A Molecular Motif Required for the Activation of Rat Neutrophil

Jan 24, 2002 - ... γ-isomers of hexachlorocyclohexane (HCCH), p,p'-dichlorodiphenyltrichloroethane (DDT), dieldrin, and chlordane each induced activa...
0 downloads 0 Views 118KB Size
Download PDF
Chem. Res. Toxicol. 2002, 15, 153-159

153

A Molecular Motif Required for the Activation of Rat Neutrophil Phospholipase A2 by Organochlorine Compounds Jesus Olivero, Steven A. Bezdecny, and Patricia E. Ganey* Department of Pharmacology and Toxicology, National Food Safety and Toxicology Center and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824 Received July 25, 2001

Organochlorine (OC) compounds are some of the main toxicants present in the food web and target several cellular systems including the nonspecific immune system. The objective of this study was to test the hypothesis that OC compounds that activate neutrophils share common structural features. Using activation of phospholipase A2 (PLA2) as a marker of neutrophil activation, isolated rat neutrophils were exposed to a variety of OC compounds. The ortho-substituted polychlorinated biphenyl 2,2′,4,4′-tetrachlorobiphenyl, the R-, δ-, and γ-isomers of hexachlorocyclohexane (HCCH), p,p′-dichlorodiphenyltrichloroethane (DDT), dieldrin, and chlordane each induced activation of PLA2 in neutrophils. β-HCCH and the nonortho-substituted 3,3′,4,4′-tetrachlorobiphenyl were without effect. PLA2 activation stimulated by each of the OC compounds was reduced by methyl arachidonyl fluorophosphonate, which inhibits both a cytosolic and a calcium-independent PLA2 (iPLA2), and by E-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL), a selective inhibitor of iPLA2. These results suggest that a fraction of the PLA2 activity stimulated by OC compounds is dependent on iPLA2. Western analysis confirmed the presence of iPLA2 in rat neutrophils. Molecular modeling techniques were used to develop structure-activity relationships for the activation of PLA2 by OC compounds. Superimposing three-dimensional structures, an electrotopological motif shared by all of the active compounds was identified. This motif was absent in the inactive β-HCCH and 3,3′,4,4′-tetrachlorobiphenyl. This motif, which we have called PHEN, is required for the activation of the neutrophil PLA2 by OC compounds and consists of a planar hydrophobic domain connected rigidly at a perpendicular angle to a halogen atom.

Introduction One of the forces that moved the industrial age was the extensive use of new chemicals that allowed the production of large quantities of food supplies and the decrease in child mortality. Among these chemicals, organochlorine (OC) compounds were of particular importance due to their versatility and low cost. These same chlorinated chemicals, such as polychlorinated biphenyls (PCBs) and chlorinated pesticides such as DDT (p,p′dichlorodiphenyltrichloroethane), dieldrin, hexachlorocyclohexanes (HCCHs), and chlordane, are recognized as food contaminants due to their accumulation in the food chain. Adding to concern about OC compounds is their diverse toxicity. The immune system, and particularly the neutrophil, has been shown to be a target for OC compounds such as PCBs (1, 2), HCCHs (3-5), DDT (6), dieldrin (7), and chlordane (8), among others. For example, PCBs activate neutrophils through complex biochemical mechanisms to induce superoxide anion generation and degranulation (1). These processes are mediated through the activation of phospholipase A2 (PLA2) (9). Toxicological testing of all the known OC food contaminants for their activity on neutrophil function is not * Address correspondence to this author at the Department of Pharmacology and Toxicology, 214 Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824. Phone: 517-4321761. Fax: 517-432-2310. E-mail: [email protected].

practical because it is expensive and time-consuming. A knowledge of the molecular structure and the biological activity of groups of related compounds will allow the development of structure-activity relationships (SARs) which can be critical not only to predict the activity of unknown, related compounds but also for risk assessment. SAR studies for various biological activities have been performed for PCBs (10-23), hydroxylated PCBs (24), cyclodienes (25), DDT (26), and other halogenated pesticides (27). In neutrophils, SAR studies have been conducted for the activity of PCBs to cause superoxide anion production (11, 28) and degranulation (29). Since these functional changes in rat neutrophils are mediated through activation of PLA2, this SAR study was conducted to find molecular features responsible for the activation of PLA2 by PCBs and other OC compounds.

