Early damage indicators in the lung I. Lactate dehydrogenase activity in the airways

The rapid determination of damage in the lung is important in developing screening methods for ranking the toxicity of inhalable pollutants. The presence of lactate dehydrogenase (LDH) activity in the airways was found to be a sensitive indicator of acute toxicity to lung cells. The airway content of LDH increased after bronchopulmonary lavage of Syrian hamsters with increasing amounts of Triton X-100, with a correlation coefficient of 0.98. No increase in iron content of the lavage fluid occurred, indicating that lysed erythrocytes were not the source of the LDH. The isoenzyme pattern of the LDH activity in the lavage fluid suggested the LDH was released from lung cells. The method of detecting early lung injury by the presence of LDH in the airways can be used in one of two ways: The toxic material to be tested may be introduced into the lung by pulmonary lavage and the released LDH may be measured in the same lavage fluid; or in animals exposed to toxicants in aersol form, a lavage can be performed after exposure to obtain a sample of the LDH in the airways.     

Isoenzymes of succinate dehydrogenase in measles virus-infected monkey kidney cells

Succinate dehydrogenase (SDH) isoenzyme patterns were analyzed in R6CA monkey kidney cells inoculated with wild and attenuated measles virus. Cell homogenates were prepared either in a sucrose--Triton X-100 mixture, or in sucrose alone. In both cases the most important SDH isoenzyme pattern alterations were observed in the homogenates of cells infected with wild measles virus at 72 hours post inoculation. Similar alterations occurred in homogenates of cells infected with the attenuated strain after a delay of about 100 hours. In all the experimental variants the use of Triton X-100 allowed the visualization of a larger number of isoenzyme bands.     

Acute toxicity of polyethylene glycol p-isooctylphenol ether in Syrian hamsters exposed by inhalation or bronchopulmonary lavage

Dose-response studies were conducted with Syrian hamsters exposed to polyethylene glycol p-isooctylphenyl ether (Triton X-100) via inhalation or bronchopulmonary lavage. Syrian hamsters were exposed to an aerosol of Triton X-100 with a mass median aerodynamic diameter of 1.5 μm and a concentration of 3.0 mg/liter. Estimated initial lung burdens of Triton X-100 ranged from 800 to 3100 μg. Hamsters were lavaged with concentrations of Triton X-100 ranging from 0.01 to 0.10% in isotonic saline resulting in initial lung burdens of Triton X-100 that ranged from 300 to 3200 μg. The $\text{LD}\text{50}\text{7}$ values were 1700 μg (1300–2100 μg, 95% confidence limits) for the inhalation study and 2100 (1900–2700) μg for the lavage study. The difference between the $\text{LD}\text{50}\text{7}$ values for the two methods of exposure was not significant. However histopathological examination revealed differences in the nature and distribution of pathologic changes observed in animals exposed by the two routes of administration. Animals exposed by inhalation died as a result of ulcerative laryngitis and laryngeal edema with only minimal pulmonary pathologic alterations. Animals exposed by lavage, where the larynx was not exposed to Triton X-100, died from pulmonary edema and acute exudative pneumonia, these results demonstrate the need for careful selection of exposure methods to meet the specific objectives of a toxicology study.     

Early damage indicators in the lung: V. Biochemical and cytological response to NO2 inhalation

