Neutrophil Activation by Polychlorinated Biphenyls: Structure–Activity Relationship

TOXICOLOGICAL SCIENCES ARTICLE NO.

46, 308 –316 (1998)

TX982551

Neutrophil Activation by Polychlorinated Biphenyls: Structure–Activity Relationship Alan P. Brown,* Jesus Olivero-Verbel,* Wendy L. Holdan,* and Patricia E. Ganey*,† *Department of Pharmacology and Toxicology and †Department of Medicine, Institute for Environmental Toxicology, and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824 Received April 1, 1998; accepted July 23, 1998

Neutrophil Activation by Polychlorinated Biphenyls: Structure– Activity Relationship. Brown, A. P., Olivero-Verbel, J., Holdan, W. L., and Ganey, P. E. (1998). Toxicol. Sci. 46, 308 –316.

along with the sum of chlorine atomic charges, are associated with this biological activity. © 1998 Society of Toxicology. Key Words: PCB; molecular model; superoxide anion; leukocyte.

Polychlorinated biphenyls (PCBs) rapidly stimulate polymorphonuclear leukocytes (neutrophils) in vitro to produce superoxide anion (O2 2 ). This response results from activation of various intracellular signal transduction pathways and appears to occur in a structure-specific fashion. Individual PCB congeners, varying in pattern and extent of chlorination, were tested for their ability to stimulate production of O2 2 and/or to enhance the response to protein kinase C activation by phorbol myristate acetate (PMA). Neutrophils were isolated from retired breeder, male, Sprague– Dawley rats and exposed to either vehicle, 10 or 50 mM PCB for 30 min at 37°C. PMA (0 or 20 ng/ml) was added for an additional 10 min, and O2 2 generated during the incubation period was measured. 2,2*-Dichlorobiphenyl (2,2*-DCB), 2,4*-DCB, or 3,3*DCB (50 mM) stimulated neutrophils to produce O2 2 . Incubation of neutrophils with 4,4*-DCB, 3,3*,4,4*,5-pentachlorobiphenyl (3,3*,4,4*,5-PeCB), 3,3*,4,5,5*-PeCB, or 2,2*,3,3*,4,4*-hexachlorobiphenyl (2,2*,3,3*,4,4*-HCB) did not result in generation of O2 2 . Of the various congeners, 2,4*-DCB elicited the greatest production of O22. Exposure to 10 mM 2,2*-DCB, 2,4*-DCB, 3,3*-DCB, or 2,2*,3,3*,4,4*-HCB prior to addition of PMA caused a significant increase in the amount of O2 2 produced, greater than that seen with either compound alone. PMA-stimulated O2 2 production was unaffected by prior exposure to 4,4*-DCB, 3,3*,4,4*,5-PeCB, or 3,3*,4,5,5*-PeCB. In separate experiments, 3,3*,4,4*,5-PeCB inhibited the amount of O2 2 produced in response to activation with either 3,3*-DCB or 2,4*-DCB. Thus, it appears that congeners which are noncoplanar are capable of stimulating neutrophil O2 2 production. Coplanar congeners with high affinity for the Ah receptor do not activate neutrophils to produce O2 2 and may inhibit this response. These results are consistent with the hypothesis that PCBs stimulate neutrophil O2 2 production by a mechanism that is structure-specific and dependent on the chlorine substitution pattern of the biphenyl rings. Molecular modeling suggested that the sum of atomic charges on chlorine atoms is the most important descriptor for congeners which stimulate O2 2 production. The angle of rotation and the difference in energy between the highest occupied molecular orbital and the lowest unoccupied molecular orbital are integrative descriptors which,

Polychlorinated biphenyls (PCBs) are chemicals which have been released into the environment, resulting in widespread and persistent contamination. They exist as 209 different congeners that vary with the chlorine substitution pattern of the biphenyl rings. Twenty of these congeners favor a coplanar configuration (Safe, 1984). Coplanar PCBs are structurally related to polychlorinated dibenzofurans and polychlorinated dibenzo-p-dioxins, the most toxic of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Many of the biochemical changes and toxic responses elicited by TCDD, coplanar PCBs, and structurally related compounds arise from interactions with the Ah receptor and subsequent alterations in gene expression (Whitlock, 1990). Structure–activity studies have shown that the Ah receptor contains a hydrophobic ligand-binding site that preferentially binds coplanar, nonpolar molecules (Landers and Bunce, 1991). Among the biological effects produced by PCBs is activation of polymorphonuclear leukocytes (neutrophils). Neutrophils are usually the first leukocyte to arrive at sites of infection or tissue injury and are a major cellular component of acute inflammatory responses (Sandborg and Smolen, 1988). As part of their function in inflammation and immunity, neutrophils become activated and generate superoxide anion (O2 2 ). PCBs and alter the genactivate neutrophils in vitro to produce O2 2 in response to other stimuli (Ganey et al., 1993). eration of O2 2 Studies examining the mechanisms by which PCBs stimulate neutrophils have revealed roles for phosphoinositide hydrolysis, activation of phospholipase A2 and tyrosine kinases, and calcium mobilization (Brown and Ganey, 1995; Tithof et al., 1995, 1996, 1997, and 1998). Some PCB congeners are effective activators of neutrophils and others are not. The ability of individual PCB congeners to stimulate O2 2 production by neutrophils is likely to be related to structural

1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.