Experimental Procedures Chemicals. [3H-5,6,8,9,11,12,14,15]-Arachidonic acid ([3H]AA; 180-240 Ci/mmol) was purchased from DuPont NEN (Boston, MA), E-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) was purchased from Biomol (Plymouth Meeting, PA), and methyl arachidonyl fluorophosphonate (MAFP) and polyclonal antiserum against the calcium-independent isoform of PLA2 (iPLA2) were from Cayman Chemical (Ann Arbor, MI). Aroclor 1242, 2,2′,4,4′-tetrachlorobiphenyl, 3,3′,4,4′tetrachlorobiphenyl, R-, β-, δ-, and γ-HCCH, dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4,5,8dimethanonaphthalene), DDT, and chlordane (1,2,4,5,6,7,8,8-

10.1021/tx0155449 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/24/2002

154

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

octachlor-2,3,3a,4,7,7a-hexahydro-4,7-methanoindane) were purchased from ChemService (West Chester, PA). Isolation of Rat Peritoneal Neutrophils. Neutrophils were isolated from the peritoneum of male, Sprague-Dawley, retired breeder rats (Charles River, Portage, MI) by glycogen elicitation (7). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaCl2. The percentage of neutrophils in the cell preparations was >95%, and the viability was >95% determined by the ability to exclude trypan blue. The isolation procedure was performed at room temperature. Exposure to Organochlorine Compounds. Stock solutions of OC compounds were prepared by dissolution in N,Ndimethylformamide (DMF). Neutrophils (2 × 106) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 × 75 mm (VWR, Chicago, IL), and 1 µL of the stock solution was added to the tubes to achieve the desired concentration. Control neutrophils received 1 µL of DMF. Determination of PLA2 Activity. Neutrophils (107/mL) were suspended in Mg2+- and Ca2+-free HBSS containing 0.1% bovine serum albumin and incubated in the presence of 0.5 µCi/ mL [3H]-AA for 2 h, gently shaking at 37 °C. Neutrophils were then washed twice with Mg2+- and Ca2+-free HBSS. The cell count was adjusted so that the final concentration of neutrophils was 2 × 106/mL. Total cellular uptake of [3H]-AA was measured in a 1 mL aliquot of suspended cells: the incorporation of [3H]AA was routinely between 80 and 88%. Release of [3H]-AA from labeled neutrophils was measured in cells treated with OC compounds for 30 min (37 °C). Studies with the inhibitor of iPLA2, BEL, were conducted by incubating the cells with BEL (25 µM) for 20 min (37 °C) and then with OC compounds for an additional 30 min at the same temperature. This concentration of BEL was chosen because it causes maximal inhibition of PCBstimulated PLA2 activity (30) and is not cytotoxic. For studies using MAFP, which inhibits both iPLA2 and cytosolic PLA2, cells were preincubated with MAFP (1 µM) for 15 min prior to exposure to OC compounds. This concentration of MAFP was chosen based on preliminary experiments in which this concentration caused maximal inhibition of PCB-stimulated PLA2 activity and was not cytotoxic. At the end of each incubation, neutrophils were placed on ice and spun in a centrifuge (4000g) at 0 °C for 10 min. The cell-free supernatant fluids were transferred to vials containing scintillation cocktail (14 mL), and the total radioactivity in each sample was determined by liquid scintillation counting. Cytotoxicity Assay. Release by neutrophils of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as an indicator of cytotoxicity. LDH activity present in 10 µL of the supernatant fluid from the PLA2 assay was measured according to the method of Bergmeyer and Bernt (31). Detection of iPLA2 in Rat Neutrophils. The presence of iPLA2 was detected in rat neutrophils by western analysis. Neutrophils (3 × 107/mL) were isolated as described above, and then suspended in ice-cold phosphate-buffered saline and sonicated. Cell lysates were spun in a centrifuge (20800g for 10 min, 4 °C) to remove cellular debris. Protein concentration was measured using the bicinchoninic acid (BCA) Assay Reagent kit from Pierce (Rockford, IL). Aliquots of cell lysates (80 µg of protein) were mixed 1:1 with sample buffer [Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 5% β-mercapatoethanol, 2.5% bromophenol blue]. The samples were denatured by boiling for 5 min at 100 °C and separated on a 10% denaturing SDS-polyacrylamide gel (constant current, 2 mA, for approximately 3 h at room temperature). Proteins were transferred electronically (constant current, 45 mA, for 1 h at 4 °C) to prepared PVDF (polyvinylidenedifluoride) membranes. Transfer of rainbow molecular weight standards run alongside lysate samples indicated the success of transfer. PVDF membranes were blocked overnight in blocking buffer [Tris-buffered saline + Tween-20 (0.2%; TBS-T) containing 5% milk] with constant rocking. Anti-iPLA2 was incubated with blots for 1 h at room temperature in blocking buffer containing 0.02% sodium

