluke that had been exposed to anthelmintic. Profiles C3 and C4 are similar with the only
only difference being that a cathodal band is located closer to the origin in C4. The GST purified by Howell et al. (1988) again shows its unique nature compared with the other samples. The origin of the flukes the purified GST was derived from is believed to be sheep but the only isoenzyme profiles showing any similarity come from GST purified from flukes isolated from a kangaroo (data not shown). However, it is clear that these GSTs are not related to the isolates studied. Subunit unalysis using SDS-PAGE Western blots of crude homogenates of individual flukes from all isolates (data not shown) separated on SDS-PAGE showed 2 bands (23/23.5 k) reported previously (Howell et d., 1988). Exposure to anthelmintics had no effect on the number of subunits observed (Fig. 5). Although a few Compton individuals also had a third band (24 kDa) slightly larger than the other two (data not shown), the prevalence of this did not change following exposure to anthelmintics. The high mol. wt bands observed (6&70 k and occasionally higher) appear to result from GST binding to other proteins rather than high mol. wt
C. M. D. MILLER efd
538
1
234567
8
9
10
11
12
1s
Isoenzyme
HI
Profile *_
I
2345678
10
9
11
12
13
ISOenZpC3
H2 -
H3 -
H4
Cl
Profile
..-
-
o-
c2
c3
-
-
c4 lYZ=i
+ %Unexposed
50
33
17
0
hUnexposed
67
17
17
0
%Exposed
17
50
17
17
%Exposed
17
50
17
17
FIG. 3. Diagram (upper) of Western blot of 10% native PAGE gel of crude homogenates of individual flukes from Hampton II. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample; 2 pg of purified GST was loaded as a control. Bands of darker intensity are shaded while fainter bands are open. The lower diagram identifies the various isoenzyme profiles found (Hl, H2, H3 and H4) and details the frequency of occurrence of each in exposed and unexposed flukes. Lanes l&6: samples from exposed flukes; lanes 7-12: sampies from unexposed flukes; lane 13: purified GST (Howell er al., 1988).
FIG. 4. Diagram (upper) of Western blot of 10% native PAGE gel of crude homogenates of individual flukes from Compton. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample: 2 pg of purified GST was loaded as a control. Bands of darker intensity are shaded while fainter bands are open. The lower diagram identifies the various isoenzyme profiles found (C 1, C2, C3 and C4) and shows the frequency of occurrence of each in unexposed and exposed samples. Lane 1: purified GST (Howell et al., 1988); lanes 2-7: samples from unexposed flukes: lanes S-13: samples from exposed flukes.
subunits. Alternatively, they could result from nonspecific binding of antibody although a negative control using serum from a color&rum-deprived uninfected sheep confirmed the specificity of the antiGST serum used (data not shown).
rafoxanide was tested on exposed and unexposed flukes from Compton, Lancashire, Hampton II and Jerangte K isolates. The IW value for both anthelmintics were determined for individual fluke homogenates and the results presented as a scattergram (Fig. 6). There appeared to be a trend towards higher closantel I,, vlaues in homogenates of exposed flukes of the relatively susceptible Compton and Lancashire iso-
Inhibition assays The inhibition
of GST
in vitro by closantel
and
GSTs and salicylanilide
resistance
in F. hepatica
539
FIG. 5. Western blot of 10% SDS-PAGE gel of crude homogenates of individual flukes from Compton and Lancashire. The blot was probed as described in Fig. 2. Approximately 40 pg of protein was loaded for each sample; 2 pg of purified GST was loaded as a control. Lane 1: purified GST (Howell et al., 1988); lanes 24: samples of exposed Compton flukes; lanes 5-7: samples of unexposed Compton flukes; lanes 8-10: samples of exposed Lancashire flukes; lanes 1 l-13: samples of unexposed Lancashire flukes.
