Photochemical products in urban mixtures enhance inflammatory responses in lung cells

Complex urban air mixtures that realistically mimic urban smog can be generated for investigating adverse health effects. "Smog chambers" have been used for over 30 yr to conduct experiments for developing and testing photochemical models that predict ambient ozone (O(3)) concentrations and aerosol chemistry. These chambers were used to generate photochemical and nonirradiated systems, which were interfaced with an in vitro exposure system to compare the inflammatory effects of complex air pollutant mixtures with and without sunlight-driven chemistry. These are preliminary experiments in a new project to study the health effects of particulate matter and associated gaseous copollutants. Briefly, two matched outdoor chambers capable of using real sunlight were utilized to generate two test atmospheres for simultaneous exposures to cultured lung cells. One chamber was used to produce a photochemically active system, which ran from sunrise to sunset, producing O(3) and the associated secondary products. A few hours after sunset, NO was added to titrate and remove completely the O(3), forming NO(2). In the second chamber, an equal amount of NO(2) and the same amount of the 55-component hydrocarbon mixture used to setup the photochemical system in the first side were injected. A549 cells, from an alveolar type II-like cell line grown on membranous support, were exposed to the photochemical mixture or the "original" NO(2)/hydrocarbon mixture for 5 h and analyzed for inflammatory response (IL-8 mRNA levels) 4 h postexposure. In addition, a variation of this experiment was conducted to compare the photochemical system producing O(3) and NO(2), with a simple mixture of only the O(3) and NO(2). Our data suggest that the photochemically altered mixtures that produced secondary products induced about two- to threefold more IL-8 mRNA than the mixture of NO(2) and hydrocarbons or O(3). These results indicate that secondary products generated through the photochemical reactions of NO(x) and hydrocarbons may significantly contribute to the inflammatory responses induced by exposure to urban smog. From previous experience with relevant experiments, we know that many of these gaseous organic products would contribute to the formation of significant secondary organic particle mass in the presence of seed particles (including road dust or combustion products). In the absence of such particles, these gaseous products remained mostly as gases. These experiments show that photochemically produced gaseous products do influence the toxic responses of the cells in the absence of particles.     

Combined respiratory effects of cold air with SO(2) or NO(2) in single 1-hour exposures of hyperventilating guinea pigs

The present study is a continuation of our previous experiments on repeated 10-min exposures of anesthetized, mechanically ventilated guinea pigs to clean cold dry air (H?linen et al., 2000a), and to cold air plus gaseous air pollutants (H?linen et al., 2000b). This time we made continuous 60-min exposures to clean cold dry air, cold air + SO(2) at 1 ppm, cold air + NO(2) at 1 ppm, and warm humid air + NO(2) at 1 ppm, and focused on responses at 10-60 min. Clean cold dry air and cold air + pollutants (n = 8-9 in each group) produced similar cooling in the guinea pig lower respiratory tract. The decreases in intratracheal temperature (T(tr)) reached a plateau at 20 min with mean maximal decreases of 9.7-11.3 degrees C from the pre-exposure control values of 36.0-37.3 degrees C. In contrast, there were progressive decreases in esophageal temperature (T(oe)) during the exposures, indicating constant conductive and evaporative heat losses from the tracheobronchial tissues. The mean maximal decreases in T(oe) were 1.2-1.4 degrees C from the preexposure control values of 37.8-38.0 degrees C. Clean cold dry air induced 4. 5-10.8% mean decreases in peak expiratory flow (PEF) at 10-60 min of exposure, which were statistically nonsignificant due to a relatively large variation between animals. Cold air + SO(2) at 1 ppm induced a mean decrease of 11.4% in PEF at 10 min (p <.05), which was spontaneously abolished during the next 10 min of exposure. Cold air + NO(2) at 1 ppm caused no decrease, but in fact small, nonsignificant increases in PEF at 30-60 min of exposure. Cold air + NO(2) at 1 ppm, and to some extent also cold air + SO(2) at 1 ppm, attenuated significantly the mechanical ventilation induced gradual decrease in tidal volume (V(T)), when compared to clean cold dry air exposure. Cold air + NO(2) at 1 ppm, but not warm humid air + NO(2) at 1 ppm, increased significantly the proportion of macrophages in the differential count of bronchoalveolar lavage fluid (BALF) white cells when compared to both clean warm humid air and cold dry air. None of the exposure conditions caused morphological or inflammatory changes in the respiratory tissues. In conclusion, continuous 60-min exposures to clean cold dry air, cold air + SO(2), and cold air + NO(2) produced weaker functional effects on the lower respiratory tract of guinea pigs than our previous consecutive 10-min exposures to these air conditions. After the first 10 min, there was a strong attenuation of the bronchoconstrictor responses, especially to cold air + NO(2) at 1 ppm. The small airway effects of prolonged mechanical ventilation were significantly modified by NO(2) at 1 ppm in both cold dry and warm humid breathing air. Finally, cold air + NO(2) at 1 ppm increased the proportion of macrophages in BALF white cells.     