Olivero et al. azide with constant rocking. Blots were washed 5 times with TBS-T for 10 min each. Anti-mouse antibody linked to horseradish peroxidase (1:1000) in blocking buffer was added and incubated with blots for 1 h at room temperature with constant rocking. Blots were washed 3 times with TBS-T and 3 times with TBS, 10 min each. Enhanced chemiluminescence using Amersham (Piscataway, NJ) ECL reagents was performed on blots to visualize antibody-labeled proteins. Structure-Activity Relationships. (A) Data Set. OC compounds tested for their ability to activate PLA2 were 2,2′,4,4′tetrachlorobiphenyl (2,2′,4,4′-TCB), 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB), R-, β-, δ-, and γ-HCCH, DDT, dieldrin, and chlordane. Compounds that induced a statistically significant release of arachidonic acid at 25 µM when compared to that elicited by the vehicle control (DMF) were considered active. (B) Computational Details. Molecular modeling procedures used in this study were performed using Hyperchem 5.1 (Hypercube Inc., 1996). The compounds in the data set were entered as two-dimensional sketches into Hyperchem and stored as atomic coordinates. The presence of a torsional angle in the PCB structures and DDT generates different conformers, among which some are less energetically favorable. Molecule geometries for these compounds were submitted to a conformational search to obtain a conformer with the lowest energy (i.e., the most stable conformer). Full optimization geometry for the best conformer for PCBs, DDT, and the other OC compounds was determined using the semiempirical method AM1 (32) running on Hyperchem. Electronic properties were calculated from single-point calculations at the ab initio level STO-3G (33). Other properties from the energetically minimized structure, such as the molecular weight and the total solvent-accessible surface area, were calculated using the subroutine, QSAR properties, implemented in ChemPlus/Hyperchem 5.1. (C) Model Construction. Relationships between the molecular structure and the ability to activate PLA2 for the OC compounds in the data set were established following a structurebased design approach. The intrinsic biological/toxicological activity or potency can be a function of the three-dimensional shape of the molecule and of the electronic charge distribution and lipophilicity. These are also determinant factors in the molecular affinity for a receptor. Accordingly, comparison of conformational profiles of various molecules to a template may reveal those overlapping or similar conformational shapes and electronic features that are common to the molecules that elicit a particular effect. Using 2,2′,4,4′-TCB as a template, general similarities were searched by superposition of selected atoms of each OC compound on their chemically equivalent atoms on 2,2′,4,4′-TCB using Hyperchem Software. 2,2′,4,4′-TCB was used as a template because its activation of PLA2 has been characterized (30). Initially, the file for the optimized 2,2,′4,4′-TCB molecule was opened and merged with the targeted optimized geometry. For each molecule, three atoms were selected, and the function “RMS overlay” was used to perform the superpositions. The program translates the centers of the atoms to be fitted to the centroid of the corresponding atom in the reference molecule. The best fit was obtained when the structure superposition gave the lowest root-mean-square deviation value between selected pairs of equivalent atoms using 2,2′,4,4′-TCB as a reference. After visual comparison of the superpositions of 2,2′,4,4′-TCB with the active and the inactive OC compounds, the molecular features, both topological and electronic, present or absent in both groups were determined (SAR model). To validate the model, noncytotoxic doses of different OC and organobromine compounds were tested to see if they activated neutrophil PLA2 following the structural rules presented by the SAR model.

Results PLA2 Activity Induced by Organochlorine Compounds. Aroclor 1242 is a mixture of PCB congeners for which the activation of rat neutrophil PLA2 is well

Motif Required for Activation of PLA2 by Organochlorines

Figure 1. Dose-response relationships for phospholipase A2 activity (filled circles) and cytotoxicity (open circles) in neutrophils exposed to (A) 2,2′,4,4′-tetrachlorobiphenyl or (B) 3,3′,4,4′tetrachlorobiphenyl. Neutrophils were labeled with [3H]-AA and incubated with the concentrations of PCBs shown, as stated under Experimental Procedures. The release of [3H]-AA into the medium at each concentration was compared with release from neutrophils treated with Aroclor 1242 (10 µg/mL) under the same experimental conditions. Values are presented as percentage of activity of Aroclor 1242. LDH released at each concentration was compared with the LDH released from the same number of neutrophils lysed with 0.01% Triton X-100 and is presented as percentage of total LDH activity. Results are expressed as means ( SEM for four different experiments performed in triplicate. *, Significantly different from vehicle control (DMF).

Figure 2. Dose-response relationships for phospholipase A2 activity (filled circles) and cytotoxicity (open circles) in neutrophils exposed to (A) R-HCCH, (B) β-HCCH, (C) δ-HCCH, or (D) γ-HCCH. Experiments were conducted as described in the legend to Figure 1. Results are expressed as means ( SEM for four different experiments performed in triplicate. *, Significantly different from vehicle control (DMF).

