Methamidophos induces cytotoxicity and oxidative stress in human peripheral blood mononuclear cells

Previous studies have shown that organophosphate pesticide (OP) exposure is associated with oxidative stress. Methamidophos (MET) is an OP widely used in agriculture, which is regarded as a highly toxic pesticide and it is a potent inhibitor of acetylcholinesterase. The aim of this study was to evaluate whether MET can induce oxidative stress at low concentrations in primary cultures of human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy individuals were exposed to MET (0–80 mg/L) for 0–72 h. We performed the MTT and neutral‐red assays to assess the cytotoxicity. As indicators of oxidative stress, the levels of reactive oxygen species (ROS) were assessed using flow cytometry, and the malondialdehyde (MDA) and reduced glutathione (GSH) levels were determined. MET decreased the viability of PBMCs in a dose‐dependent manner. At concentrations of 3, 10, or 20 mg/L for 24 h, MET increased the ROS production significantly compared with the vehicle control. Similarly, MET increased the levels of MDA at the same concentrations that increased ROS (10 and 20 mg/L); however, no changes in GSH levels were observed. These results suggest that MET increased the generation of oxidative stress in PBMCs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 147–155, 2017.     

Immunomodulatory properties of characellide A on human peripheral blood mononuclear cells

Inflammopharmacology - Marine sponges and their associated microbiota are multicellular animals known to produce metabolites with interesting pharmacological properties playing a pivotal role...     

Anti-inflammatory effect of spironolactone on human peripheral blood mononuclear cells

We evaluated the effect of alacepril, CV-11974, and spironolactone on the production of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) in cultured human peripheral blood mononuclear cells stimulated with angiotensin (Ang) II. Alacepril, CV-11974, and spironolactone significantly reduced the enhanced production of MCP-1 and TNF-alpha induced by exogenous Ang II. Specifically, 10 muM of spironolactone significantly reduced cytokine production, compared to the same dose of alacepril or CV-11974. These findings indicate that spironolactone may contribute to ameliorate the prognosis of patients with cardiovascular diseases by reducing Ang II-induced inflammation, although further exploration including determining the mechanisms would be required.     

Characterization of peripheral benzodiazepine receptors in human blood mononuclear cells

In the present study, peripheral-type benzodiazepine receptors in human circulating mononuclear cells were characterized, using [3H]PK 11195 as specific ligand. The specific binding was saturable, with a Bmax of 14 pmol/mg protein and a Kd of 7 nM. The pharmacological characterization, using different displacing drugs, indicated a mitochondrial type of peripheral benzodiazepine receptor since it was not coupled to the GABA receptor and was displaced by protoporphyrin IX. These data indicate that human circulating mononuclear cells possess benzodiazepine recognition sites, similar to non-neuronal receptors. The role of these receptors and possible modifications in different diseases need to be investigated.     

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.     

Effect of cordycepin on interleukin-10 production of human peripheral blood mononuclear cells

Therapeutic options for controlling autoimmune diseases are still very limited. Interleukin-10 has been reported to be a promising approach to therapeutic intervention. In the search for a drug which results in the selective upregulation of interleukin-10, we investigated the immunoregulative effects of cordycepin. We have measured interleukin-10 and interleukin-2 secretion of human peripheral blood mononuclear cells that were incubated with cordycepin and assessed the influence of cordycepin on the expression of interleukin-10 mRNA, the proliferative response and the expression of surface markers on T lymphocytes. In addition, the subsets of interleukin-10-secreting cells, the influence of anti-interleukin-10 neutralizing antibody and cytotoxicity of cordycepin were evaluated. Our results suggest that cordycepin has a significantly upregulative effect on interleukin-10 production and interleukin-10 mRNA expression. Interleukin-10-producing cells included in CD4+, CD8+, CD19+, CD56+ and CD14+ cells. At the same time, cordycepin inhibited phytohaemagglutinin-induced interleukin-2 production and proliferation of peripheral blood mononuclear cells. A restricted T lymphocyte activation was also reflected by a reduced expression of the surface markers CD25, CD45RO, CD54, CD71 and HLA DR. Anti-interleukin-10 neutralizing antibody could not completely block the suppressive effect of cordycepin on production of interleukin-2. Cordycepin in the effective concentration presented slight cytotoxicity but did not increase apoptosis. These results indicate that cordycepin exerts immunoregulative effects. Further research on it may provide an approach for the development of novel immunomodulatory drugs which directly alter the secretion of cytokines.     

