Activation of Neutrophil Calcium-Dependent and -Independent Phospholipases A2 by Organochlorine Compounds

Patricia K. Tithof*,1, Jesus Olivero{dagger}, Kirsten Ruehle{dagger} and Patricia E. Ganey{dagger}

* College of Veterinary Medicine, The University of Tennessee, 2407 River Drive. Knoxville, Tennessee 37996-4500; and {dagger} Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan

Received April 7, 1999; accepted August 15, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of reactive oxygen species by organochlorine pesticides has been implicated in the toxicity and carcinogenicity of these compounds; however, the mechanism by which these agents stimulate the production of oxygen radicals is unknown. Phospholipase A2 (PLA2)-mediated release of arachidonic acid has been shown to play an essential role in superoxide anion (O) production in neutrophils exposed to various physiologic and pharmacologic agents. Therefore, studies were performed to determine if the organochlorine pesticides, lindane and dieldrin, activate neutrophils to produce O by a mechanism that requires PLA2. Production of Oand 3H-AA release increased with similar kinetics and concentration-response relations in neutrophils activated with either dieldrin or lindane. Significant release of 3H -AA was seen in neutrophils stimulated with dieldrin or lindane in calcium-free medium and in the presence of the intracellular calcium chelator BAPTA-AM, suggesting that these agents stimulate a PLA2 that does not require calcium for activation. In addition, both O production and 3H-AA release were inhibited in a concentration-dependent manner by BEL, a mechanism-based inhibitor of calcium-independent PLA2. These data suggest that dieldrin and lindane stimulate O production by a mechanism that involves PLA2. However, release of 3H-AA was not abrogated completely by BEL nor, in the case of dieldrin, preserved entirely in the absence of calcium. This suggests that more than one isoform of PLA2 is activated by dieldrin and by lindane, and that one isoform is calcium-dependent.

Key Words: arachidonic acid; phospholipase A2; organochlorine compounds; neutrophils; superoxide anion; NADPH oxidase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of man-made chemicals have been released into the environment that have carcinogenic and toxic potential. Among these chemicals are the persistent, bioaccumulative, organohalogen compounds, which include insecticides such as dieldrin as well as lindane, and industrial chemicals such as polychlorinated biphenyls (PCBs). These compounds share in common the ability to cause oxidative stress by stimulating the production of reactive oxygen species (ROS), and this mechanism has been implicated in the immunotoxicity, hepatotoxicity, and carcinogenicity of these chemicals (Bachowski et al., 1997Go; Junqueira et al., 1986Go, 1994Go; Klaunig et al., 1998Go). Lindane ({gamma}-hexachlorocyclohexane) causes an increase in activity of the pro-oxidant enzyme NADPH oxidase in isolated neutrophils (Kuhns et al., 1986Go) and in liver microsomal preparations (Junqueira et al., 1986Go, 1994Go). In the liver, this pro-oxidant activity is accompanied by lipid peroxidation and hepatocellular injury (Junqueira et al., 1986Go, 1994Go). Similarly, dieldrin induces a state of oxidative stress in liver by causing an increase in the production of ROS with a concomitant decrease in antioxidant concentrations and an increase in hepatic DNA synthesis. Treatment with anti-oxidants attenuates all of these changes, suggesting the involvement of oxidant mechanisms in the toxic and tumor-promoting effects of dieldrin (Bachowski et al., 1997Go; Klaunig et al., 1995Go). In addition, dieldrin has been shown to activate the NADPH oxidase to produce superoxide anion (O) in isolated neutrophils, and this activity has been implicated in the immunotoxicity of this insecticide (Hewett et al., 1988). Although several studies have implicated the role of oxygen radicals in the toxic and carcinogenic effects of organochlorine pesticides, little work has been done to determine the cellular mechanism by which these chemicals act to promote pro-oxidant activity.

Our laboratory research has focused on the mechanism by which a related group of compounds, the PCBs, causes ROS formation in neutrophils (Ganey et al., 1993Go; Tithof et al., 1995Go, 1996Go, 1997Go, 1998Go). Activation of neutrophils by PCBs is complex and involves multiple enzymes including protein tyrosine kinases (Tithof et al., 1997Go), phospholipase C (Tithof et al., 1995Go), and phospholipase A2 (Tithof et al., 1996Go). Activation of phospholipase A2 (PLA2) by PCBs results in the release of large amounts of arachidonic acid (AA), which is essential for activation of the O-generating NADPH oxidase (Tithof et al., 1996Go, 1998Go). More recently, we have focused on characterization of the PLA2 that is activated by PCBs (Tithof et al., 1998Go).

