磷砷四氯化碳对大鼠肝切片乳酸脱氢酶同功酶影响的研究

利用肝切片技术探讨P_4、NaAsO_2、CCl_4染毒大鼠肝切片及培养液中LDH同功酶的剂量效应关系。研究表明P_4、CCl_4 染毒组LDH_5 泄漏存在剂量效应关系;介质中LDH_5和LDH活性升高比ALT明显,LDH_5和LDH升高不伴随切片匀浆酶活性下降与毒物对酶的直接作用无关;LDH_5似乎较其它组分更易泄漏。NaAsO_2染毒组肝切片匀浆及培养液LDH同功酶无明显变化,介质中LDH活性升高较ALT明显。     

Antioxidant potential of tea reduces arsenite induced oxidative stress in Swiss albino mice

Environmental arsenic (As) is a potent human carcinogen and groundwater As contamination is a major health concern in West Bengal, India. Oxidative stress has been one of the prime factors in As-induced carcinogenicity. Generation of reactive oxygen species (ROS), beyond the body’s endogenous antioxidant balance cause a severe imbalance of the cellular antioxidant defence mechanism. Tea, a popular beverage has excellent chemopreventive and antioxidant properties. In this study it was investigated whether these flavonoids could ameliorate the arsenite (As III) induced oxidative stress in Swiss albino mice. Bio-monitoring with comet assay elicited that the increase in genotoxicity caused by As III was counteracted by both black tea and green tea. Elevated levels of lipid peroxides and protein carbonyl by As III were effectively reduced with green as well as black tea. They also exhibited protective action against the As III induced depletion of antioxidants like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) in mice liver tissue. Thus the tea polyphenols by virtue of their antioxidant potential may be used as an effective agent to reduce the As III induced oxidative stress in Swiss albino mice.     

Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest

Arsenic induces clinical remission in patients with acute promyelocytic leukemia and has potential for treatment of other cancers. The current study examines factors influencing sensitivity to arsenic using human malignant melanoma cell lines. A375 and SK-Mel-2 cells were sensitive to clinically achievable concentrations of arsenite, whereas SK-Mel-3 and SK-Mel-28 cells required supratherapeutic levels for toxicity. Inhibition of glutathione synthesis, glutathione S-transferase (GST) activity, and multidrug resistance protein (MRP) transporter function attenuated arsenite resistance, consistent with studies suggesting that arsenite is extruded from the cell as a glutathione conjugate by MRP-1. However, MRP-1 was not overexpressed in resistant lines and GST-pi was only slightly elevated. ICP-MS analysis indicated that arsenite-resistant SK-Mel-28 cells did not accumulate less arsenic than arsenite-sensitive A375 cells, suggesting that resistance was not attributable to reduced arsenic accumulation but rather to intrinsic properties of resistant cell lines. The mode of arsenite-induced cell death was apoptosis. Arsenite-induced apoptosis is associated with cell cycle alterations. Cell cycle analysis revealed arsenite-sensitive cells arrested in mitosis whereas arsenite-resistant cells did not, suggesting that induction of mitotic arrest occurs at lower intracellular arsenic concentrations. Higher intracellular arsenic levels induced cell cycle arrest in the S-phase and G(2)-phase in SK-Mel-3 and SK-Mel-28 cells, respectively. The lack of arsenite-induced mitotic arrest in resistant cell lines was associated with a weakened spindle checkpoint resulting from reduced expression of spindle checkpoint protein BUBR1. These data suggest that arsenite has potential for treatment of solid tumors but a functional spindle checkpoint is a prerequisite for a positive response to its clinical application.     

Quercetin uptake and metabolism by murine peritoneal macrophages in vitro

Quercetin (Q), a bioflavonoid ubiquitously distributed in vegetables, fruits, leaves, and grains, can be absorbed, transported, and excreted after oral intake. However, little is known about Q uptake and metabolism by macrophages. To clarify the puzzle, Q at its noncytotoxic concentration (44μM) was incubated without or with mouse peritoneal macrophages for different time periods. Medium alone, extracellular, and intracellular fluids of macrophages were collected to detect changes in Q and its possible metabolites using high-performance liquid chromatography. The results showed that Q was unstable and easily oxidized in either the absence or the presence of macrophages. The remaining Q and its metabolites, including isorhamnetin and an unknown Q metabolite [possibly Q⁻ (O-semiquinone)], might be absorbed by macrophages. The percentage of maximal Q uptake by macrophages was found to be 2.28% immediately after incubation; however, Q uptake might persist for about 24 hours. Q uptake by macrophages was greater than the uptake of its methylated derivative isorhamnetin. As Q or its metabolites entered macrophages, those compounds were metabolized primarily into isorhamnetin, kaempferol, or unknown endogenous Q metabolites. The present study, which aimed to clarify cellular uptake and metabolism of Q by macrophages, may have great potential for future practical applications for human health and immunopharmacology.     

