Bioconjugate Chem. 1996, 7, 396−400
396
ARTICLES Binding and Transport of Benzo[a]pyrene by Blood Plasma Lipoproteins: The Possible Role of Apolipoprotein B in This Process Lev M. Polyakov,* Marina I. Chasovskikh, and Lev E. Panin Laboratory of Medical Biotechnology, Institute of Biochemistry, Russian Academy of Medical Sciences, Novosibirsk, Russia. Received August 5, 1995X
The role of plasma lipoproteins as carriers in the transport of benzo[a]pyrene was assessed in in vitro and in vivo studies. Addition of [3H]benzo[a]pyrene to rat plasma resulted in binding of the xenobiotic to lipoproteins. Studies of labeled benzo[a]pyrene distribution in rat blood plasma by the method of ultracentrifugation have given the following results: high-density lipoproteins, 40%; low-density lipoproteins, 14%; very-low-density lipoproteins, 23%; other plasma proteins, 23%. Complexes of benzo[a]pyrene-lipoproteins were isolated by gel filtration with Sephadex G-25 and used for intravenous injection in rats. Biodistribution studies have shown different localization of benzo[a]pyrene in rat organs and tissues depending on lipoprotein classes. A high amount of radioactivity was bound by the liver and adrenals when all classes of lipoproteins were used, but especially with high-density lipoproteins. High levels of benzo[a]pyrene were measured in the kidneys. Equilibrium dissociation constants for complexes of benzo[a]pyrene with high-density lipoproteins and low-density lipoproteins were obtained (Kd 4.1 × 10-5 and 1.5 × 10-5 M, respectively). Binding and distribution of the protein component of lipoproteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 80% of the radioactivity recovered from the gel was localized in the area of apolipoprotein B. After isolation and purification of apolipoprotein B, the equilibrium dissociation constant for complexes of benzo[a]pyrene with apolipoprotein B was obtained, and its value indicated that apolipoprotein B might be the main protein carrier for benzo[a]pyrene.
INTRODUCTION
Blood plasma lipoproteins (LFs)1 are involved in one of the specialized forms of biological transport. Besides the basic function of lipid transport, LPs also bind and transport a number of biologically active compounds: vitamins (1, 2), steroid and thyroid hormones (3, 4), and xenobiotics, including some drugs (5, 6). It is known that LPs are carriers in the bloodstream for many chemical carcinogens such as 2,3,7,8-tetrachloro-p-benzodioxin (7), 3-methylcholanthrene (8), and p-(dimethylamino)azobenzene (9). However, the mechanisms of carcinogen delivery to the cells and their internalization by the cell are not well understood. The lipophilicity of transported carcinogens is considered to play the major role in the ligand binding. Most lipophilic compounds are mainly bound by LP fractions (10). Besides the lipophilicity, there are other factors which can influence the interaction between LP particles and lipophilic carcinogens. For example, dihydroxylated * Address correspondence to this author at: Institute of Biochemistry, Russian Academy of Medical Sciences, 630117, Novosibirsk-117, Timakov Street, 2, Russia. X Abstract published in Advance ACS Abstracts, March 1, 1996. 1 Abbreviations: BP, benzo[a]pyrene; LP, lipoprotein; HDL, high-density lipoprotein (1.063 < density < 1.210 g/L); LDL, low-density lipoprotein (1.019 < density < 1.063 g/L); VLDL, very-low-density lipoprotein (density < 1.019 g/L); apoB, apolipoprotein B; SDS, sodium dodecyl sulfate; PAG, polyacrylamide gel; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid.
