Long-term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages.

To investigate how alveolar macrophages adapt themselves to oxidative pollutants in the long term, rats were exposed to a strong oxidant, ozone (O3), or a weak oxidant, nitrogen dioxide (NO2), for a maximum duration of 12 wk. After exposures, alveolar macrophages were collected by pulmonary lavage. Throughout 11 wk of exposure to 0.2 ppm O3, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase of the peroxidative metabolic pathway and pyruvate kinase and hexokinase of the glycolytic pathway were 40-70% elevated over the controls in alveolar macrophages. The population of alveolar macrophages was consistently 60% higher than the controls. The small-sized macrophages, immature macrophages, preferentially increased. To the contrary, the thymidine incorporation per cell was always 20-30% lower than in the controls, although the total incorporation remained unchanged. No infiltration of polymorphonuclear leukocytes occurred. By 12 wk of exposures to 1.2 and 4.0 ppm NO2, the population of alveolar macrophages increased 30% over the control. Among the enzymes examined, however, only the G6PDH activity increased 10% for 4.0 ppm NO2. No increase in the enzyme activities occurred for 1.2 ppm NO2. Based on these results, alveolar macrophages adapt themselves to the long-term exposure of O3 or NO2 by recruiting immature macrophages through an apparent influx of monocytes. During the exposure to O3, the peroxidative metabolic and glycolytic pathways are enhanced persistently in alveolar macrophages, whereas both pathways were not enhanced by the exposures to NO2.     

Nitrate formation in rats exposed to nitrogen dioxide

Sprague-Dawley rats exposed to atmospheres containing low levels of nitrogen dioxide (NO₂) for 24 hr had increased levels of nitrate in their urine on the day of exposure and on the 3 subsequent days. The total increase in urinary nitrate was linearly related to the nitrogen dioxide concentration administered. We recovered in urine 8.4 ± 1.1 μmol nitrate/ppm $\text{NO}{}_2\text{24}\text{-hr}$ exposure (slope ± 95% confidence limits) for 185-g rats. Both the linearity and magnitude of this effect imply that reaction with respiratory tract water is not a major pathway of NO₂ absorption in the lung. Instead, our observations support the hypothesis that the major interaction of NO₂ in the lung is with readily oxidizable tissue components to form nitrite. We estimate that 9.6 μmol of nitrite is formed in the respiratory tract of the rat per ppm NO₂ per 24-hr exposure. We also estimate that humans breathing air containing 0.1 ppm NO₂ have about 3.6 mg of nitrite formed in their respiratory tract per day.     

Effect of exposure to nitrogen dioxide on alveolar macrophage-mediated immunosuppressive activity in rats

Nitrogen dioxide (NO2), a major component of air pollutants, induces inflammatory responses in the lungs. Resident alveolar macrophages (AM) play an immunosuppressive role in the lungs via suppression of lymphocyte proliferation, and nitric oxide (NO) plays a crucial role in this immunosuppressive activity. Microenvironmental changes within the alveoli during inflammatory responses, however, can inhibit this immunosuppressive activity of AM. The present study was designed to clarify the effect of NO2 exposure on the immunosuppressive activity of and NO production by AM in rats. Wistar rats were exposed to 10 ppm NO2 for 3, 14 or 28 days, after which bronchoalveolar lavage fluid (BALF) was taken as a sample of the alveolar microenvironment. Suppression of concanavalin A-induced lymphocyte proliferation and NO production by AM were markedly inhibited by BALF from NO2-exposed rats (NO2-BALF). The inhibitory effect of NO2-BALF at 28-days exposure was stronger than that of NO2-BALF at 3 or 14 days exposure. In conclusion, AM-mediated immunosuppressive activity was inhibited by the NO2-induced changes of the alveolar microenvironment through the inhibition of NO production.     

