Comparison of cytotoxicity and genotoxicity induced by the extracts of methanol and gasoline engine exhausts.

Gasoline engine exhaust has been considered a major source of air pollution in China, and methanol is considered as a potential substitute for gasoline fuel. In this study, the genotoxicity and cytotoxicity of organic extracts of condensate, particulate matters (PM) and semivolatile organic compounds (SVOC) of gasoline and absolute methanol engine exhaust were examined by using MTT assay, micronucleus assay, comet assay and Ames test. The results have showed that gasoline engine exhaust exhibited stronger cytotoxicity to human lung carcinoma cell lines (A549 cell) than methanol engine exhaust. Furthermore, gasoline engine exhaust increased micronucleus formation, induced DNA damage in A549 cells and increased TA98 revertants in the presence of metabolic activating enzymes in a concentration-dependent manner. In contrast, methanol engine exhaust failed to exhibit these adverse effects. The results suggest methanol may be used as a cleaner fuel for automobile.     

Extract of motorcycle exhaust particles induced macrophages apoptosis by calcium-dependent manner

Large survey and experiments have reported that environment pollutants from fossil fuel combustion would cause immune system deleterious by enhancement of allergic reaction and damage to respiratory tract. In this study, we reported that the extract of motorcycle exhaust particles (MEP) might affect the immune system by inducing cell apoptosis on macrophages. The motorcycle exhaust particles were collected from a two-stoke engine and their cytotoxic effect on macrophages was investigated. We found MEP is cytotoxic and induced apoptosis in RAW 264.7 cells, murine peritoneal macrophage, and rat alveolar macrophage. Pretreatment with mitochondria permeability transition inhibitor (cyclosporin A), intracellular (BAPTA-AM) and extracellular (EGTA) Ca(2+) chelator, and antioxidants (NAC, GSH, catalase, SOD) attenuated the MEP-induced cell apoptosis, and BAPTA-AM was the most effective one. Utilized Fura-2/AM loaded RAW 264.7 cells to directly detect the change of intracellular Ca(2+) concentration ([Ca(2+)](i)), we found that MEP could induce a sustained increase of [Ca(2+)](i). The raise of [Ca(2+)](i) induced by MEP could be completely blocked by the intracellular Ca(2+) chelator, BAPTA-AM, however, only partially inhibited by the extracellular Ca(2+) chelator, EGTA. These results suggested that both influx of extracellular Ca(2+) and release of Ca(2+) from the internal storage were involved. We also found that MEP caused a decrease of mitochondria membrane potential and an increase of oxidative stress in RAW 264.7 cells. In conclusion, we found that the particles, collected from the motorcycle exhaust, contain chemicals that will induce apoptosis of macrophage in calcium-dependent manner.     

Indium oxide nanoparticles induce lung intercellular toxicity between bronchial epithelial cells and macrophages

Concerns have been raised over the safety and health of industrial workers exposed to indium oxide nanoparticles (IO-NPs) when working. IO-NPs were previously shown in vitro and in vivo to be cytotoxic, but the mechanism of pathogenesis was unclear. In this study, the effects of IO-NPs on lung cells associated with respiratory and immune barriers and the toxic effects of intercellular cascades were studied. Here IO-NPs had acute toxicity to Wistar rats over a time course (5 days post-intratracheal instillation). Following treatment epithelial cells (16HBE) or macrophages (RAW264.7) with IO-NPs or IO fine particles (IO-FPs), the damage of 16HBE cells caused by IO-NPs was serious, mainly in the mitochondrial and rough endoplasmic reticulum. The lactate dehydrogenase level also showed that cytotoxicity in vitro was more serious for IO-NPs compared with IO-FPs. The level of In³⁺ (examined by inductively coupled plasma mass spectrometry) in 16HBE cells was 10 times higher than that in RAW cells. In³⁺, releasing from IO-NPs absorbed by 16HBE cells, could not only significantly inhibit the phagocytosis and migration of macrophages (P < .0001), but also stimulate RAW cells to secrete high levels of inflammatory cytokines. IO-NPs can directly damage pulmonary epithelial cells. The In³⁺ released by epithelial cells affect the phagocytosis and migration of macrophages, which may be a new point for the decrease in the clearance of alveolar surfactants and the development of IO-related pulmonary alveolar proteinosis.     

