Hemocyanin lipid uptake in Polybetes pythagoricus is altered by fenitrothion

Pesticide Biochemistry and Physiology 86 (2006) 57–62 www.elsevier.com/locate/ypest

Hemocyanin lipid uptake in Polybetes pythagoricus is altered by fenitrothion Monica Cunningham ¤, Fernando Garcia, Horacio Garda, Ricardo Pollero Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de Investigaciones CientiWcas y Técnicas (CONICET)-Universidad Nacional de La Plata (UNLP), (1900) La Plata, Argentina Received 24 August 2005; accepted 2 November 2005 Available online 6 March 2006

Abstract The spider very high density lipoprotein (VHDL), which contains hemocyanin as the major apoprotein, transports most of the circulating lipids. In this work, the eVect of the pesticide fenitrothion (FS) on the ability of VHDL-apoproteins to uptake diVerent lipids was investigated. For this, VHDL was delipidated using Triton X-100 and recombined with diVerent radiolabeled lipids in the presence or the absence of FS. The oligomeric structural integrity was maintained after delipidation as shown by non-denaturating PAGE. In the presence of the insecticide, palmitic acid uptake decreased by 28.2 and 62.4% after treating the apolipoprotein with 10 and 20 ppm FS, respectively. Decreases in the uptake of cholesterol, triolein, and phosphatidylcholine caused by FS were 29, 23, and 31% using 10 ppm, and 40, 44, and 29% using 20 ppm FS, respectively. Fluorescence measurements with the hydrophobic probes diphenylhexatriene (DPH) and diphenylhexatrienyl-propionic acid (DPH-PA) indicate that FS induces a red shift, decreases the intensity and increases the anisotropy of the emission of these probes in the VHDL. These results indicate that insecticide binding to the lipoprotein enhances the environment polarity and restricts the mobility of these probes at their binding site. These changes at the hydrophobic VHDL binding sites could lead to the decreased aYnity for lipids and hydrophobic ligands. It is inferred that FS could alter the normal lipid exchange between this lipoprotein and tissues. © 2006 Elsevier Inc. All rights reserved. Keywords: Hemocyanin; Fenitrothion; Lipoproteins; Arachnids; Lipid uptake; Fluorescent hydrophobic probes

1. Introduction Pesticides frequently exert toxic eVects on non-target organisms. Some lipophylic insecticides tend to accumulate in membranes, modifying their physicochemical properties and functions, such as permeability, lipid order, and dynamics. It was demonstrated that small amounts of organophosphate insecticides alter several properties of either natural vertebrate [1–3] or invertebrate [4] membranes, as well as artiWcial phospholipid bilayers [5]. Recently, we have demonstrated that the organophosphate insecticide (phosphorothioic acid, O,O-dimethyl-O-methyl-4-nitro*

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phenyl ester-FS) not only aVects membranes, but also the structure of lipoproteins. FS incorporates into several circulating invertebrate lipoproteins, altering their lipid dynamics, penetration of water into the lipid phase and, in some cases, their ability to exchange lipids with the tissues. DiVerential alterations in physical properties were found in shrimp lipoproteins with diVerent lipid and apoprotein compositions. These alterations were observed in circulating lipoproteins [6] as well as in a vitellinic one [7]. Changes evoked by FS aVected not only the free fatty acid exchange with tissues, but also the transfer of some acylglycerides [8]. Three hemolymph lipoproteins (HDL1, HDL2, and VHDL) isolated from plasma of Polybetes pythagoricus were characterized [10,11]. Changes in lipid dynamics were found in the HDLs with diVerent basal lipid order as well as lipid/

