Maria Hammer

UPPER AIRWAY AND PULMONARY EFFECTS OF OXIDATION PRODUCTS OF (+)- α -PINENE, d -LIMONENE, AND ISOPRENE IN BALB/ c MICE

The oxidation products (OPs) of ozone and the unsaturated hydrocarbons d -limonene, (+)-α -pinene, and isoprene have previously been shown to cause upper airway irritation in mice during 30-min acute exposures. This study evaluated the effects of OPs and the hydrocarbons themselves on the upper airways, the conducting airways, and the lungs over a longer exposure period. The time course of development of effects and the reversibility of effects were investigated; in addition, we assessed possible exacerbation of sensory responses of the airways to the unreacted hydrocarbons. Respiratory parameters in male BALB/ c mice were monitored via head-out plethysmography. Exposures to OPs or hydrocarbons were for 60 min, followed by a 30-min challenge period with air or hydrocarbon, and a 15-min recovery period with air only. Experiments were also performed where limonene/ozone exposures were separated 6 h from the challenge period. Ozone concentration in the reaction mixture was 3.4 ppm, and concentrations of hydrocarbons were 47 ppm (α -pinene), 51 ppm (d -limonene), and 465 ppm (isoprene). Due to reaction, the ozone concentration at the point of exposure was less than 0.35 ppm; exposure to 0.30 ppm ozone for 60 min did not produce effects different from air-exposed control animals. As previously established, upper airway irritation was a prominent effect of OP exposure. In addition, over the longer exposure period we observed the development of airflow limitation that persisted for at least 45 min postexposure. All effects from limonene/ozone exposures were reversible within 6 h. Exposures to OPs did not cause enhanced upper airway irritation during challenge with the hydrocarbons, indicating that a 1-h exposure to OPs did not increase the sensitivity of the upper respiratory system. However, airflow limitation was exacerbated in animals exposed to d -limonene alone immediately following exposure to limonene OPs. These findings suggest that terpene/ozone reaction products may have moderate-lasting adverse effects on both the upper airways and pulmonary regions. This may be important in the context of the etiology or exacerbation of lower airway symptoms in office workers, or of occupational asthma in workers involved in industrial cleaning operations.

Acute airway effects of formaldehyde and ozone in BALB/c mice

1 Concentration and time-effect relationships of formaldehyde and ozone on the airways were investigated in BALB/c mice. The effects were obtained by continuous monitoring of the respiratory rate, tidal volume, expiratory flow rate, time of inspiration, time of expiration, and respiratory patterns. 2 With concentrations up to 4 p.p.m., formaldehyde showed mainly sensory irritation effects of the upper airways that decrease the respiratory rate from a trigeminal reflex. The no-effect level (NOEL) was about 0.3 p.p.m. This value is close to the human NOEL, which is about 0.08 p.p.m. 3 Ozone caused rapid, shallow breathing in BALB/c mice. Later on, the respiratory rate decreased due to another vagal response that indicated an incipient lung oedema. The NOEL in mice was about p.p.m. during 30 min of ozone exposure. No major effect occurs in resting humans at about 0.4 p.p.m. 4 Thus, the upper airway irritant, formaldehyde, and the deep lung irritant, ozone, showed the same types of respiratory effects in humans and in BALB/c mice. Also, the sensitivity was nearly identical. Continuous monitoring of respiratory effects in BALB/c mice, therefore, may be a valuable method for the study of effects of other environmental pollutants, which, however, should be confirmed in further studies.