lates, i.e. the number of individuals present with high Iso values was greater following exposure but this was not significant P > 0.05, Mann-Whitney U-test). There appeared to be no differences between closantel Iso values among flukes of the relatively resistant Hampton II and Jerangle K isolates following exposure. Chemotherapy appeared to have the opposite effect on rafoxanide Is0 values among individual homogenates from Compton and Hampton II isolates. There was a trend towards lower Iso values following exposure but the changes were not statistically significant. There were no significant differences between rafoxanide I,, values among flukes of the Lancashire and Jerangle K isolates. DISCUSSION two striking features of GST in F. hepatica. First, there is a strong positive correlation between GST activity (using CDNB) as second substrate) and the efficacy of rafoxanide and closantel in eliminating flukes. In other words, isolates This study
has revealed
in which the efficacies of rafoxanide and closantel were low, generally had low levels of GST activity. This is surprising since GST has generally been found to be overexpressed in drug-resistant cells/organisms (Pickett & Lu, 1989), including H. contortus (Kawalek et al., 1984) and is therefore more usually associated with drug resistance rather than susceptibility. However, most examples of overexpression of GST in association with drug resistance appear to be due to induction of novel isoenzymes (Pickett & Lu, 1989). This phenomenon was not observed in this study. Analysis of the scattergram in Fig. 1 shows a pattern in which susceptible isolates exhibit a wide range of activities among individuals whereas resistant isolates have a narrow range of low activities. Hence, selection may act to eliminate individuals with high GST activity. Evidence that this hypothesis may be correct is provided in Table 1 and further supported by Figs. 3,4 and 6. Thus, comparison of GST activity in adult flukes of isolates exposed to rafoxanide and closantel as juveniles with that in unexposed flukes from the same population generally showed a significant
540
C. M. D. MILLERC~ al. r
,
lides, and two possibilities are apparent. First, Brophy, Southan & Barrett (1989) have shown that GSTs of Moniesiu c.upansa bind anthelmintics rather than conjugate them and it is possible that the same is true of the GSTs of F.~~~~~jc~ (Brophy et al., 1990). Binding of the drug may increase uptake into the parasite and, in the absence of any apparent selection l l v for decreased binding of drug by the enzyme, a 4 mechanism whereby drug uptake could be reduced is through lowered levels of GST. Therefore, lower levels of GST may limit the amount of drug entering the parasite, thereby increasing the chances of parasite survival. Another possibility is that the products of mctabolism of the anthelmintics are more toxic than the drug 0 itself. There is some evidence that not all metabolites V produced by GST conjugation are less toxic than the original compound, particularly for halogenated hydrocarbons (Pickett & Lu, 1989). Metabolism of V 0 halogenated hydrocarbons may also result in the T release of free radicals. For example, in the reaction of GST with CDNB, the initial step in the breakdown of V CDNB involves the replacement of Cl atom with glutathione; a byproduct is HCI. However. the possibility exists of generation of Cl free radicals (Cl.) or aromatic ring-centered free radicals which may be more toxic than the original compound. Most U E u E U E U E flukicides contain halogens and may undergo similar FIG.6. Inhibition of Iluke GST in virro with closantel and metabolism. For example, carbon tetrachloride (a tafoxanide. The I,, values (i.e. that concentration of anthelmintic at which 50% of GST activity is inhibited) were successful flukicide) is metabolised in the liver to determined for each Auke and plotted as a scattergram. produce toxic radical species (Cl.) which cause severe isolates are: Compton (V), Lancashire(C). I-Iampton II (V) membrane damage, probably via lipid peroxidation and Jerangle K (+). (Prichard, 1978). In these circumstances it would also be advantageous to have lower levels of GST and be less efficient at metabolizing the drug. Either possibidecrease in GST activity and a restriction in the range lity discussed could explain the low levels of GST in of activities following exposure (Table I) (the drug-resistant flukes observed in this study. exceptions Hampton II and Jerangle K will be Another interesting point arising from this study is discussed below). In addition, analysis of isoenzyme the apparent specificity of the effect seen. The decrease profiles of exposed and unexposed Compton and in GST activity was confined to isolates exposed to Hampton II flukes showed a change in the proportions rafoxanide and closantel; isolates exposed to other in the population of existing isoenzymes following anthelmintics do not appear to give rise to lower levels exposure rather than induction of new isoenzymes. of GST among surviving fluke populations. The effect Induction cannot be ruled out, however, as native on GST activity may be related to the mode of action PAGE/Western blotting simply shows the presence/ of the drug and its pharmacokinetics in the host. absence of isoenzymes. Therefore, an increase in Rafoxanide and closantel bind very strongly to plasma production of an isoenzyme, particularly one with protein and persist at subtherapeutic concentrations in perhaps low activity using CDNB as second substrate, the host for up to 90 days following treatment, will not be detected. There were also no significant whereas oxyclozanide and luxabendazole reach peak changes in I,, values with closantel and rafoxanide, in the blood very quickly and are which possibly indicates that the ability of F.hepatica concentration excreted equally rapidly (Lacey, 1988; Boray, 1990). GST to bind salicylanilides is unaffected by exposure Triclabendazole is metabolized in the liver into sulfone of isolates to these anthelmintics. and sulphoxide metabolites which bind to serum These results may be indicative of the kind of interaction that occurs between GST and salicylanialbumin and have an extended circulation in this form, I
1
I
GSTs and salic~laniljde although the actual mode of action is unknown (Boray, 1990). Thus, the different modes of action of, and metabolites produced by, the different anthelmintics may require different responses on the part of the parasite. It seems that F. ~e~uf~cff GST is diffcrcnti~lly responsive to different anthelmintics, as would be expected in an enzyme that is multifunctional, and could be expected to be the major line of defence in F. hepuficu against toxins. This plasticity and responsiveness on the part of GST underscores the second striking feature of GST in F. l7epffti~ff - the large amount of variation found in activities and isoenzyme profiles both within and between isolates. Variation appears to be an important feature and may help to explain the anomalies in the data, i.e. Jerdngle K flukes show no difference in activity and Hampton II flukes show a significant increase following exposure to closantel. Different isolates may respond differently to the same stimulus. Individual GST subunits possess a unique substrate specificity and relative concentrations will determine a fluke’s detoxification capacity. Therefore, the repertoire of GST isoenzymes present in the isolates may play a large part in deterlnining the overall response of the parasite population to anthelmintic pressure. In conclusion, there appears to be potential for GST activity to bc used as a marker of closantcl and rafoxanide resistance in F. hepatica. However, the large amount of variation found and the apparent plasticity of the response to various stimuli suggests a multiplicity of functions. There appears to be greater selective pressure to maintain genetic variation and, therefore, the capacity to respond to a variety of situations than to maintain a low level ofGST activity. Evidence that selection for increased variation overrides anthelmintic selection for lower levels of GST activity comes from samples of Hampton If taken during the course of the study. Early in the study exposed flukes had significantly lower levels of GST activity compared with unexposed flukes. Midway through the study exposed flukes showed no significant difference in GST activity compared with unexposed flukes while in the final samples taken exposed flukes had significantly higher levels of GST activity than unexposed flukes (Table 1, C. M. D. Miller, M. J. Howell & J. C. Boray, unpublished observations). Maintaining a high level of variation within a population may help to promote survival of enough parasites to produce a generation able to respond to a variety of circumstances. GST activity is clearly influenced by a multitude of factors indicating that the role of GST in detoxification in F. hepatica is almost certainly a complex phenomenon requiring further studies before GST can be established as a field
resistance
in F. iz~p~r~~u
marker for salicylanilide long
resistance
541 in F. izeputicain the
term.