In an extension of earlier work on the usefulness of analysis of pulmonary lavage fluid as a probe to detect lung injury, we have examined lavage fluid from animals with a multifocal, terminal bronchiolitis induced by exposure to an oxidant gas. Syrian hamsters were exposed to concentrations of 0, 12, 17, and 22 ppm NO₂ gas for 48 hr. Bronchopulmonary lavage fluids were profiled biochemically and cytologically to determine (1) the indicators of a multifocal, deep lung injury that could be detected in the lung washings and (2) the lowest level of this type of injury that could be detected by the lavage fluid screen. Lung homogenates were assayed for the enzymatic activities measured in lavage fluid and the lungs were evaluated histologically. Highest response for all parameters measured was at 2 days (end of the exposure) when the lavage fluid showed dose-dependent elevations in lactate dehydrogenase, alkaline phosphatase, acid phosphatase, glutathione reductase, and glutathione peroxidase activities, sialic acid, and total protein content, as well as increases in macrophage and neutrophil cell counts. By far the most sensitive indicator of this type of injury, as measured by the lavage fluid screen, was the neutrophil cell count, which showed a 10-fold increase even at the lowest level of exposure. The greatest change seen in the biochemical parameters measured in lavage fluid was the increase in sialic acid and protein content. There was good correlation between the degree of alteration of biochemical and cytologic indicators of injury seen in the lavage fluid and the morphological alterations seen in tissue.     

LUNG INJURY,INFLAMMATION, AND INFLAMMATORY STIMULI IN RATS EXPOSED TO OZONE

The effects of ozone (O₃) on airway epithelia, inflammation, and expression of inflammatory stimuli were investigated to delineate the mechanisms of inflam matory reactions relevant to lung injury. Because the airway responses to O₃ develop gradually, this investigation included a time-sequence analysis. Rats exposed for 3 h to 1 ppm O₃ were studied at 4-h intervals up to 20 h postexposure. Bronchoalveolar lavage fluid (BAL) was analyzed for albumin as an indicator of increased permeability, polymorphonuclear leukocytes (PMNs) to assess the inflammatory status, macrophage inflammatory protein-2 (MIP-2, an inflammatory chemokine), and cell adhesion molecules for their role in inflammation and PMN functions. The time-related increase in album in was matched by a similar significant increase for PMNs, MIP-2, and intercellular adhesion molecule-1 (ICAM-1). However, no marked change occurred for b-2 integrin (CD-18) and leukotriene B₄ (LTB₄). The results establish a temporal correlation of epithelial permeability with changes in inflammatory activity and stimuli responsible for PMN recruitment in the lung. The observations of elevated MIP-2 and ICAM-1 levels are consistent with their role in injury and inflammation. An early expression of MIP-2 mRNA in BAL cells, that is, immediately post O₃ exposure, and the peak increase in BAL MIP-2 levels 4 h later support the chem otactic role of MIP-2 in PMN recruitm ent at 4- and 12-h time points. The rapid drop in MIP-2 and ICAM-1 levels appears to signal the termination of inflammatory cell recruitment, which is accompanied by an onset of recovery.     

Oxygen toxicity: Protection of the lung by bacterial lipopolysaccharide (endotoxin)

Adult rats were exposed to > 95% O₂ for a 72-hr period and treated with small dosages of endotoxin (250 μg/kg/day, 1/100th LD50) during the exposure or with a single dose of endotoxin (500 μg/kg) administered just prior to the hyperoxic exposure. Endotoxin treatment increased the survival rate in hyperoxia from 12/44 (27%) for untreated rats to 38/40 (95%) for the treated animals (p < 0.05). In addition, the endotoxin-treated animals had significantly decreased O₂-induced pulmonary edema and pleural fluid accumulation compared to the untreated rats (p < 0.05). Histological examination by light microscopy demonstrated marked lung damage in the untreated. hyperoxia-exposed animal lungs (perivascular, interstitial, and alveolar edema and lung hemorrhage) but only minimal changes in the lungs of the endotoxin-treated group of animals exposed to hyperoxia. Transmission electron microscopy showed a protective action of endotoxin treatment on the pulmonary capillary endothelium, compatible with the decreased exudative changes (edema) observed in the O₂-exposed endotoxin-treated lungs. Indomethacin and methylprednisolone pretreatment failed to alter the protective action of endotoxin versus pulmonary O₂ toxicity.     