Acute airway effects of ozone-initiated d-limonene chemistry : Importance of gaseous products

There are concerns about ozone-initiated chemistry, because the formation of gaseous oxidation products and ultrafine particles may increase complaints, morbidity and mortality. Here we address the question whether the gaseous products or the ultrafine particles from the ozone-initiated chemistry of limonene, a common and abundant indoor pollutant, cause acute airway effects. The effects on the airways by d-limonene, a ca. 16s old ozone/d-limonene mixture, and clean air were evaluated by a mice bioassay, from which sensory irritation of the upper airways, airflow limitation, and pulmonary irritation can be obtained. A denuder was inserted to separate the ultrafine particles from the gaseous products prior to the exposure chamber. Reduction of mean respiratory frequency (>30%) and 230% increase of time of brake were observed without denuder, during 30min exposure, to the ozonolyzed d-limonene mixture, which are indicative of prominent sensory effects. The initial concentrations (ppm) were 40 d-limonene and 4 ozone. The exposure concentrations (ppm) were about 35 d-limonene and 0.05 ozone. Formaldehyde and residual d-limonene, the salient sensory irritants, accounted for up to three-fourth of the sensory irritation. The upper airway effects reversed to baseline upon cessation of exposure. An effect on the conducting airways was also significant, which did not reverse completely upon cessation. Airway effects were absent with the denuder inserted, which did not alter the size distribution of ultrafine particles ( approximately 10mg/m(3)), significantly. The result was statistically indistinguishable from clean dry air. It is concluded that ultrafine particles that are generated from ozone-initiated d-limonene chemistry and denuded are not causative of sensory effects in the airways.

Upper airway irritation of terpene/ozone oxidation products (TOPS). Dependence on reaction time, relative humidity and initial ozone concentration

Recently, we reported the formation of unidentified strong upper airway irritants in reaction mixtures of terpenes and ozone. The identified products included aldehydes, ketones and carboxylic acids. Here we report the effects of variation of reaction time, relative humidity and initial ozone concentration on irritant formation in a flow reaction system using R-(+)-limonene and isoprene. Upper airway irritation was measured in mice as reduction of the respiratory rate. For both substances maximum irritation was observed for low humidity (<2% RH)/short time (16-30 s) reaction mixtures, and both moderate humidity ( approximately 32% RH) and longer reaction times (60-90 s) resulted in significantly less irritation. These results suggest that some unidentified intermediates react with water vapor to give less irritating products. Irritation measured at four ozone concentrations (0.5, 1, 2 and 3.5 ppm) using low humidity/short time reaction conditions for limonene (50 ppm) and isoprene (500 ppm) revealed that at 0.5 ppm, irritation was at the same level as that for the pure terpenes, indicating that at 0.5 ppm ozone the combined irritant effect was near the no effect level for the product mixture.

Effects of mono-2-ethylhexyl phthalate on the respiratory tract in BALB/c mice

Airborne mono-2-ethylhexyl phthalate (MEHP) was studied for acute airway effects using a bioassay with BALB/c mice. Concentration-and time-dependent effects were obtained by continuous monitoring of the breathing pattern during exposure to 0.3-43.6 mg/m3 MEHP for 60 min. Additionally, inflammatory effects of MEHP were studied from bronchoalveolar lavage (BAL) fluid. MEHP showed no upper airway irritating effect. Lower airway irritation was apparent from a concentration-dependent decrease in tidal volume (shallow respiration) with a no-observed effect level (NOEL) of 0.3 mg/m3. The respiratory rate reached a maximum at about 8 mg/m3, demonstrating a rapid shallow breathing pattern. At concentrations above 4.9 mg/m3, the time of pause, another marker of lung irritation, increased concentration-dependently, resulting in a decrease in respiratory rate at high exposure levels. BAL fluid obtained from 0 to 72 hours after a 60 min exposure to 30 mg/m3 MEHP showed that the number of macrophage reached maximum about 16 hours after exposure. The NOEL was 1.7 mg/m3. BAL content of neutrophils, lymphocytes, eosinophils and epithelial cells was normal after exposure to 30 or 1.7 mg/m3 MEHP. Based on worst case inhalation scenario in the general population, no airway irritation is expected from non-occupational levels of MEHP originating from DEHP.