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apoprotein ratio after FS treatment. A consequence of these structural alterations was a misfunction in the oxygen binding capacity [9]. The very high density lipoprotein VHDL has a relative density of 1.21–1.24 g/ml, 3.55% (w/w) of lipids and hemocyanin as the major apolipoprotein. In a previous work, the delipidated spider VHDL was used to evaluate its capacity to bind diVerent lipid classes by measuring some kinetic parameters [12]. The use of lipoprotein systems with few controlled variables such as those constructed with only one lipid component should be suitable for carrying out studies dealing with their interaction with xenobiotics. On the basis of our experience on lipid binding to hemocyanin, models of artiWcial lipoproteins containing the VHDL apoproteins and only one lipid class were prepared to perform the present work. VHDL from P. pythagoricus was delipidated and, in the presence of FS, lipoprotein particles were reconstituted with phosphatidylcholine, triolein, cholesterol or palmitic acid. To determine the eVect of FS on the lipid–apoprotein interaction as well as on the lipoprotein functionality, changes evoked by the pesticide in the delipidated VHDL binding capacity of each lipid class were evaluated. 2. Materials and methods 2.1. Sampling and isolation of lipoproteins Hemolymph from wild specimens of P. pythagoricus was obtained as described in a previous work [10]. Plasma lipoproteins were isolated by density gradient ultracentrifugation. Aliquots of blue plasma were overlayered on NaBr solution (density 1.26 g/ml) containing Trasylol (FBA Pharmaceuticals, NY) as protease inhibitor, and centrifuged at 178,000g for 22 h in a Beckman L8 70 M centrifuge, using a SW 60 Ti rotor. As we assumed that the plasma density was 1.006 g/ml, a saline solution of the same density was run simultaneously as blank. The total volume of the tubes was fractionated from top to bottom into 0.3 ml aliquots, and the protein content of each fraction was monitored spectrophotometrically at 280 nm. Those fractions corresponding to VHDL (density 1.21– 1.24 g/ml) were collected. 2.2. Delipidation VHDL was delipidated using Triton-X-100 (Sigma Chemical Co., St. Louis, MO) 0.02 g/g protein. The mixture was incubated with shaking at room temperature for 1 h. According to Mangum et al. [13], the detergent was eliminated using macroreticular adsorbent beads (Bio-Beads SM-2, Bio-Rad Laboratories, CA) 1 g/g protein ratio, keeping the system with shaking at room temperature for 1 h. The beads were separated by Wltration on lipid-free glass Wber. An aliquot of the delipidated sample was extracted using organic solvents and analyzed by TLC-FID in an Iatroscan apparatus [10] to estimate the delipidation eYciency.

2.3. Protein analysis Total protein concentration of native and delipidated VHDL were measured colorimetrically by the method of Lowry et al. [14]. Samples were then analyzed by electrophoresis under native and dissociating conditions. Analyses in non-dissociating conditions were performed using 4–23% PAGE. Protein subunits were analyzed by SDS–PAGE using a gradient of 4–23% acrylamide [15]. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma Chemical Co., St. Louis, MO). Molecular weight standards (HMW, Pharmacia, Uppsala, Sweden) were run in parallel lines. 2.4. Fluorescence measurements Native or delipidated VHDL (0.3 mg in 3 ml of 50 mM potassium phosphate buVer, pH 7.4) were mixed with few microliters of concentrated DMSO solutions of DPH or DPH-PA (Wnal concentration 2 M ). Corrections for nonspeciWc Xuorescence and light scattering contributions were made by using blanks prepared in the same way as the samples but in the absence of the Xuorescent probes. Ethanol solutions of fenitrothion (FS) were added at Wnal concentrations of 0, 10, and 20 ppm to delipidated VHDL. Samples were gently swirled at room temperature for 2 h to promote the incorporation and equilibrium of FS and probes in the system assayed. Steady-state Xuorescence measurements were made in a Perkin-Elmer LS55 Luminescence Spectrometer (Norwalk, CT, USA). Emission spectra were acquired by using 361 nm excitation wavelength (bandpass 2.5 nm) and scanning the emission wavelength between 400 and 550 nm (bandpass 2.5 nm). Measurements of steady-state anisotropy (rs) were done according to Lakowicz et al. [16,17] with modiWcations [6,9]. Excitation and emission wavelengths were 361 and 430 nm, respectively (bandpass 2.5 nm). These measurements were made at room temperature. 2.5. Lipoprotein fraction reconstitution Lipoprotein fractions were reconstituted in vitro with delipidated VHDL (1.29 mg) and [1-14C]palmitic acid (57 mCi/mM), [4-14C]cholesterol (51.3 mCi/mM), ([dipalmitoyl-1-14C]phosphatidylcholine 113.4 mCi/mM) or [carboxyl-14C]triolein (112.0 mCi/mM) which were provided by New England Nuclear (Boston, MA). All of them had previously been treated with 0,10 and 20 ppm FS. To reconstitute lipoproteins with palmitic acid, the delipidated VHDL (50 mM buVer phosphate, pH 7.4), was added to the ammonium salt of the free fatty acid in a protein/lipid molar ratio of 1/1000. Mixtures were incubated in a water bath at 28 °C for 5 min, followed by vortexing for 1 min. This procedure was performed seven times. For the reconstitution of lipoproteins with cholesterol and triacylglycerols, sodium cholate (Sigma Chemical Co., St. Louis, MO) was used as lipid emulsiWer following the Matz and

