Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid

Abstract

Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.

Figure 1

Enhanced production of ROS of uncertain identity in THP-1 monocytes following treatment with OC chemicals or exogenous AA: DCFH-DA fluorescence assay. (A) Time course of DCF-derived mean fluorescence intensity in monocytes exposed to trans-nonachlor (top panel) and dieldrin (bottom panel) for up to 16 h. Data represent mean values of duplicate measurements. THP-1 monocytes were preloaded with DCFH-DA (50 μM) for 30 min and then treated with 10 μM OC for the indicated amount of time. The vehicle was ethanol (0.1% v/v). Area under curve (AUC) values indicates the time-integrated fluorescence intensity. (B) Representative histogram of DCF-derived fluorescence in vehicle-treated and 10 μM trans-nonachlor (TN)-treated monocytes. The abscissa represents fluorescence intensity, and ordinate represents cell number. (C) Mean fluorescence intensity following treatment of monocytes with vehicle (ethanol, 0.1% v/v), 10 μM TN, or 10 μM arachidonic acid (AA) for 2 h. Data represents the mean ± SD of three experiments. *, p < 0.05; **, p < 0.01; one-way ANOVA with Tukey’s Studentized range test.

Figure 2

Enhanced production of ROS of uncertain identity in THP-1 monocytes following treatment with trans-nonachlor: hydroethidine fluorescence assay. Rate of hydroethidine (HE) oxidation in THP-1 monocytes treated with 10 μM TN in the presence or absence of Nox inhibitors (A) DPI (50 μM) and (C) VAS-2870 (10 μM) was determined by a fluorometric kinetic assay (λ_excit, 490 nm; λ_emis, 565 nm). Production of HE-derived oxidation products in intact cells was monitored for 33–60 min in a fluorescence plate reader. (B, D) Treatment with 10 μM TN caused a significant increase in the rate of HE oxidation (slopes of curves shown in A and C) compared to that in vehicle (ethanol)-treated cells, which could be attenuated by concomitant treatment with either 50 μM DPI (B) or 10 μM VAS-2870 (D). Data represent the mean ± SD (or individual data points) of three experiments. *, p < 0.05; one-way ANOVA with Tukey’s Studentized range test. RFU, relative fluorescence units; TN, trans-nonachlor.

Figure 3

Determination of superoxide levels following trans-nonachlor treatment. (A) HPLC-MS analysis of 2-hydroxyethidium (2-OH-Et⁺, m/z 330), the superoxide-dependent oxidation product of HE, in a xanthine/xanthine oxidase (XO) cell-free system. (B) 2-OH-Et⁺ production in THP-1 monocytes treated with 10 μM TN in the presence or absence of 50 μM DPI, measured by UPLC-MS analysis. DPI suppressed superoxide production in TN-exposed monocytes. (C) UPLC-MS/MS chromatograms of 2-OH-Et⁺ (m/z 330 > 255) following treatments with vehicle (a), 5 μM TN (b), and 5 μM TN + 2.5 μM VAS-2870 (c). (D) Profiles of 2-OH-Et⁺, Et⁺, and Et⁺-Et⁺ following treatment of THP-1 cells with vehicle or indicated amount of trans-nonachlor. The relative amounts of each analyte were normalized to the level detected in vehicle-treated cells. (E) Relative levels of 2-OH-Et⁺ detected in THP-1 cells treated with 5 μM trans-nonachlor in the absence or presence of indicated amount of VAS-2870. The difference in elution time for 2-OH-Et⁺ peaks in panels (A–C) reflects that the HPLC-MS system was used in (A) with a Thermo C18-column (100 × 2.1 mm), whereas UPLC-MS system was used with a Mac-Mod ACE C18-column (100 × 2.1 mm) (B) or Phenomenex C6 phenyl column (50 × 2.1 mm) (C–E). Data represents the mean ± SD of at least three experiments. *, p < 0.05 relative to TN alone (B); *, p < 0.05 relative to vehicle control (D, E); †, p < 0.05 relative to 5 μM TN without the inhibitor (E).

Figure 4

trans-Nonachlor-induced production of ROS of uncertain identity in J774 macrophaghes and HL-60 cells. (A) Murine J774a.1 macrophages and (B) human HL-60 cells treated with 10 μM TN exhibited a significant increase in the rate of HE oxidation compared to that in vehicle (ethanol)-treated cells, as measured by the fluorometric kinetic assay, further demonstrating the ability of TN to induce reactive oxygen flux in phagocytes. Data represents the mean ± SD of two to three experiments. *, p < 0.05; **, p < 0.01; one-way ANOVA with Tukey’s Studentized range test. RFU, relative fluorescence units; TN, trans-nonachlor; VAS, VAS-2870.

Figure 5

OC chemical treatment stimulates arachidonic acid (AA) liberation. (A) Release of AA from phospholipids into the cell culture medium was quantified by UPLC-MS/MS to evaluate phospholipase A₂ (PLA₂) activity following OC treatment. Generic phospholipid structure is shown with sites modified by phospholipases indicated. R₁ and R₂ represent acyl groups; R₃ represents the polar headgroup. (B) AA release from THP-1 monocytes treated for 12 h with either vehicle (ethanol), 10 μM dieldrin (Diel), or 10 μM TN. (C) Dose–response curve of monocytes treated for 12 h with increasing concentrations of TN. A dose-dependent increase in AA levels was observed, with an apparent threshold for this effect observed between 0.1 and 1 μM. (D) Time course of AA release from monocytes treated for 2 to 12 h with 1 μM TN. Elevated AA liberation compared to that from the vehicle control was evident at all time points. Data in panels B–D represent the mean ± SD of two to three experiments. *, p < 0.05; **, p < 0.01; one-way ANOVA with Tukey’s Studentized range test. Vehicle, ethanol (0.1% v/v); TN, trans-nonachlor.