Several different isoforms of PLA2 have been described. These include a large-molecular-weight cytosolic PLA2, which is calcium-dependent and selective for arachidonic acid at the sn-2 position of phospholipids (Alonso et al., 1986Go; Gronich et al., 1988Go; Kramer et al., 1991Go) and small-molecular-weight, calcium-dependent enzymes that are arachidonoyl-nonselective (14 kDa secretory PLA2 and 14 kDa pancreatic PLA2) (Dennis et al., 1991Go; Mayer and Marshall, 1993Go). In addition, a number of PLA2 enzymes have been described which are activated by a mechanism that does not require calcium. These include a calcium-independent enzyme found in cardiac myocytes, which is selective for arachidonic acid (Hazen et al., 1991bGo), and an enzyme in macrophage-like p388D1 cells which is not (Ackermann et al., 1994Go).

Recently, we demonstrated that the PLA2 activated by PCBs is an isoform not previously described in neutrophils (Tithof et al., 1998Go). Specifically, the enzyme is activated in the absence of calcium, is selective for arachidonate at the sn-2 position, and is inhibited by bromoenol lactone (BEL), an agent that is selective for calcium-independent PLA2 isoforms (Hazen et al., 1991bGo). Activation of this PLA2 isoform may play an important role in the mechanism by which other chlorinated compounds cause ROS formation. Therefore, the purpose of the present study was to test the hypothesis that dieldrin and lindane stimulate O production in neutrophils by a mechanism that involves calcium-independent PLA2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Dieldrin was obtained from Aldrich (Milwaukee, WI; purity ~90%). Lindane ({gamma}-hexachlorocyclohexane; purity ~99%), cytochrome C, and superoxide dismutase (SOD) were obtained from Sigma Chemical Company (St. Louis, MO). [3H][5,6,8,9,11,12,14,15]AA (3H-AA; 180–240 Ci/mmol) was purchased from DuPont NEN (Boston, MA). One,2-bis-(o-aminophenoxy)-ethane, N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) was obtained from Calbiochem (San Diego, CA). E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) was purchased from Biomol (Plymouth Meeting, PA). For all experiments, dieldrin, lindane, and BEL were dissolved in DMSO and diluted so that the final concentration of DMSO was <1%.

Neutrophil isolation.
Glycogen-elicited neutrophils were obtained from the peritoneal cavity of male Sprague-Dawley, retired-breeder rats as described previously (Ganey et al., 1993Go). Rats were anesthetized with diethyl ether, and were injected intraperitoneally with 30 ml of 1% glycogen. After 4 h, the rats were anesthetized again and killed by decapitation. The peritoneal cavity was washed with 30 ml of heparinized (1 U/ml) 0.1 M phosphate-buffered saline (PBS). The solution obtained from the peritoneal cavity was collected, filtered through gauze, and centrifuged at 500 x g for 7 min. Contaminating red blood cells were lysed with 15 ml of 0.15 M NH4Cl, and neutrophils were suspended to a final volume of 50 ml with PBS and centrifuged for 7 min at 300 x g. Cells were washed once with 0.1 M PBS and resuspended in Hanks' balanced salt solution (HBSS).

Labeling of neutrophils with 3H-arachidonic acid.
Neutrophils were suspended in Ca2+- and Mg2+-free HBSS containing 0.1% bovine serum albumin (BSA) and were labeled for 90 min at 37°C with 0.5 µCi/ml 3H-arachidonic acid (3H-AA). Suspended neutrophils were prelabeled in the absence of Ca2+ and Mg2+ to prevent cellular aggregation. At the end of the labeling period, neutrophils were washed x2 and resuspended in HBSS containing 0.1% BSA, according to the method of Volpi et al., (1984). At the end of the incubation period, an aliquot of cells was subjected to scintillation counting to determine cellular uptake of radiolabel: uptake was routinely ~70% of added 3H-AA. In addition, the distribution of label within the various phospholipids was determined by thin-layer chromatography. Approximately 22% was distributed into phosphatidylinositol, 8% into phosphatidylserine, 53% into phosphatidylcholine, and 17% into phosphatidylethanol, indicating uniform distribution of label amongst membrane phospholipids. At the end of the labeling period, neutrophils were washed x2 and resuspended in HBSS containing 0.1% BSA.

