Effects of Endocrine Disruptor Compounds, Alone or in Combination, on Human Macrophage-Like THP-1 Cell Response.

The aim of the present study was to evaluate the immunological effects on human macrophages of four endocrine disruptor compounds (EDCs) using the differentiated human THP-1 cell line as a model. We studied first the effects of these EDCs, including Bisphenol A (BPA), di-ethylhexyl-phthalate (DEHP), dibutyl phthalate (DBP) and 4-tert-octylphenol (4-OP), either alone or in combination, on cytokine secretion, and phagocytosis. We then determined whether or not these effects were mediated by estrogen receptors via MAPK pathways. It was found that all four EDCs studied reduced strongly the phagocytosis of the differentiated THP-1 cells and that several of these EDCs disturbed also TNF-α, IL-1 ß and IL-8 cytokine secretions. Furthermore, relative to control treatment, decreased ERK 1/2 phosphorylation was always associated with EDCs treatments-either alone or in certain combinations (at 0.1 µM for each condition). Lastly, as treatments by an estrogen receptor antagonist suppressed the negative effects on ERK 1/2 phosphorylation observed in cells treated either alone with BPA, DEHP, 4-OP or with the combined treatment of BPA and DEHP, we suggested that estrogen receptor-dependent pathway is involved in mediating the effects of EDCs on human immune system. Altogether, these results advocate that EDCs can disturb human immune response at very low concentrations.

Effects of 4-nonylphenol and/or diisononylphthalate on THP-1 cells: impact of endocrine disruptors on human immune system parameters.

The aim of the present work is to investigate the link between two endocrine disruptor compounds (EDCs), which are chemicals that interfere with the hormone system in human and wildlife, and the human immune response through a study of their effects on the THP-1 human cell line which was used as a model for macrophages. We used two EDCs, diisononylphthalate (DiP) and 4-n-nonylphenol (NP) alone or in combination in order to evaluate the effects of these compounds on several parameters of the immune response - cytokine secretion, phagocytosis and the putative implication of the estrogen receptors - by studying the level of MAPK activation. NP and DiP strongly reduced phagocytosis and modify cytokine secretions. Indeed, differentiated THP-1 cell exposures (i) to 5 and 10 microM of combination of NP and DiP induced an IL-8 level in the medium respectively of 28.9 and 45 percent higher than the level obtained for the control (untreated cells), (ii) to combination of NP and DiP at 10 microM induced an increase of IL-1ß level in comparison to the control level, (iii) to combination of NP and DiP induced an increase of TNF-α level whatever the concentration of EDCs tested (between 0 and 10 microM). Lastly, differentiated THP-1 cell exposure to NP, DiP alone or in combination (2 microM for each condition) induced a decrease of ERK1/2 phosphorylation in comparison to ERK1/2 phosphorylation level of the control. Moreover, differentiated THP-1 cell treatments by ICI-182780 (an estrogen receptor antagonist) supressed the EDC effects on ERK1/2 phosphorylation level which indicates an estrogen receptor-dependent pathway. These results suggest that EDCs have the ability to alter the human immune function, maybe by interfering with endocrine balance.

Oxidative stress induction by nanoparticles in THP-1 cells with 4-HNE production: stress biomarker or oxidative stress signalling molecule?

The aim of this study was to investigate whether carbon black (CB) nanoparticles might induce toxicity to monocytic cells in vitro via an oxidative stress mechanism involving formation of the lipid peroxidation product 4-hydroxynonenal (4-HNE) and the subsequent role of 4-HNE in inducing further cytotoxic effects. ROS production in cells by CB nanoparticles was shown by the oxidation of DCFH after a short time exposure. These particles induced the formation of 4-HNE-protein adducts and significant modification of glutathione content corresponding to an increase of oxidized glutathione form (GSSG) and a decrease of total glutathione (GSX) content. These results attest to an oxidative stress induced by the carbon black nanoparticles, although no induction of HO-1 protein expression was detected. Concerning the effects of a direct exposure to 4-HNE, our results showed that 4-HNE is not cytotoxic for concentrations lower than 12.5 microM. By contrast, it provokes a very high cytotoxicity for concentrations above 25 microM. An induction of HO-1 expression was observed from concentrations above 5 microM of 4-HNE. Finally, glutathione content decreased significantly from 5 microM of 4-HNE but no modification was observed under this concentration. The discrepancy between effects of carbon black nanoparticles and 4-HNE on the intracellular markers of oxidative stress suggests that 4-HNE is not directly implied in the signalling of oxidative toxicity of nanoparticles but is an effective biomarker of oxidative effects of nanoparticles.

Effect of polycyclic aromatic hydrocarbons and carbon black particles on pro-inflammatory cytokine secretion: impact of PAH coating onto particles.

