Søren Thor Larsen

In vivo Biology and Toxicology of Fullerenes and Their Derivatives

Fullerenes represent a group of nanoparticles discovered in 1985. They are spherical molecules consisting entirely of carbon atoms (C(x)) to which side chains can be added, furnishing compounds with widely different properties. Fullerenes interact with biological systems, for example, by enzyme inhibition, causing phototoxic reactions, being scavengers of reactive oxygen species and free radicals, in addition to being able to initiate free radical reactions. Absorption, distribution and excretion strongly depend on the properties of the side chains. The pristine C(60) has a very long biological half-life, whereas the most water-soluble derivatives are eliminated from the exposed animals within weeks. A long biological half-life raises concern about bioaccumulation and long-term effects. In general, the acute oral, dermal and airway toxicity is low. However, few relevant experimental studies of repeated dose toxicity, reproductive toxicity and carcinogenic effect are available. The data suggest that direct DNA damaging effects are low, but formation of reactive oxygen species may cause inflammation and genetic damage. Apparently, it is dose-dependent whether a beneficial or an adverse effect occurs.

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.

Di-(2-ethylhexyl) phthalate possesses an adjuvant effect in a subcutaneous injection model with BALB/c mice

The prevalence of allergic airway diseases is rapidly increasing in Western Europe and North America and the introduction of anthropogenic chemicals may explain a part of this increase. Recently, our group found that degradation products from several commonly used phthalate plasticizers possess adjuvant activity in an animal model. Mono-2-ethylhexyl phthalate, which is the degradation product of di-(2-ethylhexyl) phthalate (DEHP), was among these substances. These findings prompted the study of the adjuvant activity of the parent compound itself. Thus, DEHP was studied in a model using ovalbumin (OA) as the model antigen. OA was injected subcutaneously in the neck region of BALB/cJ mice with or without DEHP. The levels of OA-specific IgE, IgG1 and IgG2a antibodies in sera were measured by ELISA. Adjuvant effect, defined as a statistically significant increase in antibody level, was observed with IgG1 at a concentration of 2000 microg DEHP/ml after both one and two boosters.

Adjuvant and immuno-suppressive effect of six monophthalates in a subcutaneous injection model with BALB/c mice

The prevalence of allergic airway diseases is rapidly increasing in Western Europe and North America. This increase in disease prevalence may be associated with environmental pollutants. The present study investigated the adjuvant and immuno-suppressive effect of a series of monophthalates which are considered to be important metabolites of commonly used phthalate plasticizers. The effects were studied in a screening model. Ovalbumin (OA), used as the model antigen, was injected subcutaneously in the neck region of BALB/cJ mice with or without one of the test substances, mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MiNP) or mono-iso-decyl phthalate (MiDP). The levels of OA-specific IgE, IgG1 and IgG2a in sera were measured by ELISA. Immuno-suppressive effect, defined as a statistically significant reduction in IgE or IgG1 antibody production, was observed with MEHP (1000 microg/ml, IgE and IgG1), MnOP (1000 microg/ml, IgE and IgG1), MiNP (1000 microg/ml, IgE and 10 microg/ml, IgG1) and MiDP (100 microg/ml, IgE and IgG1). Adjuvant effect, defined as a statistically significant increase in IgE or IgG1 antibody level, occurred with MEHP (10 microg/ml, IgE), MnOP (100 microg/ml, and 10 microg/ml, IgG1) and MiNP (100 microg/ml, IgE). No statistically significant immune modulating effect was seen with MBnP and MnBP.

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.

Investigation of the adjuvant and immuno-suppressive effects of benzyl butyl phthalate, phthalic acid and benzyl alcohol in a murine injection model

In a recent study, di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono-2-ethylhexyl phthalate, were shown to possess adjuvant effect [Toxicology 169 (2001) 37; Toxicology Letters 125 (2001) 11]. The present study investigates the adjuvant effect of another important commercial phthalate plasticizer, benzyl butyl phthalate (BBP) as well as its degradation products, phthalic acid and benzyl alcohol (BA) in a murine model. The model antigen, ovalbumin (OA), was injected either alone (OA control group), together with one of the test substances (test group) or together with aluminium hydroxide, which served as the positive adjuvant control. The mice were boosted either once or twice with OA before blood was collected and assayed for the content of OA-specific IgE, IgG1 and IgG2a antibodies by ELISA methods. Adjuvant effect was defined as a statistically significant increased antibody level in the test groups compared with the OA control group. Conversely, if the antibody production in a test group was significantly lower than the OA control group, it was deemed to be immunosuppression. This study demonstrated that BBP, in contrast to DEHP, did not possess adjuvant effect. Furthermore, immunosuppression was apparent in the case of BA. The study also demonstrated that if the injections give rise to formation of wounds, it may cause false positive results.

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.

Effects of methacrolein on the respiratory tract in mice

The acute respiratory effects of airborne exposure to methacrolein were studied in a recent refinement of the standard test method with mice (ASTM, 1984. American Society for Testing and Materials, Philadelphia). Irritation of the upper respiratory tract caused a concentration-dependent decrease in the respiratory rate of 2-26 ppm methacrolein. In this range, only a minor airflow limitation occurred in the lower respiratory tract, suggesting that the main effect of methacrolein is sensory irritation. During exposure, the sensory irritation response maintained the same level, i.e. no desensitisation occurred. The concentration 10.4 ppm methacrolein reduced the respiratory rate by 50% (RD50). The extrapolated threshold for the respiratory depressing effect, RD0, was 1.3 ppm. The sensory irritation effect of methacrolein was compared with results from closely related compounds in order to elucidate the mechanism of the interaction between methacrolein and the sensory irritant receptor.