S1043-1802(96)00005-5 CCC: $12.00
derivatives of BP display an affinity for HDL higher than expected, considering the HDL lipid volume. Serum albumin plays a minor role in the binding of lipophilic carcinogens in the living organisms; 5% of unmetabolized BP associates with albumin and 95% associates with LPs (11). Interactions of lipophilic carcinogens with polar lipids of LPs and with apolipoproteins may be involved in the mechanism of modulation of the toxicity and in the uptake of the xenobiotics by cells. However, the role of the protein component of LPs in the binding of carcinogens is still obscure. Studies on the interaction of BP and other polycyclic aromatic hydrocarbons with proteins may be very useful for understanding the molecular mechanisms of carcinogenesis. In the present study we have obtained some quantitative characteristics of the interactions between different LP classes with BP and show a possible role of apoB in this process. In addition, the distribution of labeled BP in rat organs and tissues was investigated using different LP classes as BP carriers. EXPERIMENTAL PROCEDURES
Isolation of Lipoproteins. Plasma LPs were isolated by sequential ultracentrifugation in potassium bromide (KBr) solution in the presence of 5 mM EDTA Na2 (Serva, Germany) as described previously (12), using a Beckman L-75 centrifuge and 75 Ti rotor. HDL, LDL, and VLDL fractions were dialyzed overnight against 0.15 M NaCl, 5 mM Tris-HCl, pH 8.0, 5 mM EDTA Na2, and 10 mM NaN3 at 4 °C. Distribution of [3H]BP among LP Fractions. 3 [ H]BP (Amersham, England, specific activity 50 Ci/ © 1996 American Chemical Society
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Binding and Transport of BP
mmol) in 50 µL of dimethyl sulfoxide was added to rat plasma (20 mL) before the centrifugation; the radioactivity of [3H]BP in this solution was 0.05 µCi/mL of plasma. This solution was shaken up and incubated for 30 min, and then LP fractions were isolated as described (12). Radioactivity of each fraction was determined in a MarkIII spectrometer in toluene scintillation fluid. Counting effectiveness was 40%. The LP fractions were further purified by chromatography with a Sephadex G-25 (Pharmacia, Sweden) column (1.6 × 32 cm) in a buffer containing 5 mM TrisHCl, pH 8.0, and 5 mM 2-mercaptoethanol at a flow rate of 0.5 mL/min. Fractions (2 mL) were collected, and the elution profile was recorded by UV detector 2151 (LKB, Sweden). The amount of radioactivity in each fraction was determined in toluene scintillation fluid. Biodistribution of [3H]BP in Rat Tissues. Male Wistar rats weighing 160-180 g were used for in vivo experiments. About 1 µCi of [3H]BP bound to LPs in 0.5 mL of a 0.15 M NaCl solution was injected into the tail vein. The procedure was performed under ether anesthesia. Rats were scarificed 30 min after injection and perfused for 5 min with cold 0.15 M NaCl via aorta and v. porta. Pieces (100-150 mg) of required tissues were removed, weighed, and homogenized on ice in 1 mL of saline using a motor-driven Teflon Potter-Elvehjem homogenizer for 1 min at 3000 rpm. From this homogenate was loaded 100 µL of each tissue sample onto glass microfiber filters (GF/C, Whatman, England), which were dried. Radioactivity was measured in 5 mL of toluene scintillation fluid. Binding and Distribution of [3H]BP among ApoLP. LPs (100 µL) were incubated with 10 µL of [3H]BP (about 1.3 × 105 cpm, in dimethyl sulfoxide) for 1 h at room temperature. Then, 10 µL of 20% SDS (Serva, Germany) and 20 µL of glycerol were added to each sample, followed by warming for 5 min at 90 °C. The apolipoproteins (30 µL, about 50 µg) were separated by SDS-PAGE (12. 5%) as described by Laemmli (13). After electrophoresis, every lane of gel was divided lengthwise into two parts, one of which was stained with 0.05%. Coomassie brilliant blue G-250 in 50% methanol and 10% acetic acid; the unstained part was sliced into 4-mm slices and dissolved in NCS tissue solubilizer (Amersham, England) until the samples were cleared (usually 2-3 h). The radioactivity of the gel slice samples was determined in a Mark-III liquid scintillation spectrometer. Radioactivity profiles for the gels were constructed from the radioactivity present in the gel slice samples. Binding Analysis. The quantitative characteristics of the stability of the LP-BP bioconjugates were obtained using the cellulose circles method developed in our laboratory (14). This method was as follows: round cellulose circles prepared from Whatman 3MM (Whatman, England) were soaked with 50 µL of dimethyl sulfoxide solution containing a constant quantity of [3H]BP (1 µg or 15 000 cpm) and increasing amounts of unlabeled BP (from 0 to 50 µg on every circle). The circles were dried and incubated in 1 mL of LP solution in PBS (10 mM, 0.15 M NaCl, pH 7.4) or apoB in 0.02% SDS solution of PBS at 20 °C for 24 h. Control incubations of cellulose circles treated with increasing amounts of unlabeled ligands were performed in the presence of BSA. The protein concentration in these solutions was 1-3 g/L. The circles were dried, and radioactivity was determined in 5 mL of toluene scintillation fluid for every circle. The results were used for equilibrium dissociation constant (Kd) calculations. Isolation and Purification of apoB. Rat LDLs were delipidated by extracting with a cooled methanol-
Table 1. Percentage Binding of BP to Lipoprotein Fractions (Isolated by Ultracentrifugation) LP fractions
radioactivity (%)
plasma amounts of LPs (in %)a
VLDL LDL HDL other plasma proteins
23 14 40 23
14 17 69
a
According to Chapman (16).
