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Binding of lindane to locust haemolymph proteins
03~-~92192 $5.00+ 0.00 0 1991Pergamon Press plc
IOlC, No. 1, pp. 137-142,1992
Camp. Biochem. Physiol. Vol.
Printed in Great Britain
BINDING
OF LINDANE TO LOCUST PROTEINS
HAEMOLYMPH
PIERRE BRETON, DICK J. VAN DER HORST,* JAN M. VAN DDDRN and A. M. TH. BEENAKKERS Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands (Telephone 30-533084) (Received 13 lMarch 1991) Abstract-l. Within the scope of effects of sublethal doses of lindane on reproductive physiology of the migratory locust, binding of the insecticide to hemolymph proteins was studied after incubation of locust haemol~ph with [i4C]-lindane. 2. In male haemolymph, radiolabehed lindane is principaliy associated with the major plasma lipoprotein, high density Iipophorin (HDLp), whereas in female haemolymph radiolabel is additionally recovered in vitellogenin. 3. Elevation of the haemolymph titer of vitellogenin by ovariectomy as well as by additional treatment of locusts with the juvenile hormone analogue methoprene (ZR-515) results in increased recovery of lindane in vitellogenin, whereas recovery in HDLp is consecutively decreased.
INTRODUCTION Lindane (y-hexachlorocyclohexane) last organochlorine insecticides still countries, although its application quently discussed (Picot, 1983). Utility
is one of the used in many has been fre-
and efficacy of lindane are due to neurotoxic properties, whereas its persistence is relatively low in comparison to other substances from the same group of insecticides. Studies on the mode of action of lindane, particularly on the central nervous system, have indicated that its primary effect is exerted at the synaptic transmission (Joy, 1982). Different target sites or mechanisms may, however, be additionally involved, leaving the ultimate mode of action of the compound as yet unresolved. The transport of the toxicant in an organism could be important in the understanding of the toxicity of the xenobiotic. Transport of insecticides in target organisms has been investigated extensively during the last two decennaries. Lethal effects due to neurotoxic action were firstly explained by transport of insecticides through the tracheal system, permitting fast access of the insecticide to the brain (Gerolt, 1983). However, implication of the haemolymph as circulatory system is also considered to be an important component of the insect response to toxicants. Binding of insecticides to macromolecules in the haemolymph has been reported (Skalsky and Guthrie, 1977; Nath and Mehrotra, 1982) and it is generally accepted that the principal insect haemolymph lipoprotein (high density lipophorin, HDLp) binds toxins, particularly the hydrophobic ones, since a considerable part of the macromolecule constitutes lipids (Shapiro et al., 1988). Haunerland and Bowers (1986) showed that the proteins responsible for the binding of xenobiotics in larval ~eZio~h~s zeu include
*To whom correspondence
should be addressed.
both arylphorin (a class of larval proteins containing 2-5% of lipids) and HDLp; the distribution of the insecticide between both proteins being dependent on its polarity. The binding of insecticides to haemolymph components is even more important when the intoxication levels are decreased and become sublethal. In such a situation the haemolymph proteins may increase adsorption of xenobiotics from cuticule or midgut, sequester insecticides or deliver them to detoxification sites, or to sites of intoxication, and finally increase the complexity of the metabolism of the xenobiotic in the organism. Sublethal doses of lindane applied to the fly Musca domestica appeared to stimulate the fecundity and fertility, leading to an accrescence of the population in the next generation (Ramade, 1967). In the tobacco hornworm, Munduca sex@ Helling et al. (1986) showed binding of lindane to the juvenile hormone binding protein (JHBP) and demonstrated a competition between Iindane and JH III. The present paper investigates the binding of lindane to haemolymph protein components in Locusta migratoriu, focusing particularly on vitellogenin and HDLp, the two li~proteins implicated in reproductive physiology (Ryan, 1990). MATERIALS AND METHODS Locusta migruroriu were reared in the laboratory under crowded conditions as described earlier (Van der Horst et al., 1978). Only adult locusts were used on different days after ecdysis. To increase the amount of vitellogenin in the haemol~ph of females, in some ex~~ments ovariectomy was performed on day 4 after ecdysis under carbon dioxide anaesthesia through a slit in the fourth abdominal segment, sealing the wound with paraffin wax. Additionally, some of these insects received 100 pg of the juvenile hormone analogue, metboprene (ZR-5 15) in 2 ~1 acetone by topical application on the abdomen on day 9 and day 13. From both experimental groups, haemolymph was collected on day 14.
137
138
PIERRE BRETON ef al.
Haemolymph samples (50 pi/insect) were collected from a puncture in the ventral neck membrane with a microsyringe as described earlier (Van der Horst et al., 1984) and immediately pooled in ice-cold buffer (PH 7.5) containing 130mM NaCl, 5mM KCl, 1.9mM NaH,PO,, 1.7mM K,HPO,, 10 mM EDTA, 10pM phenylmethane sulfonyl fluoride (PMSF) and 50 mM diisopropyl fluorophosphate (DFP); to which 0.02% Na-azide and few crystals of phenylthiourea were added. Haemolymph (final dilution: 1: 1 by vol) was centrifuged at 12,000 g for 10 min at 4°C to remove haemocytes, and the supernatant was used for the incubation experiments.