characterized (9, 30). Activation of PLA2 by Aroclor 1242 is consistent from experiment to experiment and results in release of between 25 and 35% of total incorporated radioactivity. For this reason, in studies examining the activation of PLA2 by OC compounds, values for each of the OC compounds were normalized to the response to Aroclor 1242 and are presented as a percentage of that response. The dose-response curves for the activation of rat neutrophil PLA2 by OC compounds showed clear differences in activity. 2,2′,4,4′-TCB activated PLA2 at concentrations >1 µM (Figure 1A). Its activity at concentrations >10 µM was similar to that elicited by Aroclor 1242 (10 µg/mL). Concentrations of 2,2′,4,4′-TCB greater than 50 µM produced some cytotoxicity (≈20% LDH release). 3,3′,4,4′-TCB failed to activate PLA2 at concentrations up to 25 µM (Figure 1B). Greater concentrations were not compatible with the buffer and precipitated. R-, δ-, and γ-HCCH-activated PLA2 at concentrations equal to or greater than 10 µM (Figure 2A,C,D). Of these HCCHs, R-HCCH was the least efficacious. No cytotoxicity was observed for any of these OC compounds at the

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 155

maximal concentrations tested (100 µM). Conversely, β-HCCH (Figure 2B) did not activate PLA2 over the concentration range tested (0-100 µM). DDT, dieldrin, and chlordane each activated PLA2 at concentrations equal to or greater than 10 µM (Figure 3A-C). Only dieldrin presented significant cytotoxicity (>20%). This occurred at concentrations greater than 50 µM. There are several isoforms of PLA2, and one that has been implicated in activation of neutrophils by Aroclor 1242 is an iPLA2 that is sensitive to inhibition by BEL (9, 30). Previous studies have demonstrated that PCBstimulated activation of PLA2 occurs in the absence of extracellular calcium, is only slightly diminished by inhibitors of intracellular calcium, and is reduced by about 80% by the inhibitor of iPLA2, BEL (30, 34). Furthermore, Aroclor 1242 activates PLA2 in calciumfree cytosolic preparations of rat neutrophils (30). To verify the presence of this isoform of PLA2 in rat neutrophils, western anaylsis was performed using a polyclonal anti-iPLA2 antiserum. Calcium-independent PLA2 activity has been ascribed to proteins with molecular sizes of about 40 kDa (35) and about 80 kDa (36, 37). Two bands were revealed on western analysis of rat neutrophils, one at about 41 kDa and another at about 78 kDa (data not shown). Thus, iPLA2 is present in rat neutrophils. Neutrophil PLA2 activity elicited by each of the active compounds was decreased by BEL (Figure 4A). Pretreatment with MAFP, an inhibitor of both iPLA2 and a cytosolic PLA2, reduced PLA2 activity in response to each of the OC compounds (Figure 4B). Neither BEL nor MAFP caused significant toxicity in the presence of the OC compounds (data not shown). Structure-Activity Relationships. Molecular structures for the OC compounds used in this study are diverse, and apparently they have few particular similarities. Selected electronic and topological properties of these compounds are shown in Table 1. It is evident that global properties such as the energy of the frontier orbitals, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), and dipolar moment vary considerably among the compounds. Interestingly, the inactive compounds 3,3′,4,4′-TCB and β-HCCH have the lowest dipolar moment values. To look for common structural features responsible for the activity of this diverse group of OC compounds on PLA2, the structure of the ortho-chlorinated PCB, 2,2′,4,4′TCB, an activator of PLA2, was used as a template to superimpose the structures of the other OC compounds. Results reveal a motif common to OC compounds that activate PLA2 that is absent in inactive OC compounds (Figure 5). The motif is formed by a planar, hydrophobic (PH) substructure linked perpendicularly to an electronegative (EN) atom such as a halogen. For example, R-HCCH can fit on one phenyl group of 2,2′,4,4′-TCB and position a chlorine in close proximity to the o-chlorine on the other phenyl ring. Similarly, DDT can be superimposed to position a chlorine perpendicular to a hydrophobic plane. Dieldrin and chlordane lack phenyl rings, but they each have a planar-like domain that fits onto one phenyl group of the PCB, and when this is done a chlorine atom is positioned in the vicinity of the o-chlorine of the other phenyl group on the PCB. Although both of the inactive OC compounds, 3,3′,4,4′-TCB and β-HCCH, can be superimposed on 2,2′,4,4′-TCB through their

156

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

Olivero et al.

Figure 3. Dose-response relationships for phospholipase A2 activity (filled circles) and cytotoxicity (open circles) in neutrophils exposed to (A) DDT, (B) dieldrin, or (C) chlordane. Experiments were conducted as described in the legend to Figure 1. Results are expressed as means ( SEM for four different experiments performed in triplicate. *, Significantly different from vehicle control (DMF).