M. Cunningham et al. / Pesticide Biochemistry and Physiology 86 (2006) 57–62

Jonas procedure [18]. The protein/lipid molar ratio was 1/ 100 for cholesterol and 1/5000 for triacylglycerol. Lipoproteins reconstituted with palmitic acid, triacylglycerol or cholesterol, were analyzed by FPLC on Superdex 200 HR 10/30 molecular exclusion columns (Pharmacia, Uppsala, Sweden), using 50 mM potassium phosphate buVer, 150 mM sodium chloride pH 7.4, at a Xow rate of 0.4 ml/min. Protein elution from the column was monitored by absorption at 280 nm using a Merck-Hitachi L 4200 UV/VIS detector. Radioactivity of lipids bound to the protein was detected with a radiometer detector (Radiomatic™ 500 TR Series, Packard Instruments, Downers Grove, IL). A liquid scintillation cocktail (Ultima Flo-M, Packard Instruments) was mixed with the eluate at a Wnal Xow rate of 1.2 ml/min. The binding of phosphatidylcholine (PC) to delipidated VHDL was done by using PC-labeled liposomes. A chloroform solution containing 14C PC (12.3 nmol, 1.4 Ci) were dry evaporated, hydrated with 1 ml of 50 mM potassium phosphate buVer, pH 7.4, vortexed and extruded through two polycarbonate membranes with pore diameters of 100 and 200 nm (Avestin Inc., Ottawa, Canada). FS-treated delipidated VHDL was incubated with [14C-PC]liposomes at 37 °C for 30 min in 0.5 ml of 50 mM potassium buVer, pH 7.4. The protein/lipid molar ratio was 1/300. After incubation, the labeled lipoprotein was isolated from the remaining liposomes by density gradient ultracentrifugation as described elsewhere. The total volume of the tubes was fractionated from top to bottom into 0.2 ml aliquots, and the protein content of each fraction was monitored spectrophotometrically at 280 nm. Radioactivity was measured in each fraction by liquid scintillation counting in a Wallac 1214 Rack Beta apparatus. 3. Results Total VHDL delipidation was conWrmed by sample extraction with solvents and its analysis by TLC-FID, evidencing lipid removal. Aliquots of native and delipidated VHDL were analyzed by electrophoresis. The electropherogram in native conditions showed bands of about 500, 420, and 70 kDa in both fractions. In dissociating conditions the two samples were composed of a major apoprotein band of 67 kDa corresponding to hemocyanin, and two minor bands of greater Mr, 105 and 121 kDa, the typical range of the non-respiratory proteins of spiders [19]. Thus, it was corroborated that both native and delipidated VHDL showed the same electrophoretic behavior under native or denaturing conditions. The neutral hydrophobic probe DPH and the negatively charged related compound DPH-PA were used to obtain information about the inXuence of delipidation and FS on the properties of the hydrophobic binding sites in VHDL. Fig. 1 shows that no signiWcant diVerences in the DPH emission intensity or steady-state anisotropy (rs) were found between native or delipidated VHDL, indicating that delipidation does not aVect the binding capacity nor the

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Fig. 1. Fluorescence anisotropy (A) and relative emission intensity (B) of DPH and DPH-PA in native or delipidated VHDL (in the presence or absence of 20 ppm FS). Values represent the average of three diVerent determinations §SD. Student’s t test was used to compare the signiWcance of the diVerences with respect to the samples with and without FS, as well as between FS-untreated native and delipidated VHDL: *P < 0.05.

properties of the DPH binding site. On the other hand, the Xuorescence emission of DPH-PA is somewhat higher in delipidated than in native VHDL, fact that can be due to a higher uptake of this fatty acid Xuorescent analogue. The DPH-PA emission anisotropy, however, is not aVected by the lipidation state of the protein, indicating that the presence of lipids evokes no change in the rotational mobility of this probe at its VHDL binding site. The addition of 20 ppm FS to delipidated VHDL led to a large decrease of the Xuorescence intensity of DPH (59%) and DPH-PA (68%) (Fig. 1B), and to a moderate increase in their steady state anisotropies (35 and 20% for DPH and DPH-PA, respectively) as shown in Fig. 1A. FS also produces a red shift in the emission of these probes, mainly for DPH. The wavelength of the emission maximum is shifted from 423.5 to 433.5 nm for DPH, and from 424 to 426 nm for DPH-PA, indicating that FS treatment increases the probes environment polarity at their binding site/s in the VHDL. Radioactive free fatty acid (palmitic acid), free sterol (cholesterol), triacylglycerol (triolein), and phosphoglyceride (phosphatidylcholine) were bound separately to delipidated VHDL, previously incubated with fenitrothion. Reconstituted lipoproteins were analyzed by radio-FPLC. Simultaneous records of mass and radioactivity showed, in the four preparations, that radiolabeled lipids were