Effects of R-(+)-and S-(-)-limonene on the respiratory tract in mice

The effects of airborne R-(+)- and S-(-)-limonene were studied in conscious BALB/c mice by continuous monitoring respiratory rate (f), tidal volume (VT) and mid-expiratory flow rate (VD) during an exposure period of 30 min. Both enantiomers decreasedffrom a trigeminal reflex, i.e., due to sensory irritation. The exposure concentration decreasingf by 50% (RD50) in the first 10 min of the exposure period was estimated to be 1076 ppm for R-(+)-limonene and 1467 ppmforS-(-)-limonene. Resultsforsensoryirritation ofR –(+)-limonene in BALB/c mice and humans are in close agreement. The reported sensory irritation threshold is above 80 ppm in humans while the no – observed – effect level was estimated to be 100 ppm in mice. The enantiomers were devoid of pulmonary irritation or general anesthetic effects with R-(+)-limonene <1599 ppm and S-(-)-limonene <2421 ppm. R-(+)-limonene did not influence VT below 629 ppm. S-(-)-limonene increased VT above 1900 ppm. Both enantiomers induced a mild bronchoconstrictive effect above 1000 ppm.

Polyhydroxylated C60 Fullerene (Fullerenol) Attenuates Neutrophilic Lung Inflammation in Mice

Inflammation is crucial to eliminate pathogens and promoterepair of injured tissue. However, excessive or persistentinflammation can contribute to tissue injury and thepathogenesis and exacerbation of diseases, includinginflammatory lung diseases, such as chronic obstructivepulmonary disease [1] and silicosis [2]. Neutrophilic inflam-mation is an important aspect of chronic obstructive pulmonarydisease [1,3,4] and silicosis [2,5]. Thus, in human beings, arelationship between exposure to respirable silica in coalmine dust and pulmonary inflammation is seen, resulting inan elevated neutrophil count in bronchoalveolar lavage fluid(BALF) [6]. Exposure to silica can cause silicosis, where thesevere inflammation in the lung appears to be an initiatingstep in the development of the disease [7]. The quartzparticles can in itself generate reactive oxygen species (ROS),but additional inflammatory injuries appear to be a result ofthe influx of inflammatory cells [2]. The cell-generated ROSand nitric oxide radicals are hallmarks of the toxicity of thequartz particles [8,9], and quartz-induced inflammation ischaracterised by, for example, neutrophilic inflammation inrodents [10]. The importance of neutrophils in the developmentof inflammatory lung diseases has been reported in rodents,where exposure to quartz resulted in induced influx ofneutrophils [2,4,11–15]. Furthermore, it was shown that treatmentwith anti-macrophage inflammatory protein 2 (MIP-2)antiserum prior to quartz exposure attenuated neutrophilinflux [16], suggesting that MIP-2 can play an importantrole in quartz-induced neutrophilic lung inflammation.Development of novel anti-inflammatory drugs is animportant issue. One mechanism by which inflammationcan be attenuated is by elimination of ROS and free radicals[17,18]. Fullerenes, a recently discovered allotrope of carbon[19], have attracted much attention in pharmacology asreviewed [20]. Fullerenes, also termed buckminsterfullerenesor simply ‘bucky balls’ [21], are molecules consisting of 60or more carbon atoms arranged in a soccer ball-like struc-ture. The pristine C

Airway effects of repeated exposures to ozone-initiated limonene oxidation products as model of indoor air mixtures

Repeated low-level indoor air exposure to volatile organic compounds (VOCs) may influence the reporting of sensory irritation in the eyes and airways. The ozone-initiated reaction products of limonene, an abundant VOC, were used as a model of indoor air mixtures to study upper airway (sensory) irritation, bronchoconstrictive and alveolar level effects after repeated exposures. Mice were exposed 1h/day for 10 consecutive days to: air, limonene (52 ppm/289 mg/m(3)); ozone (0.1 ppm/0.2mg/m(3)); a reaction mixture of limonene (52±8 ppm) and ozone (0.5, 2.5 and 3.9 ppm) resulting in ~0.05 ppm residual ozone. Neither the limonene nor the ozone exposures alone showed consistent effects on the respiratory parameters. In the limonene/ozone groups, the respiratory rate decreased concentration-dependently with an extrapolated no-effect-level of ~0.3 ppm admixed ozone. Both sensory irritation and airflow limitation were conspicuous effects of the mixtures; sensory irritation appeared rapidly and airflow limitation developed slowly during each exposure. The effects of these parameters did not change with increasing number of exposures. No firm conclusion could be drawn about alveolar level effects. Cells in bronchoalveolar lavage were unchanged irrespective of exposure to air, ozone, and limonene with and without ozone. In conclusion, the study indicated that repeated exposures to ozone-initiated limonene mixtures did not cause sensitization of sensory irritation and airflow limitation. Bronchoalveolar lavage after exposures to ozone, and limonene with and without ozone, respectively, did not show airway inflammation.