chloroform (1:1, v/v) solution at 4 °C for 24 h and then by washing with ether alone. Then 10-15 mg of dried proteins was dissolved in 2% SDS solution, warmed for 5 min at 90 °C, and fractionated on a Sepharose 4B (Pharmacia, Sweden) column (1.6 × 100 cm) in a buffer containing 5 mM Tris-HCl, pH 8.0, 0.05% SDS, 0.01% NaN3, and 1 mM phenylmethanesulfonyl fluoride at a flow rate of 9 mL/h. The purity of the proteins obtained was evaluated by SDS-PAGE. The first peak contained both forms of apoB (B-100 and B-48). Immunization and Separation of Antibodies to apoB. Purified apoB in 0.1% SDS solution was used for immunization of rabbits. Injections were performed subcutaneously (in multiple points of upper back part) with 0.5 mL of apoB (100 µg) and 0.5 mL of complete Freund’s adjuvant (Difco Laboratories). The same dose was repeated 10 days later in incomplete Freund’s adjuvant. The last immunization was performed by intravenous injection of 100 µg of apoB without Freund’s adjuvant. Antibodies were obtained from the blood serum by repeated precipitation with 50% (NH4)2SO4 followed by dialysis against PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) containing 0.1% NaN3. The last purification step was ionic exchange chromatography on DEAESepharose (Pharmacia, Sweden). The column (1.6 × 20 cm) was equilibrated for 1 h at room temperature with PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) containing 0.1% NaN3. The antibodies were then eluted with PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) containing 0.1% NaN3 at a 1 mL/min flow rate. The purified antibodies were frozen and stored at -70 °C. Immunoblotting. Samples of rat HDL (50 µg of protein) were fractioned by SDS electrophoresis using a 12.5% gel. After fractionation, the apolipoproteins were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Germany) by a semidry method using flat coal electrodes (15). The protein transfer was carried out during 1 h at 0.8 mA/cm2. After blocking with 0.5% BSA, the nitrocellulose membrane was placed into a 1000-fold diluted solution of antibody to apoB in PBS and incubated for 1 h. After washing with PBS containing 0.5% BSA, the membrane was incubated for 1 h with a 1000-fold dilution of antirabbit IgG conjugated with horseradish peroxidase (Bio-Rad) in the same solution as that used in the incubation with the first antibody, and washed thoroughly. The antigenantibody complex was then identified by the color development after incubation of the membrane with a solution containing 1 mg/mL 3,3-diaminobenzidine, 1 mg/mL imidazole, and 1 µL/mL 30% H2O2. RESULTS AND DISCUSSION
Addition of [3H]-labeled BP to rat plasma followed by ultracentrifugation has demonstrated that most of the radioactivity (about 77%) was associated with LP fractions. The greatest amount of radioactivity was found in HDLs while the smallest amount was in LDLs. The VLDL fraction had an intermediate amount of radioactivity (Table 1). After ultracentrifugation LP fractions
398 Bioconjugate Chem., Vol. 7, No. 4, 1996
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Figure 1. Elution profiles obtained with gel filtration chromatography of [3H]BP complex and HDL on Sephadex G-25, as detected by radioactivity (- - -) and UV absorbance at 280 nm (s). Elution was performed with 5 mM Tris-HCl buffer, pH 8.0, containing 5 mM 2-mercaptoethanol at flow rate of 0.5 mL/min. Fractions (2 mL) were collected, and the radioactivity was determined. Table 2. Biodistribution of [3H]BP in Rats after Intravenous Injection of [3H]BP-LP Complexes (in cpm/mg Weight Tissues)a liver lung heart spleen kidney adrenals thymus
VLDL
LDL
HDL
27.9 ( 4.1 6.6 ( 1.6 3.1 ( 0.1 1.9 ( 0.3 11.5 ( 2.0 24.7 ( 2.3 4.1 ( 0.9
25.9 ( 2.6 5.9 ( 1.0 2.5 ( 0.2 3.7 ( 0.5 9.1 ( 1.0 29.1 ( 3.7 3.8 ( 0.4
40.8 ( 9.5 9.6 ( 1.1 2.3 ( 0.7 5.9 ( 1.0 26.3 ( 3.8 47.5 ( 9.5 6.4 ( 1.3
a Values quoted are mean ( standard deviation. Number of animals in every case was not less than five.