Cell‐specific immune‐modulation of cadmium on murine macrophages and mast cell lines in vitro

Toxic trace metals are widespread contaminants that are potentially immunotoxic even at environmentally low exposure levels. They can modulate the immunity to infections, e.g., in wildlife species living in contaminated areas. The diverse immune cell types can be differentially affected by the exposure leading to the modulation of specific protective mechanisms. Macrophages and mast cells, part of the innate immune system, trigger immune responses and perform particular effector functions. The present study compared toxicological and functional effects of cadmium in two models of murine macrophages (RAW264.7 and NR8383 cell lines) and two models of murine mast cells (MC/9 and RBL‐2H3 cell lines). Cadmium was selected as a model compound because its known potential to induce reactive oxygen species and its relevance as an environmental contaminant. Mechanisms of toxicity, such as redox imbalance and apoptosis induction were measured in stationary cells, while functional outcome effects were measured in activated cells. Cadmium‐depleted glutathione antioxidant in all four cell lines tested although reactive oxygen species was not significantly increased. Mast cells had full dose‐response depletion of glutathione below cytotoxic levels while in macrophages the depletion was not complete. Functional endpoints tumour necrosis factor‐alpha and nitrite production in lipopolysaccharide‐activated macrophages were increased by cadmium exposure. In contrast, mast cell lipopolysaccharide‐induced tumour necrosis factor‐alpha and IgE‐mediated histamine release were reduced by cadmium. These data indicate potentially differential effects of cadmium among murine innate immune cell types, where mast cells would be more susceptible to oxidative stress and their function might be at a higher risk to be modulated compared to macrophages.     

Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells

Paraquat dichloride (N,N-dimethyl-4-4′-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 μM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.     

Anti-inflammatory and anti-nociceptive effects of the methanol extract of Fomes fomentarius

In an attempt to find bioactive natural products with an anti-inflammatory activity, we evaluated the effects of the methanol extract of Fomes fomentarius (MEFF) on in vivo anti-inflammatory and anti-nociceptive activities. MEFF (50, 100 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed MEFF analgesic activity, as determined by an acetic acid-induced writhing test and a hot plate test in mice. To investigate the mechanism of the anti-inflammatory action of MEFF, we examined the effect of MEFF on lipopolysaccharide (LPS)-induced responses in murine macrophages cell line RAW 264.7. MEFF potently inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-alpha (TNF-alpha) in LPS-stimulated RAW 264.7 macrophages. Consistent with these observations, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) levels were reduced by MEFF in a dose-dependent manner. Furthermore, MEFF suppressed nuclear factor-kappaB (NF-kappaB) activation in LPS-stimulated RAW 264.7 macrophages. These findings suggest that the anti-inflammatory and anti-nociceptive properties of the methanol extract of MEFF may result from the inhibition of iNOS and COX-2 expression through the down-regulation of NF-kappaB binding activity.     

Role of 5-lipoxygenase pathway in the regulation of RAW 264.7 macrophage proliferation

Arachidonic acid (AA) metabolites control cell proliferation, among other physiologic functions. RAW 264.7 macrophages can metabolise AA through the cyclooxygenase and lipoxygenase (LOX) pathways. We aimed to study the role of AA-metabolites derived from 5-LOX in the control of RAW 264.7 macrophage growth. Our results show that zileuton, a specific 5-LOX inhibitor, and nordihydroguaiaretic acid (NDGA), a non-specific LOX inhibitor, inhibit cell proliferation and [(3)H]-thymidine incorporation in a concentration-dependent fashion. Growth inhibition induced by NDGA can be explained by an apoptotic process, while zileuton does not seem to induce apoptosis. Moreover, these treatments delay the cell cycle, as analysed by flow cytometry. On the other hand, the leukotriene (LT) B(4) receptor antagonist U-75302, the LTD(4) receptor antagonists LY-171883 and MK-571, and the cysteinyl-LT receptor antagonist REV-5901 also inhibit cell proliferation and [(3)H]-thymidine incorporation in a concentration-dependent manner, and delay the RAW 264.7 cell cycle. However, these antagonists did not induce annexin V staining, caspase activation or DNA fragmentation. Furthermore, we demonstrated that exogenous addition of LTB(4) or LTD(4) revert the cell growth inhibition induced by zileuton or the leukotriene receptor antagonists mentioned above. Finally, we observed that LTB(4) and LTD(4), in the absence of growth factors, have pro-proliferative effects on macrophages, and we obtained preliminary evidences that this effect could be through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, our results show that the interaction between LTB(4) and LTD(4) with its respective receptor is involved in the control of RAW 264.7 macrophage growth.     