乌司他丁对小鼠肺泡巨噬细胞TREM蛋白表达的影响研究

目的 研究乌司他丁对脂多糖(lipopoly saccharide,LPS)刺激后的小鼠肺泡巨噬细胞(AM)表面TREM蛋白表达的影响,并探讨乌司他丁在AM介导的炎症反应中的作用及机制.方法 培养肺泡巨噬细胞,分为对照组、LPS干预组、LPS+乌司他丁(LU)组.对照组不予处理,LPS干预组给予100 ng/mL LPS,LU组给予100ng/mL LPS+ 10 000 U/mL乌司他丁,之后分别在3h、12h、24 h用流式细胞仪(FACS)检测AM表面TREM1、TREM2蛋白的表达水平.ELISA检测AM培养上清液中TNF-α表达水平.结果 对照组TREM1、TREM2各时间点的表达差异无统计学意义(P＞0.05).LPS干预组TREM1的表达在3h上升到最高,24 h时接近正常;TREM2在3h下降到最低,之后上升.LU组TREM1的表达均明显下降,24h下降至最低,与LPS组对比差异有统计学意义(P＜0.05);TREM2的表达均明显升高,在24 h升至最高,与LPS组对比差异有统计学意义(P＜0.05).TNF-α的表达对照组各时间点差异无统计学意义(P＞0.05);LPS组在3h达到最高,之后下降,24 h时接近正常;LU组均明显下降,与LPS组对比差异有统计学意义(P＜0.05).结论 乌司他丁可能通过调节TREM蛋白在LPS致AM介导的炎症反应中起重要作用.     

Petroleum hydrocarbon toxicity in vitro: effect of n-alkanes,benzene and toluene on pulmonary alveolar macrophages and lysosomal enzymes of the lung

The in vitro effects of straight chain alkanes (nC₆-nC₁₀), benzene and toluene on pulmonary alveolar macrophages (PAM) of rats and rabbits was studied. The concentrations used ranged from 0.02 to 1.0 mM. All hydrocarbons used in the study were cytotoxic to isolated cultured PAM cells in a dose-dependent manner. The LC₅₀ for these hydrocarbons towards rat PAM cells was estimated to be 1.0 mM for nC₈, 2 mM for nC₇, 5 mM for nC₉ and 10 mM for nC₆, nC₁₀, benzene and toluene. Rabbit PAM cells were more sensitive to the hydrocarbons, resulting in an LC₅₀ half that for rat PAM cells. Hydrocarbons also caused extracellular release of the lysosomal enzymes cathepsin D (EC 3.4.23.5) and cathepsin B (EC 3.4.22.1) in a manner corresponding with cell damage. There was more cathepsin D activity released from cells than cathepsin B. In addition, hydrocarbons also caused the release of cathepsin B and D from isolated lysosomes, and there was 10–15% more enzyme activity released in the culture medium of lysosomes exposed to concentrations of 0.5 and 1.0 mM compared to PAM cell cultures of either rats or rabbits. Hydrocarbons also caused loss of cell respiration and stimulated a dose-dependent and a time-dependent increase in lipid peroxidation. The two alkanes nC₇ and nC₈ caused the greatest increase in lipid peroxidation and the greatest loss of cell respiration. The results indicate that there is a relationship between chain length of alkanes and their cytotoxicity to PAM cells. The results also demonstrate a good correlation between decreased cell viability, increased lysosomal enzyme release, decreased cell respiration and increased lipid peroxidation in response to the hydrocarbons.     

Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts

 Many studies have shown that oxygen radicals can be produced during arsenic metabolism. We report here that in human fibroblasts (HFW cells) sodium arsenite exposure caused increased formation of fluorescent dichlorofluorescein (DCF) by oxidation of the nonfluorescent form. The enhanced DCF fluorescence was inhibited by a radical scavenger, butylated hydroxytoluene. The effects of sodium arsenite treatment on cellular antioxidant activities were then examined. Treatment of HFW cells with sodium arsenite resulted in a significant increase in heme oxygenase activity and ferritin level. Sodium arsenite-enhanced heme oxygenase synthesis was inhibited by co-treatment of cells with the antioxidants sodium azide and dimethyl sulfoxide. Furthermore, sodium arsenite treatment did not apparently affect glucose-6-phosphate dehydrogenase activity, but resulted in significantly increased glutathione levels and superoxide dismutase activity, slightly decreased glutathione peroxidase activity, and significantly decreased catalase activity. Sodium arsenite toxicity was partly reduced by addition of catalase to the culture medium. These results imply that arsenite can enhance oxidative stress in HFW cells. Received: 24 November 1994 / Accepted: 31 January 1995     

Intracellular influx of calcium induced by quartz particles in alveolar macrophages

Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca²⁺). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca²⁺]_i and the relationship between [Ca²⁺]_i and ROS production remains unknown. Quartz particle has been found to increase [Ca²⁺]_i and activate the generation of ROS. Our hypothesis is that [Ca²⁺]_i increase induced by quartz particle is from extracellular Ca²⁺ through the Ca²⁺ channel, and [Ca²⁺]_i increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO₂) particles and used laser scanning confocal microscopy to measure [Ca²⁺]_i and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca²⁺]_I and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca²⁺ with ethylene glycol tetraacetic acid and releasing intracellular Ca²⁺ with thapsigargin, we found that 72.7% of the [Ca²⁺]_i increase was due to the influx of Ca²⁺ from the extracellular environment, via Ca²⁺ channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH·), and removing surface iron from the quartz particles to reduce OH· generation, we observed a reduced level of ROS generation, whereas the increase in [Ca²⁺]_i was unaffected. When using EGTA to reduce [Ca²⁺]_i, we observed a decrease in ROS production. This study suggests that the [Ca²⁺]_i influx was independent of OH· production, and the [Ca²⁺]_i increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca²⁺ content and cellular injury as well as silica exposure.     

Apoptotic cell death following exposure to fluoride in rat alveolar macrophages

 Since inhaled fluoride is implicated in the acute respiratory failure, cytotoxic effects of fluoride on alveolar macrophages, primary target cells of inhaled toxicants, were investigated. The LC₅₀ of sodium fluoride was estimated to be 0.41 mM, while 1 mM sodium chloride, bromide and iodide had virtually no effects on the viability of alveolar macrophages. Photomicroscopic observation revealed that nuclei of the fluoride-exposed alveolar macrophages were fragmented. The ladder formation was observed when DNA isolated from fluoride-exposed alveolar macrophages was electrophoresed in agarose gel. These results suggest that cytotoxicity of fluoride is associated with apoptosis in rat alveolar macrophages. Received: 21 June 1995 / Accepted: 18 September 1995     

L-Arginine-dependent production of nitrogen oxides by rat pulmonary macrophages

Rat alveolar and pleural macrophages incubated with lipopolysaccharide, opsonized zymosan or recombinant interferon-γ, but not with recombinant tumor necrosis factor-, produced nitrite dose and time depcndently. This production depends on the presence and amount of L-arginine in the culture medium. The precursor of the nitrite was demonstrated as being nitric oxide, by bleaching of ferredoxin at 410 nm when added to the culture medium. Addition of N^G-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, and cycloheximide, a protein synthesis inhibitor, to the medium resulted in a decrease of nitrite production. Glucocorticoids were able to block the induction of nitrite production in alveolar macrophages. These data indicate that pulmonary macrophages are capable of secreting L-arginine-derived nitrogen oxides.     

Alveolar macrophages suppress non-specific inflammation caused by inhalation challenge with trimellitic anhydride conjugated to albumin

Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to affect TMA-induced asthma-like symptoms in the Brown Norway rat (Valstar et al., Toxicol. Appl. Pharmacology 211:20–29, 2006). TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA conjugated to albumin is able to induce asthma-like symptoms and if these are affected by AM depletion. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day prior to the inhalation challenge the rats were treated intratracheally with either empty liposomes or liposomes containing clodronate (dichloromethylene diphosphonate) to specifically deplete the lungs of AMs. On day 21, all groups of rats were challenged by inhalation of TMA-BSA. Breathing frequency, tidal volume, and minute ventilation were measured before, during, within 1 h, and 24 h after challenge and the gross respiratory rate score was determined during challenge. Total and TMA-specific IgE levels were determined in serum and lung lavage fluid and parameters of inflammation and tissue damage were assessed in lung lavage fluid and/or lung tissue 24 h after challenge. Sensitization with TMA had no effect on the lung function before challenge, but TMA-BSA challenge resulted in an early asthmatic response as compared to the non-sensitized rats, irrespective of AM depletion. AM depletion had no effect on the sensitization-induced serum and lung lavage fluid IgE levels. TMA-BSA inhalation did not induce airway inflammation and tissue damage irrespective of sensitization, unless AM were depleted. Data indicate that AMs inhibit immunologically non-specific damage and inflammatory cell influx into the lungs as caused by TMA-BSA inhalation. Since effects of inhalation challenge with TMA-BSA are partly different from those of TMA, challenge with the latter is to be preferred for hazard identification.     