Thin-layer chromatography After incubation with [‘Y+lindane, fractions were extracted with hexane (1: 1 by vol). The hexane phase was evaporated under a gentle stream of N, to dryness, and applied in a small volume of hexane to a silica gel G thin-layer plate which was run in cyclohexane:chloroform (80: 20 by vol) as used by the manufacturer (Amersham) to verify the radiochemical purity of batches. Radioactive areas on plates were located by means of a Berthold LB 2842
thin-layer chromatogram scanner. RESULTS
Incubation of haemolymph with lindane The haemolymph supernatant was incubated in a shaking waterbath at room temperature with [‘Y+lindane (Amersham International; spec. act. 64 mCi/mmol). Radioactivity of samples of haemolymph or isolated proteins was measured in LSC-Cocktail for aqueous samples (Packard), using a Packard liquid scintillation spectrometer type 4550. Isolation of haemolymph proteins Gel filtration chromatography of haemolymph proteins was performed at 4”C, essentially as described by Van der Horst et al. (1979), using a glass column (100 x 2.6 cm i.d.) packed with Ultrogel AcA 22 (IBF, Villeneuve la Garenne, France). The buffer used for the collection of the haemolymph however supplemented with 5 mM EDTA was used to elute the proteins. Density gradient ultracentrifugation of haemolymph proteins was performed essentially according to Shapiro et al. (1984). Solid KBr was added to a final concentration of 44.3 g KBr/lOOml (density 1.31 g/ml), 18 ml of the solution was placed in a 37ml centrifuge tube and overlayed with 0.9% NaCl (density 1.007 g/ml). Tubes were centrifuged at 49,000 rpm for 4 hr at 4°C in a Sorvall TV 850 vertical rotor. For antibody production, vitellogenin was isolated from haemolymph of ovariectomized female locusts treated with methoprene (see above) by DEAE-cellulose column chromatography according to the procedure of Chinzei et al. (1981).
Figure 1A shows the elution profile resulting from gel filtration of haemolymph (7 ml) from 14 days old female locusts after incubation with 1 PCi [‘4C]-lindane for 24 hr. Radioactivity distribution over the different protein fractions (Fig. 1B) shows that label was only associated with the first protein peak (A). In locust haemolymph, the high density lipophorin (HDLp or A,, mol. wt .~450,000) is recovered in the leading edge of the A-peak, whereas other high molecular weight proteins constitute the trailing edge. (Van der Horst et al., 1979, 1987). In female locusts, vitellogenin (mol. wt -550,000) (Chen et al., 1978; Kempa-Tomm et al., 1990) would be recovered in the same peak. Therefore, after concentration of fractions 90-120 of the proteins of female haemolymph (see Fig. 1) by ultrafiltration in an Amicon cell (PM10 membrane), density gradient ultracentrifugation was used to separate the HDLp from the other protein components in the A-peak. The radioactivity profile (Fig. 2) indicates a nearly equal repartition of label between HDLp and the higher density protein fraction. Nearly identical results were obtained using hemolymph of 12 days old female locusts (results not shown).
Gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) of haemolymph proteins was performed in gradient gels (410%) at 20mA until the Bromophenol Blue dye front neared the bottom of the gel. The proteins were fixed and stained with Coomassie Brillant Blue R 250. In some experiments, pieces of gel containing the separated proteins were cut out, dissolved in Lumasolve (Packard) overnight at 50°C whereafter the radioactivity was counted. In other experiments proteins were electrophoretically transferred to nitrocellulose paper for 4 hr at 80 V (400 mA) as described previously (Schulz et al., 1987). Nitrocellulose blots were stained with 0.05% Amido black 10B and destained in methanol-acetic acid-water (45 : 10: 45 by vol) and used for immunostaining.
A
1.6-
A
1.6-
j
salt
:::: l.O0% 0.6s B
0.4 0.2I
C
: 60
100 120
140 160 Fraction’nr
P
Immunological procedures The preparation of a polyclonal (rabbit) antiserum specific for HDLp was described earlier (Schulz ef al., 1991). Using identical techniques, a polyclonal antiserum specific for locust vitellogenin was obtained. Both antisera were used for immunostaining. Additionally, enzyme-linked immunosorbent assays (ELISA) or locust haemolymph proteins were carried out as described before (Schulz et al., 1987). Determination of protein and lipid Protein concentrations were estimated by the Lowry method as modified by Schacterle and Pollack (1973). Concentrations of total lipids in haemolymph were determined by the vanillin method as modified by Holwerda et al. (1977).
d
60 65 90 95 100106110115120125130135140 Fraction number
Fig. 1. Separation of hemolymph proteins on Ultrogel AcA 22 after incubation of haemolymph from lCday-old female locusts with [WI-labelled lindane. A: elution profile. Peaks are numbered in order of elution; B: radioactivity of the eluted protein fractions.
