Risk assessment of consumer spray products using in vitro lung surfactant function inhibition, exposure modelling and chemical analysis.

Consumer spray products release aerosols that can potentially be inhaled and reach the deep parts of the lungs. A thin layer of liquid, containing a mixture of proteins and lipids known as lung surfactant, coats the alveoli. Inhibition of lung surfactant function can lead to acute loss of lung function. We focused on two groups of spray products; 8 cleaning and 13 impregnation products, and in the context of risk assessment, used an in vitro method for assessing inhibition of lung surfactant function. Original spray-cans were used to generate aerosols to measure aerodynamic particle size distribution. We recreated a real-life exposure scenario to estimate the alveolar deposited dose. Most impregnation products inhibited lung surfactant function at the lowest aerosolization rate, whereas only two cleaning products inhibited function at the highest rates. We used inhibitory dose and estimated alveolar deposition to calculate the margin of safety (MoS). The MoS for the inhibitory products was ≤1 for the impregnation products, while much larger for the cleaning products (>880). This risk assessment focused on the risk of lung surfactant function disruption and provides knowledge on an endpoint of lung toxicity that is not investigated by the currently available OECD test guidelines.

Use of thermal desorption gas chromatography-olfactometry/mass spectrometry for the comparison of identified and unidentified odor active compounds emitted from building products containing linseed oil.

The emission of odor active volatile organic compounds (VOCs) from a floor oil based on linseed oil, the linseed oil itself and a low-odor linseed oil was investigated by thermal desorption gas chromatography combined with olfactometry and mass spectrometry (TD-GC-O/MS). The oils were applied to filters and conditioned in the micro emission cell, FLEC, for 1-3days at ambient temperature, an air exchange rate of 26.9h(-1) and a 30% relative humidity. These conditions resulted in dynamic headspace concentrations and composition of the odor active VOCs that may be similar to real indoor setting. Emission samples for TD-GC-O/MS analysis from the FLEC were on Tenax TA. Although many volatile VOCs were detected by MS, only the odor active VOCs are reported here. In total, 142 odor active VOCs were detected in the emissions from the oils. About 50 of the odor active VOCs were identified or tentatively identified by GC-MS. While 92 VOCs were detected from the oil used in the floor oil, only 13 were detected in the low-odor linseed oil. The major odor active VOCs were aldehydes and carboxylic acids. Spearmen rank correlation of the GC-O profiles showed that the odor profile of the linseed oil likely influenced the odor profile of the floor oil based on this linseed oil.

The impact of information on perceived air quality--'organic' vs. 'synthetic' building materials.

UNLABELLED: As indoor air quality complaints cannot be explained satisfactorily and building materials can be a major source of indoor air pollution, we hypothesized that emissions from building materials perceived as unfamiliar or annoying odors may contribute to such complaints. To test this hypothesis, emissions from indoor building materials containing linseed oil (organic) and comparable synthetic (synthetic) materials were evaluated by a naïve sensory panel for evaluation of odor intensity (OI) and odor acceptability (OA). The building materials were concealed in ventilated climate chambers of the CLIMPAQ type. When information was provided about the identity and type of building material during the evaluation, i.e. by labeling the materials in test chambers either as 'organic' or 'synthetic', the OI was significantly lower for all the 'organic' materials compared with evaluations without information. Similarly, OA was increased significantly for most 'organic' samples, but not the 'synthetic' ones. The major effect is probably that OA is increased when the panel is given information about the odor source. PRACTICAL IMPLICATIONS: As providing information about the source of odors can increase their acceptability, complaints about indoor air quality may be decreased if occupants of buildings are well informed about odorous emissions from the new building materials or new activities in their indoor environment.

Organic compounds in office environments - sensory irritation, odor, measurements and the role of reactive chemistry.