Combined respiratory effects of cold air with SO(2) or NO(2) in repeated 10-minute exposures of hyperventilating guinea pigs

Previous studies in asthmatic subjects and guinea pigs have demonstrated attenuation of bronchoconstriction in repeated exposures to clean cold dry air. In the present animal study, we have simulated short-lasting human exposures to subfreezing urban air containing sulfur dioxide (SO(2)) and nitrogen dioxide (NO(2)). The anesthetized, paralyzed, and mechanically ventilated guinea pigs had 4 consecutive 10-min exposures either to clean cold dry air or to cold air with graded concentrations of SO(2) (0-5 ppm) or NO(2) (0-4 ppm). Peak expiratory flow (PEF) and tidal volume (V(T)) were continuously measured both during and after highly controlled exposures. Bronchoalveolar lavage fluid (BALF) and histological samples were obtained after finishing the consecutive exposures. Cold air + SO(2) at 1 and 2.5 ppm (n = 12) produced immediate concentration-dependent increases in the lung function responses compared to the preceding single exposure to clean cold dry air in the same animals (DeltaPEF = -32.7 +/- 6.1% and -35.6 +/- 6.5% vs. -27.0 +/- 3.1%; DeltaV(T) = -22.4 +/- 4.4% and -28.3 +/- 4.7% vs. -18.1 +/- 2.9%). In a multivariate analysis, these responses were significantly larger than the attenuated lung function responses to the corresponding second and third clean cold dry air exposures (p <. 05). The fourth exposure to cold air + SO(2) at 5 ppm produced a smaller response (DeltaPEF = -25.3 +/- 4.8% and DeltaV(T) = -17.8 +/- 3.7%) than cold air with the lower SO(2) concentrations. Cold air + NO(2) at 1 and 2.5 ppm (n = 12) produced roughly similar lung function responses to the preceding single exposure to clean cold dry air in the same animals, and there was no significant attenuation of bronchoconstriction as with the consecutive exposures to clean cold dry air. The largest decreases in lung functions (DeltaPEF = -33.8 +/- 6.7% and DeltaV(T) = -26.2 +/- 6.8%) were recorded during the fourth exposure, which was to cold air + NO(2) at 4 ppm. In the cold air + SO(2) group, there was a significantly lower proportion of macrophages in the differential count of BALF white cells compared to the clean cold dry air group. In addition, there was eosinophilic infiltration within and below the tracheal epithelium in all guinea pigs exposed to either clean cold dry air, cold air + SO(2), or cold air + NO(2). In conclusion, the addition of moderate concentrations of SO(2) or NO(2) to clean cold dry air counteracted the attenuation of bronchoconstriction induced by repeated cold dry air exposures in guinea pigs. Cold air + SO(2) also decreased the proportion of macrophages in BALF white cells.     

