The term “sick building syndrome” (SBS) has been used to describe situations in which no specific illness or cause, aside from time spent indoors, explains adverse health effects experienced by building occupants; however, the specific cause of the related symptoms has yet to be identified (USEPA, 1991
). Increasing evidence suggests that some of these health effects may be attributed to reactive indoor air chemistry, such as ozone-terpene reactions (Weschler, 2006
The dicarbonyls tested in these studies, with the exception of diacetyl, are oxygenation reaction products detected in indoor chemistry experiments. There is extensive literature available on the adverse health effects associated with glutaraldehyde (a dicarbonyl) exposure (Gannon et al., 1995
; Rideout et al., 2005
; Waters et al., 2003
), supporting its selection for testing in this system. Likewise, diacetyl is a known sensitizer, and epidemiological studies have found that exposed workers have twice the expected rates of physician-diagnosed asthma compared to the general population (Anderson et al., 2007
; Kreiss et al., 2002
; Mendell et al., 2002
). Additionally, diacetyl has received considerable attention in the investigations of worker exposure to butter flavorings (Hubbs et al., 2008
). 4-OPA has not been investigated as extensively as glutaraldehyde and diacetyl; however, recent data suggest that exposure may be responsible for certain health complaints including increased lip and skin dryness (Strom-Tejsen et al., 2007
). Research investigating the exposure-related health effects for glyoxal and methyl glyoxal is lacking.
This work describes a novel in vitro
exposure method, utilizing the VitroCell module, for the analysis of oxygenated reaction products found in the indoor environment. The system has many advantages over what has previously been described in the literature, which lack a realistic exposure atmosphere and require the addition of chemicals directly to the cell media or the evaporation of a volatile liquid (Bakand et al., 2006
; Wichmann et al., 2005
). The VitroCell exposure systems allows for continuous chemical exposures while maintaining cell viability and avoiding dehydration of the cells. Airway epithelial cells were chosen for these studies since they would be expected to be the first cells exposed to these volatized compounds in the respiratory tract and are known to produce inflammatory cytokines capable of modulating immune cell activation.
These studies investigated high concentrations (15–65 ppm) of individual dicarbonyls to establish if exposure to these compounds generated a biological effect. Because these compounds have not been measured by conventional indoor air sampling methods, a “typical” concentration is not truly known. However, based on simulated indoor chemistry data, concentrations of 10–100’s of ppb (~2.5–25 × 1011 molecules/cm) are anticipated. Results demonstrated that exposure to the structurally similar dicarbonyls caused alterations in both inflammatory cytokine mRNA and protein expression in A549 cells. There also appears to be a trend between the sensitization potential and inflammatory cytokine expression for these chemicals. Of the five dicarbonyls tested, 4-OPA is considered to be the strongest sensitizer based on the lowest EC3 value () in the LLNA. In these studies 4-OPA was also the only chemical to significantly alter all cytokines investigated and generated the highest expression levels for IL-6 and GM-CSF. Glutaraldehye, considered to be the second strongest sensitizer, significantly altered three of the four cytokines, inducing the highest expression of TNF-α.
IL-8, IL-6, TNF-α, and GM-CSF are proinflammatory mediators produced by macrophages and epithelial cells with suggested roles in asthma pathogenesis (Bautista et al., 2009
; Macedo et al., 2009
; Saha et al., 2009
). Epidemiological studies have associated occupational asthma with increases in IL-8 (Zhang et al., 2009
), IL-6 (Piirila et al., 2008
), GM-CSF, and TNF-α (Ogawa et al., 2006
The structurally similar dicarbonyls evaluated in this system have the potential to be oxygenated reaction products. Methyl glyoxal, 6-hydroxyhept-5-en-2-one (not synthesized), and 4-OPA are generated through the reaction of ozone + α-terpineol; glyoxal is generated by reactions with geraniol, β-ionone, and citronellol (Forester et al., 2007
; Ham et al., 2006b
); and glutaraldehyde is generated through indoor air reactions of cyclohexene + ozone (Aschmann et al., 2003
; Harrison et al., 2007
). Although not described in the literature as a reaction product based on its structure, diacetyl could be a reaction product of ozone reacting with a compound having a carbon-carbon double bond connected to a methyl group and a methyl ketone (-C(=O)CH3
) group. The frequent incidence of ozone in indoor ventilated air along with the ubiquitous presence of terpenes in the indoor environment favors the occurrence of ozone-terpene indoor chemistry, which generates these types of reaction products. Ozone-terpene reactions are extremely complex and can generate a variety of products such as dicarboxylic acid, dicarbonyls, and oligomers. Ozone-terpene reactions also produce the hydroxyl radical (OH), which reacts with terpenes in addition to a wide range of hydrocarbons, possibly further contributing to the formation of indoor oxygenated reaction products.