Aldosterone synthesis and cytokine production in human peripheral blood mononuclear cells

Previously, we reported that spironolactone reduced cytokine production in cultured human peripheral blood mononuclear cells (PBMCs) with angiotensin (Ang) II stimulation. To address the mechanisms underlying this effect, we examined the contribution of aldosterone to cytokine production in cultured human PBMCs with Ang II stimulation. PBMCs expressed the messenger RNA (mRNA) of Ang II type 1 receptor (AT1R) and mineralocorticoid receptor (MR) both spontaneously and after Ang II stimulation, but expressed Ang II type 2 receptor (AT2R) under neither condition. After 24 h of incubation, exogenous Ang II induced the expression of CYP11B2 (a key enzyme of aldosterone synthesis) mRNA and caused aldosterone synthesis. CV-11974 (an AT1R antagonist) reduced Ang II-induced aldosterone synthesis, whereas PD-123319 (an AT2R antagonist) had no effect. The concentration of aldosterone peaked earlier than those of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha). After 48 h of incubation (under the influence of synthesized aldosterone), CV-11974 and spironolactone significantly reduced the Ang II-enhanced production of MCP-1 and TNF-alpha, whereas PD-123319 also had no effect. In conclusion, Ang II induces aldosterone synthesis through AT1R and enhances cytokine production through an AT1R-dependent mechanism and, at least partly, through a MR-dependent mechanism in human PBMCs.     

Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL60) and human peripheral blood mononuclear cells

Dehydrocrotonin (DHC) is a diterpene lactone obtained from Croton cajucara (Sacaca). Dimethylamide-crotonin (DCR), a DHC derivative, has a similar inhibitory effect on leukemic HL60 cells than its parent compound evaluated by different endpoints of cytotoxicity. No cytotoxicity or morphological alterations associated with apoptosis were detected in human peripheral blood mononuclear cells (PBMC) after treatment with up to 400 micro M DCR in presence of phytohemaglutinin (5 micro g/ml). Based on morphological changes and the pattern of DNA fragmentation, DHC and DCR were found to induce apoptosis and terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells, but these compounds did not show any toxic effect in PBMC. Thus, DCR and DHC inhibit HL60 cell growth in vitro partly by inducing apoptosis and cell differentiation, but does not cause serious damage to immune cells according to our experimental conditions.     

Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells

Titanium dioxide nanoparticles (TiO₂‐NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO₂‐NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO₂‐NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum‐free medium, for which there is little information regarding the cytotoxic effects of TiO₂‐NPs. Our results provide evidence that PBMCs treated with TiO₂‐NPs (at concentrations ≥25 μg ml^?1) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin‐6 and tumor necrosis factor‐α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase‐2 and interleukin‐1β were also observed in PBMCs treated with TiO₂‐NPs at concentrations ≥125 μg ml^?1. Our data presented here clearly indicate that the concentration of TiO₂‐NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright © 2016 John Wiley & Sons, Ltd.     

TiO2 nanoparticles and bulk material stimulate human peripheral blood mononuclear cells

Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure to nanoparticles has risen. Because of their small 1–100 nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system. In this study, we investigated the effects of TiO₂ nanoparticles and bulk material in the in vitro model of human peripheral blood mononuclear cells (PBMC) and cytokine-induced neopterin formation and tryptophan breakdown was monitored. Both biochemical processes are closely related to the course of diseases like infections, atherogenesis and neurodegeneration. OCTi60 (25 nm diameter) TiO₂ nanoparticles and bulk material increased neopterin production in unstimulated PBMC and stimulated cells significantly, the effects were stronger for OCTi60 compared to bulk material, while P25 TiO₂ (25 nm diameter) nanoparticles had only little influence. No effect of TiO₂ nanoparticles on tryptophan breakdown was detected in unstimulated cells, whereas in stimulated cells, IDO activity and IFN-γ production were suppressed but only at the highest concentrations tested. Because neopterin was stimulated and tryptophan breakdown was suppressed in parallel, data suggests that the total effect of particles would be strongly pro-inflammatory.     

Effects of ozone on isolated peripheral blood mononuclear cells.