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configuration, which is determined by the chlorine substitution pattern on the biphenyl rings. Previous studies have demonstrated that congeners with ortho substitution such as 2,29,4,49tetrachlorobiphenyl (2,29,4,49-TCB) and 2,3,4,5-TCB activate neutrophils, whereas congeners without ortho substitution (e.g., 3,39,4,49-TCB) do not (Ganey et al., 1993; Brown and Ganey, 1995; Tithof et al., 1996). Since both 2,29,4,49-TCB and 2,3,4,5-TCB have low affinity for the Ah receptor, whereas 3,39,4,49-TCB has high affinity for the receptor (Safe et al., 1985), these results suggest that neutrophil activation occurs through a mechanism independent of the Ah receptor. In the present study, a more detailed examination of the relationship between PCB structure and ability to activate neutrophils was undertaken. PCB congeners with various chlorination patterns were tested for their ability to stimulate production of O2 2 and to enhance O2 2 generation in response to activation of protein kinase C. Molecular modeling was conducted to identify molecular descriptors which associate with stimulation of neutrophil O2 2 production. MATERIALS AND METHODS Chemicals Individual polychlorinated biphenyl congeners (.99% pure) were purchased from ChemService (West Chester, PA). Glycogen (Type II from oyster), phorbol myristate acetate (PMA), superoxide dismutase (SOD), dimethylformamide (DMF), and ferricytochrome C were purchased from Sigma Chemical Company (St. Louis, MO). Isolation of Rat Neutrophils Neutrophils were isolated from the peritoneum of male, Sprague–Dawley, retired breeder rats (CD-Crl:CD (SD)BR VAF/Plus; Charles River Laboratories, Portage, MI) by glycogen elicitation as described previously (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.4. The final concentration of cells in all of the assays was 2 3 106 cells/ml. Superoxide Anion Production Neutrophils (2 3 106 cells) were suspended in borosilicate glass test tubes, 12 3 75 mm (VWR, Chicago, IL), in a final volume of 1 ml HBSS and exposed to either vehicle (1 ml DMF) or 10 or 50 mM PCB for 30 min at 37°C. Stock solutions of the PCBs were prepared in DMF, and 1 ml of the stock solution of PCB was added to the cells to achieve the desired concentration. These PCB concentrations were chosen based on results of previous studies examining the ability of PCBs to activate neutrophils in the absence of significant cytotoxicity (Ganey et al., 1993; Brown and Ganey, 1995). PMA (0 or 20 ng/ml) was then added for an additional 10 min (37°C). O2 2 generated during this 40-min period was measured by the SOD-sensitive reduction of ferricytochrome C (Babior et al., 1973). In a separate series of experiments, neutrophils were exposed to PCB congeners alone or in combination for 40 min at 37°C, and O2 2 production was determined. Cytotoxicity Assay Release by neutrophils of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium was used as a marker of cytotoxicity. Neutrophils were exposed to either vehicle (DMF) or to PCB congeners alone or in

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combination for 40 min at 37°C. The cells were centrifuged immediately at 4°C, and the cell-free supernatant fluids were collected. LDH activity in the supernatant fluids was measured according to the method of Bergmeyer and Bernt (Bergmeyer and Bernt, 1974). Release of LDH into the medium was expressed as the percentage of total LDH activity that was present in an equivalent number of cells lysed with 1% Triton X-100. Statistical Analysis All results are presented as means 6 the standard error of the mean (SEM). For all results presented, N represents the number of individual experiments using neutrophils isolated from individual animals. Unless otherwise stated, data were analyzed by a two-way analysis of variance (ANOVA), and individual comparisons were performed using the least significant difference test. The criterion for statistical significance was p , 0.05. Analysis of Structure–Activity Relationships Descriptor generation. Chemical structures of the seven PCB congeners were entered into a computer program which then searched for the conformation of least energy for each congener in aqueous solution. Based on these structures, electronic, geometrical, and topological molecular descriptors were calculated for each congener. To accomplish this, PCB congener structures were entered into the Spartan Program (Wavefunction, Inc., Spartan Version 4, 1995, Irvine, CA) running on a Silicon Graphics Workstation. Conformer search was conducted at the semiempirical level Austin Model 1 (AM1) (Dewar et al., 1985) using water as a solvent, and this preliminary conformation was used to perform Ab Initio 3-21G(*) full optimization (Pietro et al., 1982) and single-point properties calculations. The following electronic descriptors were obtained from single-point calculations: the most positive charge (MPC) and the most negative charge (MNC) in the molecule accounting for nonspecific interactions between molecules; the sum of the atomic charges for all of the carbon atoms (¥CAC) and the sum of the atomic charges for all of the chlorine atoms (¥ClAC) as a measure of localized electronic density which may mediate electrostatic, intermolecular interactions; and the energy of the highest occupied molecular orbital (HOMOE) and the energy of the lowest unoccupied molecular orbital (LUMOE), which have been recognized as important descriptors in electron transfer from a donor to an acceptor molecule through the formation of charge transfer complexes (Franke, 1984). In general, charge-based descriptors are considered as chemical reactivity indices which may mediate weak, intermolecular interactions (Karelson and Lobanov, 1996). In addition, the difference between HOMOE and LUMOE, the HOMO–LUMO gap, was included as a descriptor. The torsional angle 2,1,19,29(u) obtained from the Ab Initio calculation was used as a geometrical descriptor. Topological descriptors included molecular connectivity values which describe molecular size and shape. These were calculated with a PC 586/66 MHz using PCDM, a program written by Olivero et al. (1995). Calculated connectivity values included Randic (Randic, 1975) and Kier and Hall indices (Kier and Hall, 1986).