Binding of lindane to locust haemolymph proteins
Fraction
139
number
Fig. 2. Radioactivity distribution over HDLp (a) and the higher density proteins (b) after density gradient ultracentrifugation of the A-peak of Fig. 1. Similar ultracentrifugation experiments on binding of the radiolabelled insecticide to haemolymph of male locusts (12 days old) showed, however, that although radiolabel in HDLp was comparable, the higher density protein fraction contained considerably less radioactivity. The same holds for younger (9-day-old) females. This suggests that in mature female locusts this high density fraction contains a female-specific protein able to bind lindane. Using Ouchterlony immunodiffusion, it appeared that vitellogenin was present in this high density protein fraction, as was to be expected since the vitellogenin titer in Locusta females peaks at day 12 of the adult life (Chinzei and Wyatt, 1985) (results not shown). Incubation time (0.54 hr) has hardly any effect on the distribution of the radiolabelled lindane over the protein fractions in the haemol~ph {Fig. 3); only a very low variation is observed, Apparently, the process of association of the insecticide with protein is rather fast. To test the importance of the vitellogenin titer on the binding capacity of lindane in the haemol~ph, an incubation time of 1 hr was taken. Haemolymph was collected from four different groups of locusts (all 14 days old), in which increasing levels of vitellogenin were expected: 1. Males, 2. Control females, 3. Ovariectomized females, and 4. Ova~ecto~zed and methoprene-treated females (see Materials and Methods). Haemolymph samples were incubated with 1 y Ci [i4C]-lindane and subsequently ultracentrifuged. The radiolabel profile of the separation between HDLp and the higher density proteins (in-
Fig. 4. Ultracentrifugation of haemolymph from four different groups of locusts (all 14 days after adult ecdysis) incubated for 1 hr with radiolabelled lindane. 0-O: males; +---0: control females; A-----A: ovari~omi~d females; A---& ovariectomized + methoprene-treated females. A: radiolabel distribution over the different fractions; B: protein contents.
eluding vitellogenin) is shown in Fig. 4A; protein determination of the fractions is presented in Fig. 4B. From these data it appears that when the amount of vitellogenin increases, the radiolabel associated with this fraction also increases, at the expense of the radioactivity present in HDLp. The level of HDLp is equivalent in all four samples. The presence of vitellogenin in the female haemolymph samples is demonstrated by ELISA in Fig. 5. When haemolymph samples of the four experimental groups were subjected to native PAGE and blotted to nitrocellulose paper (Fig. 6A), radioscanning revealed that the most of the radiolabel was recovered at the top of the blot (Fig. 6B), however, not associated with detectable protein bands: both HDLp and vitellogenin (which were identified by immunoblotting, Fig. 6C) contained hardly any label. Apparently, the electrophoretic techniques used detach most of
Incubi&n time ih)
Fig. 3. Proportional distribution of radioactivity in HDLp (a) and the higher density proteins (b) separated by density gradient ultracent~fugation of total haemolymph samples after incubation with r~iola~lled lindane for OS-4 hr.
Fig. 5. ELISA of locust haemolymph (10 ng per well) using the polycional antiserum specific for vitellogenin. The four different locust groups tested are as in Fig. 4.
PIERREBRETONet al.
140
HDLP-
5
6
Fig. 6. Electrophoresis of haemolymph proteins (500 pug)on native gradient PAGE. A: blot of the gel onto nitrocellulose. The four experimental groups of locusts as in Fig. 4 were used. Lane 1: males; lane 2: control females; lane 3: ovariectomized females; lane 4: ovariectomized + methoprene-treated females; B: radioscanning of lane 4 of the blot, illustrating that the [14C]-lindane is no longer associated with protein bands; C: identification of HDLp (lane 5) and vitellogenin (Vg; lane 6) by immunoblotting, using polyclonal antisera specific for HDLp and Vg. the insecticide from the protein; additionally the radioscanning may be not sufficiently sensitive to detect remaining radiolabel in the protein bands. Therefore, the gel was directly stained after native PAGE, the different bands cut out and their radiolabe1 counted by ~intillation spectrometry. This procedure rest&d in recovery of radioactivity, which was located virtually only in HDLp and vitellogenin (Table 1); total recovery however was only very low in comparison to the initial radiolabel present in the samples. Finally it was verified that the radioactivity present in the separated fractions is still attributable to the
lindane, and that there is no degradation of the insecticide in the incubation system. After extraction of fractions with hexane, thin-layer chromatography was performed with authentic lindane as a reference (Fig. 7). DISCUSSION The data presented lindane to specifically
here show lipid-binding
the binding of locust haemo-
Table 1. Proportional recovery (in %) of radiolabel in HDLp and vitellogenin after separation of haemolymph proteins by native gradient PAGE and cutting out individual protein bands from the gel Group 1’ 2 3 4
HDLp 100 91.3 56.0 54.1
Viteliogenin 8.7 44.0 45.3
*I: males; 2: control females; 3: ovariectomized females; 4: ovariectomized + methoprenetreated females.