DIFFERENTIAL SUSCEPTIBILITY TO OXIDANT EXPOSURE IN INBRED STRAINS OF MICE: NITROGEN DIOXIDE VERSUS OZONE

The contribution of genetic background to the pathogenesis of airway responses to environmental agents including air pollutants is becoming increasingly clear. Characterization of genetic mechanisms of response to these agents may assist in the identification of susceptible individuals and populations. The primary objective of this investigation was to utilize inbred strains of mice to determine (1) whether there was significant genetic contribution in susceptibility to lung injury and inflammation induced by single and repeated acute exposures to nitrogen dioxide (NO2) and (2) whether similar genetic factors control sus- ceptibility to lung injury induced by NO2 and another oxidant, ozone (O3). Nine strains of inbred mice (male, 5-6 wk) were studied: 129/ J, A/ J, AKR/ J, BALB/ cJ, C3H/ HeJ, C57BL/6J, DBA/2J, SJL/J, and SWR/J. Each was exposed for 3 h to filtered air (controls) or 15 ppm NO2, and cellular inflammation, epithelial injury, and cytotoxicity were measured 2, 6, and 24 h thereafter. NO2 exposure caused significant increases in cytotoxicity and lavageable macrophages, epithelial cells, polymorphonuclear leukocytes, and protein in all strains. Interstrain variation in each of these effects indicated that genetic background contributed a significant portion of the variance in responses to this oxidant. Two strains that were differentially susceptible to 3-h exposure to 15 ppm NO2\[C57BL/6J (B6), C3H/HeJ (C3)] were also exposed for 6 h/ day to 10 ppm NO2 on 5 consecutive days. Each of the responses to NO2 was completely adapted after 5 days in resistant C3 mice. Only the lavageable total protein response was adapted in susceptible B6 mice. To determine whether mechanisms of susceptibility to NO2 and O3 were the same, each strain was exposed for 3 h to filtered air or 2 ppm O3 and inflammation was assessed 6 and 24 h thereafter. Strain distribution patterns (SDPs) for responses to each oxidant were not significantly concordant and indicated that susceptibility mechanisms were different. Results of these studies suggest that there is a strong genetic component to NO2 susceptibility that is partially adaptable and significantly different from O3 susceptibility.     

Mutagenicity and cytotoxicity of 2-chlorobenzylidene malonitrile (CS) and metabolites in V79 Chinese hamster cells

The genotoxicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated using the induction of gene mutations, micronuclei and DNA repair synthesis as biological endpoints. CS efficiently induced micronuclei and mutants resistant to 6-thioguanine in these cells, but it did not elicit DNA repair synthesis. Induction of micronuclei and mutants showed very similar courses of concentration dependence, suggesting that both events were caused by the same mechanism. The hydrolysis products of CS, o-chlorobenzaldehyde and malononitrile dit not induce micronuclei and were much less cytotoxic than CS. The observation of heritable genetic changes in cells exposed to CS in the absence of detectable DNA damage suggests that the genetic effects of CS are not caused by an interaction of the compound or its hydrolysis products with DNA. It appears more likely that the mutagenic activity is the consequence of effects of CS on the mitotic apparatus of the cells causing chromosomal aneuploidy.     

Effects of oxygen on the reactivity of nitrogen oxide species including peroxynitrite

This paper describes the O(2)-dependent control of the reactivity of nitrogen oxide species for the production of biologically important nitrated and nitrosated compounds. In this study, the effects of O(2) on the reactivity of NO, NO(2), and ONOO(-)/ONOOH for nitration of tyrosine (Tyr) and nitrosation of glutathione (GSH) and morpholine (MOR) were examined. NO produced S-nitrosoglutathione (GSNO) and N-nitrosomorpholine (NMOR) through the formation of N(2)O(3) under aerobic conditions, and NO(2) produced 3-nitrotyrosine (3-NO(2)Tyr), GSNO, and NMOR. Transnitrosation from GSNO to MOR was observed only in the presence of O(2). Although preformed ONOO(-)/ONOOH produced all the products under aerobic conditions, the formation of 3-NO(2)Tyr and GSNO was markedly reduced and the formation of NMOR was enhanced under anaerobic conditions. The reactivity of the CO(2) adduct of ONOO(-) was similarly dependent on O(2). 3-NO(2)Tyr was produced effectively by reaction with ONOO(-)/ONOOH at the O(2) concentration of 270 microM and by reaction with its CO(2) adduct at O(2) concentrations greater than 5 microM. Generation of.OH from ONOO(-)/ONOOH was suppressed under anaerobic conditions. The reactivity of ONOO(-)/ONOOH and.OH generation from ONOO(-) were reversibly controlled by the O(2) concentration.     

