Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that affect a number of cellular systems, including neutrophils. It has been demonstrated that noncoplanar PCBs (i.e., ortho- substituted PCBs) alter function of primary rat neutrophils. The objectives of these experiments were to determine if responses in a human, neutrophil-like cell line exposed to PCBs were similar to those reported for rat neutrophils and to explore further PCB-mediated alterations in neutrophil function. The human promyelocytic leukemia cell line (HL-60) was differentiated with DMSO to a neutrophil-like phenotype. Treatment of differentiated HL-60 cells with 2,2',4,4'-tetrachlorobiphenyl, a noncoplanar, ortho-substituted PCB congener, caused an increase in f-Met-Leu-Phe-induced degranulation, as measured by release of myeloperoxidase (MPO). Treatment with the coplanar, non-ortho-substituted congener 3,3',4,4'-tetrachlorobiphenyl had no effect on MPO release. 2,2',4,4'-Tetrachlorobiphenyl caused a time- and dose-dependent release of [3H]-arachidonic acid (3H-AA). A significant increase in 3H-AA release was observed after 60 min of exposure, and concentrations of 10 microM or larger increased 3H-AA release. In contrast, 3,3',4,4'-tetrachlorobiphenyl had no effect on 3H-AA release. The effect of PCBs on mRNA levels for cyclooxygenase-2 (COX-2) was examined using semiquantitative RT-PCR. COX-2 mRNA was significantly elevated in response to 2,2',4,4'-tetrachlorobiphenyl in a concentration-dependent manner. COX-2 expression was maximal by 30 min of exposure to 2,2',4,4'-tetrachlorobiphenyl. COX-2 protein and activity were also increased after exposure to 2,2',4,4'-tetrachlorobiphenyl; COX-1 protein and activity were unaffected. 3,3',4,4'-Tetrachlorobiphenyl did not increase COX-2 mRNA levels. These results demonstrate that a noncoplanar PCB alters the functional status of granulocytic HL-60 cells, causing enhanced degranulation and upregulation of COX-2, whereas a coplanar PCB lacks this activity. These data suggest that noncoplanar PCBs alter HL-60 cell function and COX-2 expression via an Ah-receptor-independent mechanism.
Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2′,4,4′,-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A 2 with subsequent release of arachidonic acid (AA), and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 minutes. The increase in COX-2 mRNA was associated with release of 3 H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A 2 , reduced 3 H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCBmediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3 H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB-mediated increases in superoxide anion were prevented by the NADPH oxidase inhibitor apocynin or the free radical scavenger 4-hydroxy TEMPO, but neither of these inhibitors affected the 2244-TCB-induced changes in COX-2 mRNA levels or 3 H-AA release. Taken together these data suggest that p38 MAP kinase-dependent activation of NF-κB is critical for the 2244-TCB-mediated upregulation of COX-2 mRNA.
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 A(2) (PLA(2)) 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 alpha-, delta-, and gamma-isomers of hexachlorocyclohexane (HCCH), p,p'-dichlorodiphenyltrichloroethane (DDT), dieldrin, and chlordane each induced activation of PLA(2) in neutrophils. Beta-HCCH and the non-ortho-substituted 3,3',4,4'-tetrachlorobiphenyl were without effect. PLA(2) activation stimulated by each of the OC compounds was reduced by methyl arachidonyl fluorophosphonate, which inhibits both a cytosolic and a calcium-independent PLA(2) (iPLA(2)), and by E-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL), a selective inhibitor of iPLA(2). These results suggest that a fraction of the PLA(2) activity stimulated by OC compounds is dependent on iPLA(2). Western analysis confirmed the presence of iPLA(2) in rat neutrophils. Molecular modeling techniques were used to develop structure-activity relationships for the activation of PLA(2) 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 beta-HCCH and 3,3',4,4'-tetrachlorobiphenyl. This motif, which we have called PHEN, is required for the activation of the neutrophil PLA(2) by OC compounds and consists of a planar hydrophobic domain connected rigidly at a perpendicular angle to a halogen atom.
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