IL-6在巨噬细胞向M2型极化过程中的诱导作用

目的 将小鼠巨噬细胞系RAW264.7细胞和骨髓瘤细胞系KM3细胞共培养,探讨IL-6在巨噬细胞向M2型极化的过程的作用.方法 取对数生长期细胞,分为3组:A组(KM3细胞组),B组(RAW264.7细胞组),C组(RAW264.7细胞+KM3细胞组).分别予以ACTA(IL-6特异性抑制剂激活素A)或rIL-6(重组人IL-6)处理后,检测肿瘤相关M2型巨噬细胞表达标志F4/80+ CD206+的比例.RT-PCR和Westernblot方法检测各组细胞因子CCL22、IL-10、IL-12、TNF-αmRNA和蛋白表达量.ELISA法检测各组细胞培养上清中IL-6的含量.结果 RAW264.7和KM3细胞共培养24 h后,与对照组相比,M2 型巨噬细胞比例显著增加(P<0.05);予以ACTA处理后M2型巨噬细胞表达明显下降(P<0.05);而予以rIL-6处理后M2型巨噬细胞表达明显上调(P<0.05).与B组(RAW264.7细胞组)相比,C组(RAW264.7细胞+KM3细胞)M2型巨噬细胞相关细胞因子CCL22,IL-10的mRNA和蛋白表达水平明显上调(P<0.05),而M1型巨噬细胞相关因子IL-12,TNF-α的mRNA和蛋白表达水平明显下调(P<0.05).与A组、B组相比,C组细胞上清液中IL-6含量在24 h、48 h、72 h时间点均明显上调(P<0.05).通过浓度梯度改变RAW264.7细胞或KM3细胞的数量,发现RAW264.7细胞数量的改变显著影响了IL-6的表达水平.结论 将RAW264.7细胞和KM3细胞共培养后,后者能诱导RAW264.7细胞向肿瘤相关M2型巨噬细胞转化, IL-6在上述转化过程中发挥重要作用.     

Production of Cytokine and NO by RAW 264.7 Macrophages and PBMC In Vitro Incubation with Flavonoids

Flavonoids, a group of low molecular weight phenylbenzopyrones, have various pharmacological properties including antioxidant activity, anticancer, and immunomodulatory effects. In the present study, lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate/phytohemagglutinin (PMA/PHA) were used as stimulants for RAW 264.7 macrophages and human peripheral blood mononuclear cell (hPBMC), and tumor necrosis factor (TNF)-alpha and interleukin (IL)-2 productions were measured. In addition, flavonoids were examined for their effects on LPS-induced NO production in RAW 264.7 macrophages. The results showed that all compounds were not strongly cytotoxic at the tested concentrations on hPBMC and RAW 264.7 macrophages. On immunomodulatory properties, catechin, epigallocatechin (EGC), naringenin, and fisetin repressed NO production and TNF-alpha secretion. Furthermore, catechin, epigallocatechin gallate (EGCG), epicatechin (EC), luteolin, chrysin, quercetin, and galangin increased IL-2 secretion while EGC, apigenin, and fisetin inhibited the secretion. These results indicated that flavonoids have the capacity to modulate the immune response and have a potential anti-inflammatory activity. There was no obvious structure-activity relationship regard to the chemical composition of the flavonoids and their cell biological effects.     