Toxicity of ozone and nitrogen dioxide to alveolar macrophages: comparative study revealing differences in their mechanism of toxic action

The toxicity of ozone and nitrogen dioxide is generally ascribed to their oxidative potential. In this study their toxic mechanism of action was compared using an intact cell model. Rat alveolar macrophages were exposed by means of gas diffusion through a Teflon film. In this in vitro system, ozone appeared to be 10 times more toxic than nitrogen dioxide. alpha-Tocopherol protected equally well against ozone and nitrogen dioxide. It was demonstrated that alpha-tocopherol provided its protection by its action as a radical scavenger and not by its stabilizing structural membrane effect, as (1) concentrations of alpha-tocopherol that already provided optimal protection against ozone and nitrogen dioxide did not influence the membrane fluidity of alveolar macrophages and (2) neither one of the structural alpha-tocopherol analogs tested (phytol and the methyl ether of alpha-tocopherol) could provide a protection against ozone or nitrogen dioxide comparable to the one provided by alpha-tocopherol. It was concluded that reactive intermediates scavenged by alpha-tocopherol are important in the toxic mechanism of both ozone and nitrogen dioxide induced cell damage. However, further results presented strongly confirmed that the kind of radicals and/or reactive intermediates, and thus the toxic reaction mechanism involved, must be different in ozone- and nitrogen dioxide-induced cell damage. This was concluded from the observations that showed that (1) vitamin C provided significantly better protection against nitrogen dioxide than against an equally toxic dose of ozone, (2) glutathione depletion affected the cellular sensitivity toward ozone to a significantly greater extent than the sensitivity towards nitrogen dioxide, and (3) the scavenging action of alpha-tocopherol was accompanied by a significantly greater reduction in its cellular level during nitrogen dioxide exposure than during exposure to ozone. One of the possibilities compatible with the results presented in this study might be that lipid (peroxyl) free radicals formed in a radical-mediated peroxidative pathway, resulting in a substantial breakdown of cellular alpha-tocopherol, are involved in nitrogen dioxide-induced cell damage, and that lipid ozonides, scavenged by alpha-tocopherol as well, are involved in ozone-induced cell damage.     

Activation and increment of alveolar macrophages induced by nitrogen dioxide

Male Wistar rats were exposed to 4 ppm nitrogen dioxide (NO2) for 10 d, and at intervals alveolar macrophages were collected by pulmonary lavage. A metabolic enhancement of alveolar macrophages was observed on d 4 of exposure. The specific activities of glucose-6-phosphate dehydrogenase and glutathione peroxidase of the peroxidative metabolic pathway increased to 1.29-fold (p less than 0.001) and 1.17-fold (p less than 0.05) those of the control values, respectively. The specific activities of succinate-cytochrome c reductase of the mitochondrial respiratory system and pyruvate kinase of the glycolytic pathway also increased to 1.17-fold (p less than 0.01) and 1.20-fold (p less than 0.01) those of the control values, respectively. In addition, the incorporation of [3H]leucine and [14C]thymidine into alveolar macrophages were elevated to 1.77-fold (p less than 0.001) and 1.84-fold (p less than 0.01) those of the control values, respectively. The activities of all enzymes tested decreased to control levels by d 10. The number of alveolar macrophages collected from exposed animals increased to 1.24-fold (p less than 0.01) that of the control value on d 7 and was maintained at a significantly higher level until d 10. Alveolar macrophages were heterogeneous in size (7-21 micron in diameter), and most of them were distributed between 11 and 17 micron in diameter. Exposures to 4 ppm NO2 increased significantly the cells of 9-13 micron in diameter on the seventh day. These results show that exposures to 4 ppm NO2 cause a metabolic enhancement and subsequent increase in alveolar macrophages.     