In order to more closely simulate an indoor environment, the products of an α-terpineol + ozone reaction were also evaluated in these studies. However, the trend of increased inflammatory cytokines did not carry over when the cells were exposed to the α-terpineol + ozone reaction. One possible explanation for the noncongruent results is dicarbonyl exposure concentrations as the α-terpineol + ozone reaction used in the latter studies generated much lower concentrations of 4-OPA and methyl glyoxal than those used in initial experiments. However, consistent with results described in the literature, changes in gene expression were observed after individual exposures to terpenes and ozone (Wilkins et al., 2003). It is important to note that this work is not focused on the known effects caused by exposure to the parent compounds, but rather seeks to identify enhanced alterations in inflammatory responses, caused by oxygenated reaction products.
While the results from the individual dicarbonyl and ozone/α-terpineol reaction product studies were not consistent, limited exposure conditions and complexity of the ozone-terpene reaction may also be important factors that can influence the effects of dicarbonyl exposure in an actual indoor environment. While individuals often spend the majority of a 24-h day indoors, a 6-h exposure was the longest duration the cells would tolerate in this in vitro system. In addition, for these studies, cells were exposed only to the oxygenated reaction products generated from the α-terpineol + ozone reaction. In an indoor environment, individuals are exposed to numerous potential oxygenated reaction products generated from ozone-initiated reactions. While there is a likely possibility that products can be generated from these types of reactions, the identification can be challenging. For example, the dicarbonyls generated from the reaction of ozone and α-terpineol only account for ~34% of the total reaction products. The “missing” chemicals generated from these indoor air reactions () along with the detailed oxidation pathways still need to be identified and investigated to advance our knowledge of indoor air exposure-related health effects.
While the concentrations of the individual chemicals in the Teflon chambers are higher than those likely to be found in most indoor environments, it is important to consider the cumulative effect of these structurally similar indoor contaminants. Comparable results were observed in these studies for all dicarbonyls tested suggesting the exposure-related health effects and biological mode of action may also be similar. Given the abundance of oxygenated reaction products possible in the indoor environment along with the potential for similar types of health effects, exposure to small amounts of individual chemicals could cause amplified responses when present in a mixture. Structure-activity relationships (SARs), which utilizes a combination of computational biology and statistics, have been developed to identify a link between structure and biological activity. There has been increased attention and promising evidence, focused on the development of models to predict respiratory sensitizers based on chemical structure (Jarvis et al., 2005
There has been an unexplained increase in the incidence of asthma and allergies in the developed world over the last 30–40 years. More air-tight buildings, a wide range of new building materials, association of VOCs with respiratory conditions, and a greater amount of time spent indoors suggest the possibility that exposure to VOCs may contribute to this increase of adverse health effects. Attempts have been made to regulate VOCs exposures based on chemical class, mixtures, and suspected health effects by organizations including the Health Council of the Netherlands (2000)
, Commission of the European Communities (1992)
, and the World Health Organization (2000) Air Quality Guidelines for Europe
, but given the complexities and variability of the indoor environment along with the large number of potential VOCs and reaction products, this has proven to be a problematic task.
These results show that dicarbonyls stimulate the release of proinflammatory mediators from lung epithelial cells and suggest that oxygenated reaction products may be contributing to the adverse health effects associated with indoor air exposure. In addition, consistent elevations in inflammatory cytokines obtained for the structurally similar compounds suggest that the effects may be caused by exposure to the summation of small amounts of reaction products. The novel system described here may provide an in vitro method based on the expression of inflammatory cytokine by airway epithelial cells to screen contaminated indoor environments and help to clarify the culprits responsible for the symptoms associated with SBS and the other diverse health complaints of workers in those environments.