were passed through a Sephadex G-25 column. The BP radioactivity peak always eluted with LP fractions (Figure 1). After gel filtration, the total binding [3H]BP by LPs and the radioactivity ratio between LP fractions was practically unchanged. The low concentration of LDLs in rats, compared to HDLs and VLDLs (16), partially explains this phenomenon. Besides, both lipid and protein parts of LP particles participate in the BP binding process, and the contribution of these parts varies between different classes of LPs and depends on their ratio in LP particles and on their nature. LPs containing labeled BP were used to transport [3H]BP in rats. BP-LP complexes were injected into the rat’s tail vein. Biodistribution studies showed different [3H]BP localization in the rat’s organs and tissues depending on the LP class (Table 2). It is worth mentioning that administration of [3H]Bp complexed with chylomicrons under similar experimental condition resulted in a homogeneous distribution between rat organs and tissues (17). When a Bp-VLDL complex was used, the [3H]BP uptake by the tissues was the greatest in liver and adrenals, followed by the kidney and the lung. Less radioactivity was found in the heart and the spleen. Earlier, we observed high uptake of [125I]VLDLs by liver and adrenals (18). High levels of BP were observed in adrenals when we used all classes of LPs, but especially when HDL was used. This result may be explained by the fact that cholesterol of HDL is used for steroid hormone biosynthesis in rat adrenals (19). It is probably for this reason that adrenals and testicles are the most
Figure 2. Distribution of [3H]BP among apolipoproteins of rat HDL (A) and LDL (B) after SDS-PAGE. LPs (100 µL) were incubated with [3H]BP (about 1.3 × 105 cpm) as described in Experimental Procedures, and 30 µL of apolipoproteins (about 50 µg total proteins) was separated by SDS-PAGE (12.5%). After electrophoresis, the gel was sliced into 4 mm sections, and the radioactivity in each slice was determined. The arrows indicate the localization of each apolipoprotein: B, apoB (Mr 340 kDa); A-IV, apoA-IV (Mr 42 kDa); E, apoE (Mr 33 kDa); A-I, apoA-I (Mr 26 kDa); Cs, apoC’s, (Mr about 10-14 kDa).
Figure 3. Demonstration of apoB in the rat HDL by immunoblotting. Lane 1 and 2, SDS-PAGE pattern of molecular weight standards and rat HDL, respectively. Lane 2 shows apolipoproteins of rat HDL containing apoB (Mr 340 kDa), apoA-I (Mr 26 kDa), and apoC’s (Mr about 10-14 kDa). Lane 3 shows an immunoblot with antiserum to rat apoB after transferring proteins from gel 2 to nitrocellulose as described in the Experimental Procedures. The arrow indicates positive reactive corresponding to the apoB band.
sensitive, target for toxic action of some compounds, like chlordecon (20). The high level of radioactivity in the liver may explain the main role of this organ in both the synthesis and catabolism of all LP classes. The high level of [3H]BP in the kidney, especially when HDL was used, is noteworthy. This is consistent with previous investigations that showed the importance of kidneys in the catabolism of apoA-I and apoE from HDLs
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Figure 4. Saturation curve (left panel) and Scatchard plot (right panel) for binding [3H]BP to rat LDL. Experiments were performed in the presence of the increasing amounts of unlabeled ligands as described under Experimental Procedures. Control incubations of cellulose (circles) treated with increasing amounts of unlabeled ligands with BSA showed no binding of BP (4). Each point represents the average of a duplicate determination. The equilibrium dissociation constants (Kd) was calculated assuming the average molecular weight of 3000 kDa for rat LDL.