We have investigated the release of cytokines from isolated peripheral human blood mononuclear cells (PBMC) exposed to various ozone concentrations for 10 min and the release of both proinflammatory and immunosuppressive cytokine after 24, 48 and 76 h incubation. Ozonation was performed by exposing for 10 min equal cell numbers and volumes of cell suspension to equal volumes of a gas mixture (1:1 ratio) composed of oxygen-ozone with precise ozone concentrations ranging from 1.0 up to 80 microg/ml (0.02 up to 1.68 mM). Markers of oxidative stress showed a significant relationship between ozone doses and both lipid peroxidation and protein thiol groups content. With the exception of the lowest ozone concentration, the cytokine production of PBMC was depressed particularly at concentrations from 40 mug/ml upwards. There was no significant effect on IL-6 production between exposed or unexposed cells, up to 72 h of incubation. IL-4 production was markedly affected by ozone exposure, showing a marked decrease even at the lowest ozone concentration (2.5 microg/ml) already after 24 h incubation. On the other hand, production of IFN-gamma and TNF-alpha was slightly stimulated by the lowest ozone dose either at all times or only after 72 h incubation, respectively. Analysis of the proliferation index (PI) is consistent with these results showing that, while the lowest concentration stimulates it, progressively increasing O(3) concentrations inhibit the PI. These data show that there is a significant relationship between cytokine production and ozone concentrations and that PBMC are very sensitive to oxidation particularly in presence of serum with low antioxidant capacity.     

Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells

Immunomodulation by fungal compounds can be determined by the capacity of the compounds to influence the cytokine production by human peripheral blood mononuclear cells (hPBMC). These activities include mitogenicity, stimulation and activation of immune effector cells. Eight mushroom strains (Agaricus blazei, Coprinus comatus, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Volvariella volvacea, Lentinus edodes, and Pleurotus ostreatus) were tested for the immunomodulating activity of the isolated protein fractions and polysaccharides fractions present in mycelia and culture liquid. The fungal proteins and polysaccharides have been investigated for their in vitro effect on the cytokine profile (IFN-gamma, IL-4, IL-10, IL-12 and TNF-alpha) of unstimulated or hPBMC stimulated with the polyclonal stimulations PMA/Ca-I, ConA or LPS. In addition to their influence on the cytokine profile, the hemagglutination activity of the fungal proteins on rabbit red blood cells was determined. Proteins from V. volvacea and G. lucidum showed immunomodulating activity without the presence of any mitogen, however, neither of them decreased the production of IL-4 and IFN-gamma in combination with a stimulus. All used stimuli resulted in an induction of IL-12 in the presence of the protein extracts, suggesting a direct effect on monocytes. This effect might lead to the indirect immunomodulation of T cell activation and cytokine production. In addition, both protein extracts showed more hemagglutination activity after trypsin treatment of the rabbit red blood cells, indicating the presence of carbohydrate-binding proteins, like lectins and FIPs. In conclusion, the protein extracts of V. volvacea and G. lucidum contain immunomodulating activity by acting directly on monocytes and thereby modulating T cell activation. Further purification of the fungal extracts is needed to clarify whether there are FIPs or lectins present that are responsible for this immunomodulating activity.     

细胞因子实时定量PCR检测方法的建立和应用

目的 建立细胞因子mRNA实时定量PCR的检测方法,并探讨人单个核细胞(PBMC)在激发型CD3单克隆抗体作用下,细胞因子mRNA的表达.方法 分离健康个体的PBMC,体外与激发型CD3单克隆抗体共培养,应用实时定量PCR检测不同共培养时间点Th1型(IL-2,IFN-γ)和Th2型(IL-10)细胞因子mRNA的表达.结果 激发型CD3抗体能显著上调PBMC胞浆IL-2,IFN-γ和IL-10 mRNA的表达.在共培养的12 h,IL-2 mRNA的含量显著上升并达到峰值14 000 copies/ml,随着培养时间的延长快速下降.IFN-γ和IL-10 mRNA在共培养的24～48 h达到峰值,分别为110 000 copies/ml和15 000 copies/ml.不同细胞因子其胞浆mRNA水平的基础值有明显差异.结论 实时定量PCR能在基因转录水平敏感和特异地反映微量生物活性因子的表达和调节.因此,该技术在基础和临床免疫研究中,具有良好的应用价值.     