Formation of N-nitrosamines and N-nitramines by the reaction of secondary amines with peroxynitrite and other reactive nitrogen species: comparison with nitrotyrosine formation

Reactive nitrogen species, including nitrogen oxides (N(2)O(3) and N(2)O(4)), peroxynitrite (ONOO(-)), and nitryl chloride (NO(2)Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R(2)N(*)), which react with nitric oxide ((*)NO) or nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO(*) and superoxide anion (O(2)(*)(-)), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H(2)O(2), with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO(2) plus H(2)O(2) with myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.     

Nitrosation and nitration of 2-amino-3-methylimidazo[4,5-f]quinoline by reactive nitrogen oxygen species

Both cooked red meat intake and chronic inflammation/infection are thought to play a role in the etiology of colon cancer. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) is formed during cooking of red meat and may be involved in initiation of colon cancer. Reactive nitrogen oxygen species (RNOS), components of the inflammatory response, contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible chemical transformation of IQ by RNOS. RNOS were generated by various conditions to react with (14)C-IQ, and samples were evaluated by HPLC. Myeloperoxidase (MPO)-catalyzed reaction was dependent upon both H(2)O(2) and NO(2)(-). This reaction produced an azo-IQ dimer and IQ dimer along with two nitrated IQ products identified by ESI/MS. 2-Nitro-IQ was not detected. Product formation was inhibited by 2 mM cyanide. Reduction in nitrated products observed with 100 mM chloride was not altered with 0.5 mM taurine. Nitrated products were also produced by other conditions, ONOO(-) and NO(2)(-) + HOCl, which generate nitrogen dioxide radical. In contrast, conditions which generate N(2)O(3), such as diethylamine NONOate, produced only small amounts of nitrated products with the major product identified by MS and NMR as N-nitroso-IQ. MPO activation of IQ to bind DNA was dependent upon both H(2)O(2) and NO(2)(-). RNOS generated by ONOO(-) and DEA NONOate also activated IQ DNA binding. The nitrated IQ products were not activated by MPO to bind DNA. In contrast, N-nitroso-IQ was activated to bind DNA by MPO +/- NO(2)(-). HOCl activated N-nitroso-IQ, but not IQ. RAW cells produced N-nitroso-IQ and increased amounts of NO(2)(-)/NO(3)(-), when incubated with 0.1 mM IQ and stimulated with lipopolysaccharide and interferon gamma. Results demonstrate chemical transformation and activation of IQ by RNOS and activation of its N-nitroso product by biological oxidants, events which may contribute to initiation of colon cancer.     