RESULTS

In the absence of any stimulus, O2 2 production by rat neutrophils was negligible (Figs. 1–3), which is consistent with previous reports (Ganey et al., 1993). Exposure to 50 mM but not 10 mM 2,29-dichlorobiphenyl (2,29-DCB) caused significant production of O2 2 in the absence of PMA (Fig. 1A). Neutrophils exposed to vehicle (DMF) prior to stimulation with PMA, an activator of protein kinase C (Tauber, 1987), produced a significant amount of O2 2 , as demonstrated previously (Ganey et al., 1993). Exposure to 10 mM 2,29-DCB prior

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O2 2 in response to 10 mM but not to 50 mM 3,39-DCB was elevated significantly by addition of PMA. O2 2 generation was not stimulated by exposure of neutrophils to either 10 or 50 mM 4,49-DCB (Fig. 2A). Addition of PMA resulted in significant generation of O2 2 which was unaffected by prior exposure to 4,49-DCB. Similar results were seen with 3,39,4,49,5-pentachlorobiphenyl (3,39,4,49,5-PeCB; Fig. 2B) and 3,39,4,5,59-PeCB (Fig. 2C): neutrophils exposed to either of these congeners alone were not stimulated to produce O2 2 , and neither PCB congener increased the response to PMA. Exposure of neutrophils to either 10 or 50 mM 2,29,3,39,4,49hexachlorobiphenyl (2,29,3,39,4,49-HCB) did not result in generation of O2 2 (Fig. 3). Prior exposure to 2,29,3,39,4,49-HCB significantly increased the amount of O2 2 produced upon addition of PMA.

FIG. 1. Superoxide anion (O2 2 ) production during exposure to PCB congeners which activated neutrophils in the absence of PMA and increased the response to PMA. Neutrophils were exposed to 0, 10, or 50 mM of (A) 2,29-DCB, (B) 2,49-DCB, or (C) 3,39-DCB in the presence (filled circles) and absence (open circles) of PMA as described under Materials and Methods. O2 2 produced during the incubation period was determined. N 5 3–5. a, Significantly different from respective value in the absence of PMA. b, Significantly different from respective value in the absence of DCB.

to addition of PMA resulted in a significant increase in the amount of O2 2 produced, greater than that seen with either compound alone. O2 2 production by neutrophils exposed to 50 mM 2,29-DCB before addition of PMA was not different from that seen with either stimulus alone. Neutrophils exposed to 50 mM but not 10 mM 2,49-DCB produced a significant amount of O2 2 (Fig. 1B). Exposure to 10 mM 2,49-DCB prior to addition of PMA resulted in a significant increase in the amount of O2 2 generated compared to that produced by PMA alone. Exposure to 50 mM 2,49-DCB prior to PMA produced an equivalent amount of O2 2 as with 50 mM 2,49-DCB alone. Exposure of neutrophils to 50 mM but not 10 mM 3,39-DCB elicited significant production of O2 2 (Fig. 1C). Generation of

FIG. 2. Superoxide anion (O2 2 ) production during exposure to PCB congeners which did not activate neutrophils or alter the response to PMA. Neutrophils were exposed to 0, 10, or 50 mM of (A) 4,49-DCB, (B) 3,39,4,49,5PeCB, or (C) 3,39,4,5,59-PeCB in the presence (filled circles) and absence (open circles) of PMA as described under Materials and Methods. O2 2 produced during the incubation period was determined. N 5 3– 6. a, Significantly different from respective value in the absence of PMA.

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TABLE 2 Lactate Dehydrogenase Released (% Total) from Neutrophils 3,39,4,49,5-PeCB (mM) Stimulus

0

50

Vehicle 3,39-DCB 2,49-DCB

7.1 6 1.5 15.6 6 3.4 13.8 6 1.9

8.4 6 0.8 10.4 6 0.2 13.9 6 2.0

Note. Rat neutrophils were exposed to vehicle (DMF), 50 mM 3,39-DCB, or 50 mM 2,49-DCB in the presence or absence of 3,39,4,49,5-PeCB for 40 min at 37°C. LDH released into the medium was measured and reported as the percentage of total cellular LDH activity as described under Materials and Methods. N 5 3. No significant differences were observed. FIG. 3. Superoxide anion (O2 2 ) production during exposure to a PCB congener which increased the response to PMA but alone did not activate neutrophils. Neutrophils were exposed to 0, 10, or 50 mM 2,29,3,39,4,49-HCB in the presence (filled circles) and absence (open circles) of PMA as described under Material and Methods. O2 2 produced during the incubation period was determined. N 5 4 – 6. a, Significantly different from respective value in the absence of PMA. b, Significantly different from respective value in the absence of 2,29,3,39,4,49-HCB.