Anti-inflammatory effects of indomethacin's methyl ester derivative and induction of apoptosis in HL-60 Cells

Indomethacin is used as an anti-inflammatory drug and a nonselective cyclooxygenase inhibitor. When indomethacin in methanol was photo-irradiated with an Hg lamp, methyl ester, ethyl ester, and gamma-lactone derivatives of indomethacin were produced. In the present study, we found that the methyl ester derivative of indomethacin (M-IN) could more potently inhibit prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) protein expression from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells than indomethacin, similar to the effect of a non-steroidal anti-inflammatory drugs (NSAID). On the other hand, the results showed that M-IN with an IC(50) value maintained at 36.9 microg/ml for 12 h exhibited stronger cytotoxicity than ethyl ester, gamma-lactone derivatives of indomethacin, and indomethacin in promyelocytic leukemia HL-60 cells. Moreover, a series of biochemical analyses determined that M-IN caused apoptotic bodies, DNA fragmentation, and enhanced PARP and pro-caspase 3 degradation in HL-60 cells. These above results indicate that the photosynthesized product, M-IN, had stronger anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and cytotoxicity effects in HL-60 cells than the parent drug, indomethacin.     

Anti-inflammatory effects of diaporisoindole B in LPS-stimulated RAW 264.7 macrophage cells via MyD88 activated NF-κB and MAPKs pathways

Diaporisoindole B (DPB), an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, has been proved to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-challenged RAW 264.7 mouse macrophages, showing potent anti-inflammatory effects. In this study, we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages. The results showed that DPB (3.125, 6.2, 12.5 and 25 μM) could significantly reduce LPS-induced levels of PGE2, and inhibit the expressions of iNOS and COX-2 in a dose-dependent manner. In addition, DPB also inhibited LPS-induced production of inflammatory cytokines, including TNF-α, IL-1β, IL-6. Moreover, we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects. DPB could affect LPS-mediated nuclear factor kappa B (NF-κB) signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88 (MyD88) at the protein level. Additionally, DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38. Therefore, DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.     

S-亚硝基-N-乙酰-DL-青霉安对巨噬细胞诱导型一氧化氮合酶的影响

目的 观察S-亚硝基-N-乙酰-DL-青霉胺（SNAP）对RAW 264.7诱导型一氧化氮合酶（iNOS）表达的影响,探讨NO在动脉粥样硬化（炎症）过程中的作用.方法 以RAW 264.7巨噬细胞为研究对象,分为空白对照组、SNAP组,采用不同浓度（30、100、300、400、500μmol/L）的SNAP对巨噬细胞进行干预24 h,应用RT-PCR法检测RAW 264.7巨噬细胞iNOS mRNA的表达,采用Western blotting技术检测iNOS蛋白的表达.结果 与空白对照组比较,不同浓度SNAP（30、100、300、400、500 μmol/L）组iNOS mRNA的表达比空白对照组均明显降低（P＜0.05）,不同浓度SNAP（30、100、300 μmol/L）组iNOS蛋白表达明显低于空白对照组（P＜0.05）.结论 NO可以抑制巨噬细胞自身iNOS mRNA基因转录,降低iNOS的合成而发挥负反馈作用.     

Immunomodulatory effect of arctigenin, a lignan compound, on tumour necrosis factor-alpha and nitric oxide production, and lymphocyte proliferation

We have investigated the immunomodulatory effects of arctigenin, a dibenzyl butyrolactone lignan compound, on tumour necrosis factor (TNF)-alpha and nitric oxide (NO) production, and lymphocyte proliferation. Arctigenin inhibited strongly TNF-alpha production by lipopolysaccharide-stimulated murine macrophage RAW264.7 and differentiated human macrophage U937 with IC50 values of 5.0 and 3.9 microM, respectively, without displaying cytotoxicity. The TNF-alpha inhibitory effect of arctigenin in lipopolysaccharide-triggered RAW264.7 cells was increased by co-treatment with several known TNF-alpha inhibitors. It also potently attenuated T and B cell proliferation stimulated by concanavalin A and lipopolysaccharide in a dose-dependent manner with IC50 values of 2.9 and 14.6 microM, respectively. In contrast, the compound showed a different pattern in lipopolysaccharide- and interferon (IFN)-gamma-induced NO production from RAW264.7 cells. Arctigenin inhibited NO release by IFN-gamma signal, whereas it significantly enhanced lipopolysaccharide-triggered NO production in RAW264.7 cells. The results suggested that arctigenin may regulate immune responses in activated macrophages and lymphocytes including TNF-alpha and NO production and lymphocyte proliferation.     