COMBINED RESPIRATORY EFFECTS OF COLD AIR WITH SO2 OR NO2 IN SINGLE 1-HOUR EXPOSURES OF HYPERVENTILATING GUINEA PIGS

The present study is a continuation of our previous experiments on repeated 10-min exposures of anesthetized, mechanically ventilated guinea pigs to clean cold dry air (Hälinen et al., 2000a), and to cold air plus gaseous air pollutants (Hälinen et al., 2000b). This time we made continuous 60-min exposures to clean cold dry air, cold air + SO₂ at 1 ppm, cold air + NO₂ at 1 ppm, and warm humid air + NO₂ at 1 ppm, and focused on responses at 10-60 min. Clean cold dry air and cold air + pollutants (n = 8-9 in each group) produced similar cooling in the guinea pig lower respiratory tract. The decreases in intratracheal temperature (T_tr) reached a plateau at 20 min with mean maximal decreases of 9.7-11.3°C from the pre-exposure control values of 36.0-37.3°C. In contrast, there were progressive decreases in esophageal temperature (T_oe) during the exposures, indicating constant conductive and evaporative heat losses from the tracheobronchial tissues. The mean maximal decreases in T_oe were 1.2-1.4°C from the preexposure control values of 37.8-38.0°C. Clean cold dry air induced 4.5-10.8% mean decreases in peak expiratory flow (PEF) at 10-60 min of exposure, which were statistically nonsignificant due to a relatively large variation between animals. Cold air + SO₂ at 1 ppm induced a mean decrease of 11.4% in PEF at 10 min (p &lt; .05), which was spontaneously abolished during the next 10 min of exposure. Cold air + NO₂ at 1 ppm caused no decrease, but in fact small, nonsignificant increases in PEF at 30-60 min of exposure. Cold air + NO₂ at 1 ppm, and to some extent also cold air + SO₂ at 1 ppm, attenuated significantly the mechanical ventilation induced gradual decrease in tidal volume (V_T), when compared to clean cold dry air exposure. Cold air + NO₂ at 1 ppm, but not warm humid air + NO2 at 1 ppm, increased significantly the proportion of macrophages in the differential count of bronchoalveolar lavage fluid (BALF) white cells when compared to both clean warm humid air and cold dry air. None of the exposure conditions caused morphological or inflammatory changes in the respiratory tissues. In conclusion, continuous 60-min exposures to clean cold dry air, cold air + SO₂, and cold air + NO₂ produced weaker functional effects on the lower respiratory tract of guinea pigs than our previous consecutive 10-min exposures to these air conditions. After the first 10 min, there was a strong attenuation of the bronchoconstrictor responses, especially to cold air + NO₂ at 1 ppm. The small airway effects of prolonged mechanical ventilation were significantly modified by NO₂ at 1 ppm in both cold dry and warm humid breathing air. Finally, cold air + NO₂ at 1 ppm increased the proportion of macrophages in BALF white cells.     

Cytogenetic damage induced by acrylamide and glycidamide in mammalian cells: correlation with specific glycidamide-DNA adducts.

Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite playing a central role in AA genotoxicity. In this work we investigated DNA damage induced by AA and GA in mammalian cells, using V79 Chinese hamster cells. For this purpose, we evaluated two cytogenetic end points, chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), as well as the levels of specific GA-DNA adducts, namely, N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) using high-performance liquid chromatography coupled with tandem mass spectrometry. GA was more cytotoxic and clastogenic than AA. Both AA and GA induced CAs (breaks and gaps) and decreased the mitotic index. GA induced SCEs in a dose-responsive manner; with AA, SCEs were increased at only the highest dose tested (2mM). A linear dose-response relationship was observed between the GA concentration and the levels of N7-GA-Gua. This adduct was detected for concentrations as low as 1 microM GA. N3-GA-Ade was also detected, but only at very high GA concentrations (>or= 250 microM). There was a very strong correlation between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction. Such correlation was not apparent for CAs. These data suggest that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts; however, other mechanisms must be involved in the induction of CAs.     

Mutagenesis of the supF gene of pSP189 replicating in AD293 cells cocultivated with activated macrophages: roles of nitric oxide and reactive oxygen species