Fig. 7. Perspective view of thin-layer chromatogram scanning of the radioactivity originally associated with the high density protein fraction (1) and HDLp (2) from Fig. 2, and extracted with hexane, in comparison to labelled lindane (Amersham) (3) and labelled lindane diluted with cold lindane (4).
Binding of lindane to locu1st haemolymph proteins lymph components. After incubation of haemolymph of male locusts with radiolabelled lindane, HDLp particularly, appeared to be involved in the binding of the insecticide, whereas in females the radiolabel is additionally recovered in vitellogenin. Experimentally, the haemolymph vitellogenin titer was increased by ovariectomy, whereas additional treatment with the juvenile hormone analogue methoprene was applied to stimulate vitellogenin synthesis. Interestingly, whereas the level of HDLp was not affected in these insects, the binding of lindane to this component decreases when the vitellogenin concentration (and binding capacity) increases. In insect oocytes, vitellogenin is selectively sequestered by receptor-mediated endocytosis, a process first proposed for mosquito oocytes by Roth and Porter (1964). The vitellogenin-binding protein from locust ovaries has been recently isolated (Roehrkasten et al., 1989). The accumulation of lindane in maturing oocytes will result in part from this pathway, and additionally from the insecticide bound to HDLp which is also taken up and provides a major source of the lipid reserves in the eggs (Kawooya and Law, 1988). Still, vitellogenin remains of major interest since, for instance, after application of sublethal doses of lindane to Musca domestica, the level of haemolymph proteins, particularly that of female-specific proteins, was decreased (Le Bras et al., 1973). Since lindane is known to bind to different receptors (Abalis et al., 1985), to act on cholinergic synapses (Beeman, 1982) and to affect the fluidity of membranes (Antunes-Madeira and Madeira, 1989), it can be stated that only by integration of studies on the actions of the insecticide on its different target sites may the effect of sublethal doses of lindane on reproduction be understood. thank A. Th. M. Van den Broek for technical assistance on the experiments involving ovariectomy and topical application of the juvenile hormone analogue. Methoprene (ZR-515) was a generous gift from Dr G. B. Staal, Zoicon Corporation, Palo Alto (CA). A visiting grant to P.B. from the Dutch Ministery for Education is gratefully acknowledged. Acknowledgements-We
141
Gerolt P. (1983) Insecticides: their route of entry, mechanism of transport and mode of action. Biol. Rev. 58, 233-274.
Haunerland N. H. and Bowers W. S. (1986) Binding of insecticides to lipophorin and arylphorin, two hemolymph proteins of Heliothis zea. Arch. Insect Physiol. Biochem.
3, 87-96.
Helling D. J., Brownie C. and Guthrie F. E. (1986) Binding of insecticides by hemolymph proteins of the tobacco hornworm, Manduca sexta (L.). Pestic. Biochem. Physiol. 25, 125-I 32.
Holwerda D. A., Van Doom J. M. and Beenakkers A. M. Th. (1977) Characterization of the adipokinetic and hyperglycaemic substances from the locust corpus cardia&m_&ect Biochem. 7, 151-157. Joy R. M. (1982) Mode of action of lindane, dieldrin and related insecticides in the central nervous system. Neurobehav.
Toxicol.
Teratol. 4, 813-823.
Kawoova J. K. and Law J. H. (1988) Role of lioonhorin in lipid -transport to the insec‘t egi. J. biol. %em. 263, 8748-8753.
Kempa-Tomm S., Hoffmann K. H. and Engelmann F. (1990) Vitellogenins and vitellins of the mediterranean field cricket, Gryllus bimaculatus: isolation, characterization and quantification. Physiol. Entomol. 15, 167-178. Le Bras S., Echaubard M. and Ramade F. (1973) Variations de la protCinhCmie chez Musca domestica au tours d’un cycle gonotrophique dans les conditions normales et apr&s action d’agents toxiaues. Bull. Sot. 2001. 98. 385403. Nath A. an> MehroGa K. N. (1982) Binding of [“‘Cl p, p’-DDT with haemolymph protein of the desert locust Schistocerca gregaria (F.). Indian J. exp. Biol. 20, 701-703.
Picot A. (1983) Le lindane un insecticide en sursis? Ln Recherche
14, 1584-1587.
Ramade F. (1967) Contribution $ l’Ctude du mode d’action de certains insecticides de synthbse, plus particullrement du lindane, et des phtnomines de rksistance B ces composes chez Musca domestica (L.). Ann. Inst. Nat. Agron. Paris, tome V, N.S., l-268. Roehrkasten A., Ferenz H.-J., Buschmann-Gebhardt B. and Hafer J. (1989) Isolation of the vitellogenin-binding protein from locust ovaries. Arch. Insect Biochem. Physiol. 10, 141-149. Roth T. F. and Porter K. R. (1964) Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. J. Cell Biol. 20, 313-332.