Abstract Sensory irritation and odor effects of organic compounds in indoor environments are reviewed. It is proposed to subdivide volatile organic compounds (VOCs) into four categories: (i) chemically non-reactive, (ii) chemically 'reactive', (iii) biologically reactive (i.e. form chemical bonds to receptor sites in mucous membranes) and (iv) toxic compounds. Chemically non-reactive VOCs are considered non-irritants at typical indoor air levels. However, compounds with low odor thresholds contribute to the overall perception of the indoor air quality. Reported sensory irritation may be the result of odor annoyance. It appears that odor thresholds for many VOCs probably are considerably lower than previously reported. This explains why many building materials persistently are perceived as odorous, although the concentrations of the detected organic compounds are close to or below their reported odor thresholds. Ozone reacts with certain alkenes to form a gas and aerosol phase of oxidation products, some of which are sensory irritants. However, all of the sensory irritating species have not yet been identified and whether the secondary aerosols (ultrafine and fine particles) contribute to sensory irritation requires investigation. Low relative humidity may exacerbate the sensory irritation impact. Practical Implications Certain odors, in addition to odor annoyance, may result in psychological effects and distraction from work. Some building materials continually cause perceivable odors, because the odor thresholds of the emitted compounds are low. Some oxidation products of alkenes (e.g. terpenes) may contribute to eye and airway symptoms under certain conditions and low relative humidity.

Emission of phthalates from PVC and other materials.

The main objective of this study was to generate quantitative and qualitative emission data on phthalates from different materials. To achieve this the existing (Chamber for Laboratory Investigations of Materials, Pollution and Air Quality) Climpaq-based procedure for simplified measurements of emissions of plasticizer from PVC and other plasticized materials was modified. It was applied to a range of products. Some of them were suspected of contributing to the indoor concentration of plasticizers. The emissions from PVC flooring, polyolefine flooring, a refrigerator list, two electric cables, PVC skirting and floor wax were studied in separate Climpaqs. The emission from the PVC flooring in the Climpaq was compared with results from the ultra-small chamber Field and Laboratory Emission Cell (FLEC). Sampling and analysis methods were optimized to measure plasticizers. Samples were taken in exhaust air from the chambers after 6, 35, 62, 105, and 150 days from the start of the experiment. PVC flooring was tested for an additional 100 days. Polyolefine covered with wax resulted in an air concentration of 22 microg/m3 of dibutylphthalate (DBP), which is two orders of magnitude larger than any other materials, but did not emit di(2-ethylhexyl)phthalate (DEHP). The other materials resulted in max concentration of approximately 1 microg/m3 of DEHP and low emissions of DBP. The concentration of DEHP in each chamber increased slowly to a rather stable level which was reached after 150 days. DBP concentrations in the chambers with PVC skirting, PVC flooring, polyolefine and floor wax reached their quasi-static equilibrium after 60 days. The modified method did not create sufficient data for the calculation of emission rates. Adsorption of emission on chamber surfaces made it impossible to use the first part of the experiment for emission rate calculation. When the concentration had stabilized, it was found to be almost identical and independent of chamber and ventilation rate. Emission rates were reduced at high concentrations probably because the concentration in the material was near equilibrium with the concentration in the chamber air.

Sensory evaluation of emissions from selected building products exposed to ozone.

The interaction of ozone with eight different building products was studied in test chambers. The products were plasterboard, two types of paints on plasterboard, two types of carpet, linoleum, pinewood, and melamine-covered particleboard. Four months of conditioning prior to the experiment had left the products with a low emission. The products' ability to remove ozone from the air covered a wide range. For three of the products (plasterboard with paint, carpet, and pinewood), it was shown that the removal was primarily due to interactions in the products' surfaces and only to a minor extent due to gas-phase reactions. Sensory evaluations were carried out for five of the products, with different ozone-removal potentials. A sensory panel assessed the emissions from sets of two specimens of each product; one specimen was exposed to a high, but realistic, ozone concentration (10 or 80 ppb) and one specimen was exposed to no ozone (background level < 3 ppb). The panel assessed odor intensity and was asked to choose which odor of the two specimens they preferred. The perceivable changes in emissions due to exposure of the products to ozone depended on the type of product. The greatest effect was seen for carpet. Carpet was the only product that showed significantly higher odor intensity when exposed to ozone. Besides, the effect of ozone on preference was strongest for carpet and resulted in a clear negative sensory evaluation. A similar but less pronounced effect was seen for pinewood and plasterboard with paint. No clear preference was seen for melamine and linoleum.