Dysregulated production of nitric oxide (NO*) and reactive oxygen species by inflammatory cells contributes to mutagenesis and carcinogenesis. We have characterized mutagenesis in the target supF gene of pSP189 replicating in AD293 cells cocultivated with mouse macrophage-like RAW264.7 cells activated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Activated macrophages produced substantial amounts of NO*, superoxide anion (O2*-), and hydrogen peroxide (H2O2) over 12-72 h periods. A time-dependent decrease in total cell number and a 3.7-fold increase in supF mutation frequency (MF), compared with unstimulated controls, were observed at 72 h. The increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate (NMA), an NO* synthase inhibitor, and also by superoxide dismutase (SOD) and catalase (CAT); cotreatment with NMA and SOD/CAT suppressed mutagenesis by 87% at 72 h. Mutations in supF were mainly multiple sequence changes (47%) and single base pair substitutions (51%) following IFN-gammaLPS activation. Following cotreatment with NMA alone or together with SOD/CAT, however, single base pair substitutions were prevalent (70 and 85%); decreased multiple mutations were observed (24 and 11%). Almost all single base pair substitutions induced under all exposure conditions occurred at G:C base pairs (87.8-94.6%). Whereas those induced by all treatments consisted predominantly of G:C to T:A transversions, G:C to T:A and A:T to T:A transversions were less frequent following treatment with NMA alone or with SOD/CAT compared to those induced by activated macrophages without additional treatment. Our results strongly suggest that ONOO- or its derivatives generated by reaction of NO* with O2*- may have been a major contributor to the observed mutagenesis by the activated macrophages, and mitigating their effects might serve a preventive function in ameliorating cancer risks associated with prolonged inflammation.     

Tracheal and bronchoalveolar permeability changes in rats inhaling oxidant atmospheres during rest or exercise

Permeability of tracheal and bronchoalveolar airways of rats was measured and used to examine the effects of inhaled oxidant-containing atmospheres. The atmospheres studied were (a) ozone (O3) at 0.6 ppm (1.2 mg/m3) or 0.8 ppm (1.6 mg/m3); (b) nitrogen dioxide (NO2) at 6 ppm (11.3 mg/m3) or 12 ppm (22.6 mg/m3); (c) O3 + NO2 at 0.6 ppm (1.2 mg/m3) and 2.5 ppm (4.7 mg/m3), respectively; and (d) a 7-component particle and gas mixture (complex atmosphere) representing urban air pollution in a photochemical environment. The rats were exposed for 2 h. The effects of exercise during exposure were evaluated by exposing additional groups in an enclosed treadmill. Exposure of resting rats to 0.8 ppm O3 increased tracheal permeability to DTPA and bronchoalveolar permeability to diethylenetriamine pentaacetate (DTPA) and bovine serum albumin (BSA) at 1 h after the exposure. Bronchoalveolar, but not tracheal, permeability remained elevated at 24 h after the exposure. Exercise during exposure to O3 increased permeability to both tracers in the tracheal and the bronchoalveolar zones, and prolonged the duration of increased permeability in the tracheal zone from 1 h to 24 h, and in the bronchoalveolar zone from 24 h to 48 h. Permeability in the tracheal and bronchoalveolar zones of rats exposed at rest to 6 or 12 ppm NO2 did not differ from controls. However, rats exposed during exercise to 12 ppm NO2 for 2 h developed a significant increase in tracheal and bronchoalveolar permeability to DTPA and BSA at 1 h, but not at 24 or 48 h, after exposure. Exposure at rest to 0.6 ppm O3 plus 2.5 ppm NO2 significantly increased bronchoalveolar permeability at 1 and 24 h after exposure, although exposure at rest to 0.6 ppm O3 alone increased bronchoalveolar permeability only at 1 h after exposure. Exposure to O3 + NO2 during exercise led to significantly greater permeability to DTPA than did exercising exposure to O3 alone. Resting rats exposed to a complex gas/aerosol atmosphere composed of the above O3 and NO2 concentrations, plus 5 ppm (13.1 mg/m3) sulfur dioxide (SO2) and an aerosol of insoluble colloidal Fe2O3 with an aerosol of manganese, ferric, and ammonium salts, demonstrated increased permeability at 1 and 24 h after exposure. Nitric acid vapor was formed in both the O3 + NO2 atmosphere and the complex gas/aerosol atmosphere.(ABSTRACT TRUNCATED AT 400 WORDS)     

绞股蓝总皂苷对光化学诱导大鼠大脑中动脉栓塞性脑缺血损伤的保护作用

目的研究绞股蓝总皂苷(Gyp)对栓塞性脑缺血损伤的保护作用。方法应用光化学诱导大鼠大脑中动脉栓塞模型,观察Gyp预防性给药后脑组织超氧化物歧化酶(SOD)活力,脂质过氧化产物TBARS,钠、钾、钙和水的含量以及缺血区范围的变化。结果Gyp能缩小光化学反应后的缺血区面积,降低TBARS含量,提高SOD活性,降低缺血区Na     

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.     