Functions, structures and Triton X-100 effect for the catalytic subunits of heterodimeric phospholipases A2 from Vipera nikolskii venom

Phospholipases A₂ (PLA₂s) from snake venoms have diverse pharmacological functions including neurotoxicity, and more studies are necessary to understand relevant mechanisms. Here we report the different crystal structures for two enzymatically active basic subunits (HDP-1P and HDP-2P) of heterodimeric neurotoxic PLA₂s isolated from Vipera nikolskii venom. Structural comparisons with similar PLA₂s clearly show some flexible regions which might be important for the catalytic function and neurotoxicity. Unexpectedly, Triton X-100 molecule bound in the hydrophobic channel of HDP-1P and HDP-2P was observed, and its binding induced conformational changes in the Ca²⁺ binding loop. Enzymatic activity measurements indicated that Triton X-100 decreased the activity of PLA₂, although with comparatively low inhibitory activity. For the first time exocytosis experiments in pancreatic β cells were used to confirm the presynaptic neurotoxicity of relevant snake PLA₂. These experiments also indicated that Triton X-100 inhibited the influence of HDP-1P on exocytosis, but the inhibition was smaller than that of MJ33, a phospholipid-analogue inhibitor of PLA₂. Our studies performed at a cellular level are in good agreement with earlier findings that enzymatic activity of the snake presynaptic PLA₂ neurotoxins is essential for effective block of nerve terminals.     

Acute lung inflammation in rats induced by phorbol myristate acetate (PMA)

Intratracheal administration of PMA produces acute lung injury in part due to the generation of O2-derived free radicals. This study evaluated the role of the antioxidant enzyme superoxide dismutase (SOD) in PMA-induced lung injury in the rat. PMA was instilled into rats intratracheally (20-60 micrograms/kg), and the lungs were lavaged 4 hr later. Total number of cells recovered from lavage after PMA treatment was not different from the total number recovered from controls; lavagable PMNs increased in a dose-dependent manner. Albumin in lavage fluid (an index of lung vascular permeability) was significantly increased at 60 micrograms/kg PMA. SOD (10,000 U) + PMA (60 micrograms/kg) reduced the albumin level but significantly increased both total number of cells and number of PMNs recovered from lavage fluid. To investigate the possibility that SOD decreases the ability of PMNs to adhere, PMN aggregation was measured in vitro. The results indicated that 10,000 U SOD can inhibit PMA-induced aggregation by 50%. In contrast, aggregation to other stimuli (e.g., fMet-Leu-Phe, A23187) was unaffected by SOD. We conclude SOD prevents PMA-induced lung permeability and diminishes PMN adherence.     

Early damage indicators in the lungs. IV. Biochemical and cytologic response of the lung to lavage with metal salts

A rapid screening test for estimating the acute toxicity level of substances in the lung has been developed and evaluated using metal salts as the toxic agents. In the test, animals were exposed in vivo to the metal salts by bronchopulmonary lavage, and toxicity was determined by the enzymatic and cytologic response observed in the airways 1 day after exposure. The airway response was determined by analysis of bronchopulmonary lavage fluid for lactate dehydrogenase, acid and alkaline phosphatase, and β-glucuronidase activity as well as for total sialic acid and protein content and total and differential cell counts. These values showed a dose-dependent response in the lavage fluid. The enzymatic response of the lung tissue itself was not as marked as that seen in the lavage fluid. Histopathological evaluations of lung tissue were made in order to correlate morphological change with the airway response. The test allowed a rapid determination of the lung dose of metal salt which caused acute toxicity. Based on the biochemical response in the airway, the relative toxicity of the heavy-metal-containing compounds tested was: CdCl₂>SeO₂, NH₄VO₃, NiCl₂>CrCl₃.     

Chemokine receptor CXCR3 is important for lung tissue damage and airway remodeling induced by short-term exposure to cigarette smoking in mice

Aim:

To investigate the role of chemokine receptor CXCR3 in cigarette smoking (CS)-induced pulmonary damage.