Determination of fatty acid release from prelabeled neutrophils.
Cumulative release of 3H-AA was measured in cells stimulated for 20 min at 37°C with various concentrations of lindane (0, 10, 25, 50, and 100 µM) or dieldrin (0, 1, 10, 25, 50, and 100 µM). To determine the kinetics of release of arachidonate, accumulation of 3H-AA in the medium was determined in neutrophils stimulated for various times (1, 5, 10, or 30 min) with lindane (100 µM) or dieldrin (100 µM). To determine the role of calcium-independent PLA2 in lindane- and dieldrin-induced release of AA, neutrophils were preincubated for 15 min with the inhibitor of calcium-independent PLA2, BEL (0.1–20 µM). This was followed by stimulation with lindane or dieldrin for 20 min. Neutrophils were stimulated with lindane or dieldrin for 20 min in the presence or absence of extracellular calcium, and in the presence of 0.5 mM EGTA, to determine the role of extracellular calcium in stimulated release of AA. To determine the role of intracellular calcium in release of AA, neutrophils were loaded with the cell-permeant calcium chelator BAPTA-AM (50 µM) in calcium-free medium for 60 min prior to stimulation. This concentration of BAPTA-AM has been shown previously to effectively chelate intracellular calcium in neutrophils (O'Flaherty et al., 1991Go). At the end of the incubations, neutrophils were placed on ice and centrifuged, and radioactivity in the cell-free supernatant fluids was determined by liquid scintillation counting.

Generation and detection of O.
Superoxide anion (O) production was measured in neutrophils stimulated as describe above. Cumulative O production was measured as the reduction of cytochrome C in the presence or absence of superoxide dismutase (SOD) (Babior et al., 1973Go). For every sample, two tubes were incubated, one to which SOD (840 U/ml) was added before stimulation and one to which SOD (840 U/ml) was added at the end of incubation. The amount of cytochrome C reduced was estimated from the difference in absorbance (550 nm) between the cell-free supernatant fluids in the two tubes, using an extinction coefficient of 18.5 cm-1mM-1.

Determination of cytotoxicity.
Cytotoxicity was determined in neutrophils exposed to dieldrin or lindane by measuring activity of the cytosolic enzyme, lactate dehydrogenase (LDH), in cell-free supernatant fluids as described previously (Tithof et al., 1995Go).

Statistical analysis.
Data are expressed as mean ± SEM. Data were analyzed by analysis of variance, and group means were compared using Student-Newman-Keuls' test. Appropriate transformations were performed on all data that did not follow a normal distribution (e.g., percent data). If transformation failed to normalize the data, nonparametric statistics (Mann-Whitney rank sum test) were used. For all studies, the criterion for statistical significance was p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3H-arachidonic acid release and O production in response to lindane and dieldrin.
Exposure of neutrophils to lindane or dieldrin (Fig. 1Go) resulted in an increase in the release of 3H-arachidonate; however, minimal amounts of 3H-AA were released from unstimulated neutrophils. The minimum concentrations of dieldrin or lindane that caused statistically significant release of 3H-AA were 10 and 5 µM, respectively. These concentrations have also been reported to be the minimum concentrations for statistically significant O generation in neutrophils upon exposure to lindane (Kuhns et al., 1986Go) or dieldrin (Hewett and Roth, 1988Go). Generation of O was not observed in neutrophils treated with vehicle; however, both lindane (Fig. 1Go, top panel) and dieldrin (Fig. 1Go, bottom panel) stimulated the production of O. The concentration-response relations were similar for 3H-AA release and O generation in neutrophils exposed to either agent.



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FIG. 1. Concentration-dependent generation of superoxide anion (O) and release of 3H -arachidonic acid in rat peritoneal neutrophils stimulated with lindane (top panel) or dieldrin (bottom panel). Cumulative release of 3H -arachidonate and generation of O were measured as described in Materials and Methods. Release of 3H-AA into the medium was expressed as percent of total incorporated cellular radioactivity; n = 3–5; a, significantly different from respective value obtained in the absence of stimulation.

 
Significant release of 3H-AA was observed within 1 min after stimulation with either lindane or dieldrin (Fig. 2Go) with no further increases after this time. Generation of O was first significant 5 min after stimulation with either lindane or dieldrin. No further accumulation was seen after that time in neutrophils exposed to lindane; however, in the presence of dieldrin, O production continued to increase through 10 min. The release of 3H-AA and generation of O did not result from cytotoxic effects of either lindane or dieldrin. As can be seen in Table 1Go, no significant release of LDH was observed in neutrophils treated with lindane at any concentration. Dieldrin caused significant release of LDH only at 100 µM, a concentration well above the concentration that caused 3H-AA release and O production.