Discussion

Figure 4. Inhibition of PLA2 activity induced by OC compounds by pretreatment with (A) BEL or (B) MAFP. Neutrophils were labeled with [3H]-AA and incubated with (A) 25 µM BEL for 20 min at 37 °C or (B) 1 µM MAFP for 15 min. This was followed by incubation with OC compounds for 30 min at 37 °C. The release of [3H]-AA into the medium at each concentration was compared with the release by the same compound in the absence of inhibitor. Results are expressed as means ( SEM from 3-4 different experiments performed in triplicate. *, Significantly different from value in the absence of inhibitor (i.e., 100%).

planar motifs, they fail to match a chlorine with the analogue in the ortho-position of the template PCB. Interestingly, BEL, the inhibitor of iPLA2, could also be superimposed onto 2,2′,4,4′-TCB (not shown), revealing a motif similar to active OC compounds with the exception that an oxygen atom, not a halogen, is positioned near the chlorine in the PCB. We have called this motif in OC compounds “PHEN”. External validation of the model with a limited number of halogenated compounds revealed that the three compounds that contain a PHEN motif, 2,4′-dichlorobiphenyl, 2,2′-dibromobiphenyl, and aldrin, activated PLA2 (Table 2). On the other hand, 4,4′-dichlorobiphenyl and 4,4′dibromobiphenyl, which do not contain a PHEN motif, did not stimulate neutrophil PLA2.

In this study, the use of an empirical model to classify chlorinated compounds as activators of neutrophil PLA2 has been described. Seven of the nine OC compounds tested initially activated neutrophil PLA2. For each of these, MAFP inhibited activation by at least 30%, and BEL inhibited activity by at least 50%. MAFP has been reported to inhibit both the cytosolic PLA2 and iPLA2 (38-42). BEL has been used as a selective inhibitor of the iPLA2 (43). Results of western analysis demonstrated the presence of iPLA2 in rat neutrophils. Taken together with the results presented in Figure 4, these observations suggest that at least part of the arachidonic acid released by OC compounds arises from the activity of iPLA2 and that other isoforms are likely to be involved. These results for dieldrin and for γ-HCCH are consistent with those previously published (44). In addition to identifying OC compounds as activators of neutrophil PLA2, an SAR study was performed, and results of that study suggest that a molecular substructure, PHEN, within these OC compounds is required for activation of PLA2. It is not known whether PHEN is involved in the activation of iPLA2 or whether it is necessary for the residual release of arachidonic acid that was insensitive to the pharmacological inhibitors. The approach of looking at substructures associated with biological activity has been applied successfully to study the effects of dieldrin in GABA (γ-aminobutyric acid) channel functioning (45) and carcinogenicity of OC compounds (46). It is important to mention that one advantage of this approach is that it considers simultaneously both topological and electronic features of the molecule and does not require additional interpretation from molecular descriptors obtained from classical quantitative SAR models. The identification of PHEN in active OC compounds of different homologous series suggests that this may be a useful model for searching for compounds with the capacity to activate PLA2. It is tempting to summarize the SAR study in terms of structural requirements that determine the activity of OC compounds toward neutrophil PLA2. Three factors are necessary to define the PHEN motif present in OC compounds. First is the presence of a planar structure that is mainly hydrophobic (PH) and measures approximately 5.0 Å (R-HCCH). The second is the presence of an electronegative (EN) point (halogen atom) located perpendicular to one corner of the planar structure within 1.8 Å (R-HCCH) to 2.7 Å (2,2′,4,4′-TCB). The third part is a rigid connector or bridge between PH and EN. This bridge is important to generate some rigidity to keep the

Motif Required for Activation of PLA2 by Organochlorines

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 157

Table 1. Molecular Descriptors for OCs in the Data Set molecular descriptorsa molecules

MW (g/mol)

SA

(Å2)

DM (D)

MPC

MNC

HOMO (eV)

LUMO (eV)

2,2′,4,′4-TCBc R-HCCHd δ-HCCH γ-HCCH DDT dieldrin chlordane

291.99 290.83 290.83 290.83 354.49 380.91 409.78

395.6 396.4 402.2 388.3 460.4 370.5 445.20

Activeb 2.573 2.707 2.788 3.782 1.490 2.953 2.934

0.098 0.124 0.125 0.127 0.118 0.095 0.112

-0.108 -0.134 -0.126 -0.146 -0.117 -0.215 -0.145

-8.053 -10.943 -10.966 -10.656 -7.757 -8.733 -9.088

5.645 7.103 7.150 6.781 5.578 5.302 5.048

3,3′,4,′4-TCB β-HCCH

291.99 290.83

418.9 411.4

Inactive 1.273 0.088 0.000 0.114

-0.095 -0.116

-7.430 -10.990

4.608 7.012

a Abbreviations: MW, molecular weight; SA, solvent-accessible surface area; DM, dipolar moment; MPC and MNC, most positive and most negative charge in the molecule, respectively; HOMO, energy of the highest occupied molecular orbital; LUMO, energy of the lowest unoccupied molecular orbital. b Active as defined by producing a significant increase in release of [3H]-AA from neutrophils. Inactive as defined by lack of a significant increase in release of [3H]-AA. c TCB, tetrachlorobiphenyl. d HCCH, hexachlorocyclohexane.