Methods:

CXCR3 knockout (CXCR3-/-) mice were used. Differences in airspace enlargement, mRNA expression of matrix metalloproteinases (MMPs), transforming growth factor (TGF) β1, CXCL10 in lung homogenates, and CXCL10 content in bronchoalveolar lavage (BAL) fluids and homogenates were compared between CXCR3-/- mice and wild-type (WT) mice three days after three-day CS exposures.

Results:

The linear intercept was significantly less in CXCR3-/- mice than in WT mice (30.1±0.9 μm vs 40.3±2.4 μm, P<0.01). Morphologically, collagen was deposited less around airways and vessels in CXCR3-/- mice. The lung hydroxyproline content was significantly lower in CXCR3-/- mice than in WT mice (6.0±1.0 μg/mL vs 12.0±1.6 μg/mL, P<0.05). Profoundly lower mRNA expression of MMP2, MMP12, TGFβ1, and CXCL10 was seen in lung homogenates from CXCR3-/- mice. CXCL10 concentrations in BAL fluid and lung homogenates were significantly lower in CXCR3-/- mice than in WT mice (BAL fluid: 19.3±1.4 pg/mL vs 24.8±1.6 pg/mL, P<0.05; lung homogenates: 76.6±7.0 pg/mL vs 119.5±15.9 pg/mL, P<0.05).

Conclusion:

CXCR3 is important in mediating lung tissue damage and airway remodeling following a short-term CS insult, possibly through up-regulation of CXCL10 and inducement of mRNA expression of MMPs. Targeting CXCR3 may be helpful for prevention of CS-induced pulmonary pathology.     

Regulation of ozone‐induced lung inflammation by the epidermal growth factor receptor in mice

Human exposure to the highly reactive oxidant gas Ozone (O₃) is associated with inflammatory responses in the airway epithelium. The mechanisms responsible have not been fully elucidated. Epidermal growth factor receptor (EGFR) has previously been shown to play a critical role in the pathogenesis of lung inflammation. To define the role of EGFR in O₃‐induced lung inflammation in mice. 40 BALB/c mice were exposed to filtered air (FA) or (0.25, 0.5, 1.00 ppm) O₃ for 3 h per day for 7 consecutive days. Levels of reactive oxygen species (ROS), EGF, and transforming growth factor α (TGF‐α) in the bronchoalveolar lavage fluid (BALF) of mice were measured using ELISA. BALB/c mice were intratracheally instilled with the EGFR kinase inhibitor PD153035 2 h prior to O₃ exposure and every other day thereafter. Phosphorylation of EGFR (Y1068) in lung sections was determined using immunohistochemical staining and western blot 24 h after exposure. Inhalation of O₃ induced pronounced lung inflammation in a dose‐dependent manner. Levels of ROS, TGF‐α, and total proteins and cells in the BALF of mice exposed to 0.5 ppm or 1.0 ppm of O₃ were markedly elevated relative to those in the BALF of the mice exposed to FA. In addition, exposure to O₃ induced EGFR(Y1068) phosphorylation in the airway epithelium. Administration of PD153035 resulted in a significantly reduced lung inflammation as well as EGFR phosphorylation induced by O₃ exposure. Inhalation of O₃ leads to inflammatory responses that are dependent on the activation the EGFR in the airway epithelium. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2016–2027, 2016.     

Toxicological Interactions in the Respiratory System after Inhalation of Ozone and Sulfuric Acid Aerosol Mixtures