Influence of polyunsaturated fatty acid supplementation and membrane fluidity on ozone and nitrogen dioxide sensitivity of rat alveolar macrophages

The phospholipid polyunsaturated fatty acid (PUFA) content and the membrane fluidity of rat alveolar macrophages were modified dose-dependently and in different ways. This was done to study the importance of both membrane characteristics for the cellular sensitivity toward ozone and nitrogen dioxide. Cells preincubated with arachidonic acid (20:4) complexed to bovine serum albumin (BSA) demonstrated an increased in vitro sensitivity versus ozone and nitrogen dioxide. The phenomenon was only observed at the highest 20:4 concentrations tested, whereas the membrane fluidity of the 20:4-treated cells already showed a maximum increase at lower preincubation concentrations. Hence it could be concluded that the increased ozone and nitrogen dioxide sensitivity of PUFA-enriched cells is not caused by their increased membrane fluidity, resulting in an increased accessibility of sensitive cellular fatty acid moieties or amino acid residues. This conclusion receives further support from other observations. These results strongly support the involvement of lipid oxidation in the mechanism(s) of toxic action of both ozone and nitrogen dioxide in an intact cell system.     

Intermittent inhalation of nitrogen dioxide: effects on rabbit alveolar macrophages

Rabbits were exposed to 0.3 or 1 ppm NO2 for 2 h/d and the effects on alveolar macrophages recovered by bronchopulmonary lavage at 3 time points during the exposure series were assessed. Cells were examined 24 h after 2, 6, or 13 exposures, i.e., on d 3, 7, and 14. There was no change in the viability or numbers of macrophages recovered at any time, nor was an inflammatory response produced. The ability of macrophages to attach to a surface was not affected by exposure. Random mobility was reduced at d 3 in the 0.3 ppm exposure series, but was not different from control after any of the 1 ppm exposures. The phagocytic index, i.e., the fraction of viable cells able to phagocytize latex particles, was not affected by NO2 exposure. However, phagocytic capacity--i.e., the numbers of phagocytizing cells that internalized specific numbers of particles--was different from control on d 3 with both levels of NO2. At 0.3 ppm, particle uptake was reduced, while at 1 ppm it was enhanced in that a greater fraction of cells contained a large number of latex particles. The results show that phagocytic capacity and random mobility are sensitive indices of response to NO2 and that adaptation to the exposures apparently occurred in that both phagocytosis and mobility returned to control levels by d 7 and remained there through the duration of the exposure series.     

Relationship between survival times of rats exposed to lethal level of nitrogen dioxide and arylhydrocarbon hydroxylase activity in lungs

A previous paper has shown that survival times of rats exposed to lethal levels of NO2 were prolonged by administration of enzyme-inducing agents prior to NO2 exposure, and that the survival time of female rats was longer than that of male rats. In order to investigate a mechanism on prolongation of survival times by administration of enzyme-inducing agents and on sex-difference, a relationship between survival times and microsomal enzyme activity in lungs of rats administered enzyme-inducing agents was examined. The survival times of rats exposed to 65 ppm NO2 were prolonged with increase of 3MC doses, and AHH activity in lungs of rats rose with increase of 3MC doses. A significant correlation between the survival time and AHH activity was observed. Induction of AHH activity of female rats administered 3MC was higher than that of male rats. NADPH-dependent cytochrome c reductase activity in lungs of male and female rats did not change with 3MC administration, and a significant relationship between the survival time and NADPH-dependent cytochrome c reductase activity was not observed. These results suggest that cytochrome P1-450 system in lung microsomes may have protective action against the toxicity of NO2.     

Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation

Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causes thymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have investigated the effect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite-induced cytotoxicity. TPEN (10 microM) abolished cell death induced by authentic peroxynitrite (25 microM) and the peroxynitrite generating agent 3-morpholinosidnonimine (SIN-1, 250 microM). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective effect of the chelator. TPEN (10 microM) markedly reduced the peroxynitrite-induced decrease of mitochondrial transmembrane potential, secondary superoxide production and mitochondrial membrane damage, indicating that it acts proximal to mitochondrial alterations. Although TPEN (1 - 300 microM) did not scavenge peroxynitrite, it inhibited PARS activation in a dose-dependent manner. The cytoprotective effect of TPEN is only partly mediated via PARS inhibition, as the chelator also protected PARS-deficient thymocytes from peroxynitrite-induced death. While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 microM), similar to other agents that inhibit PARS, enhanced apoptosis (at 5-6 h after exposure), as characterized by phosphatydilserine exposure, caspase activation and DNA fragmentation. In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exerts protective effects against peroxynitrite-induced necrosis. Its effects are, in part, mediated by inhibition of PARS.     