Figure 6. Representation of the PHEN motif of OC compounds interacting with the toxicophore. Table 2. External Validation for SAR Model: Activation of PLA2 by Organochlorine and Related Compoundsa

Figure 5. Molecular structures of OC compounds. Structures of each of the OC compounds used in the initial, molecular modeling study are shown with the PH (polar, hydrobic) and EN (electronegative) motifs identified. Stippled rectangles outline the PH. Hydrogen atoms are represented by small, light substitutions on the rings; chlorine atoms by larger, darker substitutions. Dieldrin also has one oxygen atom, which is slightly darker in color compared to carbon atoms.

EN atom perpendicular to PH. A diagram of the PHEN motif with a hypothetical binding structure is shown in Figure 6. Given that ortho-chlorinated PCBs, and not non-orthochlorinated PCBs, activate PLA2 (9), the steric rotational impediment that ortho-substitution confers may be necessary for activity. It is consistent with the model that activation may depend on the possibility of the 2-chlorine of PCBs interacting with a positive site in a hypothetical toxicophore. This toxicophore is the critical, local, molecular fragment that is responsible for the activity of OC compounds in neutrophils. It is interesting that BEL, a compound that binds covalently to the active site of

molecule

PHEN motif

PLA2 activity

2,4′-dichlorobiphenyl 4,4′-dichlorobiphenyl 2,2′-dibromobiphenyl 4,4′-dibromobiphenyl aldrin

yes no yes no yes

yes no yes no yes

a Compounds were tested for the ability to activate neutrophil PLA2 as described under Experimental Procedures. The PHEN motif was identified as described for OC compounds.

iPLA2 (43), also contains a similar motif but instead of a halogen it has an oxygen atom (lactone) perpendicular to the PH. This may create the conditions for the initial activation of the enzyme. Instantaneously, the active site of the enzyme reacts with the lactone group, forming a covalent bond that locks the molecule into the catalytic site making the enzyme inactive (36). Although it is tempting to speculate that the toxicophore for PHENcontaining compounds might be present in this iPLA2, direct binding studies must be conducted to understand fully the mechanism of activation of PLA2 by OC compounds. One interesting and important observation from these studies is the possibility of similar biological activity among OC compounds for which structural similarity is not obvious from two-dimensional rendering. The molecular modeling data showed that DDT, dieldrin, chlordane, R-HCCH, δ-HCCH, and γ-HCCH are likely to

158

Chem. Res. Toxicol., Vol. 15, No. 2, 2002

behave as PCB-like compounds in some biochemical systems and vice-versa. A shortcoming of this SAR model is that it cannot provide information on the potencies of the OC compounds. Consequently, secondary parameters in addition to the PHEN motif, such as energy of orbitals, charge density, and number of chlorines, are necessary for potency prediction. Furthermore, it should be stated that the activity predicted from SAR methods is an estimate, and that the geometry from quantum calculations may not be entirely correct. However, the concept that quantum chemical approaches can help to clarify mechanistic questions involving chemical-receptor interactions has been reinforced (47). In addition, it has been suggested that SARs could and should be used in the hazard assessment process (48). Accordingly, the biochemical activity of OC compounds having the PHEN motif should be considered when assessing potential toxicological effects. Based on this model we predict that all of the orthosubstituted PCBs and ortho-brominated biphenyls, as well as other chemicals having the PHEN motif substructure, are likely to activate neutrophil PLA2. This has been demonstrated in a limited way in this study. On the other hand, compounds such as polychlorinated naphthalenes, dioxins, or other non-ortho-substituted PCBs are not likely to activate neutrophil PLA2. It may also be concluded that OC and organobromine compounds having the PHEN motif, and, therefore, a good likelihood to activate neutrophil PLA2, will consequently induce neutrophil activation to produce superoxide anion. Thus, this represents another biological activity of these chemicals.

Acknowledgment. We thank Cathy Rondelli for technical assistance. This work was supported by Grant ES04911 from the NIH. J.O. was sponsored by a COLCIENCIAS-FULBRIGHT-LASPAUScholarship,Bogota´, Colombia, and by the University of Cartagena, Cartagena, Colombia (South America).

Olivero et al.