It has been suggested that the organic fraction of particulate matter in air pollution has a major role in the toxicity of this pollutant, notably via its effects on inflammation. The major organic compounds adsorbed onto these particles are polycyclic aromatic hydrocarbons (PAH), among which benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), and pyrene (Pyr) are quantitatively the most important. Generally, cells or organisms are exposed to organic extracts of the particles rather than the native particles in order to study the effects of these PAH. In this study, B[a]P, B[b]F, and Pyr were tested alone and/or adsorbed onto carbon black (CB) particles differing in size in order to evaluate their impact on cytokine production (with or without LPS stimulation) by THP-1 macrophage-like cells. PAH induced significant secretion of IL-1beta, IL-8, and IL-12 after 24 or 48 hr of treatment, an effect reinforced by LPS stimulation; no effect on IL-10 secretion was noted. Fine CB particles (260 nm diameter) induced secretion of each cytokine. In general, coating the CB with PAH did not modify the effect of the CB alone; the exception was that LPS-induced IL-1beta secretion was reduced. In contrast, ultrafine CB (14 nm diameter: ufCB) caused a decrease in cytokine secretion; this effect was modified by PAH coating. For example, PAH coating on ufCB amplified the inhibitory effect of ufCB against IL-1beta secretion but did not modify IL-8 formation. Moreover, PAH coating on ufCB tended to minimize the effect of LPS stimulation; this included (i) inhibition of the decrease in IL-12 secretion induced by uncoated ufCB and (ii) stimulation of IL-10 production. It was concluded that adsorption of PAH onto these particles could decrease their bioavailability and so their abilities to affect cell cytokine production. The results also showed that when PAH were adsorbed onto the fine particles, any observed increases in cytokine secretion consistently appeared to be due to the particles themselves. In contrast, while ufCB alone almost uniformly led to decreases in cytokine formation by the cells, the added presence of the test PAHs led to variable effects - depending on whether stimulation with LPS took place or not. Thus, while some PAHs likely to be associated with PM are clearly immunomodulants, their ultimate effects in situ will likely depend on the properties of the particles themselves, in particular, their size.

Oxidative stress induction by short time exposure to ozone on THP-1 cells.

Ozone is a major component of air pollution mainly formed by photochemical reactions of nitrogen oxides with volatile organic compounds and/or carbon monoxide. Numerous studies have shown the association between ozone exposure with pulmonary injuries. This pollutant is a strong oxidant exerting its biological action either by direct reaction with target molecules or by generating reactive oxygen species which result in its biological effects and its toxicity. In order to study the effects of an induced oxidative stress by ozone on THP-1 cell, a human macrophage-like cell line, we used an in vitro system which has been previously used to study the rapid responses to ozone exposure. Using this system, THP-1 cells were subjected to short time exposure (30 min) followed by different incubation times ranging from 4 to 24 h. Our results show that ozone exposure provokes an alteration of the cell membrane translating an induction of lipid peroxidation resulting in a 3.2-fold increase of thiobarbituric reactive substances (TBARS), an increase by 35% of heme oxygenase-1 (HO-1) expression, and significant modifications of the redox status evaluated by glutathione measurement and of antioxidant enzyme activities in THP-1 cells. Our in vitro model constitutes a very interesting tool for the measurement of ozone effect on rapid modifications induced by this pollutant as well as intracellular modifications due to an oxidative stress.

Modification of membrane markers on THP-1 cells after ozone exposure in the presence or absence of fMLP.

The impacts of ozone and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the detection of membrane markers on non-differentiated THP-1 cells were evaluated for in vitro exposures. Several markers, specific for monocytes and macrophages, were identified on the THP-1 cells, allowing their use as a model for alveolar macrophages. Ozone exposure modified not only the detection of membrane markers, especially CD13 and CD14, monocyte and macrophage markers, but also the detection of the specific receptor for fMLP, formyl peptide receptor (FPR). Activation by fMLP also reduced the detection of the CD antigens, and a combined exposure to ozone and fMLP amplified this decrease, probably due to an additive effect of these chemicals. Overall, these results suggest important membrane rearrangements for short-term treatments to ozone and/or fMLP.

Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO, H2O2).

Alveolar macrophages secrete numerous mediators, playing an important role in host defence. Among these mediators, nitric oxide (NO) and hydrogen peroxide (H2O2) are both involved in bactericidal killing and trigger the release of other cellular metabolites. We have analyzed the effect of an atmosphere polluted with ozone (0.03-0.5 ppm v/v) on the monocytic cell line THP-1, as a model for alveolar macrophages, in vitro. NO and H2O2 were chosen to evaluate cell response to ozone. Cell injury was evaluated using lactate dehydrogenase (LDH) liberation into the medium. An exposure to 0.5 ppm ozone proved to be more toxic to the cells, than 0.1 or 0.03 ppm, evidenced by more LDH being liberated and cytotoxicity reaching values up to 64%. For all ozone concentrations, H2O2 production reached a peak value after 10-15 min of exposure, after which the concentration of extracellular H2O2 production diminished rapidly. The highest NO concentrations were measured with 0.5 ppm ozone, reaching a maximum value of 1460 nmol/L per 5 x 10(6) cells, which is 1.55 times higher than for nonexposed cells. Lower concentrations barely induced higher NO concentrations compared to nonexposed cells. The results indicate that ozone effects not only the viability of human monocytes but also the release of antibacterial and defense signaling molecules and suggest that ozone-mediated cytotoxicity may be related to the secretion of NO and H2O2.

A new approach to evaluate toxicity of gases on mobile cells in culture.

A novel technique is described that measures the degree of toxicity of short-term exposure to gaseous pollutants or other chemical compounds on cultured cells, in 30 min. This technique, based on the study of the mobility properties of activated macrophages, consists of an image analysis procedure incorporating a specific exposure chamber (EC). The EC, which is developed from commercial culture flasks (50 ml, 25 cm(2) of culture surface), was first used to maintain cells in culture conditions, overnight, prior to the assay. In order to measure toxicity, it was then connected to the gaseous pollutant or chemical source. After exposing the culture medium and cells to the gas stream for 10 min, fMLP, a chemotactic factor, was added and the mobility of the macrophages measured by superimposing sequential analogue images captured by a CCD camera that were digitised and analysed using a software developed for this purpose. For example, the effect of ozone on macrophage-like cell (THP-1) was investigated. After exposure to 0.1 and 0.5 ppm, cells lost, respectively 79% and 90% of their mobility, compared to the control sample.