Acute and Subchronic Airway Inflammation after Intratracheal Instillation of Quartz and Titanium Dioxide Agglomerates in Mice

This study investigated the acute and subchronic inflammatory effects of micrometer-size (micro-size) and nanometer-size (nano-size) particles after intratracheal (i.t.) installation in mice. The role of the type of compound, polymorphism, and size of the particles was investigated. Studied compounds were the two micro-size reference quartzes, SRM1878a and DQ12, a micro- and nano-size rutile titanium dioxide (TiO2), a nano-size anatase, and an amorphous TiO2. Particles were administered by a single i.t. instillation in mice at a fixed dose of 5, 50, and 500 μg, respectively. Inflammation was evaluated from the bronchoalveolar lavage fluid (BALF) content of inflammatory cells, the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), as well as from lung histology. Evaluations were at 24 h (acute effects) and 3 months (subchronic effects) after instillations. Both types of quartz induced a dose-dependent acute increase of neutrophils, IL-6, and total protein in BALF. Limited subchronic inflammation was observed. All types of TiO2 induced a dose-dependent acute increase of neutrophils in BALF. In the acute phase, micro- and nano-size rutile and nano-size amorphous TiO2 induced elevated levels of IL-6 and total protein in BALF at the highest dose. At the nano-size rutile and amorphous TiO2, subchronic lung inflammation was apparent from a dose-dependent increase in BALF macrophages. Histology showed little inflammation overall. The two types of quartz showed virtually similar inflammatory effects. Nearly similar effects were observed for two sizes of rutile TiO2. Differences were seen between the different polymorphs of nano-size TiO2, with rutile being the most inflammogenic and amorphous being the most potent in regard to acute tissue damage.

Human reference values for acute airway effects of five common ozone-initiated terpene reaction products in indoor air

Ozone-initiated monoterpene reaction products have been hypothesized to cause eye and airway complaints in office environments and some have been proposed to cause skin irritation and sensitization. The respiratory effects of 60 min exposures to five common oxidation products from abundant terpenoids (e.g. limonene), used as solvent and fragrance in common household products or present in skin lipids (e.g. squalene), were studied in a head out mouse bioassay. This allowed determination of acute upper airway (sensory) irritation, airflow limitation in the conducting airways, and pulmonary irritation in the alveolar region. Derived human reference values (RFs) for sensory irritation were 1.3, 0.16 and 0.3 ppm, respectively, for 4-acetyl-1-methylcyclohexene ( 0.2 ppm) [corrected], 3-isopropenyl-6-oxo-heptanal (IPOH), and 6-methyl-5-heptene-2-one (6-MHO). Derived RFs for airflow limitation were 0.8, 0.45, 0.03, and 0.5 ppm, respectively, for dihydrocarvone (DHC), 0.2 ppm [corrected], 4-oxo-pentanal (0.3 ppm) [corrected], and 6-MHO. Pulmonary irritation was unobserved as a critical effect. The RFs indicate that the oxidation products would not contribute substantially to sensory irritation in eyes and upper airways in office environments. Reported concentrations in offices of 6-MHO and 0.3 ppm [corrected]would not result in airflow limitation. However, based upon the RFs for IPOH and 0.3 ppm [corrected], precautionary actions should be considered that disfavor their formation in excess.