Acknol~icrlRrme,lr,s-Thanks to Doreen DeBono for technical assistance and Dr Nick Smith for many helpful comments. This work was funded, including the award of an Australian Wool Corporation Postgraduate Scholarship to C. M. D. Miller, by the Australian Wool Corporation. REFERENCES BORAY J. C. & DE BONO D. 1989. Drug resistance in &sciokc heputic~. In: Au.~truliun Adwut7c~.s in ~~~~~jfiur~ Scknc~ (Edited by OUTERIIX P. M. & RICHARDS R. B.), pp. 166169. Australian Veterinary Association, Sydney. BORAY J. C. 1990. Drug resistance in Fusciolu hepaticu In: Reristanw of Parasites to Antiparositic Drugs. Round table conference at the VIIth international Congress of Parasitology Paris August 1990 (Edited by BOKAV J. C., MARTIN P. J. & ROUSH R. T.), pp. 51 60. MSD AGVET Rahway, New Jersey. BRADFORD M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana~vticul Biochemistry 72: 248-254. BROFHY P. M., PAPAUOPOULOS A., TOUKAKI M., COLKS B., KORTING W. &BARRETT J. 1989. Purification ofcytosolic glutathione transferases from Schistowphalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation. Molecular and Biochemicul Parasitology 36: 187-196. BROPHY P. M.. SOUTHAN C.&BARRETT J. 1989. Giutathione transferases in the tapeworm Monkin espnnsa. Biochemical Journal 262: 939-946. BROFHY P. M., CROWLEY P. & BARRETT J. 1990. Detoxification reactions of Fusciolu heputicu cytosolic glutathione transferases. Molecular and Biochemical Para.sitol0g.y 39: 155-l 62. CLARK A. G. 1989. The comparative enzymology of the glu~dthion~ S-Iran&erases from non-vertebrate organisms. ~~~~parati~,e ~iff~hernistr~ urtd Physiology 92B: 419446. DARGIE J. D. 1986. The impact on production and mechanisms of pathogenesis of trematode infections in cattle and sheep. In: Parasi/olog,y-- Quo Vadit?Proceedings of the Sixth International Congress of Parasitology, Brisbane, 1986 (Edited by HOWELL M. .I.), pp. 453-463. Australian Academy of Science, Canberra. HOWELL M. J., BOARD P. G. & BORAV J. C. 1988. Glutathione S-transferases in Fusciola hepatica. Jotrmul of Parasitology 74: 7 15-718. JAFFE J. J. & LAMBERT R. A. 1986. Glutathione S-transferase in adult Dirqjilariu immitis and Brugia pahangi. Molecular and Biochemical Parasitoiog.)’ 20: 199 206. KAWALEK J. C., REW R. S. & HEAVNER J. 1984. Glutathion~ S-transferase, a possible drug-metabolizing enzyme, in Harmonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. lnternationul Journaljbr Purusitology 14: 173-175. LAC~Y E. 1988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to
C. M. D. MILLER rr al.
542
benzimidazoles. International Journcrl,fi,r Parasitology 18: 885-936. MANNERVIK B. & DANIELSON U. H. 1988. Glutathione transferases structure and catalytic activity. CRC Critical
Reviews in Biochemistry
PICKETTCB. & Lu A.Y.H. gene structure,
regulation
Review of Biochemisrry
23: 283 337.
1989. GlutathioneS-transferases: and biological function. Annual 58: 743 -764.
PRKIOUS W.Y.
& BARRETI J. 1989. Xenobiotic metabolism in helminths. Parasitology Today 5: 1% 160. PRICHARDR.K. 1978. Sheep anthelmintics In: T/w Epidemiology and Control
o~Gastro-inrrstinai
Parasites
of’Sheep
iti
(Edited by DONALD A.D., SOUTH~OTTW.H. & DINEEN J.K.), pp. 75-107. Division of Animal Health, CSIRO, Melbourne.
Australia