'EUROPART'. Airborne particles in the indoor environment. A European interdisciplinary review of scientific evidence on associations between exposure to particles in buildings and health effects.

The relevance of particle mass, surface area or number concentration as risk indicators for health effects in non-industrial buildings has been assessed by a European interdisciplinary group of researchers (called EUROPART) by reviewing papers identified in Medline, Toxline, and OSH. Studies dealing with dermal effects or cancer or specifically addressing environmental tobacco smoke, house dust-mite, cockroach or animal allergens, microorganisms and pesticides were excluded. A total of 70 papers were reviewed, and eight were identified for the final review: Five experimental studies involving mainly healthy subjects, two cross-sectional office studies and one longitudinal study among elderly on cardiovascular effects. From most studies, no definite conclusions could be drawn. Overall, the group concluded that there is inadequate scientific evidence that airborne, indoor particulate mass or number concentrations can be used as generally applicable risk indicators of health effects in non-industrial buildings and consequently that there is inadequate scientific evidence for establishing limit values or guidelines for particulate mass or number concentrations.

Formation of strong airway irritants in mixtures of isoprene/ozone and isoprene/ozone/nitrogen dioxide.

We evaluated the airway irritation of isoprene, isoprene/ozone, and isoprene/ozone/nitrogen dioxide mixtures using a mouse bioassay, from which we calculated sensory irritation, bronchial constriction, and pulmonary irritation. We observed significant sensory irritation (approximately 50% reduction of mean respiratory rate) by dynamically exposing the mice, over 30 min, to mixtures of isoprene and O3 or isoprene, O3, and NO2. The starting concentrations were approximately 4 ppm O3 and 500 ppm isoprene (+ approximately 4 ppm NO2. The reaction mixtures after approximately 30 sec contained < 0.2 ppm O3. Addition of the effects of the residual reactants and the identified stable irritant products (formaldehyde, formic acid, acetic acid, methacrolein, and methylvinyl ketone) could explain only partially the observed sensory irritation. This suggests that one or more strong airway irritants were formed. It is thus possible that oxidation reactions of common unsaturated compounds may be relevant for indoor air quality.

Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) gene in human erythroleukemia cells.

Methylation of mammalian DNA by the DNA methyltransferase enzyme (dnmt-1) at CpG dinucleotide sequences has been recognized as an important epigenetic control mechanism in regulating the expression of cellular genes (Yen, R. W., Vertino, P. M., Nelkin, B. D., Yu, J. J., el-Deiry, W., Cumaraswamy, A., Lennon, G. G., Trask, B. J., Celano, P., and Baylin, S. B. (1992) Nucleic Acids Res. 20, 2287-2291; Ramchandani, S., Bigey, P., and Szyf, M. (1998) Biol. Chem. 379, 535-5401). Here we show that interleukin (IL)-6 regulates the methyltransferase promoter and resulting enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor (Spyropoulos, D. D., Pharr, P. N., Lavenburg, K. R., Jackers, P., Papas, T. S., Ogawa, M., and Watson, D. K. (1998) Mol. Cell. Biol. 15, 5643-5652). The data suggest that inflammatory cytokines such as IL-6 may exert many epigenetic changes in cells via the regulation of the methyltransferase gene. Furthermore, IL-6 regulation of transcription factors like Fli-1, which can help to direct cells along opposing differentiation pathways, may in fact be reflected in part by their ability to regulate the methylation of cellular genes.

Chemical and biological evaluation of a reaction mixture of R-(+)-limonene/ozone: formation of strong airway irritants.