A factorial design study was performed to examine the acute effects of inhaled acid particles alone and in mixtures with ozone to test the hypothesis that acid particles and ozone would act synergistically. Sprague-Dawley rats were exposed nose-only for a single 4-h period to all 9 possible combinations of purified air and 2 concentrations each of O₃ (0.3 and 0.6 ppm) and submicrometer (0.3 μm mass median diameter [MMD]) sulfuric acid aerosols H₂SO₄ (0.5 and 1.0 mg/m³). Respiratory-tract injury and impairment of alveolar macrophage functions were evaluated. Two-way analyses of variance were used to test for significance of main effects and statistical interactions, and Tukey multiple comparison tests were used to test the significance of differences between group mean values. Addition of H₂SO₄ to O₃-containing atmospheres resulted in significant H₂SO₄ concentration-dependent reductions in O₃-induced inflammatory responses, and H₂SO₄, alone and in combination with O₃, depressed some functions of innate immunity. DNA synthesis in nasal, tracheal, and lung tissue following pollutant exposure, which is an index of injury or killing of epithelial cells, was significantly increased by O₃ but not by H₂SO₄ when administered alone, compared to purified air. When administered with O₃, H₂SO₄ did not reduce the effects of O₃ on DNA synthesis in the trachea or the lung, but did reduce the DNA synthesis response to O₃ in the nose. No significant changes in antibody-directed Fc receptor (FcR) binding of sheep red blood cells by alveolar macrophages were observed, but macrophage phagocytic activity was significantly reduced by the pollutant exposures. In summary, the results of this study indicate significant interactions between O₃ and H₂SO₄ in concurrent exposures; however, the findings do not support the hypothesis that O₃ and H₂SO₄ act synergistically in rats after single 4-h exposures.     

An in vivo hamster bioassay to assess the toxicity of particulates for the lungs

We have developed a short-term bioassay to predict the toxicity of particulates for the lungs using hamsters exposed by intratracheal instillation. After exposure the animals were killed, their lungs were lavaged, and the pulmonary damage was characterized by cellular and biochemical assays of lavage fluid: (a) changes in in situ phagocytic ability of macrophages; (b) damage to the air-blood barrier shown by increases in albumin and red blood cells; (c) inflammation shown by increases in polymorphonuclear neutrophils (PMNs) and macrophages; and (d) cellular damage, measured by the levels of lactate dehydrogenase (LDH), β-N-acetylglucosaminidase, peroxidase, and elastase in the extracellular supernatant fraction of the lavage fluid. The system was calibrated using toxic α-quartz and two nontoxic dusts, aluminum oxide and iron oxide. Increases in albumin and red blood cells one day after exposure were greater following quartz than aluminum oxide and iron oxide; in contrast, a large part of the LDH increase was a nonspecific response to increased dust within the lungs. Most of the indicators, including red blood cell numbers, glucosaminidase, and peroxidase, either approached or were at control levels 4 days after exposure to iron oxide or α-quartz. In α-quartz-exposed animals, macrophage and PMN numbers were more elevated at 4 days that at 1 day and remained elevated for at least 14 days. In contrast, in iron oxide-exposed hamsters, macrophage numbers did not differ from control levels and PMN numbers approached control levels with time. The ability to cause a prolonged infiltration of macrophages and PMNs may be an important determinant of the toxicity of mineral dusts.     

Siderite (FeCO3) and magnetite (Fe3O4) overload-dependent pulmonary toxicity is determined by the poorly soluble particle not the iron content

The two poorly soluble iron containing solid aerosols of siderite (FeCO₃) and magnetite (Fe₃O₄) were compared in a 4-week inhalation study on rats at similar particle mass concentrations of approximately 30 or 100?mg/m³. The particle size distributions were essentially identical (MMAD ≈1.4 μm). The iron-based concentrations were 12 or 38 and 22 or 66?mg Fe/m³ for FeCO₃ and Fe₃O₄, respectively. Modeled and empirically determined iron lung burdens were compared with endpoints suggestive of pulmonary inflammation by determinations in bronchoalveolar lavage (BAL) and oxidative stress in lung tissue during a postexposure period of 3 months. The objective of study was to identify the most germane exposure metrics, that are the concentration of elemental iron (mg Fe/m³), total particle mass (mg PM/m³) or particle volume (μl PM/m³) and their associations with the effects observed. From this analysis it was apparent that the intensity of pulmonary inflammation was clearly dependent on the concentration of particle-mass or -volume and not of iron. Despite its lower iron content, the exposure to FeCO₃ caused a more pronounced and sustained inflammation as compared to Fe₃O₄. Similarly, borderline evidence of increased oxidative stress and inflammation occurred especially following exposure to FeCO₃ at moderate lung overload levels. The in situ analysis of 8-oxoguanine in epithelial cells of alveolar and bronchiolar regions supports the conclusion that both FeCO₃ and Fe₃O₄ particles are effectively endocytosed by macrophages as opposed to epithelial cells. Evidence of intracellular or nuclear sources of redox-active iron did not exist. In summary, this mechanistic study supports previous conclusions, namely that the repeated inhalation exposure of rats to highly respirable pigment-type iron oxides cause nonspecific pulmonary inflammation which shows a clear dependence on the particle volume-dependent lung overload rather than any increased dissolution and/or bioavailability of redox-active iron.     