The effect of a coplanar congener which alone did not activate neutrophils on the response to two congeners which did stimulate neutrophils was examined. Neutrophils were exposed to either 3,39-DCB or 2,49-DCB for 40 min in the presence or absence of 3,39,4,49,5-PeCB (Table 1). 3,39,4,49,5PeCB inhibited the production of O2 2 in response to 3,39-DCB and 2,49-DCB by 91 and 57%, respectively. Coincubation with 3,39,4,49,5-PeCB did not affect release of LDH from neutrophils exposed to either of the DCB congeners (Table 2). The relationship among the absolute values for the HOMO– LUMO gap, torsional angle, ¥ClAC, and the difference between O2 2 produced in response to 0 and 50 mM PCB in the presence of PMA (i.e., “biological response”) is shown graphically in Fig. 4. As illustrated, the molecular descriptor ¥ClAC TABLE 1 Inhibition of Neutrophil Superoxide Anion Production (nmoles O2 2 Produced) By 3,3*,4,4*,5-Pentachlorobiphenyl 3,39,4,49,5-PeCB (mM) Stimulus

0

50

% Inhibition

Vehicle 3,39-DCB 2,49-DCB

1.6 6 0.2 28.0 6 5.1 36.8 6 5.0

2.9 6 1.1 2.5 6 1.0* 16.0 6 3.7*

— 91 57

Note. Rat neutrophils were exposed to vehicle (DMF), 50 mM 3,39-DCB, or 50 mM 2,49-DCB in the presence or absence of 3,39,4,49,5-PeCB for 40 min at 37°C, and O2 2 produced during the incubation period was determined. N 5 4. * Significantly different from respective value in the absence of 3,39,4,49,5PeCB (Student’s t test).

was inversely related to this biological response. Congeners for which ¥ClAC is large (4,49-DCB, 3,39,4,5,59-PeCB, and 3,39,4,49,5-PeCB) produced little biological response, whereas those congeners for which this molecular descriptor is small (3,39-DCB, 2,49-DCB, and 2,29,3,39,4,49-HCB) caused an increase in O2 2 production at 50 mM in the presence of PMA. The exception is 2,29-DCB for which both the biological response (at 50 mM) and the molecular descriptor were small. Torsional angle did not appear to influence this response because the value for this parameter was small for a congener which produced a significant response (i.e., 3,39-DCB) and for congeners (4,49-DCB, 3,39,4,5,59-PeCB, and 3,39,4,49,5-PeCB) which did not elicit a response. Likewise, torsional angle was large for congeners which did (2,49 DCB and 2,29,3,39,4,49HCB) and did not (2,29-DCB) induce O2 2 production under these conditions. All of the molecules with small biological activities also had small HOMO–LUMO gaps, whereas three of the four congeners with large HOMO–LUMO gaps elicited O2 2 production. Once again, the exception was 2,29-DCB. Relationships between biological activity (dependent variable) and molecular descriptors (independent variables) were established by the stepwise multiple regression technique (Jandel Corp.). The biological response used for modeling was the difference in the amount of O2 2 produced at 0 and 50 mM PCB, and models were generated for data obtained in the absence and in the presence of PMA. The best regression models were selected on the basis of the greatest multiple correlation coefficient (R) obtained between the experimental and predicted data, the lowest standard error of estimation (SE) and the highest F value from the variance analysis. The most important descriptor for models in the absence or presence of PMA was the reciprocal of ¥ClAC. The model for O2 2 production in the absence of PMA was improved by the addition of torsional angle as a descriptor (R 5 0.930 using both descriptors compared to 0.538 with 1/¥ClAC alone). The addition of the HOMO–LUMO gap as a descriptor improved the prediction model for data obtained in the presence of PMA (R 5 0.964 using both descriptors compared to 0.574 with 1/¥ClAC alone).

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FIG. 4. Relationship among the specific response produced at 50 mM PCB in the presence of PMA, HOMO–LUMO gap, ¥ClAC, and torsional angle for PCB congeners examined in this study. The length of the line from the center represents the absolute value for each parameter.