Effects of gallium on immune stimulation and apoptosis induction in human peripheral blood mononuclear cells

Gallium is commonly used in the semiconductor industry and medical field. Biologically, gallium is able to interrupt iron metabolism. Exposure to gallium has been shown to affect the human immune system. The purpose of this study was to investigate the in vitro biological effects of different gallium concentrations on cultured human peripheral blood mononuclear cells (PBMCs) in terms of cell growth, cytokine release, and apoptosis induction. In addition, the in vivo effects of gallium were analyzed by Wistar rat model. Our results revealed that low concentrations (1-10 microg/ml) of gallium promoted cells to enter the S phase of cell cycle and enhanced cellular release of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma, both in vitro and in vivo. In contrast, high concentrations of gallium (50-100 microg/ml) induced apoptosis. Furthermore, gallium-induced cytokine release and apoptosis could be inhibited by iron-saturated transferrin (Tf-Fe). These results suggest that the concentration-dependent effects of gallium on PBMCs are related to iron metabolism.     

Biochemical effects of KH 1060 and anti-TNF monoclonal antibody on human peripheral blood mononuclear cells

The aim of this study was to investigate whether the vitamin D analogue KH 1060 could exert a suppressive action on Tumor necrosis factor-alpha (TNF-alpha). The chimeric anti-TNF-alpha monoclonal antibody (anti-TNF), alone or in combination with KH 1060, was also used. KH 1060 (0.01, 0.1, 1 nM) significantly inhibited cell proliferation, determined after 5 days by [3H]thymidine incorporation, when peripheral blood mononuclear cells (PBMC), obtained from healthy subjects, were stimulated with phytohaemagglutinin (PHA) and incubated for 24 h in the absence and in the presence of lipopolysaccharide (LPS). In the same experimental conditions, anti-TNF exerted a significant inhibition on PBMC proliferation, at the lowest doses (0.001, 0.01 microg/ml) in the absence of LPS, and at 0.001, 1, 10 microg/ml in its presence. A synergistic inhibition was registered combining KH 1060 and anti-TNF, at well-defined concentrations. 0.1 nM KH 1060 produced a significant decrease in TNF-alpha levels, determined by ELISA, although less remarkable than in the presence of anti-TNF. This decrease was synergistic, associating 0.1 nM KH 1060 and 0.1 microg/ml anti-TNF. VDR protein levels were increased by 0.1 nM KH 1060, 0.1 microg/ml anti-TNF or their combination. The protein levels of two oncogenes, Bax and Bcl-2, remained unchanged, when PBMC were incubated with KH 1060, anti-TNF or their combination in the absence of LPS, while, in its presence, an increase was registered. The demonstrated anti-TNF-alpha effect of KH 1060 may suggest for this compound an immunosuppressive action and the possibility to synergistically act with other drugs.     

The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells

1. The effects of unfractionated heparin (UH) and a selectively O-desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated. 2. For comparison, the effects of poly-L-glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two different molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied. 3. UH (50 - 1000 u ml(-1)) significantly (P<0.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL-1beta (100 u ml(-1)), TNF-alpha (1000 u ml(-1)) or LPS (100 microg ml(-1)), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such effects on adhesion occurred with limited influence on expression of relevant endothelial adhesion molecules (ICAM-1 and VCAM-1). 4. UH (100 - 1000 u ml(-1)), when added to prestimulated HUVECs, significantly (P<0.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any effect on adhesion molecule expression. In contrast, the opposite effect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell specific. 5. The effects of UH on HPBMNC adhesion were shared by the non-anticoagulant ODSH (600 - 6000 microg ml(-1)) but not by sulphated dextrans or PGA (300 - 6000 microg ml(-1)). 6. Heparin affects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, effects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics.     