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Fig. 2. Uptake of [1-14C]palmitic acid (A), [414C]cholesterol (B), [carboxyl-14C]triolein (C) and [dipalmitoyl-1-14C]phosphatidylcholine (D) by hemocyanin previously treated with 10 and 20 ppm fenitrothion. P values were calculated by the Student’s t test comparing the FS-treated samples with the controls: **P < 0.01, * P < 0.05.

associated to the hemocyanin form of Mr 420 kDa, previously identiWed as hexamer [11]. Fig. 2 displays the uptake of palmitic acid, cholesterol, triolein, and phosphatidylcholine by delipidated VHDL previously treated with 10 and 20 ppm fenitrothion. In the presence of the insecticide, palmitic acid uptake decreased by 28.2 and 62.4% after treating the apolipoprotein with 10 and 20 ppm fenitrothion, respectively (Fig. 2A). The uptake of the other lipids assayed was also decreased by the insecticide (Figs. 2B–D). 4. Discussion The analysis of P. pythagoricus VHDL using molecular exclusion columns showed previously the same chromatographic proWles either in the native lipoprotein or in the delipidated state [12]. In the present study, we found that both states have the same electrophoretic proWles either under non-denaturating or denaturing conditions. It is evident that the presence or absence of lipids bound to VHDL does not alter the chromatographic or electrophoretic behavior either of its oligomers (hexamers) or its subunits. Thus, the techniques used for the delipidation process maintain the subunit composition and the oligomeric structure of VHDL, and they seem to be appropriate for this study. These structural properties are also invariable after the addition of diVerent lipid classes, supporting the reconstitution approach used in this work.

Steady-state Xuorescence measurements performed in native and delipidated VHDL indicate that delipidation does not alter the DPH or DPH-PA emission properties, suggesting that lipids naturally bound to hemocyanin (about 3% w/w) do not aVect the environment of these probes at their binding sites. This evidences that hemocyanin does not lose its structural integrity after lipid removal. However, changes in its secondary structure cannot be discarded, as reported by Zatta et al. [20] for the hemocyanin of other invertebrates. The inXuence of FS on the Xuorescence emission characteristics of DPH and DPH-PA bound to VHDL indicates that this insecticide alters the conformation and properties of the binding site/s for these hydrophobic compounds. The red shifted emissions in the presence of FS indicate an increased polarity of the probe environments at their binding site/s. We have previously observed in several membranes and lipoproteins that FS produces a decrease in the Xuorescence lifetime of these probes, a fact attributed to an increased water penetration into the lipid phase since no direct quenching is evoked by FS [6,7,9]. Although this VHDL, specially when delipidated, constitutes a very diVerent system compared to membranes or lipid-rich lipoproteins, it could be possible that a similar increment in water penetration can be produced by FS in DPH or DPH-PA binding sites, leading to an enhanced polarity of the probes environment. In turn, the increased hydration and polarity of the binding site/s could lead to a decreased aYnity of VHDL for these hydrophobic probes as well as for the