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

References (1) Ganey, P., Sirois, J., Denison, M., Robinson, J., and Roth, R. (1993) Neutrophil function after exposure to polychlorinated biphenyls in vitro. Environ. Health Perspect. 101, 430-434. (2) Silkworth, J. B., and Grabstein, E. M. (1982) Polychlorinated biphenyl immunotoxicity: dependence on isomer planarity and the Ah gene complex. Toxicol. Appl. Pharmacol. 65, 109-115. (3) Dunstan, R. H., Roberts, T. K., Donohoe, M., McGregor, N. R., Hope, D., Taylor, W. G., Watkins, J. A., Murdoch, R. N., and Butt, H. L. (1996) Bioaccumulated chlorinated hydrocarbons and red/ white blood cell parameters. Biochem. Mol. Med. 58, 77-84. (4) English, D., Schell, M., Siakotos, A., and Gabig, T. G. (1986) Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction. J. Immunol. 137, 283-290. (5) Kuhns, D. B., Kaplan, S. S., and Basford, R. E. (1986) Hexachlorocyclohexanes, potent stimuli of O2- production and calcium release in human polymorphonuclear leukocytes. Blood 68, 535540. (6) Sitarska, E., Winnicka, A., and Klucinski, W. (1990) Effect of organochlorine pesticides on the phagocytic activity of bovine milk cells. Zentralbl Veterninarmed. 37, 471-476. (7) Hewett, J. A., and Roth, R. A. (1988) Dieldrin activates rat neutrophils in vitro. Toxicol. Appl. Pharmacol. 96, 269-278. (8) Miyagi, T., Lam, K. M., Chuang, L. F., and Chuang, R. Y. (1998) Suppression of chemokine-induced chemotaxis of monkey neutrophils and monocytes by chlorinated hydrocarbon insecticides. In Vivo 12, 441-446. (9) Tithof, P. K., Schiamberg, E., Peters-Golden, M., and Ganey, P. E. (1996) Phospholipase A2 is involved in the mechanism of

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

activation of neutrophils by polychlorinated biphenyls. Environ. Health Perspect. 104, 52-58. Van der Burght, A. S., Clijsters, P. J., Horbach, G. J., Andersson, P. L., Tysklind, M., and van den Berg, M. (1999) Structuredependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of cynomolgus monkeys (Macaca fascicularis). Toxicol. Appl. Pharmacol. 155, 13-23. Brown, A. P., Olivero-Verbel, J., Holdan, W. L., and Ganey, P. E. (1998) Neutrophil activation by polychlorinated biphenyls: Structure-activity relationship. Toxicol. Sci. 46, 308-316. Kodavanti, P. R., and Ward, T. R. (1998) Interactive effects of environmentally relevant polychlorinated biphenyls and dioxins on [3H]phorbol ester binding in rat cerebellar granule cells. Environ. Health Perspect. 106, 479-486. Kodavanti, P. R., and Tilson, H. A. (1997) Structure-activity relationships of potentially neurotoxic PCB congeners in the rat. Neurotoxicology 18, 425-442. Kodavanti, P. R., Shafer, T. J., Ward, T. R., Mundy, W. R., Freudenrich, T., Harry, G. J., and Tilson, H. A. (1994) Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells. Brain Res. 662, 75-82. Kodavanti, P. R., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1995) Increased [3H]phorbol ester binding in rat cerebellar granule cells by polychlorinated biphenyl mixtures and congeners: structure-activity relationships. Toxicol. Appl. Pharmacol. 130, 140-148. Kodavanti, P. R., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996) Inhibition of microsomal and mitochondrial Ca2+-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners. Structure-activity relationships. Arch. Toxicol. 70, 150-157. Mekenyan, O. G., Veith, G. D., Call, D. J., and Ankley, G. T. (1996) A QSAR evaluation of Ah receptor binding of halogenated aromatic xenobiotics. Environ. Health Perspect. 104, 1302-1310. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., and Kafafi, A. G. (1993) Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor. Environ. Health Perspect. 101, 422-428. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., Abd-Elazem, I. S., and Kafafi, A. G. (1993) Affinities for the aryl hydrocarbon receptor, potencies as aryl hydrocarbon hydroxylase inducers and relative toxicities of polychlorinated biphenyls. A congener specific approach. Carcinogenesis 14, 2063-2071. Matthews, H. B., and Anderson, M. W. (1975) The distribution and excretion of 2,4,5,2′,5′-pentachlorobiphenyl in the rat. Drug. Metab. Dispos. 3, 371-380. McKinney, J. D., and Waller, C. L. (1994) Polychlorinated biphenyls as hormonally active structural analogues. Environ. Health Perspect. 102, 290-297. Seegal, R. F., Bush, B., and Shain, W. (1990) Lightly chlorinated ortho-substituted PCB congeners decrease dopamine in nonhuman primate brain and in tissue culture. Toxicol. Appl. Pharmacol. 106, 136-144. Svendsgaard, D. J., Ward, T. R., Tilson, H. A., and Kodavanti, P. R. (1997) Empirical modeling of an in vitro activity of polychlorinated biphenyl congeners and mixtures. Environ. Health Perspect. 105, 1106-1115. Connor, K., Ramamoorthy, K., Moore, M., Mustain, M., Chen, I., Safe, S., Zacharewski, T., Gillesby, B., Joyeux, A., and Balaguer, P. (1997) Hydroxylated polychlorinated biphenyls (PCBs) as estrogens and antiestrogens: structure-activity relationships. Toxicol. Appl. Pharmacol. 145, 111-123. Rohr, U., Konig, W., and Selenka, F. (1985) Effect of pesticides on the release of histamine, chemotactic factors and leukotrienes from rat mast cells and human basophils. Zentralbl. Bakteriol. Mikrobiol. Hyg. 181, 469-486. Coats, J. R. (1990) Mechanisms of toxic action and structureactivity relationships for organochlorine and synthetic pyrethroid insecticides. Environ. Health Perspect. 87, 255-262. Moser, G. J., Robinette, C. L., and Smart, R. C. (1993) Characterization of skin tumor promotion by mirex: structure-activity relationships, sexual dimorphism and presence of Ha-ras mutation. Carcinogenesis 14, 1155-1160. Voie, O. A., Tysklind, M., Andersson, P. L., and Fonnum, F. (2000) Activation of respiratory burst in human granulocytes by polychlorinated biphenyls: A structure-activity study. Toxicol. Appl. Pharmacol. 167, 118-124. Olivero-Verbel, J., and Ganey, P. E. (1998) Effect of three polychlorinated biphenyls on f-met-leu-phe-induced degranulation in rat neutrophils. Toxicol. Lett. 98, 195-202. Tithof, P. K., Peters-Golden, M., and Ganey, P. E. (1998) Distinct phospholipases A2 regulate the release of arachidonic acid for