Pretreatment with EDU decreases rat lung cellular responses to ozone

The phenylurea compound EDU (N-[2-(2-oxo-1-imidazolindinyl)ethyl]-N'-phenylurea) has been shown to protect plants from the damaging effects of ozone exposure. Models of rat lung injury, based on acute exposure to 2 ppm ozone for 3 hr and on exposure to 0.85 ppm ozone for 2 days, were used to determine whether EDU pretreatment of rats protected lungs from oxidant injury. Rats were pretreated with 100 mg/kg body wt EDU by ip administration for 2 days prior to and on the days of ozone exposure. No adverse toxicological effects of EDU pretreatment were observed. Lung superoxide dismutase (SOD) and catalase (CAT) activities were significantly enhanced from 636 to 882 U/lung and from 599 to 856 U/lung, respectively. One day following acute exposure (2 ppm for 3 hr), an ozone-induced increase of polymorphonuclear leukocytes (PMNs) from 0.01 to 1.18 million cells/lung was decreased to 0.68 million by EDU pretreatment. No alteration occurred in the degree of lung permeability indicated by increased lavage fluid albumin. EDU pretreatment also significantly decreased ozone-induced increases in PMN recovery after 2 days exposure to 0.85 ppm ozone from 5.54 to 2.12 million cells/lung. However, in this second case, EDU pretreatment reduced the observed ozone damage, indicated by a decrease in lavage fluid albumin and by a decrease in the macrophage and lymphocyte infiltration associated with this length of ozone exposure. The observation that EDU-treated cultured pulmonary arterial endothelial cells increased SOD and CAT activities identified a potential lung site of EDU interaction. These data demonstrated that although EDU pretreatment appears not to prevent initial ozone damage, it does reduce the infiltration of PMNs and might therefore prevent amplification of the injury associated with this cell type.     

Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system.     

Characterization of a phosphatidic acid phosphatase from rat brain cell membranes

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [³²P] phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0. Mg²⁺ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca²⁺ at concentrations of 0.1 to 1 mmol/1. Zn²⁺ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/1 Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The K _m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V _max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M _r of 320000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D. Correspondence to: Ariane Hoer at the above address     

Age-Dependent Effect of Ozone on Pulmonary Eicosanoid Metabolism in Rabbits and Rats