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Ryan R. 0. (1990) Dynamics of insect lipophorin metabolism. J. Lipid Res. 31, 1725-1739. Schacterle G. R. and Pollack R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biological material. Analyt. Biochem. 51,
Abalis I. M., Eldefrawi M. E. and Eldefrawi A. T. (1985) High-affinity stereospecific binding of cyclodiene insecticides and y-hexachlorocyclohexane to y-amino butyric acid receptors of rat brain. Pestic. Biochem. Physiol. 24,
Schulz T. K. F., Van der Horst D. J., Amesz H., Voorma H. 0. and Beenakkers A. M. Th. (1987) Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust. Arch. Insect Biochem. Physiol. 6,
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Antunes-Madeira M. C. and Madeira V. M. C. (1989) Membrane fluidity as affected by the insecticide lindane. Biochim.
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Beemann R. W. (1982) Recent advances in mode of action of insecticides. A. Rev. Entomol. 27, 253-281. Chen T. T., Strahlendorf P. W. and Wvatt G. R. (1978) Vitellin and vitellogenin from locusts :Locusta m&a,: ria). J. biol. Chem. 253. 5325-5331. Chintei Y. and Wyatt 6. R. (1985) Vitellogenin titre in haemolymph of Locusta migratoria in normal adults, after ovariectomy, and in response to methoprene. J. Insect Physiol.
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Chinzei Y., Chino H. and Wyatt G. R. (1981) Purification of vitellogenin and vitellin from Locusra migratoria. Insect Biochem.
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Schulz T. K. F., Van der Horst D. J. and Beenakkers A. M. Th. (1991) Binding of locust high density lipophorin to fat body proteins monitored by an enzymelinked immunosorbant assay. Biol. Chem. Hoppe-Seyler 372, 5-12.
Shapiro J. P. (1988) Lipid transport Enromol.
in insects. A. Reo.
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Shapiro J. P., Keim P. S. and Law J. H. (1984) Structural studies on lipophorin: an insect lipoprotein. J. biol. Chem. 259, 3680-3685.
Skalsky H. L. and Guthrie F. E. (1977) Affinities of parathion, DDT, dieldrin, and carbarvl for macromolecules in the blood of the rat and American cockroach and the competitive interaction of steroids. Pestic. Biochem.
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Van der Horst D. J., Baljet A. M. C., Beenakkers A. M. Th. and Van Handel E. (1978) Turnover of locust haemolymph diglycerides during flight and rest. Insect Biochem. 8, 369-373. Van der Horst D. J., Van Doom J. M. and Beenakkers A. M. Th. (1979) Effects of the adipokinetic hormone on the release and turnover of haemolymph diglycerides and on the formation of the diglyceride-transporting lipoprotein system during locust flight. Insect Biochem. 9, 627435.
Van der Horst D. J., Van Doom J. M. and Beenakkers A. M. Th. (1984) Hormone-induced rearrangement of locust haemolymph lipoproteins: the involvement of glycoprotein C,. Insect Biochem. 14, 495-504. Van der Horst D. J., Beenakkers A. M. Th., Van Doom J. M., Gerritse K. and Schulz T. K. F. (1987) Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts. Insect Biochem. 17, 799-808.
IOlC, No. 1, pp. 137-142,1992
Camp. Biochem. Physiol. Vol.
Printed in Great Britain
BINDING
OF LINDANE TO LOCUST PROTEINS
HAEMOLYMPH
PIERRE BRETON, DICK J. VAN DER HORST,* JAN M. VAN DDDRN and A. M. TH. BEENAKKERS Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands (Telephone 30-533084) (Received 13 lMarch 1991) Abstract-l. Within the scope of effects of sublethal doses of lindane on reproductive physiology of the migratory locust, binding of the insecticide to hemolymph proteins was studied after incubation of locust haemol~ph with [i4C]-lindane. 2. In male haemolymph, radiolabehed lindane is principaliy associated with the major plasma lipoprotein, high density Iipophorin (HDLp), whereas in female haemolymph radiolabel is additionally recovered in vitellogenin. 3. Elevation of the haemolymph titer of vitellogenin by ovariectomy as well as by additional treatment of locusts with the juvenile hormone analogue methoprene (ZR-515) results in increased recovery of lindane in vitellogenin, whereas recovery in HDLp is consecutively decreased.
INTRODUCTION Lindane (y-hexachlorocyclohexane) last organochlorine insecticides still countries, although its application quently discussed (Picot, 1983). Utility
is one of the used in many has been fre-
and efficacy of lindane are due to neurotoxic properties, whereas its persistence is relatively low in comparison to other substances from the same group of insecticides. Studies on the mode of action of lindane, particularly on the central nervous system, have indicated that its primary effect is exerted at the synaptic transmission (Joy, 1982). Different target sites or mechanisms may, however, be additionally involved, leaving the ultimate mode of action of the compound as yet unresolved. The transport of the toxicant in an organism could be important in the understanding of the toxicity of the xenobiotic. Transport of insecticides in target organisms has been investigated extensively during the last two decennaries. Lethal effects due to neurotoxic action were firstly explained by transport of insecticides through the tracheal system, permitting fast access of the insecticide to the brain (Gerolt, 1983). However, implication of the haemolymph as circulatory system is also considered to be an important component of the insect response to toxicants. Binding of insecticides to macromolecules in the haemolymph has been reported (Skalsky and Guthrie, 1977; Nath and Mehrotra, 1982) and it is generally accepted that the principal insect haemolymph lipoprotein (high density lipophorin, HDLp) binds toxins, particularly the hydrophobic ones, since a considerable part of the macromolecule constitutes lipids (Shapiro et al., 1988). Haunerland and Bowers (1986) showed that the proteins responsible for the binding of xenobiotics in larval ~eZio~h~s zeu include
*To whom correspondence
should be addressed.