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FIG. 2. Kinetics of release of 3H-arachidonate and generation of superoxide anion (O) in neutrophils stimulated with 100 µM lindane (top panel) or 100 µM dieldrin (bottom panel). Cumulative release of 3H-arachidonate and generation of O were measured as described in Materials and Methods; n = 3; a, significantly different from respective value obtained in the absence of stimulation.

 

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TABLE 1 Release of Lactate Dehydrogenase (LDH) from Neutrophils Exposed to Lindane or Dieldrin
 
The role of calcium in release of 3H-AA.
In the presence of extracellular calcium, 24.3 ± 2.6% and 49.7 ± 4.7% of total incorporated 3H-arachidonate was released into the medium in cells stimulated with lindane or dieldrin, respectively (Fig. 3Go). Removal of calcium from the extracellular medium caused a 27% decrease in 3H-AA release from lindane-treated cells and a 51% decrease in 3H-AA released from dieldrin-treated cells. This decrease was statistically significant for dieldrin but not for lindane. No further decrease in 3H-AA release was observed in cells pretreated with BAPTA-AM.



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FIG. 3. Calcium-dependence of 3H-arachidonate release in neutrophils stimulated with lindane or dieldrin. The release of 3H-arachidonate was measured in neutrophils suspended in HBSS with or without CaCl2, and also after pretreatment with BAPTA-AM (50 µM) or its vehicle. The concentration of lindane and dieldrin used for stimulation was 100 µM. n = 3; a, significantly different from response obtained in the absence of stimulation; b, significantly different from response obtained in the presence of 1.6 mM CaCl2.

 
Effect of the calcium-independent PLA2 inhibitor, BEL, on 3H-AA release and O production.
Pretreatment with the calcium-independent PLA2 inhibitor, BEL, caused a dose-dependent inhibition of lindane-induced release of 3H-AA (Fig. 4Go). The response to lindane was decreased by 56% in cells pretreated with 20 µM BEL. Neutrophils stimulated with lindane in the absence of BEL produced 14.4 ± 1.4 nmol O/106 cells/20 min. At concentrations that significantly attenuated the release of 3H-AA, BEL also inhibited the production of O in lindane-treated neutrophils. No O was produced by unstimulated neutrophils, either in the absence or the presence of BEL (data not shown).



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FIG. 4. Effect of bromoenol lactone (BEL) on release of 3H-arachidonate and generation of O in neutrophils stimulated with lindane. Neutrophils were incubated in the presence or absence of BEL for 15 min, and cumulative O production (n = 5–6) and 3H-arachidonate release (n = 8) were measured 20 min after treatment with 100 µM lindane; a, significantly different from value obtained in the absence of BEL.

 
Dieldrin-induced release of 3H-AA was inhibited in cells pretreated with BEL (Fig. 5Go). At 20 µM, BEL caused a 39% decrease in 3H-AA release. In the absence of BEL, dieldrin-exposed neutrophils produced 19.5 ± 2.9 nmol O/106 cells/20 min. Dieldrin-induced O production was inhibited by concentrations of BEL greater than 0.1 µM.



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FIG. 5. Inhibition by bromoenol lactone (BEL) of O generation and 3H-arachidonate release in neutrophils stimulated with dieldrin. Neutrophils were incubated in the presence or absence of BEL for 15 min and cumulative O production (n = 5–6) and 3H-arachidonate release (n = 8) were measured 20 min after treatment with 100 µM dieldrin; a, significantly different from value obtained in the absence of BEL.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of isolated rat neutrophils to dieldrin or lindane resulted in significant release of 3H-arachidonic acid from pre-labeled cells. A similar release of AA has been reported in macrophages, renal tubular cell cultures, and myometrial smooth muscle cells exposed to lindane (Criswell and Loch-Caruso, 1995Go; Meade et al., 1984Go; Puente-Fraga et al., 1995Go). The concentration-response relations for dieldrin- or lindane-induced release of 3H-AA were similar to those for production of O (Fig. 1Go). In addition, significant accumulation of 3H-AA occurred within 1 min after stimulation with either pesticide, and significant production of O was not observed until 5 min. These data are consistent with the hypothesis that accumulation of arachidonate is important in activation of the NADPH oxidase. In addition, they are in agreement with previous studies indicating that PLA2-mediated release of arachidonic acid plays an essential role in the generation of O by neutrophils (Dana et al., 1994Go; Henderson et al., 1993Go, 1995Go; Tithof et al., 1996Go, 1998Go).