The airway irritation of a reaction mixture of R-(+)-limonene and ozone was evaluated by a mouse bioassay in which sensory irritation, bronchoconstriction and pulmonary irritation were measured. Significant sensory irritation (33% reduction of mean respiratory rate) was observed by dynamic exposure of the mice, during 30 min, to a ca. 16 s old reaction mixture of ozone and limonene. The initial concentrations were nominally 4 ppm O3 and 48 ppm limonene. After reaction, the residual O3 was <0.03 ppm. Conventional analytical chemical methods were used to measure the formation of readily identified and stable products. Besides the expected products, 1-methyl-4-acetylcyclohexene (AMCH), 3-isopropenyl-6-oxoheptanal (IPOH), formaldehyde and formic acid, autooxidation products of limonene and a series of compounds including acetone, acrolein and acetic acid, which may or may not be artefacts, were identified. Addition of the sensory irritation effects of the residual reactants and all the identified compounds could not explain the observed sensory irritation effect. This suggests that one or more strong airway irritants were formed. Since limonene is common in the indoor air, and ozone is infiltrated from outdoors and/or produced indoors (e.g., by photocopiers), such oxidation reactions may be relevant for indoor air quality.

Determination of ozone removal rates by selected building products using the FLEC emission cell.

Ozone removal by 16 aged (older than 1-120 months) but unused building products or materials was studied in a test system that included the field and laboratory emission cell (FLEC). The ozone removal was studied at 50 +/- 1 ppb ozone, a relative humidity of 50 +/- 5%, a temperature of 21 +/- 2 degrees C, and an air flow rate of 900 +/- 10 mL min(-1) through the FLEC (air velocity ca. 3 cm s(-1)). The ozone removal increased rapidly during the first 1-2 min and either remained at a constant level or decreased asymptotically to reach a steady state-like value. The ozone removal profiles for a given material showed good repeatability during replicate experiments. Ozone deposition velocities for the building products were calculated to be between 0.0007 cm s(-1) (lacquered ash) and 0.8 cm s(-1) (unpainted gypsum board).

Transcriptional activation analysis using bioluminescent reporter assays.

Analysis of promoter and enhancer DNA sequences provides the researcher with valuable information regarding the expression patterns of genes. Insertion of small DNA fragments containing the regulatory sequence of interest into vectors carrying reporter genes allows for the accurate quantitative analysis of the gene's expression patterns and responses to various stimuli. The use of bioluminescent reporter genes provides a simple, rapid, and inexpensive system that generates virtually no toxic or radioactive waste products. In addition, bioluminescent reporter vectors are more sensitive than previous methods such as the chloramphenicol acetyl transferase (CAT) systems, that require the use of hazardous chemicals and isotopically labeled reagents.

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 decreasedf from a trigeminal reflex, i.e., due to sensory irritation. The exposure concentration decreasing f by 50% (RD50) in the first 10 min of the exposure period was estimated to be 1,076 ppm for R-(+)-limonene and 1,467 ppm for S-(-)-limonene. Results for sensory irritation of R-(+)-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 < or =1,599 ppm and S-(-)-limonene < or =2,421 ppm. R-(+)-limonene did not influence VT below 629 ppm. S-(-)-limonene increased VT above 1,900 ppm. Both enantiomers induced a mild bronchoconstrictive effect above 1,000 ppm.

Formation of strong airway irritants in terpene/ozone mixtures.

The American Society for Testing and Materials (ASTM) mouse bioassay, which quantifies airway irritation from reduction in the respiratory rate, was used to find evidence for the formation of highly irritating substances in reactions of ozone with terpenes (common indoor volatile organic compounds (VOCs)). No-observed-effect-levels (NOELs) and concentration-effect relationships were established for ozone, (+)-alpha-pinene and R-(+)-limonene, isoprene, and some of their major reaction products. Reaction mixtures of excess terpene and ozone considerably below their NOEL concentrations resulted in significant upper airway irritation. The reduction of the respiratory rate was from 30% to about 50%, lowest for the alpha-pinene and highest for the isoprene mixture. Chemical analysis of reaction mixtures by conventional methods showed that readily identified stable products and residual reactants at the concentrations found could not account for the observed reductions of the respiratory rate, assuming additivity of the reaction products. The results suggest that, in addition to known irritants (formaldehyde, acrolein, methacrolein, methyl vinylketone), one or more strong airway irritant(s) of unknown structure(s) were formed. Future indoor air quality (IAQ) guidelines for unsaturated VOCs (e.g., terpenes) and their emission from building products may require the consideration of reactions with oxidants, like ozone. Similarly, effects of ozone-emitting equipment should be re-evaluated.