DISCUSSION

Much of the research into the biological effects of PCBs has focused on TCDD-like activity of these chemicals such as cytochrome P-450 induction, hepatotoxicity, thymic atrophy, etc. (Safe, 1994). A primary structural requirement for TCDDlike effects of PCB congeners is the tendency toward coplanarity of the two phenyl rings, such that the PCB molecule attains a structure with dimensions approximating 3 3 10 Å. Conditions favoring this structure include substitution with chlorine at four meta and/or para sites and lack of substitution at ortho positions. This structure provides steric fit and net polarizability which increase binding affinity for the Ah receptor (McKinney and Singh, 1981). More recently, quantum chemical calculations revealed that electronic-derived physicochemical properties such as electron affinities, entropies, and electronic energy gaps are also important parameters governing the interaction of PCBs with the Ah receptor (Kafafi et al., 1993a, b). There is growing evidence that congeners which do not favor a coplanar configuration also have biological activity (Ganey et al., 1993; Shain et al., 1991; Kodavanti et al., 1995),

although the structural requirements for these effects are incompletely understood. We have demonstrated previously that activation of neutrophils by PCBs does not appear to be mediated through the Ah receptor, despite the presence of these receptors in rat neutrophils (Ganey et al., 1993). The current study was undertaken to explore more extensively which PCB congeners elicit neutrophil activation and to identify structural requirements for these effects. What emerged from examination of the effects of these congeners on neutrophil function in this and previous studies were three patterns of response. In the first, PCBs (i.e., 2,29-DCB, 2,49-DCB, 3,39-DCB, 2,29,4,49-TCB, and 2,3,4,5-TCB (Brown and Ganey, 1995)) stimulated neutrophils to produce O2 2 in the absence of PMA and increased the response to PMA (Table 3, Fig. 1). The second pattern was characterized by 2,29,3,39,4,49HCB, which alone did not activate neutrophils but potentiated PMA-induced O2 2 production (Fig. 3). In the third pattern, PCBs (i.e., 4,49-DCB, 3,39,4,5,59-PeCB, 3,39,4,49,5-PeCB, and 3,39,4,49TCB (Ganey et al., 1993)) neither elicited O2 2 production nor changed the response to PMA (Fig. 2). Results of previous studies suggested that substitution at the

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TABLE 3 Physical Properties of Various PCB Congeners and Their Effects on Function of Neutrophils

Congener

Stimulates O2 2 production?

Potentiates PMA response?

Kcalc (M)a

Log KOWb

u 2,1,19,29

2,29-DCB 2,49-DCB 2,29,4,49-TCB 2,3,4,5-TCB 3,39-DCB 2,29,3,39,4,49-HCB 4,49-DCB 3,39,4,5,59-PeCB 3,39,4,49-TCB 3,39,4,49,5-PeCB

Yes Yes Yesc Yesd Yes No No No Noc No

Yes Yes Yesc Yesd Yes Yes No No Noc No

6.9 3 1021 1.9 3 1023 5.2 3 1024 1.0 3 1024 9.4 3 1025 2.7 3 1025 1.3 3 1026 1.0 3 1027 7.1 3 1028 3.3 3 1028

4.9 5.1 5.9 5.9 5.3 7.0 5.3 NR 6.1 NR

90.4 73.2 90.2 59.9 52.3 90.2 51.7 53.0 52.4 52.4

Note. NR, not reported. Kcalc (M) is the calculated in vitro dissociation constant of PCB–Ah receptor complexes in rat liver cytosols (from Kafafi et al., 1993b). b KOW is the octanol/water partition coefficient (from Shiu and Mackay, 1986). c From Ganey et al., 1993. d From Brown and Ganey, 1995. a

ortho position may be a requirement for neutrophil activation by PCBs because 2,29,4,49-TCB and 2,3,4,5-TCB, but not 3,39,4,49-TCB, influenced neutrophil function (Ganey et al., 1993; Brown and Ganey, 1995). All of the ortho-substituted congeners examined in this study stimulated neutrophils to produce O2 2 and/or altered the response to PMA (Table 3). The hypothesis that ortho substitution is required for activation of neutrophils by PCBs was refuted, however, by results of studies in which 3,39-DCB caused generation of O2 2 by itself and increased the response to PMA (Fig. 1C). Examination of octanol/water partition coefficients (Table 3) for individual congeners revealed that the propensity to activate neutrophils did not correlate with hydrophobicity. For example, the values for log KOW are the same for 3,39-DCB and 4,49-DCB: The former both stimulated O2 2 production and increased the response to PMA, whereas the latter did not. Based on the log KOW values, at the concentrations used most of the PCBs would be expected to partition into the lipid membranes of the neutrophils. Thus, it is not surprising that differences in biological activity among congeners are not related to different partitioning within the cells. The relation of the octanol/water partition coefficient to activity has been examined in other systems. Similar to results observed here, this parameter did not correlate with the decrease in dopamine content of PC12 cells (Shain et al., 1991) and in rat hepatoma cells hydrophobicity was found to modulate interactions of PCBs with the Ah receptor (Safe et al., 1985). Alteration of neutrophil function by PCBs also did not correlate with the degree of chlorination because congeners with as few as two and as many as six chlorines potentiated the response to PMA. Activation of neutrophils by PCBs appears to be related to a low affinity for the Ah receptor, based on the in vitro dissociation constant (Table 3). This suggests that congeners which