One PLA2 isoform activated upon exposure of neutrophils to lindane or dieldrin is likely to be the same as, or similar to the calcium-independent enzyme that is activated by PCBs (Tithof et al., 1998Go). Evidence for this conclusion is based on the findings that lindane and dieldrin caused the release of AA in the presence of calcium-free medium and in cells loaded with the intracellular calcium chelator, BAPTA-AM (Fig. 3Go). In cells stimulated with lindane, AA release was not significantly different in the presence or absence of calcium, suggesting that the predominant isoform of PLA2 activated by lindane in rat neutrophils is a calcium-independent isoform. Lindane-induced inhibition of gap junctional intercellular communication in rat, myometrial smooth muscle cells is dependent on release of AA and is not inhibited by removal of extracellular calcium, thus indirectly implicating activation of a calcium-independent PLA2 in that system as well (Criswell and Loch-Caruso, 1995Go; Criswell et al., 1995Go). Although omission of extracellular calcium did not cause a significant decrease in release of AA, pretreatment with BEL, an agent that inhibits many calcium-independent isoforms of PLA2 with 1000-fold greater potency than its effect on calcium-dependent enzymes (Hazen et al., 1991bGo), decreased AA release by only 56%. These results indicate that nearly half of the AA released arises from a calcium-independent mechanism other than BEL-sensitive PLA2. Several isoforms of calcium-independent PLA2 have been characterized that are not inhibited by BEL. These include acidic calcium-independent PLA2 isoforms isolated from lung and several other organs (Akiba et al., 1994Go; Kim et al., 1998Go), and a cytosolic isoform that is expressed in rat ventricular myocytes (Liu and McHowat, 1998Go). Lindane may cause the release of AA by activating more than one isoform of calcium-independent PLA2 having different sensitivities to BEL. Alternatively, phospholipase D may be the source of the BEL-insensitive portion of the released AA. Activation of phospholipase D can occur in a calcium-independent manner (English et al., 1991Go), and AA can be liberated from some phospholipids by this enzyme (Song and Foster, 1993Go).

Dieldrin caused significant accumulation of arachidonate both in the presence and absence of calcium, but in contrast to lindane, the amount of AA released was significantly less under calcium-free conditions. In fact, less than 50% of the AA released in response to dieldrin could be attributed to calcium-independent phospholipase A2 activity. These results suggest that dieldrin activates more than one isoform of PLA2 that differ in their requirements for calcium. This conclusion is corroborated by results with BEL. Release of AA was inhibited only partially by either preincubation with BEL (40% inhibition at 20 µM) or by omission of extracellular calcium (51% inhibition), supporting the interpretation that both calcium-dependent and calcium-independent isoforms of PLA2 are involved.

Despite incomplete inhibition of AA release by BEL, this inhibitor nearly abolished O production in response to either lindane or dieldrin (Figs. 4 and 5GoGo). There are several possible explanations for these results. One explanation is that both dieldrin and lindane activate more than one isoform of PLA2, and these enzymes release AA for different cellular functions. The portion of released AA that was inhibited by BEL may be required for activation of the NADPH oxidase, while the portion of released AA that was not inhibited by BEL may be linked to cellular functions other than generation of O (e.g., eicosanoid production). Although eicosanoid production in response to lindane or dieldrin has not been reported for neutrophils, both agents have been shown to cause the release of eicosanoids from other cell types (Forgue et al., 1990Go; Kacew and Singhal, 1974Go). Furthermore, in neutrophils, two different PLA2 isoforms are responsible for O generation and eicosanoid production. One PLA2 is a calcium-independent isoform that is activated by PCBs and causes the release of AA for activation of the NADPH oxidase. The other PLA2 isoform is a calcium-dependent enzyme that is activated by A23187 and results in the release of AA for subsequent production of prostaglandins and leukotrienes (Tithof et al., 1998Go). Thus, BEL may totally abolish O generation without completely inhibiting AA release by inhibiting only the isoform(s) of PLA2 that is (are) linked to NADPH oxidase activity.