Effects of exercise exposure on toxic interactions between inhaled oxidant and aldehyde air pollutants

Respiratory tract injury resulting from inhalation of mixtures of ozone (O3) and nitrogen dioxide (NO2) and of O3 and formaldehyde (HCHO) was studied in Sprague-Dawley rats under exposure conditions of rest and exercise. Focal inflammatory injury induced in lung parenchyma by O3 exposure was measured morphometrically and HCHO injury to the nasal respiratory epithelium was measured by cell turnover using tritium-labeled thymidine. Mixtures of O3 (0.35 or 0.6 ppm) with NO2 (respectively 0.6 or 2.5 ppm) doubled the level of lung injury produced by O3 alone in resting exposures to the higher concentrations and in exercising exposures to the lower concentrations. Formaldehyde (10 ppm) mixed with O3 (0.6 ppm) resulted in reduced lung injury compared to O3 alone in resting exposures, but exercise exposure to the mixture did not show an antagonistic interaction. Nasal epithelial injury from HCHO exposure was enhanced when O3 was present in a mixture. Mixtures of O3 and NO2 at high and low concentrations formed respectively 0.73 and 0.02 ppm nitric acid (HNO3) vapor. Chemical interactions among the oxidants, HNO3, and other reaction products (N2O5 and nitrate radical) and lung tissue may be the basis for the O3-NO2 synergism. Increased dose and dose rate associated with exercise exposure may explain the presence of synergistic interaction at lower concentrations than observed in resting exposure. No oxidation products were detected in O3-HCHO mixtures, and the antagonistic interaction observed in lung tissue during resting exposure may result from irritant breathing pattern interactions.     

Experimental studies on tumor promotion by nitrogen dioxide

The effects of nitrogen dioxide (NO2) on promotion of lung tumorigenesis induced by N-bis(2-hydroxypropyl) nitrosamine (BHPN) were investigated in male Wistar rats. In a preliminary study, the highest non-effective dose of BHPN was found to be 0.5 g per kg body weight. Rats were given a single intraperitoneal injection of BHPN at a dose of 0.5 g per kg body weight or saline at 6 weeks of age, and then exposed to clean air, 0.04 ppm, 0.4 ppm or 4 ppm of NO2 for 17 months, respectively. The incidence of pulmonary tumors in rats exposed to BHPN plus 4 ppm of NO2 was 12.5%; the tumors were adenomas and adenocarcinomas. Adenomas were found in 4 out of 40 rats (10%) and adenocarcinomas were found in 1 out of 40 rats (2.5%). The tumor incidence in the lungs of rats kept in BHPN plus clean air and BHPN plus 0.04 ppm of NO2 was 2.5% (1/40). In both groups adenomas were found. There was no significant difference in tumor incidence between animals exposed to BHPN plus clean air and to BHPN plus 4 ppm of NO2. No lung tumors were found in the group of BHPN plus 0.4 ppm NO2 and in animals exposed to NO2 without BHPN treatment. A high incidence of alveolar cell hyperplasia was observed in the lungs of rats injected with BHPN, and the effect of NO2 on development of alveolar cell hyperplasia was slight. On the other hand, marked bronchiolar mucosal hyperplasia was found in 17 out of 40 rats (42.5%) in the group of BHPN plus 4 ppm of NO2, and in 1 out of 40 rats (2.5%) in each of the group exposed to clean air, 0.04 ppm or 0.4 ppm of NO2 with BHPN treatment, respectively. The hyperplasia in lungs of rats exposed to 4 ppm of NO2 without BHPN treatment was slighter than that in lung of rat exposed to 4 ppm of NO2 with BHPN treatment. On the other hand, tumor incidence in the nasal cavity of rats in each of group exposed to clean air and NO2 with BHPN treatment was 97-100%. Incidence of tumors in other organs in the groups exposed to clean air and NO2 with and without BHPN treatment was very low, and NO2 had no effect on tumor development in the nasal cavity and other organs whether animals were treated with BHPN or not.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiovascular effects of peroxynitrite