favor a noncoplanar structure are more likely than coplanar congeners to activate neutrophils in vitro. Para substitution of 3,39-DCB to yield 3,39,4,49-TCB abolished activity to stimulate neutrophils; however, para substitution of 2,29-DCB (to yield 2,29,4,49-TCB) did not diminish the potential to activate neutrophils. 3,39,4,49-TCB attains a coplanar structure more readily than the diortho-substituted 2,29,4,49-TCB because ortho substitution substantially raises the energy barrier to rotation about the bond between the phenyl rings (McKinney and Waller, 1994). Thus, this observation supports the hypothesis that noncoplanar structure is important in the ability of PCBs to activate neutrophils. Similarly, ortho substitution of 3,39,4,49TCB conferred the property to increase generation of O2 2 in response to PMA (Fig. 3). Despite the apparent importance of noncoplanarity in the ability of PCBs to influence O2 2 production, results of molecular modeling argue against a structure–activity relationship based solely on coplanarity of the molecules. Examination of Table 3 reveals that the value for torsional angle (u) for 3,39-DCB, a congener which activated neutrophils, is similar to the value for a number of congeners which did not. Similar results are depicted in Fig. 4 for the specific responses observed at a PCB concentration of 50 mM in the presence of PMA. Although torsional angle alone did not correlate with O2 2 production, inclusion of this parameter with the reciprocal of ¥ClAC in a regression model for the response in the absence of PMA significantly improved the regression coefficient. Thus, angle of rotation about the bond between phenyl rings may be an important parameter for PCB-induced O2 2 production under some, but not all, conditions, and other factors influence this response as well. In fact, results of regression analysis suggest that the reciprocal of ¥ClAC is the most important descriptor for the re-

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sponse to PCBs in the presence or absence of PMA. The sum of atomic charges is an important descriptor in other models of toxicity (Katritzky and Gordeeva, 1993) as well as in some pharmacological activities (Cocchi et al., 1995). The regression model for biological activity in the presence of PMA was strengthened by addition of the value for the HOMO–LUMO gap, which correlates strongly (R 5 0.953, p 5 0.001) with torsional angle. The HOMO–LUMO gap is a measure of the capacity of a molecule to transfer one electron from the HOMO to the LUMO, and its magnitude is related directly to molecular stability (Mekenyan et al., 1994). A model described by SClAC and the HOMO–LUMO gap suggests that PCB-induced O2 2 production may be mediated through electrostatic interactions which involve an electron transfer complex between the PCB congener and a cellular molecule(s) and that this interaction is favored for PCB congeners with greater molecular stability. Studies in vitro investigating the neurotoxicity of individual PCB congeners have demonstrated similar structure–activity relationships as seen in the current study. Congeners with chlorine atoms in the ortho position decreased cellular dopamine content in PC12 cells (Shain et al., 1991) and increased binding of phorbol esters in rat cerebellar granular cells (Kodavanti et al., 1995), whereas congeners without ortho substitution that are coplanar were inactive. Similar to our studies in which 3,39-DCB stimulated O2 2 production by neutrophils, 3,39-DCB decreased PC12 dopamine content and increased phorbol ester binding in cerebellar granular cells (Shain et al., 1991; Kodavanti et al., 1995). Studies with isolated rat cerebellar microsomes and mitochondria demonstrated that congeners with chlorine substitution at the ortho positions were the most potent in inhibiting Ca12 sequestration (Kodavanti et al., 1996). Similarly, in sarcoplasmic reticulum from skeletal and cardiac tissue ortho-substituted, and not coplanar, PCB congeners increased Ca12 release and binding of ryanodine to ryanodine-sensitive Ca12 release channels (Wong and Pessah, 1996). The mechanisms by which noncoplanar PCBs induce biological effects are a subject of current investigation. In studies presented here, both the ability to stimulate O2 2 production and the capacity to alter the response to activation of protein kinase C by PMA were examined. Generation of O2 2 by neutrophils is effected by an NADPH oxidase which is assembled and activated in the plasma membrane, and this process involves a number of intracellular signal transduction pathways (Sadler and Badwey, 1988). PMA initiates O2 2 production through activation of protein kinase C (PKC) (Tauber, 1987). The observation that 3,39,4,49,5-PeCB decreased the response to 2,49-DCB and 3,39-DCB but not to PMA suggests that the mechanism by which these two DCBs activate neutrophils differs from the mechanism of activation by PMA. In support of this hypothesis, previous studies have demonstrated that PCBs activate O2 2 production by a mechanism which is depen-