Activation of alveolar macrophages and peripheral red blood cells in rats exposed to fibers/particles

The cytotoxic and oxidative responses of crocidolite and chrysotile asbestos fibers and ultrafine titanium dioxide (UF–TiO₂) particles were measured in alveolar macrophages (AM) and peripheral red blood cells (RBC) of rat after 30 days with a single intratracheal exposure (5 mg). The following responses were observed one month after fiber/particle instillation: (1) AM population increased; (2) lactate dehydrogenase and acid phosphatase activities in cell free lung lavage fluid increased; (3) substances that react with hydrogen peroxide or thiobarbituric acid were elevated in both AM and peripheral RBC; (4) glutathione peroxidase, glutathione reductase, and catalase were altered in both AM and peripheral RBC; (5) glutathione and ascorbic acid decreased in both AM and peripheral RBC. A significant difference from negative controls was noted in all responses of the two fiber-exposed groups, and in most responses of the UF–TiO₂-exposed group. The level of responses to the three test substances suggested a decreasing order of toxicity, with crocidolite>chrysotile>UF–TiO₂.     

Ultrastructural changes of tracheal epithelium and alveolar macrophages of rats exposed to mosquito coil smoke

The volatile organic compounds of mosquito coil smoke were analysed by a combination of gas chromatography and mass spectrometry. 67 volatile organic compounds were detected and 46, including allethrin, phenol, benzene, toluene, xylene, etc. could be identified. Allethrin, a common name for (+/-)-3-allyl-2-methyl-4-oxocyclopent-2-enyl (+/-)-cis,trans-chrysanthemate, is a synthetic pyrethroid, which is used as an insecticide or repellent in subtropical countries. The vapors also contain other aromatic and aliphatic hydrocarbons which are combustion products of other mosquito coil constituents, including wood dust, coconut flour and other fillers and dyes. An exposure to the mosquito coil smoke for 60 days (8 h per day, 6 days per week) resulted in a focal deciliation of the tracheal epithelium, metaplasia of epithelial cells, and morphological alterations of the alveolar macrophages of exposed rats.     

The role of poly(ADP-ribose) synthetase inhibition in preventing endotoxemia-induced intestinal epithelial apoptosis.

In this lipopolysaccharide (LPS)-induced endotoxemia model, the effects of 3-aminobenzamide (3-AB), a poly(ADP-ribose) synthetase (PARS) inhibitor, on ileal apoptosis were evaluated by light microscopy and M30 cell death staining. Moreover, the relationship between Bcl-2, iNOS expression, and serum nitrate (NO(3)(-)) levels were investigated. Thirty-two male Wistar rats, weighing 180-220g were randomly divided into four groups. The group I (control; n=8) received saline and group II (sepsis; n=8) received 10 mg kg(-1) LPS intraperitoneally. 3-AB was given to the group IV (S+3-AB; n=8) 20 min before giving LPS and to the group III (C+3-AB; n=8) 20 min before giving saline. Six hours later, blood and ileum samples were taken. Endotoxemic group exhibited significant apoptosis in intestinal epithelial cells and the immunohistochemical examination with M30 was demonstrated that the 3-AB reduced the LPS-induced intestinal apoptosis. Serum NO(3)(-) level was increased in endotoxemic group, whereas the elevation of NO(3)(-) level was prevented in LPS+3-AB group (P<0.05). The increased iNOS expression observed in the LPS group was also prevented by 3-AB. Compared with the endotoxemic group, ileal epithelial columnar cells from LPS+3-AB group had a dense Bcl-2 staining which was almost identical with control. In conclusion, 3-AB decreases LPS-induced apoptosis in ileum by preventing LPS-induced depletion of Bcl-2 and blocking iNOS gene. Modification of Bcl-2 expression by PARS inhibitors should further be investigated as a new therapeutic alternatives in septic states.     

Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity.

Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.     

Adhesion molecules of blood polymorphonuclear leukocytes and alveolar macrophages in rats: modulation by exposure to ozone.

1. Exposure to elevated levels of ozone results in an infiltration of polymorphonuclear leukocytes (PMNs) into the lungs. The purpose of this study was to investigate whether the ozone-induced inflammatory process is preceded by a change in the expression of adhesion molecules (integrins and selectins) in peripheral blood PMNs and alveolar macrophages in rats. 2. Female Sprague Dawley rats were exposed to air or ozone (1 p.p.m., 2 h). Bronchoalveolar lavage (BAL) was carried out and blood was collected via intracardiac puncture at 0 or 18 h after the exposure. There were no PMN in the BAL fluid at time 0 after the 2 h exposure to ozone. The expression of cell adhesion molecules from the integrin family (represented by CD18) on alveolar macrophages (AM) was lowered. The expression of cell adhesion molecules from the selectin family (represented by CD62L) on blood PMN was not affected by exposure to ozone, while the expression of integrins (CD11b) on blood PMN was lowered. 3. This effect was confirmed by experiments in which plasma of ozone-exposed animals was incubated with PMN from peripheral blood obtained from non-exposed animals. In these experiments, the expression of CD11b on PMNs of non-exposed animals was lower after incubation with plasma from ozone-exposed animals. 4. Our experiments suggest the presence of factor(s) in blood, which cause a decrease in the expression of CD11b on PMNs.     

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。     

Effects of inhibitors of the activity of poly (ADP-ribose) synthetase on the organ injury and dysfunction caused by haemorrhagic shock

1 Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA, which are caused by reactive oxygen species (ROS). Here we investigate the effects of the PARS inhibitors 3-aminobenzamide (3-AB), nicotinamide and 1,5-dihydroxyisoquinoline (ISO) on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat. 2 Haemorrhage (sufficient to lower mean arterial blood pressure to 50 mmHg for 90 min) and subsequent resuscitation with shed blood resulted (within 4 h after resuscitation) in a delayed fall in blood pressure to 66+/-4 mmHg (control, n=13). This circulatory failure was not affected by administration (5 min prior to resuscitation) of 3-AB (10 mg kg-1 i.v., n=7), nicotinamide (10 mg kg-1 i.v., n=6) or ISO (3 mg kg-1 i.v., n=6). 3 Haemorrhage and resuscitation also resulted in rises in the serum levels of urea and creatinine. This renal dysfunction was attenuated by 3-AB and nicotinamide, but not by nicotinic acid (n=7), an inactive analogue of nicotinamide. Although ISO (n=6) also attenuated the renal dysfunction caused by haemorrhage and resuscitation, its vehicle (10% DMSO, n=4) had the same effect. 4 Haemorrhagic shock resulted in enhanced serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lipase, indicating the development of hepatocellular and pancreatic injury, respectively. Similarly, haemorrhagic shock also resulted in an increase in the serum levels of creatine kinase (CK) indicating the development of neuromuscular injury. This was attenuated by 3-AB and nicotinamide, but not by nicotinic acid. Although ISO also attenuated the liver, pancreatic and neuromuscular injury caused by haemorrhagic shock, its vehicle had the same effect. 5 Thus, activation of PARS contributes to the organ injury and dysfunction caused by haemorrhage and resuscitation in the rat.     