both arylphorin (a class of larval proteins containing 2-5% of lipids) and HDLp; the distribution of the insecticide between both proteins being dependent on its polarity. The binding of insecticides to haemolymph components is even more important when the intoxication levels are decreased and become sublethal. In such a situation the haemolymph proteins may increase adsorption of xenobiotics from cuticule or midgut, sequester insecticides or deliver them to detoxification sites, or to sites of intoxication, and finally increase the complexity of the metabolism of the xenobiotic in the organism. Sublethal doses of lindane applied to the fly Musca domestica appeared to stimulate the fecundity and fertility, leading to an accrescence of the population in the next generation (Ramade, 1967). In the tobacco hornworm, Munduca sex@ Helling et al. (1986) showed binding of lindane to the juvenile hormone binding protein (JHBP) and demonstrated a competition between Iindane and JH III. The present paper investigates the binding of lindane to haemolymph protein components in Locusta migratoriu, focusing particularly on vitellogenin and HDLp, the two li~proteins implicated in reproductive physiology (Ryan, 1990). MATERIALS AND METHODS Locusta migruroriu were reared in the laboratory under crowded conditions as described earlier (Van der Horst et al., 1978). Only adult locusts were used on different days after ecdysis. To increase the amount of vitellogenin in the haemol~ph of females, in some ex~~ments ovariectomy was performed on day 4 after ecdysis under carbon dioxide anaesthesia through a slit in the fourth abdominal segment, sealing the wound with paraffin wax. Additionally, some of these insects received 100 pg of the juvenile hormone analogue, metboprene (ZR-5 15) in 2 ~1 acetone by topical application on the abdomen on day 9 and day 13. From both experimental groups, haemolymph was collected on day 14.
137
138
PIERRE BRETON ef al.
Haemolymph samples (50 pi/insect) were collected from a puncture in the ventral neck membrane with a microsyringe as described earlier (Van der Horst et al., 1984) and immediately pooled in ice-cold buffer (PH 7.5) containing 130mM NaCl, 5mM KCl, 1.9mM NaH,PO,, 1.7mM K,HPO,, 10 mM EDTA, 10pM phenylmethane sulfonyl fluoride (PMSF) and 50 mM diisopropyl fluorophosphate (DFP); to which 0.02% Na-azide and few crystals of phenylthiourea were added. Haemolymph (final dilution: 1: 1 by vol) was centrifuged at 12,000 g for 10 min at 4°C to remove haemocytes, and the supernatant was used for the incubation experiments.
Thin-layer chromatography After incubation with [‘Y+lindane, fractions were extracted with hexane (1: 1 by vol). The hexane phase was evaporated under a gentle stream of N, to dryness, and applied in a small volume of hexane to a silica gel G thin-layer plate which was run in cyclohexane:chloroform (80: 20 by vol) as used by the manufacturer (Amersham) to verify the radiochemical purity of batches. Radioactive areas on plates were located by means of a Berthold LB 2842
thin-layer chromatogram scanner. RESULTS
Incubation of haemolymph with lindane The haemolymph supernatant was incubated in a shaking waterbath at room temperature with [‘Y+lindane (Amersham International; spec. act. 64 mCi/mmol). Radioactivity of samples of haemolymph or isolated proteins was measured in LSC-Cocktail for aqueous samples (Packard), using a Packard liquid scintillation spectrometer type 4550. Isolation of haemolymph proteins Gel filtration chromatography of haemolymph proteins was performed at 4”C, essentially as described by Van der Horst et al. (1979), using a glass column (100 x 2.6 cm i.d.) packed with Ultrogel AcA 22 (IBF, Villeneuve la Garenne, France). The buffer used for the collection of the haemolymph however supplemented with 5 mM EDTA was used to elute the proteins. Density gradient ultracentrifugation of haemolymph proteins was performed essentially according to Shapiro et al. (1984). Solid KBr was added to a final concentration of 44.3 g KBr/lOOml (density 1.31 g/ml), 18 ml of the solution was placed in a 37ml centrifuge tube and overlayed with 0.9% NaCl (density 1.007 g/ml). Tubes were centrifuged at 49,000 rpm for 4 hr at 4°C in a Sorvall TV 850 vertical rotor. For antibody production, vitellogenin was isolated from haemolymph of ovariectomized female locusts treated with methoprene (see above) by DEAE-cellulose column chromatography according to the procedure of Chinzei et al. (1981).