1. Peroxynitrite (PN) is formed in biological systems from the reaction of nitric oxide (*NO) with superoxide (O2(-)*) and both exist as free radicals. By itself, PN is not a free radical, but it can generate nitrogen dioxide (*NO2) and carbonate radical (CO3(-)*) upon reaction with CO2. 2. The reaction of CO2 constitutes a major pathway for the disposition of PN produced in vivo and this is based on the rapid reaction of PN anion with CO2 and the availability of CO2 in both intra- and extracellular fluids. The free radicals *NO2 and CO3(-)*, in combination with *NO, generated from nitric oxide synthase, can bring about oxidation of critical biological targets resulting in tissue injury. However, the reactions of *NO2, CO3(-)* and *NO with carbohydrates, protein and non-protein thiols, phenols, indoles and uric acid could result in the formation of a number of nitration and nitrosation products in the vasculature. These products serve as long-acting *NO donors and, therefore, contribute to vasorelaxant properties, protective effects on the heart, inhibition of leucocyte-endothelial cell interactions and reduction of reperfusion injury. 3. Herein, we review the chemistry of PN, the observations that the effects of PN could be mediated by formation of an *NO donor-like substance and review the physiological and beneficial effects of PN.     

Genotoxic and cytotoxic evaluation of the herbicide flurochloridone on Chinese hamster ovary (CHO-K1) cells

The in vitro effects of flurochloridone (FLC) and its formulations Twin Gold Pack® (25% a.i.) and Rainbow® (25% a.i.) were evaluated on Chinese hamster ovary (CHO-K1) cells by genotoxicity [sister chromatid exchange (SCE)] and cytotoxicity [cell-cycle progression, proliferative rate index (PRI), mitotic index (MI), MTT, and neutral red] end points. Cells were treated for 24 h within the 0.25–15 μg/ml concentration range. FLC and Twin Pack Gold® induced a significant and equivalent increase in SCEs regardless of the concentration. Rainbow®-induced SCEs at concentrations higher than 2.5 μg/ml; however, the increases were always lower than those induced by FLC and Twin Pack Gold®. For all compounds, the PRI decreased as a function of the concentration titrated into cultures. Whereas only the highest FLC and Twin Pack Gold® concentrations induced a significant reduction of the MI, all tested Rainbow® concentrations induced MI inhibition. Overall, the results demonstrated that although all compounds were not able to reduce the lysosomal activity, the mitochondrial activity was diminished when the highest concentrations were employed. These observations represent the first study analyzing the genotoxic and cytotoxic effects exerted by FLC and two formulated products on mammalian cells in vitro, at least on CHO-K1 cells.     

Effects of short-term, single and combined exposure to low-level NO2 and O3 on lung tissue enzyme activities in rats

To examine the pulmonary effects of relatively low levels of NO2 and O3, and test for any possible interaction in their effects, we exposed 3-mo-old male Sprague-Dawley rats, free of specific pathogens, to either filtered room air (control) or 1.20 ppm (2256 micrograms/m3) NO2, 0.30 ppm (588 micrograms/m3) O3, or a combination of the two oxidants continuously for 3 d. We studied a series of parameters in the lung, including lung weight, and enzyme activities related to NADPH generation, sulfhydryl metabolism, and cellular detoxification. The results showed that relative to control, exposure to NO2 caused small but nonsignificant changes in all the parameters; O3 caused significant increases in all the parameters except for superoxide dismutase; and a combination of NO2 and O3 caused increases in all the parameters, and the increases were greater than those caused by NO2 or O3 alone. Statistical analysis of the data showed that the effects of combined exposure were synergistic for 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, glutathione reductase, and superoxide dismutase activities, and additive for glutathione peroxidase and disulfide reductase activities, but indifferent from those of O3 exposure for other enzyme activities.