Role of the poly(ADP-ribose)polymerase activity in vancomycin-induced renal injury

The aim of the present study was to investigate the role of poly(ADP-ribose)polymerase (PARP) activity in vancomycin (VCM)-induced renal injury and to determine whether 1,5-isoquinelinediol (ISO), a PARP inhibitor agent, could be offered as an alternative therapy in VCM-induced renal impairment. Rats were divided into four groups as follows: (i) control (Group 1); (ii) VCM-treated (Group 2); (iii) VCM plus ISO-treated (Group 3); and (iv) ISO-treated (Group 4). VCM (200 mg/kg, i.p., twice daily) was administered to Groups 2 and 3 for 7 days. ISO (3 mg/kg/day, i.p.) treatment was started 24 h before the first administration of VCM and continued for 8 days. After the 14th VCM injection, the animals were placed in metabolic cages to collect urine samples. All the rats were sacrificed by decapitation, blood samples were taken in tubes and kidneys were excised immediately. Blood urea nitrogen (BUN) and plasma creatinine, and urinary N-acetyl-β-d-glucosaminidase (NAG, a marker of renal tubular injury) were used as markers of VCM-induced renal injury in rats. Light microscopy was used to evaluate semi-quantitative analysis of the kidney sections. Poly(ADP-ribose) (PAR, the product of activated PARP) and PARP-1 expressions in renal tissues were demonstrated by immunohistochemistry and Western blot. VCM administration increased BUN levels from 8.07 ± 0.75 mg/dL to 53.87 ± 10.11 mg/dL. The plasma creatinine levels were 0.8 ± 0.04 mg/dL and 3.38 ± 0.51 mg/dL for the control and VCM-treated groups, respectively. Also, urinary excretion of NAG was increased after VCM injection. Besides, there was a significant dilatation of the renal tubules, eosinophilic casts within some tubules, desquamation and vacuolization of renal tubule epithelium, and interstitial tissue inflammation in VCM-treated rats. In VCM-treated rats, both PAR and PARP-1 expressions were increased in renal tubular cells. ISO treatment attenuated VCM-induced renal injury, as indicated by BUN and plasma creatinine levels, urinary NAG excretion, and renal histology. PARP inhibitor treatment also decreased PAR and PARP-1 protein expressions similar to that of controls. Herewith, the overactivation of the PARP pathway may have a role in VCM-induced renal impairment and pharmacological inhibition of this pathway might be an effective intervention to prevent VCM-induced acute renal injury.     

Inhibitors of the activity of poly (ADP-ribose) synthetase reduce the cell death caused by hydrogen peroxide in human cardiac myoblasts

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS). Inhibitors of PARS activity reduce the degree of reperfusion injury of the heart in vivo and in vitro. Here we investigate the role of PARS in the cell death of human cardiac myoblasts caused by hydrogen peroxide.
2. Exposure of human cardiac myoblasts to hydrogen peroxide caused a time- and concentration-dependent reduction in mitochondrial respiration (cell injury), an increase in cell death (LDH release), as well as an increase in PARS activity.
3. The PARS inhibitors 3-aminobenzamide (3 mM), 1,5-dehydroxyisoquinoline (300 μM) or nicotinamide (3 mM) attenuated the cell injury and death as well as the increase in PARS activity caused by hydrogen peroxide (3 mM; 4 h for cell injury/death, 60 min for PARS activity) in human cardiac myoblasts. In contrast, the inactive analogues 3-aminobenzoic acid (3 mM) or nicotinic acid (3 mM) were without effect.
4. The iron chelator deferoxamine (1–10 mM) caused a concentration-dependent reduction in the cell injury and death caused by hydrogen peroxide in these human cardiac myoblasts.
5. Thus, the cell injury/death caused by hydrogen peroxide in human cardiac myoblasts is secondary to the formation of hydroxyl radicals and due to an increase in PARS activity. We therefore propose that activation of PARS contributes to the cell injury/cell death associated with oxidant stress in the heart.

     

Time-dependent apoptosis of alveolar macrophages from rats exposed to bleomycin: involvement of tnf receptor 2

Tumor necrosis factor-alpha (TNF-a) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-a-mediated toxicity.     

Role of peroxynitrite and activation of poly (ADP-ribose) synthetase in the vascular failure induced by zymosan-activated plasma

1. Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. Injection of zymosan into experimental animals is known to produce an intense inflammatory response. Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis. The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes.
2. Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6–24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting.
3. Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These effects were reduced by inhibition of NOS with N^G-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS.
4. Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction).
5. Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP. Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle.
6. The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO. These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation. The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.