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 1 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.

Mutation of polyadenylation signals generates murine retroviruses that produce fused virus-cell RNA transcripts at high frequency.

Retroviruses act as insertional mutagens and can also capture cellular sequences through a mechanism which initially requires the generation of RNA transcripts which fail to cleave and polyadenylate correctly. The correct termination of retroviral transcripts at the 3' LTR R/U5 junction is primarily dependent on the canonical AAUAAA polyadenylation signal, so we have analyzed the effect of mutating the polyadenylation signal sequences on the properties of a selectable murine retroviral vector. Mutation of consensus polyadenylation signal sequences in the 5' and/or 3' proviral LTRs demonstrated that a UA to GG change generated larger sized virus-specific RNA, consistent with loss of normal polyadenylation. Cell clones infected with viruses generated by proviral constructs containing this mutation in the 5' LTR express either normal-length or elongated viral RNA. Fused transcripts contained the mutant polyadenylation signal, while sequence analysis was consistent with the hypothesis that premature 5' to 3' primer strand transfer was responsible for the high frequency (80%) of wild-type polyadenylation. Cells infected by viruses from constructs mutated in both 5' and 3' proviral LTRs expressed poly(A)+ viral RNA between 0.3 and 3 kb larger than normal virus in 100% of infected clones, and sequence analysis of clones derived from either infected rodent or human cells confirmed that these transcripts contained both viral and adjacent cellular sequences. While mutant virus exhibits no increased ability to alter cell phenotypes, the read-through transcripts contain both unique and repetitive cell-derived sequences and can easily be recovered using PCR techniques, suggesting that these viruses may serve as effective tools for rapidly cloning cellular sequences and generating random genomic markers for gene mapping.

Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL.

Members of the ETS gene family are known to be expressed in hematopoietic tissues and cell lines, and there is increasing evidence that ETS proteins may play a role in normal hematopoietic cell development. We demonstrate that ETS-1 can contribute to the development of an erythroid phenotype in vitro. The pluripotent erythroleukemic K562 and HEL cell lines express messages for a number of ETS genes, but only c-ETS-1 levels are elevated in response to treatment with hemin or cytosine arabinofuranoside (Ara-C), agents which induce erythroid differentiation. Furthermore, ETS-1 antisense oligonucleotides inhibit hemoglobinization of cells treated with Ara-C or hemin, and K562 and HEL cells infected with retrovirus expressing the c-ETS-1 gene exhibit a significant increase in erythroid character (as indicated by benzidine staining for hemoglobin (Hb) and surface marker analysis), a dramatic increase in responsiveness to hemin or Ara-C, and a decreased rate of proliferation (20-40% of control rates). In contrast, infection with virus expressing ETS-2 or vector sequences only causes no detectable changes in the proliferation or erythroid character of either the HEL or K562 cell lines. These data indicate a role for ETS-1 in erythroid differentiation.

Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.

The human leukemia cell line K562 can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the megakaryocytic pathway, generating morphological changes and increased expression of lineage-specific surface markers. We report that TPA-treated K562 cells also express higher levels of FLI-1/ERGB, a member of the ETS family of transcription factors. Furthermore, introduction of a retroviral construct expressing human FLI-1/ERGB into K562 cells induces changes similar to those seen following TPA treatment, including increased adherence to the surface of the culture vessel and altered size and morphology. Infected cells exhibit higher levels of the megakaryocyte marker CD41a and, to a lesser extent, CD49b. These markers, as well as virally encoded FLI-1/ERGB-specific RNA and protein, are expressed at the highest levels in the attached cell population, while the growth rate of adherent cells is reduced, and the fraction of cells in G0-G1 is increased. FLI-1/ERGB virus-infected cells also exhibit increased expression of hemoglobin, a marker of erythroid differentiation. Our results suggest FLI-1/ERGB plays a role in controlling differentiation and gene expression along the megakaryocyte/platelet pathway, and further implicate ETS-related genes in the control of multiple developmentally regulated hematopoietic genes.