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natural lipids. The enhanced hydration of the binding site/s can also be partially responsible for the decrease observed in the DPH and DPH-PA Xuorescence intensities. However, the relatively large eVect of FS on the emission intensities suggests that these probes are released from their binding sites. This possibility is strengthened by the fact that FS reduces the uptake of several lipid ligands. The increased polarity of DPH and DPH-PA environment produced by FS could also decrease their Xuorescence lifetimes, explaining, at least in part, the increment observed in emission anisotropies. In fact, if the decrease in emission intensity was only due to increased environment hydration and polarity, the Xuorescence lifetimes would decrease proportionally to the quantum yield and Xuorescence intensities. Then, Perrin equation [17] can predict that the observed decreases in emission intensities are large enough to explain the increases observed in steady state anisotropies, without assuming any change in the rotational mobility. However, if as mentioned above, binding of DPH and DPH-PA was decreased in the presence of FS, and this is the main reason for the low emission intensities, then the increased anisotropies would also indicate some restriction in the probes rotational mobility at their binding site/s. Thus, the present results also suggest that FS might decrease the Xexibility of the VHDL hydrophobic site/s sensed by DPH and DPH-PA, this can be relevant for determining a decreased aYnity for hydrophobic lipid ligands. Given the hydrophobic character of FS, there would be a competition between these hydrophobic probes and natural lipids for the same sites in the protein. However, we cannot discard the possibility that FS binding to a diVerent sites can result in distortions and lower aYnity observed at DHP or DPH-PA binding site/s. Phosphatidylcholine, triolein, cholesterol, and palmitic acid were chosen for these assays, since they are the main lipid classes in the native VHDL isolated from P. pythagoricus plasma [11]. We have previously demonstrated that VHDL binds these four lipid classes with diVerent aYnity and capacity [12]. Based on these results, hemocyanin was incubated at lipid saturated concentrations. These in vitro assays demonstrated that FS binding to VHDL decreases its ability to uptake these four lipid classes, although they cannot discriminate between a decreased binding aYnity or capacity (number of binding sites). Further kinetic studies will be necessary to assess whether the decreased lipid uptake is due to a competence with FS for the same binding site. Among the four lipids assayed, palmitate was the most aVected and phosphatidylcholine the least by FS in their binding to VHDL. This minor eVect of FS on phosphatidylcholine could be related with its relatively high aYnity as previously demonstrated [12]. However, FS produced a similar impairment in the uptake of lipids with very diVerent aYnities and polarities as cholesterol and triacylglycerol. Despite these data, a clear idea based on lipid polarity could not be drawn. It is not possible to discard the existence of multiple sites responsible for the binding of lipids with diVerent structure and polarity. In this respect, it was

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previously shown that only 70% of cholesterol bound to VHDL could be replaced by increasing phosphatidylcholine concentration [12], suggesting the existence of at least two binding sites for cholesterol, one of them able to be occupied also by phosphatidylcholine. The possible existence of diVerent binding sites raised the question whether they were diVerentially aVected by FS or not. Although only palmitate the decrease in the uptake was statistically signiWcant, other lipids it seemed to be greater when the pesticide concentration was doubled. This suggests blockade of lipid binding site/s by FS, and a saturation of VHDL hydrophobic regions with lipophillic molecules. In brief, our results indicate that FS alters the hydration and Xexibility of VHDL binding site/s for hydrophobic Xuorescent probes as well as the ability to bind these probes and natural lipid ligands. This has also been assessed in the lipovitellin of the crustacean Macrobrachium borellii, in which structural changes were correlated with functional alterations produced by the same pesticide [7]. An important role in lipid transport and exchange among tissues is suggested for P. pythagoricus VHDL, since it binds more than two thirds of the circulating lipids in hemolymph [11]. Such function may be seriously aVected by structural alterations modifying its lipid binding properties as those evoked by FS. These observations could be relevant in two aspects related to physiology and toxicology, respectively. This insecticide might produce alterations in the lipid exchange between the VHDL and tissues of P. pythagoricus, and the VHDL could be the carrier for the transport and distribution of this toxin to diVerent tissues. Acknowledgments M.C., F.G., and H.G. are member of Carrera del Investigador CONICET, Argentina. R.P. is a member of Carrera del Investigador CICBA, Argentina. The present work was partially funded by CONICET PIP no 02101. References [1] M.C. Antunes-Madeira, R.A. Videira, V.M. Madeira, EVects of parathion on membrane organization and its implications for the mechanisms of toxicity, Biochim. Biophys. Acta 1190 (1994) 149–154. [2] R.A. Videira, M.C. Antunes-Madeira, J.B. Custodio, V.M. Madeira, Partition of DDE in synthetic and native membranes determined by ultraviolet derivative spectroscopy, Biochim. Biophys. Acta. 1238 (1995) 22–28. [3] J. Blasiak, Changes in the Xuidity of model lipid membranes evoked by the organophosphorus insecticide methylbromfenvinfos, Acta. Biochim. Pol. 40 (1993) 39–41. [4] M.R. Gonzalez-Baró, H. Garda, R.J. Pollero, EVect of fenitrothion on hepatopancreas microsomal membrane Xuidity in Macrobrachium borellii, Pest. Biochem. Physiol. 58 (1997) 133–143. [5] M.R. Gonzalez-Baró, H. Garda, R.J. Pollero, EVect of fenitrothion on dipalmitoyl and 1-palmitoyl and 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, Biochim. Biophys. Acta 1468 (2000) 304–310. [6] C.F. García, M.L. Cunningham, M.R. Gonzalez-Baró, H. Garda, R. Pollero, EVect of fenithothion on the physical properties of crustacean lipoproteins, Lipids 37 (2002) 673–678.

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