Figure 6

Increased bioactive lipid liberation following OC chemical treatment. Eicosanoid concentrations in culture medium were assessed by UPLC-MS/MS analysis. (A) UPLC-MS/MS chromatograms of prostaglandin E₂ (PGE₂; m/z 351 > 271) following treatments with vehicle (a), 10 μM TN (b), and 10 μM TN + 20 μM ATK (c). (B) Prostaglandin E₂ and (C) thromboxane B₂ levels in the culture medium were significantly elevated following either 10 μM dieldrin or 10 μM TN treatments. Pretreatment with 20 μM ATK prior to OC exposure effectively blocked the production of AA-derived eicosanoids. Data represents the mean ± SD of two separate experiments. *, p < 0.05; one-way ANOVA with Tukey’s Studentized range test. Vehicle, ethanol (0.1% v/v); TN, trans-nonachlor. Arachidonic acid levels in culture medium following treatment with 5 μM trans-nonachlor in the absence or presence of indicated amounts of ATK (D) or BEL (E). Data represents the mean ± SD of three separate experiments. *, p < 0.05 relative to vehicle control; †, p < 0.05 relative to 5 μM TN without the inhibitor.

Figure 7

OC chemical treatment stimulates p47^phox serine phosphorylation. (A) Western blot analysis of p47^phox serine phosphorylation in THP-1 monocytes treated with indicated amounts of trans-nonachlor for 12 h. (B) Western blot analysis of p47^phox phosphorylation in THP-1 monocytes treated with 1 μM TN for 2 to 12 h. p47^phox serine phosphorylation was increased in monocytes treated with 1 μM TN compared to that with vehicle (ethanol, 0.1% v/v) at 2, 4, 8, and 12 h, which is indicated by the values beneath the blot. TN, trans-nonachlor; IP, immunoprecipitation.

Figure 8

OC-stimulated p47^phox serine phosphorylation: concentration–response. Western blot analysis of p47^phox phosphorylation in THP-1 monocytes treated with vehicle (ethanol, 0.1% v/v) or the indicated amounts of TN for 12 h. Ordinate depicts phospho-p47^phox band density normalized to p47^phox. Treatment with 1, 2.5, 5, and 10 μM TN resulted in markedly increased p47^phox phosphorylation relative to that in the vehicle control, whereas treatment with 20 μM ATK prior to TN exposure blocked the induction of p47^phox serine phosphorylation at all concentrations. Data represents the mean ± SD of duplicate experiments. *, p < 0.05; **, p < 0.01; one-way ANOVA with Tukey’s Studentized range test. TN, trans-nonachlor; IP, immunoprecipitation.

Figure 9

Enhanced p47^phox membrane translocation following OC exposure. Western blot analysis of THP-1 cell membranes after cell treatments with TN (1–10 μM), AA (50 μM), or vehicle (0.1% v/v ethanol) for 4 h. Arachidonic acid is a known inducer of p47^phox translocation and served as a positive control. Treatment with 10 μM TN resulted in a significant increase in the p47^phox content of the membrane fraction compared to that in the vehicle control (ethanol, 0.1% v/v), indicative of activation of the NAPDH oxidase enzyme complex. Data represents the mean ± SD of duplicate experiments. *, p < 0.05; one-way ANOVA with Tukey’s Studentized range test. TN, trans-nonachlor; PDI, protein disulfide isomerase.

Figure 10

Production of ROS of uncertain identity in THP-1 monocytes following trans-nonachlor exposure is attenuated by PLA₂ inhibitor ATK. (A) THP-1 monocytes were pretreated with 20 μM ATK for 1 h, followed by treatment with 10 μM trans-nonachlor for 2 h. One hour after the cells were treated with trans-nonachlor, HE was added and the extent of HE oxidation was monitored for an additional 60 min by a fluorometric kinetic assay (λ_excit, 490 nm; λ_emis, 565 nm). (B) Treatment with 10 μM TN caused a significant increase in the rate of HE oxidation (slopes of curves shown in A) compared to that in vehicle (ethanol)-treated cells, which could be attenuated by 20 μM ATK. Data represents the mean ± SD of three experiments. *, p < 0.05; one-way ANOVA with Tukey’s test. RFU, relative fluorescence units; TN, trans-nonachlor.

Figure 11

Proposed scheme for cPLA₂/AA-mediated and Nox-derived superoxide in monocytes following treatment with organochlorine chemicals or extracellular arachidonic acid. Molecules denoted in red indicate those that were assayed in the current study. Calcium ions and stress kinases are known to activate cytosolic PLA₂. AA, arachidonic acid; COX1/2, cyclooxygenase 1 and 2; ERK, extracellular-signal regulated kinases; JNK, c-jun N-terminal kinases; MAPK, mitogen-activated protein kinases; PKC, protein kinase C; cPLA₂, cytosolic phospholipase A₂.