Motif Required for Activation of PLA2 by Organochlorines

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 159

eicosanoid production and superoxide anion generation in neutrophils. J. Immunol. 160, 953-960. Bergmeyer, H., and Bernt, E. (1974) Lactate dehydrogenase UV assay with pyruvate and NADH. In Methods of Enzymatic Analysis (Bergmeyer, H. V., Ed.) pp 524-579, Academic Press, New York. Dewar, M., Zoebisch, E., Healy, E., and Stewart, J. (1985) AM1: A new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107, 3902-3909. Hehre, W. J., Stewart, R. F., and Pople, J. A. (1969) Self-consisted molecular orbital methods. I. Use of Gaussian expansion of Slatertype atomic orbitals. J. Chem. Phys. 51, 2657-2664. Olivero, J., and Ganey, P. E. (2001) Participation of Ca2+/ calmodulin during activation of rat neutrophils by polychlorinated biphenyls. Biochem. Pharmacol. 62, 1125-1132. Hazen, S. L., Hall, C. R., Ford, D. A., and Gross, R. W. (1993) Isolation of a human myocardial cytosolic phospholipase A2 isoform. Fast atom bombardment mass spectroscopic and reversephase high-pressure liquid chromatography identification of choline and ethanolamine glycerophospholipid substrates. J. Clin. Invest. 91, 2513-2522. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J. Biol. Chem. 270, 445-450. Tang, J., Kriz, W., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs. J. Biol. Chem. 272, 8567-8575. Surette, M. E., Dallaire, N., Jean, N., Picard, S., and Borgeat, P. (1998) Mechanisms of priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils. FASEB J. 14, 1521-1531. Mollapour, E., Linch D. C., and Roberts, P. J. (2001) Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42 (ERK2) and p38 (SAPK) and by methyl arachidonyl fluorophosphonate, the dual inhibitor of

cytosolic and calcium-independent phospholipase A2. Blood 97, 2469-2477. Marshall, J., Krump, E., Lindsay, T., Downey, G., Ford, D. A., Zhu, P., Walker, P., and Rubin, B. (2000) Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils. J. Immunol. 164, 20842091. Lio, Y. C., Reynolds, L. J., Balsinde, J., and Dennis, E. A. (1996) Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochim. Biophys. Acta 1302, 55-60. Balsinde, J., and Dennis, E. A. (1997) Function and inhibition of intracellular calcium-independent phospholipase A2. J. Biol. Chem. 272, 16069-16072. Hazen, S. L., Zupan, L. A., Weiss, R. H., Getman, D. P., and Gross, R. W. (1991) Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J. Biol. Chem. 266, 7227-7232. Tithof, P. K., Olivero, J., Ruehle, K., and Ganey, P. E. (2000) Activation of neutrophil calcium-dependent and -independent phospholipases A2 by organochlorine compounds. Toxicol. Sci. 53, 40-47. Matsumura, F. (1985) Involvement of picrotoxinin receptor in the action of cyclodiene insecticides. Neurotoxicology 6, 139-163. King, R. D., and Srinivasan, A. (1996) Prediction of rodent carcinogenicity bioassays from molecular structure using inductive logic programming. Environ. Health Prospect. 104S, 10311040. Randic, M. (1991) Resolution of ambiguities in structure-property studies by use of orthogonal descriptors. J. Chem. Inf. Comput. Sci. 31, 311-320. Wagner, P. M., Nabholz, J. V., and Kent, R. J. (1995) The new chemicals process at the Environmental Protection Agency (EPA): Structure-activity relationships for hazard identification and risk assessment. Toxicol. Lett. 79, 67-73.

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42) (43)

(44)

(45) (46)

(47) (48)

TX0155449