dent on calcium, whereas PMA stimulates O2 2 generation by a calcium-independent mechanism (Brown and Ganey, 1995). The fact that some congeners (e.g., 2,29,3,39,4,49-HCB) did not alone stimulate O2 2 production but did increase the response to PMA raises the possibility that these two events occur by different mechanisms. 2,29,3,39,4,49-HCB may invoke a signal transduction pathway which does not activate NADPH oxidase but enhances O2 2 generation once the enzyme is assembled and activated. Alternatively, the same mechanism may be responsible for both responses. For example, in the case of 2,49-DCB, a signal transduction pathway may be activated which is sufficient at large PCB concentrations (50 mM) to activate NADPH oxidase in the absence of PMA but at smaller concentrations (10 mM) requires additional pathways to be activated by PMA to cause generation of O2 2 . It has been demonstrated that noncoplanar PCBs increase phorbol ester binding in cerebellar granular cells (Kodavanti et al., 1995), so that one mechanism by which these congeners potentiate the response to PMA in neutrophils may be by increasing the accessibility or affinity of PKC for PMA. In the current study, 3,39,4,49,5-PeCB was inactive in stimulating O2 2 production by neutrophils but inhibited the response to stimulation of neutrophils with either 3,39-DCB or 2,49-DCB. This inhibitory effect was unrelated to cytotoxicity, and the mechanism(s) involved is unknown. Previous studies have shown that [3H]TCDD bound specifically to cytosol from rat neutrophils, suggesting the presence of Ah receptors (Ganey et al., 1993). It is possible that congeners with dioxin-like characteristics and high binding affinities for the Ah receptor may elicit Ah receptor-dependent responses which downregulate activation of neutrophils. For example, activation of neutrophils is dependent on the sequential phosphorylation and dephosphorylation of several proteins in the signalling cascade. Recent evidence suggests that binding of TCDD to the Ah receptor elicits protein tyrosine phosphorylation through activation of a protein tyrosine kinase, pp60src (Enan and Matsumura, 1996; Blankenship and Matsumura, 1997; Ma et al., 1992). It is conceivable that activation of a protein tyrosine kinase may interfere with the signaling required for PCBinduced O2 2 production. If this is the case, the inhibitory effect may not extend to agents which stimulate neutrophils via direct activation of PKC, such as PMA (Fig. 2B). These results, in which a PCB congener inhibited the biological effect of another, are similar in nature to those observed for estrogenic activity of PCBs (Connor et al., 1997; Jansen et al., 1993). For example, whereas Aroclor 1242 and 2,29,5,59-TCB demonstrated estrogenic effects in rats, 3,39,4,49-TCB did not and inhibited this effect of Aroclor 1242 (Jansen et al., 1993). In summary, the results of this study suggest that chlorines in the ortho or meta position are required for stimulation of neutrophil O2 2 production in vitro. This pattern of substitution may provide characteristics of atomic charge distribution on chlorine atoms, torsional angle, and HOMO–LUMO gap that

NEUTROPHIL ACTIVATION BY POLYCHLORINATED BIPHENYLS

result in a configuration capable of activation of neutrophils. By contrast, coplanar congeners may act in an inhibitory fashion to downregulate O2 2 production in the presence of a second neutrophil stimulus. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Henry Ng. This research was supported by NIH Grant ES04911. A.P.B. was supported in part by a National Research Service Award from the National Institute of Environmental Health Sciences, NIH Grant ES05684.

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Karelson, M. and Lobanov, V. S. (1996). Quantum-chemical descriptors in QSAR/QSPR studies. Chem. Rev. 96, 1027–1043. Katritzky, A. R., and Gordeeva, E. V. (1993). Traditional topological indices vs electronic, geometrical, and combined molecular descriptors in QSAR/ QSPR research. J. Chem. Inf. Comput. Sci. 33, 835– 857. Kier, L., and Hall, H. L. (1986). Molecular Connectivity in Structure–Activity Analysis. Wiley, New York. Kodavanti, P. R. S., 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.

REFERENCES

Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996). Inhibition of microsomal and mitochondrial Ca21-sequestion in rat cerebellum by polychlorinated biphenyl mixtures and congeners: Structure–activity relationships. Arch. Toxicol. 70, 150 –157.

Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973). Biological defense mechanisms: The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52, 741–744.

Landers, J. P., and Bunce, N. J. (1991). The Ah receptor and the mechanism of dioxin toxicity. Biochem. J. 276, 273–287.

Bergmeyer, H. V., and Bernt, E. (1974). Lactate dehydrogenase UV assay with pyruvate and NADH. In Methods of Enzymatic Analysis (H. V. Bergmeyer, Ed.), pp. 524 –579. Academic Press, New York. Blankenship, A., and Matsumura, F. (1997). 2,3,7,8-Tetrachlorodibenzo-pdioxin-induced activation of a protein tyrosine kinase, pp60src, in murine hepatic cytosol using a cell-free system. Mol. Pharmacol. 52, 667– 675. Brown, A. P., and Ganey, P. E. (1995). Neutrophil degranulation and superoxide production induced by polychlorinated biphenyls are calcium dependent. Toxicol. Appl. Pharmacol. 131, 198 –205. Cocchi, M., Menziani, C., Fanelli, F., and de Benedetti, P. G. (1995). Theoretical quantitative structure–activity relationship analysis of congeneric and non-congeneric a1 adrenoreceptor antagonists: A chemometric study. J. Mol. Struct. (Theochem.) 331, 79 –93. Connor, K., Ramamoorthy, K., Moore, M., Maustain, 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. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F., and Stewart, J. J. P. (1985). AM1: A new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107, 3902–3909. Enan, E., and Matsumura, F. (1996). Identification of c-Src as the integral component of the cytosolic Ah receptor complex, transducing the signal of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the protein phosphorylation pathway. Biochem. Pharmacol. 52, 1599 –1612. Franke, R. (1984). Theoretical Drug Design Methods. Elsevier, New York. Ganey, P. E., Sirois, J. E., Denison, M., Robinson, J. P., and Roth, R. A. (1993). Neutrophil function after exposure to polychlorinated biphenyls in vitro. Environ. Health Perspect. 101(5), 430 – 434. Hewett, J. A., and Roth, R. A. (1988). Dieldrin activates rat neutrophils in vitro. Toxicol. Appl. Pharmacol. 96, 269 –278. Jansen, H. T., Cooke, P. S., Porcelli, J., Liu, T. C., and Hansen, L. G. (1993). Estrogenic and antiestrogenic actions of PCBs in the female rat: In vitro and in vivo studies. Reprod. Toxicol. 7, 237–248. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., Abd-Elazem, I. S., and Kafafi, A. G. (1993a). 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. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., and Kafafi, A. G. (1993b). Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor. Environ. Health Perspect. 101, 422– 428.