Figure 1A shows the elution profile resulting from gel filtration of haemolymph (7 ml) from 14 days old female locusts after incubation with 1 PCi [‘4C]-lindane for 24 hr. Radioactivity distribution over the different protein fractions (Fig. 1B) shows that label was only associated with the first protein peak (A). In locust haemolymph, the high density lipophorin (HDLp or A,, mol. wt .~450,000) is recovered in the leading edge of the A-peak, whereas other high molecular weight proteins constitute the trailing edge. (Van der Horst et al., 1979, 1987). In female locusts, vitellogenin (mol. wt -550,000) (Chen et al., 1978; Kempa-Tomm et al., 1990) would be recovered in the same peak. Therefore, after concentration of fractions 90-120 of the proteins of female haemolymph (see Fig. 1) by ultrafiltration in an Amicon cell (PM10 membrane), density gradient ultracentrifugation was used to separate the HDLp from the other protein components in the A-peak. The radioactivity profile (Fig. 2) indicates a nearly equal repartition of label between HDLp and the higher density protein fraction. Nearly identical results were obtained using hemolymph of 12 days old female locusts (results not shown).
Gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) of haemolymph proteins was performed in gradient gels (410%) at 20mA until the Bromophenol Blue dye front neared the bottom of the gel. The proteins were fixed and stained with Coomassie Brillant Blue R 250. In some experiments, pieces of gel containing the separated proteins were cut out, dissolved in Lumasolve (Packard) overnight at 50°C whereafter the radioactivity was counted. In other experiments proteins were electrophoretically transferred to nitrocellulose paper for 4 hr at 80 V (400 mA) as described previously (Schulz et al., 1987). Nitrocellulose blots were stained with 0.05% Amido black 10B and destained in methanol-acetic acid-water (45 : 10: 45 by vol) and used for immunostaining.
A
1.6-
A
1.6-
j
salt
:::: l.O0% 0.6s B
0.4 0.2I
C
: 60
100 120
140 160 Fraction’nr
P
Immunological procedures The preparation of a polyclonal (rabbit) antiserum specific for HDLp was described earlier (Schulz ef al., 1991). Using identical techniques, a polyclonal antiserum specific for locust vitellogenin was obtained. Both antisera were used for immunostaining. Additionally, enzyme-linked immunosorbent assays (ELISA) or locust haemolymph proteins were carried out as described before (Schulz et al., 1987). Determination of protein and lipid Protein concentrations were estimated by the Lowry method as modified by Schacterle and Pollack (1973). Concentrations of total lipids in haemolymph were determined by the vanillin method as modified by Holwerda et al. (1977).
d
60 65 90 95 100106110115120125130135140 Fraction number
Fig. 1. Separation of hemolymph proteins on Ultrogel AcA 22 after incubation of haemolymph from lCday-old female locusts with [WI-labelled lindane. A: elution profile. Peaks are numbered in order of elution; B: radioactivity of the eluted protein fractions.
Binding of lindane to locust haemolymph proteins
Fraction
139
number
Fig. 2. Radioactivity distribution over HDLp (a) and the higher density proteins (b) after density gradient ultracentrifugation of the A-peak of Fig. 1. Similar ultracentrifugation experiments on binding of the radiolabelled insecticide to haemolymph of male locusts (12 days old) showed, however, that although radiolabel in HDLp was comparable, the higher density protein fraction contained considerably less radioactivity. The same holds for younger (9-day-old) females. This suggests that in mature female locusts this high density fraction contains a female-specific protein able to bind lindane. Using Ouchterlony immunodiffusion, it appeared that vitellogenin was present in this high density protein fraction, as was to be expected since the vitellogenin titer in Locusta females peaks at day 12 of the adult life (Chinzei and Wyatt, 1985) (results not shown). Incubation time (0.54 hr) has hardly any effect on the distribution of the radiolabelled lindane over the protein fractions in the haemol~ph {Fig. 3); only a very low variation is observed, Apparently, the process of association of the insecticide with protein is rather fast. To test the importance of the vitellogenin titer on the binding capacity of lindane in the haemol~ph, an incubation time of 1 hr was taken. Haemolymph was collected from four different groups of locusts (all 14 days old), in which increasing levels of vitellogenin were expected: 1. Males, 2. Control females, 3. Ovariectomized females, and 4. Ova~ecto~zed and methoprene-treated females (see Materials and Methods). Haemolymph samples were incubated with 1 y Ci [i4C]-lindane and subsequently ultracentrifuged. The radiolabel profile of the separation between HDLp and the higher density proteins (in-
Fig. 4. Ultracentrifugation of haemolymph from four different groups of locusts (all 14 days after adult ecdysis) incubated for 1 hr with radiolabelled lindane. 0-O: males; +---0: control females; A-----A: ovari~omi~d females; A---& ovariectomized + methoprene-treated females. A: radiolabel distribution over the different fractions; B: protein contents.