An alternative explanation for the complete inhibition of O production, despite incomplete inhibition of AA release in the presence of BEL may lie in the mechanisms of BEL's action. Although BEL selectively inhibits calcium-independent isoforms among phospholipases A2, it does have other activity. At concentrations which inhibit calcium-independent PLA2, BEL also inhibits phosphatidic acid phosphohydrolase (PAP) (Balsinde and Dennis, 1996Go), an enzyme that catalyzes the production of diacylglycerol from phosphatidic acid, which arises as a result of phospholipase D activation. Activation of PAP and phospholipase D has been implicated in the production of O in neutrophils stimulated with phorbol myristate acetate (PMA) or f-met-leu-phe (fMLP) (English and Taylor, 1991Go; Perry et al., 1992Go). However, these conclusions were based solely on the findings that O production was attenuated by the inhibitor of PAP, DL-propranolol. This inhibitor also demonstrated the ability to decrease the activity of protein kinase C (Sozzani et al., 1992Go), an enzyme that is known conclusively not only to be targeted by fMLP and PMA (Castagna et al., 1982Go; Nishizuka, 1984Go), but also to play an essential role in the assembly and activation of the NADPH oxidase (Tauber, 1987Go). Thus, interpretation of results, using propranolol to implicate PLD in a mechanism, is obscured by simultaneous inhibition of protein kinase C. Since most, if not all studies that suggest a role for PAP in production of O have been based on the use of propranolol, it is improbable that BEL's action to inhibit O production by neutrophils stimulated with dieldrin or lindane occurred through a mechanism involving this pathway (Sozzani et al., 1992Go).

Although mobilization of AA for the generation of O occurs in a calcium-independent manner in neutrophils exposed to dieldrin or lindane, generation of O does not. Omission of extracellular calcium reduces O production in dieldrin-treated neutrophils by greater than 90% (Hewett and Roth, 1988Go). Likewise, incubation with the intracellular calcium antagonist, TMB-8 (Kuhns et al., 1986Go), or omission of calcium from the extracellular medium (English et al., 1986Go) attenuates O generation in lindane-treated neutrophils. These results suggest that the requirement for calcium may occur distal to PLA2 activation in the signal transduction pathway. Since arachidonic acid has been shown to act as a calcium ionophore (Ramanadham et al., 1993Go; Soliven et al., 1993Go), it is possible that PLA2-mediated AA release causes intracellular calcium to rise by mobilizing extracellular or intracellular stores.

Organochlorine pesticides have been shown to be potent neurotoxicants. These compounds exert their neurotoxicity by binding to the GABAA receptor-Cl- ionophore complex, which mediates inhibitory synaptic neurotransmission in the central nervous system (Ghiasuddin and Matsumura, 1982Go). Lindane has also been shown to alter calcium-channel activity, and this alteration has been implicated in the neurotoxicity (Rosa et al., 1997Go), immunotoxicity (Meera et al., 1993Go), and alterations in reproductive function (Criswell et al., 1994Go) associated with this class of compound. The neural and reproductive effects occur in the nanomolar to micromolar range. However, the effects on the immune system occur at somewhat higher concentrations. For example, significant inhibition of gap junctional communication and release of 3H-AA from rat myometrium can be seen after exposure to 100 nM lindane (Criswell and Loch-Caruso, 1995Go). However, significantly higher concentrations were required to cause O production and 3H-AA release in phagocytic cells (Kuhns et al., 1986Go). The concentrations used in this study and in previous in vitro studies (Kuhns et al., 1986Go) are within the range shown to cause immunotoxicity in animal models (Koner et al., 1998Go).

Calcium-independent PLA2 isoforms have been implicated in a variety of disease states in which oxidative stress has been implicated (Goligorsky et al., 1993Go; Hazen et al., 1991aGo; Kuo et al., 1995Go; McHowat et al., 1998Go; Ross et al., 1997Go). Since both dieldrin and lindane have been shown to cause parenchymal cell injury by inducing oxidative stress (Bachowski et al., 1997Go; Junqueira et al., 1986Go, 1994Go), it is plausible that activation of calcium-independent PLA2 is involved in this process. This possibility warrants further investigation.


    ACKNOWLEDGMENTS
 
This work was supported by NIH grant ESO4911. Jesus Olivero is sponsored by a Colciencias-Fulbright-Laspau scholarship (Bogota, Columbia) and by the Universidad de Cartagena, Cartagena, Columbia.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (423) 974-2215. E-mail: ptithof{at}utkux.utcc.utk.edu. Back


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 ABSTRACT
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 DISCUSSION
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