Eudesmin inhibits tumor necrosis factor-α production and T cell proliferation

Possible antiinflammatory effects of eudesmin were examined by assessing the effects on tumor necrosis factor (TNF)-alpha production and lymphocyte proliferation as well as cytotoxicity against murine and human macrophages. The compound significantly inhibited TNF-alpha production by lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 without displaying cytotoxicity suggesting that eudesmin may inhibit TNF-alpha production without any interference of normal cell function. It also significantly attenuated T cell proliferation stimulated by concanavalin A (Con A) in a dose-dependent manner.     

Induction of cell death in RAW 264.7 cells by alpha-lactalbumin.

Alpha-lactalbumin (alpha-LA), a major human milk whey protein, has been reported to exhibit bactericidal properties, immune suppressive effects, anti-proliferation and apoptosis in transformed cells; however, little is known about its anti-inflammation and related molecular mechanism. In this study we investigated the effects of alpha-LA on macrophages. We found that treatment with high concentration alpha-LA (> or = 100 microg/ml) could result in a time- and dose-dependent decrease in growth activity, morphological changes, increase in hypodiploid DNA population, and DNA fragmentation in RAW 264.7 cells. We also found that high dose alpha-LA could induce cellular apoptosis and necrosis, as determined by Annexin V binding assay. The alpha-LA could enhance the expression levels of cytochrome c, active caspase 3, active caspase 8, extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) activation without changing the protein levels, but suppress the protein level of Bcl-2. The broad-spectrum caspase inhibitor, Boc-D-fmk, failed to block cell death, indicating that alpha-LA-induced cell death was modulated in a caspase-independent manner. In addition, the ERK1/2 inhibitor, PD98059, could partially rescue alpha-LA-induced cell death, while the JNK inhibitor, SP600125, could weakly protect cells from death. Our results suggested that activation of ERK1/2 might mediate alpha-LA-induced cell death in RAW 264.7 cells.     

Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production

Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.     

Air pollution particulate SRM 1648 causes oxidative stress in RAW 264.7 macrophages leading to production of prostaglandin E2, a potential Th2 mediator

Particulates in air pollution have been strongly associated with asthma symptoms. These particulates are a conglomeration of many components, including metals, polyaromatic hydrocarbons, and lipopolysaccharide, that may cause oxidative stress upon uptake by alveolar macrophages. The objective of this study was to assess whether uptake of a model air particulate (SRM 1648) causes oxidative stress in macrophages resulting in the production of the eicosanoid mediator prostaglandin E(2) (PGE(2)) that might exacerbate asthma. SRM 1648 suspended in phosphate-buffered saline (PBS) was introduced into wells with plated RAW 264.7 monocyte/macrophages. Following incubation of SRM 1648 with RAW 264.7 macrophages, prostaglandin E(2) was measured by enzyme immunosorbent assay (EIA), and oxidative stress was assessed by the levels of intracellular reduced glutathione (GSH) as well as by the oxidation of dihydrodichlorofluorescein (H(2)DCFDA) to the fluorescent dichlorofluoresecein (DCF). The results indicated that SRM 1648 caused oxidative stress in RAW 264.7 macrophages, as shown by a compensatory increase in GSH levels in comparison to the controls of titanium dioxide and media alone. Prostaglandin E(2) levels significantly increased at the 3-, 6-, and 12-h time points. Introduction of GSH ester to buffer against oxidative stress was able to block the elevation of PGE(2). The data show that SRM 1648 causes oxidative stress in RAW 264.7 macrophages resulting in formation of the potential Th2 mediator prostaglandin E(2).