Ma, X., Mufti, N. A., and Babish, J. G. (1992). Protein tyrosine phosphorylation as an indicator of 2,3,7,8-tetrachloro-p-dioxin exposure in vivo and in vitro. Biochem. Biophys. Res. Commun. 189, 59 – 65. McKinney, J. D., and Singh, P. (1981). Structure–activity relationships in halogenated biphenyls: Unifying hypothesis for structural specificity. Chem.–Biol. Interact. 33, 271–283. McKinney, J. D., and Waller, C. L. (1994). Polychlorinated biphenyls as hormonally active structural analogues. Environ. Health Perspect. 102, 290 –297. Mekenyan, O. G., Ankley, G. T., Veith, G. D., and Call, D. J. (1994). QSARs for photoinduced toxicity. I. Acute lethality of polycyclic aromatic hydrocarbons to Daphnia magna. Chemosphere 28, 567–582. Olivero, J., Vivas, R., Payares, P., Dı´az, D., and Mercado, J. (1995). PCDM Version 2, Universidad de Cartagena, Cartagena, Columbia. Pietro, W. J., Francl, M. M., Hehre, W. J., DeFrees, D. J., Pople, J. A., and Binkley, J. S. (1982). Self-consistent molecular orbital methods. 24. Supplemented small split-valence basis sets for second-row elements. J. Am. Chem. Soc. 104, 5039 –5048. Randic, M. (1975). On characterization of molecular branching. J. Am. Chem. Soc. 97, 6609 – 6615. Sadler, K. L., and Badwey, J. A. (1988). Second messengers involved in superoxide production by neutrophils. Hematol./Oncol. Clin. N. Am. 2, 185–200. Safe, S. (1984). Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs): Biochemistry, toxicology, and mechanism of action. CRC Crit. Rev. Toxicol. 13, 319 –395. Safe, S., Bandiera, S., Sawyer, T., Bozena, Z., Mason, G., Romkes, M., Denomme, M. A., Sparling, J., Okey, A. B., and Fujita, T. (1985). Effects of structure on binding to the 2,3,7,8-TCDD receptor protein and AHH induction—Halogenated biphenyls. Environ. Health Perspect. 61, 21–33. Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. CRC Crit. Rev. Toxicol. 24, 87–149. Sandborg, R. R., and Smolen, J. E. (1988). Biology of disease: Early biochemical events in leukocyte activation. Lab. Invest. 59(3), 300 –320. Shain, W., Bush, B., and Seegal, R. (1991). Neurotoxicity of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation. Toxicol. Appl. Pharmacol. 111, 33– 42. Shiu, W. Y., and Mackay, D. (1986). A critical review of aqueous solubilities, vapor pressures, Henry’s law constants, and octanol–water partition coefficients of the polychlorinated biphenyls. J. Phys. Chem. Ref. Data 15, 911–929. Tauber, A. I. (1987). Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 69(3), 711–720.

316

BROWN ET AL.

Tithof, P. K., Contreras, M. L., and Ganey, P. E. (1995). Aroclor 1242 stimulates the production of inositol phosphates in polymorphonuclear neutrophils. Toxicol. Appl. Pharmacol. 131, 136 –143. Tithof, P. K., Peters-Golden, M., and Ganey, P. E. (1998). Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils. J. Immunol. 160, 953–960. Tithof, P. K., Schiamberg, E., Peters-Golden, M., and Ganey, P. E. (1996). Phospholipase A2 is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls. Environ. Health Perspect. 104, 52–58.

Tithof, P. K., Watts, S., and Ganey, P. E. (1997). Protein tyrosine kinase involvement in the production of superoxide anion by neutrophils exposed to Aroclor 1242, a mixture of polychlorinated biphenyls. Biochem. Pharmacol. 53, 1833–1842. Whitlock, J. P., Jr. (1990). Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu. Rev. Pharmacol. Toxicol. 30, 251–277. Wong, P. W., and Pessah, I. N. (1996). Ortho-substituted polychlorinated biphenyls alter calcium regulation by a ryanodine receptor-mediated mechanism: Structural specificity toward skeletal- and cardiac-type microsomal calcium release channels. Mol. Pharmacol. 49, 740 –751.