Inability of rat alveolar macrophages to recycle l-citrulline to l-arginine despite induction of argininosuccinate synthetase mRNA and protein, and inhibition of nitric oxide synthesis by exogenous l-citrulline

In the present study it was tested whether rat alveolar macrophages (AMΦ) convert l-citrulline to l-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of l-arginine. Rat AMΦ (0.5×10⁶ cells/well, cultured for 20 h in the absence or presence of 1 μg/ml lipopolysaccharides, LPS), were incubated for 6 h in amino acid-free Krebs solution and nitrite accumulation was determined as a measure of NO synthesis. After culture in the absence of LPS, nitrite in the incubation media was at the detection limit, independent of the addition of l-arginine or l-citrulline. AMΦ, cultured in the presence of LPS, produced about 4 nmol per 10⁶ cells and 6 h nitrite, and l-arginine enhanced nitrite accumulation in a concentration-dependent manner, maximally about threefold (EC₅₀: 55 μM). In LPS-treated AMΦ l-citrulline (up to 10 mM) failed to enhance nitrite accumulation, but rather inhibited it by about 50% in the presence of 100 μM l-arginine, i.e. when NO synthesis was enhanced. l-Arginine in the culture medium was ³H-labelled and its metabolism analysed by HPLC. In medium of AMΦ exposed to LPS [³H]-l-arginine was reduced by about 60% after a 20-h culture period and this was almost balanced by an almost equal increase in [³H]-l-citrulline and [³H]-l-ornithine, i.e. l-arginine was markedly consumed. When [¹⁴C]-l-citrulline was added to the culture medium of AMΦ, no significant formation of [¹⁴C]-l-arginine could be detected. On the other hand, argininosuccinate synthetase mRNA (by RT-PCR) and protein (by Western blot) was marginally detectable in control AMΦ, but clearly induced after exposure to LPS. Finally, l-citrulline was shown to inhibit l-arginine uptake in a concentration dependent manner, by about 50% at 10 mM. In conclusion, although the expression of argininosuccinate synthetase in rat AMΦ can be induced by LPS, AMΦ appear not to be able to recycle significant amounts of l-citrulline to l-arginine to maintain sustained NO synthesis. On the contrary, at high concentrations l-citrulline can reduce NO synthesis, and this effect appears to be caused by inhibitory effects on l-arginine uptake. Received: 21 April 1998 / Accepted: 2 October 1998     

Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes

Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers. The objectives of the current study were to examine the effect of aldehydes originating from CS on intracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells.Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaT keratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possible intracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase (LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein) assay and GSH (glutathione) oxidation.Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to 1 μmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CS and acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels compared to controls. Activity of intracellular LDH was preserved.α,β-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role in intracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels.     

Atorvastatin protected from paraquat-induced cytotoxicity in alveolar macrophages via down-regulation of TLR-4

The current study designed to clarify the mechanism of paraquat-induced cytotoxicity and protective effects of Atorvastatin on freshly isolated alveolar macrophages (AMs). AMs were collected via bronchoalveolar lavage and exposed to various concentrations of paraquat in the presence and absence of atorvastatin for 24 h. Cell viability, myeloperoxidase activity; nitric oxide generation and total antioxidant capacity were assessed. Expression of TLR-4 at mRNA and protein levels were studied by using PCR and western blot methods Atorvastatin enhanced the paraquat-reduced cell viability and reduced the paraquat-induced myeloperoxidase activity and nitric oxide production. Moreover, atorvastatin down-regulated by 60% the paraquat up-regulated expression of TLR-4 at protein and mRNA level. Our results suggest that, AMs in vitro model could be a novel cytological tool for studies on paraquat poisoning and therapy regimens. Additionally, atorvastatin cytoprotective effects on paraquat-induced cytotoxicity partly attribute to its anti-myeloperoxidase, antioxidant properties, which might be regulated via TLR-4 expression.     

Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants

Today nanosciences are experiencing massive investment worldwide although research on toxicological aspects of these nano-sized particles has just begun and to date, no clear guidelines exist to quantify the effects. In the present study, we focus on carbon nanotubes (CNTs), which represent one of the most widely investigated carbon nanoparticles. The present data indicate that CNTs are able to cross the cell membrane of rat macrophages (NR8383) and, therefore, might have an influence on cell physiology and function. NR8383 and human A549 lung cells were incubated with commercial single-walled (NT-1) and multi-walled (NT-2, NT-3) CNTs, carbon black and quartz as reference particles as well as an acid-treated single-walled CNT preparation (SWCNT a.t.) with reduced metal catalyst content. We did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all CNT products. None of the CNTs induced the inflammatory mediators NO, TNF-alpha and IL-8. A rising tendency of TNF-alpha release from LPS-primed cells due to CNT treatment could be observed. We detected however, a dose- and time-dependent increase of intracellular reactive oxygen species and a decrease of the mitochondrial membrane potential with the commercial CNTs in both cell types after particle treatment whereas incubation with the purified CNTs (SWCNT a.t.) had no effect. This leads us to the conclusion that metal traces associated with the commercial nanotubes are responsible for the biological effects.     

Oxidative stress and apoptotic changes in murine splenocytes exposed to cadmium

Cadmium being a potent immunotoxicant, affects both humoral and cell mediated immunity. However, its effect on spleen is not clearly understood. Hence, to delineate the action of Cd, mouse splenic lymphocytes were exposed to Cd (10, 25 and 50 microM) for 60 min, 1.5, 3, 6 and 18 h. At 6 h, apoptosis was reflected by DNA fragmentation, increased sub-G1 population (apoptotic DNA) and apoptotic cells (Annexin V binding assay). The early stage markers of apoptosis, i.e. decreased mitochondrial membrane potential and caspase-3 activation were observed as early as 1.5 h by the highest dose of Cd (50 microM). Significant ROS production by 25 and 50 microM Cd at 60 min occurred prior to the lowering of mitochondrial membrane potential, suggesting involvement of ROS in causing mitochondrial membrane damage. N-acetylcysteine and pyrrolidine dithiocarbamate (thiol antioxidants) lowered the sub-G(1) population, inhibited the ROS generation and raised the GSH levels induced by Cd. Buthionine sulfoximine (GSH depletor) on the other hand, enhanced the ROS production as well as the sub-G1 fraction. These results imply that ROS is a critical mediator of Cd-induced apoptosis and that cadmium may compromise splenic immune function by accelerating apoptosis.     

Time-dependence of lung injury in mice acutely exposed to cylindrospermopsin

Cylindrospermopsin is a cyanobacterial toxin of increasing environmental importance, as it can lead to disease if orally or intravenously absorbed. However, its in vivo lung impairment has not been documented. Thus, we aimed at verifying whether cylindrospermopsin can induce lung injury and establish its putative dependence on the time elapsed since exposure.BALB/c mice were intratracheally injected with either saline (NaCl 0.9%, 50 μL, SAL group, n = 12) or a sublethal dose (70 μg/kg) of semi-purified extract of cylindrospermopsin (CYN groups, n = 52). Lung mechanics, histological and biochemical analyses, and cylindrospermopsin presence in lungs and liver were determined in independent groups at 2, 8, 24, 48, and 96 h after cylindrospermopsin instillation.There was a significant increase in static elastance at 24 and 48 h after exposure to cylindrospermopsin, while viscoelastic component of elastance and viscoelastic pressure rose at 48 h. Alveolar collapse augmented in CYN groups at 8 h. A significant increase in polymorphonuclear influx into lung parenchyma, as well as a higher myeloperoxidase activity started off at 24 h. Exposure to cylindrospermopsin increased lipid peroxidation and superoxide dismutase activity and reduced catalase activity in CYN groups. The toxin was detected in lungs and liver of all CYN mice.In conclusion, cylindrospermopsin exposure impaired lung mechanics, which was preceded by lung parenchyma inflammation and oxidative stress.     

氰戊菊酯染毒大鼠肺泡巨噬细胞超微结构的定量分析

根据立体学原理和方法,对染毒氰戊菊酯大鼠肺泡巨噬细胞超微结构改变进行了定量分析。主要结构参数包括细胞核、细胞浆、线粒体、溶酶体、高尔基体、粗面和滑面内质网以及脂滴的体密度,脂滴、溶酶体、线粒体外膜的面密度和数密度。绝大多数参数呈现明显的剂量依赖关系和时间反应特征。