(21). In the rat 39% of apoA-I is catabolized in the proximal tube of the kidney (22). On the whole, the BP radioactivity biodistribution correlates with tissue uptake of the LPs (18). The binding and distribution of BP between different protein components of rat LPs were studied by means of SDS-PAGE (Figure 2). The diagram shows the distribution of [3H]BP among apolipoproteins of rat HDLs and LDLs (according to localization of every apolipoprotein after SDS-PAGE). More than 80% of the radioactivity recovered from the gels was localized in the area of apoB. It is obvious that BP localization at the area of apoB may result from two main reasons: formation of stable apoB-BP complexes and covalent interaction. However, unmetabolized BP under the conditions of our experiment (see Experimental Procedures) does not show the covalent interactions with proteins (23). We can speculate only one reason to explain the localization of BP at the apoB area: that is the formation of apoB-BP complexes. The question may be raised concerning the stability of the apoB-BP complexes toward heating in SDS. The heating of LPs in SDS at 90 °C for 5 min actually results in removal of most of the lipids, but some part of lipids remains associated with the proteins. It is known that even treatment of LDLs with organic solvent (diethyl ether) resulted in just partial delipidation (24). Besides, Benvenga and co-workers (4) have found that after SDSPAGE radiolabeled thyroxine was still associated with the apoB-100 and its fragments (B-76 and B-21). In general, we can suppose with confidence that some part of BP remains associated with hydrophobic domains of the apoB and is sufficient for determination of radioactivity in the PAG. The presence of apoB in the rat HDL was unexpected and was demonstrated by immunoblotting with specific antibodies to apoB (Figure 3). Our data have been confirmed by other investigators, although only with respect to the newly synthesized HDLs. ApoB-containing rat HDL particles represents one-third of the newly synthesized apoB-48 (25). The BP binding capacity of native LDLs, HLDs, and apoB isolated from rat blood plasma was estimated quantitatively by the cellulose circles method (14). Fig-
Table 3. Equilibrium Dissociation Constants (Kd) for Complexes of BP with LDL, HDL, and Purified ApoB Obtained by the Method of Cellulose Circles (10)a complex
Kd, M
LDL-BP HDL-BP apoB-BP
(1.5 ( 0.37) × 10-5 (4.1 ( 1.04) × 10-5 (2.5 ( 0.62) × 10-5
a The following molecular weights and protein composition percentage were used for computation: rat LDL, Mr 3000 kDa, 20% protein; rat HDL, Mr 300 kDa, 40% protein; rat apoB, Mr 340 kDa. Each point is the average of two experiments. Control incubations of cellulose circles treated with increasing amounts of unlabeled ligands with BSA showed no binding of BP.
ure 4 shows a typical saturation curve for LDLs and a Scatchard plot analysis of binding data. Control incubations of cellulose circles treated with increasing amounts of unlabeled ligands with bovine serum albumin showed no binding of BP. Equilibrium dissociation constants for native LDLs, HLDs, and purified apoB are presented in Table 3. The fact that these values are similar support the fact that the main carrier protein for Bp may be apoB. In summary, it seems apparent that at least one possible pathway of Bp transport into the cell is mediated by apoB-containing LPs through specific receptors for apoB. ApoB may also be involved in the binding and transport of other related compounds. Our results support, the carrier role of apolipoproteins in the blood plasma transport of BP and other hydrophobic carcinogens. LITERATURE CITED (1) Blomhoff, R., Green, M. H., Berg, T., and Norum, K. R. (1990) Transport and storage of vitamin A. Science 250, 399404. (2) Dueland, S., Pedersen, J. I., Helgerud, P., and Drevon, C. A. (1982) Transport of vitamin D3 from rat intestine. J. Biol. Chem. 257, 146-150. (3) Panin, L. E., Polyakov, L. M., Rozumenko, A. A., and Biushkina, N. G. (1988) Transport of steroid hormones by means of blood serum lipoproteins. Voprosy meditsinskoi khimii (Russia) 34, 5, 56-58. (4) Benvenga, S., Cahnmann, H. J., Gregg, R., and Robbins, J. (1989) Binding of thyroxine to human plasma low density
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BC960005E