eluding vitellogenin) is shown in Fig. 4A; protein determination of the fractions is presented in Fig. 4B. From these data it appears that when the amount of vitellogenin increases, the radiolabel associated with this fraction also increases, at the expense of the radioactivity present in HDLp. The level of HDLp is equivalent in all four samples. The presence of vitellogenin in the female haemolymph samples is demonstrated by ELISA in Fig. 5. When haemolymph samples of the four experimental groups were subjected to native PAGE and blotted to nitrocellulose paper (Fig. 6A), radioscanning revealed that the most of the radiolabel was recovered at the top of the blot (Fig. 6B), however, not associated with detectable protein bands: both HDLp and vitellogenin (which were identified by immunoblotting, Fig. 6C) contained hardly any label. Apparently, the electrophoretic techniques used detach most of
Incubi&n time ih)
Fig. 3. Proportional distribution of radioactivity in HDLp (a) and the higher density proteins (b) separated by density gradient ultracent~fugation of total haemolymph samples after incubation with r~iola~lled lindane for OS-4 hr.
Fig. 5. ELISA of locust haemolymph (10 ng per well) using the polycional antiserum specific for vitellogenin. The four different locust groups tested are as in Fig. 4.
PIERREBRETONet al.
140
HDLP-
5
6
Fig. 6. Electrophoresis of haemolymph proteins (500 pug)on native gradient PAGE. A: blot of the gel onto nitrocellulose. The four experimental groups of locusts as in Fig. 4 were used. Lane 1: males; lane 2: control females; lane 3: ovariectomized females; lane 4: ovariectomized + methoprene-treated females; B: radioscanning of lane 4 of the blot, illustrating that the [14C]-lindane is no longer associated with protein bands; C: identification of HDLp (lane 5) and vitellogenin (Vg; lane 6) by immunoblotting, using polyclonal antisera specific for HDLp and Vg. the insecticide from the protein; additionally the radioscanning may be not sufficiently sensitive to detect remaining radiolabel in the protein bands. Therefore, the gel was directly stained after native PAGE, the different bands cut out and their radiolabe1 counted by ~intillation spectrometry. This procedure rest&d in recovery of radioactivity, which was located virtually only in HDLp and vitellogenin (Table 1); total recovery however was only very low in comparison to the initial radiolabel present in the samples. Finally it was verified that the radioactivity present in the separated fractions is still attributable to the
lindane, and that there is no degradation of the insecticide in the incubation system. After extraction of fractions with hexane, thin-layer chromatography was performed with authentic lindane as a reference (Fig. 7). DISCUSSION The data presented lindane to specifically
here show lipid-binding
the binding of locust haemo-
Table 1. Proportional recovery (in %) of radiolabel in HDLp and vitellogenin after separation of haemolymph proteins by native gradient PAGE and cutting out individual protein bands from the gel Group 1’ 2 3 4
HDLp 100 91.3 56.0 54.1
Viteliogenin 8.7 44.0 45.3
*I: males; 2: control females; 3: ovariectomized females; 4: ovariectomized + methoprenetreated females.
Fig. 7. Perspective view of thin-layer chromatogram scanning of the radioactivity originally associated with the high density protein fraction (1) and HDLp (2) from Fig. 2, and extracted with hexane, in comparison to labelled lindane (Amersham) (3) and labelled lindane diluted with cold lindane (4).
Binding of lindane to locu1st haemolymph proteins lymph components. After incubation of haemolymph of male locusts with radiolabelled lindane, HDLp particularly, appeared to be involved in the binding of the insecticide, whereas in females the radiolabel is additionally recovered in vitellogenin. Experimentally, the haemolymph vitellogenin titer was increased by ovariectomy, whereas additional treatment with the juvenile hormone analogue methoprene was applied to stimulate vitellogenin synthesis. Interestingly, whereas the level of HDLp was not affected in these insects, the binding of lindane to this component decreases when the vitellogenin concentration (and binding capacity) increases. In insect oocytes, vitellogenin is selectively sequestered by receptor-mediated endocytosis, a process first proposed for mosquito oocytes by Roth and Porter (1964). The vitellogenin-binding protein from locust ovaries has been recently isolated (Roehrkasten et al., 1989). The accumulation of lindane in maturing oocytes will result in part from this pathway, and additionally from the insecticide bound to HDLp which is also taken up and provides a major source of the lipid reserves in the eggs (Kawooya and Law, 1988). Still, vitellogenin remains of major interest since, for instance, after application of sublethal doses of lindane to Musca domestica, the level of haemolymph proteins, particularly that of female-specific proteins, was decreased (Le Bras et al., 1973). Since lindane is known to bind to different receptors (Abalis et al., 1985), to act on cholinergic synapses (Beeman, 1982) and to affect the fluidity of membranes (Antunes-Madeira and Madeira, 1989), it can be stated that only by integration of studies on the actions of the insecticide on its different target sites may the effect of sublethal doses of lindane on reproduction be understood. thank A. Th. M. Van den Broek for technical assistance on the experiments involving ovariectomy and topical application of the juvenile hormone analogue. Methoprene (ZR-515) was a generous gift from Dr G. B. Staal, Zoicon Corporation, Palo Alto (CA). A visiting grant to P.B. from the Dutch Ministery for Education is gratefully acknowledged. Acknowledgements-We
141
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