Age-Dependent Effect of Ozone on Pulmonary Eicosanoid Metabolismin Rabbits and Rats. GUNNISON, A. F., FINKELSTEIN, I., WEIDEMAN,P., SU, W.-Y., SOBO, M., AND SCHLESINGER, R. B. (1990). Fundam.Appl Toxicol. 15, 779–790. Acute exposures to ozone havepreviously been shown to cause quantitative changes in the spectrumof arachidonic acid (AA) metabolites in lung lavage fluid. Sinceage appears to be an important variable in the toxicity of inhaledozone, we investigated its effect on ozone-induced changes inpulmonary eicosanoid metabolism. Rats and rabbits ranging inage from neonates to young adults were exposed either to airor to 1 ppm ozone for 2 hr. Lung lavage fluid was collectedwithin 1 hr following exposure and analyzed for its contentof selected eicosanoids. In both species, there was a pronouncedeffect of age on ozone-induced pulmonary eicosanoid metabolism.Ozone-exposed animals at the youngest ages examined had severalfoldgreater amounts of two products of the cyclooxygenase pathway,prostaglandin E₂ (PGE₂) and prostaglandin F_2a (PGF_2a), thandid age-matched controls. This effect lessened and eventuallydisappeared as the animals grew toward adulthood. In rabbits,ozone also induced increases in 6-keto-prostaglandin F_1a andthromboxane B₂, but these changes were of lesser magnitude andevident only in the youngest rabbits exposed. There was no observedeffect of ozone on lung lavage content of leukothriene B₄. Indicesof nonspecific pulmonary damage, i.e., protein concentrationin lung lavage fluid and total number and viability of lavagedlung cells, were affected by ozone exposure, but not in an age-dependentmanner that correlated with changes in pulmonary eicosanoidmetabolism. In vitro ozone exposure of lung macrophages fromnaive rabbits of the same age range as those exposed in vivodemonstrated that ozone is capable of stimulating the elaborationof PGF_2a and especially PGE₂. However, the increase in lavagefluid PGE₂ and PGF_2a caused by ozone inhalation could not beattributed to macrophage metabolism conclusively since elaborationof PGE₂ and PGF_2a by cultured macrophages was not enhanced byprior in vivo ozone exposure. In an ancillary study it was shownthat 15-hydroxyprostaglandin dehydrogenase (PGDH) activity inrabbit lung homogenates was not affected by prior exposure toozone, indicating that the increase in lung lavage fluid eicosanoidsthat occurred in these animals could not be explained by inhibitionof PGDH.     

Pharmacological modulation of C‐X‐C motif chemokine receptor 4 influences development of acute respiratory distress syndrome after lung ischaemia–reperfusion injury

Activation of C‐X‐C motif chemokine receptor 4 (CXCR4) has been reported to result in lung protective effects in various experimental models. The effects of pharmacological CXCR4 modulation on the development of acute respiratory distress syndrome (ARDS) after lung injury, however, are unknown. Thus, we studied whether blockade and activation of CXCR4 influences development of ARDS in a unilateral lung ischaemia–reperfusion injury rat model. Anaesthetized, mechanically ventilated animals underwent right lung ischaemia (series 1, 30 minutes; series 2, 60 minutes) followed by reperfusion for 300 minutes. In series 1, animals were treated with vehicle or 0.7 μmol/kg of AMD3100 (CXCR4 antagonist) and in series 2 with vehicle, 0.7 or 3.5 μmol/kg ubiquitin (non‐cognate CXCR4 agonist) within 5 minutes of reperfusion. AMD3100 significantly reduced PaO₂/FiO₂ ratios, converted mild ARDS with vehicle treatment into moderate ARDS (PaO₂/FiO₂ ratio<200) and increased histological lung injury. Ubiquitin dose‐dependently increased PaO₂/FiO₂ ratios, converted moderate‐to‐severe into mild‐to‐moderate ARDS and reduced protein content of bronchoalveolar lavage fluid (BALF). Measurements of cytokine levels (TNFα, IL‐6, IL‐10) in lung homogenates and BALF showed that AMD3100 reduced IL‐10 levels in homogenates from post‐ischaemic lungs, whereas ubiquitin dose‐dependently increased IL‐10 levels in BALF from post‐ischaemic lungs. Our findings establish a cause‐effect relationship for the effects of pharmacological CXCR4 modulation on the development of ARDS after lung ischaemia–reperfusion injury. These data further suggest CXCR4 as a new drug target to reduce the incidence and attenuate the severity of ARDS after lung injury.     

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

Abstract

1.?Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined.

2.?Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains.

3.?In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben.

4.?Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity.

5.?Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes.

6.?Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens.

7.?Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.