Metabolism of tenofovir and didanosine in quiescent or stimulated human peripheral blood mononuclear cells

OBJECTIVE: As tenofovir disoproxil fumarate substantially increases plasma concentrations of didanosine in patients with human immunodeficiency virus-1 infection, we sought to determine whether tenofovir and didanosine showed a similar intracellular interaction in human peripheral blood mononuclear cells (PBMCs). DESIGN: Comparative in vitro incubation of two antiretrovirals in lymphocytes. SETTING: Clinical research laboratory. MATERIAL: Radiolabeled tenofovir and didanosine in human PBMCs. MEASUREMENTS AND MAIN RESULTS: Phosphorylation of 2 and 20 microM didanosine to dideoxyadenosine triphosphate (ddATP) was determined in quiescent and stimulated PBMCs in the presence or absence of 5 microM tenofovir. Similarly, phosphorylation of 5 microM tenofovir to tenofovir diphosphate (TFVpp) was examined in the presence or absence of 2 and 20 microM didanosine. Intracellular amounts of ddATP and TFVpp were determined by incubating PBMCs with radiolabeled tenofovir or didanosine alone and together for up to 16 hours and then separating the anabolites by high-performance liquid chromatography for quantitation. The presence of tenofovir did not affect the amount of ddATP in quiescent or stimulated PBMCs with 2 or 20 microM didanosine. In addition, didanosine did not alter the amount of TFVpp that formed. The amount of ddATP was modestly (1.5-3-fold) but consistently higher in stimulated than in quiescent PBMCs, but the amount of TFVpp did not differ. CONCLUSION: There is no significant interaction between tenofovir and didanosine in human PBMCs as determined by the extent of formation of the phosphorylated anabolites. This suggests that adjusting didanosine dosage, when given with tenofovir, to achieve similar didanosine plasma concentrations, may be sufficient to accommodate the systemic drug interaction.     

Effect of albumin on adenylate cyclase receptor-related signal transduction of human peripheral blood mononuclear cells.

In the present study we investigated in vitro the effect of human serum albumin (HSA) on receptor-stimulated cAMP production in isolated human peripheral blood mononuclear cells (PBMC). The cAMP production is strongly correlated with the pH of the medium during long incubations with albumin. Adenylate cyclase is stimulated by receptor agonists like histamine, forskolin, prostaglandin E2 and the beta-adrenergic agonist isoprenaline, in the presence or absence of HSA. This protein, at concentrations above 0.1%, dose-dependently inhibits both basal and agonist-stimulated cAMP levels in PBMC. In the presence of 0.5% HSA a significant reduction of 30-60% (cell batch dependent) is induced, a reduction which is not incubation time dependent. Washing the cells after a period of incubation with 2% HSA does not reverse the HSA-induced cAMP inhibition. Oleic acid-evoked conformational changes in HSA were not capable of influencing the inhibition processes of HSA on the isoprenaline-stimulated cAMP production. Structure-controlled interactions between HSA and membrane or adenylate cyclase are therefore unlikely. Bovine serum albumin and chicken albumin had different effects upon the agonist-stimulated cAMP production as compared with HSA. At this moment no explanation for this behavior can be provided. The findings indicate that albumin may inhibit non-specifically cAMP production in PBMC and possibly influences membrane-controlled processes.     

Microarray analysis of gene expression in peripheral blood mononuclear cells from dioxin-exposed human subjects

Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen and exerts toxic effects on the skin (chloracne). Effects on reproductive, immunological, and endocrine systems have also been observed in animal models. TCDD acts through the aryl hydrocarbon receptor (AhR) pathway influencing largely unknown gene networks. An industrial accident in Seveso, Italy in 1976 exposed thousands of people to substantial quantities of TCDD. Twenty years after the exposure, this study examines global gene expression in the mononuclear cells of 26 Seveso female never smokers, with similar age, alcohol consumption, use of medications, and background plasma levels of 22 dioxin congeners unrelated to the Seveso accident. Plasma dioxin levels were still elevated in the exposed subjects. We performed analyses in two different comparison groups. The first included high-exposed study subjects compared with individuals with background TCDD levels (average plasma levels 99.4 and 6.7ppt, respectively); the second compared subjects who developed chloracne after the accident, and those who did not develop this disease. Overall, we observed a modest alteration of gene expression based on dioxin levels or on chloracne status. In the comparison between high levels and background levels of TCDD, four histone genes were up-regulated and modified expression of HIST1H3H was confirmed by real-time PCR. In the comparison between chloracne case-control subjects, five hemoglobin genes were up-regulated. Pathway analysis revealed two major networks for each comparison, involving cell proliferation, apoptosis, immunological and hematological disease, and other pathways. Further examination of the role of these genes in dioxin induced-toxicity is warranted.